Subhojit Datta

Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh]

BackgroundPigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping.ResultsIn this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population.ConclusionWe developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus Cajanus. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.

Genetic Patterns of Domestication in Pigeonpea (Cajanus cajan (L.) Millsp.) and Wild Cajanus Relatives

Pigeonpea (Cajanus cajan) is an annual or short-lived perennial food legume of acute regional importance, providing significant protein to the human diet in less developed regions of Asia and Africa. Due to its narrow genetic base, pigeonpea improvement is increasingly reliant on introgression of valuable traits from wild forms, a practice that would benefit from knowledge of its domestication history and relationships to wild species. Here we use 752 single nucleotide polymorphisms (SNPs) derived from 670 low copy orthologous genes to clarify the evolutionary history of pigeonpea (79 accessions) and its wild relatives (31 accessions). We identified three well-supported lineages that are geographically clustered and congruent with previous nuclear and plastid sequence-based phylogenies. Among all species analyzed Cajanus cajanifolius is the most probable progenitor of cultivated pigeonpea. Multiple lines of evidence suggest recent gene flow between cultivated and non-cultivated forms, as well as historical gene flow between diverged but sympatric species. Evidence supports that primary domestication occurred in India, with a second and more recent nested population bottleneck focused in tropical regions that is the likely consequence of pigeonpea breeding. We find abundant allelic variation and genetic diversity among the wild relatives, with the exception of wild species from Australia for which we report a third bottleneck unrelated to domestication within India. Domesticated C. cajan possess 75% less allelic diversity than the progenitor clade of wild Indian species, indicating a severe “domestication bottleneck” during pigeonpea domestication.

Genomics-assisted breeding for drought tolerance in chickpea

Terminal drought is one of the major constraints in chickpea (Cicer arietinum L.), causing more than 50% production losses. With the objective of accelerating genetic understanding and crop improvement through genomics-assisted breeding, a draft genome sequence has been assembled for the CDC Frontier variety. In this context, 544.73Mb of sequence data were assembled, capturing of 73.8% of the genome in scaffolds. In addition, large-scale genomic resources including several thousand simple sequence repeats and several million single nucleotide polymorphisms, high-density diversity array technology (15360 clones) and Illumina GoldenGate assay genotyping platforms, high-density genetic maps and transcriptome assemblies have been developed. In parallel, by using linkage mapping approach, one genomic region harbouring quantitative trait loci for several drought tolerance traits has been identified and successfully introgressed in three leading chickpea varieties (e.g. JG 11, Chefe, KAK 2) by using a marker-assisted backcrossing approach. A multilocation evaluation of these marker-assisted backcrossing lines provided several lines with 10-24% higher yield than the respective recurrent parents.Modern breeding approaches like marker-assisted recurrent selection and genomic selection are being deployed for enhancing drought tolerance in chickpea. Some novel mapping populations such as multiparent advanced generation intercross and nested association mapping populations are also being developed for trait mapping at higher resolution, as well as for enhancing the genetic base of chickpea. Such advances in genomics and genomics-assisted breeding will accelerate precision and efficiency in breeding for stress tolerance in chickpea.

Cross-genera amplification of informative microsatellite markers from common bean and lentil for the assessment of genetic diversity in pigeonpea.

A total of 24 pigeonpea (Cajanus cajan L. Millspaugh) cultivars representing different maturity groups were evaluated for genetic diversity analysis using 10 pigeonpea specific and 66 cross-genera microsatellite markers. Of the cross-genera microsatellite markers, only 12 showed amplification. A total of 45 alleles were amplified by the 22 markers. Nine markers showed 100 % polymorphism. Markers Lc 14, BMd 48 and CCB 9 amplified maximum number (5) of alleles each. One genotype specific unique band in Pusa 9 was generated by markers CCB 8. Maximum genetic diversity (74 %) was observed between cultivars MA 3 and CO 6, while the minimum diversity (12 %) was observed between NDA 1 and DA 11. The average diversity among the cultivars was estimated to be 45.6 %. SSR primers from pigeonpea were found to be more polymorphic (37 %) as compared to common bean and lentil markers. The arithmetic mean heterozygosity (Hav) and marker index (MI) were found to be 0.014 and 0.03, respectively, indicating the potential of common bean and lentil microsatellite markers for genetic mapping, diversity analysis and genotyping in Cajanus.

