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Heat stress has a significant impact on the climatic adaptation of flax, a cool-season economic crop. Genome-wide DNA methylation patterns are crucial for understanding how flax cultivars respond to heat adversities. It is worth noting that the DNA methylome in flax has yet to be investigated at the nucleotide level. Although heat stress above 40°C caused oxidative damage in flax leaves, 5-azacytidine, a hypomethylating agent, reduced this effect by 15%-24%. Differences in the expression of the LuMET1 (DNA methyltransferase) gene suggested that DNA methylation/demethylation may play a major role in the flax heat stress response. Thus, whole-genome bisulfite sequencing-derived DNA methylation profiles in flax, with or without heat stress and 5-azaC, were developed and analyzed here. In response to heat stress, a high percentage of significant differentially methylated regions (DMRs), particularly hypomethylated DMRs, were identified in the CHH nucleotide sequence context (H = A/T/C). Some of these DMRs overlapped with transposable element insertions. The majority of DMRs were discovered in intergenic regions, but several DMR loci were also found near genes relevant to heat stress response and epigenetic processes. These DMRs, in particular, are linked to CpG islands, implying a possible role in promoter methylation and gene silencing. The DMRs discovered in this study are crucial for understanding and identifying the key players in heat stress response in flax, which will help in developing climate-smart flax varieties.

Additional file 2: Sup Table 1. Transgenic chickpea lines and Generation Advancement. Sup Table 2. Segregation of AtDREB1a in T1 progenies of four independent chickpea events. Sup Table 3. ANOVA for RWC and OA of transgenic event E5 (T2). Sup Table 4. ANOVA for RWC and OA of transgenic event E17 (T2). Sup Table 5. ANOVA for RWC and OA of transgenic event E19 (T2). Sup Table 6. ANOVA for RWC and OA of transgenic event E22 (T2). Sup Table 7. ANOVA for RWC of transgenic chickpea lines (T3). Sup Table 8. ANOVA for OP of transgenic chickpea lines (T3). Sup Table 9. ANOVA for MSI of transgenic chickpea lines (T3). Sup Table 10. ANOVA for chlorophyll content (SPAD values) of transgenic chickpea lines (T3). Sup Table 11. ANOVA for ETR of transgenic chickpea lines (T3). Sup Table 12. ANOVA for CID of transgenic chickpea lines (T3). Sup Table 13. ANOVA for seed yield of transgenic chickpea lines, under WW and WS conditions (T3).

Additional file 1: Sup Fig. 1. T-DNA of the AtDREB1a construct. Sup Fig. 2. Different stages of genetic transformation in chickpea (C. arietinum L.), A. De-coated DCP 92–3 seed, B. Prepared CWHEA explant, C. Explants dipped in Agrobacterium suspension, D. Explants in Whatman filter paper, E. Co-cultivated explants in SIM 1 media, F. Germinated explants in SIM 2 media, G. Explant having multiple shoots, H. Kanamycin resistant shoot (Ready for grafting), I. Micrografting, J. Mature fertile plant. Sup Fig. 3. Full image of Southern Blot analyses (1F). Sup Fig. 4. Full image of RT-PCR of transgenic chickpea lines and control (1G). (PPT 3221 kb)

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.

India does not have a clear stand on the release and consumption of genetically modified crops (food). The only approved crop is Bt-cotton, which has put India on the global map as a cotton exporting country. Even so, Bt-brinjal is under moratorium and GM mustard is prevented from undergoing commercial trial. All these decisions are not based on sound scientific principles. Activism against has successfully prevented exploitation of a powerful technology that can contribute to India's food and nutrition security. This article attempts to give a balanced perspective of genetic modification technology as one of the serious options to be considered on case to case basis. Ambivalence will seriously affect India's food security in the future.

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.

With the objective to study the geographical distribution pattern and pathotype classification, isolates from 12 major pigeonpea growing states of India were examined at morphological and molecular levels. Two DNA based internal transcribe spacer (ITS) region derived primers FDP 3 (ITS1/ITS2), FDP 25 (mRNA, LOC100383610) and two elongation factors FDP 4 (F98-BKR5) and FDP 29 (M9968PY) were employed to genetically differentiate the isolates. As a result, each marker system gave an average of 3 alleles/marker. The higher efficiency of ITS over EF-1α marker was revealed using detailed comparative analysis that included various parameters like gene diversity index, effective marker ratio, and marker index. Neighbour Joining tree analysis grouped the isolates into three major clusters and showed narrow existence of genetic divergence. Combination of genotyping data with pathological measurements indicates dominance of variant 1 in the Central zone, South zone and North East Plain Zone, while North East Plain Zone and North West Plain Zone were largely dominated by variants 2 and 1, with strong possibility of evolving other variants. The present study would help in identifying specific isolate and patterns of its distribution in various pigeonpea growing regions thereby enhancing the scope for precise resistance breeding for crop improvement.

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.