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BackgroundChickpea (Cicer arietinumL.) is the second most widely grown pulse and drought (limiting water) is one of the major constraints leading to about 40–50% yield losses annually. Dehydration responsive element binding proteins (DREBs) are important plant transcription factors that regulate the expression of many stress-inducible genes and play a critical role in improving the abiotic stress tolerance. Transgenic chickpea lines harbouring transcription factor, Dehydration Responsive Element-Binding protein 1A fromArabidopsis thaliana(AtDREB1agene) driven by stress inducible promoterrd29awere developed, with the intent of enhancing drought tolerance in chickpea.Performance of the progenies of one transgenic event and control were assessed based on key physiological traits imparting drought tolerance such as plant water relation characteristics, chlorophyll retention, photosynthesis, membrane stability and water use efficiency under water stressed conditions.ResultsFour transgenic chickpea lines harbouring stress inducibleAtDREB1awere generated with transformation efficiency of 0.1%. The integration, transmission and regulated expression were confirmed by Polymerase Chain Reaction (PCR), Southern Blot hybridization and Reverse Transcriptase polymerase chain reaction (RT-PCR), respectively. Transgenic chickpea lines exhibitedhigherrelative water content,longerchlorophyll retention capacity andhigherosmotic adjustment under severe drought stress (stress level 4), as compared to control. The enhanced drought tolerance in transgenic chickpea lines were also manifested by undeterred photosynthesis involving enhanced quantum yield of PSII, electron transport rate at saturated irradiance levels and maintaining higher relative water content in leaves under relatively severe soil water deficit. Further, lower values of carbon isotope discrimination in some transgenic chickpea lines indicated higher water use efficiency. Transgenic chickpea lines exhibiting better OA resulted in higher seed yield, with progressive increase in water stress, as compared to control.ConclusionsBased on precise phenotyping, involving non-invasive chlorophyll fluorescence imaging, carbon isotope discrimination, osmotic adjustment, higher chlorophyll retention and membrane stability index, it can be concluded thatAtDREB1atransgenic chickpea lines were better adapted to water deficit by modifying important physiological traits. The selected transgenic chickpea event would be a valuable resource that can be used in pre-breeding or directly in varietal development programs for enhanced drought tolerance under parched conditions.

Global cultivation of flax fibre and oilseed is sparse due to poor climatic adaptability. Abiotic stresses, such as drought, salinity, and heat stress are the major limiting factors of flax cultivation. Varieties tolerant to biotic and abiotic stresses are the need of the hour with a sustainable high and stable yield. Exploring candidate genes to provide wider climatic adaptability in flax is of paramount importance. The present study delineates a detailed annotation of 164 Linum usitatissimum NAC-domain transcription factor genes (LuNACs) that are scattered across all 15 chromosomes. Phylogeny-wise majority of the LuNAC proteins were categorized into recognized NAC groups. Few LuNACs remain distinct, suggesting their species–specific expansion. Analysis of the LuNAC gene and protein domain architectures established their conserved nature and support the phylogenetic grouping. The homologs of LuNAC genes revealed their expansion because of whole-genome duplication events. Potential target sites of miRNA families, including the miRNA164, were identified in LuNAC genes, suggesting that a complex regulatory mechanism might be associated with abiotic stress tolerance in flax. In silico gene expression, deep GO analysis, functional inference from homologs, and RT-qPCR of selected LuNAC genes revealed their functional involvement in growth and development and in response to diverse abiotic stresses in flax. The LuNAC003 gene from the senescence-related subfamily was responsive to multiple stress conditions. All the above findings on LuNAC genes may promote them as candidate genes for further functional studies or utilize them in flax genetic improvement programs for improved fibre and seed oil productions, even under adverse environmental conditions.

