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Complex genetic structure of african cassava and genetic architecture of key traits

Presentation at RTB Annual Review and Planning Meeting (Entebbe, Uganda, 29 Sep-3 Oct 2014)

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Complex genetic structure of african cassava and genetic architecture of key traits

  1. 1. Complex genetic structure of African cassava and genetic architecture of key traits Ismail Y. Rabbi IITA Ibadan – Nigeria 2014 Annual RTB Meeting
  2. 2. Team members and funding acknowledgements IITA Cornell University Peter Kulakow, Jean-Luc Jannink, Melaku Gedil, Martha Hamblin, Elizabeth Parkes, Charlotte Acharya, Lava Kumar, Delphine Ly, Nzola Mahungu, Puna Ramu, Rachid Hanna, Pheneas Ntewaruhunga, Edward Kanju, Morag Ferguson, US DoE-JGI and UC Berkeley Dan Rokhsar, Simon Prochnik, Jessen Bredesen, Cindy Ha CIAT Luis-Augusto Becerra National Programs NRCRI – Chiedozie Egesi NaCCRI – Robert Kawuki, Yona Baguma, Funding: CRP-RTB/HarvestPlus/BMGF and DFID
  3. 3. Objective: increase genetic gain through use of molecular markers in cassava breeding • Limited use of markers in cassava research. • Advances in next-generation sequencing should change (and is changing) this situation.
  4. 4. PstI sites ApekI sites GBS 96-plex Protocol (cont.) Primers ..... ..... .. .. . .. . . ... .. .. . Clean-up .. .. . . ... .. . . . . . . . .. ... . .. . .. . .. . . ... .. . . . . . . . . . . . .. . . . . .. . . ... ... .. . . . . . ....... . . . . .. . . . . . . ... . .. . . .. . .. . ... ... . .. ... . . . . . . . ...... . ... . . . .. . . .. . .. . . . 1. Plate DNA & adapter pair 4. Pool DNAs Primers .. .. . . . . . . . . . .. . ... .. . . . . . .. . . . . . . .. . . . . . . Primers . .. . ... . . ... ... . . .. .... .. .. . . . .. . . . . . .. . . . . . 5. PCR ..... .. .. . .. . Sample digestion, barcode and 2. Digest DNA with RE 3. Ligate adapters Illumina F and R adapters (may be done simultaneously) ApeKI (5 base-cutter) Clean-up Clean-up 6. Evaluate fragment sizes GBS 96-plex Protocol (cont.) ... ...... .. . . 1. Plate DNA & adapter pair 4. Pool DNAs 5. PCR 2. Digest DNA with RE 3. Ligate adapters (may be done simultaneously) A ApeKI (5 base-cutter) 6. Evaluate fragment sizes GBS 96-plex Protocol (cont.) ... ...... . .. . . . . ... . ... . . ... .. 1. Plate DNA & adapter pair 4. Pool DNAs 5. PCR 2. Digest DNA with RE 3. Ligate adapters (may be done simultaneously) ApeKI (5 base-cutter) 6. Evaluate fragment sizes Sample DNA A/G A A G G G G A A A A A A G A G G A A A A G G Step 1 Sample digestion using enzyme Step 2 Barcode adapter ligation Step 3 Pooling of samples, PCR amplification and sequencing Reference sequence Step 4 DNA sequences aligned to reference genome, sorted by barcodes and SNPs called. DNA barcode Universal forward primer Digestion site Digestion site Pooling PCR Illumina sequencing Sequence sorting (bioinformatics) Technical overview
  5. 5. Application of GBS in cassava improvement research areas 1. Understanding genetic diversity and population structure for targeted breeding strategy (e.g. heterotic grouping and hybrid breeding) 2. Development of genomic resources (e.g. snp markers, annotated reference genomes, genetic linkage maps). 3. Determining genetic architecture of target traits (e.g. disease resistance, nutrition). 4. Genomic selection for accelerated breeding.
  6. 6. Population structure and genetic diversity of African cassava • Very little is known about the population structure, levels of diversity, and ancestry of African cassava. • Such information is required for targeted breeding strategy (e.g. heterotic grouping and hybrid breeding). • Most historical studies relied on at most few dozen markers and limited germplasm set.
