Analysis of diversity in rice (Oryza sativa L.) using random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers

Molecular markers are useful tool for assessing genetic variations and resolving genotype identity. In the current study, genetic diversity among 20 rice genotypes was assessed using the random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR). In RAPD analysis, 20 primers generated a total of 116 bands of which 114 were polymorphic. The number of amplification products produced by each primer varied from 4 to 7 with an average of 5.8 bands per primer. Twenty (20) SSR primers generated a total of 65 alleles with an average 3.2 alleles per primer. Genetic diversity of 20 genotypes estimated by polymorphic information content (PIC) value ranged from 0.62 to 0.97 in SSR and 0.33 to 0.88 with RAPD analysis. The cluster dendrogram by SSR revealed two major clusters. Rajeshwari was the only genotype in cluster I. The cluster II further divided into two sub clusters IIA and IIB. II A consisted of 17 genotypes while II B consisted of two genotypes (Apo and Kalakeni). The information generated from this study can be used to maximize selection of diverse parents and broaden the germplasm base for the future rice breeding programs.


INTRODUCTION
Rice (Oryza spp.) is one of the most important food crops in the world, being planted on almost 11% of the Earth's cultivated land area over a wide number of ecosystems (Cuevas and Fitzgerald, 2012). Rice belongs to the genus Oryza and has two cultivated and 22 wild species.
Rice is an ideal model plant for the study of grass genetics and genome organization due to its diploid genetics, relatively small genome size of 430 Mb (Causse et al., 1994), significant level of genetic polymorphism (McCouch et al., 1998), large amount of well conserved genetically diverse material (about 100,000 accessions world wide) and the availability of widely collected compatible wild species. The success of breeding program also depends upon the amount of genetic variability present in the population and extent to which the desirable traits are heritable. The assessment of phenotype may not be a reliable measure of genetic differences. The rapid development of biotechnology allows easy analysis of a large number of loci distributed throughout the genome of plants. Molecular markers have proven to be powerful tool in the assessment of genetic variation and in the elucidation of genetic relationships within and among species (Matin et al., 2012). Molecular markers originate from different parts of the genome including coding and non-coding regions and can cover either the full genome or large genomic segments. Morphological traits are controlled by a relatively small number of loci (Onaga et al., 2013) however; they serve as a valuable guide for effective collection and use of genetic resources. Molecular markers (RAPD) without prior knowledge of DNA sequences are especially useful for unzipping the variations in species with low genetic variability. RAPD markers are considered to be unbiased and neutral markers for genetic mapping applications, in population genetics, taxonomy as well as for genetic diagnostics. Microsatellites are the popular molecular markers in rice for various applications in genetics and breeding. SSR markers are important tool for estimation of genetic variation and identification of germplasm. These markers have some merits like quickness, simplicity, rich polymorphism and stability, thus being widely used in molecular map construction and gene mapping, construction of fingerprints and genetic purity test (Ma et al., 2011), analysis of germ plasm diversity (Jin et al., 2010 ), utilization of heterosis, especially in identification of species with closer genetic relationship. SSR markers are more popular in rice because they are highly informative, mostly monolocus, co dominant, easily analyzed and cost effective (Prabakaran et al., 2010). Using PCR rapid amplification and gel electrophoresis of high resolving power, we can test SSR length polymorphism rapidly and economically. In the current studies, RAPD, SSR and combined analysis was performed to assess the genetic diversity among 20 genotypes of rice.

Plant material
Leaf samples of 20 rice genotypes (25 days after transplanting.) were collected from the Genetics Division, Indian Agriculture research institute, Delhi, India from wet season 2010. The details of rice genotypes are presented in Table 1.

DNA extraction
Total DNA was extracted from fresh leaves by the cetyl tri-methyl ammonium bromide (CTAB) method (Murray and Thompson, 1980). The quality and concentration of extracted DNA were estimated by using a UV-Vis spectrophotometer. The DNA was spooled out, washed twice with 70% ethanol and dissolved in TE (10 mM Tris, 0.1 mM EDTA, pH 8.0) containing 25 μg/ml RNase-A, incubated at 37°C for 30 min and extracted with chloroform:isoamyl alcohol (24:1 v/v). DNA was re-precipitated and dissolved in TE buffer. DNA was checked for its quality and quantity by 0.8% agarose gel electrophoresis.

PCR analysis and gel electrophoresis
In case of SSR, a set of 20 primers (Chakravarthi et al., 2006) were used. The details of primers used for SSR analysis are presented in Table 2. The PCR reaction was carried out using Taq polymerase (Genei) in 20 ml reaction volume containing 1.5X PCR buffer, 2 mM MgCl 2 , 0.02 mM of each dNTPs, 1 mM of forward and reverse primers, 0.5 µl (3 unit) Taq polymerase and 50 ng genomic DNA. Profile used was as follows: an initial hot start and denaturing step at 95°C for 5 min followed by 35 cycles at 94°C for 1 min, appropriate annealing temperature 55°C for 1 min, and primer elongation at 72°C for 2 min. Final extension step at 72°C for 7 min was performed.
RAPD analysis was performed according to Williams et al. (1990) with minor modifications. The primers used for RAPD are given in Table 3. PCR reactions were carried out in 20 μl volume containing 50 ng of total genomic DNA, 10 pmol primer, 200 μM dNTPs, 2 mM MgCl2, 2.5X PCR buffer and 0.4 μl ( 3 units) AmpliTaq Polymerase. Twenty (20) 10-mer oligonucleotide random primers were selected for analysis. These primers were obtained from Banglore Genei, India. Amplification was performed in a Astec thermal cycler with the following profile: 94°C for 4 min (initial denaturation), 94°C for 1 min, 32°C for 1 min, 72°C for 2 min for 42 cycles with a final extension at 72°C for 7 min.
The RAPD-PCR products were analyzed directly on 1.5% agarose gels in TAE buffer while SSR-PCR products were analyzed on 4% agarose gel, visualized by staining with ethidium bromide under short-wave UV light. DNA ladder used in the electrophoresis was of 100 bp. Agrose gel electrophoresis for PCR products of RM-222, SSR marker and OPA-07, RAPD marker is shown in Figure 4 and 5 respectively.