Corchorus capsularis (white jute) and C. olitorius (dark jute) are the two principal cultivated species of jute that produce natural bast fiber of commercial importance. We have identified 4509 simple sequence repeat (SSR) loci from 34,163 unigene sequences of C. capsularis to develop a non-redundant set of 2079 flanking primer pairs. Among the SSRs, trinucleotide repeats were most frequent (60%) followed by dinucleotide repeats (37.6%). Annotation of the SSR-containing unigenes revealed their putative functions in various biological and molecular processes, including responses to biotic and abiotic signals. Eighteen expressed gene-derived SSR (eSSR) markers were successfully mapped to the existing single-nucleotide polymorphism (SNP) linkage map of jute, providing additional anchor points. Amplification of 72% of the 74 randomly selected primer pairs was successful in a panel of 24 jute accessions, comprising five and twelve accessions of C. capsularis and C. olitorius , respectively, and seven wild jute species. Forty-three primer pairs produced an average of 2.7 alleles and 58.1% polymorphism in a panel of 24 jute accessions. The mean PIC value was 0.34 but some markers showed PIC values higher than 0.5, suggesting that these markers can efficiently measure genetic diversity and serve for mapping of quantitative trait loci (QTLs) in jute. A primer polymorphism survey with parents of a wide-hybridized population between a cultivated jute and its wild relative revealed their efficacy for interspecific hybrid identification. For ready accessibility of jute eSSR primers, we compiled all information in a user-friendly web database, JuteMarkerdb ( http://jutemarkerdb.icar.gov.in /) for the first time in jute. This eSSR resource in jute is expected to be of use in characterization of germplasm, interspecific hybrid and variety identification, and marker-assisted breeding of superior-quality jute.

In jute (Corchorus spp.), phenylpropanoid biosynthesis pathway is associated with lignocellulosic bast fibre development and production of various phytochemicals. Development of phenylpropanoid-related genic markers is thus a key component of gene-specific precision molecular breeding in jute. We identified a total of 12,772 SSRs in 10,041 unigenes and designed genic SSR markers associated with phenylpropanoid biosynthesis from bast transcriptome of Corchorus capsularis cv. ‘JRC-212’. A total of 39 and 14 SSR markers were developed from key genes of phenylpropanoid biosynthesis and bast fibre formation, respectively. Further, we developed 457 SSR markers associated with different regulatory gene sequences. Of these, 137 markers were for transcription factors such as WRKY, MYB, MYB-related, bHLH and zinc finger, which are key regulators and/or modulators of the biosynthesis of phenylpropanoids. Additionally, 600 SSR primer pairs were also designed for other unigenes containing SSR. Diversity analysis based on a set of 41 genic SSR markers in 35 genotypes revealed high polymorphism. The genic SSRs described here are expected to be useful for molecular breeding targeting fibre quality, phytochemical-related and stress tolerance traits in jute.

Here, we present the draft genome (377.3 Mbp) of Corchorus olitorious cv. JRO-524 (Navin), which is a leading dark jute variety developed from a cross between African (cv. Sudan Green) and indigenous (cv. JRO-632) types. We predicted from the draft genome a total of 57,087 protein-coding genes with annotated functions. We identified a large number of 1765 disease resistance-like and defense response genes in the jute genome. The annotated genes showed the highest sequence similarities with that of Theobroma cacao followed by Gossypium raimondii. Seven chromosome-scale genetically anchored pseudomolecules were constructed with a total size of 8.53 Mbp and used for synteny analyses with the cocoa and cotton genomes. Like other plant species, gypsy and copia retrotransposons were the most abundant classes of repeat elements in jute. The raw data of our study are available in SRA database of NCBI with accession number SRX1506532. The genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession LLWS00000000, and the version described in this paper will be the first version (LLWS01000000).

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.

Production of transgenic pigeonpea is becoming increasingly important, but the methods currently employed in production and subsequent screening still requires improvement. Here, we describe Agrobacterium-mediated genetic transformation of pigeonpea with reporter uidA (gus) gene and selectable marker, neomycin phospho-transferase (nptII) gene. Histochemical assay demonstrate localization of gus activity in cells and transformed plants. Overall, a transformation frequency of 0.33% was achieved using the protocol. Grafting of in vitro-regenerated healthy shoots indicates higher survival percent (72.6%), when stock and scion are of the same variety. Seeds harvested from primary transgenic plants can be screened based on lateral root inhibition strategy. Approximately 87% of the screened T1 plants were found to be PCR positive. In conclusion, in vitro grafting of transgenic pigeonpea shoots leads to better plant establishment and screening based on lateral root inhibition leads to quick identification of positive segregants.

