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Subject: search.filters.subject.Microsatellites

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    Development, characterization and cross-species transferability of genomic SSR markers in berseem (Trifolium alexandrinum L.), an important multi-cut annual forage legume
    (Springer, 2015) Verma, Priyanka; Chandra, Amaresh; Roy, Ajoy Kumar; Malaviya, Devendra Ram; Kaushal, Pankaj; Pandey, Dinesh; Bhatia, Sabhyata
    Egyptian clover (Trifolium alexandrinum), a multi-cut forage legume, is widely grown as an annual feed crop. Genetic improvement in this legume is largely hampered due to limited availability of genomic resources. Hence, a genomic library enriched for GA/CT motif was constructed to develop SSR primers. As a result, 667 SSR primer pairs were developed and 111 were validated in T. alexandrinum cv. Wardan s-99-1. Among these, 45 primer pairs were used for the analysis of genetic diversity among 86 Trifolium accessions, resulting in 220 alleles with an average of 4.88 alleles per locus. Polymorphic information content for these markers ranged from 0.040 to 0.819 with an average of 0.445. The observed and expected heterozygosity for individual loci ranged from 0.39 to 0.89 and 0.368 to 0.80 with an average of 0.675 and 0.659, respectively. The markers exhibited varied levels of cross-species transferability ranging from 93.33 % across T. resupinatum to 40.0 % across T. balansae. Phylogenetic relationships were analyzed using the unweighted pair group method with arithmetic averages (UPGMA)-based dendrogram which was able to establish the uniqueness of each genotype and grouped them into two major clusters. To the best of our knowledge, the SSR markers reported here are among the first to be reported in this important yet neglected forage legume. They were assessed to be highly efficient and polymorphic and found to be useful for distinguishing the closely related genotypes of Trifolium. This large resource of SSR markers would therefore facilitate new avenues for T. alexandrinum genomics.
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    Discovery of molecular markers for Fusarium wilt via transcriptome sequencing of chickpea cultivars
    (Springer, 2015) Jain, Mukesh; Pole, Anil K.; Singh, Vikash K.; Ravikumar, Ramapura L.; Garg, Rohini
    The wilt caused by Fusarium oxysporum f.sp. Ciceris is a major problem for chickpea. The large-scale discovery of DNA polymorphisms in different cultivars can provide insights into the genetic basis of phenotypic differences. In the present study, we sequenced the transcriptomes of four chickpea cultivars with contrasting response to Fusarium infection, including susceptible JG62 and resistant/tolerant ICCV2, K850 and WR315. Based on the analysis of their transcriptome sequences, we identified at least 303 polymorphic microsatellites, of which 64 % could be physically mapped on the chickpea genome. Further, we identified a total of 14,462 single nucleotide polymorphisms (SNPs) and 1864 insertions/deletions (InDels) among the chickpea cultivars analysed. More than 50 % of the SNPs and 19 % of the InDels were detected in the predicted coding regions, and about 46 % of these SNPs resulted in nonsynonymous changes. In addition, we identified at least 60 large-effect SNPs and 248 InDels that affected the integrity of the encoded protein. Several defence-related genes involved in different cellular processes were found to harbour nonsynonymous and/or large-effect SNPs and/or InDels. These data will provide functional markers and promising target genes for wilt resistance and present a valuable resource for molecular breeding for this important trait in chickpea.
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    Exploring genetic variability within lentil (Lens culinaris Medik.) and across related legumes using a newly developed set of microsatellite markers
    (Springer, 2014) Verma, Priyanka; Sharma, Tilak R.; Srivastava, Prem S.; Abdin, M. Z.; Bhatia, Sabhyata
    Lentil (Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2-5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement.
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    Expanding the repertoire of microsatellite markers for polymorphism studies in Indian accessions of mung bean (Vigna radiata L. Wilczek)
    (Springer, 2014) Shrivastava, Divya; Verma, Priyanka; Bhatia, Sabhyata
    Limited availability of validated, polymorphic microsatellite markers in mung bean (Vigna radiata), an important food legume of India, has been a major hurdle towards its improvement and higher yield. The present study was undertaken in order to develop a new set of microsatellite markers and utilize them for the analysis of genetic diversity within mung bean accessions from India. A GA/CT enriched library was constructed from V. radiata which resulted in 1,250 putative recombinant clones of which 850 were sequenced. SSR motifs were identified and their flanking sequences were utilized to design 328 SSR primer pairs. Of these, 48 SSR markers were employed for assessing genetic diversity among 76 mung bean accessions from various geographical locations in India. Two hundred and thirty four alleles with an average of 4.85 alleles per locus were detected at 48 loci. The polymorphic information content (PIC) per locus varied from 0.1 to 0.88 (average: 0.49 per locus). The observed and expected heterozygosities ranged from 0.40 to 0.95 and 0.40 to 0.81 respectively. Based on Jaccard's similarity matrix, a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) analysis which revealed that one accession from Bundi, Rajasthan was clustered out separately while remaining accessions were grouped into two major clusters. The markers generated in this study will help in expanding the repertoire of the available SSR markers thereby facilitating analysis of genetic diversity, molecular mapping and ultimately broadening the scope for genetic improvement of this legume.
