Molecular and genetics studies in the SMIP(Strategic Musa Improvement Project) II project,Where is IITA in the improvement of bananas,Next steps in banana improvement and delivery to farmers

1. Genetic improvement of bananas (banana and plantain)
at IITA: Current status and the way forward
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
Vroh, Bi Irie

2. 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
-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

3. Constraints and Opportunities
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

4. 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

5. 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)

6. 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
Parents A1A2 x A3B1
Progenies A1A3/ A1A2A3/ A2B1/ A1A2B1/ etc. (Breeder???)
-Ploidy analysis
-Genome specific molecular probes
Accurate decisions for breeding

7. Results of diversity analysis, marker screening and design
Markers specific to the A and B genomes of Musa found
Screening of diversity panels
A genome
B genome
DNA markers differentiating the A from the B genome
3 PCR markers available

8. Use of genome specific probes

9. 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

10. Analysis of somaclonal variations in bananas
-Methylation-Sensitive Amplification Polymorphism (MSAP)
Accession Accession description Genome b Provider
Name composition
Calcutta 4 aWild Musa acuminata subsp. AA Philippine
Burmanica (Clone C4)
Montpellier aWild Musa balbisiana (clone BB India
MPL)
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

11. Characterization of somaclonal variations in banana germplasm
Results
2nd round amplification of variant band by PCR
-In vitro culture of meristems and sub-cultures
&
-MSAP techniques
Sequencing
-Putative functions of the variation assessed
-Sequences registered in public genomic databases
Examples:
GenBank Acc# ET165586 to ET165601 (US_NCBI)
-Plants tagged & transferred to the field to link
genomic variations to phenotypes
Sequence of a variant fragment in Bluggoe (ABB genome)
-% variations: -5.6% in vitroplants

12. Comparison to Genomic databases
Genbank Hits in TIGR* plant Putative Functions
registration transcript assemblies
number (protein)
ET165586 None -
ET165587 BE033387 Cellulose, callose, starch formation, energy
(Sucrose synthase) production [38]
ET165588 BX254671(Hypothetical protein) Unknown
ET165589 TA21638_47664 Biosynthesis of di-, oligo- and
(Galactosyltransferasefamily polysaccharides.
protein) (e.g. plant cell wall) [31]
ET165590 TA54845_3847 Repair of the 3′ terminal sequence of tRNA
(Nucleotidyl transferase) molecules [39] Protein synthesis
ET165591 BF053442 Cytochrome P450-dependent hydroxylase
(Cinnamic acid hydroxylase) involved
in the biosynthesis of rosmarinic acid [33].
Antimicrobial, antiviral, antibacterial
properties.
Role in Plant defense against
ET165592 TA4172_4679(Cytochrome P450 Biosynthesis of secondary products,
like_TBP) hormones, defense
compounds, detoxification of herbicides
(http://arabidopsis-
p450.biotec.uiuc.edu/About_P450s.shtml)
ET165593 None -
ET165594 TA1_3933(Putative senescence- Cell, organ or whole plant death
associated protein)
-See GenBank at www.NCBI.nlm.nih.gov
-Vroh-Bi et al. Plant Science (submitted)

13. Genetic Research and Breeding at IITA
Analysis of resistance Characterization of the Breeding for
to pests and diseases Pathogens resistance/Tolerance
(e.g. black Sigatoka, nematodes)
-Genetic diversity -Development of
-Understanding the resistant varieties
genetics of resistance -Identification
-Identifying major genes -Early diagnostics -Delivery to farmers
& manipulation in crosses
Increased efficiency
Increased efficiency in breeding & in disease Food security and
in breeding management Improved livelihood
In collaboration with Ranajit, Danny and Biodun

14. 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

15. Genetic analysis of resistance to black Sigatoka
AB's
Results
50
100
Agbagba
1064_89
80 MPL 40
1064_53
Leaf area infected (%)
Calcutta 4
1064_37
60 30
FREQ
40 20
20
10
0
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
5 10 15 20 25 30 35 40 45
AUDPC MIDPOINT
Days after inoculation
FREQ
The resistance is quantitative (e.g. distribution of AUDPC)
-in M. acuminata selfed progenies
-in M. acuminata x M. balbisiana progenies

16. Analysis of the segregating populations
3 genes in each parental species with assumptions
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
4 genes with assumptions
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