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.

We designed a set of 580 simple sequence repeat markers; 506 from transcription factor-coding genes, and 74 from long non-coding RNAs and designated them as regulatory gene-derived simple sequence repeat (ReG-SSR) markers. From this set, we could anchor 559 ReG-SSR markers on 15 flax chromosomes with an average marker distance of 0.56 Mb. Thirty-one polymorphic ReG-SSR primers, amplifying SSR loci length of at least 20 bp were chosen from 134 screened primers. This primer set was used to characterize a diversity panel of 93 flax accessions. The panel included 33 accessions from India, including released varieties, dual-purpose lines and landraces, and 60 fiber flax accessions from the global core collection. Thirty-one ReG-SSR markers generated 76 alleles, with an average of 2.5 alleles per primer and a mean allele frequency of 0.77. These markers recorded 0.32 average gene diversity, 0.26 polymorphism information content and 1.35% null alleles. All the 31 ReG-SSR loci were found selectively neutral and showed no evidence of population reduction. A model-based clustering analysis separated the flax accessions into two sub-populations—Indian and global, with some accessions showing admixtures. The distinct clustering pattern of the Indian accessions compared to the global accessions, conforms to the principal coordinate analysis, genetic dissimilarity-based unweighted neighbor-joining tree and analysis of molecular variance. Fourteen flax accessions with 99.3% allelic richness were found optimum to adopt in breeding programs. In summary, the genome-wide ReG-SSR markers will serve as a functional marker resource for genetic and phenotypic relationship studies, marker-assisted selections, and provide a basis for selection of accessions from the Indian and global gene pool in fiber flax breeding programs.

Grass pea (Lathyrus sativus) is an important food-legume crop for resource-poor farmers in the developing world. However, given its cultivation in the most underprivileged regions, the crop has not received appropriate scientific attention particularly from the genomic perspective, thereby giving it a status of genomic orphan. Nevertheless, some recent studies have attempted to develop modern molecular tools to strengthen the genetic and genomic research. In the present investigation, a comprehensive collection comprising 176 accessions was analysed using EST-simple sequence repeats (SSRs). The SSR analysis revealed existence of a total of 51 alleles with an average polymorphic information content value of 0.35. A moderate level of gene diversity was noticed that ranged from 0.04 to 0.73 with an average of 0.43. Noticeably, two distinct subpopulations were recovered using cluster analysis. In addition, the presence of admixtures in population reflected the strong possibilities of gene flow between the accessions across the geographical boundary. In summary, we provide additional insights about the informativeness of available EST-SSR markers along with an extended understanding of relatedness, genetic structure and gene flow in an under-researched legume crop.

Domain swapping and generation of chimeric insecticidal crystal protein (ICP) 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 (Domain 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 monosulphate (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 programme.

article i nfo Advances in next-generation sequencing and genotyping technologies have enabled generation of large-scale genomic resources such as molecular markers, transcript reads and BAC-end sequences (BESs) in chickpea, pigeonpea and groundnut, three major legume crops of the semi-arid tropics. Comprehensive transcriptome assemblies and genome sequences have either been developed or underway in these crops. Based on these resources, dense genetic maps, QTL maps as well as physical maps for these legume species have also been developed. As a result, these crops have graduated from 'orphan' or 'less-studied' crops to 'genomic resources rich' crops. This article summarizes the above-mentioned advances in genomics and genomics-assisted breeding applications in the form of marker-assisted selection (MAS) for hybrid purity assessment in pigeonpea; marker-assisted backcrossing (MABC) for introgressing QTL region for drought-tolerance related traits, Fusarium wilt (FW) resistance and Ascochyta blight (AB) resistance in chickpea; late leaf spot (LLS), leaf rust and nematode resistance in groundnut. We critically present the case of use of other modern breeding approaches like marker-assisted recurrent selection (MARS) and genomic selection (GS) to utilize the full potential of genomics-assisted breeding for developing superior cultivars with enhanced tolerance to various environmental stresses. In addition, this article recommends the use of advanced-backcross (AB-backcross) breeding and development of specialized populations such as multi-parents advanced generation intercross (MAGIC) for creating new variations that will help in developing superior lines with broadened genetic base. In summary, we propose the use of integrated genomics and breeding approach in these legume crops to enhance crop productivity in marginal environments ensuring food security in developing countries.

