Sunil Archak

Analysis of genetic diversity of Indian mango cultivars using RAPD markers

Summary Random amplified polymorphic DNA analysis was carried out in 29 Indian mango cultivars comprising popular landraces and some advanced cultivars. PCR amplification with 24 primers generated 314 bands, 91.4% of which were polymorphic. Jaccard’s similarity between pairs of cultivars ranged between 0.318 and 0.75 with a mean of 0.565. A UPGMA dendrogram showed the majority of the cultivars from northern and eastern regions of India clustering together and separate from southern and western cultivars. Analysis of molecular variance revealed that 94.7% of the genetic diversity in mango existed within regions. However, differences among regions were significant; northern and eastern regions formed one zone and western and southern regions formed another zone of mango diversity in India.

DNA fingerprinting of Indian cashew (Anacardium occidentale L.) varieties using RAPD and ISSR techniques

Indian cashew breeding programme has produced 24 selections and 11 hybrids with increased yield and excellent nut characters. Molecular profiles of these varieties were developed using a combination of five RAPD and four ISSR primers pre-selected for maximum discrimination and repeatability. A total of 94 markers were generated which discriminated all the varieties with a probability of identical match by chance of2.8 × 10-11. There was no correlation between the relationships based on molecular data and the pedigree of the varieties. Narrow range of average similarity values among major cashew breeding centres with only 3.6% of molecular variance partitioned between them was attributed to the exchange of genetic material in developing varieties. Difference in the average similarity coefficients between selections and hybrids was low indicating the need and scope for identification of more parental lines in enhancing the effectiveness of hybridisation programme.

InSatDb: a microsatellite database of fully sequenced insect genomes

InSatDb presents an interactive interface to query information regarding microsatellite characteristics per se of five fully sequenced insect genomes (fruit-fly, honeybee, malarial mosquito, red-flour beetle and silkworm). InSatDb allows users to obtain microsatellites annotated with size (in base pairs and repeat units); genomic location (exon, intron, up-stream or transposon); nature (perfect or imperfect); and sequence composition (repeat motif and GC%). One can access microsatellite cluster (compound repeats) information and a list of microsatellites with conserved flanking sequences (microsatellite family or paralogs). InSatDb is complete with the insects information, web links to find details, methodology and a tutorial. A separate ‘Analysis’ section illustrates the comparative genomic analysis that can be carried out using the output. InSatDb is available at .

Computational prediction of rice (Oryza sativa) miRNA targets

Bioinformatic approaches have complemented experimental efforts to inventorize plant miRNA targets. We carried out global computational analysis of rice (Oryza sativa) transcriptome to generate a comprehensive list of putative miRNA targets. Our predictions (684 unique transcripts) showed that rice miRNAs mediate regulation of diverse functions including transcription (41%), catalysis (28%), binding (18%), and transporter activity (11%). Among the predicted targets, 61.7% hits were in coding regions and nearly 72% targets had a solitary miRNA hit. The study predicted more than 70 novel targets of 34 miRNAs putatively regulating functions like stress-response, catalysis, and binding. It was observed that more than half (55%) of the targets were conserved between O. sativa indica and O. sativa japonica. Members of 31 miRNA families were found to possess conserved targets between rice and at least one of other grass family members. About 44% of the unique targets were common between two dissimilar miRNA prediction algorithms. Such an extent of cross-species conservation and algorithmic consensus confers confidence in the list of rice miRNA targets predicted in this study.

Genetic analyses of Casuarinas using ISSR and FISSR markers

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and 12 superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay. The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA)n and its complementary nucleotide (GT)n repeat motifs amplified relatively higher number of bands with an average of 6.0 ± 3.5 and 6.3 ± 1.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5′ or 3′ end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C.equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT)4 distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.

Analysis of genetic diversity in Piper nigrum L. using RAPD markers

RAPD analysis was conducted in 22 cultivars of P. nigrum(black pepper) from South India and one accession each of P. longum and P. colubrinum. Twenty-four primers generated 372 RAPD markers of which 367 were polymorphic. Jaccards similarity between pairs of accessions ranged between 0.11 and 0.66 with a mean of 0.38. Among P. nigrum cultivars, the similarity ranged between 0.20 and 0.66 and the mean was 0.42. The existence of wide genetic diversity as revealed in the present study is supported by earlier reports of extensive inter- and intrapopulation morphological variability in pepper cultivars from South India. UPGMA dendrogram and PCO plot revealed P. colubrinum to be most distant of the three species. Genetic proximity among P. nigrum cultivars could be related to their phenotypic similarities or geographical distribution. Greater divergence was observed among landraces than among advanced cultivars. Landraces grown in southern parts of coastal India and those grown in more northern parts were grouped in separate clusters of the dendrogram.

