Marwan Diapari

Genome wide SNP identification in chickpea for use in development of a high density genetic map and improvement of chickpea reference genome assembly

BackgroundIn the whole genome sequencing, genetic map provides an essential framework for accurate and efficient genome assembly and validation. The main objectives of this study were to develop a high-density genetic map using RAD-Seq (Restriction-site Associated DNA Sequencing) genotyping-by-sequencing (RAD-Seq GBS) and Illumina GoldenGate assays, and to examine the alignment of the current map with the kabuli chickpea genome assembly.ResultsGenic single nucleotide polymorphisms (SNPs) totaling 51,632 SNPs were identified by 454 transcriptome sequencing of Cicer arietinum and Cicer reticulatum genotypes. Subsequently, an Illumina GoldenGate assay for 1,536 SNPs was developed. A total of 1,519 SNPs were successfully assayed across 92 recombinant inbred lines (RILs), of which 761 SNPs were polymorphic between the two parents. In addition, the next generation sequencing (NGS)-based GBS was applied to the same population generating 29,464 high quality SNPs. These SNPs were clustered into 626 recombination bins based on common segregation patterns. Data from the two approaches were used for the construction of a genetic map using a population derived from an intraspecific cross. The map consisted of 1,336 SNPs including 604 RAD recombination bins and 732 SNPs from Illumina GoldenGate assay. The map covered 653 cM of the chickpea genome with an average distance between adjacent markers of 0.5 cM. To date, this is the most extensive genetic map of chickpea using an intraspecific population. The alignment of the map with the CDC Frontier genome assembly revealed an overall conserved marker order; however, a few local inconsistencies within the Cicer arietinum pseudochromosome 1 (Ca1), Ca5 and Ca8 were detected. The map enabled the alignment of 215 unplaced scaffolds from the CDC Frontier draft genome assembly. The alignment also revealed varying degrees of recombination rates and hotspots across the chickpea genome.ConclusionsA high-density genetic map using RAD-Seq GBS and Illumina GoldenGate assay was developed and aligned with the existing kabuli chickpea draft genome sequence. The analysis revealed an overall conserved marker order, although some localized inversions between draft genome assembly and the genetic map were detected. The current analysis provides an insight of the recombination rates and hotspots across the chickpea genome.

Gene‑based SNP discovery and genetic mapping in pea

Key messageGene-based SNPs were identified and mapped in pea using five recombinant inbred line populations segregating for traits of agronomic importance.AbstractPea (Pisum sativum L.) is one of the world’s oldest domesticated crops and has been a model system in plant biology and genetics since the work of Gregor Mendel. Pea is the second most widely grown pulse crop in the world following common bean. The importance of pea as a food crop is growing due to its combination of moderate protein concentration, slowly digestible starch, high dietary fiber concentration, and its richness in micronutrients; however, pea has lagged behind other major crops in harnessing recent advances in molecular biology, genomics and bioinformatics, partly due to its large genome size with a large proportion of repetitive sequence, and to the relatively limited investment in research in this crop globally. The objective of this research was the development of a genome-wide transcriptome-based pea single-nucleotide polymorphism (SNP) marker platform using next-generation sequencing technology. A total of 1,536 polymorphic SNP loci selected from over 20,000 non-redundant SNPs identified using deep transcriptome sequencing of eight diverse Pisum accessions were used for genotyping in five RIL populations using an Illumina GoldenGate assay. The first high-density pea SNP map defining all seven linkage groups was generated by integrating with previously published anchor markers. Syntenic relationships of this map with the model legume Medicago truncatula and lentil (Lens culinaris Medik.) maps were established. The genic SNP map establishes a foundation for future molecular breeding efforts by enabling both the identification and tracking of introgression of genomic regions harbouring QTLs related to agronomic and seed quality traits.

Classification and characterization of species within the genus lens using genotyping-by-sequencing (GBS).

