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Research Program Genetic Gains (RPGG) Review Meeting 2021: Identification of markers and genomic regions associated with aflatoxin resistance in peanut (2016- 2020) By Dr Rajeev K Varshney Team

  1. Identification of markers and genomic regions associated with aflatoxin resistance in peanut (2016- 2020) Copyright © 2019 Mars Wrigley — Confidential Principal Investigator Rajeev Varshney Project Team: Manish Pandey Hari Sudini P Janila Anu Chitkineni
  2. Copyright © 2019 Mars Wrigley — Confidential 2 Project Background Although breeders have been doing their best to produce peanuts with higher resistance to aflatoxin, their efforts have not been very successful. One of the main reasons for this is limited knowledge and information available on the three mechanisms of resistance to aflatoxin in peanut and their associated genes: (1) resistance to in vitro seed colonization (IVSC), (2) resistance to pre-harvest seed infection, and (3) resistance to pre-harvest aflatoxin production (PAC). All of these resistance types are very important in developing stable aflatoxin resistant genotypes. Genetic resistance to aflatoxin contamination has also been difficult to detect consistently due to low heritability and problems with phenotyping methods. Resistance is highly influenced by the environmental conditions and soil biome, and it is essential to ensure high quality genotypic and phenotypic data on diverse genetic populations for conducting high resolution trait mapping. Also in the past, only limited number of lines (mainly germplasm and breeding material) were used for screening for aflatoxin research. A comprehensive approach is key to unlocking this complex trait. In this project, we used large and novel germplasm collections e.g. MAGIC population with maximum recombinations from 8 resistant parents and a reference set for screening in aflatoxin sick plot in 2 replications and 2 years. In addition, whole genome sequencing was conducted on both germplasm sets. Such large-scale sequencing and phenotyping dataset were used for identification of superior lines and markers/haplotypes associated with aflatoxin contamination traits (ACTs).
  3. Reference set (300 lines) with rich genetic diversity for GWAS analysis Reference set represents global genetic diversity of groundnut Global / base collection Six seasons phenotyping data on Aspergillus infection, PAC, aflatoxin production ICRISAT reference set Wild accessions, landraces and breeding lines Core collection ICRISAT Minicore collection (184), also part of Reference Set
  4. MAGIC population for combining three resistance mechanisms and fine mapping Eight founder parents Completed phenotyping for A. flavus infection % and aflatoxin content for two seasons in MAGIC lines (2 replications) • IVSC resistance: ICGV 88145, ICGV 12014, ICGV 89104, ICG 51 • PAC resistance : ICGV 91278 and 55-437 • Aflatoxin production resistance: VRR 245 and U 47-5
  5. Genomics and transcriptomics approaches for understanding three resistance mechanisms Genome-wide association study (GWAS) Multiparent advanced generation intercross (MAGIC) Transcriptome analysis In Vitro Seed colonization (IVSC) Pre-harvest aflatoxin contamination (PAC) Aflatoxin production (AP)
  6. Identification of promising lines with low A. flavus infection % and aflatoxin content during post-rainy 2017-18 and 2018-19 Preparation of inoculum to impose infection in field Preparation of 0.1% Mercuric chloride Plating seeds for scoring Sorghum seeds inoculated with AF-14 strain Field inoculation with A. flavus sick plot Harvesting of 812 Sick plot lines during post-rainy season Sterilization with 0.1% mercuric chloride Scoring for infection percentage 66 promising MAGIC lines in 2019 with minimum A. flavus infection % and aflatoxin content selected based on the two seasons of phenotyping in sick plot experiment of 812 MAGIC lines
  7. Artificial inoculation J 11 JL 24 A. flavus toxigenic strains mixture 24hr after inoculation 48hr after inoculation 72hr after inoculation Sample collection U4-7-5 JL 24 Scarification In vitro In vitro A. flavus toxigenic strains mixture Artificial inoculation 2 days after inoculation Experimentalset-up Transcriptome analysis RNA isolation Mechanism II: Field seed colonization resistanceMechanism I: In vitro colonization resistance Mechanism III: Resistance to aflatoxin production Artificial inoculation in soil During harvesting (120 DAS) ICGV 91278, ICGV 91284, ICGV 91315, ICGV 91324, ICGV 93305, ICGV 94379, J 11 and JL 24 Understanding three different mechanisms of Aflatoxin resistance in groundnut In vivo A. flavus toxigenic strains mixture 3 days after inoculation 1 day after inoculation 7 days after inoculation The transcriptome analysis for IVSC, PAC and AP provided insights on candidate genes and pathways as well as cross-talk between host and pathogen.
  8. GWAS on cultivated samples (263) Unique genes for A. flavus infection %, and aflatoxin content (µg/kg) on the basis of MTA Haplotypes identified for genes related to A. flavus infection % and aflatoxin content (µg/kg) Heterozygosity was removed followed by elimination of the genes consisting single haplotype Superior haplotypes for A. flavus infection % and aflatoxin content Flowchart for identification of superior haplotypes for the genes associated with low A. flavus infection % and aflatoxin content Superior haplotypes were identified for 37 genes for A. flavus infection % and for 17 genes for aflatoxin content based on haplo-pheno analysis
  9. Summary… Multiple aflatoxin resistant/breeding lines available for further yield trials Superior haplotypes and suitable donor genotypes identified for A. flavus infection % and aflatoxin content Haplotype-based breeding can be initiated for improving aflatoxin resistance in genetic background elite varieties Transcriptome approaches provided improved understanding on resistance to IVSC, PAC and AP
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  11. Thank You
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