Genetic improvement of bananas (banana and plantain)

at IITA: Current status and the way forward

Vroh, Bi Irie

1. Molecular and genetics studies in the SMIP II project

2. Where is IITA in the improvement of bananas

3. Next steps in banana improvement and delivery to farmers

-World production of bananas is around 100 million tons/annum

-One third of the production comes from sub-Saharan Africa (SSA)

-Provides >25% carbohydrates and 10% of the daily calories for >70 millions

people in Africa

-Grown mostly by smallholders in SSA

Some facts on bananas (Musa spp.)

Cultivated bananas are derived from two wild banana species

(M. acuminata and M. balbisiana)

-Dessert bananas

-Cooking bananas (Plantain bananas, East African highland bananas, other

cooking bananas)

-Main cultivated species are triploid, sterile, highly difficult to improve by breeding

-Although susceptible to diseases, the main landraces of cooking bananas are still

the most preferred by farmers

Major constraints to genetic improvement by conventional breeding

-Major cultivars are triploid

-Completely or partially male and female sterile

-Major cultivars are parthenocarpic

Major constraint to sustainable production is susceptibility to diseases

-Leaf spot diseases (e.g. black Sigatoka, yellow sigatoka)

-Nematodes

-Fusarium wilt

-Etc.

Major constraints to the application of modern genomic tools

-Weak knowledge of the genomes

-Weak knowledge of Musa and pathogens’ genomes

-Low number of molecular markers

-Lack of mapping populations of good sizes

Constraints and Opportunities

The Strategic Musa Improvement Project (SMIP) of IITA (1997-2001 & 2002-2006)

Funded by: Directorate General for Development Cooperation (Belgium)

-Conventional breeding

-Agronomy

-Biotechnology

Breeding and delivery of resistant varieties of cooking bananas

Molecular and genetic targets

Molecular characterization

-Musa genomes

-Somaclonal variations

-Pathogens

-Mycosphaerella fungi

-Nematodes

Genetics of agronomic traits and molecular markers

-Resistance to black Sigatoka disease

-Parthenocarpy

-Dwarfism/Plant height

-Earliness

-Apical dominance

Chemical mutagenesis using ethane methyl sulfonate (EMS)

Identification of Musa genomes

-Main cooking bananas are AAA, AAB or ABB, with the A and the B sub-genomes

of Musa carrying different agronomic traits

-Production of unreduced gametes in Musa crosses

-Breeding programs need to identify the ploidy and the genome

composition of progenies for efficient decision making

A1A2 x A3B1

A1A3/ A1A2A3/ A2B1/ A1A2B1/ etc. (Breeder???)Progenies

Parents

-Ploidy analysis

-Genome specific molecular probes

Accurate decisions for breeding

Markers specific to the A and B genomes of Musa found

A genome

B genome

DNA markers differentiating the A from the B genome

Screening of diversity panels

3 PCR markers available

Results of diversity analysis, marker screening and design

Use of genome specific probes

Analysis of somaclonal variations in bananas

-Genetic integrity is of primary importance in germplasm conservation, plant breeding and

variety dissemination

-IITA conserves bananas and other vegetatively propagated crops in vitro

-Germplasm is maintained over years and distributed to national and international requesters

-Unexpected variations can occur during in vitro maintenance (somaclonal)

to generate off-types with or without agronomic value

-Somaclonal variations are of genetic and epigenetic origins but the molecular basis remains to be

elucidated

-To provide true-to-type materials it is critical to track those variations and to possibly link them

to traits

Check for somaclonal variations in germplasm

Analysis of somaclonal variations in bananas

-Methylation-Sensitive Amplification Polymorphism (MSAP)

Accession

Name

Accession description Genome

composition

b Provider

Calcutta 4 a Wild Musa acuminata subsp.