  7. 7. Populations genotyped so far Population N Description IITA improved varieties 1055 Improved genotypes cloned since 1970s IITA-GRC 278 IITA genebank’s core collection NRCRI 383 National Root Crops Research Institute (Nigeria) IITA regional breeding 543 Breeding germplasm/landraces from DRC, Tanzania, Ghana, Malawi, Zambia, Cameroon IITA landrace collection 809 Landraces assembled from sub-Saharan Africa since early 1980’s CIAT* 285 Total 3353 *** Sequencing just completed, analysis in progress 60326 SNP loci from ApekI GBS
  8. 8. Population structure/ancestry of African cassava 100% 50% 0% West Africa, East Africa, Central Africa • Admixture analysis detected about nine subpopulation (divergent founders). • Most clones derive their ancestry from more than two subpopulations • Improved varieties typically show more ancestries (expected?)
  9. 9. Identification of genetic duplicates: Reduce cost of germplasm maintenance Zambia_M86_0016 Zambia_00_0093_ZAMBIA Tanzania_NDL_2003_111 MexMglaziovii_MExMGlaziovii_003 Tanzania_NDL_2005_1471 Zambia_TME_594_ZAMBIA Cameroon_Le_blanc_Bakou Cameroon_Doumbouno(Dongali) Ghana_BANKYE_(6MONTHS) Cameroon_Tibedop(Betare) Zambia_MWAKA_UMO_3 Cameroon_Obala Ghana_DAGYEAEBANKYE Zambia_NULUMINO Zambia_192_034_ZAMBIA Ghana_AFS_2000_053 Ghana_SW_2000_210 Ghana_OGhFaFn_a2_0B0D0__0986_141 Ghana_UCC_2001_138 Ghana_AKS_2000_052 Ghana_NKS_2000_024 Tanzania_Lwakitangaza Zambia_MUKUNTA Zambia_KABALA Zambia_UNKNOWN_4B5 Ghana_DNA_2000_008 Ghana_KW_2000_161 GGCORE_mm990477 Cameroon_MI_alor Cameroon_Mi_alot Cameroon_Be_ysrgp Ghana_BD_96_121 Cameroon_Libongo(s alando) Cameroon_Mbouyombo(salando) Cameroon_Ba_Mabongo Cameroon_Var_Atin_odzoe Ghana_BD_96_186 DRC_VUABIKA_VUABIKA Cameroon_MI_kolo_2 Cameroon_Babank Cameroon_Banya Cameroon_EP_chou Cameroon_Etoko_abomo(cook) Ghana_UCC_2001_015 Cameroon_NM_RC Cameroon_Ni_Panya Ghana_BD_96087 Cameroon_Pendere(Betare) Cameroon_Dg_Agric Cameroon_Ni_locale_musate Cameroon_Be_arougnchong Cameroon_BPA_mukonde Cameroon_Bg_re d Cameroon_Mboumpe Cameroon_AO_YSRP Cameroon_Bbe_rc Cameroon_Libongo(Betare) Cameroon_Bititi(Betare) Cameroon_Peau_rouge (petit_Koundji) Cameroon_Ni_mogheuti Cameroon_TL_WC(bs_v1) Cameroon_Yara(moun_bourou) Cameroon_Feofouo Cameroon_NO_WC_RSS Cameroon_Monembong Ghana_AFS_2000_131 Cameroon_Etuku Cameroon_Ajimbi(petit_Koundji) Cameroon_Be_white_root Cameroon_Napol_odo(ka ya_II) Cameroon_Yokadouma_foufoue ng Cameroon_Pk_peau_rouge Cameroon_Mi_mambo Cameroon_BI_mwende YM_White_GSRP Cameroon_Be_peppep Cameroon_Cathalan(nkonagena) Cameroon_Yahoroup(Bi djoro) Cameroon_SO_Akossi Cameroon_Yahoroup(ngoura gadi) Cameroon_Campou_Monatele Cameroon_BSGP_Kotbotie_Essere Cameroon_NI_marigo Cameroon_LMR Cameroon_Mambo(Widikum) Cameroon_Dg_nkolo_bitcho GGCORE_caricass_1 DRC_KIZELANA DRC_LEMBE DRC_NSOKOMBU DRC_DINKONDO DRC_NSUMAKANI DRC_KIMBILA_1 DRC_KIMBODILA DRC_KIMUISI_KIMUISI DRC_94_0330 DRC_DIYILU DRC_MANDOMBE DRC_KIYIZILA_2 DRC_MULUTU DRC_SADISA DRC_MVUAMA(83_138) DRC_KINSAKI_NSAKI DRC_BOMA DRC_NGUNGILA DRC_KIMONEKENE DRC_ZIZILA