Chromosomes and molecular cytogenetics of oil palm: impact for breeding and genetics PIPOC
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Oil palm molecular cytogenetics & genomics KLCC 7 th October 2015 Pat Heslop-Harrison [email protected] www.molcyt.com Twitter, YouTube and Slideshare
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Transcript of Chromosomes and molecular cytogenetics of oil palm: impact for breeding and genetics PIPOC
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
1062015
Oil Palm
32 chromosomes DAPI
TTTAGGG telomere
45S rDNA (1 major pair + minor)
5S rDNA (1 major + minor)
Telomere (TTTAGGG)n
Centromere function
Genes small proportion
The Karyotype and Repetitive DNA
Several repeats are broadly proximal others cover whole arms
The Karyotype and repetitive DNA
SSRs tend to be distal in oil palm
Organellar
Repetitive DNA sequences
Plant Nuclear Genome
DNA Retro-
Telomeres Microsatellites
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
Organellar
Viral
Transgenes
Genes
Dispersed Transposable
Elements
Tandem
Centro-meres
Structural
Telo-meres
Micro-satellites
Repeated genes
Sub-telomeric
rRNA
Blocks
Others
Organelle sequences from chloroplasts or
mitochondria
Sequences from viruses Agrobacterium or other
vectors
Transgenes introduced with molecular biology
methods
Genes regulatory and non-coding single copy sequences
Dispersed repeats Transposable Elements
Repetitive DNA sequences
Plant Nuclear Genome
Tandem repeats
DNA transposons copied and
moved via DNA
Retrotransposons amplifying via an RNA intermediate
Centromeric repeats
Structural components of chromosomes
Telomeric repeats
Simple sequence repeats or
microsatellites
Repeated genes
Subtelomeric repeats
45S and 5S rRNA genes
Blocks of tandem repeats at discrete chromosomal loci
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
httpmolcytorg20120818plant-nuclear-genome-composition
Other genes
Plant genome components ndash full for Slideshare
Repetitive DNA
The major genomic component
Actively evolving amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing
Current methods mask out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization
variation and consequences of these sequences
Repeats in assemblies cause problems
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
1062015
Oil Palm
32 chromosomes DAPI
TTTAGGG telomere
45S rDNA (1 major pair + minor)
5S rDNA (1 major + minor)
Telomere (TTTAGGG)n
Centromere function
Genes small proportion
The Karyotype and Repetitive DNA
Several repeats are broadly proximal others cover whole arms
The Karyotype and repetitive DNA
SSRs tend to be distal in oil palm
Organellar
Repetitive DNA sequences
Plant Nuclear Genome
DNA Retro-
Telomeres Microsatellites
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
Organellar
Viral
Transgenes
Genes
Dispersed Transposable
Elements
Tandem
Centro-meres
Structural
Telo-meres
Micro-satellites
Repeated genes
Sub-telomeric
rRNA
Blocks
Others
Organelle sequences from chloroplasts or
mitochondria
Sequences from viruses Agrobacterium or other
vectors
Transgenes introduced with molecular biology
methods
Genes regulatory and non-coding single copy sequences
Dispersed repeats Transposable Elements
Repetitive DNA sequences
Plant Nuclear Genome
Tandem repeats
DNA transposons copied and
moved via DNA
Retrotransposons amplifying via an RNA intermediate
Centromeric repeats
Structural components of chromosomes
Telomeric repeats
Simple sequence repeats or
microsatellites
Repeated genes
Subtelomeric repeats
45S and 5S rRNA genes
Blocks of tandem repeats at discrete chromosomal loci
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
httpmolcytorg20120818plant-nuclear-genome-composition
Other genes
Plant genome components ndash full for Slideshare
Repetitive DNA
The major genomic component
Actively evolving amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing
Current methods mask out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization
variation and consequences of these sequences
Repeats in assemblies cause problems
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Telomere (TTTAGGG)n
Centromere function
Genes small proportion
The Karyotype and Repetitive DNA
Several repeats are broadly proximal others cover whole arms
The Karyotype and repetitive DNA
SSRs tend to be distal in oil palm
Organellar
Repetitive DNA sequences
Plant Nuclear Genome
DNA Retro-
Telomeres Microsatellites
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
Organellar
Viral
Transgenes
Genes
Dispersed Transposable
Elements
Tandem
Centro-meres
Structural
Telo-meres
Micro-satellites
Repeated genes
Sub-telomeric
rRNA
Blocks
Others
Organelle sequences from chloroplasts or
mitochondria
Sequences from viruses Agrobacterium or other
vectors
Transgenes introduced with molecular biology
methods
Genes regulatory and non-coding single copy sequences
Dispersed repeats Transposable Elements
Repetitive DNA sequences
Plant Nuclear Genome
Tandem repeats
DNA transposons copied and
