Rakesh Singh

Comparison of SSR and SNP markers in estimation of genetic diversity and population structure of Indian rice varieties.

Simple sequence repeat (SSR) and Single Nucleotide Polymorphic (SNP), the two most robust markers for identifying rice varieties were compared for assessment of genetic diversity and population structure. Total 375 varieties of rice from various regions of India archived at the Indian National GeneBank, NBPGR, New Delhi, were analyzed using thirty six genetic markers, each of hypervariable SSR (HvSSR) and SNP which were distributed across 12 rice chromosomes. A total of 80 alleles were amplified with the SSR markers with an average of 2.22 alleles per locus whereas, 72 alleles were amplified with SNP markers. Polymorphic information content (PIC) values for HvSSR ranged from 0.04 to 0.5 with an average of 0.25. In the case of SNP markers, PIC values ranged from 0.03 to 0.37 with an average of 0.23. Genetic relatedness among the varieties was studied; utilizing an unrooted tree all the genotypes were grouped into three major clusters with both SSR and SNP markers. Analysis of molecular variance (AMOVA) indicated that maximum diversity was partitioned between and within individual level but not between populations. Principal coordinate analysis (PCoA) with SSR markers showed that genotypes were uniformly distributed across the two axes with 13.33% of cumulative variation whereas, in case of SNP markers varieties were grouped into three broad groups across two axes with 45.20% of cumulative variation. Population structure were tested using K values from 1 to 20, but there was no clear population structure, therefore Ln(PD) derived Δk was plotted against the K to determine the number of populations. In case of SSR maximum Δk was at K=5 whereas, in case of SNP maximum Δk was found at K=15, suggesting that resolution of population was higher with SNP markers, but SSR were more efficient for diversity analysis.

Genetic and biochemical stability assessment of plants regenerated from cryopreserved shoot tips of a commercially valuable medicinal herb Bacopa monnieri (L.) Wettst

Shoot tips from four accessions (IC249250, IC 426442, IC 375976, and IC468878) of Bacopa monnieri (L.) Wettst., a commercially valuable memory revitalizing medicinal plant, were cryopreserved using a vitrification technique. Depending on the genotype, 0 to 20% plant regeneration without intermediary callus was achieved from cryopreserved shoot tips. Genetic stability of plants derived from cryopreserved shoot tips was assessed using biochemical and molecular markers. The regenerated plants from non-frozen controls and cryopreserved shoot tips exhibited morphological similarity to respective parental material when transferred to soil. On the basis of ten random amplified polymorphic DNA (RAPD) and bacoside A content using HPLC analysis, no significant reproducible variation was observed between the controls and in vitro-cryopreserved plants. Thus, after cryopreservation treatment, the regenerated plants exhibited molecular and biochemical genetic stability.

In Vitro Propagation and Conservation of Bacopa monnieri L.

Bacopa monnieri L. (common name brahmi) is a traditional and renowned Indian medicinal plant with high commercial value for its memory revitalizer potential. Demand for this herb has further escalated due to popularization of various brahmi-based drugs coupled with reported anticancer property. Insufficient seed availability and problems associated with seed propagation including short seed viability are the major constraints of seed conservation in the gene banks. In vitro clonal propagation, a prerequisite for in vitro conservation by enhanced axillary branching was standardized. We have developed a simple, single step protocol for in vitro establishment, propagation and medium-term conservation of B. monnieri. Single node explants, cultured on Murashige and Skoogs medium supplemented with BA (0.2 mg/L), exhibited shoot proliferation without callus formation. Rooting was achieved on the same medium. The in vitro raised plants were successfully transferred to soil with ~80 % survival. On the same medium, shoots could also be conserved for 12 months with high survival and genetic stability was maintained as revealed by molecular markers. The protocol optimized in the present study has been applied for culture establishment, shoot multiplication and medium-term conservation of several Bacopa germplasm, procured from different agro-ecological regions of India.

Identification, analysis and development of salt responsive candidate gene based SSR markers in wheat

BackgroundSalinity severely limits wheat production in many parts of the world. Development of salt tolerant varieties represents the most practical option for enhancing wheat production from these areas. Application of marker assisted selection may assist in fast tracking development of salt tolerant wheat varieties. However, SSR markers available in the public domain are not specifically targeted to functional regions of wheat genome, therefore large numbers of these need to be analysed for identification of markers associated with traits of interest. With the availability of a fully annotated wheat genome assembly, it is possible to develop SSR markers specifically targeted to genic regions. We performed extensive analysis to identify candidate gene based SSRs and assessed their utility in characterizing molecular diversity in a panel of wheat genotypes.ResultsOur analysis revealed, 161 SSR motifs in 94 salt tolerance candidate genes of wheat. These SSR motifs were nearly equally distributed on the three wheat sub-genomes; 29.8% in A, 35.7% in B and 34.4% in D sub-genome. The maximum number of SSR motifs was present in exons (31.1%) followed by promoters (29.8%), 5’UTRs (21.1%), introns (14.3%) and 3’UTRs (3.7%). Out of the 65 candidate gene based SSR markers selected for validation, 30 were found polymorphic based on initial screening and employed for characterizing genetic diversity in a panel of wheat genotypes including salt tolerant and susceptible lines. These markers generated an average of 2.83 alleles/locus. Phylogenetic analysis revealed four clusters. Salt susceptible genotypes were mainly represented in clusters I and III, whereas high and moderate salt tolerant genotypes were distributed in the remaining two clusters. Population structure analysis revealed two sub-populations, sub-population 1 contained the majority of salt tolerant whereas sub-population 2 contained majority of susceptible genotypes. Moreover, we observed reasonably higher transferability of SSR markers to related wheat species.ConclusionWe have developed salt responsive gene based SSRs in wheat for the first time. These were highly useful in unravelling functional diversity among wheat genotypes with varying responses to salt stress. The identified gene based SSR markers will be valuable genomic resources for genetic/association mapping of salinity tolerance in wheat.

Genetic diversity and population structure analysis of Kala bhat ( Glycine max (L.) Merrill) genotypes using SSR markers

BackgroundKala bhat (Black soybean) is an important legume crop in Uttarakhand state, India, due to its nutritional and medicinal properties. In the current study, the genetic variabilities present in Kala bhat were estimated using SSR markers and its variability was compared with other improved soybean varieties cultivated in Uttarakhand state, India.ResultsSeventy-five genotypes cultivated in different districts of Uttarakhand were collected, and molecular analysis was done using 21 SSR markers. A total of 60 alleles were amplified with an average of 2.85 alleles per locus. The mean value of gene diversity and PIC was estimated to be 0.43 and 0.36, respectively. The unrooted phylogenetic tree grouped soybean genotypes into three major clusters, where, yellow seed coat (improved varieties) genotypes were grouped in one cluster, while reddish brown (improved varieties) and Kala bhat showed intermixing. Population structure divided the soybean genotypes into six different populations. AMOVA analysis showed 12% variance among the population, 66% variance among individual and 22% variance was observed within individuals. Principal Coordinate Analysis (PCoA) also showed that yellow seed coat genotypes were grouped in one cluster, whereas, the Kala bhat showed scattered distribution and few genotypes of Kala bhat showed grouping with red and yellow genotypes.ConclusionsThe different genetic diversity parameters used in the present study indicate that Kala bhat genotypes were more diverse than the yellow seed coat and brown seed coat colour genotypes. Therefore, Kala bhat genotypes can be a good source for the soybean breeding programme due to its better genetic diversity as well as its medicinal properties.