Characterization of Genetic Diversity of Rice Genotypes Showing Resistance to Rice Leaf Folder, Cnapholocrocis medinalis Using SSR Markers

Rice (Oryza sativa L.) is one of the most significant cereals and is the staple food for more than 2 billion people, mostly in developing countries. India leads the world in rice with 44.06 million hectares with a production of 105.32 million tonnes, but productivity is only 75 % of the world average of 4.02 tonnes ha -1 (Directorate of Rice Development, 2014). Insect pests are the major constraints in enhancing rice productivity which cause 20-30 % loss, besides diseases and weeds (Behura et al., 2011). Nearly 300 spp. of insect pests are attacking the paddy crop at various stages and among them only 23 species cause notable damage (Pasalu and Katti, 2006).


Introduction
Rice (Oryza sativa L.) is one of the most significant cereals and is the staple food for more than 2 billion people, mostly in developing countries. India leads the world in rice with 44.06 million hectares with a production of 105.32 million tonnes, but productivity is only 75 % of the world average of 4.02 tonnes ha -1 (Directorate of Rice Development, 2014). Insect pests are the major constraints in enhancing rice productivity which cause 20-30 % loss, besides diseases and weeds (Behura et al., 2011). Nearly 300 spp. of insect pests are attacking the paddy crop at various stages and among them only 23 species cause notable damage (Pasalu and Katti, 2006).

The
Rice leaffolder, Cnaphalocrocis medinalis earlier considered as a minor pest has gained the status of major pest with the wide spread of high yielding rice varieties, changes in the cultural practices, multiple cropping patterns, reduced genetic variability of high yielding rice varieties and application of high levels of nitrogenous fertilizers (Nanda et al., 2000). For managing the pest, host plant resistance is one of the components in Integrated Pest Management (IPM) in which identification of phenotypic, bio chemical and molecular markers are included. Among the different classes of molecular markers, SSR markers are often chosen as the preferred markers for a variety of applications in breeding because of their multi-allelic nature, co-dominant inheritance, relative abundance and extensive genome coverage (Gupta and Varshney, 2000). It is essential to know the genetic diversity at molecular level for developing a resistant variety among the available genotypes. Keeping these points in view, present study was undertaken with the objective of estimating genetic diversity in rice genotypes, which showed resistance to rice leaf folder in field level, using SSR markers.

Field screening of rice genotypes against Rice leaf folder
Fifty rice genotypes collected from different locations were raised under natural field conditions at ARS, Nellore during kharif 2009 and 2010. Two rows of 25 hills in each row were transplanted for each entry with two replications. TN1 was used as susceptible check. All the recommended agronomic practices except leaf folder protection measures were adopted during the experiment. Observations on leaf folder damage were recorded on five randomly selected plants in each genotype at 25, 40 and 70 DAT. Per cent damage was calculated by using the following formula. and Highly Susceptible ( >75% ) by following IRRI Standard Evaluation System (SES) for rice,1980. A total of twenty rice genotypes which exhibited resistant and moderately susceptible reaction against rice leaf folder were characterized using SSR markers.

DNA extraction and SSR marker analysis
The total DNA of each rice genotypes was extracted from leaves using the modified CTAB method (Murray and Thomson, 1980). Then the quality and quantity of DNA was verified by spectrophotometrically and also by using Nano drop procedure.
PCR was performed in 25 µl of reaction mixture using 2.5µl of 10X PCR reaction buffer, 2.5µl of 25 mM MgCl 2, 0.5µl of 10 mM dNTPs, 2 pmol of each forward and reverse primer, 0.2µl of 3U/µl of Taq DNA polymerase (Genei, Bangalore), 100-200ng of genomic DNA and 18.9µl sterile distilled water. The amplification was performed in a PCR machine (Eppendorf Programmable thermal controller, Hamburg, Germany). PCR amplifications profile was with initial denaturation of DNA at 94 0 C for 5min followed by 35 cycles of amplification at 94 0 C for 1 min with annealing temperatures of 54-60 0 C for 1min, extension step at 72 0 C for 2min and final extension was made at 72 0 C for 5min. Then the PCR product was finally kept at 4 0 C for electrophoresis. The annealing temperatures were optimized individually for each primer (Table 2). Before loading PCR Products in vertical gels, amplification was checked on 2.0% agarose gel. Based on agarose gel electrophoresis of amplification product, the separation of DNA fragments was carried out using 9% nondenaturing polyacrylamide gel electrophoresis (PAGE).
After PCR amplification, 3μl of bromo phenol blue dye was added to 25μl reaction mixture. Then 25μl of this reaction mixture was loaded by using eppendorf micropipette on each lane of 13-track gel of 9% non-denaturing PAGE and as the base pair marker, 50kb DNA ladder was loaded on one corner of the gel. Composition of 30ml of 9% PAGE gel was 16.5ml of distilled water, 4.5ml of 10X TBE, 9ml of acrylamide-methylbisacrylamide 29:2:0:8 (V/V), 40μl of TEMED and 180μl of 10% APS (ammonium per sulphate). Electrophoresis was done at 100 volts for 3-4 hours in 1X TBE running buffer. PCR products were visualized by using silver staining protocol (Kolodny, 1984).
The banding pattern was observed under white lamp trans-illuminator using gel documentation system (Alpha Innotech, USA) in an auto exposure mode and the gel picture was documented. Clearly resolved, un ambiguous bands were scored visually for their presence or absence with each primer. The scores were obtained in the form of matrix with '1' and '0', which indicates the presence and absence of bands in each variety respectively. The data matrix thus generated was used to calculate Jaccard's similarity coefficient for each pair-wise comparison. The coefficients were calculated Insilico following Jaccard (1908), using the following formula. a Similarity coefficient = ----n where, a = Number of matching bands for each pair of comparisons n = Total number of bands observed in two samples.
The similarity co-efficients were subjected to Unweighted Pair-Group Method of Arithmetical averages (UPGMA) cluster analysis to for grouping the genotypes based on their overall similarities. Statistical Package for Social Sciences (SPSS) package was used for the cluster analysis and subsequent dendrogram preparation.