moved via DNA
Retrotransposons amplifying via an RNA intermediate
Centromeric repeats
Structural components of chromosomes
Telomeric repeats
Simple sequence repeats or
microsatellites
Repeated genes
Subtelomeric repeats
45S and 5S rRNA genes
Blocks of tandem repeats at discrete chromosomal loci
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
httpmolcytorg20120818plant-nuclear-genome-composition
Other genes
Plant genome components ndash full for Slideshare
Repetitive DNA
The major genomic component
Actively evolving amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing
Current methods mask out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization
variation and consequences of these sequences
Repeats in assemblies cause problems
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
The Karyotype and Repetitive DNA
Several repeats are broadly proximal others cover whole arms
The Karyotype and repetitive DNA
SSRs tend to be distal in oil palm
Organellar
Repetitive DNA sequences
Plant Nuclear Genome
DNA Retro-
Telomeres Microsatellites
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
Organellar
Viral
Transgenes
Genes
Dispersed Transposable
Elements
Tandem
Centro-meres
Structural
Telo-meres
Micro-satellites
Repeated genes
Sub-telomeric
rRNA
Blocks
Others
Organelle sequences from chloroplasts or
mitochondria
Sequences from viruses Agrobacterium or other
vectors
Transgenes introduced with molecular biology
methods
Genes regulatory and non-coding single copy sequences
Dispersed repeats Transposable Elements
Repetitive DNA sequences
Plant Nuclear Genome
Tandem repeats
DNA transposons copied and
moved via DNA
Retrotransposons amplifying via an RNA intermediate
Centromeric repeats
Structural components of chromosomes
Telomeric repeats
Simple sequence repeats or
microsatellites
Repeated genes
Subtelomeric repeats
45S and 5S rRNA genes
Blocks of tandem repeats at discrete chromosomal loci
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
httpmolcytorg20120818plant-nuclear-genome-composition
Other genes
Plant genome components ndash full for Slideshare
Repetitive DNA
The major genomic component
Actively evolving amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing
Current methods mask out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization
variation and consequences of these sequences
Repeats in assemblies cause problems
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
The Karyotype and repetitive DNA
SSRs tend to be distal in oil palm
Organellar
Repetitive DNA sequences
Plant Nuclear Genome
DNA Retro-
Telomeres Microsatellites
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
Organellar
Viral
Transgenes
Genes
Dispersed Transposable
Elements
Tandem
Centro-meres
Structural
Telo-meres
Micro-satellites
Repeated genes
Sub-telomeric
rRNA
Blocks
Others
Organelle sequences from chloroplasts or
mitochondria
Sequences from viruses Agrobacterium or other
vectors
Transgenes introduced with molecular biology
methods
Genes regulatory and non-coding single copy sequences
Dispersed repeats Transposable Elements
Repetitive DNA sequences
Plant Nuclear Genome
Tandem repeats
DNA transposons copied and
moved via DNA
Retrotransposons amplifying via an RNA intermediate
Centromeric repeats
Structural components of chromosomes
Telomeric repeats
Simple sequence repeats or
microsatellites
Repeated genes
Subtelomeric repeats
45S and 5S rRNA genes
Blocks of tandem repeats at discrete chromosomal loci
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
httpmolcytorg20120818plant-nuclear-genome-composition
Other genes
Plant genome components ndash full for Slideshare
Repetitive DNA
The major genomic component
Actively evolving amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing
Current methods mask out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization
variation and consequences of these sequences
Repeats in assemblies cause problems
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Organellar
Repetitive DNA sequences
Plant Nuclear Genome
DNA Retro-
Telomeres Microsatellites
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
Organellar
Viral
Transgenes
Genes
Dispersed Transposable
Elements
Tandem
Centro-meres
Structural
Telo-meres
Micro-satellites
Repeated genes
Sub-telomeric
rRNA
Blocks
Others
Organelle sequences from chloroplasts or
mitochondria
Sequences from viruses Agrobacterium or other
vectors
Transgenes introduced with molecular biology
methods
Genes regulatory and non-coding single copy sequences
Dispersed repeats Transposable Elements
Repetitive DNA sequences
Plant Nuclear Genome
Tandem repeats
DNA transposons copied and
moved via DNA
Retrotransposons amplifying via an RNA intermediate
Centromeric repeats
Structural components of chromosomes
Telomeric repeats
Simple sequence repeats or
microsatellites
Repeated genes
Subtelomeric repeats
45S and 5S rRNA genes
Blocks of tandem repeats at discrete chromosomal loci
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