DRC_PAPAYI DRC_DISANKA DRC_MAMBIKA_2 DRC_NGAYI_NGAYI DRC_AKOKOLIA DRC_KIVIEKI DRC_KIVANGA DRC_MOYINDO DRC_NDEKE_LUKA DRC_NTITI DRC_RAV DRC_MBABA DRC_BOLILO DRC_MUKULU_DADA DRC_KIYIZILA_1 DRC_BUTAMU DRC_KIMBUNGU_2 DRC_MANDUDI DRC_KULA_NSATU DRC_NGONGA_NABUTU_2 DRC_NGABIDU DRC_KINTUENGE DRC_BATENDE DRC_SALONGO_2 DRC_KAYEBU DRC_NAMBIYO_MBIYO DRC_LIYAYI DRC_NKENI DRC_ETIENNE DRC_MAMBIKA_1 DRC_DOLODUA Ghana_UCC_2001_011 Ghana_KS12000196 Ghana_1235 Ghana_AFS_2000_048 Ghana_AGA_97208 Ghana_AFS_2000_043 Ghana_WCH_2000_004(6M ONTHS) Ghana_BD_96010 Ghana_BD_96184_SUS_30572 Ghana_KW_2000_148 Ghana_BD_96_136 Ghana_BD_96_087 Ghana_BD_96_178 Ghana_I011412_GHANA Ghana_NKABOM Ghana_TA_97_123 Ghana_TA_97_034 Ghana_AFS_2000_027 Ghana_AFS_2000_081 Ghana_UCC_2001_157 Ghana_NKZ_2000_109 Ghana_SW_2000_146 Ghana_NKZ_2000_011 Ghana_TA_97_137 Ghana_AFS_2000_050 Ghana_SW_2000_095 Ghana_AFS_2000_071 Ghana_OFF_2000_022 Ghana_NKZ_2000_034 Ghana_UCC_2001_062 Ghana_UCC_2001_325 Ghana_DMA_2000_070_A Ghana_H_008 Ghana_SW_2000_220 Ghana_KSI_2000_036 Ghana_AFS_2000_011 Ghana_DMA_2000_070_B Ghana_UCC_2001_432 Ghana_DMA_2000_008 Ghana_KSI_2000_151 Ghana_OFF_2000_025 Ghana_SW_2000_187 Ghana_BD_96_154 AFS_2000_020 Ghana_UCC_2001_153 Ghana_WENCHI Ghana_I011371_GHANA Ghana_BD_96_021 Ghana_KSI_2000_191 Ghana_SW_2000_077 Ghana_UCC_2001_417 GGCORE_i011371 GGCORE_i011551 GGCORE_i011797 GGCORE_i30572-2 GGCORE_i940330 GGCORE_i960249 GGCORE_i960590 GGCORE_i990313 Malawi_Chisalanzo Malawi_Chiwombola Malawi_Chikwela Malawi_Phoso Malawi_Mbundumali MexMglaziovii_MExMGlaziovii_001 MexMglaziovii_MExMGlaziovii_002 MexMglaziovii_MExMGlaziovii_004 GGCORE_mm990276 Tanzania_Maktub Tanzania_46106_27 Tanzania_Sepide Tanzania_Kipusa Tanzania_Mwari Tanzania_Namikonga Tanzania_NDL_2003_031 Tanzania_Lwabahimba Tanzania_Rubona Tanzania_Liongo_Kigoma Tanzania_Aipin_Valenca Tanzania_Kalingisi Tanzania_Ging'hi_Magu Tanzania_KBH_2006_29 Tanzania_KBH_2006_74 Tanzania_KBH_2006_22 Tanzania_MIGYERA Tanzania_KIROBA Tanzania_KBH2006_94 Tanzania_KBH2002_505 GGCORE_z970474 Zambia_KASAI Zambia_CHILA Zambia_MANYOPOLA Zambia_KATOBA_MPUTA Zambia_KARIBA Zambia_MWAKA_UMO_1 Zambia_MWAKA_UMO_2 Zambia_BANGWEULU_1 Zambia_CHIMAMBA Zambia_NAMUNYONGO Zambia_BUNGABUTU Zambia_KALABA Zambia_KASWESHI Zambia_NAKAMBUMA Zambia_NDELE_MUKAKA Zambia_UNKNOWN_1 Zambia_MULOSHA Zambia_NDOLA Zambia_MANGABWA Zambia_NAJOYCE Zambia_LUSAKA Zambia_MABUMBA_1 Zambia_NAKAPAYI Zambia_UNKNOWN_1B Zambia_UNKNOWN_3 Zambia_UNKNOWN_1BG Zambia_NAKASHI_MBI Zambia_TANKENA Zambia_BANGWEULU_2 Zambia_KASONTA Zambia_TOMO Zambia_MABUMBA_2 Zambia_L9_304_36 Zambia_MH95_0414_ZAMBIA Zambia_99_0359_ZAMBIA Zambia_14(2)1425_Z AMBIA Zambia_00_1093_ZAMBIA Zambia_96_1432_ZAMBIA Zambia_L9_304_73 Zambia_L9_92_032 Zambia_MM96_1757_ZAMBIA Zambia_01_1551_ZAMBIA Zambia_01_1649_ZAMBIA Zambia_MM96_1759_ZAMBIA Zambia_L9_304_26 Zambia_180_142_ZAMBIA Zambia_L9_304_175 Zambia_96_1565_ZAMBIA Zambia_L9_304_101 Zambia_99_3575_ZAMBIA Zambia_L9_304_5 Zambia_I92_0342_Z AMBIA IITA GRC Zambia DRC Cameroon Ghana Tanzania Histogram of RTB.IBD Distance (1−PSA) Frequency 0.00 0.05 0.10 0.15 0.20 0.25 0 50000 100000 150000 Duplicate distance threshold
