Measuring genetic variation of tarnished plant bug, Lygus lineolaris, over temporal and spatial...
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Transcript of Measuring genetic variation of tarnished plant bug, Lygus lineolaris, over temporal and spatial...
Measuring genetic variation Measuring genetic variation of tarnished plant bug, of tarnished plant bug, Lygus lineolarisLygus lineolaris, over , over
temporal and spatial scalestemporal and spatial scales
O. P. Perera, Jeff Gore, Gordon O. P. Perera, Jeff Gore, Gordon Snodgrass & Brian Scheffler & Craig Snodgrass & Brian Scheffler & Craig
AbelAbel
USDA-ARS, Stoneville, MississippiUSDA-ARS, Stoneville, Mississippi
IntroductionIntroduction
IntroductionIntroduction
Very few markers suitable for Very few markers suitable for population genetic studies population genetic studies availableavailable
No microsatellite markers No microsatellite markers for H. virescens, Helicoverpa zea, or Lygus species
IntroductionIntroduction
Simple Sequence Repeat (SSR) Simple Sequence Repeat (SSR) or microsatellite markers are or microsatellite markers are widely used in population widely used in population geneticsgenetics
Highly variable DNA regionsHighly variable DNA regionsCo-dominant markersCo-dominant markersRepeats of two to six Repeats of two to six
nucleotides are commonly usednucleotides are commonly used
MethodsMethods
Partial genomic libraries for Partial genomic libraries for each specieseach species
Enrich the library for SSR Enrich the library for SSR sequences using biotin sequences using biotin labeled repeat oligos and labeled repeat oligos and magnetic beads followed by magnetic beads followed by PCR amplificationPCR amplification
MethodsMethodsOligo GroupOligo Group
(Hyb. (Hyb. Temp.)Temp.)
Oligonucleotide SequencesOligonucleotide Sequences
Group 1Group 1
(58(58C)C)(AC)(AC)1313, (AACC), (AACC)55, (AACG), (AACG)55, , (AAGC)(AAGC)55, (AAGG), (AAGG)55, (ATCC), (ATCC)55
Group 2Group 2
(52(52C)C)(AG)(AG)1212, (AAC), (AAC)66, (AAG), (AAG)88, (ACT), (ACT)1212, , (ATC)(ATC)88
Group 3Group 3
(48(48C)C)
(AAAC)(AAAC)66 , (AAAG) , (AAAG)66, (AAAG), (AAAG)66, , (AATC)(AATC)66, (AATG), (AATG)66, (ACAG), (ACAG)66, , (ACCT)(ACCT)88, (ACTC), (ACTC)66, (ACTG), (ACTG)66
Group 4Group 4
(42(42C)C)(AAAT)(AAAT)88, (AACT), (AACT)88, (AAGT), (AAGT)88, , (ACAT)(ACAT)88, (AGAT), (AGAT)88
Genomic DNA
Digest with 4-base cutter
Ligate adapter to ends
Denature & hybridize withBiotin labeled SSR oligos
Magnetically capturerepeat sequenceshybridized to biotinlabeled SSR oligos
Perform a 2nd roundof Enrichment
PCR amplifyEnriched sequences
Clone & sequence
Validate primer pairs
Analyze sequences and design primer pairs
MethodsMethods
Genotyping with ABI3730xl Genotyping with ABI3730xl instrument and GeneMapper instrument and GeneMapper softwaresoftware
Initial screening with pooled DNA Initial screening with pooled DNA samples of laboratory insectssamples of laboratory insects
Selected primer pairs used for Selected primer pairs used for analyzing field samplesanalyzing field samples
Data analysis with PopGene v1.32Data analysis with PopGene v1.32
Results: Results: H. virescensH. virescens
DNA sequences of 192 clonesDNA sequences of 192 clones147 clones contained repeats147 clones contained repeats96 primer pairs synthesized96 primer pairs synthesized20 polymorphic primer pairs 20 polymorphic primer pairs
producing 1 or 2 peaks per producing 1 or 2 peaks per individual identifiedindividual identified
Results: Results: H. zeaH. zea
DNA sequences of 192 DNA sequences of 192 clonesclones
34 primer pairs synthesized 34 primer pairs synthesized for 34 unique clonesfor 34 unique clones
12 polymorphic primer pairs 12 polymorphic primer pairs producing 1 or 2 peaks per producing 1 or 2 peaks per individual identifiedindividual identified
Repeat typeRepeat Sequence H. virescens H. zea
Di-nucleotide AC 9 1
AG 6 0
Tri-nucleotide
AAG 5 0
AGT 3 0
ATA 1 0
ATG 39 3
GTT 1 0
TAA 1 2
Tetra-nucleotide ACAG 53 14
ACAT 1 3
ACGG 3 0
CAAA 9 7
CAAC 4 0
CATA 4 0
GATG 2 0
GTCA 1 1
GTTC 1 0
TAAA 1 0
TTAA 1 0
Penta-nucleotide
CTAAC 1 0
TCCTC 1 0
Octa-nucleotide TTTGTCTG 1 1
Total 147 32
Repeat type Repeat Sequence H. virescens H. zea
LocusLocus (Repeat Sequence)(Repeat Sequence)Repeat #Repeat #Amplicon size range Amplicon size range
(bp)(bp)
11 (TGA)(TGA)77 169-188169-188
22 (AACA)(AACA)44 151-169151-169
33 (CAT)(CAT)77 167-177167-177
44 (TCA)(TCA)44 151-160151-160
55 (TGA)(TGA)44 165-224165-224
66 (TGA)(TGA)44 168-202168-202
77 (TC)(TC)1010 171-191171-191
88 (TGAC)(TGAC)44 159-180159-180
The sequence and the number of repeat units at each polymorphic locus and the amplicon size range observed in the test population of H. virescens (data for 8 loci shown).
