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).

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

10. Financial support

11. Thank
You