  10. 10. Application of GBS in cassava improvement 1. Understanding genetic diversity and population structure for targeted breeding strategy (e.g. heterotic grouping and hybrid breeding) 2. Development of genomic resources (e.g. snp markers, annotated reference genomes, genetic linkage maps). 3. Determining genetic architecture of target traits (e.g. disease resistance, nutrition). 4. Genomic selection for accelerated breeding. 10/30/2014
  11. 11. Mapping populations genotyped through RTB/other projects Population Cross N Genotyping IITA_MP1 TMSI961089AxTMEB117 205 IGD IITA_MP2 TMEB117 x TMSI961089A 207 IGD IITA_MP3* TMS I011412 x TMS4(2)1425 177 IGD, Berkeley IITA_MP4* TMS30001 x I961089A 271 Berkeley IITA_MP5* TMSI961089A x TMS30001 243 Berkeley IITA_MP6 I011371xTMEB117 300 Berkeley * Contributed to the cassava consensus map consortium
  12. 12. High-density GBS SNP maps Genetic map PstI (GACGTC); 770 SNPs ApekI (GCWGC); 6756 SNPs 0 50 100 150 Genetic map Chromosome Location (cM) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 0 20 40 60 80 100 120 Chromosome Location (cM) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 IITA_MP1 IITA_MP3 Crop Science Vol. 54 (2014) Virus Research (2014)
  13. 13. Improvement of reference genome using high-density linkage maps • High-quality genome assembly needed for QTL, GS, GWAS, genetic transformation. • Current cassava genome is assembled into 12977 pieces (scaffolds). • 10 high-density GBS SNP maps used to anchor the cassava reference genome. • 71.9% of the assembled genome has been placed on the 18 cassava chromosomes. • Work done as part of the cassava genetic map consortium
  14. 14. Application of GBS in cassava improvement 1. Understanding genetic diversity and population structure for targeted breeding strategy (e.g. heterotic grouping and hybrid breeding) 2. Development of genomic resources (e.g. snp markers, annotated reference genomes, genetic linkage maps). 3. Determining genetic architecture of target traits (e.g. disease resistance, nutrition). a. QTL mapping using bi-parental populations 4. Genomic selection for accelerated breeding. 10/30/2014
  15. 15. QTL mapping in bi-parental populations • Mapping population: – Full-sib F1 populations derived from pairs of non-inbred parents • Phenotyping (>2 years): – Susceptibility to Cassava Mosaic Disease – Carotenoid accumulation (b-carotene) in storage roots – Anthocyanin pigmentations – Number of harvested roots – root weight per plot. • Genotyping: – GBS using PstI Restriction enzyme (n = 770 SNPs) • Data analysis: – Genetic map – Joinmap® – Phenotype data analysis - R/lme4 – QTL mapping using R/qtl
  16. 16. Log of odds ratio QTL mapping in bi-parental populations Rabbi et al. Crop Science vol. 54:
  17. 17. Major locus underlying carotenoid accumulation in cassava roots 80 60 40 20 0 S7520_912057 PULPCOL Phytoene synthase II linkage group lod 2011 2012 combined 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Rabbi et al. Crop Science vol. 54:
