Genomic tools in groundnut breeding program: Status and perspectives

Groundnut, a nutrient-rich food legume, is cultivated world over. It is valued for its good quality cooking oil, energy and protein rich food, and nutrient-rich fodder. Globally, groundnut improvement programs have developed varieties to meet the preferences of farmers, traders, processors, and consumers. Enhanced yield, tolerance to biotic and abiotic stresses and quality parameters have been the target traits. Spurt in genetic information of groundnut was facilitated by development of molecular markers, genetic, and physical maps, generation of expressed sequence tags (EST), discovery of genes, and identification of quantitative trait loci (QTL) for some important biotic and abiotic stresses and quality traits. The first groundnut variety developed using marker assisted breeding (MAB) was registered in 2003. Since then, USA, China, Japan, and India have begun to use genomic tools in routine groundnut improvement programs. Introgression lines that combine foliar fungal disease resistance and early maturity were developed using MAB. Establishment of marker-trait associations (MTA) paved way to integrate genomic tools in groundnut breeding for accelerated genetic gain. Genomic Selection (GS) tools are employed to improve drought tolerance and pod yield, governed by several minor effect QTLs. Draft genome sequence and low cost genotyping tools such as genotyping by sequencing (GBS) are expected to accelerate use of genomic tools to enhance genetic gains for target traits in groundnut

Single seed-based high-throughput genotyping and rapid generation advancement for accelerated groundnut genetics and breeding research

The groundnut breeding program at International Crops Research Institute for the Semi-Arid Tropics routinely performs marker-based early generation selection (MEGS) in thousands of segregating populations. The existing MEGS includes planting of segregating populations in fields or glasshouses, label tagging, and sample collection using leaf-punch from 20–25 day old plants followed by genotyping with 10 single nucleotide polymorphisms based early generation selection marker panels in a high throughput genotyping (HTPG) platform. The entire process is laborious, time consuming, and costly. Therefore, in order to save the time of the breeder and to reduce the cost during MEGS, we optimized a single seed chipping (SSC) process based MEGS protocol and deployed on large scale by genotyping >3000 samples from ongoing groundnut breeding program. In SSC-based MEGS, we used a small portion of cotyledon by slicing-off the posterior end of the single seed and transferred to the 96-deep well plate for DNA isolation and genotyping at HTPG platform. The chipped seeds were placed in 96-well seed-box in the same order of 96-well DNA sampling plate to enable tracking back to the selected individual seed. A high germination rate of 95–99% from the chipped seeds indicated that slicing of seeds from posterior end does not significantly affect germination percentage. In addition, we could successfully advance 3.5 generations in a year using a low-cost rapid generation turnover glass-house facility as compared to routine practice of two generations in field conditions. The integration of SSC based genotyping and rapid generation advancement (RGA) could significantly reduce the operational requirement of person-hours and expenses, and save a period of 6–8 months in groundnut genetics and breeding research

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

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

Improvement of three popular Indian groundnut varieties for foliar disease resistance and high oleic acid using SSR markers and SNP array in marker-assisted backcrossing

Foliar fungal diseases (rust and late leaf spot) incur large yield losses, in addition to the deterioration of fodder quality in groundnut worldwide. High oleic acid has emerged as a key market trait in groundnut, as it increases the shelf life of the produce/products in addition to providing health benefits to consumers. Marker-assisted backcrossing (MABC) is the most successful approach to introgressing or pyramiding one or more traits using trait-linked markers. We used MABC to improve three popular Indian cultivars (GJG 9, GG 20, and GJGHPS 1) for foliar disease resistance (FDR) and high oleic acid content. A total of 22 BC3F4 and 30 BC2F4 introgression lines (ILs) for FDR and 46 BC3F4 and 41 BC2F4 ILs for high oleic acid were developed. Recurrent parent genome analysis using the 58 K Axiom_Arachis array identified several lines showing upto 94% of genome recovery among second and third backcross progenies. Phenotyping of these ILs revealed FDR scores comparable to the resistant parent, GPBD 4, and ILs with high (~80%) oleic acid in addition to high genome recovery. These ILs provide further opportunities for pyramiding FDR and high oleic acid in all three genetic backgrounds as well as for conducting multi-location yield trials for further evaluation and release for cultivation in target regions of India

Genomic regions associated with resistance to peanut bud necrosis disease (PBND) in a recombinant inbred line (RIL) population

