EFFICIENCY OF RAPD AND ISSR MARKERS FOR GENOTYPE FINGERPRINTING AND GENETIC DIVERSITY STUDIES IN CANOLA (Brassica napus)

Abstract

Agreat deal of research has been focused on oil crops of various plants, especially members of the mustard family (Brassicaceae) such as species of Brassica. Canola (rapeseed; Brassica napus L. genome AACC, 2n=38) arises from spontaneous hybridization between turnip (Brassica rapa) (AA, 2n= 20) and cabbage (Brassica oleracea) (CC, 2n=18). It is now the second largest oilseed crop over the world after soybean (Glycine max) providing 13% of the world supplies (Abbas et al., 2009). Canola is primarily used for food and feed, but has recently gained an increasing interest as a source for bio-products (e.g., biodiesel). Besides that, the Food and Drug Administration (FDA) approved canola oil with a Qualified Health Claim (QHC) due to its ability to reduce the risk of coronary heart disease (Miller-Cebert et al., 2009).
Like any other crop species, to improve quality and quantity of Brassica spp., presence of sufficient genetic diver- sity is very important. In the breeding process, significant improvement of quality and production was achieved, as well as utilization of rapeseed oil in human nutrition. However, genetic variability in this important crop is restricted with regard to many characters of value for breeding process (Marjanovic-jeromela et al., 2009). The success in breeding programs of a crop species largely relies on the presence of sufficient genetic diversity in the germplasm and knowledge about the characteristics of the genotypes and their genetic relationship. Various methods have been elaborated for this purpose. Pedigree analysis is the most widely used method for estimating the degree of similarity between varieties or populations, but the necessary information on ancestry is not always accurate or available. Application of morphological traits is hindered by their limited number and by the modifying effect of environmental factors in some cases. The spread of DNA markers has allowed the genome to be analyzed directly, thus eliminating errors caused by environmental factors. By using these markers, the genome can be characterized with great accuracy. In addition to the estimation of degrees of relationship between different varieties, a further important use of these markers is to distinguish between genotypes. Numerous molecular markers have been used for variety identification in various plant species, which allow cultivar identification in early stages of plant development, being neutral to environmental effects (Mohammadi, 2002; Meszaros et al., 2007; Moghaddam et al., 2009). A variety of molecular markers including Restriction Fragment Length Polymorphism (RFLP) (Thormann et al., 1994), Inter-Simple Sequence Repeats (ISSR) (Carolyn et al., 2000; Rudolph et al., 2002), amplified fragment length polymorphism (AFLP) (Sandip et al., 1999; Seyis et al., 2003; Jiang et al., 2007) and random amplified polymorphic DNA (RAPD) (Ashik Rabbani et al., 1998; Lazaro and Aguinagalde, 1998; Divaret et al., 1999), have been used to study the extent of genetic variation among the diverse group of important crop species in the genus Brassica (Afiah et al., 2007; Marjanovic-jeromela et al., 2009).
In this study, RAPD and ISSR markers based on the polymerase chain reaction (PCR) were applied. The value of RAPD analysis for efficient germplasm management in plants is already known (Young, 2000; Jaroslava et al., 2002). The technique is quick, easy and requires less time. This detects nucleotide sequence polymorphisms using a single primer of arbitrary nucleotide sequence (Welsh and McClelland, 1990; Williams et al., 1990). ISSR permits detection of polymorphisms in inter-microsatellite loci, using a primer designed from dinucleotide or trinucleotide simple sequence repeats.
Only a few papers comparing re- sults obtained by different molecular genetic methods have been published in the case of Brassica napus L. The capability of individual methods to differentiate the analyzed canola genotypes is described here. The present study was therefore undertaken (1) to determine the efficiency of RAPD and ISSR markers for estimating the genetic diversity and (2) to estimate the genetic diversity of canola genotypes based on molecular characterization. For this purpose, 10 canola (Brassica napus L.) genotypes were analyzed and the results of genetic distances estimated by ISSR and RAPD markers were compared.