  18. 18. High-resolution mapping of the CMD2 locus 40 30 20 10 0 CMD1S S5214_30876 linkage group lod CMD2 locus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Resistant parent Susceptible parent
  19. 19. Narrow genetic base for major gene resistance to CMD? a 50 S5214_780931 40 30 20 10 0 -Log10(P) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 50 40 30 20 10 0 0 30 60 90 120 150 IITA TMS 011412 IITA TMS 4(2)1425 scaffold 5214 Position (cM) b -Log10(P) a. Genome-wide scan for linkage between CMD resistance and 6756 SNPs across 18 cassava linkage groups. b. A detailed view of the of linkage group 16 Rabbi et al. Virus Research 2014 DOI: 10.1016/j.virusres.2013.12.028
  20. 20. Anchoring previously-mapped resistance loci S4175_365840 S4175_207572 S4175_58407 S4175_116369 S4175_207488 S4175_241208 S4175_208108 S4175_64514 S4175_62342 S4175_181543 S4175_255324 S7933_3549 S7933_41036 S5214_1427030 S5214_1273806 S5214_1274892 S5214_1073280 S5214_1084049 S5214_831886 S5214_780931 S5214_776689 S5214_514589 S5214_472282 S5214_566592 S6906_46617 S6906_46672 S6906_119465 S6906_119507 S6906_359407 S6906_359424 S6906_359496 20 40 60 80 100 120 CMD Resistance LG16P1F2 r^2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 NS198 PVE: 11% SSRY106 PVE: 30% SSRY28 PVE: 60-70% NS158 and NS169 PVE: N/A All markers linked to dominant, major gene resistance to CMD from different genetic backgrounds occur in same chromosome! Linkage group 16
  21. 21. Application of GBS in cassava improvement 1. Understanding genetic diversity and population structure for targeted breeding strategy (e.g. heterotic grouping and hybrid breeding) 2. Development of genomic resources (e.g. snp markers, annotated reference genomes, genetic linkage maps). 3. Determining genetic architecture of target traits (e.g. disease/pest resistance, plant morphology, nutrition) b. Genome-wide association 4. Genomic selection for accelerated breeding. 10/30/2014
  22. 22. What next? • So far, most of the applications of GBS SNPs relate to germplasm characterization and QTL discovery. • Next step is to change gears to ‘applications’ by using the discovered information in the crops’ improvement. – Heterotic pattern/grouping (on-going) – Genomic selection and MAS • www.nextgencassava.org • www.cassavabase.org
  23. 23. Team members and funding acknowledgements IITA Cornell University Peter Kulakow, Jean-Luc Jannink, Melaku Gedil, Martha Hamblin, Elizabeth Parkes, Charlotte Acharya, Lava Kumar, Delphine Ly, Nzola Mahungu, Puna Ramu, Rachid Hanna, Pheneas Ntewaruhunga, Edward Kanju, Morag Ferguson, US DoE-JGI and UC Berkeley Dan Rokhsar, Simon Prochnik, Jessen Bredesen, Cindy Ha CIAT Luis-Augusto Becerra National Programs NRCRI – Chiedozie Egesi NaCCRI – Robert Kawuki, Yona Baguma, Funding: CRP-RTB/HarvestPlus/BMGF and DFID
  24. 24. Also, check out our poster on tracking of cassava varieties in farmers fields • • • Thank you !!!

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