Genetic mapping of legume orthologs reveals high conservation of synteny between lentil species and the sequenced genomes of Medicago and chickpea.

Lentil (Lens culinaris Medik.) is a global food crop with increasing importance for food security in south Asia and other regions. Lens ervoides, a wild relative of cultivated lentil, is an important source of agronomic trait variation. Lens is a member of the galegoid clade of the Papilionoideae family, which includes other important dietary legumes such as chickpea (Cicer arietinum) and pea (Pisum sativum), and the sequenced model legume Medicago truncatula. Understanding the genetic structure of Lens spp. in relation to more fully sequenced legumes would allow leveraging of genomic resources. A set of 1107 TOG-based amplicons were identified in L. ervoides and a subset thereof used to design SNP markers for mapping. A map of L. ervoides consisting of 377 SNP markers spread across seven linkage groups was developed using a GoldenGate genotyping array and single SNP marker assays. Comparison with maps of M. truncatula and L. culinaris documented considerable shared synteny and led to the identification of a few major translocations and a major inversion that distinguish Lens from M. truncatula, as well as a translocation that distinguishes L. culinaris from L. ervoides. The identification of chromosome-level differences among Lens spp. will aid in the understanding of introgression of genes from L. ervoides into cultivated L. culinaris, furthering genetic research and breeding applications in lentil

Prioritization of candidate genes in "QTL-hotspot" region for drought tolerance in chickpea (Cicer arietinum L.)

A combination of two approaches, namely QTL analysis and gene enrichment analysis were used to identify candidate genes in the "QTL-hotspot" region for drought tolerance present on the Ca4 pseudomolecule in chickpea. In the first approach, a high-density bin map was developed using 53,223 single nucleotide polymorphisms (SNPs) identified in the recombinant inbred line (RIL) population of ICC 4958 (drought tolerant) and ICC 1882 (drought sensitive) cross. QTL analysis using recombination bins as markers along with the phenotyping data for 17 drought tolerance related traits obtained over 1-5 seasons and 1-5 locations split the "QTL-hotspot" region into two subregions namely "QTL-hotspot_a" (15 genes) and "QTL-hotspot_b" (11 genes). In the second approach, gene enrichment analysis using significant marker trait associations based on SNPs from the Ca4 pseudomolecule with the above mentioned phenotyping data, and the candidate genes from the refined "QTL-hotspot" region showed enrichment for 23 genes. Twelve genes were found common in both approaches. Functional validation using quantitative real-time PCR (qRT-PCR) indicated four promising candidate genes having functional implications on the effect of "QTL-hotspot" for drought tolerance in chickpea.Sandip M Kale, Deepa Jaganathan, Pradeep Ruperao, Charles Chen, Ramu Punna, Himabindu Kudapa, Mahendar Thudi, Manish Roorkiwal, Mohan AVSK Katta, Dadakhalandar Doddamani, Vanika Garg, P B Kavi Kishor, Pooran M Gaur, Henry T Nguyen, Jacqueline Batley, David Edwards, Tim Sutton and Rajeev K Varshne

Development of genomic resources for Rhodes grass (Chloris gayana), draft genome and annotated variant discovery

Genomic resources for grasses, especially warm-season grasses are limited despite their commercial and environmental importance. Here, we report the first annotated draft whole genome sequence for diploid Rhodes grass (Chloris gayana), a tropical C4 species. Generated using long read nanopore sequencing and assembled using the Flye software package, the assembled genome is 603 Mbp in size and comprises 5,233 fragments that were annotated using the GenSas pipeline. The annotated genome has 46,087 predicted genes corresponding to 92.0% of the expected genomic content present via BUSCO analysis. Gene ontology terms and repetitive elements are identified and discussed. An additional 94 individual plant genotypes originating from three diploid and two tetraploid Rhodes grass cultivars were short-read whole genome resequenced (WGR) to generate a single nucleotide polymorphism (SNP) resource for the species that can be used to elucidate inter- and intra-cultivar relationships across both ploidy levels. A total of 75,777 high quality SNPs were used to generate a phylogenetic tree, highlighting the diversity present within the cultivars which agreed with the known breeding history. Differentiation was observed between diploid and tetraploid cultivars. The WGR data were also used to provide insights into the nature and evolution of the tetraploid status of the species, with results largely agreeing with the published literature that the tetraploids are autotetraploid

Development and bin mapping of gene-associated interspecific SNPs for cotton (Gossypium hirsutum L.) introgression breeding efforts

