Manish K. Pandey

Single Nucleotide Polymorphism-based Genetic Diversity in the Reference Set of Peanut (Arachis spp.) by Developing and Applying Cost-Effective Kompetitive Allele Specific Polymerase Chain Reaction Genotyping Assays

Kompetitive allele‐specific polymerase chain reaction (KASP) assays have emerged as cost‐effective marker assays especially for molecular breeding applications. Therefore, a set of 96 informative single nucleotide polymorphisms (SNPs) was used to develop KASP assays in groundnut or peanut (Arachis spp.). Developed assays were designated as groundnut KASP assay markers (GKAMs) and screened on 94 genotypes (validation set) that included parental lines of 27 mapping populations, seven synthetic autotetraploid and amphidiploid lines, and 19 wild species accessions. As a result, 90 GKAMs could be validated and 73 GKAMs showed polymorphism in the validation set. Validated GKAMs were screened on 280 diverse genotypes of the reference set for estimating diversity features and elucidating genetic relationships. Cluster analysis of marker allelic data grouped accessions according to their genome type, subspecies, and botanical variety. The subspecies Arachis hypogaea L. subsp. fastigiata Waldron and A. hypogaea subsp. hypogaea formed distinct cluster; however, some overlaps were found indicating their frequent intercrossing during the course of evolution. The wild species, having diploid genomes, were grouped into a single cluster. The average polymorphism information content value for polymorphic GKAMs was 0.32 in the validation set and 0.31 in the reference set. These validated and highly informative GKAMs may be useful for genetics and breeding applications in Arachis species.

Advances in Arachis genomics for peanut improvement

Peanut genomics is very challenging due to its inherent problem of genetic architecture. Blockage of gene flow from diploid wild relatives to the tetraploid; cultivated peanut, recent polyploidization combined with self pollination, and the narrow genetic base of the primary genepool have resulted in low genetic diversity that has remained a major bottleneck for genetic improvement of peanut. Harnessing the rich source of wild relatives has been negligible due to differences in ploidy level as well as genetic drag and undesirable alleles for low yield. Lack of appropriate genomic resources has severely hampered molecular breeding activities, and this crop remains among the less-studied crops. The last five years, however, have witnessed accelerated development of genomic resources such as development of molecular markers, genetic and physical maps, generation of expressed sequenced tags (ESTs), development of mutant resources, and functional genomics platforms that facilitate the identification of QTLs and discovery of genes associated with tolerance/resistance to abiotic and biotic stresses and agronomic traits. Molecular breeding has been initiated for several traits for development of superior genotypes. The genome or at least gene space sequence is expected to be available in near future and this will further accelerate use of biotechnological approaches for peanut improvement.

Integrated consensus map of cultivated peanut and wild relatives reveals structures of the A and B genomes of Arachis and divergence of the legume genomes.

The complex, tetraploid genome structure of peanut (Arachis hypogaea) has obstructed advances in genetics and genomics in the species. The aim of this study is to understand the genome structure of Arachis by developing a high-density integrated consensus map. Three recombinant inbred line populations derived from crosses between the A genome diploid species, Arachis duranensis and Arachis stenosperma; the B genome diploid species, Arachis ipaënsis and Arachis magna; and between the AB genome tetraploids, A. hypogaea and an artificial amphidiploid (A. ipaënsis × A. duranensis)4×, were used to construct genetic linkage maps: 10 linkage groups (LGs) of 544 cM with 597 loci for the A genome; 10 LGs of 461 cM with 798 loci for the B genome; and 20 LGs of 1442 cM with 1469 loci for the AB genome. The resultant maps plus 13 published maps were integrated into a consensus map covering 2651 cM with 3693 marker loci which was anchored to 20 consensus LGs corresponding to the A and B genomes. The comparative genomics with genome sequences of Cajanus cajan, Glycine max, Lotus japonicus, and Medicago truncatula revealed that the Arachis genome has segmented synteny relationship to the other legumes. The comparative maps in legumes, integrated tetraploid consensus maps, and genome-specific diploid maps will increase the genetic and genomic understanding of Arachis and should facilitate molecular breeding.