Burmanica (Clone C4)

AA Philippine

Montpellier a Wild Musa balbisiana (clone

MPL)

BB India

Neypoovan Diploid landrace AB Burundi

Agbagba Plantain banana landrace AAB Nigeria

Bluggoe Cooking banana landrace ABB Cameroon

Note:a Musa acuminata and M. balbisiana are the progenitors of the cultivated bananas

b From the IITA Musa database www.IITA.org

Characterization of somaclonal variations in banana germplasm

-Putative functions of the variation assessed

-Sequences registered in public genomic databases

Examples:

GenBank Acc# ET165586 to ET165601 (US_NCBI)

2nd round amplification of variant band by PCR

Sequence of a variant fragment in Bluggoe (ABB genome)

-In vitro culture of meristems and sub-cultures

&-MSAP techniques

-Plants tagged & transferred to the field to link

genomic variations to phenotypes

Sequencing

Results

-% variations: -5.6% in vitroplants

Genbank

registration

number

Hits in TIGR* plant

transcript assemblies

(protein)

Putative Functions

ET165586 None -

ET165587 BE033387

(Sucrose synthase)

Cellulose, callose, starch formation, energy

production [38]

ET165588 BX254671(Hypothetical protein) Unknown

ET165589 TA21638_47664

(Galactosyltransferasefamily

protein)

Biosynthesis of di-, oligo- and

polysaccharides.

(e.g. plant cell wall) [31]

ET165590 TA54845_3847

(Nucleotidyl transferase)

Repair of the 3′ terminal sequence of tRNA

molecules [39] Protein synthesis

ET165591 BF053442

(Cinnamic acid hydroxylase)

Cytochrome P450-dependent hydroxylase

involved

in the biosynthesis of rosmarinic acid [33].

Antimicrobial, antiviral, antibacterial

properties.

Role in Plant defense against

ET165592 TA4172_4679(Cytochrome P450

like_TBP)

Biosynthesis of secondary products,

hormones, defense

compounds, detoxification of herbicides

(http://arabidopsis-

p450.biotec.uiuc.edu/About_P450s.shtml)

ET165593 None -

ET165594 TA1_3933(Putative senescence-

associated protein)

Cell, organ or whole plant death

Comparison to Genomic databases

-See GenBank at www.NCBI.nlm.nih.gov

-Vroh-Bi et al. Plant Science (submitted)

Genetic Research and Breeding at IITA

Breeding for

resistance/Tolerance

-Development of

resistant varieties

-Delivery to farmers

Food security and

Improved livelihood

Characterization of the

Pathogens

-Genetic diversity

-Identification

-Early diagnostics

Increased efficiency

in breeding & in disease

management

-Understanding the

genetics of resistance

-Identifying major genes

& manipulation in crosses

Increased efficiency

in breeding

Analysis of resistance

to pests and diseases(e.g. black Sigatoka, nematodes)

In collaboration with Ranajit, Danny and Biodun

Understanding the genetics of resistance to BS

-Two segregating populations

-M. acuminata Calcutta 4 selfed (AA)

-M. acuminata Calcutta 4 x M. balbisiana Montpellier (AB)

-False horn plantain banana (Agbagba) as susceptible control

-M. acuminata C4 as resistance reference

-M. balbisiana Montpellier included

-Infection of detached leaf in vitro

-Analysis of segregating proportions

Genetics of BS resistance

Days after inoculation

5 10 15 20 25 30 35 40 45

Lea

f a

rea

in

fecte

d (

%)

0

20

40

60

80

100

Agbagba

1064_89

MPL

1064_53

Calcutta 4

1064_37

Genetic analysis of resistance to black Sigatoka

AB's

AUDPC MIDPOINT

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

FR

EQ

0

10

20

30

40

50

FREQ

The resistance is quantitative (e.g. distribution of AUDPC)