httpmolcytorg20120818plant-nuclear-genome-composition
Other genes
Plant genome components ndash full for Slideshare
Repetitive DNA
The major genomic component
Actively evolving amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing
Current methods mask out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization
variation and consequences of these sequences
Repeats in assemblies cause problems
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Organelle sequences from chloroplasts or
mitochondria
Sequences from viruses Agrobacterium or other
vectors
Transgenes introduced with molecular biology
methods
Genes regulatory and non-coding single copy sequences
Dispersed repeats Transposable Elements
Repetitive DNA sequences
Plant Nuclear Genome
Tandem repeats
DNA transposons copied and
moved via DNA
Retrotransposons amplifying via an RNA intermediate
Centromeric repeats
Structural components of chromosomes
Telomeric repeats
Simple sequence repeats or
microsatellites
Repeated genes
Subtelomeric repeats
45S and 5S rRNA genes
Blocks of tandem repeats at discrete chromosomal loci
DNA sequence components of the plant nuclear genome Heslop-Harrison amp Schmidt 2012 Encyclopedia of Life Sciences
httpmolcytorg20120818plant-nuclear-genome-composition
Other genes
Plant genome components ndash full for Slideshare
Repetitive DNA
The major genomic component
Actively evolving amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing
Current methods mask out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization
variation and consequences of these sequences
Repeats in assemblies cause problems
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Repetitive DNA
The major genomic component
Actively evolving amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing
Current methods mask out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization
variation and consequences of these sequences
Repeats in assemblies cause problems
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al 2015 Chromosome Research
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Green Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
15
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Green Transposable element locations in diploid Brachiaria tropical forage grass
Fabiola Santos et al 2015 Chromosome Research
16
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Solanaceae ndash potato tomato peppers petunia Petunia Leader Cris Kuhlemeier with Quattrocchio Sims Mueller et al
Repetitive DNA analysis Katja Richert-Poumlggeler Trude Schwarzacher Pat Heslop-Harrison
P inflata P hybrida Paxillaris
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Please do not photograph Petunia data confidential
Reference sequences Hansen amp Heslop-Harrison Adv Bot Res 2004 httpwwwleacukblts32pubshhretrospdf
Petunia genome paper Kuhlemeier et al
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Major Genomic Components
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements
Genes
Typical Fraction
10
5
10
15
50
10
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Epigenetics Phenotype appears 5 years after tissue culture
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
06102015
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
January 23 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of Methylation
Preliminary results with anti-methyl-cytosine indicate differences between ortet right more methylated) amp mantled regenerants (left less methylated)
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
January 23 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation Status
Changes seen in retroelements in culture some reverting in regenerants
New DNA methylation pattern
Expression of copia
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Modulation of Methylation
McrBC - shows substantial reduction in methylation in tissue culture lines
Cuts methylated DNA
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
1062015
Modulation of Methylation
McrBC digests probed with gypsy clones
present only in N and T lines
Similar with copia probe
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Variation of LINEs between varieties
1 Species 1 2-12 Species 2 13 Musa HindIII digests
M EcoRI+HindIII
Similar for gypsy-elements and EnSpm transposons
Yes between varieties Maybe between parent and regenerants
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Gypsy-element pEgKB7 Ortet | Regenerant
Marginal differences seen in HpaII (cuts CCGG) and MspI (cuts CmCGG too) digests
Consistent with HPLC data
Tracks
HindIII HaeIII ApaI RsaI HpaII MspI
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
January 23 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture anti-methyl-cytosine ortet right more methylated)
mantled regenerants (left less methylated)
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA (Retro)transponsons and Dispersion copia gypsy LINE EnSpm various characteristic copy numbers and variation copia and LINEs show lsquoactivationrsquo in culture