An International Reference Consensus Genetic Map with 897 Marker Loci Based on 11 Mapping Populations for Tetraploid Groundnut (Arachis hypogaea L.)

Only a few genetic maps based on recombinant inbred line (RIL) and backcross (BC) populations have been developed for tetraploid groundnut. The marker density, however, is not very satisfactory especially in the context of large genome size (2800 Mb/1C) and 20 linkage groups (LGs). Therefore, using marker segregation data for 10 RILs and one BC population from the international groundnut community, with the help of common markers across different populations, a reference consensus genetic map has been developed. This map is comprised of 897 marker loci including 895 simple sequence repeat (SSR) and 2 cleaved amplified polymorphic sequence (CAPS) loci distributed on 20 LGs (a01–a10 and b01–b10) spanning a map distance of 3, 863.6 cM with an average map density of 4.4 cM. The highest numbers of markers (70) were integrated on a01 and the least number of markers (21) on b09. The marker density, however, was lowest (6.4 cM) on a08 and highest (2.5 cM) on a01. The reference consensus map has been divided into 20 cM long 203 BINs. These BINs carry 1 (a10_02, a10_08 and a10_09) to 20 (a10_04) loci with an average of 4 marker loci per BIN. Although the polymorphism information content (PIC) value was available for 526 markers in 190 BINs, 36 and 111 BINs have at least one marker with >0.70 and >0.50 PIC values, respectively. This information will be useful for selecting highly informative and uniformly distributed markers for developing new genetic maps, background selection and diversity analysis. Most importantly, this reference consensus map will serve as a reliable reference for aligning new genetic and physical maps, performing QTL analysis in a multi-populations design, evaluating the genetic background effect on QTL expression, and serving other genetic and molecular breeding activities in groundnut.

Legume Crops Phylogeny and Genetic Diversity for Science and Breeding

Economically, legumes (Fabaceae) represent the second most important family of crop plants after the grass family, Poaceae. Grain legumes account for 27% of world crop production and provide 33% of the dietary protein consumed by humans, while pasture and forage legumes provide vital part of animal feed. Fabaceae, the third largest family of flowering plants, has traditionally been divided into the following three subfamilies: Caesalpinioideae, Mimosoideae, and Papilionoideae, all together with 800 genera and 20,000 species. The latter subfamily contains most of the major cultivated food and feed crops. Among the grain legumes are some of mankinds earliest crop plants, whose domestication parallelled that of cereals: Soybean in China; faba bean, lentil, chickpea and pea in the Fertile Crescent of the Near East; cowpeas and bambara groundnut in Africa; soybean and mungbeans in East Asia; pigeonpea and the grams in South Asia; and common bean, lima bean, scarlet runner bean, tepary bean and lupin in Central and South America. The importance of legumes is evidenced by their high representation in ex situ germplasm collections, with more than 1,000,000 accessions worldwide. A detailed knowledge of the phylogenetic relationships of the Fabaceae is essential for understanding the origin and diversification of this economically and ecologically important family of angiosperms. This review aims to combine the phylogenetic and genetic diversity approaches to better illustrate the origin, domestication history and preserved germplasm of major legume crops from 13 genera of six tribes and to indicate further potential both for science and agriculture.

Draft genome of the peanut A-genome progenitor (Arachis duranensis) provides insights into geocarpy, oil biosynthesis, and allergens

