Bunyamin Tar’an

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.

Gene-based SNP discovery in tepary bean (Phaseolus acutifolius) and common bean (P. vulgaris) for diversity analysis and comparative mapping

BackgroundCommon bean (Phaseolus vulgaris) is an important grain legume and there has been a recent resurgence in interest in its relative, tepary bean (P. acutifolius), owing to this species’ ability to better withstand abiotic stresses. Genomic resources are scarce for this minor crop species and a better knowledge of the genome-level relationship between these two species would facilitate improvement in both. High-throughput genotyping has facilitated large-scale single nucleotide polymorphism (SNP) identification leading to the development of molecular markers with associated sequence information that can be used to place them in the context of a full genome assembly.ResultsTranscript-based SNPs were identified from six common bean and two tepary bean accessions and a subset were used to generate a 768-SNP Illumina GoldenGate assay for each species. The tepary bean assay was used to assess diversity in wild and cultivated tepary bean and to generate the first gene-based map of the tepary bean genome. Genotypic analyses of the diversity panel showed a clear separation between domesticated and cultivated tepary beans, two distinct groups within the domesticated types, and P. parvifolius was confirmed to be distinct. The genetic map of tepary bean was compared to the common bean genome assembly to demonstrate high levels of collinearity between the two species with differences limited to a few intra-chromosomal rearrangements.ConclusionsThe development of the first set of genomic resources specifically for tepary bean has allowed for greater insight into the structure of this species and its relationship to its agriculturally more prominent relative, common bean. These resources will be helpful in the development of efficient breeding strategies for both species and will facilitate the introgression of agriculturally important traits from one crop into the other.

CDC Meadow field pea

CDC Meadow, a yellow cotyledon field pea (Pisum sativum L.) cultivar, was released in 2006 by the Crop Development Centre, University of Saskatchewan for distribution to Select seed growers in Saskatchewan and Alberta through the Variety Release Committee of the Saskatchewan Pulse Growers. CDC Meadow has a semileafless leaf type, good lodging resistance, powdery mildew resistance, medium-sized, round seeds, and good yielding ability. CDC Meadow is adapted to the field pea growing regions of western Canada. Key words: Field pea, Pisum sativum L., cultivar description

QTL mapping of early flowering and resistance to ascochyta blight in chickpea

In western Canada, chickpea (Cicer arietinum L.) production is challenged by short growing seasons and infestations with ascochyta blight. Research was conducted to determine the genetic basis of the association between flowering time and reaction to ascochyta blight in chickpea. Ninety-two chickpea recombinant inbred lines (RILs) developed from a cross between ICCV 96029 and CDC Frontier were evaluated for flowering responses and ascochyta blight reactions in growth chambers and fields at multiple locations and during several years. A wide range of variation was exhibited by the RILs for days to flower, days to maturity, node of first flowering, plant height, and ascochyta blight resistance. Moderate to high broad sense heritability was estimated for ascochyta blight reaction (H(2) = 0.14-0.34) and for days to flowering (H(2) = 0.45-0.87) depending on the environments. Negative correlations were observed among the RILs for days to flowering and ascochyta blight resistance, ranging from r = -0.21 (P < 0.05) to -0.58 (P < 0.0001). A genetic linkage map consisting of eight linkage groups was developed using 349 SNP markers. Seven QTLs for days to flowering were identified that individually explained 9%-44% of the phenotypic variation. Eight QTLs were identified for ascochyta blight resistance that explained phenotypic variation ranging from 10% to 19%. Clusters of QTLs for days to flowering and ascochyta blight resistances were found on chromosome 3 at the interval of 8.6-23.11 cM and on chromosome 8 at the interval of 53.88-62.33 cM.

Genetic Analysis of NBS-LRR Gene Family in Chickpea and Their Expression Profiles in Response to Ascochyta Blight Infection

Ascochyta blight is one of the major diseases of chickpea worldwide. The genetic resistance to ascochyta blight in chickpea is complex and governed by multiple QTLs. However, the molecular mechanism of quantitative disease resistance to ascochyta blight and the genes underlying these QTLs are still unknown. Most often disease resistance is determined by resistance (R) genes. The most predominant R-genes contain nucleotide binding site and leucine rich repeat (NBS-LRR) domains. A total of 121 NBS-LRR genes were identified in the chickpea genome. Ninety-eight of these genes contained all essential conserved domains while 23 genes were truncated. The NBS-LRR genes were grouped into eight distinct classes based on their domain architecture. Phylogenetic analysis grouped these genes into two major clusters based on their structural variation, the first cluster with toll or interleukin-1 like receptor (TIR) domain and the second cluster either with or without a coiled-coil domain. The NBS-LRR genes are distributed unevenly across the eight chickpea chromosomes and nearly 50% of the genes are present in clusters. Thirty of the NBS-LRR genes were co-localized with nine of the previously reported ascochyta blight QTLs and were tested as potential candidate genes for ascochyta blight resistance. Expression pattern of these genes was studied in two resistant (CDC Corinne and CDC Luna) and one susceptible (ICCV 96029) genotypes at different time points after ascochyta blight infection using real-time quantitative PCR. Twenty-seven NBS-LRR genes showed differential expression in response to ascochyta blight infection in at least one genotype at one time point. Among these 27 genes, the majority of the NBS-LRR genes showed differential expression after inoculation in both resistant and susceptible genotypes which indicates the involvement of these genes in response to ascochyta blight infection. Five NBS-LRR genes showed genotype specific expression. Our study provides a new insight of NBS-LRR gene family in chickpea and the potential involvement of NBS-LRR genes in response to ascochyta blight infection.