BACKGROUND: Cotton (Gossypium spp.) is the largest producer of natural fibers for textile and is an important crop worldwide. Crop production is comprised primarily of G. hirsutum L., an allotetraploid. However, elite cultivars express very small amounts of variation due to the species monophyletic origin, domestication and further bottlenecks due to selection. Conversely, wild cotton species harbor extensive genetic diversity of prospective utility to improve many beneficial agronomic traits, fiber characteristics, and resistance to disease and drought. Introgression of traits from wild species can provide a natural way to incorporate advantageous traits through breeding to generate higher-producing cotton cultivars and more sustainable production systems. Interspecific introgression efforts by conventional methods are very time-consuming and costly, but can be expedited using marker-assisted selection. RESULTS: Using transcriptome sequencing we have developed the first gene-associated single nucleotide polymorphism (SNP) markers for wild cotton species G. tomentosum, G. mustelinum, G. armourianum and G. longicalyx. Markers were also developed for a secondary cultivated species G. barbadense cv. 3–79. A total of 62,832 non-redundant SNP markers were developed from the five wild species which can be utilized for interspecific germplasm introgression into cultivated G. hirsutum and are directly associated with genes. Over 500 of the G. barbadense markers have been validated by whole-genome radiation hybrid mapping. Overall 1,060 SNPs from the five different species have been screened and shown to produce acceptable genotyping assays. CONCLUSIONS: This large set of 62,832 SNPs relative to cultivated G. hirsutum will allow for the first high-density mapping of genes from five wild species that affect traits of interest, including beneficial agronomic and fiber characteristics. Upon mapping, the markers can be utilized for marker-assisted introgression of new germplasm into cultivated cotton and in subsequent breeding of agronomically adapted types, including cultivar development. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-945) contains supplementary material, which is available to authorized users

Identification of Candidate Genes Associated With Resistance Against Race 0 of Colletotrichum lentis In Lens ervoides

Lens ervoides is a potential source of resistance to anthracnose caused by the pathogen Colletotrichum lentis. Transcriptome sequencing was performed on the resistant LR-66-528 and susceptible LR-66-524 recombinant inbred lines (RILs) of L. ervoides infected with the aggressive race 0 isolate CT-30 of C. lentis to unravel the genetic control underlying the genetic responses against this pathogen. The inoculated samples were harvested at 6, 12, 24, 48, 72, 96 and 144 hours post-inoculation (hpi) for molecular studies. Results of quantitative PCR to estimate fungal biomass revealed that 24, 48, 72 and 96 hpi were interesting time-points for studying disease development because of exponential trends of fungal growth during this period. Subsequent comparison of gene expression based on RNA-Seq at 24, 48, 72 and 96 hpi with that of mock (non-inoculated) samples showed that 3,091 disease responsive genes. Among them, 477 were differentially expressed genes (DEGs) (fold change >2, Padj < 0.05) between the resistant and susceptible RILs. Based on expression profiling, these DEGs were clustered into six expression clusters (C1-C6). In Cluster C1, 56 genes were up-regulated in the susceptible RIL whereas in C2, 79 genes were up-regulated in that RIL, mainly at 96 hpi. Cluster C3 contained 91 genes that were up-regulated in the resistant RIL LR-66-528 at 24, 72 and 96 hpi. A total of 97 genes in C4 were significantly up-regulated in LR-66-524 at 24 and 48 hpi. Cluster C5 with 51 genes was the smallest cluster with genes up-regulated in the resistant LR-66-528 and down-regulated in the susceptible LR-66-524, as were 95 genes in Cluster C6. DEGs were functionally annotated to identify those with known functions in disease resistance proteins, such as LRR and NB-ARC domain disease resistance protein, Protein Detoxification, LRR receptor-like kinase family proteins, and Wall-associated Ser/Thr Kinases. The expression of 21 of these genes was validated using RT-qPCR, which confirmed up- or down-regulation as in the RNA-Seq data. Comparison of DEGs and genes in QTLs associated with resistance to anthracnose revealed that nine DEGs were located in the resistance QTL region of chromosome 2, ten in the QTL region of chromosome 5 and three in the QTL region of chromosome 7 of L. ervoides. The identified candidate genes associated with resistance should be valuable targets for the future gene function analyses

Development of Genomic Markers and Mapping Tools for Assembling the Allotetraploid Gossypium hirsutum L. Draft Genome Sequence

