Publications of NIPGR Scientists

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    Development of 5123 intron-length polymorphic markers for large-scale genotyping applications in foxtail millet
    (Oxford University Press, 2014) Muthamilarasan, Mehanathan; Venkata Suresh, B.; Pandey, Garima; Kumari, Kajal; Parida, Swarup K.; Prasad, Manoj
    Generating genomic resources in terms of molecular markers is imperative in molecular breeding for crop improvement. Though development and application of microsatellite markers in large-scale was reported in the model crop foxtail millet, no such large-scale study was conducted for intron-length polymorphic (ILP) markers. Considering this, we developed 5123 ILP markers, of which 4049 were physically mapped onto 9 chromosomes of foxtail millet. BLAST analysis of 5123 expressed sequence tags (ESTs) suggested the function for ∼71.5% ESTs and grouped them into 5 different functional categories. About 440 selected primer pairs representing the foxtail millet genome and the different functional groups showed high-level of cross-genera amplification at an average of ∼85% in eight millets and five non-millet species. The efficacy of the ILP markers for distinguishing the foxtail millet is demonstrated by observed heterozygosity (0.20) and Nei's average gene diversity (0.22). In silico comparative mapping of physically mapped ILP markers demonstrated substantial percentage of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (∼50%), maize (∼46%), rice (∼21%) and Brachypodium (∼21%) chromosomes. Hence, for the first time, we developed large-scale ILP markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.
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    Genome-wide development and use of microsatellite markers for large-scale genotyping applications in foxtail millet [Setaria italica (L.)]
    (Oxford University Press, 2013) Pandey, Garima; Misra, Gopal; Kumari, Kajal; Gupta, Sarika; Parida, Swarup K.; Chattopadhyay, Debasis; Prasad, Manoj
    The availability of well-validated informative co-dominant microsatellite markers and saturated genetic linkage map has been limited in foxtail millet (Setaria italica L.). In view of this, we conducted a genome-wide analysis and identified 28 342 microsatellite repeat-motifs spanning 405.3 Mb of foxtail millet genome. The trinucleotide repeats (∼48%) was prevalent when compared with dinucleotide repeats (∼46%). Of the 28 342 microsatellites, 21 294 (∼75%) primer pairs were successfully designed, and a total of 15 573 markers were physically mapped on 9 chromosomes of foxtail millet. About 159 markers were validated successfully in 8 accessions of Setaria sp. with ∼67% polymorphic potential. The high percentage (89.3%) of cross-genera transferability across millet and non-millet species with higher transferability percentage in bioenergy grasses (∼79%, Switchgrass and ∼93%, Pearl millet) signifies their importance in studying the bioenergy grasses. In silico comparative mapping of 15 573 foxtail millet microsatellite markers against the mapping data of sorghum (16.9%), maize (14.5%) and rice (6.4%) indicated syntenic relationships among the chromosomes of foxtail millet and target species. The results, thus, demonstrate the immense applicability of developed microsatellite markers in germplasm characterization, phylogenetics, construction of genetic linkage map for gene/quantitative trait loci discovery, comparative mapping in foxtail millet, including other millets and bioenergy grass species.
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    Development and utilization of novel SSRs in foxtail millet [Setaria italica (L.) P. Beauv.]
    (John Wiley & Sons, 2013) Gupta, Sarika; Kumari, Kajal; Muthamilarasan, Mehanathan; Subramanian, Alagesan; Prasad, Manoj
    Although the foxtail millet [Setaria italica (L.) P. Beauv.] is recently regarded as a model crop for studying functional genomics of biofuel grasses, its genetic improvement to some extent was limited due to the non-availability of molecular markers, particularly the microsatellite markers and the saturated genetic linkage map. Considering this, we attempted to generate a significant number of microsatellite markers in cultivar ‘Prasad’. Two hundred and fifty-six clones were sequenced to generate 41.82-kb high-quality sequences retrieved from genomic library enriched with dinucleotide repeat motifs. Microsatellites were identified in 194 (76%) of the 256 positive clones, and 64 primer pairs (pp) were successfully designed from 95 (49%) unique SSR-containing clones. The 67.4% primer designing ability, 100% PCR amplification efficiency and 45.3% polymorphic potential in the parents of F2 mapping population established the efficacy of genomic microsatellites. All the 64 microsatellite markers displayed high level of cross-species amplification (~67%) in 10 millets and non-millets species. These experimental findings suggest the utility and efficacy of SSRs in diverse genotyping applications, resolving QTLs, phylogenetic relationships and transferability in several important grass species.
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    Development and utilization of novel intron length polymorphic markers in foxtail millet [Setaria italica (L.) P. Beauv.]
    (NRC Res Press, 2011) Gupta, Sarika; Kumari, Kajal; Das, Jyotirmoy; Lata, Charu; Puranik, Swati; Prasad, Manoj
    Introns are noncoding sequences in a gene that are transcribed to precursor mRNA but spliced out during mRNA maturation and are abundant in eukaryotic genomes. The availability of codominant molecular markers and saturated genetic linkage maps have been limited in foxtail millet (Setaria italica (L.) P. Beauv.). Here, we describe the development of 98 novel intron length polymorphic (ILP) markers in foxtail millet using sequence information of the model plant rice. A total of 575 nonredundant expressed sequence tag (EST) sequences were obtained, of which 327 and 248 unique sequences were from dehydration- and salinity-stressed suppression subtractive hybridization libraries, respectively. The BLAST analysis of 98 EST sequences suggests a nearly defined function for about 64% of them, and they were grouped into 11 different functional categories. All 98 ILP primer pairs showed a high level of cross-species amplification in two millets and two nonmil- lets species ranging from 90% to 100%, with a mean of ∼97%. The mean observed heterozygosity and Nei’s average gene diversity 0.016 and 0.171, respectively, established the efficiency of the ILP markers for distinguishing the foxtail millet accessions. Based on 26 ILP markers, a reasonable dendrogram of 45 foxtail millet accessions was constructed, demonstrating the utility of ILP markers in germplasm characterizations and genomic relationships in millets and nonmillets species.
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    Development and characterization of genic SSR-markers in Medicago truncatula and its transferability in leguminous and non-leguminous species
    (NRC Res Press, 2009)
    Expressed sequence tag (EST)-derived simple sequence repeat (eSSR) markers are important resources for gene discovery and comparative mapping aimed at crop improvement. In this study, we developed eSSR markers for Medicago truncatula and assessed their cross-species transferability. We detected 36,847 non-redundant sequences ("unigenes") from 198,642 M. truncatula EST sequences. Mining of microsatellites from the 36,847 unigene sequences (representing approximately 25.8 Mb) revealed 14,637 eSSRs in 11,750 SSR-containing ESTs, and primer pairs were successfully designed for 4,636 (39.5%). Of the 14 637 eSSRs, 82.6% were mononucleotide repeats and the rest (in descending order of abundance) were tri-, di-, penta-, and tetranucleotide repeats. When less stringent SSR detection criteria were used, the frequency of dinucleotide repeat motifs increased more than twofold, and the frequencies of di- (11%) and trinucleotide motifs (10.6%) were almost equal. This demonstrates that the eSSR frequency and distribution were related to the choice of search criteria. Forty-one randomly selected primer pairs were validated, and their transferability in three leguminous and three non-leguminous species was assessed. The markers showed a high level of transferability in the leguminous (53%-71%) and non-leguminous (33%-44%) species. The validation studies thus demonstrate the utility of the Medicago eSSRs in assessing genomic relationships in both leguminous and non-leguminous species.