High-throughput diagnostic markers for foliar fungal disease resistance and high oleic acid content in groundnut.

BACKGROUND: Foliar diseases namely late leaf spot (LLS) and leaf rust (LR) reduce yield and deteriorate fodder quality in groundnut. Also the high oleic acid content has emerged as one of the most important traits for industries and consumers due to its increased shelf life and health benefits. RESULTS: Genetic mapping combined with pooled sequencing approaches identified candidate resistance genes (LLSR1 and LLSR2 for LLS and LR1 for LR) for both foliar fungal diseases. The LLS-A02 locus housed LLSR1 gene for LLS resistance, while, LLS-A03 housed LLSR2 and LR1 genes for LLS and LR resistance, respectively. A total of 49 KASPs markers were developed from the genomic regions of important disease resistance genes, such as NBS-LRR, purple acid phosphatase, pentatricopeptide repeat-containing protein, and serine/threonine-protein phosphatase. Among the 49 KASP markers, 41 KASPs were validated successfully on a validation panel of contrasting germplasm and breeding lines. Of the 41 validated KASPs, 39 KASPs were designed for rust and LLS resistance, while two KASPs were developed using fatty acid desaturase (FAD) genes to control high oleic acid levels. These validated KASP markers have been extensively used by various groundnut breeding programs across the world which led to development of thousands of advanced breeding lines and few of them also released for commercial cultivation. CONCLUSION: In this study, high-throughput and cost-effective KASP assays were developed, validated and successfully deployed to improve the resistance against foliar fungal diseases and oleic acid in groundnut. So far deployment of allele-specific and KASP diagnostic markers facilitated development and release of two rust- and LLS-resistant varieties and five high-oleic acid groundnut varieties in India. These validated markers provide opportunities for routine deployment in groundnut breeding programs.

Whole-genome resequencing-based QTL-seq identified candidate genes and molecular markers for fresh seed dormancy in groundnut.

The subspecies fastigiata of cultivated groundnut lost fresh seed dormancy (FSD) during domestication and human-made selection. Groundnut varieties lacking FSD experience precocious seed germination during harvest imposing severe losses. Development of easy-to-use genetic markers enables early-generation selection in different molecular breeding approaches. In this context, one recombinant inbred lines (RIL) population (ICGV 00350 × ICGV 97045) segregating for FSD was used for deploying QTL-seq approach for identification of key genomic regions and candidate genes. Whole-genome sequencing (WGS) data (87.93 Gbp) were generated and analysed for the dormant parent (ICGV 97045) and two DNA pools (dormant and nondormant). After analysis of resequenced data from the pooled samples with dormant parent (reference genome), we calculated delta-SNP index and identified a total of 10,759 genomewide high-confidence SNPs. Two candidate genomic regions spanning 2.4 Mb and 0.74 Mb on the B05 and A09 pseudomolecules, respectively, were identified controlling FSD. Two candidate genes-RING-H2 finger protein and zeaxanthin epoxidase-were identified in these two regions, which significantly express during seed development and control abscisic acid (ABA) accumulation. QTL-seq study presented here laid out development of a marker, GMFSD1, which was validated on a diverse panel and could be used in molecular breeding to improve dormancy in groundnut.

High-density genetic map using whole-genome resequencing for fine mapping and candidate gene discovery for disease resistance in peanut.

Whole-genome resequencing (WGRS) of mapping populations has facilitated development of high-density genetic maps essential for fine mapping and candidate gene discovery for traits of interest in crop species. Leaf spots, including early leaf spot (ELS) and late leaf spot (LLS), and Tomato spotted wilt virus (TSWV) are devastating diseases in peanut causing significant yield loss. We generated WGRS data on a recombinant inbred line population, developed a SNP-based high-density genetic map, and conducted fine mapping, candidate gene discovery and marker validation for ELS, LLS and TSWV. The first sequence-based high-density map was constructed with 8869 SNPs assigned to 20 linkage groups, representing 20 chromosomes, for the 'T' population (Tifrunner × GT-C20) with a map length of 3120 cM and an average distance of 1.45 cM. The quantitative trait locus (QTL) analysis using high-density genetic map and multiple season phenotyping data identified 35 main-effect QTLs with phenotypic variation explained (PVE) from 6.32% to 47.63%. Among major-effect QTLs mapped, there were two QTLs for ELS on B05 with 47.42% PVE and B03 with 47.38% PVE, two QTLs for LLS on A05 with 47.63% and B03 with 34.03% PVE and one QTL for TSWV on B09 with 40.71% PVE. The epistasis and environment interaction analyses identified significant environmental effects on these traits. The identified QTL regions had disease resistance genes including R-genes and transcription factors. KASP markers were developed for major QTLs and validated in the population and are ready for further deployment in genomics-assisted breeding in peanut.