-in M. acuminata selfed progenies

-in M. acuminata x M. balbisiana progenies

Results

Analysis of the segregating populations

AABBCC AABBCc AABbCC AABbCc AaBBCC AaBBcC AaBbCC AaBbCc

AABBCc AABBcc AABbCc AABbcc AaBBCc AaBBcc AaBbCc AaBbcc

AABbCC AABbCc AAbbCC AAbbCc AaBbCC AaBbCc AabbCC AabbCc

AABbCc AABbCc AAbbCc AAbbcc AaBbCc AaBbcc AabbCc Aabbcc

AaBBCC AaBBCc AaBbCC AaBbCc aaBBCC aaBBCc aaBbCC aaBbCc

AaBBCc AaBBcc AaBbCc AaBbcc aaBBCc aaBBcc aaBbCc aaBbcc

AaBbCC AaBbCc AabbCC AabbCc aaBbCC aaBbCc aabbCC aabbCc

AaBbCc AaBbcc AabbCc Aabbcc aaBbCc aaBbcc aabbCc aabbcc

DDEEIIFF DDEEIIFf DDEEIiFF DDEEIiFf DDEeIIFF DDEeIIFf DDEeIiFF DDEeIiFf DdEEIIFF DdEEIIFf DdEEIiFF DdEEIiFf DdEeIIFF DdEeIIFf DdEeIiFF DdEeIiFf

DDEEIIFf DDEEIIff DDEEIiFf DDEEIiff DDeeIIFf DDEeIIff DDEeIiFf DDEeIiff DdEEIIFf DdEEIIff DdEEIiFf DdEEiiFf DdEeIIFf DdEeIIff DdEeIiFf DdEeIiff

DDEEIiFF DDEEIiFf DDEEiiFF DDEEiiFf DDEeIiFF DDEeIiFf DDEeIiFF DDEeIiFf DdEEIiFF DdEEIiFf DdEEiiFF DdEEiiFf DdEeIiFF DdEeIiFf DdEeiiFF DdEeiiFf

DDEEIiFf DDEEIiff DDEEiiFf DDEEiiff DDEeIiFf DDEeIiff DDEeiiFf DDEeiiff DdEEIiFf DdEEIiff DdEEiiFf DdEEiiff DdEeIiFf DdEeIiff DdEeiiFf DdEeiiff

DDEEIIFF DDEEIIff DDEEIiFf DDEEIiff DDEeIIFf DDEeIIff DDeeIiFf DDEeIiff DdEEIIFf DdEEIIff DdEEIiFf DdEEIiff DdEeIIFf DdEeIIff DdEeIiFf DdEeIiff

DDEeIiFf DDEeIIff DDEeIiFf DDEeIiff DDeeIIFf DDeeIIff DDeeIiFf DDeeIiff DdEeIIFf DdEEIIff DdEeIiFf DdEeIiff DdeeIIFf DdeeIIff DdeeiiFf DdeeIiff

DDEeIiFF DDEeIiFf DDEeiiFF DDEeiiFf DDeeIiFF DDeeIiFf DDeeiiFF DDeeiiFf DdEeIiFF DdEeIiFf DdEeiiFF DdEeiiFf DdeeIiFF DdeeiiFf DdeeiiFF DdeeiiFf

DDEeIiFf DDEeIiff DDEeiiFf DDEeiiff DDeeIiFf DDeeIiff DDeeiiFf DDeeiiff DdEeIiFf DdEeIiFf DdEeiiFf DdEeiiff DdeeIiFf DdeeiiFf DdeeiiFf Ddeeiiff

DdEEIIFF DdEEIIFf DdEEIiFF DdEEIiFf DdEeIIFF DdEeIIFf DdEeIiFF DdEeIiFf ddEEIIFF ddEEIIFf ddEEIiFF ddEEIiFf ddEeIIFF ddEeIiFf ddEeIiFF ddEeIiFf

DdEEIIFf DdEEIIff DdEEIiFf DdEEIiff DdEeIIFf DdEeIIff DdEeIiFf DdEeIiff ddEEIIFf ddEEIIff ddEEIiFf ddEEIiff ddEeIIFf ddEeIiff ddEeIiFf ddEeIiff

DdEEIiFF DdEEIiFf DdEEiiFF DdEEiiFf DdEeIiFF DdEeIiFf DdEeiiFF DdEeiiFf ddEEIiFF ddEEIiFf ddEEiiFF ddEEiiFf ddEeIiFF ddEeiiFf ddEeiiFF ddEeiiFf

DdEEIiFf DdEEIiff DdEEiiFf DdEEiiff DdEeIiFf DdEeIiff DdEeiiFf DdEeiiff ddEEIiFf ddEEIiff ddEEiiFf ddEEiiff ddEeIiFf ddEeiiff ddEeiiFf ddEeiiff