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

In-vitro regeneration in fieldpea was achieved from immature embryonic axes and cotyledonary node explants of six genotypes on modified MS media supplemented with different concentration of plant growth regulators, 6-Benzylamino purine (BAP) and Naphthalene acetic acid (NAA). The best regeneration response, leading to multiple shoot formation efficiency (22.34 shoots/explant) was observed in the medium supplemented with 1.0 mg/L BAP and 0.2 mg/L NAA and best frequency (67.55 ± 4.74) was achieved on medium containing 2.0 mg/L BAP and 0.4 mg/L NAA. The shoots were subcultured on a medium supplemented with a combination of 1.0 mg/L GA3, 2.0 mg/L BAP and 0.4 mg/L NAA, which resulted in elongation of 85 % of shoots. Rooting attempted from the elongated shoots, on half strength MS medium and supplemented with three different auxins IBA, IAA and NAA separately, exhibited similar results. Alternatively, micro-grafting of in vitro regenerated shoots onto pre-germinated root stocks raised in green house facility was attempted with high success rate (75 %). The grafted plants could be successfully hardened, fertigated with Hoagland solution and distilled water in a ratio of (1:10) for acclimatization and further development. All the genotypes tested, produced multiple shoots that could be established to mature fertile plant, hence, the medium combinations used were found to be genotype neutral.

Pigeonpea [Cajanus cajan (L.) Millspaugh] is an important food legume of the semi-arid tropics (SAT) sustaining livelihood of millions of people. Stagnant and unstable yield per hectare all over the world is the characteristic feature of this crop. This is primarily ascribed to its susceptibility/sensitivity to a number of biotic and abiotic factors. Among biotic factors, insects such as pod borer (Helicoverpa armigera), pod fly (Melanoagromyza obtusa) and spotted borer (Maruca vitrata) substantially damage the crop and result in significant economic losses. Management of these insects by genetic means has always been considered environment friendly approach. However, genetic improvement has always been impeded by limited genetic variability in the primary gene pool of pigeonpea. Wild species present in the secondary and tertiary gene pools have been reported to carry resistance for such insects. However, transfer of resistance through conventional backcrossing has not been much successful. It calls for gene introgression through marker assisted backcrossing (MABC) or advanced backcross breeding (AB breeding). In this review, we have attempted to assess the progress made through conventional and molecular breeding and suggested the ways to move further towards genetic enhancement for insects resistance in pigeonpea

Chickpea (Cicer arietinum L.) is a major food legume crops for the tropics and sub-tropical regions and is the main dietary protein source for vegetarians in developing countries. Besides several abiotic factors, its production is constrained by insect pests, as well as many fungal diseases. The success of any attempt to produce resistant varieties through genetic engineering to a large extent depends on the availability of efficient and reproducible regeneration and transformation procedures. Further, the transformation techniques can be more successfully applied if the ontogeny of shoot development is well understood. Ontogeny of shoot development from axillary meristem explants (AME) in chickpea has been studied with the optimization of conditions for high-frequency multiple shoot induction. Preculture of seeds in TDZ significantly enhances the frequency of multiple shoot induction from the explants. Ontogeny and early events of multiple shoot induction revealed direct adventitious origin of the shoots. The understanding of the regeneration process could be further utilized in the designing of efficient transformation methods.

With 1 figure and 1 table Abstract To study the conservation of microsatellite regions, a set of 137 microsatellite markers developed from Phaseolus, Cajanus, Lens and Cicer genera of Leguminosae family were tested for their transferability across 16 genotypes of Phaseolus belonging to diverse collections from South America and Asia. Considerable transferability was observed with markers derived from Cajanus (60%), Lens (46%) and Cicer (28%). Of the total 122 loci were amplified, 82 cross-species polymorphic amplicons were obtained. Maximum number of alleles per marker was six (Cicer markers). Polymorphism information content values ranged from 0.12 to 0.96 with Cajanus, 0.13 to 0.74 with Lens and 0.30 to 0.93 with Cicer markers. Unweighted pair group method employing arithmetic averages cluster analysis of Phaseolus genotypes showed clear demarcation between the commercial cultivars falling in separate cluster with respect to their seed size and maturity. Transferability of genomic SSRs was different from that of expressed sequence tag-derived genic microsatellite markers.

