Plant materials and DNA extraction
In the present investigation, seeds material of twenty superior productive mutants (Table 1) along with their parent Sujata and OBGG-52, obtained from the harvested material of M
5 generation were taken for SSR analysis. Seedlings were grown in pots and fresh young leaf samples were collected from 10-20 days old seedlings before sunrise and washed thoroughly in distilled water and stored at -20°C for further analysis. Total genomic DNA from the leaves was isolated by using the standard CTAB (cetyl trimethyl ammonium bromide) DNA extraction method (
Doyle and Doyle, 1990).
Purification of DNA
For purification of DNA, extracted DNA was treated with RNAse-A at a concentration of 60 µg/ml of DNA and kept for incubation at 37°C for an hour with continuous shaking in the water bath. After one hour it was removed from the water bath and an equal volume of chloroform: isoamyl alcohol (24:l) was added and mixed thoroughly. The solution was then centrifuged at l0000 rpm for 10 minutes and the upper aqueous phase was pipetted out. Starting from the addition of chloroform-isoamyl alcohol, the entire process was repeated twice. For further purification, the DNA solution was washed with phenol:chloroform: isoamyl alcohol (25:24:l) and subsequently thrice with chloroform: isoamyl alcohol (24:l). The upper aqueous phase was separated after centrifugation as per the procedure described earlier and mixed with l/10
th volume of 3M sodium acetate (pH 4.8). DNA was precipitated by adding 2.5 volumes of chilled absolute ethanol and into a pellet by centrifugation at 12000 rpm. The pellet was washed twice with 70% ethanol carefully and dried under vacuum. DNA quality was confirmed by electrophoresis on 0.8% agarose gel and quantified by using a UV-vis spectrophotometer. After quantification, the DNA was diluted in TE buffer (pH 8.0) to a working concentration of 25 ng/µl for PCR analysis and stored at -20°C.
SSR primer amplification
For SSR analysis, PCR amplifications were carried out in a total volume of 20 ml containing 50 ng of template DNA, 1 unit of
Taq DNA polymerase, 0.25 mM each dNTP, 0.25 mM primer (each forward and reverse) in 1× PCR reaction buffer. Template DNA was initially pre-denaturated at 94°C for 3 min. followed by 35 cycles at 94°C for one min denaturation, primer annealing between 46-53°C (depending upon primer) for one min and extension at 72°C for one min. Finally, incubated at 72°C for ten minutes to complete the primer extension process. The obtained PCR products were used for electrophoresis on 1.5% agarose gels stained with ethidium bromide at 70 V for 3 hours and photographed under UV light using Gel Doc system (Genai image system, UVITech, UK). The gel doc photograph was used for documentation and scoring the bands.
Data analysis
The potential of the marker for estimating genetic variability and diversity was examined by assessing the marker informativeness through scoring the amplified bands by SSR -PCR amplification based on the presence (1) or absence (0) of bands for each primer. Primer banding characteristics such as the total number of bands, number of polymorphic bands and percentage of polymorphic bands were obtained. In order to analyze the suitability of SSR markers for a genetic profile evaluation, the performance of SSR markers used was measured by estimating the polymorphic information content (PIC), marker index (MI) and resolving power (Rp). The PIC which measures the informativeness of SSR markers was calculated for each SSR marker according to the formula:
PIC = 1 - nΣ pi²
Where,
n = Total number of alleles detected for a locus of a marker.
pi = Frequency of the i
th allele
(Smith et al., 1997).
Marker index (MI) is a statistical parameter used to estimate the total utility of the maker system. The MI was determined as the product of PIC value and EMR (
Prevost and Wilkinson, 1999). The effective multiplex ratio (EMR) of a primer is calculated as the total number of polymorphic loci multiplied by the proportion of polymorphic loci per their total number. Resolving power (Rp) is a parameter used to characterize the ability of the primer/marker combination to detect the differences between a large number of genotypes and was calculated as,
Rp = ΣIb
Where,
I
b = Amplicon informativeness and calculated by the formula.
Ib = 1 - [2 × (0.5 - p)]
Where,
p = Proportion of genotypes containing the band.
The binary data matrix was converted into a genetic similarity coefficient between pairs of accessions using the Jaccard’s similarity coefficient by NTSYS-pc (Numerical taxonomy system, version 2.2 (
Rohlf, 2005). The SIMQUAL Programme was used to calculate the Jaccard’s similarity coefficient, a common estimator of genetic identity and was calculated as follows (
Jaccard, 1908).
Where,
N
AB = Number of bands shared by samples.
N
A and N
B = Amplified fragments in sample A and B respectively.
The matrix of Jaccard’s similarity coefficients was subjected to the unweighted pair group method with arithmetic averages (UPGMA) to generate a dendrogram for cluster analysis which depicting the similarity and diversity among genotypes (
Sokal and Michener, 1958;
Sneath and Sokal, 1973).