Actual number of alleles Actual number of alleles ranged from 4 to 22ranged from 4 to 22
Effective number of alleles Effective number of alleles ranged from 3 to 12ranged from 3 to 12
Rare alleles contributed to Rare alleles contributed to reduced number of effective reduced number of effective allelesalleles
LocLocusus
SamplSample Sizee Size HHoo HHee
11 156156 0.24360.2436 0.72850.7285
22 134134 0.26870.2687 0.48870.4887
33 146146 0.21920.2192 0.79990.7999
44 162162 0.27160.2716 0.57720.5772
55 8686 0.25580.2558 0.82440.8244
66 138138 0.30430.3043 0.92750.9275
77 148148 0.28380.2838 0.85570.8557
88 168168 0.30950.3095 0.73750.7375
MeaMeann 142142 0.26960.2696 0.74240.7424
St. St. DevDev 0.03030.0303 0.14590.1459
LocuLocuss
(Repeat (Repeat Sequence)Sequence)Repeat #Repeat #
Amplicon size Amplicon size range (bp)range (bp)
11 (TGA)(TGA)77 249-269249-269
22 (AGTC)(AGTC)44 116-129116-129
33 (ACAT)(ACAT)66 156-172156-172
44 (GTTT)(GTTT)44 144-232144-232
55 (GTTT)(GTTT)66 386-398386-398
66 (ATT)(ATT)44 133-136133-136
77 (TCTG)(TCTG)44 117-121117-121
88 (TCTG)(TCTG)66 211-252211-252
99 (ATG)(ATG)66 137-144137-144
The sequence and the number of repeat units at each polymorphic locus and the amplicon size range observed in the test population of H. zea (data for 9 loci shown).
Actual number of alleles Actual number of alleles ranged from 2 to 6ranged from 2 to 6
Effective number of alleles Effective number of alleles ranged from 1.3 to 4.8ranged from 1.3 to 4.8
Locus Sample Size Ho He
1 182 0.1099 0.397
2 192 0.4688 0.5027
3 192 0.3125 0.3047
4 124 0.0806 0.0774
5 192 0.4062 0.5188
6 186 0.1075 0.1031
7 164 0.122 0.1158
8 190 0.1684 0.3736
9 144 0.0417 0.067
Mean 174 0.2020 0.2733
St. Dev. 0.1543 0.1851
Lygus lineolarisLygus lineolaris microsatellites microsatellites
Eight loci suitable for population genetics Eight loci suitable for population genetics selected so farselected so far
192 insects from 2 populations analyzed192 insects from 2 populations analyzed Number of alleles ranged from 4 to 21Number of alleles ranged from 4 to 21 Effective number of alleles ranged from 1.2 Effective number of alleles ranged from 1.2
to 11.0to 11.0
LocusRepeat Sequence Sample size HoHo HoHo
1-15 (CA)8 358 0.2011 0.1909
1-26 (CA) 7 376 0.2181 0.2368
1-43 (CA) 9 368 0.8152 0.9095
2-15 (AGT) 4 374 0.5882 0.6167
2-47 (AGA) 6 370 0.6162 0.809
3-16 (CA) 7 372 0.5914 0.7241
3-60 (GAT) 7 372 0.7473 0.8527
3-91 (GAT) 6 362 0.6077 0.7556
Mean 369 0.5482 0.6369
St_Dev 0.2243 0.2756
Locus Sample Size Fis Fit Fst Nm*
1-15 358 -0.055 -0.055 0.0006 388.75
1-26 376 0.0748 0.08 0.0057 43.828
1-43 368 0.0845 0.1018 0.0189 12.985
2-15 374 0.0357 0.0477 0.0124 19.842
2-47 370 0.2404 0.2417 0.0017 145.37
3-16 372 0.177 0.1827 0.007 35.677
3-60 372 0.1198 0.1218 0.0022 111.56
3-91 362 0.1923 0.1938 0.0018 135.83
Mean 369 0.1329 0.1391 0.0071 35.094
Inbreeding Coefficients for 2 Lygus populations
* Nm = Gene flow estimated from Fst = 0.25(1 - Fst)/Fst
Other Research in Other Research in ProgressProgress
Development of microsatellites forDevelopment of microsatellites for L. L. hesperushesperus, , Sirex noctillioSirex noctillio, , Solenopsis invictaSolenopsis invicta and fungi carried by and fungi carried by SirexSirex species species ((AmylosteriumAmylosterium sp.) sp.)
Development of SNP assays for gut-specific Development of SNP assays for gut-specific genes (EcoTILLING).genes (EcoTILLING).
Sequencing of mtDNA genomes of cotton Sequencing of mtDNA genomes of cotton pest species.pest species.
Future workFuture work
Isolate and evaluate more Isolate and evaluate more SSR loci, possibly to obtain SSR loci, possibly to obtain 1-3 loci/linkage group1-3 loci/linkage group
Develop SSR based linkage Develop SSR based linkage maps maps
AcknowledgementsAcknowledgements
CollaboratorsCarlos Blanco Brian Scheffler
Technical SupportMid-South Area Genome Center
Linda Ballard Mary Duke X. Liu Sheron Simpson
SIMRUTorey Looft