Significance We present a draft genome of the peanut A-genome progenitor, Arachis duranensis, providing details on total genes present in the genome. Genome analysis suggests that the peanut lineage was affected by at least three polyploidizations since the origin of eudicots. Resequencing of synthetic Arachis tetraploids reveals extensive gene conversion since their formation by human hands. The A. duranensis genome provides a major source of candidate genes for fructification, oil biosynthesis, and allergens, expanding knowledge of understudied areas of plant biology and human health impacts of plants. This study also provides millions of structural variations that can be used as genetic markers for the development of improved peanut varieties through genomics-assisted breeding. Peanut or groundnut (Arachis hypogaea L.), a legume of South American origin, has high seed oil content (45–56%) and is a staple crop in semiarid tropical and subtropical regions, partially because of drought tolerance conferred by its geocarpic reproductive strategy. We present a draft genome of the peanut A-genome progenitor, Arachis duranensis, and 50,324 protein-coding gene models. Patterns of gene duplication suggest the peanut lineage has been affected by at least three polyploidizations since the origin of eudicots. Resequencing of synthetic Arachis tetraploids reveals extensive gene conversion in only three seed-to-seed generations since their formation by human hands, indicating that this process begins virtually immediately following polyploid formation. Expansion of some specific gene families suggests roles in the unusual subterranean fructification of Arachis. For example, the S1Fa-like transcription factor family has 126 Arachis members, in contrast to no more than five members in other examined plant species, and is more highly expressed in roots and etiolated seedlings than green leaves. The A. duranensis genome provides a major source of candidate genes for fructification, oil biosynthesis, and allergens, expanding knowledge of understudied areas of plant biology and human health impacts of plants, informing peanut genetic improvement and aiding deeper sequencing of Arachis diversity.

Emerging Genomic Tools for Legume Breeding: Current Status and Future Prospects

Legumes play a vital role in ensuring global nutritional food security and improving soil quality through nitrogen fixation. Accelerated higher genetic gains is required to meet the demand of ever increasing global population. In recent years, speedy developments have been witnessed in legume genomics due to advancements in next-generation sequencing (NGS) and high-throughput genotyping technologies. Reference genome sequences for many legume crops have been reported in the last 5 years. The availability of the draft genome sequences and re-sequencing of elite genotypes for several important legume crops have made it possible to identify structural variations at large scale. Availability of large-scale genomic resources and low-cost and high-throughput genotyping technologies are enhancing the efficiency and resolution of genetic mapping and marker-trait association studies. Most importantly, deployment of molecular breeding approaches has resulted in development of improved lines in some legume crops such as chickpea and groundnut. In order to support genomics-driven crop improvement at a fast pace, the deployment of breeder-friendly genomics and decision support tools seems appear to be critical in breeding programs in developing countries. This review provides an overview of emerging genomics and informatics tools/approaches that will be the key driving force for accelerating genomics-assisted breeding and ultimately ensuring nutritional and food security in developing countries.

Advances in genetics and molecular breeding of three legume crops of semi-arid tropics using next-generation sequencing and high-throughput genotyping technologies.

Molecular markers are the most powerful genomic tools to increase the efficiency and precision of breeding practices for crop improvement. Progress in the development of genomic resources in the leading legume crops of the semi-arid tropics (SAT), namely, chickpea (Cicer arietinum), pigeonpea (Cajanus cajan) and groundnut (Arachis hypogaea), as compared to other crop species like cereals, has been very slow. With the advances in next-generation sequencing (NGS) and high-throughput (HTP) genotyping methods, there is a shift in development of genomic resources including molecular markers in these crops. For instance, 2,000 to 3,000 novel simple sequence repeats (SSR) markers have been developed each for chickpea, pigeonpea and groundnut. Based on Sanger, 454/FLX and Illumina transcript reads, transcriptome assemblies have been developed for chickpea (44,845 transcript assembly contigs, or TACs) and pigeonpea (21,434 TACs). Illumina sequencing of some parental genotypes of mapping populations has resulted in the development of 120 million reads for chickpea and 128.9 million reads for pigeonpea. Alignment of these Illumina reads with respective transcriptome assemblies have provided >10,000 SNPs each in chickpea and pigeonpea. A variety of SNP genotyping platforms including GoldenGate, VeraCode and Competitive Allele Specific PCR (KASPar) assays have been developed in chickpea and pigeonpea. By using above resources, the first-generation or comprehensive genetic maps have been developed in the three legume species mentioned above. Analysis of phenotyping data together with genotyping data has provided candidate markers for drought-tolerance-related root traits in chickpea, resistance to foliar diseases in groundnut and sterility mosaic disease (SMD) and fertility restoration in pigeonpea. Together with these trait-associated markers along with those already available, molecular breeding programmes have been initiated for enhancing drought tolerance, resistance to fusarium wilt and ascochyta blight in chickpea and resistance to foliar diseases in groundnut. These trait-associated robust markers along with other genomic resources including genetic maps and genomic resources will certainly accelerate crop improvement programmes in the SAT legumes.