DdEeIIFF DdEeIIFf DdEeIiFF DdEeIiFf DdeeIIFF DdeeIIFf DdeeIiFF DdeeIiFf ddEeIIFF ddEeIIFf ddEeIiFF ddEeIiFf ddeeIIFF ddeeIiFf ddeeiiFF ddeeIiFf

DdEeIIFf DdEeIIff DdEeIiFf DdEeIiff DdeeIIFf DdeeIIff DdeeIiFf DdeeIiff ddEeIIFf ddEeIIff ddEeIiFf ddEeIiff ddeeIIFf ddeeIiff ddeeIiFf ddeeIiff

DdEeIiFF DdEeIiFf DdEeiiFF DdEeiiFf DdeeIiFF DdeeIiFf DdeeiiFF DdeeiiFf ddEeIiFF ddEeIiFf ddEeiiFF ddEeiiFf ddeeIiFF ddeeiiFf ddeeiiFF ddeeiiFf

DdEeIiFf DdEeIiff DdEeiiFf DdEeiiff DdeeIiFf DdeeIiff DdeeiiFf Ddeeiiff ddEeIiFf ddEeIiff ddEeiiFf ddEeiiff ddeeIiFf ddeeiiff ddeeiiFf ddeeiiff

4 genes with assumptions

3 genes in each parental species with assumptions

-Heterozygosity at all loci

-Incomplete dominance

-Complementary actions of genes

-Additive actions of alleles within loci

Mode of action of the genes

Segregation ratio is 27R:37S in both populations

Three “major” recessive genes in M. acuminata (C4)

Three “major” recessive genes in M. balbisiana (Montpellier)

Conclusions

Back to the origin of triploid edible bananas and plantains

?

Testing and validating the trihybrid model

-Dessert bananas (AAA): unreduced AA x reduced A

-African highland bananas (AAA): unreduced AA x reduced A

-Plantain bananas (AAB): unreduced AA x reduced B

-Other cooking bananas (ABB): reduced A x unreduced BB

Triploid bananas: born to be susceptible to BS

Susceptibility in dessert bananas (AAA genome)

Case 1: resistant unreduced x resistant reduced (14.28% resistant)

Case 2: resistant unreduced x susceptible reduced (14.28% resistant)

Case 3: susceptible unreduced x susceptible reduced (100% susceptible)

X

Susceptible acuminata

unreduced

AAbbCC

AAbbCc

AAbbcc

AABbCC

AABbCc

AABbcc

aaBBCC

aaBBCc

aaBBcc

aABBCC

aABBCc

aAbBBcc

AABBCC

AABBCc

AABBcc

AaBbCC

Susceptible

acuminata reduced

AbC

Abc

ABc

aBC

aBc

ABC

abc

All dessert bananas are susceptible to BLSD

Most likely scenario is Case 3

Susceptibility in plantain bananas (AAB genome)

Case 1: resistant unreduced x resistant balbisiana reduced (25% resistant)

Case 2: susceptible unreduced x resistant balbisiana reduced (100% susceptible)

Susceptible acuminata

unreduced

AAbbCC

AAbbCc

AAbbcc

AABbCC

AABbCc

AABbcc

aaBBCC

aaBBCc

aaBBcc

aABBCC

aABBCc

aAbBcc

AABBCC

AABBCc

AABBcc

AaBbCC

balbisiana

reduced

ABC

ABc

AbC

aBC

aBc

abC

abc

X

Plantain bananas (AAB) are susceptible to

BLSD

Most likely scenario is Case 2

Can we go back to synthesize resistant edible triploids?

Case 1: Susceptible acuminata reduced x trihybrid balbisiana (14.28% resistant)

Case 2: Resistant acuminata reduced x trihybrid balbisiana (12.5% resistant)

Case 1: resistant unreduced x resistant reduced (14.28% resistant)

Case 2: resistant unreduced x susceptible reduced (14.28% resistant)

Case 3: susceptible unreduced x susceptible reduced (100% susceptible)

Dessert and EAH bananas (AAA)

Plantain bananas (AAB)

Other cooking bananas of ABB genome

Case 1: resistant unreduced x resistant balbisiana reduced (25% resistant)

Case 2: susceptible unreduced x resistant balbisiana reduced (100% susceptible)

Pathway model Reaction

mfR1mfR1/--/--

--/mfR2mfR2/--

--/--/mfR3mfR3Else (inhibition of either one step) SusceptibleX

P1mfr1

P2mfr2

Resistant mfr1-/mfr2-/mfr3-P3mfr3

Genotypes

Proposed pathway model for the genetics of host response to black Sigatoka and corresponding genotypic

combinations in the diploid species. P1, P2, and P3 are precursors.

