Dissecting the genomic regions, candidate genes and pathways using multi-locus genome-wide association study for stem rot disease resistance in groundnut

Abstract

Stem rot, caused by Sclerotium rolfsii Sacc., is a devastating soil-borne disease causing up to 80% yield losses in groundnut globally. To dissect the genetic basis of resistance, we evaluated a diverse minicore germplasm panel over 3 years in stem rot sick-field conditions. Multi-locus genome-wide association study with the 58K single nucleotide polymorphisms (SNPs) Axiom_Arachis array genotyping identified 13 significant genomic regions associated with resistance across eight chromosomes with logarithm of the odds (LOD) scores ranging from 4.5 to 12.4 and R² values between 6.9% and 58%. Within these regions, 145 candidate genes were implicated, including wall-associated receptor kinases, lucine-rich repeat and NB-ARC domain proteins, and peroxidase superfamily proteins. These genes orchestrate resistance through pathogen perception (e.g., receptor-like kinases), direct inhibition (R genes), toxin detoxification, and activation of transcription factors driving protective compound synthesis for cell recovery. If these defenses are compromised, a hypersensitive response-mediated apoptosis is triggered. Notably, resistance was exclusive to Virginia-type groundnut. The identified candidate genes showed strong correlation with RNA-seq data from stem rot-infected plants, reinforcing their role in the transcriptional defense response. Three kompetitive allele-specific PCR markers, namely, SnpAH00614 (on auxin-related gene AhSR001), SnpAH00625 (on histidine triad protein gene AhSR002), and SnpAH00626 (on E3 ubiquitin ligase gene AhSR003), were validated, confirming their significant contribution to stem rot resistance. These markers may facilitate the development of stem rot-resistant varieties through direct application in breeding programs through marker-assisted selection.

FIGURE 1

Histogram for showing the distribution of phenotypic data and Manhattan plots representing significant marker trait associations (MTAs) for the stem rot disease resistance during different years of screening. PDI, percent disease incidence; PM, percent mortality.

FIGURE 2

Reaction of minicore to stem rot disease with increase in favorable alleles. An increase in the number of favorable alleles increased the amount of resistance offered by the genotype. A drastic increase in resistance was observed with an increase in favorable alleles from 5 to10, and after 10, the amount of resistance was almost similar. This shows single specific segment (gene) cannot offer resistance to stem rot disease, and it requires these 13 favorable alleles to provide stable resistance. Different color highlights are provided for 10, 11, 12, 13 favorable alleles.

FIGURE 3

Representation of allele combinations required by genotype to show resistance to stem rot disease. Genotypes showing the extreme phenotype for the percent mortality were compared for the allelic differences for the identified 13 significant single nucleotide polymorphisms (SNPs), showing that all the resistant genotypes showed favorable alleles and susceptible genotypes showed unfavorable alleles. It was also found that all the resistance genotypes belong to Virginia type groundnut. MTA, marker trait association.

FIGURE 4

Gene ontology enrichment (A, B, C), pathway (D), and protein enrichment (E) for the genes that are found through genome‐wide association study (GWAS) for stem rot resistance along with the representation of comparative reaction of stem rot resistant and susceptible lines (F). Enrichment analysis showing the genes in significant correlation with gene ontology (GO) terms into biological processes, molecular function, and cellular components. Stem rot screening showing the intensity of disease at the time of sick field screening (F).

FIGURE 5

Pathway representing the involvement of identified candidate genes in the formation of different molecules providing the potential for a plant to withstand stem rot disease. All the genes/enzymes mentioned in red boxes are the genes found in this study. The compounds produced by the plant to defend and protect the cell from pathogen and their toxic chemicals (A), plant–pathogen interaction and different receptor‐like kinases (RLKs) produced and different responses by the plant (B), and the final decision taken by the plant if it is unable to protect the cell during severe infection, that is, autophagy (C).

FIGURE 6

Allele mining and validation of identified single nucleotide polymorphisms (SNPs) for stem rot resistance. (A) Allele mining for favorable alleles in a population of 524 genotypes, (B) genotypes selected having favorable alleles from allele mining forwarded for phenotypic confirmation, (C) phenotyping resulted in showing very low lesion/no lesion on the stem on treatment with oxalic acid assay, and (D) second stage validation using kompetitive allele‐specific PCR (KASP) assays showing finalized three markers for marker‐assisted selection for stem rot resistance. LOD, logarithm of the odds.