Methylation Modulation
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
The Molecular Cytogenetics of Somaclonal Variation in Oil Palm Karyotypes Repetitive DNA Retrotransponsons and Dispersion Methylation Modulation CCGG HPLC McrBC antibodies Retroelement differences and expression
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Application of molecular cytogenetics
that to oil palm
lsquoAssessing the presencersquo DNA sequence chromosomal distribution diversity
lsquoBeyond the sequencersquo The contribution of methylation chromatin packing recombination and retroelements to genome behaviour
lsquoGenome archaeologyrsquo What has happened during evolution and what changes now
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
EvolutionEpigeneticsDevelopment
Phenotype Multiple abnormalities
Genetic changes non-reverting
Changes seen some reverting
(MaleFemale)
Normal Differentiation
Cause Chromosomal loss deletion or
translocation Gene mutation base pair
changes Telomere shortening
(Retro)transposon insertion Retrotransposon activation
SSR expansion Methylation
Heterochromatinization Chromatin remodelling
Histone modification
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
There is more information in the genome than in the sequence alone
lsquoEpigeneticsrsquo
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Oil palm molecular
cytogenetics amp genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phhmolcytcom wwwmolcytcom
Twitter YouTube and Slideshare
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Retroelements transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation
36
Plant Mol Biol 2003 May52(1)69-79
Kubis SE1 Castilho AM Vershinin AV Heslop-Harrison JS We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed and
plants regenerated from tissue culture that show mantling an abnormality leading to flower abortion Using PCR assays reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of EnSpm transposons were cloned Sequence analysis revealed the presence of a major family of LINEs in oil palm with other diverged copies Gypsy-like retrotransposons form a single homologous group whereas EnSpm transposons are present in several diverged families Southern analysis revealed their presence in low (LINEs) to medium (gypsy and EnSpm) copy numbers in oil palm and in situ hybridization showed a limited number of distinct loci for each class of transposable element No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected but different levels of sequence methylation were observed During tissue culture McrBC digestion revealed the genome-wide reduction in DNA methylation which was restored to near-normal levels in regenerated trees HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent The genomic organization of the transposable DNA elements in different oil palm species accessions and individual regenerated trees was investigated revealing only minor differences The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Repetitive DNA and the Chromosomes in the Genome of Oil Palm (Elaeis guineensis)
A CAST ILHO A VERSH IN IN and J S HESLOP -HARRISON
Annals of Botany 85 837-844 2000 doi101006anbo20001145 Like most plant genomes much of the oil palm genome (Elaeis guineensis L 2n 32) consists of repetitive DNA
sequences We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing Southern and in situ hybridization Three unrelated repetitive sequence families with no homology to known sequences showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms while simple sequence repeats of DNA (GA GATA and CAC) were clustered in the distal parts Copia-like retroelements were dispersed throughout the genome with a concentration in proximal regions but were not as abundant as in species with larger genomes Among tandemly repeated sequences a major 18S-25S rDNA site was present on a single pair of chromosomal sites often on a satellite with no visible connection to its parent chromosome A major 5S rDNA site was located on another chromosome pair variable numbers of minor sites of both rDNA families were also detected The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms but no intercalary sites of ampliregcation were detected No other major families of tandemly repeated sequences were found The molecular cytogenetic analysis and chromosome ampliregcation patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants Annals of Botany
Key words Oil palm genome evolution chromosome evolution repetitive DNA retrotransposons genetic markers
37
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
From Chromosome to Nucleus
Pat Heslop-Harrison phh4leacuk wwwmolcytcom
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Find and use biodiversity
39
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
Outputs
ndash CROPS
ndash Fixed energy
40
Inputs
ndashLight
ndashHeat
ndashWater
ndashGasses
ndashNutrients
ndash Light
ndash Heat
ndash Water
ndash Gasses
ndash Nutrients