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.

Fine Mapping of QTLs for Ascochyta Blight Resistance in Pea Using Heterogeneous Inbred Families

Ascochyta blight (AB) is an important disease of pea which can cause severe grain yield loss under wet conditions. In our previous study, we identified two quantitative trait loci (QTLs) abIII-1 and abI-IV-2 for AB resistance and these QTLs were consistent across locations and/or years in an inter-specific pea population (PR-19) developed from a cross between Alfetta (Pisum sativum) and P651 (P. fulvum). The objectives of this study were to fine map the abIII-1 and abI-IV-2 QTLs using a high density single nucleotide polymorphism (SNP)-based genetic linkage map and analyze identified markers in heterogeneous inbred family (HIF) populations. Selective genotyping of 51 PR-19 recombinant inbred lines was performed using genotyping-by-sequencing (GBS) and the resulting high density genetic linkage map was used to identify eight new SNP markers within the abI-IV-2 QTL, whereas no additional SNPs were identified within the abIII-1 QTL. Two HIF populations HIF-224 (143 lines) and HIF-173 (126 lines) were developed from F6 RILs PR-19-224 and PR-19-173, respectively. The HIF populations evaluated under field conditions in 2015 and 2016 showed a wide range of variation for reaction to AB resistance. Lodging score had significant positive (P < 0.001) correlation with AB scores. HIFs were genotyped using SNP markers within targeted QTLs. The genotypic and phenotypic data of the HIFs were used to identify two new QTLs, abI-IV-2.1 and abI-IV-2.2 for AB resistance within the abI-IV-2 QTL. These QTLs individually explained 5.5 to 14% of the total phenotypic variation. Resistance to lodging was also associated with these two QTLs. Identified SNP markers will be useful in marker assisted selection for development of pea cultivars with improved AB resistance.

Determination of Photoperiod-Sensitive Phase in Chickpea (Cicer arietinum L.)

Photoperiod is one of the major environmental factors determining time to flower initiation and first flower appearance in plants. In chickpea, photoperiod sensitivity, expressed as delayed to flower under short days (SD) as compared to long days (LD), may change with the growth stage of the crop. Photoperiod-sensitive and -insensitive phases were identified by experiments in which individual plants were reciprocally transferred in a time series from LD to SD and vice versa in growth chambers. Eight chickpea accessions with differing degrees of photoperiod sensitivity were grown in two separate chambers, one of which was adjusted to LD (16 h light/8 h dark) and the other adjusted to SD (10 h light/14 h dark), with temperatures of 22/16°C (12 h light/12 h dark) in both chambers. The accessions included day-neutral (ICCV 96029 and FLIP 98-142C), intermediate (ICC 15294, ICC 8621, ILC 1687, and ICC 8855), and photoperiod-sensitive (CDC Corinne and CDC Frontier) responses. Control plants were grown continuously under the respective photoperiods. Reciprocal transfers of plants between the SD and LD photoperiod treatments were made at seven time points after sowing, customized for each accession based on previous data. Photoperiod sensitivity was detected in intermediate and photoperiod-sensitive accessions. For the day-neutral accession, ICCV 96029, there was no significant difference in the number of days to flowering of the plants grown under SD and LD as well as subsequent transfers. In photoperiod-sensitive accessions, three different phenological phases were identified: a photoperiod-insensitive pre-inductive phase, a photoperiod-sensitive inductive phase, and a photoperiod-insensitive post-inductive phase. The photoperiod-sensitive phase extends after flower initiation to full flower development. Results from this research will help to develop cultivars with shorter pre-inductive photoperiod-insensitive and photoperiod-sensitive phases to fit to regions with short growing seasons.

CDC Centennial field pea

CDC Centennial, a yellow cotyledon field pea (Pisum sativum L.) cultivar, was released in 2007 by the Crop Development Centre, University of Saskatchewan, for distribution to Select seed growers in Saskatchewan and Alberta through the Variety Release Committee of the Saskatchew an Pulse Growers. CDC Centennial has a semileafless leaf type, fair lodging resistance, powdery mildew resistance, moderately large sized, round seeds, and good yielding ability. CDC Centennial is adapted to the field pea growing regions of western Canada. Key words: Field pea, Pisum sativum L., cultivar description