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a su...

Random amplified polymorphic DNA (RAPD) markers were used to study the DNA polymorphism in Indian mungbean cultivars. A total of 60 random primers were used in the study and 33 of them generated reproducible RAPD patterns. Amplification of genomic DNA of most popular 24 Indian mungbean cultivars with these RAPD primers yielded 249 fragments that could be scored, of which 224 were polymorphic, with an average of 7.0 polymorphic fragments per primer. Number of amplified fragments with random primers ranged from 2 (OPI 9) to 17 (OPD 7). Percentage polymorphism ranged from 33% (OPX 5) to a maximum of 100% (OPX 4, OPX 6, OPX 13, OPX 15, OPX 19, OPD 5, OPD 7, OPD 20, OPI 4, OPI 6, OPI 13, OPI 14, OPI 18 and OPF 1), with an average of 90%. The Jaccard’s similarity indices based on RAPD profiles were subjected to UPGMA cluster analysis. And genotypes grouped in two major groups. Sixteen out of 24 released cultivars grouped to cluster I. This indicated the narrow genetic base in the Indian mungbean cultivars used in the study. The details of diversity analysis and possible reasons for narrow genetic base in mungbean cultivars are discussed in the present study.

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 material The online version of this article (doi:10.1007/s13562-011-0088-8) contains supplementary material, which is available to authorized users.

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.

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.

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 molecular tools in pigeonpea and should significantly benefit pigeonpea genetics and breeding. As these efforts come to fruition, and expanded (depending on funding), pigeonpea would move from an ‘orphan legume crop’ to one where genomics-assisted breeding approaches for a sustainable crop improvement are routine.

With 2 figures and 3 tables Abstract Paucity of polymorphic molecular markers in pigeonpea, Cajanus cajan (L.) Millsp., has been a major limiting factor in application of molecular tools for its genetic improvement. As the development of microsatellite markers requires considerable time, expertise and research infrastructure, transfer of markers from other related genera offers an alternative option to increase the number of available markers. Since microsatellite sequences are conserved across Fabaceae taxa, transferability of 100 chickpea (Cicer arietinum L.)-specific SSR markers was studied in two genotypes each of five wild and one cultivated species of Cajanus. The results revealed a significant transferability (46%) of chickpea microsatellites to Cajanus. In cultivated pigeonpea, chickpea-specific SSRs showed 38–39% transferability, while among wild Cajanus species, it ranged from 26% in Cajanus sericeus ICP 15760 to 40% in C. sericeus ICP 15761. The transferable primers exhibited extensive polymorphism in Cajanus with an average number of 4.11 alleles per marker. High level of polymorphism exhibited by chickpea microsatellite markers in the present study indicates their usefulness in diversity analysis, mapping agronomically important traits and marker-assisted breeding in pigeonpea.

Molecular markers have emerged as useful tools to assess the genetic diversity across crops. In lentil, molecular markers are limited. The objective of the study was to explore genetic diversity and relatedness Indian and exotic lentil accessions. Genetic diversity was studied in 25 lentil cultivars using 100 ISSR markers. Out of 100 markers, 24 amplified PCR products and total of 156 alleles were identified with a mean of 6.5 alleles per marker. Genetic similarity among the genotypes ranged from 37 to 84%. UPGMA cluster analysis revealed two main clusters, and the second cluster comprised majority of genotypes with the exception of germplasm line Precoz, which did not fall in either cluster. Eleven genotype specific unique bands were also obtained which showed amplification only in particular genotypes. These unique bands can serve as potential diagnostic markers and therefore, may be of immense importance. The polymorphic markers will enhance marker repertoire to study genetic diversity in lentil and also improve understanding about the genetic base lentil cultivars.

