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

<p>Abstract</p> <p>Background</p> <p>Pigeonpea [<it>Cajanus cajan </it>(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.</p> <p>Results</p> <p>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 <it>in silico </it>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.</p> <p>Conclusion</p> <p>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 <it>Cajanus</it>. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.</p

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Not AvailableThe acronym MAGIC that stands for ‘multiparent advanced generation intercross’ is a powerful next generation mapping population to precisely map the agronomically important quantitative trait loci. An eight parent based soybean MAGIC population was developed by employing 2-way, 4-way and 8-way intercross hybridization. The aim was to obtain MAGIC-derived breeding lines with higher yield, broader genetic base, increased diversity and variability. The 8-way and 4-way intercross hybrids so developed in the present study will be evaluated for their yield potential in the subsequent generation under changing climatic conditions (F2–F8 generation).Not Availabl

DataSheet_1_C-terminally encoded peptide-like genes are associated with the development of primary root at qRL16.1 in soybean.pdf

Root architecture traits are belowground traits that harness moisture and nutrients from the soil and are equally important to above-ground traits in crop improvement. In soybean, the root length locus qRL16.1 was previously mapped on chromosome 16. The qRL16.1 has been characterized by transcriptome analysis of roots in near-isogenic lines (NILs), gene expression analysis in a pair of lines contrasting with alleles of qRL16.1, and differential gene expression analysis in germplasm accessions contrasting with root length. Two candidate genes, Glyma.16g108500 and Glyma.16g108700, have shown relatively higher expression in longer root accessions than in shorter rooting accessions. The C-terminal domain of Glyma.16g108500 and Glyma.16g108700 is similar to the conserved domain of C-terminally encoded peptides (CEPs) that regulate root length and nutrient response in Arabidopsis. Two polymorphisms upstream of Glyma.16g108500 showed a significant association with primary root length and total root length traits in a germplasm set. Synthetic peptide assay with predicted CEP variants of Glyma.16g108500 and Glyma.16g108700 demonstrated their positive effect on primary root length. The two genes are root-specific in the early stage of soybean growth and showed differential expression only in the primary root. These genes will be useful for improving soybean to develop a deep and robust root system to withstand low moisture and nutrient regimes.</p

Table_1_C-terminally encoded peptide-like genes are associated with the development of primary root at qRL16.1 in soybean.xlsx

Root architecture traits are belowground traits that harness moisture and nutrients from the soil and are equally important to above-ground traits in crop improvement. In soybean, the root length locus qRL16.1 was previously mapped on chromosome 16. The qRL16.1 has been characterized by transcriptome analysis of roots in near-isogenic lines (NILs), gene expression analysis in a pair of lines contrasting with alleles of qRL16.1, and differential gene expression analysis in germplasm accessions contrasting with root length. Two candidate genes, Glyma.16g108500 and Glyma.16g108700, have shown relatively higher expression in longer root accessions than in shorter rooting accessions. The C-terminal domain of Glyma.16g108500 and Glyma.16g108700 is similar to the conserved domain of C-terminally encoded peptides (CEPs) that regulate root length and nutrient response in Arabidopsis. Two polymorphisms upstream of Glyma.16g108500 showed a significant association with primary root length and total root length traits in a germplasm set. Synthetic peptide assay with predicted CEP variants of Glyma.16g108500 and Glyma.16g108700 demonstrated their positive effect on primary root length. The two genes are root-specific in the early stage of soybean growth and showed differential expression only in the primary root. These genes will be useful for improving soybean to develop a deep and robust root system to withstand low moisture and nutrient regimes.</p

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Lead ArticleMicroRNAs (miRNAs) are small regulatory RNAs that play a defining role in post-transcriptional gene silencing of eukaryotes by either mRNA cleavage or translational inhibition. Plant miRNAs have been implicated in innumerable growth and developmental processes that extend beyond their ability to respond to biotic and abiotic stresses. Active in an organism’s immune defence response, host miRNAs display a propensity to target viral genomes. During viral invasion, these virus-targeting miRNAs can be identified by their altered expression. All the while, pathogenic viruses, as a result of their long-term interaction with plants, have been evolving viral suppressors of RNA silencing (VSRs), as well as viral-encoded miRNAs as a counter-defence strategy. However, the gene silencing attribute of miRNAs has been ingeniously manipulated to down-regulate the expression of any gene of interest, including VSRs, in artificial miRNA (amiRNA)-based transgenics. Since we currently have a better understanding of the intricacies of miRNA-mediated gene regulation in plant–virus interactions, the majority of miRNAs manipulated to confer antiviral resistance to date are in plants. This review will share the insights gained from the studies of plant-virus combat and from the endeavour to manipulate miRNAs, including prospective challenges in the context of the evolutionary dynamics of the viral genome. Next generation sequencing technologies and bioinformatics analysis will further delineate the molecular details of host–virus interactions. The need for appropriate environmental risk assessment principles specific to amiRNA-based virus resistance is also discussed.Not Availabl

Molecular characterization and genetic diversity analysis of soybean (Glycine max (L.) Merr.) germplasm accessions in India