17. Mode of action of the genes
Conclusions
Three “major” recessive genes in M. acuminata (C4)
Three “major” recessive genes in M. balbisiana (Montpellier)
Segregation ratio is 27R:37S in both populations
-Heterozygosity at all loci
-Incomplete dominance
-Complementary actions of genes
-Additive actions of alleles within loci

18. Back to the origin of triploid edible bananas and plantains
Triploid bananas: born to be susceptible to BS ?
-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
Testing and validating the trihybrid model

19. 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)
Susceptible acuminata Susceptible
unreduced acuminata reduced
AAbbCC AbC
AAbbCc Abc
X
AAbbcc ABc
AABbCC aBC
AABbCc aBc
AABbcc ABC
aaBBCC abc
aaBBCc
aaBBcc
aABBCC
aABBCc All dessert bananas are susceptible to BLSD
aAbBBcc
AABBCC Most likely scenario is Case 3
AABBCc
AABBcc
AaBbCC

20. 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 balbisiana
unreduced reduced
AAbbCC ABC
AAbbCc ABc
AAbbcc X AbC
AABbCC aBC
AABbCc aBc
AABbcc abC
aaBBCC abc
aaBBCc
aaBBcc
aABBCC
aABBCc
aAbBcc Plantain bananas (AAB) are susceptible to
AABBCC BLSD
AABBCc
AABBcc Most likely scenario is Case 2
AaBbCC

21. Can we go back to synthesize resistant edible triploids ?
Dessert and EAH bananas (AAA)
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)
Plantain bananas (AAB)
Case 1: resistant unreduced x resistant balbisiana reduced (25% resistant)
Case 2: susceptible unreduced x resistant balbisiana reduced (100% susceptible)
Other cooking bananas of ABB genome
Case 1: Susceptible acuminata reduced x trihybrid balbisiana (14.28% resistant)
Case 2: Resistant acuminata reduced x trihybrid balbisiana (12.5% resistant)

22. 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.
Pathway model Reaction Genotypes
mfr1 mfr2 mfr3
P1 P2 P3 Resistant mfr1-/mfr2-/mfr3-
mfR1mfR1/--/--
--/mfR2mfR2/--
Else (inhibition of either one step) X Susceptible --/--/mfR3mfR3
What breeders need from the pathway and genes above?
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)
Identification of 33 resistant genotypes (currently maintained in the field)

23. Identification of Mycosphaerella species of bananas
Causative agents of leaf spot diseases
-Black Sigatoka (M. fijiensis)
-Yellow Sigatoka (M. musicola)
-Leaf speckle disease (M. eumusae)
Sequencing and sequence comparison at specific genes
(Ribosomal, actin and tubulin genes)
Accurate diagnostics/Early detection/improved selection pressure
-Better disease management
-Better quarantine decisions
-Efficient breeding programs

24. Identification using SNPs in rDNA
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.
-Identity of isolates further confirmed at β-tubulin and actin gene sequences

25. -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)
-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

26. Analysis of parthenocarpy, the trait that makes bananas the fruits we eat
Wild Borneo (Seeded)
X
Segregation for parthenocarpy
Cultivar SF247 (Parthenocarpic)
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

27. M. acuminata malaccensis Borneo (seeded) x SF247 (AA cultivar)
Segregating population
-AFLP and SSR markers tested
-Traits scored in 180 individuals
Segregation of AFLP markers in Borneo x SF247

28. Comparative analyses using GID sequences rice, cotton, Arabidopsis and wheat
isolation in bananas
Parthenocarpic versus non-parthenocarpic accessions at GID

29. Parthenocarpy
DELLA
SNPs
Sequence comparison
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

30. 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 Root necrosis Gall Small Big
weight index index leison 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)

31. 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)
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

32. 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

33. Top ten varieties of bananas with respect to the contents (μg g-1) of total carotenoids
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
IITA hybrids
-Good nutritional value
-Good levels of tolerance to black Sigatoka

34. 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
Material testing and dissemination
-IITA has many partners and collaborators
So what’s next?

35. 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

36. 1. Shift towards improving the preferred landraces (Pre-breeding operations)
Chromosome doubling EMS treatment to induced mutations in vitro
Plantain Agbagba (AAB)
-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

37. 2. Backstopping NARS, NGOs and the private sector

38. -Training Students
Martine 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
-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)
Evaluation of drought tolerance

39. Acknowledgements
Not present in the picture:
Abdou Tenkouano, Ranajit Bandyopadhyay, Peter Ojiambo,
Claudius Cole, Danny Coyne, Bamisaye Bukola and Sandra Nnadi

40. Thank you