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Research Program Genetic Gains (RPGG) Review Meeting 2021: From Discovery to Delivery: A Story of Chickpea By Dr Mahendar Thudi

  1. From Discovery to Delivery: A Story of Chickpea Mahendar Thudi Senior Scientist (Chickpea Genomics) Theme - Genomics and Trait Discovery Research Program - Genetic Gains ICRISAT, India
  2. Contents Genomic resources Genome, germplasm sequencing, markers, genetic maps Trait mapping QTL-mapping, GWAS, QTL-seq, MutMap Integration in breeding
  3. Samples: - 5 wild species - 25 landraces - 60 breeding lines  Illumina sequencing used to generate 153.01 Gb  73.8% of the genome is captured in scaffolds  Genome analysis predicted 28,269 genes  High levels of synteny observed between chickpea and Medicago  > 81,845 SSRs and 4.4 million variants (SNPs and INDELs) Nature Biotechnology Genome sequence and re-sequence
  4. Sequencing of germplasm lines Mapping population parents Elite varieties (129) Reference set (300) MAGIC lines (1200) Composite collection (3000) Represent 10 countries – India, Canada, Australia, Ethiopia, Bangladesh, Kenya, Myanmar, Sudan, Burlgaria, Spain and ICARDA; includes 78 desi and 42 kabuli type Representing 32 countries; Whole genome re-sequencing (WGRS) of 292 accessions yielded 2.15 Tb of sequence data with the coverage of 5X to 12X 35 parental lines Multi-parent advanced generation intercross (MAGIC) population was developed using eight cultivars/elite lines (ICC 4958, ICCV 10, JAKI 9218, JG 11, JG 130, JG 16, ICCV 97105 and ICCV 00108) re-sequenced at 5.79 - 16.08X coverage 1956 accessions of the ICRISAT core collection 709 ICARDA cultivated genebank accessions, 39 advanced breeding lines and cultivars and 241 trait-specific accessions and 20 wild Cicer species
  5. Genome-wide variations identified were used for developing high density SNP arrays for genetics and breeding applications Helicoverpa resistant and susceptible genotypes. Vijay, IG 72953, ICC 506 and ICC 3137 Fusarium wilt resistant and susceptible genotypes. C 104, JG 62, WR 315 and ICCV 05530 Ascochyta blight resistant and susceptible genotypes- ICCV 04516, JG 62, Pb 7 and ICCV 05530 Resequencing parental lines of mapping populations a b c
  6.  Large number of unique SNPs in varieties released after 2002 indicate an enhancement in diversity in the primary gene pool as a result of recent breeding programs  Grouping of some desi with kabuli in Cluster II was due to inter crossing of desi and kabuli genotypes and vice versa for enhancing yield and disease resistance.  Nucleotide diversity in desi varieties (θπ = 0.684 × 10-3) is higher compared to kabuli (θπ = 0.650 × 10-3) varieties.  The high diversity among varieties released in RP3 (θπ = 0.684 × 10-3) can be attributed to involvement of multiple crosses while developing these varieties, which is evident from the pedigree information, compared to double or triple crosses in case of varieties of RP2 or predominantly direct selections from local collections or involving single or double crosses as in case of RP1. Recent breeding programs enhanced diversity
  7. Genomic resources Features Number Molecular markers SSR markers >3000 SNP markers >4 million DArT markers 15360 Maps Genetic maps 6 Physical map 1 Bin maps 1 Assembly Genome CDC Frontier Transcriptome 2 Genotyping platforms KASP assays 2006 GoldenGate 1536 Affymetrix 50K High density genetic maps
  8. Botrytis grey mould Must have traits in chickpea Drought tolerance Fusarium wilt Ascochyta blight Heat tolerance Dry root rot Product concept 1 Short- to medium-duration varieties with resistance to root diseases and tolerance to drought and heat stresses Product concept 2 Medium- to long-duration varieties with resistance to Ascochyta blight
  9. “QTL- hotspot” for drought tolerance Theor Appl Genet 2014 Accession Root depth (cm) Root dry wt (g) ICC 8261 123.3 1.25 ICC 283 91.6 0.73 ICC 4958 116.6 1.06 ICC 1882 83.9 0.71 ICC 4958 ICC 1882 ICC 283ICC 8261 ICC 4958 × ICC 1882 - 268 RILs ICC 283 × ICC 8261 - 289 RILs