Not AvailableMolecular characterization and genetic diversity among 82 soybean accessions was carried out by using 44 simple sequence repeat (SSR) markers. Of the 44 SSR markers used, 40 markers were found polymorphic among 82 soybean accessions. These 40 polymorphic markers produced a total of 119 alleles, of which five were unique alleles and four alleles were rare. The allele number for each SSR locus varied between two to four with an average of 2.97 alleles per marker. Polymorphic information content values of SSRs ranged from 0.101 to 0.742 with an average of 0.477. Jaccard’s similarity coefficient was employed to study the molecular diversity of 82 soybean accessions. The pairwise genetic similarity among 82 soybean accessions varied from 0.28 to 0.90. The dendrogram constructed based on genetic similarities among 82 soybean accessions identified three major clusters. The majority of genotypes including four improved cultivars were grouped in a single subcluster IIIa of cluster III, indicating high genetic resemblance among soybean germplasm collection in India.Not Availabl

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Not AvailableA total of 47 new soybean accessions collected were sown during kharif 2014 to evaluate and conserve in the NAGS. The ANOVA indicated significant and wide variability for the traits studied except for the primary branches and seeds per pod. To examine breeding utilities, few genetic parameters were studied for the new accessions. Except, few traits like number of seeds per pod all other traits exhibited wide range of variations. This was substantiated by the fact that seed yield per plant, number of nodes per plant, pods per plant, days to flowering and hundred seed weight showed highest PCV and GCV indicating presence of extensive variability rendering selection effective. The number of primary branches, pods per plant, seeds per pod and hundred seed weight showed positive and significant correlation with yield. The path analysis studies revealed that pods per plant and 100 seed weight were important yield components with high direct effects on improvement for seed yield.Not Availabl

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Not AvailableSoybean is a leading oilseed crop in India, which contains about 40% of protein and 20% of oil. Core collection will accelerate the management and utilization of soybean genetic resources in breeding programmes. In the present study, eight agromorphological traits of 3443 soybean germplasm were analysed for the development of core collection using the principal component score (PCS) strategy and the power core method. The PCS strategy yielded core collection (CC1) of 576 accessions, which accounted for 16.72% of the entire collection (EC). The analysis based on the power core programme resulted in CC2 of 402 accessions, which accounted for 11.67% of the EC. Statistical analysis showed similar trends for the mean and range estimated in both core collections and EC. In addition, the variance, standard deviation and coefficient of variance were in general higher in core collections than in the EC. The correlations observed in the EC in general were preserved in core collections. A total of 311 and 137 unique accessions were found in CC1 and CC2 in addition to 265 accessions that were found to be common in both core collections. These 265 common accessions were the most diverse core sets, which accounted for 7.64% of the EC. We proposed to constitute an integrated core collection (ICC) by integrating both common and unique accessions. The ICC comprised 713 accessions, which accounted for about 20.62% of the EC. Statistical analysis indicated that the ICC captured maximum variation than CC1 and CC2. Therefore, the ICC can be extensively evaluated for a large number of economically important traits for the identification of desirable genotypes and for the development of mini core collection in soybean.Not Availabl

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Molecular mapping of QTLs for plant type and earliness traits in pigeonpea (<it>Cajanus cajan</it> L. Millsp.)

Abstract Background Pigeonpea is an important grain legume of the semi-arid tropics and sub-tropical regions where it plays a crucial role in the food and nutritional security of the people. The average productivity of pigeonpea has remained very low and stagnant for over five decades due to lack of genomic information and intensive breeding efforts. Previous SSR-based linkage maps of pigeonpea used inter-specific crosses due to low inter-varietal polymorphism. Here our aim was to construct a high density intra-specific linkage map using genic-SNP markers for mapping of major quantitative trait loci (QTLs) for key agronomic traits, including plant height, number of primary and secondary branches, number of pods, days to flowering and days to maturity in pigeonpea. Results A population of 186 F2:3 lines derived from an intra-specific cross between inbred lines ‘Pusa Dwarf’ and ‘HDM04-1’ was used to construct a dense molecular linkage map of 296 genic SNP and SSR markers covering a total adjusted map length of 1520.22 cM for the 11 chromosomes of the pigeonpea genome. This is the first dense intra-specific linkage map of pigeonpea with the highest genome length coverage. Phenotypic data from the F2:3 families were used to identify thirteen QTLs for the six agronomic traits. The proportion of phenotypic variance explained by the individual QTLs ranged from 3.18% to 51.4%. Ten of these QTLs were clustered in just two genomic regions, indicating pleiotropic effects or close genetic linkage. In addition to the main effects, significant epistatic interaction effects were detected between the QTLs for number of pods per plant. Conclusions A large amount of information on transcript sequences, SSR markers and draft genome sequence is now available for pigeonpea. However, there is need to develop high density linkage maps and identify genes/QTLs for important agronomic traits for practical breeding applications. This is the first report on identification of QTLs for plant type and maturity traits in pigeonpea. The QTLs identified in this study provide a strong foundation for further validation and fine mapping for utilization in the pigeonpea improvement.</p