Lentil (Lens culinaris ssp. culinaris) is a nutritious and affordable pulse with an ancient crop domestication history. The genus Lens consists of seven taxa, however, there are many discrepancies in the taxon and gene pool classification of lentil and its wild relatives. Due to the narrow genetic basis of cultivated lentil, there is a need towards better understanding of the relationships amongst wild germplasm to assist introgression of favourable genes into lentil breeding programs. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows multiplexing of up to 384 samples or more per library to generate genome-wide single nucleotide Polymorphism (SNP) markers. In this study, we aimed to characterize our lentil germplasm collection using a two-enzyme GBS approach. We constructed two 96-plex GBS libraries with a total of 60 accessions where some accessions had several samples and each sample was sequenced in two technical replicates. We developed an automated GBS pipeline and detected a total of 266,356 genome-wide SNPs. After filtering low quality and redundant SNPs based on haplotype information, we constructed a maximum-likelihood tree using 5,389 SNPs. The phylogenetic tree grouped the germplasm collection into their respective taxa with strong support. Based on phylogenetic tree and STRUCTURE analysis, we identified four gene pools, namely L. culinaris/L. orientalis/L. tomentosus, L. lamottei/L. odemensis, L. ervoides and L. nigricans which form primary, secondary, tertiary and quaternary gene pools, respectively. We discovered sequencing bias problems likely due to DNA quality and observed severe run-to-run variation in the wild lentils. We examined the authenticity of the germplasm collection and identified 17% misclassified samples. Our study demonstrated that GBS is a promising and affordable tool for screening by plant breeders interested in crop wild relatives.

Genetic Diversity of Cultivated Lentil (Lens culinaris Medik.) and Its Relation to the World's Agro-ecological Zones

Assessment of genetic diversity and population structure of germplasm collections plays a critical role in supporting conservation and crop genetic enhancement strategies. We used a cultivated lentil (Lens culinaris Medik.) collection consisting of 352 accessions originating from 54 diverse countries to estimate genetic diversity and genetic structure using 1194 polymorphic single nucleotide polymorphism (SNP) markers which span the lentil genome. Using principal coordinate analysis, population structure analysis and UPGMA cluster analysis, the accessions were categorized into three major groups that prominently reflected geographical origin (worlds agro-ecological zones). The three clusters complemented the origins, pedigrees, and breeding histories of the germplasm. The three groups were (a) South Asia (sub-tropical savannah), (b) Mediterranean, and (c) northern temperate. Based on the results from this study, it is also clear that breeding programs still have considerable genetic diversity to mine within the cultivated lentil, as surveyed South Asian and Canadian germplasm revealed narrow genetic diversity.

Allele diversity analysis to identify SNPs associated with ascochyta blight resistance in pea

Development of pea cultivars with improved resistance to ascochyta blight disease has been hindered due to lack of strong resistance. The objective of this study was to identify single nucleotide polymorphisms (SNPs) within the candidate genes associated with ascochyta blight resistance that can be used to aid selection. A total of 54 diverse Pisum sativum accessions from eastern Europe, western Europe, Australia, and Canada were genotyped and phenotyped for disease reaction. Fifteen SNPs were detected within candidate genes associated with reaction to ascochyta blight, of which SNP loci PsDof1p308 and RGA-G3Ap103 had significant associations with ascochyta blight scores. Further, PsDof1p308 showed significant association with disease score when tested on a recombinant inbred line population (PR-15) developed from a cross between ‘CDC 1-2347-144’ and ‘CDC Meadow’. SNPs identified in this study have the potential to aid selection of pea cultivars with improved disease resistance.

Marker–Trait Association Analysis of Iron and Zinc Concentration in Lentil ( Medik.) Seeds

First study to provide knowledge on genetics of biofortification in lentils. SNP markers associated with seed Fe and Zn concentration were identified in lentils. MAS to enhance breeding programs aiming to fight global micronutrient malnutrition.

Genomic Analysis of Storage Protein Deficiency in Genetically Related Lines of Common Bean (Phaseolus vulgaris).

A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level.

Comparison of Gene Families: Seed Storage and Other Seed Proteins

Common bean (Phaseolus vulgaris) is an important source of protein and dietary fiber in human diets. Seed proteins, therefore, determine, at least in part, the nutritional value of common bean. From the very beginning of plant molecular biology, in the 1980s, common bean has been a prominent model plant to study seed storage proteins. The recent availability of several sequences for the common bean genome, coupled with seed transcriptomic and proteomic information, enables a comprehensive, in-depth view of seed protein genes in this organism. Comparisons between these sequences highlight interesting variation in lectin gene composition between the two centers of domestication. Alleles conferring storage protein deficiency may be used to improve the levels of essential sulfur amino acids and therefore protein quality. Some of the seed proteins represent anti-nutritionals, including some lectins, trypsin inhibitors, and lipoxygenases, and represent targets to be potentially removed from the genome. Other proteins have potential as bioproducts due to their biological activity against fungi or insects, including defensin D1 and albumin-1.