Novel genic microsatellite markers from Cajanus scarabaeoides and their comparative efficiency in revealing genetic diversity in pigeonpea

Paucity of molecular markers is hindering molecular breeding programmes for genetic improvement in pigeonpea, which is considered to be among the richest source of dietary protein in Asia and Africa. At the time of the start of this study, only 156 microsatellite markers were available in pigeonpea (Burns et al. 2001; Odeny et al. 2007, 2009). Recently with the publication of draft genome sequence and deep transcriptome studies, the stage has been set to enrich genomic resources to aid molecular breeding in pigeonpea (Dutta et al. 2011; Singh et al. 2012; Varshney et al. 2012). Genic microsatellites or EST-SSRs (simple sequence repeats) derived from expressed sequence tags (ESTs) are useful because these are inexpensive to develop, represent transcribed genes, and often a putative function can be assigned to them. Compared with genomic sequences, genic SSRs have several advantages as genetic markers. First, if an EST marker is found to be genetically associated with a trait of interest, it may represent the gene affecting the trait directly (Chen et al. 2001; Thiel et al. 2003). Therefore, EST-derived markers can provide opportunities for gene discovery and enhance the role of genetic markers by assaying variation in transcribed and known-function genes. Second, EST-derived

Expression of chimeric Bt gene, Cry1Aabc in transgenic pigeonpea (cv. Asha) confers resistance to gram pod borer (Helicoverpa armigera Hubner.)

The gram pod borer (Helicoverpa armigera Hubner) is the most serious insect pest of pigeonpea. It is highly susceptible to the insecticidal proteins of Bacillus thuringiensis (Bt). A codon-optimized chimeric Cry1Aabc gene of Bt driven by a constitutive promoter was introduced in pigeonpea (cv. Asha) to confer resistance against the insect. A total of eight transgenic plants could be established with transformation frequency of 0.06%. Two transgenic events were selected for advancement based on high insect mortality, single locus integration, protein expression and fertility status. Quantitative ELISA indicated high protein expression in different plant parts viz., leaves (pre and post flowering), flowers, pod walls and immature seeds. Analysis for the stable integration, expression and insect mortality (detached leaf and pod bioassay) led to identification of lines with high efficacy. These events were further advanced for the identification of a viable event by selfing to create homozygosity. The chimeric Cry1Aabc expressed in pigeonpea is effective against gram pod borer and can be utilized in transgenic variety development programme.

Expression of a Chimeric Gene Encoding Insecticidal Crystal Protein Cry1Aabc of Bacillus thuringiensis in Chickpea (Cicer arietinum L.) Confers Resistance to Gram Pod Borer (Helicoverpa armigera Hubner.)

Domain swapping and generation of chimeric insecticidal crystal protein is an emerging area of insect pest management. The lepidopteran insect pest, gram pod borer (Helicoverpa armigera H.) wreaks havoc to chickpea crop affecting production. Lepidopteran insects were reported to be controlled by Bt (cryI) genes. We designed a plant codon optimized chimeric Bt gene (cry1Aabc) using three domains from three different cry1A genes (domains I, II, and III from cry1Aa, cry1Ab, and cry1Ac, respectively) and expressed it under the control of a constitutive promoter in chickpea (cv. DCP92-3) to assess its effect on gram pod borer. A total of six transgenic chickpea shoots were established by grafting into mature fertile plants. The in vitro regenerated (organogenetic) shoots were selected based on antibiotic kanamycin monosulfate (100 mg/L) with transformation efficiency of 0.076%. Three transgenic events were extensively studied based on gene expression pattern and insect mortality across generations. Protein expression in pod walls, immature seeds and leaves (pre- and post-flowering) were estimated and expression in pre-flowering stage was found higher than that of post-flowering. Analysis for the stable integration, expression and insect mortality (detached leaf and whole plant bioassay) led to identification of efficacious transgenic chickpea lines. The chimeric cry1Aabc expressed in chickpea is effective against gram pod borer and generated events can be utilized in transgenic breeding program.