Field evaluation of rice genotypes against rice leaf folder
The mean percent leaf damage (kharif 2009 and 2010) due to leaf folder in fifty rice genotypes ranged from 11.4 to 37.4. The lowest damage was recorded in Jhitpiti (11.4%) and highest damage was observed in NLR-145 (37.4%). Based on overall leaf folder percent damage during two years, the genotypes were categorized into various groups according to Standard Evaluation System for Rice given by IRRI, 1980. Out of 50 genotypes of rice, ten genotypes were found resistant, thirty were moderately resistant and ten genotypes showed moderately susceptible reaction against leaf folder. A total of twenty rice genotypes which exhibited extreme level resistant and moderately susceptible reaction against rice leaf folder were evaluated using SSR markers (Table 1).

Primer selection and survey
In the amplification of genomic DNA of the twenty selected rice genotypes using 11 primers of SSR markers (Table 3), ten primers given reproducible and scorable bands with high percentage of polymorphism. PCR amplification with these primers was done twice before scoring for presence or absence of bands.

Banding pattern
With PAGE gel analysis among 10 primers, seven primers were found to be polymorphic. A total of 123 amplified bands were seen with 70.8 % polymorphism. The number of amplified fragments ranged from 2 to 33 with an average of 8.72 per primer (Table 3). Herrera et al., (1999) used a set of 48 microsatellites to assess the genetic diversity of 11 Venezuelan rice cultivars and a total of 203 alleles were detected and the number of alleles per marker ranged from 2 to 9, with an average of 4.23.
The maximum polymorphism in PCR reaction was observed with the primers RM 3691, RM 600, RM 14 and RM 228 and these showed 100 % polymorphism as all the bands were polymorphic with size ranging from 100 bp to 1000 bp. The primers RM-495, RM 5380 and RM -6867 showed 0 % polymorphism as all were monomorphic. Selvaraju et al., (2007) reported that the primers RM 495, RM 600, RM 14 and RM 3691 were significantly associated and alleles amplified with rice leaf folder resistance.      Table 1 and M indicates the Marker -

Fig.3 Dendrogram depicting variation among rice genotypes based on SSR analysis
A clear variation in the banding pattern was observed among resistant and moderately susceptible genotypes with the primer RM 3691. In majority of genotypes, a maximum number of 33 amplified bands at 100 bp and at 250 bp size were present (Fig. 1). In the profile of RM 14, a clear variation in banding pattern was observed with 14 scorable polymorphic bands present at 200 bp and 450 bp size (Fig. 2). In the profile of remaining primers there was a clear variation among different genotypes. The similarity index values ranged from 0 to 89 % indicating the presence of wide range of genetic diversity at molecular level among the genotypes, tested for leaf folder resistance (Table 3).

Cluster analysis
Similarity coefficients were subjected to Unweighted Pair Group Method on Arthematic average (UPGMA) using paired matrix values and are presented in figure 3. The dendrogram exhibited variable degree of relationship among the genotypes studied and indicated two major clusters. The ten resistance genotypes and other ten moderately susceptible genotypes to rice leaf folder were grouped into two different clusters indicating that they are significantly different at molecular level and among cluster-I, INRC-3021 and W1263 were very closely related and in cluster-II, NLR 145 and Sri Satya were closely related with the similarity index of 86 and 89 %, respectively.
In conclusion, results revealed that 70.8 percent polymorphism existed among twenty genotypes using SSR markers indicated the presence of wide range of genetic diversity at molecular level. Dendogram clearly distinguished the resistant and moderately susceptible genotypes for rice leaf folder, Cnapholocrocis medinalis by clearly forming into two different clusters indicating that they are significantly different at molecular level and were correlated with the field performance.
In future, Correlation studies may be conducted among different genotypes with the data of parentage for drawing more conclusions.