بررسی فیلوژنی ژنوتیپهای نخود ایکاردا با تودة بومی بیونیچ (Bivanij) غرب ایران

Abstract

Introduction Despite the fact that Iran is one of the major chickpea (Cicer arietinum L.) center of origins, limited information is available regarding chickpea genetic variation and diversity. Genetic diversity information is crucial for the choice of proper parents to establish new breeding programs. Chickpea germplasm is poor, suggesting the need for gaining enough knowledge of genetic diversity among available chickpea genotypes. A number of molecular techniques have been developed to unveil the genetic potentials of plant materials. Random Amplified Polymorpic DNA (RAPD) seems to be a reliable molecular marker to investigate the genetic diversity of chickpea genotypes in IRAN. The objectives of present research was: (1) to investigate the power of RAPD markers for estimation of genetic diversity among chickpea genotypes in west of Iran, (2) to investigate the genetic relationships between chickpea genotypes, and (3) to determine whether chickpea genotypes could be distinguished by RAPD marker data. Materials and Methods Random amplified polymorphic DNA markers (RAPD) were used to assess the genetic relationship between 18 different chickpea genotypes representing the cultivated chickpea cultivars in west of Iran. Genomic DNA was isolated according to Murray & Thompson (1980). Eighteen oligonucleotide primers were selected according to the number of literature published with the highest number of polymorphic bands. Polymerase chain reaction (PCR) was carried out in a total volume of 25 μl including 2 units of Taq DNA polymerase, 30 ng of genomic DNA template, 10 pmol of primers, 0.2 mM of dNTPs, and 2.5 μl of 10 × reaction buffer. DNA amplifications were performed in a thermocycler. The thermal profile was as follow: One time denaturation at 94◦C (5 min), followed by 40 cycles of denaturation at 94◦C (3 min), annealing at each primer proper Tm (1 min) and extension at 72◦C (2 min) and one time final extension at 72◦C (5 min). PCR products were analyzed on 1.5 % agarose gels in TBE buffer running at 100 V for 2h. The gels were stained using ethidium bromide and visualized with UV light. The reproducibility of the DNA band patterns was evaluated duplicate gel electrophoresis analysis. Only clear and repeatedly amplified RAPD DNA bands were scored as (1) for present bands and (0) for absent ones. Results and Discussion Out of 18 random RAPD primers used in this study, 17 primers amplified genomic DNA across all the genotypes. In total, 201 polymorphic bands (96.63%) out of 208 reproducibly scoreable RAPD markers were generated (OPM-05 primer did not produce any band). On average, 12.2 bands per primer were observed in RAPD analysis. Cluster analysis using Dice coefficient of similarity and UPGMA (r=0.98) method based on polymorphic fragments, grouped all eighteen genotypes into 6 groups with 77% accuracy. Based on dendrogram obtained, Bivanij (Landrace genotype) showed the least similarity with the 6th cluster. Although there was no redundancy among the genotypes tested, the majority of genotypes were clustered together. ICARDA genotypes may have been improved from an Iranian landrace. Conclusions Genetic diversity among chickpea cultivars using RAPD markers have been studied by a number of researchers. Although in most cases a low level of polymorphism with RAPD markers have been reported, this study showed a considerable amount of polymorphism. Furthermore, our results showed that cultivated chickpea cultivars in west of Iran have many genes in common. We recommend further studies to be conducted by using more number of chickpea genotypes as well as more robust molecular markers. Results of this study can be used in germplasm management/conservation practices, developing core collections and as guidance to plant geneticist and breeders for planning future explorations, and crop improvement purposes. These findings may help to avoid genetic vulnerability and erosion, keeping chickpea genetic diversity and germplasm.