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    Development of SSR and gene-targeted markers for construction of a framework linkage map of Catharanthus roseus
    (Oxford University Press, 2011) Shokeen, Bhumika; Choudhary, Shalu; Sethy, Niroj Kumar; Bhatia, Sabhyata
    BACKGROUND AND AIMS: Catharanthus roseus is a plant of great medicinal importance, yet inadequate knowledge of its genome structure and the unavailability of genomic resources have been major impediments in the development of improved varieties. The aims of this study were to develop co-dominant sequence-tagged microsatellite sites (STMS) and gene-targeted markers (GTMs) and utilize them for the construction of a framework intraspecific linkage map of C. roseus.
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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.
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    Development of sequence-tagged microsatellite site markers for chickpea (Cicer arietinum L.)
    (Wiley-Blackwell, 2006) Choudhary, Shalu; Sethy, Niroj K.; Shokeen, Bhumika; Bhatia, Sabhyata
    Microsatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence-tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)-amplified fragments. Thirty-five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross-species transferability of the sequence-tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra- and interspecific genetic linkage maps.
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    Development of STMS markers from the medicinal plant Madagascar periwinkle (Catharanthus roseus L.) G. Don
    (Wiley-Blackwell, 2005) Shokeen, Bhumika; Sethy, Niroj K.; Choudhary, Shalu; Bhatia, Sabhyata
    We report the isolation and characterization of the first set of sequence-tagged microsatellites sites (STMS) markers in Catharanthus roseus, a plant with a vast range of medicinal uses. The microsatellite loci were cloned from an enriched library constructed using degenerate primers. Based on the microsatellite motifs, seven STMS primer pairs were designed. They were used to amplify 32 accessions of C. roseus and one accession of Catharanthus trichophyllus. The primers amplified an average of 3.86 alleles per locus. The observed heterozygosity ranged from 0.2903 to 0.9688 with an average of 0.7511. The STMS markers of C. roseus also amplified corresponding loci in a related species (C. trichophyllus) suggesting conservation of the loci across the genus. These markers will prove useful for genetic diversity analysis and linkage map construction in C. roseus.