Data analysis
Pair wise comparison of genotypes, based on the presence (1) or absence (0) of unique and shared polymorphic products was used to generate similarity coefficients of Jaccord's coefficient by NTSYS-pc version 2.1 software (Rohlf, 2000). The similarity coefficient was used to construct a dendrogram by the unweighted pair group method with arithmetic averages (UPGMA) according to Rohlf (1993). The polymorphism information content (PIC) value described by Botstein et al. (1980) and modified by Anderson et al. (1993) for self-pollinated species was calculated as follows: Where, p i equals to the frequency of the ith allele and p j the frequency of the allele. Only data from polymorphic loci were used for analysis.

SSR analysis
In present study, 20 SSR primers distributed from chromosome 7 to 12 were used to estimate genetic diversity among 20 genotypes. A total of 65 alleles were detected among all genotypes. The number of alleles per locus varied from 2 to 7. The mean allele in current studies (3.25 alleles) was comparable with the results of Etemad et al. (2012) as 3.57 in rice. However, it was reported lower by Prathepha et al. (2012) and Rahman et al. (2012) with an average of 11.85 and 4.18 alleles per locus, respectively. The overall size of amplified products ranged from 100 (RM264) to 250 bp (RM286). In the current studies, 15 out of 20 SSR primer pairs generated polymorphic bands. PIC values for SSR ranged from 0.62 to 0.97 with mean value of 0.81 (Table 4) Figure 1). The dendrogram revealed two distinct clusters at a similarity coefficient level of0.63. Cluster II was the largest and included 19 genotypes while, clusters I comprised only one genotype, that is, Rajeshwari. The cluster II further divided into two sub clusters IIA and IIB. II A consisted of 17 genotypes while II B consisted of only two genotypes (Apo and Kalakeni). Apo and

RAPD analysis
Using 20 RAPD primers, a total of 116 alleles were detected among the 20 rice genotypes. The number of alleles per locus varied from 4 to 7. The average number of alleles per locus was 5.8. The overall size of amplified products ranged from 100 (OPA-10) to 1200 bp (OPA-11). All the RAPD primers used for analysis of genetic diversity and relationship generated polymorphic bands among the genotypes. The PIC value for the RAPD ranged from 0.33 to 0.88 with an average of 0.65 (Table  5). Similar results were also reported by Shiva et.al.  The highest PIC value (0.88) was observed for primers OPA-17. Cluster analysis revealed two major clusters at a cut-off similarity coefficient of 0.57 (Figure 2). Cluster I was largest and included 14 genotypes while cluster II consisted of six genotypes. Jaccard's coefficient of similarity revealed that 83.8% exist between genotypes Shravani and Abhishek. Whereas, Apo keeps very low level of similarity with Rajeshwari at similarity coefficient of 0.57%. The major cluster I further divided into two sub clusters IA and IB. The sub cluster IA consist of 10 genotypes namely Khitish, Naveen, Udaya, Sukhasamat, Shravani, Abhishek, Sambhamasuri, Dandi,Samanta and Rajeshwari with the similarity coefficient ranged between 0.62 to 0.83. While sub cluster IB consist of 4 genotypes namely Swarna, IR-64, Safri and Lolut with similarity coefficient from 0.67 to 0.78.

Combined analysis
The cluster dendrogram with combined analysis revealed two major clusters. Cluster I was the largest and included 14 genotypes, namely Swarna. IR64, Lolat, Safri, Suskhasamanat, Khitis, Naveen, Sambhamasuri, Dandi, Samanta, Rajaewari, Shravani, Abhishek and Udaya with the similarity   Rajeshwari, Udaya and Apo belong to different clusters and are genetically diverse. SSR markers showed higher PIC value compared to RAPD which indicates that SSR markers are highly informative and are reliable. In the current study, larger range of distinct values for genotypes revealed by microsatellite markers provides greater confidence for the assessments of genetic diversity and relationships, which can be used in future breeding programs. With the aid of microsatellite makers and clustering data, different distantly related rice genotypes may be combined by intercrossing genotypes, for instance, aromatic rice genotypes with non-aromatic rice genotypes from different clusters to get hybrid varieties with highest heterosis (Sajib et al., 2012). Markers with PIC values of 0.5 or higher are highly informative for genetic diversity studies and can be successfully used to distinguish the polymorphism at a specific locus. The joint use of primer is excellent way of identification of genotypes. A combination of RAPD and SSR help to provide whole genome coverage and   Table 1). M=DNA ladder (100 bp).