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    Isolation of microsatellites from Catharanthus roseus (L.) G. Don using enriched libraries
    (Springer, 2009) Bhatia, Sabhyata; Shokeen, Bhumika
    Catharanthus roseus is an indispensable source of the anticancerous alkaloids-vincristine and vinblastine, even though they are produced in trace amounts in vivo. In order to increase the yield of alkaloids, in vitro tissue culture studies are carried out which result in a large number of lines/cultures. For identification and characterization of the in vitro cultures, microsatellites in the form of STMS (Sequenced Tagged Microsatellite Sites) markers are used for identification of genetic polymorphism. STMS markers are also used for assessment of genetic diversity within natural populations as well as for construction of genetic linkage maps. Isolation of microsatellites and development of STMS markers typically involves library construction and screening, DNA sequencing, polymerase chain reaction (PCR) primer design, and PCR optimization. This chapter details two approaches for the isolation of microsatellite loci. The first approach is based on PCR using microsatellite containing primers which also have degenerate bases at the 5¢-end that act as anchors preventing the primers from slippage to the 3¢-end and the subsequent loss of polymorphism. The multi-locus PCR amplified product is cloned and sequenced. Though this method generates a large number of microsatellites, the major drawback is the high redundancy observed in this method. The second approach described in this chapter is based on the construction of a microsatellite enriched library which involves preferential cloning of the microsatellite enriched fraction of genomic DNA. This method therefore necessitates the isolation of microsatellites through hybridization with biotin labeled oligoprobe followed by their capture with streptavidin-coated magnetic beads. In comparison to the first approach, this approach yields less redundant clones with high microsatellite enrichment. Moreover enriched libraries are 40–60 times more efficient than the conventional small insert genomic libraries.
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    Isolation and characterization of microsatellite markers for analysis of molecular variation in the medicinal plant Madagascar periwinkle (Catharanthus roseus (L.) G. Don.)
    (Elsevier B.V., 2007) Shokeen, Bhumika; Sethy, Niroj Kumar; Kumar, Sushil; Bhatia, Sabhyata
    Catharanthus roseus (L.) G. Don, commonly known as Madagascar periwinkle, is a plant of great medicinal value. In this study microsatellite markers were developed and utilized for analysis of genetic diversity in C. roseus. In order to isolate microsatellite sequences, two small insert genomic libraries of C. roseus cv. Nirmal were constructed and screened with CA, CT, GC and GCG oligonucleotide repeats. Sixty-five microsatellite motifs were identified, from which 38 functional STMS primer pairs were designed and validated. Out of these, 24 STMS markers were used to evaluate the genetic polymorphism in 37 genotypes, which comprised of 32 accessions of C. roseus, a single accession each of two related species (C. trichophyllus and C. pusillus) and one accession each of three related genera (Vinca minor, Thevetia peruviana and Nerium indicum). The 24 STMS markers detected 26 loci with two markers amplifying more than one locus. A total of 124 alleles were amplified in the 37 genotypes ranging from 2 to 10 alleles with an average of 4.76 alleles per locus. The high average expected heterozygosity (H) value of 0.56 and observed heterozygosity (H) value of 0.52 established the efficiency of the STMS markers for discriminating the C. roseus genotypes. Nei and Li’s similarity coefficients were calculated and a UPGMA-based dendrogram was constructed which clearly distinguished all genotypes except two pairs. Sequence analysis of the length variant alleles at three STMS loci revealed that the variation in the copy number of repeat motifs was the major source of length polymorphism within C. roseus. However, isolated point mutations and indels in the microsatellite flanking regions (MFRs) of homologus loci from other species also contributed to size homoplasy and allelic size variation. This study is the first report of microsatellite development and utilization in C. roseus, providing significant insights into its genome structure and organization.