Genetic improvement of bananas (banana and plantain) at IITA: Current status and the way forward

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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

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Genetic improvement of bananas (banana and plantain) at IITA: Current status and the way forward

  1. 1. Genetic improvement of bananas (banana and plantain)at IITA: Current status and the way forward1. Molecular and genetics studies in the SMIP II project2. Where is IITA in the improvement of bananas3. Next steps in banana improvement and delivery to farmers Vroh, Bi Irie
  2. 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 stillthe 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 millionspeople in Africa-Grown mostly by smallholders in SSA
  3. 3. Constraints and OpportunitiesMajor constraints to genetic improvement by conventional breeding -Major cultivars are triploid -Completely or partially male and female sterile -Major cultivars are parthenocarpicMajor 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. 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. 5. Molecular and genetic targetsMolecular characterization -Musa genomes -Somaclonal variations -Pathogens -Mycosphaerella fungi -NematodesGenetics of agronomic traits and molecular markers -Resistance to black Sigatoka disease -Parthenocarpy -Dwarfism/Plant height -Earliness -Apical dominanceChemical mutagenesis using ethane methyl sulfonate (EMS)
  6. 6. Identification of Musa genomes-Main cooking bananas are AAA, AAB or ABB, with the A and the B sub-genomesof 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. 7. Results of diversity analysis, marker screening and design Markers specific to the A and B genomes of Musa found Screening of diversity panelsA genomeB genome DNA markers differentiating the A from the B genome 3 PCR markers available
  8. 8. Use of genome specific probes
  9. 9. Analysis of somaclonal variations in bananas-Genetic integrity is of primary importance in germplasm conservation, plant breeding andvariety 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 beelucidated-To provide true-to-type materials it is critical to track those variations and to possibly link themto traits Check for somaclonal variations in germplasm
  10. 10. Analysis of somaclonal variations in bananas-Methylation-Sensitive Amplification Polymorphism (MSAP)Accession Accession description Genome b ProviderName compositionCalcutta 4 aWild Musa acuminata subsp. AA Philippine Burmanica (Clone C4)Montpellier aWild Musa balbisiana (clone BB India MPL)Neypoovan Diploid landrace AB BurundiAgbagba Plantain banana landrace AAB NigeriaBluggoe Cooking banana landrace ABB CameroonNote: a Musa acuminata and M. balbisiana are the progenitors of the cultivated bananasb From the IITA Musa database www.IITA.org
  11. 11. Characterization of somaclonal variations in banana germplasmResults 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 databasesExamples:GenBank Acc# ET165586 to ET165601 (US_NCBI)-Plants tagged & transferred to the field to linkgenomic variations to phenotypes Sequence of a variant fragment in Bluggoe (ABB genome) -% variations: -5.6% in vitroplants
  12. 12. Comparison to Genomic databasesGenbank Hits in TIGR* plant Putative Functionsregistration transcript assembliesnumber (protein)ET165586 None -ET165587 BE033387 Cellulose, callose, starch formation, energy (Sucrose synthase) production [38]ET165588 BX254671(Hypothetical protein) UnknownET165589 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 synthesisET165591 BF053442 Cytochrome P450-dependent hydroxylase (Cinnamic acid hydroxylase) involved in the biosynthesis of rosmarinic acid [33]. Antimicrobial, antiviral, antibacterial properties. Role in Plant defense againstET165592 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. 13. Genetic Research and Breeding at IITAAnalysis of resistance Characterization of the Breeding forto 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. 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. 15. Genetic analysis of resistance to black Sigatoka ABsResults 50 100 Agbagba 1064_89 80 MPL 40 1064_53Leaf 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. 