Identification of conserved drought stress responsive gene-network across tissues and developmental stages in rice

Identification of genes that are coexpressed across various tissues and environmental stresses is biologically interesting, since they may play coordinated role in similar biological processes. Genes with correlated expression patterns can be best identified by using coexpression network analysis of transcriptome data. In the present study, we analyzed the temporal-spatial coordination of gene expression in root, leaf and panicle of rice under drought stress and constructed network using WGCNA and Cytoscape. Total of 2199 differentially expressed genes (DEGs) were identified in at least three or more tissues, wherein 88 genes have coordinated expression profile among all the six tissues under drought stress. These 88 highly coordinated genes were further subjected to module identification in the coexpression network. Based on chief topological properties we identified 18 hub genes such as ABC transporter, ATP-binding protein, dehydrin, protein phosphatase 2C, LTPL153 - Protease inhibitor, phosphatidylethanolaminebinding protein, lactose permease-related, NADP-dependent malic enzyme, etc. Motif enrichment analysis showed the presence of ABRE cis-elements in the promoters of > 62% of the coordinately expressed genes. Our results suggest that drought stress mediated upregulated gene expression was coordinated through an ABA-dependent signaling pathway across tissues, at least for the subset of genes identified in this study, while down regulation appears to be regulated by tissue specific pathways in rice.

Genetic analysis and historical perspective of cashew (Anacardium occidentale L.) introduction into India.

Cashew (Anacardium occidentale L.), introduced into India about 400 years ago, is distributed widely in the coastal regions as spontaneous populations as well as in cultivation. Despite the plants commercial exploitation, little is known about its actual introduction and diversification. We attempted to construct these events by investigating the level of genetic variation and genetic structure of cashew populations collected from different geographical regions of India. A total of 91 individuals from four populations were analysed using AFLP markers and morphometric data. AFLP analysis based on 354 polymorphic loci revealed Indian cashew to have low but relatively substantial genetic diversity for an introduced species (H(E) = 0.262 and I(S) = 0.404). Twenty-seven qualitative and quantitative traits also revealed the existence of considerable morphometric variation (24% around the mean values). Bayesian cluster analysis based on AFLP data did not indicate the existence of definite population differentiation. Morphometric analysis allocated 12% variation among all four populations, whereas AFLP variation observed in cashew individuals was entirely within populations. The results, supporting the possibility of cashew having been introduced into India repeatedly over a period of time but at a single location (west coast), are discussed and their implications for germplasm management are described.

Evaluation of 19,460 Wheat Accessions Conserved in the Indian National Genebank to Identify New Sources of Resistance to Rust and Spot Blotch Diseases

A comprehensive germplasm evaluation study of wheat accessions conserved in the Indian National Genebank was conducted to identify sources of rust and spot blotch resistance. Genebank accessions comprising three species of wheat–Triticum aestivum, T. durum and T. dicoccum were screened sequentially at multiple disease hotspots, during the 2011–14 crop seasons, carrying only resistant accessions to the next step of evaluation. Wheat accessions which were found to be resistant in the field were then assayed for seedling resistance and profiled using molecular markers. In the primary evaluation, 19,460 accessions were screened at Wellington (Tamil Nadu), a hotspot for wheat rusts. We identified 4925 accessions to be resistant and these were further evaluated at Gurdaspur (Punjab), a hotspot for stripe rust and at Cooch Behar (West Bengal), a hotspot for spot blotch. The second round evaluation identified 498 accessions potentially resistant to multiple rusts and 868 accessions potentially resistant to spot blotch. Evaluation of rust resistant accessions for seedling resistance against seven virulent pathotypes of three rusts under artificial epiphytotic conditions identified 137 accessions potentially resistant to multiple rusts. Molecular analysis to identify different combinations of genetic loci imparting resistance to leaf rust, stem rust, stripe rust and spot blotch using linked molecular markers, identified 45 wheat accessions containing known resistance genes against all three rusts as well as a QTL for spot blotch resistance. The resistant germplasm accessions, particularly against stripe rust, identified in this study can be excellent potential candidates to be employed for breeding resistance into the background of high yielding wheat cultivars through conventional or molecular breeding approaches, and are expected to contribute toward food security at national and global levels.

Molecular Genetic Diversity Analysis of Commercial Mango (Mangifera indica L.) Cultivars Employed as Parents in Hybrid Development in India

Mango hybridization programmes use commercial cultivars as parents. Twenty three such popular cultivars of mango belonging to different regions of India were analyzed by employing multi-locus marker techniques to measure the genetic diversity existing among them. Twelve unanchored ISSR primers (114 markers) and 15 AFLP primer-pair combinations (1,073 markers) revealed average gene diversity over loci to be 0.231 and 0.257 respectively. Mango cultivars from southern India were found to be significantly different (p<0.001). In order to decipher how these markers are inherited, three popular hybrids ‘Amrapali’, ‘Mallika’ and ‘Ratna’ were compared with their parents for band sharing information. Number of ISSR markers shared between pairs of parents and their hybrid was nearly 20% more than that of AFLP markers, endorsing the conserved nature of ISSR profiles. Our observations on (i) genetic relatedness among popular cultivars and (ii) band sharing pattern among parents and hybrids have implications on mango germplasm collection and breeding activities.