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Item 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, SabhyataBACKGROUND 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.Item Advancing the STMS genomic resources for defining new locations on the intraspecific genetic linkage map of chickpea (Cicer arietinum L.)(BioMed Central, 2011) Gaur, Rashmi; Sethy, Niroj K; Choudhary, Shalu; Shokeen, Bhumika; Gupta, Varsha; Bhatia, SabhyataChickpea (Cicer arietinum L.) is an economically important cool season grain legume crop that is valued for its nutritive seeds having high protein content. However, several biotic and abiotic stresses and the low genetic variability in the chickpea genome have continuously hindered the chickpea molecular breeding programs. STMS (Sequence Tagged Microsatellite Sites) markers which are preferred for the construction of saturated linkage maps in several crop species, have also emerged as the most efficient and reliable source for detecting allelic diversity in chickpea. However, the number of STMS markers reported in chickpea is still limited and moreover exhibit low rates of both inter and intraspecific polymorphism, thereby limiting the positions of the SSR markers especially on the intraspecific linkage maps of chickpea. Hence, this study was undertaken with the aim of developing additional STMS markers and utilizing them for advancing the genetic linkage map of chickpea which would have applications in QTL identification, MAS and for de novo assembly of high throughput whole genome sequence data.Item Development of chickpea EST-SSR markers and analysis of allelic variation across related species(Springer, 2009) Choudhary, Shalu; Sethy, Niroj Kumar; Shokeen, Bhumika; Bhatia, SabhyataDespite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea.Item Isolation of microsatellites from Catharanthus roseus (L.) G. Don using enriched libraries(Springer, 2009) Bhatia, Sabhyata; Shokeen, BhumikaCatharanthus 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.Item 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, SabhyataCatharanthus 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.Item Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis(Springer, 2006) Sethy, Niroj Kumar; Choudhary, Shalu; Shokeen, Bhumika; Bhatia, SabhyataMicrosatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)- based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.Item Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)(Springer, 2006) Sethy, Niroj Kumar; Shokeen, Bhumika; Edwards, Keith J.; Bhatia, SabhyataPaucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT)n and (CT/GA)n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers devel- oped in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.Item Development of sequence-tagged microsatellite site markers for chickpea (Cicer arietinum L.)(Wiley-Blackwell, 2006) Choudhary, Shalu; Sethy, Niroj K.; Shokeen, Bhumika; Bhatia, SabhyataMicrosatellite 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.Item 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, SabhyataWe 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.Item Isolation and characterization of sequence-tagged microsatellite sites markers in chickpea (Cicer arietinum L.)(Wiley-Blackwell, 2003) Sethy, Niroj K.; Shokeen, Bhumika; Bhatia, SabhyataIn this study we report the isolation of microsatellite sequences and their conversion to sequence-tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.