Pak. J. Bot., 44(6): 2033-2040, 2012. ESTIMATION OF GENETIC VARIABILITY AMONG ELITE WHEAT GENOTYPES USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSIS SAJIDA BIBI 1 , IMTIAZ A. KHAN 1 , M. UMAR DAHOT 2 , ABDULLAH KHATRI 1 , M. H. NAQVI 1 , M. AQUIL SIDDIQUI 1 , SHAFQAT YASMEEN 1 AND NIGHAT SEEMA 1 1 Plant Breeding and Genetics Division, Nuclear Institute of Agriculture, Tando Jam, Pakistan 2 Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro, Pakistan. Abstract Twenty four wheat varieties/lines were assessed through RAPD for genetic diversity. Of forty primers, thirteen were able to amplify the genomic DNA and yielded 269 polymorphic bands. The percentage of the polymorphic loci was 86.22%. Nei’s genetic diversity (h) ranged from 0.248 to 0.393, with an average of 0.330. Shanon’s index (I) ranged from 0.382 to 0.567, with an average of 0.487. The proportion of genetic variation among the populations (Ds) accounted for 28.58 % of the whole gentic diversity. The level of gene flow (Nm) was 1.25. Some specific RAPD bands were also identified, variety C-591, and QM-4531 contain a specific segment of 4.9kbp. Whereas SARC-1 and PKV-1600 amplified a specific DNA segment with primer A-09. Marvi-2000 contains two specific segments of 3.2kb and 200bp amplified with primer B-07. Genetically most similar genotypes were C-591 and Pasban-90 (76%) and most dissimilar genotypes were Rawal-87 and Khirman (36.1%). On the basis of results, 24 wheat varieties under study could be divided into ‘two’ groups and five clusters ‘A’ to ‘E’. Introduction Every year a number of plant cultivars are released particularly for crops of world wide economic importance, such as hexaploid wheat (Triticum aestivum L.). Breeders usually select the new genotype mainly on the basis of morphological and biochemical markers. Morphological traits are the byproduct of gene and environmental interactions. The degree of gene expression is highly influenced by the conduciveness of the environment and genetic background in which gene is present. This difference is not actually by the genetic changes and breeder who is doing selection for such traits may do wrong selection on the basis of phenotypic traits (Kumar et al., 1998; Astarini et al., 2004). Due to lack of the information, breeders have been using genetically similar parents extensively in a breeding programme leading to a narrow genetic base (Fouilloux & Bannerot, 1988; Xia et al., 2004; Rehman et al, 2002). DNA marker is a tool to provide the exact information of the genotype. Molecular characterization of cultivars is also useful to evaluate potential genetic erosion (Manifesto et al., 2001). Thus, molecular markers generally detect more variability than other markers because they are not influenced by the environment. The most common molecular markers are restriction fragment length polymorphism (RFLP) and marker based on polymerase chain reaction such as random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). The genetic variability estimated through such markers distributed throughout the genome should (in principle) provide a mean to predict hybrid performance prior to making and evaluating the actual cross (Vienne et al., 1992). Molecular markers have been used to directly assess, the genetic diversity and/or genetic distance between parental genotypes of wheat (Sasanuma et al., 1996; Mayburg et al., 1997; Sun et al., 1998), Lens species (Ahmed & Mc Neil, 1996), Alium cepa (Le-Thierry et al., 1997) and other crops. RAPD is least complicated, most user-friendly, cost and time effective (Williams et al., 1990; Karp et al., 1997; Asif et al., 2005). Therefore, RAPD analysis was conducted to assess genetic diversity and genetic relationship among local collection of wheat genotypes for marker assisted breeding. Materials and Methods Plant material: Seeds of 24 wheat genotypes viz; C-591, Pasban-90, Barani-83, Punjab-81, LU-26-S, QM-4531, QM-4934, GA-2002, Bakhar-2002, 2K-CO-50, Chakwal- 97, Rawal-87, Sarsabz, Kiran-95, Marvi-2000, Bhitai, Khirman, NIA amber, Abadgar-93, Inqilab-91, SARC-1, PKV-1600, Chakwal-86 and CM24/87 were grown in bowls at control condition at NIA Tando Jam, and seedlings were harvested after 15 days. DNA extraction and amplification: DNA was extracted from fresh leaves of wheat genotypes using MATAB method (Bibi et al., 2010). The concentration of the extracted DNA was measured with spectrophotometer (BIOMATE 3) at absorbance 260nm and 280 nm. The quality of DNA was further checked on 0.8% agrose gel. Thirteen primers from Gene Link, each ten bases in length, were used to amplify the DNA (Table 2). PCR reaction was carried out in 25μl reaction mixture and amplification reaction was performed in the Eppendorf Master Cycler according to the method described by Bibi et al., 2010. Data analysis: Data was scored as presence of band as (1) and absence of bands as (0) on the basis of size in comparison with external standards using the UVitec Cambridge UK, gel documentation and analysis system with Band Map software for calculating the bands size from RAPD amplification profile. The following parameters were generated using the population genetic software package POPGENE: the observed number of alleles (Na), the effective number of alleles (Ne), Nei’s gene diversity (h) and Shannon’s index (Nei’s, 1987).