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.

Broadening of the genetic base and systematic exploitation of heterosis in faba bean requires reliable information on the genetic diversity in the germ plasm. Three groups of faba bean inbred lines were examined by means of RAPDs (random amplified polymorphic DNAs) assays: 13 European small-seeded lines, 6 European large-seeded lines, and 9 Mediterranean lines. Out of 59 primers, 35 were informative and yielded 365 bands, 289 of which were polymorphic with a mean of 8.3 bands per primer. Monomorphic bands were omitted from the analyses and genetic distances (GD) were estimated via the coefficient of Jaccard. The mean GD among the European small-seeded lines was significantly greater than those among the lines of the other two groups. Repeatability of GD estimates was high. Cluster (UPGMA) and principal coordinate analyses identified European small-seeded lines and Mediterranean lines as distinct groups with European large-seeded lines located in between. The results are in harmony with published archaeobotanical findings. We conclude that RAPDs are useful for classification of germ plasm and identification of divergent heterotic groups in faba bean.

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety ‘Asha’. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable ‘Arhar’ simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.Electronic supplementary materialThe online version of this article (doi:10.1007/s13562-011-0088-8) contains supplementary material, which is available to authorized users.

Pigeonpea (Cajanus cajan), an important food legume crop in the semi-arid regions of the world and the second most important pulse crop in India, has an average crop productivity of 780 kg/ha. The relatively low crop yields may be attributed to non-availability of improved cultivars, poor crop husbandry and exposure to a number of biotic and abiotic stresses in pigeonpea growing regions. Narrow genetic diversity in cultivated germplasm has further hampered the effective utilization of conventional breeding as well as development and utilization of genomic tools, resulting in pigeonpea being often referred to as an ‘orphan crop legume’. To enable genomics-assisted breeding in this crop, the pigeonpea genomics initiative (PGI) was initiated in late 2006 with funding from Indian Council of Agricultural Research under the umbrella of Indo-US agricultural knowledge initiative, which was further expanded with financial support from the US National Science Foundation’s Plant Genome Research Program and the Generation Challenge Program. As a result of the PGI, the last 3 years have witnessed significant progress in development of both genetic as well as genomic resources in this crop through effective collaborations and coordination of genomics activities across several institutes and countries. For instance, 25 mapping populations segregating for a number of biotic and abiotic stresses have been developed or are under development. An 11X-genome coverage bacterial artificial chromosome (BAC) library comprising of 69,120 clones have been developed of which 50,000 clones were end sequenced to generate 87,590 BAC-end sequences (BESs). About 10,000 expressed sequence tags (ESTs) from Sanger sequencing and ca. 2 million short ESTs by 454/FLX sequencing have been generated. A variety of molecular markers have been developed from BESs, microsatellite or simple sequence repeat (SSR)-enriched libraries and mining of ESTs and genomic amplicon sequencing. Of about 21,000 SSRs identified, 6,698 SSRs are under analysis along with 670 orthologous genes using a GoldenGate SNP (single nucleotide polymorphism) genotyping platform, with large scale SNP discovery using Solexa, a next generation sequencing technology, is in progress. Similarly a diversity array technology array comprising of ca. 15,000 features has been developed. In addition, >600 unique nucleotide binding site (NBS) domain containing members of the NBS-leucine rich repeat disease resistance homologs were cloned in pigeonpea; 960 BACs containing these sequences were identified by filter hybridization, BES physical maps developed using high information content fingerprinting. To enrich the genomic resources further, sequenced soybean genome is being analyzed to establish the anchor points between pigeonpea and soybean genomes. In addition, Solexa sequencing is being used to explore the feasibility of generating whole genome sequence. In summary, the collaborative efforts of several research groups under the umbrella of PGI are making significant progress in improving molecu...

Restriction fragment length polymorphisms (RFLPs) have been proposed for characterizing genetic diversity of barley (Hordeum vulgare L.) germplasm. Forty‐eight (24 winter, 24 spring) cultivars from the European barley germplasm were assayed for RFLPs with 136 clone‐enzyme combinations (CEC) (48 DNA clones, three restriction enzymes). Objectives of this study were to (i) assess the amount variation for RFLPs in these materials, (ii) determine RFLP‐based genetic similarity estimates between unrelated cultivars within and between the winter and spring barley germplasms, and (iii) examine the usefulness of RFLPs for classification of cultivars from different germplasm pools. Thirty‐five DNA clones detected polymorphism with at least one enzyme. For the 62 polymorphic CEC, 58% of all RFLP patterns were common to both winter and spring barley, and about 20% were specific to the cultivars of each group. Genetic similarity (GS) calculated from RFLP data ranged from 0.64 to 0.93 between unrelated pairs of cultivars. Mean GS (MGS) for unrelated cultivar combinations within the group of winter (0.85) and spring (0.84) type cultivars were significantly greater than for winter × spring cultivar combinations (0.76). All winter and spring cultivars except two had a smaller MGS to cultivars from the other group than to unrelated cultivars from the same group. Principal coordinate analysis of GS estimates resulted in a separate grouping of winter and spring cultivars; further subgroups were also apparent for cultivars with a similar pedigree background. Results from our study corroborate the usefulness of RFLPs for the organization of elite barley germplasm.

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Genetic diversity and identification of simple sequence repeat markers correlated with Fusarium wilt resistance was performed in a set of 36 elite cultivated pigeonpea genotypes differing in levels of resistance to Fusarium wilt. Twenty-four polymorphic sequence repeat markers were screened across these genotypes, and amplified a total of 59 alleles with an average high polymorphic information content value of 0.52. Cluster analysis, done by UPGMA and PCA, grouped the 36 pigeonpea genotypes into two main clusters according to their Fusarium wilt reaction. Based on the Kruskal-Wallis ANOVA and simple regression analysis, six simple sequence repeat markers were found to be significantly associated with Fusarium wilt resistance. The phenotypic variation explained by these markers ranged from 23.7 to 56.4%. The present study helps in finding out feasibility of prescreened SSR markers to be used in genetic diversity analysis and their potential association with disease resistance.

In vitro grown microshoots of Picrrhiza kurrooa were encapsulated in the alginate beads. Regrowth of encapsulated microshoots, using alginate encapsulation, of P. kurrooa reached 89.33% following 3 months of storage. Amongst developing plantlets, 42.66% exhibited formation of multiple shoots at the onset of regrowth and 21.43% demonstrated simultaneous formation of shoots and roots. Healthy root formation was observed in plantlets following 2 weeks of their transfer to half-strength Murashige and Skoog medium containing 1 lM a-naphthalene acetic acid. Plants were transplanted to the greenhouse in three batches with 95% frequency of survival. The genetic fidelity of P. kurrooa plants growing out after storage in encapsulated form was ascertained by random amplified polymorphic DNA (RAPD) analysis. Molecular analysis of randomly selected plants from each batch was conducted using 45 random decamer primers. Of 45 primes tested, 14 produced scorable amplified products. Total 68 bands were observed amongst them 7.35% bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient of 0.966 thus confirming genetic stability of plants derived from encapsulated microshoots following 3 months of storage.