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 assumptionsDDEEIIFF DDEEIIFf DDEEIiFF DDEEIiFf DDEeIIFF DDEeIIFf DDEeIiFF DDEeIiFf DdEEIIFF DdEEIIFf DdEEIiFF DdEEIiFf DdEeIIFF DdEeIIFf DdEeIiFF DdEeIiFfDDEEIIFf DDEEIIff DDEEIiFf DDEEIiff DDeeIIFf DDEeIIff DDEeIiFf DDEeIiff DdEEIIFf DdEEIIff DdEEIiFf DdEEiiFf DdEeIIFf DdEeIIff DdEeIiFf DdEeIiffDDEEIiFF DDEEIiFf DDEEiiFF DDEEiiFf DDEeIiFF DDEeIiFf DDEeIiFF DDEeIiFf DdEEIiFF DdEEIiFf DdEEiiFF DdEEiiFf DdEeIiFF DdEeIiFf DdEeiiFF DdEeiiFfDDEEIiFf DDEEIiff DDEEiiFf DDEEiiff DDEeIiFf DDEeIiff DDEeiiFf DDEeiiff DdEEIiFf DdEEIiff DdEEiiFf DdEEiiff DdEeIiFf DdEeIiff DdEeiiFf DdEeiiffDDEEIIFF DDEEIIff DDEEIiFf DDEEIiff DDEeIIFf DDEeIIff DDeeIiFf DDEeIiff DdEEIIFf DdEEIIff DdEEIiFf DdEEIiff DdEeIIFf DdEeIIff DdEeIiFf DdEeIiffDDEeIiFf DDEeIIff DDEeIiFf DDEeIiff DDeeIIFf DDeeIIff DDeeIiFf DDeeIiff DdEeIIFf DdEEIIff DdEeIiFf DdEeIiff DdeeIIFf DdeeIIff DdeeiiFf DdeeIiffDDEeIiFF DDEeIiFf DDEeiiFF DDEeiiFf DDeeIiFF DDeeIiFf DDeeiiFF DDeeiiFf DdEeIiFF DdEeIiFf DdEeiiFF DdEeiiFf DdeeIiFF DdeeiiFf DdeeiiFF DdeeiiFfDDEeIiFf DDEeIiff DDEeiiFf DDEeiiff DDeeIiFf DDeeIiff DDeeiiFf DDeeiiff DdEeIiFf DdEeIiFf DdEeiiFf DdEeiiff DdeeIiFf DdeeiiFf DdeeiiFf DdeeiiffDdEEIIFF DdEEIIFf DdEEIiFF DdEEIiFf DdEeIIFF DdEeIIFf DdEeIiFF DdEeIiFf ddEEIIFF ddEEIIFf ddEEIiFF ddEEIiFf ddEeIIFF ddEeIiFf ddEeIiFF ddEeIiFfDdEEIIFf DdEEIIff DdEEIiFf DdEEIiff DdEeIIFf DdEeIIff DdEeIiFf DdEeIiff ddEEIIFf ddEEIIff ddEEIiFf ddEEIiff ddEeIIFf ddEeIiff ddEeIiFf ddEeIiffDdEEIiFF DdEEIiFf DdEEiiFF DdEEiiFf DdEeIiFF DdEeIiFf DdEeiiFF DdEeiiFf ddEEIiFF ddEEIiFf ddEEiiFF ddEEiiFf ddEeIiFF ddEeiiFf ddEeiiFF ddEeiiFfDdEEIiFf DdEEIiff DdEEiiFf DdEEiiff DdEeIiFf DdEeIiff DdEeiiFf DdEeiiff ddEEIiFf ddEEIiff ddEEiiFf ddEEiiff ddEeIiFf ddEeiiff ddEeiiFf ddEeiiffDdEeIIFF DdEeIIFf DdEeIiFF DdEeIiFf DdeeIIFF DdeeIIFf DdeeIiFF DdeeIiFf ddEeIIFF ddEeIIFf ddEeIiFF ddEeIiFf ddeeIIFF ddeeIiFf ddeeiiFF ddeeIiFfDdEeIIFf DdEeIIff DdEeIiFf DdEeIiff DdeeIIFf DdeeIIff DdeeIiFf DdeeIiff ddEeIIFf ddEeIIff ddEeIiFf ddEeIiff ddeeIIFf ddeeIiff ddeeIiFf ddeeIiffDdEeIiFF DdEeIiFf DdEeiiFF DdEeiiFf DdeeIiFF DdeeIiFf DdeeiiFF DdeeiiFf ddEeIiFF ddEeIiFf ddEeiiFF ddEeiiFf ddeeIiFF ddeeiiFf ddeeiiFF ddeeiiFfDdEeIiFf DdEeIiff DdEeiiFf DdEeiiff DdeeIiFf DdeeIiff DdeeiiFf Ddeeiiff ddEeIiFf ddEeIiff ddEeiiFf ddEeiiff ddeeIiFf ddeeiiff ddeeiiFf ddeeiiff
  17. 17. Mode of action of the genes ConclusionsThree “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. 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. 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. 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. 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 genomeCase 1: Susceptible acuminata reduced x trihybrid balbisiana (14.28% resistant)Case 2: Resistant acuminata reduced x trihybrid balbisiana (12.5% resistant)
  22. 22. Proposed pathway model for the genetics of host response to black Sigatoka and corresponding genotypiccombinations 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. 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. 24. Identification using SNPs in rDNASequence alignment of Nigerian isolates (Q1_ISO_11 to 57) on reference GenBank references ofM. fijiensis (Fiji), M. musicola (Musi), M. eumusae (Eumu), and M. musae (Musae). Inter- and intraspecific SNPvariations are highlighted. Isolates Q1_ISO_11, Q1_ISO_20, Q1_ISO_30, Q1_ISO_54, and Q1_ISO_57 wereidentified as M. eumusae. -Identity of isolates further confirmed at β-tubulin and actin gene sequences
  25. 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 inbanana production
  26. 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. 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. 28. Comparative analyses using GID sequences rice, cotton, Arabidopsis and wheat isolation in bananas Parthenocarpic versus non-parthenocarpic accessions at GID
  29. 29. Parthenocarpy DELLA SNPs Sequence comparison How useful are these words for breeders?1. Most useful agronomic traits are present in seeded wild species2. Hard shell seeds are undesired in edible bananas3. Cultivated x wild crosses result to useful progenies with unwanted seeds4. 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. 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. 31. 2. Where is IITA in genetic improvement of bananas?1. Genetic improvement at IITA has targeted mostly resistance to BS2. 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. 32. Farmer and consumer preferencesLong 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. 33. Top ten varieties of bananas with respect to the contents (μg g-1) of total carotenoids Varieties Total carotenoids Obino LEwai 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. 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 materialsMaterial testing and dissemination -IITA has many partners and collaborators So what’s next?
  35. 35. 3. Next steps in banana improvement and delivery to farmers1. 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 value2. 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. 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. 37. 2. Backstopping NARS, NGOs and the private sector
  38. 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. 39. Acknowledgements Not present in the picture: Abdou Tenkouano, Ranajit Bandyopadhyay, Peter Ojiambo, Claudius Cole, Danny Coyne, Bamisaye Bukola and Sandra Nnadi
  40. 40. Thank you

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