Parents and 318 F8 recombinant inbred lines (RILs) derived from the cross, TAG 24 × ICGV 86031 were evaluated for peanut bud necrosis disease (PBND) resistance and agronomic traits under natural infestation of thrips at a disease hotspot location for 2 years. Significant genotype, environment and genotype × environment interaction effects suggested role of environment in development and spread of the disease. Quantitative trait loci (QTL) analysis using QTL Cartographer identified a total of 14 QTL for six traits of which five QTL were for disease incidence. One quantitative trait locus q60DI located on LG_AhII was identified using both QTL Cartographer and QTL Network. Another QTL q90DI was detected with a high PVE of 12.57 using QTL Cartographer. A total of nine significant additive × additive (AA) interactions were detected for PBND disease incidence and yield traits with two and seven interactions displaying effects in favour of the parental and recombinant genotype combinations, respectively. This is the first attempt on QTL discovery associated with PBND resistance in peanut. Superior RILs identified in the study can be recycled or released as variety following further evaluations

Foliar fungal disease-resistant introgression lines of groundnut (Arachis hypogaeaL.) record higher pod and haulm yield in multilocation testing

Introgression lines (ILs) of groundnut with enhanced resistance to rust and late leaf spot (LLS) recorded increased pod and haulm yield in multilocation testing. Marker-assisted backcrossing (MABC) approach was used to introgress a genomic region containing a major QTL that explains >80% of phenotypic variance (PV) for rust resistance and 67.98% PV for LLS resistance. ILs in the genetic background of TAG 24, ICGV 91114 and JL 24 were evaluated for two seasons to select 20 best ILs based on resistance, productivity parameters and maturity duration. Multilocation evaluation of the selected ILs was conducted in three locations including disease hot spots. Background genotype, environment and genotype × environment interactions are important for expression of resistance governed by the QTL region. Six best ILs namely ICGV 13192, ICGV 13193, ICGV 13200, ICGV 13206, ICGV 13228 and ICGV 13229 were selected with 39–79% higher mean pod yield and 25–89% higher mean haulm yield over their respective recurrent parents. Pod yield increase was contributed by increase in seed mass and number of pods per plant

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Advances in crop improvement and delivery research for nutritional quality and health benefits of groundnut (Arachis hypogaea L.)

Groundnut is an important global food and oil crop that underpins agriculture-dependent livelihood strategies meeting food, nutrition, and income security. Aflatoxins, pose a major challenge to increased competitiveness of groundnut limiting access to lucrative markets and affecting populations that consume it. Other drivers of low competitiveness include allergens and limited shelf life occasioned by low oleic acid profile in the oil. Thus grain off-takers such as consumers, domestic, and export markets as well as processors need solutions to increase profitability of the grain. There are some technological solutions to these challenges and this review paper highlights advances in crop improvement to enhance groundnut grain quality and nutrient profile for food, nutrition, and economic benefits. Significant advances have been made in setting the stage for marker-assisted allele pyramiding for different aflatoxin resistance mechanisms—in vitro seed colonization, pre-harvest aflatoxin contamination, and aflatoxin production—which, together with pre- and post-harvest management practices, will go a long way in mitigating the aflatoxin menace. A breakthrough in aflatoxin control is in sight with overexpression of antifungal plant defensins, and through host-induced gene silencing in the aflatoxin biosynthetic pathway. Similarly, genomic and biochemical approaches to allergen control are in good progress, with the identification of homologs of the allergen encoding genes and development of monoclonal antibody based ELISA protocol to screen for and quantify major allergens. Double mutation of the allotetraploid homeologous genes, FAD2A and FAD2B, has shown potential for achieving >75% oleic acid as demonstrated among introgression lines. Significant advances have been made in seed systems research to bridge the gap between trait discovery, deployment, and delivery through innovative partnerships and action learning

Assessing variability for disease resistance and nutritional quality traits in an interspecific collection of groundnut (Arachis hypogaea)

Rust and late leaf spot (LLS) resistance sources involving Arachis batizocoi, A. duranensis, A. cardenasii and A. sps Manfredi‐5 were identified from field evaluation of interspecific derivatives (IDs) of groundnut in a disease nursery for two seasons. Although the sources displayed low levels of resistance compared to currently cultivated lines, they contribute allele diversity in groundnut breeding that has so far relied on alleles contributed from A. cardenasii for disease resistance. Multiple disease‐resistant and agronomically superior IDs, ICGVs 11379, 10121, 10179, 05097, 02411 and 00248 involving A. batizocoi, A. duranensis and A. cardenasii can be used in breeding for groundnut improvement. Genetic variability for resistance to rust and LLS, yield and nutritional quality traits was influenced by genotype, environment and genotype × environment interaction effects in individual and pooled analyses. In case of FAD (fatty acid desaturase)‐mutant alleles that govern high oleic trait, allele mining of IDs (110) showed that frequency of mutation in ahFAD2B is rare, whereas of ahFAD2A is common. High oleic lines were not detected among the IDs