Groundnut improvement: use of genetic and genomic tools.

Groundnut (Arachis hypogaea L.), a self-pollinated legume is an important crop cultivated in 24 million ha world over for extraction of edible oil and food uses. The kernels are rich in oil (48–50%) and protein (25–28%), and are source of several vitamins, minerals, antioxidants, biologically active polyphenols, flavonoids, and isoflavones. Improved varieties of groundnut with high yield potential were developed and released for cultivation world over. The improved varieties belong to different maturity durations and possess resistance to diseases, tolerance to drought, enhanced oil content, and improved quality traits for food uses. Conventional breeding procedures along with the tools for phenotyping were largely used in groundnut improvement programs. Mutations were used to induce variability and wide hybridization was attempted to tap variability from wild species. Low genetic variability has been a bottleneck for groundnut improvement. The vast potential of wild species, reservoir of new alleles remains under-utilized. Development of linkage maps of groundnut during the last decade was followed by identification of markers and quantitative trait loci for the target traits. Consequently, the last decade has witnessed the deployment of molecular breeding approaches to complement the ongoing groundnut improvement programs in USA, China, India, and Japan. The other potential advantages of molecular breeding are the feasibility to target multiple traits for improvement and provide tools to tap new alleles from wild species. The first groundnut variety developed through marker-assisted back-crossing is a root-knot nematode-resistant variety, NemaTAM in USA. The uptake of molecular breeding approaches in groundnut improvement programs by NARS partners in India and many African countries is slow or needs to be initiated in part due to inadequate infrastructure, high genotyping costs, and human capacities. Availability of draft genome sequence for diploid (AA and BB) and tetraploid, AABB genome species of Arachis in coming years is expected to bring low-cost genotyping to the groundnut community that will facilitate use of modern genetics and breeding approaches such as genome-wide association studies for trait mapping and genomic selection for crop improvement.

Identification of QTLs associated with oil content and mapping FAD2 genes and their relative contribution to oil quality in peanut (Arachis hypogaea L.)

BackgroundPeanut is one of the major source for human consumption worldwide and its seed contain approximately 50% oil. Improvement of oil content and quality traits (high oleic and low linoleic acid) in peanut could be accelerated by exploiting linked markers through molecular breeding. The objective of this study was to identify QTLs associated with oil content, and estimate relative contribution of FAD2 genes (ahFAD2A and ahFAD2B) to oil quality traits in two recombinant inbred line (RIL) populations.ResultsImproved genetic linkage maps were developed for S-population (SunOleic 97R × NC94022) with 206 (1780.6 cM) and T-population (Tifrunner × GT-C20) with 378 (2487.4 cM) marker loci. A total of 6 and 9 QTLs controlling oil content were identified in the S- and T-population, respectively. The contribution of each QTL towards oil content variation ranged from 3.07 to 10.23% in the S-population and from 3.93 to 14.07% in the T-population. The mapping positions for ahFAD2A (A sub-genome) and ahFAD2B (B sub-genome) genes were assigned on a09 and b09 linkage groups. The ahFAD2B gene (26.54%, 25.59% and 41.02% PVE) had higher phenotypic effect on oleic acid (C18:1), linoleic acid (C18:2), and oleic/linoleic acid ratio (O/L ratio) than ahFAD2A gene (8.08%, 6.86% and 3.78% PVE). The FAD2 genes had no effect on oil content. This study identified a total of 78 main-effect QTLs (M-QTLs) with up to 42.33% phenotypic variation (PVE) and 10 epistatic QTLs (E-QTLs) up to 3.31% PVE for oil content and quality traits.ConclusionsA total of 78 main-effect QTLs (M-QTLs) and 10 E-QTLs have been detected for oil content and oil quality traits. One major QTL (more than 10% PVE) was identified in both the populations for oil content with source alleles from NC94022 and GT-C20 parental genotypes. FAD2 genes showed high effect for oleic acid (C18:1), linoleic acid (C18:2), and O/L ratio while no effect on total oil content. The information on phenotypic effect of FAD2 genes for oleic acid, linoleic acid and O/L ratio, and oil content will be applied in breeding selection.