SSR markers associated to early leaf spot disease resistance through selective genotyping and single marker analysis in groundnut (Arachis hypogaea L.).

Groundnut (Arachis hypogaea L.) is an important oilseed and food crop of the world. Breeding for disease resistance is one of major objectives in groundnut breeding. Early leaf spot (ELS) is one of the major destructive diseases worldwide and in West Africa, particularly in Burkina Faso causing significant yield losses. Conventional breeding approaches have been employed to develop improved varieties resistant to ELS. Molecular dissection of resistance traits using QTL analysis can improve the efficiency of resistance breeding. In the present study, an ELS susceptible genotype QH243C and an ELS resistant genotype NAMA were crossed and the F2 population genotypic and F3 progenies phenotypic data were used for marker-trait association analysis. Parents were surveyed with 179 simple sequence repeat (SSR) markers out of which 103 SSR markers were found to be polymorphic between the parents. These polymorphic markers were utilized to genotype the F2 population followed by marker-trait analysis through single marker analysis (SMA) and selective genotyping of the population using 23 resistant and 23 susceptible genotypes. The SMA revealed 13 markers while the selective genotyping method identified 8 markers associated with ELS resistance. Four markers (GM1911, GM1883, GM1000 and Seq13E09) were found common between the two trait mapping methods. These four markers could be employed in genomics-assisted breeding for selection of ELS resistant genotypes in groundnut breeding.

Molecular Mapping of Oil Content and Fatty Acids Using Dense Genetic Maps in Groundnut (Arachis hypogaea L.).

Enhancing seed oil content with desirable fatty acid composition is one of the most important objectives of groundnut breeding programs globally. Genomics-assisted breeding facilitates combining multiple traits faster, however, requires linked markers. In this context, we have developed two different F2 mapping populations, one for oil content (OC-population, ICGV 07368 × ICGV 06420) and another for fatty acid composition (FA-population, ICGV 06420 × SunOleic 95R). These two populations were phenotyped for respective traits and genotyped using Diversity Array Technology (DArT) and DArTseq genotyping platforms. Two genetic maps were developed with 854 (OC-population) and 1,435 (FA-population) marker loci with total map distance of 3,526 and 1,869 cM, respectively. Quantitative trait locus (QTL) analysis using genotyping and phenotyping data identified eight QTLs for oil content including two major QTLs, qOc-A10 and qOc-A02, with 22.11 and 10.37% phenotypic variance explained (PVE), respectively. For seven different fatty acids, a total of 21 QTLs with 7.6-78.6% PVE were identified and 20 of these QTLs were of major effect. Two mutant alleles, ahFAD2B and ahFAD2A, also had 18.44 and 10.78% PVE for palmitic acid, in addition to oleic (33.8 and 17.4% PVE) and linoleic (41.0 and 19.5% PVE) acids. Furthermore, four QTL clusters harboring more than three QTLs for fatty acids were identified on the three LGs. The QTLs identified in this study could be further dissected for candidate gene discovery and development of diagnostic markers for breeding improved groundnut varieties with high oil content and desirable oil quality.

Genome-Wide Discovery and Deployment of Insertions and Deletions Markers Provided Greater Insights on Species, Genomes, and Sections Relationships in the Genus Arachis.

