Black gram [V. mungo (L.) Hepper] is an important legume crop extensively grown in south and south-east Asia, where it is a major source of dietary protein for its predominantly vegetarian population. However, lack of genomic information and markers has become a limitation for genetic improvement of this crop. Here, we report the transcriptome sequencing of the immature seeds of black gram cv. TU94-2, by Illumina paired end sequencing technology to generate transcriptome sequences for gene discovery and genic-SSR marker development. A total of 17.2 million paired-end reads were generated and 48,291 transcript contigs (TCS) were assembled with an average length of 443 bp. Based on sequence similarity search, 33,766 TCS showed significant similarity to known proteins. Among these, only 29,564 TCS were annotated with gene ontology (GO) functional categories. A total number of 138 unique KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified, of which majority of TCS are grouped into purine metabolism (678) followed by pyrimidine metabolism (263). A total of 48,291 TCS were searched for SSRs and 1,840 SSRs were identified in 1,572 TCS with an average frequency of one SSR per 11.9 kb. The tri-nucleotide repeats were most abundant (35%) followed by di-nucleotide repeats (32%). PCR primer pairs were successfully designed for 933 SSR loci. Sequences analyses indicate that about 64.4% and 35.6% of the SSR motifs were present in the coding sequences (CDS) and untranslated regions (UTRs) respectively. Tri-nucleotide repeats (57.3%) were preferentially present in the CDS. The rate of successful amplification and polymorphism were investigated using selected primers among 18 black gram accessions. Genic-SSR markers developed from the Illumina paired end sequencing of black gram immature seed transcriptome will provide a valuable resource for genetic diversity, evolution, linkage mapping, comparative genomics and marker-assisted selection in black gram.
Detached mungbean (Vigna radiata L.Wilczek) leaves were inoculated with a conidial suspension of a local isolate (TI-1) of the powdery mildew pathogen (Erysiphe polygoni DC) under controlled environment conditions. Based on the latent period and severity of the infection, a rating scale of 0-5 was used to classify the host pathogen interactions. Reactions 0, 1 and 2 were considered resistant and referred to as R0, R1 and R2 while 3, 4 and 5 were classified as susceptible (S). RUM lines (resistant to powdery mildew) and their derivatives are crossed with several susceptible (reaction types 3-5) genotypes and the inheritance of the resistance was studied in the F1, F2 and F3 generations. The results showed that powdery mildew resistance in mungbean is governed by two dominant genes designated as Pm-1 and Pm-2. When both Pm-1 and Pm-2 were present, an R0 reaction was observed after inoculation with TI-1. The resistant reaction was R1 when only Pm-1 was present and R2 in the presence of Pm-2. In the absence of both Pm-1 and Pm-2, susceptible reactions 3, 4 and 5 were observed.
Myo-inositol metabolism plays a significant role in plant growth and development, and is also used as a precursor for many important metabolites, such as ascorbate, pinitol, and phytate. Phytate (inositol hexakisphosphate) is the major storage pool for phosphate in the seeds. It is utilized during seed germination and growth of the developing embryo. In addition, it is implicated in protection against oxidative stress. In the present study, a panel of chickpea accessions was used for an association analysis. Association analysis accounting for population structure and relative kinship identified alleles of a simple sequence repeat marker, NCPGR90, that are associated with both phytic acid content and drought tolerance. These alleles varied with respect to the dinucleotide CT repeats present within the marker. NCPGR90 located to the 5'UTR of chickpea myo-inositol monophosphatase gene (CaIMP) and showed transcript length variation in drought-tolerant and drought-susceptible accessions. CaIMP from a drought-tolerant accession with a smaller repeat was almost 2-fold upregulated as compared to a susceptible accession having a longer repeat, even under control non-stressed conditions. This study suggests an evolution of simple sequence repeat length variation in CaIMP, which might be regulating phytic acid levels to confer drought tolerance in natural populations of chickpea.
Mutations in the widely conserved Arabidopsis Terminal Flower 1 (TFL1) gene and its homologs have been demonstrated to result in determinacy across genera, the knowledge of which is lacking in cowpea. Understanding the molecular events leading to determinacy of apical meristems could hasten development of cowpea varieties with suitable ideotypes. Isolation and characterization of a novel mutation in cowpea TFL1 homolog (VuTFL1) affecting determinacy is reported here for the first time. Cowpea TFL1 homolog was amplified using primers designed based on conserved sequences in related genera and sequence variation was analysed in three gamma ray-induced determinate mutants, their indeterminate parent "EC394763" and two indeterminate varieties. The analyses of sequence variation exposed a novel SNP distinguishing the determinate mutants from the indeterminate types. The non-synonymous point mutation in exon 4 at position 1,176 resulted from transversion of cytosine (C) to adenine (A) leading to an amino acid change (Pro-136 to His) in determinate mutants. The effect of the mutation on protein function and stability was predicted to be detrimental using different bioinformatics/computational tools. The functionally significant novel substitution mutation is hypothesized to affect determinacy in the cowpea mutants. Development of suitable regeneration protocols in this hitherto recalcitrant crop and subsequent complementation assay in mutants or over-expressing assay in parents could decisively conclude the role of the SNP in regulating determinacy in these cowpea mutants.
Mungbean (Vigna radiata L. Wilczek) is a short-duration legume crop cultivated for seeds that are rich in protein and carbohydrates. Mungbeans contain phytic acid (PA), an anti-nutritional factor that is the main storage form of organic phosphorus in seeds. It is a strong inhibitor against the absorption of nutrients including iron, zinc, calcium and magnesium in monogastric animals. Genotypes with low phytic acid (lpa) in seed may show increased assimilation of nutrients and be useful in breeding lpa cultivars. The present study was conducted to identify lpa sources, genetic variation, heritability, and association with seed coat color, inorganic phosphorus (IP), and seed size in 102 mungbean genotypes including released varieties, land races, mutants, and wild species grown in two seasons: summer 2011 and rabi 2012. PA and IP in dry seeds were estimated by modified colorimetric method and Chen's modified method, respectively. PA, IP, and 100-seed weight differed significantly in the two seasons. PA content in 102 genotypes ranged from 5.74 to 18.98 mg g −1 and 5.85 to 20.02 mg g −1 in summer 2011 and rabi 2012, respectively. High heritability was found for PA (0.87 and 0.86) and seed size (0.82 and 0.83) but low heritability for IP (0.61 and 0.60). A negative correlation was found between PA and seed size (r = −0.183 and −0.267). Yellow and green seed coat genotypes contained significantly less PA than black seed coat genotypes. Cluster analysis revealed the distinctness of wild species, land races and cultivated varieties on the basis of PA content. The genotypes YBSM (6.001 mg g −1 ) and JL-781 (6.179 mg g −1 ) showed lowest PA. These lpa sources can be used to develop high-yielding mungbean cultivars with low phytic acid.
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