  10. Fine mapping drought using MAGIC population 8 parents: A) ICC 4958, B) JAKI 9218, C) JG 130, D) ICCV 00108, E) ICCV 97105, F) ICCV 10, G) JG 11, H) JG 16 F1s raised and selfed in green house SSD method SSD method F2s raised and selfed in green house 1200 F3 progenies raised in field 1200 F4 progenies raised in field 1200 F5 progenies will be raised in field 1200 F6 progenies will be raised in field SSD method SSD method Average of year 2013-2014 Year 2014 Year 2013 In collaboration with Drs Gaur, Srinivasan &Rajeev Varshney
  11. (a) SNP locus (Ca2_2323872) associated with yield under rainfed conditions, present in the candidate gene (Ca_12546) predicted to be responsible for AB resistance, the most important yield limiting factor in Australia, Canada and USA; (b) Structure of gene Ca_12546 present on pseudomolecule Ca2; (c) A total of 17 haplotypes were identified for this gene. One mutation at position 2323917 in gene (Ca_12546) results in the loss of start codon that seems to make the genotype susceptible to AB. Genome-wide scan for yield under heat stress Novel marker trait associations based on genome wide SNPs From 429 lines
  12. QTL-seq for identification of genomic regions for 100 seed weight (100SDW) ICC 4958 100SDW (26.27g) ICC 1882 100SDW (12.22g) RILs X 15 Low RILs (12.22 to 12.24g) 15 High RILs (25.27 to 30.80g) Low 100SDW pool High 100SDW pool ICC 4958 Reference guided assembly 45,053,978 17.10X Mapping of reads to CDC Frontier Mapping of bulk reads on the reference guided assembly for calculation of Δ SNP Index 17,795,370 9.97 X 20,670,924 11.5 X Plant Biotechnology Journal, 2016 CcLG04CcLG01 CaLG01: • 3.07 to 4.15 Mb (1.08 Mb) • 5 SNPs with SNP index of ‘0’ and ‘1’ • SNP Effect: No candidate genes CaLG04: • 11.12 to 13.82 Mb (2.70 Mb) • 21 SNPs with SNP index of ‘0’ and ‘1’ • SNP Effect: 5 SNPs in 4 genes
  13. Mapping dry root rot resistance CaLG01 CaLG02 CaLG03 CaLG04 CaLG05 CaLG06 CaLG07 CaLG08  The cost effective SNP genotyping platform, comprising of 50,590 high quality non-redundant SNPs tiled on to Affymetrix® Axiom® genotyping array was used for genotyping BG 212 × ICCV 08305  A high density genetic map with 13,110 SNP markers developed using RIL population.  Phenotyped under field conditions as well as controlled environment facilities for two season (2015-16 and 2016-17). One QTL explaining 9% of phenotypic variation was identified on linkage group 8. Funded by Government of Karnataka
  14. Major QTLs for heat tolerance RIL population developed using DCP 92-3 (heat sensitive) and ICCV 92944 (heat tolerant) Genetic map comprising 788 SNPs Traits Total QTLs PVE (%) 100 seed weight 4 31.30-37.23 Biological yield/plant 8 6.92-11.16 Cell membrane stability 3 7.75-11.37 Chlorophyll content 7 6.78-33.52 Days to flowering initiation 4 7.48-8.96 Days to maturity 3 8.96-18.13 Days to pod filling 3 9.38-11.97 Days to pod initiation 6 5.88-43.49 Harvest index 8 7.10-39.31 Nitrogen balance index 10 7.39-13.93 Normalized difference vegetation index 16 6.69-34.02 Number of pods per plant 1 Seed yield/plant 4 6.66-18 Dr Uday Jha, ICAR-IIPR, Kanpur
  15. Co-localization of Fe and Zn QTLs in “QTL-hotspot” Three QTLs for seed-Fe and Zn concentration (CaqFe4.4, CaqFe4.5 and CaqZn4.1) were co-located in the “QTL-hotspot” region, on CaLG04, Mapping nutritional traits
  16. TraitName Linkage group PVE(%) DF CaLG08 14.23 DF50 CaLG07 10.4 DF50 CaLG08 10.46 DF50 CaLG08 10.09 DM CaLG06 13.55 HSW CaLG01 10.43 HI CaLG06 11.11 HI CaLG08 11.05 BGM1 CaLG05 17.86 BMG2 CaLG04 11.14 In collaboration with Drs. Shivali Sharma and Mamta Sharma Genomic regions for botrytis grey mould resistance  AB-QTL population developed from ICCV 10 × BGM 2013-85  Genotyped using 56 K Affymetrix® Axiom® genotyping array  Genetic map spanning 890 cM developed with 7079 markers
  17. MutMap: early flowering and enhanced seed size Two MutMap F2 populations in field during crop season 2018-19 JRF employed in the project collecting phenotyping data during crop season 2018-19 Early flowering No flowering Early flowering lines setting pods and maturing early