1. Assess the individual contribution of each gene to BS resistance

2. Tag the genes phenotypically

3. Tag the genes with molecular markers (QTL mapping)

What breeders need from the pathway and genes above?

Identification of 33 resistant genotypes (currently maintained in the field)

Causative agents of leaf spot diseases

-Black Sigatoka (M. fijiensis)

-Yellow Sigatoka (M. musicola)

-Leaf speckle disease (M. eumusae)

-Better disease management

-Better quarantine decisions

-Efficient breeding programs

Accurate diagnostics/Early detection/improved selection pressure

Identification of Mycosphaerella species of bananas

Sequencing and sequence comparison at specific genes

(Ribosomal, actin and tubulin genes)

Sequence alignment of Nigerian isolates (Q1_ISO_11 to 57) on reference GenBank references of

M. fijiensis (Fiji), M. musicola (Musi), M. eumusae (Eumu), and M. musae (Musae). Inter- and intraspecific SNP

variations are highlighted. Isolates Q1_ISO_11, Q1_ISO_20, Q1_ISO_30, Q1_ISO_54, and Q1_ISO_57 were

identified as M. eumusae.

Identification using SNPs in rDNA

-Identity of isolates further confirmed at β-tubulin and actin gene sequences

-IITA opportunity grant for diagnostic tools

-Additionnal collections in the Ivory Coast

-Possibility to link with CARBAP collections (Cameroon, Gabon, Congo)

-Possible link with the international program on reduction of pesticides in

banana production

-M. fijiensis is the predominant in Nigeria

-M. eumusae is present

-M. musicola is absent

-Sequences registered in GenBank at www.ncbi.nlm.nih.gov

-Conclusions compiled in Zandjanakou-Tachin et al. Plant pathology (Revised)

Analysis of parthenocarpy, the trait that makes bananas the fruits we eat

1. AFLP analysis of a segregating population

2. Comparison of parthenocarpic diploids to non-parthenocarpic diploids

-Comparative analysis at specific genes (GID and DELLA)

-Sequencing and SNP analysis

Cultivar SF247 (Parthenocarpic)

Wild Borneo (Seeded)

Segregation for parthenocarpy

X

Segregation of AFLP markers in Borneo x SF247

M. acuminata malaccensis Borneo (seeded) x SF247 (AA cultivar)

Segregating population

-AFLP and SSR markers tested

-Traits scored in 180 individuals

Parthenocarpic versus non-parthenocarpic accessions at GID

Comparative analyses using GID sequences rice, cotton, Arabidopsis and wheat

isolation in bananas

DELLA

SNPs

Sequence comparison

Parthenocarpy

How useful are these words for breeders?

1. Most useful agronomic traits are present in seeded wild species

2. Hard shell seeds are undesired in edible bananas

3. Cultivated x wild crosses result to useful progenies with unwanted seeds

4. Bananas are long cycle crops (average of 1 year from planting to production)

5. DNA markers (AFLP, SNP etc.) will be very useful in selecting against

seeds at the seedling stage, therefore accelerating selection for new varieties

Characterization of Nematodes

1. Collections across Nigeria

2. Optimization of DNA extraction from single

nematodes (Water or TE)