All the essential nutrients in the human diet are ultimately derived from plants. However, most of the major food crops lack certain essential vitamins and minerals. Although a diversified diet regime provides adequate nutrition, majority of the population in developing countries relies on staple crops, such as rice, wheat, maize or cassava, which lacks full complement of essential nutrients. Malnutrition, thus, is a significant humanitarian issue in most of the developing world. A pertinent way to address this challenge is through biofortification of crops to increase their essential nutrient content. Transgenic approaches offer the most rapid and precise way to develop high-nutrient crops, thus complementing mineral fertilization and conventional breeding towards ameliorating the scourge.

Chickpea (Cicer arietinum) is among the founder crops domesticated in the Fertile Crescent. One of two major forms of chickpea, the so-called kabuli type, has white flowers and light-colored seed coats, properties not known to exist in the wild progenitor. The origin of the kabuli form has been enigmatic. We genotyped a collection of wild and cultivated chickpea genotypes with 538 single nucleotide polymorphisms (SNPs) and examined patterns of molecular diversity relative to geographical sources and market types. In addition, we examined sequence and expression variation in candidate anthocyanin biosynthetic pathway genes. A reduction in genetic diversity and extensive genetic admixture distinguish cultivated chickpea from its wild progenitor species. Among germplasm, the kabuli form is polyphyletic. We identified a basic helix-loop-helix (bHLH) transcription factor at chickpea's B locus that conditions flower and seed colors, orthologous to Mendel's A gene of garden pea, whose loss of function is associated invariantly with the kabuli type of chickpea. From the polyphyletic distribution of the kabuli form in germplasm, an absence of nested variation within the bHLH gene and invariant association of loss of function of bHLH among the kabuli type, we conclude that the kabuli form arose multiple times during the phase of phenotypic diversification after initial domestication of cultivated chickpea.

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.

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

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, 544.73 Mb 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 (15 360 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.

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 × C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM—ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2–21 alleles and polymorphic information content value 0.04–0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago. Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1265-1) contains supplementary material, which is available to authorized users.

Fungal pathogens and plant parasitic nematodes are prominent in causing diseases in legume crops. Chemical control of nematodes and fungi are not very effective and also cause product contamination. Molecular biology has made it possible to identify and clone genes associated with pathogenesis related proteins, anti-fungal proteins and compounds, phytoalexins and enzymes controlling oxidative burst, etc. which lead to a number of transgenic plant developments. It has also been possible to overexpress them constitutively in several transgenic crop systems. In a majority of cases it was proved that these anti-fungal proteins are very effective against various fungal pathogens. RFLP (Restriction Fragment Length Polymorphism) has made it possible to generate high-density maps of various commercially important crops including peas. However, progress of molecular mapping and gene tagging is limited due to availability of low degree of polymorphism using conventional DNA markers like RAPD and RFLP. In recent years, development of microsatellite based markers and AFLP has produced adequate polymorphism in a number of grain legumes like chickpea which otherwise was shown to be low polymorphic. Besides, there are also some successful examples of gene tagging using these markers for fungal diseases and nematode resistance in grain legumes.

Protease inhibitors are natural defense proteins that inhibit the proteolytic activity of proteases. Legume protease inhibitors are diverse in their molecular mechanism and many of them have successfully been deployed in crop plants to confer resistance against insect pests. Nine combinations of degenerated primers,designed, based on “multiple aligned” gene sequences from NCBI Gene bank database were used to amplify the protease inhibitor genes in of ten different legume crops (two varieties each)viz.fieldpea,chickpea, lentil, lathyrus, pigeonpea , frenchbean,mungbean ,clusterbean , horsegramand cowpea .The primer combination BBF2/BBR2 amplified the expectedamplicon of 800 bp invarities ofmungbean,horsegramand cowpea. These amplicons obtained from each genotype were further ligated to the pJET1.2/blunt vector and transformed into XL1 blue strain of E.coli. Out of 41 clones sequenced, two clone sequences obtained from cowpea (DCS 6) showed homology with substilisin-like serine protease inhibitor of Desulfovibria vulgaris and serine/threonine protein kinase of Epichlocfastucae.These genes can be transformed into Agrobacterium based binary vectors and used for transformation of crops. Therefore these findings offer new genes for transgenic development against lepidopteron pests.

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.

Background Pigeonpea [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. Results In 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. Conclusion We 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.