Cotton (Gossypium spp.) is the largest producer of natural textile fibers. Most worldwide and domestic cotton fiber production is based on cultivars of G. hirsutum L., an allotetraploid. Genetic improvement of cotton remains constrained by alarmingly low levels of genetic diversity, inadequate genomic tools for genetic analysis and manipulation, and the difficulty of effectively harnessing the vastly greater genetic diversity harbored by other Gossypium species. Development of large numbers of single nucleotide polymorphisms (SNPs) for use in intraspecific and interspecific populations will allow for cotton germplasm diversity characterization, high-throughput genotyping, marker-assisted breeding, germplasm introgression of advantageous traits from wild species, and high-density genetic mapping. My research has been focused on utilizing next generation sequencing data for intraspecific and interspecific SNP marker development, validation, and creation of high-throughput genotyping methods to advance cotton research. I used transcriptome sequencing to develop and map the first gene-associated SNPs for five species, G. barbadense (Pima cotton), G. tomentosum, G. mustelinum, G. armourianum, and G. longicalyx. A total of 62,832 non-redundant SNPs were developed. These can be utilized for interspecific germplasm introgression into cultivated G. hirsutum, as well as for subsequent genetic analysis and manipulation. To create SNP-based resources for integrated physical mapping, I used BAC-end sequences (BESs) and resequecing data for 12 G. hirsutum lines, a Pima line and G. longicalyx to derive 132,262 intraspecific and 693,769 interspecific SNPs located in BESs. These SNP data sets were used to help build the first high-throughput genotyping array for cotton, the CottonSNP63K, which now provides a standardized platform for global cotton research. I applied the array to two F2 populations and produced the first two high-density SNP maps for cotton, one intraspecific and one interspecific. By resequencing two interspecific F1 hypo-aneuploids, I also demonstrated that the chromosome-wide changes in SNP genotypes enable highly effective mass-localization of BACs to individual cotton chromosomes. These efforts provide additional validation and placement methods that can be directly integrated with the physical map being constructed for G. hirsutum and enable the production of a high-quality draft genome sequence for cultivated cotton. I used transcriptome sequencing to develop and map the first gene-associated SNPs for five species, G. barbadense (Pima cotton), G. tomentosum, G. mustelinum, G. armourianum, and G. longicalyx. A total of 62,832 non-redundant SNPs were developed. These can be utilized for interspecific germplasm introgression into cultivated G. hirsutum, as well as for subsequent genetic analysis and manipulation. To create SNP-based resources for integrated physical mapping, I used BAC-end sequences (BESs) and resequecing data for 12 G. hirsutum lines, a Pima line and G. longicalyx to derive 132,262 intraspecific and 693,769 interspecific SNPs located in BESs. These SNP data sets were used to help build the first high-throughput genotyping array for cotton, the CottonSNP63K, which now provides a standardized platform for global cotton research. I applied the array to two F2 populations and produced the first two high-density SNP maps for cotton, one intraspecific and one interspecific. By resequencing two interspecific F1 hypo-aneuploids, I also demonstrated that the chromosome-wide changes in SNP genotypes enable highly effective mass-localization of BACs to individual cotton chromosomes. These efforts provide additional validation and placement methods that can be directly integrated with the physical map being constructed for G. hirsutum and enable the production of a high-quality draft genome sequence for cultivated cotton

Fine mapping of the “QTL-hotspot” region for drought tolerance in Chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is the third most important grain legume cultivated in the arid and semi-arid regions of the world. Drought is one of the major constraints leading up to 50% production losses in chickpea. In order to understand the basics of drought tolerance, two recombinant inbred line (RIL) mapping populations (ICC 4958 × ICC 1882 and ICC 283 × ICC 8261) segregating for root traits were developed and a promising “QTL-hotspot” region was reported on these populations. With an objective to fine map this region, two approaches were adopted, i) genotyping-by sequencing (GBS) and ii) skim sequencing. GBS approach enabled identification of 828 single nucleotide polymorphism (SNP) markers. A high-density genetic map was developed, comprising 1,007 marker loci including 49 SNP markers in the “QTL-hotspot” region and spanning a distance of 727.29 cM. QTL analysis using the extended genetic map along with precise phenotyping data generated earlier, re-estimated the “QTL-hotspot” from 29 cM to 14 cM. In addition, these 49 SNPs were converted into cleaved amplified polymorphic sequence (CAPS)/derived CAPS (dCAPS) markers which can be used in marker assisted breeding. An ultra-high-density bin map was developed using 53,223 SNPs obtained through skim sequencing approach and its analysis with the phenotyping data, split the “QTL-hotspot” region into two sub-regions namely “QTL-hotspot_a” of 139.22 kb with 15 genes and “QTL-hotspot_b” of 153.36 kb with 11 genes. To validate and find more recombination in these regions, a large mapping population was developed. Flanking SNP markers of the two regions were converted to KASPar assays and screened on 1,911 F2 lines. Progeny testing on F2:3 lines revealed the role of “QTL-hotspot_a” in controlling 100-SDW. A total of 15 candidate genes were reported in this region. In summary, the refined region will help in precise introgression of the “QTL-hotspot” in breeding program for yield improvement under drought conditions and the reported genes can be used for further cloning studies to dissect the molecular basis of drought tolerance in chickpea