Small insertions and deletions (InDels) are the second most prevalent and the most abundant structural variations in plant genomes. In order to deploy these genetic variations for genetic analysis in genus Arachis, we conducted comparative analysis of the draft genome assemblies of both the diploid progenitor species of cultivated tetraploid groundnut (Arachis hypogaea L.) i.e., Arachis duranensis (A subgenome) and Arachis ipaënsis (B subgenome) and identified 515,223 InDels. These InDels include 269,973 insertions identified in A. ipaënsis against A. duranensis while 245,250 deletions in A. duranensis against A. ipaënsis. The majority of the InDels were of single bp (43.7%) and 2-10 bp (39.9%) while the remaining were >10 bp (16.4%). Phylogenetic analysis using genotyping data for 86 (40.19%) polymorphic markers grouped 96 diverse Arachis accessions into eight clusters mostly by the affinity of their genome. This study also provided evidence for the existence of "K" genome, although distinct from both the "A" and "B" genomes, but more similar to "B" genome. The complete homology between A. monticola and A. hypogaea tetraploid taxa showed a very similar genome composition. The above analysis has provided greater insights into the phylogenetic relationship among accessions, genomes, sub species and sections. These InDel markers are very useful resource for groundnut research community for genetic analysis and breeding applications.

QTL-seq approach identified genomic regions and diagnostic markers for rust and late leaf spot resistance in groundnut (Arachis hypogaea L.).

Rust and late leaf spot (LLS) are the two major foliar fungal diseases in groundnut, and their co-occurrence leads to significant yield loss in addition to the deterioration of fodder quality. To identify candidate genomic regions controlling resistance to rust and LLS, whole-genome resequencing (WGRS)-based approach referred as 'QTL-seq' was deployed. A total of 231.67 Gb raw and 192.10 Gb of clean sequence data were generated through WGRS of resistant parent and the resistant and susceptible bulks for rust and LLS. Sequence analysis of bulks for rust and LLS with reference-guided resistant parent assembly identified 3136 single-nucleotide polymorphisms (SNPs) for rust and 66 SNPs for LLS with the read depth of ≥7 in the identified genomic region on pseudomolecule A03. Detailed analysis identified 30 nonsynonymous SNPs affecting 25 candidate genes for rust resistance, while 14 intronic and three synonymous SNPs affecting nine candidate genes for LLS resistance. Subsequently, allele-specific diagnostic markers were identified for three SNPs for rust resistance and one SNP for LLS resistance. Genotyping of one RIL population (TAG 24 × GPBD 4) with these four diagnostic markers revealed higher phenotypic variation for these two diseases. These results suggest usefulness of QTL-seq approach in precise and rapid identification of candidate genomic regions and development of diagnostic markers for breeding applications.

Molecular breeding for introgression of fatty acid desaturase mutant alleles (ahFAD2A and ahFAD2B) enhances oil quality in high and low oil containing peanut genotypes.

High oleate peanuts have two marketable benefits, health benefits to consumers and extended shelf life of peanut products. Two mutant alleles present on linkage group a09 (ahFAD2A) and b09 (ahFAD2B) control composition of three major fatty acids, oleic, linoleic and palmitic acids which together determine peanut oil quality. In conventional breeding, selection for fatty acid composition is delayed to advanced generations. However by using DNA markers, breeders can reject large number of plants in early generations and therefore can optimize time and resources. Here, two approaches of molecular breeding namely marker-assisted backcrossing (MABC) and marker-assisted selection (MAS) were employed to transfer two FAD2 mutant alleles from SunOleic 95R into the genetic background of ICGV 06110, ICGV 06142 and ICGV 06420. In summary, 82 MABC and 387 MAS derived introgression lines (ILs) were developed using DNA markers with elevated oleic acid varying from 62 to 83%. Oleic acid increased by 0.5-1.1 folds, with concomitant reduction of linoleic acid by 0.4-1.0 folds and palmitic acid by 0.1-0.6 folds among ILs compared to recurrent parents. Finally, high oleate ILs, 27 with high oil (53-58%), and 28 ILs with low oil content (42-50%) were selected that may be released for cultivation upon further evaluation.