3. Sequencing

-Inter and intraspecific variations

-Discovery of new species

4. Diagnostics and increased efficiency in breeding

Genotpe Nema spp Root

weight

Root necrosis

index

Gall

index

Small

leison

Big

leison

8532-1 Praty. 297.21 34.67 0.00 2.00 4.00

Calcutta 4 Praty 146.46 42.25 0.00 2.00 1.75

Heva Praty 117.87 19.75 0.00 2.00 4.00

Km5 Praty 309.51 18.00 0.75 2.00 4.00

M balb Praty 363.65 22.50 0.25 2.00 3.88

Valery Praty 182.88 14.00 0.38 2.00 3.50

LSD Praty 86.03 14.49 0.72 0.00 1.40

F value Praty 9.83 5.15 1.22 0.00 2.81

5. Possibility of collaboration with CNRA (Ivory Coast)

3. Hybrids from IITA under evaluation in farmers’ fields

-EAHB background with Matoke-like characteristics: >5

-Plantain banana background: >5 hybrids of the PITA series

-Cooking bananas ABB background: BITA 3

2. Where is IITA in genetic improvement of bananas?

1. Genetic improvement at IITA has targeted mostly resistance to BS

2. Relatively narrow gene pool

(Obino Lewai, Mbi Egome and Bobby Tannap crossed by Pisang Lilin and Calcutta 4)

Farmer and consumer preferences

Long and big finger size is the deal!

Big and long finger Agbagba/Ebang/ etc.

-Have the highest market value

-Superior to new hybrids

-Susceptible to many diseases and pests

-Completely sterile

Varieties Total carotenoids

Obino L'Ewai 20.120

PITA-2 18.440

Mbi Egome 14.700

Maduranga 9.070

CRBP39 8.945

PITA-23 8.522

FHIA-23 8.307

PITA-14 6.700

PITA-16 6.290

SH3640 5.915

Agbagba (Control) 4.120

USDA – dessert bananas 0.260

USDA – plantain bananas 4.570

Top ten varieties of bananas with respect to the contents (μg g-1) of total carotenoids

-Good nutritional value

-Good levels of tolerance to black Sigatoka

IITA hybrids

IITA and the planting materials constraints

-Shortage of good planting materials prevents expansion of

banana production in SSA

-IITA has easy-to-grasp techniques for:

-Mass production of seedlings

-Sanitation of planting materials

-IITA has many partners and collaborators

Material testing and dissemination

So what’s next?

3. Next steps in banana improvement and delivery to farmers

1. Improved agronomic practices and backstopping NARS, NGO and the private

sector in:

-Mass production and distribution of landrace and hybrid seedlings

-Postharvest processing for added value

2. Shift towards improving the preferred landraces (Pre-breeding operations)

1. Sterility: can we make them fertile?

2. Production of double haploids?

3. Mutagenesis?

4. Genetic transformation?

3. Broaden the gene pool for new breeding schemes

1. Shift towards improving the preferred landraces (Pre-breeding operations)

Chromosome doubling

Plantain Agbagba (AAB)

EMS treatment to induced mutations in vitro

-Meristems treated with various concentrations of EMS

-Duration of treatment from 2 hours to 72 hours

-In vitro culture of meristems and acclimatation

Hundreds of plants produced and transferred

to the field for evaluation

2. Backstopping NARS, NGOs and the private sector

-Training StudentsMartine Tachin: PhD. student - University of Lome, Togo

Chinyere Anabgogu: MSc. student - University of Ibadan, Nigeria

Winifred Mbah: MSc. student - University of Abeokuta, Nigeria

Bassey Blessing: IT student - University of Calabar, Nigeria

Sandra Nnady: Youth Corper - University of Ebonyi State, Nigeria

Fawibe feyikemi: IT student - University of Technology

Ogbomosho, Nigeria

Elizabeth Oraeki, IT student - Federal University of Technology,

Owerri

-Training Professionals (Molecular)Prof. Zoro Arsene: University of Abobo-Adjame Ivory Coast

Dr. Claudius Cole Biodun: University of Ibadan, Nigeria

Evaluation of drought tolerance

-Hosting professionals, Communication,

Publications, Resource mobilization

-Supervision of flow cytometry operations

Cassava/yam/bananas/Vigna

-Lab works for Germplasm unit

-African Yam bean diversity (done)

-Somaclonal variation in yam (in progress)

-Vigna ploidy (in progress)

-Cowpea gene flow (in progress)

Not present in the picture:Abdou Tenkouano, Ranajit Bandyopadhyay, Peter Ojiambo,

Claudius Cole, Danny Coyne, Bamisaye Bukola and Sandra Nnadi

Acknowledgements

Thank you