Development of an efficient Agrobacterium mediated transformation system for chickpea (Cicer arietinum)

Abstract Morphologically normal and fertile transgenic chickpea plants have been regenerated through a standardized transformation protocols. This protocol is based on the infection of apical meristem explants (AME) with Agrobacterium strain EHA105. The stain, carrying pCAMBIA2301 vector contained β-glucuronidase (uidA) gene and neomycin phosphotransferase (nptII) genes. Different explants of chickpea and Agrobacterium specific conditions were standardized with the help of transient β-glucuronidase (uidA) gene expression to further optimize the transformation protocol. Pre-conditiong of the explants, vacuum infiltration and presence of acetosyringone significantly enhanced the frequency of gus expression. Positive transformants with nptII and gus genes were confirmed by PCR and histochemical gus analysis. An overall successful chickpea transformation frequency of 1.2 was achieved. This high efficiency and easy to use method may provide opportunities for the development of transgenic lines with different useful genes in chickpea in near future.

Using AFLP-RGA markers to assess genetic diversity among pigeon pea (Cajanus cajan) genotypes in relation to major diseases

Resistance gene analog (RGA)-anchored amplified fragment length polymorphism (AFLP-RGA) marker system was used in order to evaluate genetic relationships among 22 pigeon pea genotypes with varied responses to Fusarium wilt and sterility mosaic disease. Five AFLP-RGA primer combinations (E-CAG/wlrk-S, M-GTG/wlrk-S, M-GTG/wlrk-AS, E-CAT/S1-INV and E-CAG/wlrk-AS) produced 173 scorable fragments, of which 157 (90.7%) were polymorphic, with an average of 31.4 fragments per primer combination. The polymorphism rates obtained with the five primers were 83.3%, 92.0%, 92.3%, 93.0% and 93.1%, respectively. Mean polymorphic information content (PIC) values ranged from 0.24 (with E-CAT/S1-INV) to 0.30 (with E-CAG/wlrk-AS), whereas resolving power (RP) values varied from 11.06 (with M-GTG/wlrk-S) to 25.51 (with E-CAG/wlrk-AS) and marker index (MI) values ranged from 5.98 (with M-GTG/wlrk-S) to 12.30 (with E-CAG/wlrk-AS). We identified a positive correlation between MI and RP (r2=0.98, p<0.05), stronger that that observed for the comparison between PIC and RP (r2=0.88, p<0.05). That implies that either MI or RP is the best parameter for selecting more informative AFLP-RGA primer combinations. The Jaccard coefficient ranged from 0.07 to 0.72, suggesting a broad genetic base in the genotypes studied. A neighbor-joining tree, based on the unweighted pair group method with arithmetic mean, distinguished cultivated species from wild species. The grouping of resistant genotypes in different clusters would help in the selection of suitable donors for resistance breeding in pigeon pea.

A high-density intraspecific SNP linkage map of pigeonpea (Cajanas cajan L. Millsp.)

Pigeonpea (Cajanus cajan (L.) Millsp.) is a major food legume cultivated in semi-arid tropical regions including the Indian subcontinent, Africa, and Southeast Asia. It is an important source of protein, minerals, and vitamins for nearly 20% of the world population. Due to high carbon sequestration and drought tolerance, pigeonpea is an important crop for the development of climate resilient agriculture and nutritional security. However, pigeonpea productivity has remained low for decades because of limited genetic and genomic resources, and sparse utilization of landraces and wild pigeonpea germplasm. Here, we present a dense intraspecific linkage map of pigeonpea comprising 932 markers that span a total adjusted map length of 1,411.83 cM. The consensus map is based on three different linkage maps that incorporate a large number of single nucleotide polymorphism (SNP) markers derived from next generation sequencing data, using Illumina GoldenGate bead arrays, and genotyping with restriction site associated DNA (RAD) sequencing. The genotyping-by-sequencing enhanced the marker density but was met with limited success due to lack of common markers across the genotypes of mapping population. The integrated map has 547 bead-array SNP, 319 RAD-SNP, and 65 simple sequence repeat (SSR) marker loci. We also show here correspondence between our linkage map and published genome pseudomolecules of pigeonpea. The availability of a high-density linkage map will help improve the anchoring of the pigeonpea genome to its chromosomes and the mapping of genes and quantitative trait loci associated with useful agronomic traits.