  18. QTL introgression into elite cultivar JG 11  A “QTL-hotspot” containing QTLs for several root and drought tolerance traits was transferred through marker assisted backcrossing (MABC) into JG 11, a leading variety of chickpea in India from the donor parent ICC 4958.  After undertaking three backcrosses with foreground and background selection and two rounds of selfing, 29 BC3F2 plants homozygous for two markers (ICCM0249 and TAA170) were selected Dr. PM Gaur, ICRISAT
  19. Geletu, the first ever MABC variety of chickpea, in the background of JG 11 released in Ethiopia  Geletu is developed in genetic background of JG 11  Geletu was released for commercial production for wider adoption in the dry semi- arid tropics to moist agro- ecological zones in Ethiopia  The variety delivered the highest grain yield of 3822 kg/ha at Arsi Robe, Ethiopia, which translates into an yield advantage of 15% over the check variety ‘Teketay’ and 78% more than the local check. The Plant Genome, 2013
  20. Supporting NARS in development of Super Annigeri 1 (MABC-WR-SA-1) with fusarium wilt resistance  Super Annigeri 1 has 7% mean yield advantage over the Annigeri 1 in AICRP- Chickpea trials.  Super Annigeri 1 is tolerant to Fusarium wilt Dr. DM Mannur, UAS-Raichur
  21. Entries Yield in kg/ha Disease reaction (mean) Jabalpur Rewa Ganjbasoda Sagar Mean JG 74 × JG 11551 2587.2 2435.1 2099.3 2157.6 2319.8 2.25 JG 12 × JG 16-3 2587.3 2153.4 2467.6 2386.1 2398.6 2.25 JG 2016-45 2104.1 1885.4 2030.9 1843.8 1966.1 3 JG 2016-43 2053.8 1995.8 1788.7 2012.5 1962.7 3.5 JG 2016-44 2096.2 1850.5 2107.3 1789.6 1960.9 3.25 JG 24 2560.3 2399.8 2450.6 2245.4 2414 2 JG 2016-9605 2534.1 2222.8 2187.1 2489.5 2358.4 2 ICC 96029 × JG11551 2877.9 2228.4 2455.5 2951.4 2628.3 2 JG 74315-14 2631.9 2399.2 2523.2 2644.4 2549.7 2 JG 12 × JG 16 2230.5 2124.4 2155.9 2517.4 2257.1 2 JG 14 × JG 226 2578.6 2369.7 2249.6 2156.3 2338.6 2.5 JG 14 (Check ) 2286.7 1750.3 2006.4 2111.1 2038.6 3.25 JG 74 (Rec. parent ) 1130.4 1258.2 1169.2 955.2 1128.3 8 CV (%) 7.63 7.36 6.45 8.25 CD at 5% 299.22 258.30 231.42 302.37 Evaluation for yield performance of JG 74315-14 in state varietal trials in four locations has shown 125% and 25% mean increase in yield over the JG 74 (recurrent parent) and JG 14 (local check) respectively. The mean disease reaction of JG 74315-14 was 2, which is highly resistant Supporting NARS in development of improved JG 74 Dr Anitha Babbar, JNKVV, Jabalpur
  22. Supporting NARS in developing Drought tolerant and Fusarium wilt resistant lines
  23. 10 SNP panel for use in breeding 10 SNP panel comprise markers for drought tolerance, Fusarium wilt and Ascochyta blight resistance Intertek SNP ID SNP ID Trait snpCA0001 CKAM2210 Drought snpCA0022 CKAM2227 Drought snpCA0023 CKAM2228 Drought snpCA0004 CKAM2179 Drought snpCA0006 CKAM2182 Drought snpCA0021 CKAM2226 Drought snpCA0018 CKAM2223 Drought snpCA0166 FW2_30366103 Fusarium wilt snpCA0168 FW2_30366146 Fusarium wilt snpCA0027 Ca4_29092572 Ascochyta Blight NARS partners Dr. Asnake Fikre – EIAR, Ethiopia Dr. Bharadwaj- ICAR-IARI Dr Laxuman –UAS-Raichur Dr. Veera Jayalakshmi –RARS-Nandyal
  24. Towards developing markers for heat tolerance QTL-seq approach will be deployed chromo some coordinate reference base Bulk-A consensus base SNP-index Bulk-A Bulk-B consensus base SNP-index Bulk-B delta (SNP- index) Ca5 44596025 G G 0 A 1 -1 Ca5 44727346 A A 0 C 1 -1 Ca5 44897927 A A 0 T 1 -1 Ca5 44922136 A A 0 C 1 -1 Ca5 44922140 G G 0 A 1 -1 Ca5 45095467 C C 0 A 1 -1 Ca5 45415468 C C 0 T 1 -1 Major QTLs for heat tolerance are identified based on mapping population developed at IIPR (DCP92-3 × ICCV92944 ) Development of markers for heat tolerance are in progress.
  25. Acknowledgements Dr. Rajeev Varshney Chickpea genomics team Collaborators Funding support
  26. T h a n k Y o u Build back better opportunities by over coming challenges
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