Determination the level of genetic diversity in the rye germplasm using of ISSR and AFLP markers

Background and Aim: Rye by the scientific name Secale is a plant from the Poaceae family. It is one of the most important crops of Iran and is the most important cereal crop. In this study, the amount of variation in rye germplasm was evaluated using ISSR and AFLP markers. A population of ۳۹ rye samples from different regions of the country, the US and the USSR was selected and evaluated by ۸ ISSR primers and ۱۳ pairs of AFLP primers. Of the ۸ ISSR primers, ۵ were polymorphic and ۳ were monomorphic, with a total of ۱۹ polymorphic bands. The highest PIC values were for the ۵ + ۶ primer with ۰.۳ and the highest MI for the ۱ + ۶ marker with ۰.۹۶. These ۳۹ samples were also analyzed with ۱۳ pairs of AFLP primers.The total detectable bands were ۱۸۸ bands, of which ۱۷۷ were polymorphic. The highest PIC value was for ECC ۳N ۴S ۱ primer with ۰.۳۵ and the highest MI value for ECC ۳N ۴S ۵ primer with ۶.۶. Cluster analysis of molecular traits was performed based on Jaccard similarity coefficient using UPGMA for ISSR marker and Dice similarity coefficient with UPGMA for AFLP marker. The coefficient of similarity for ISSR marker data is ۶۰% and for AFLP marker is ۷۷%. According to the results of this study, it can be concluded that AFLP marker is more efficient than ISSR for detecting diversity within species of rye.Methods: In this experiment, ۳۹ rye populations were collected from different parts of the country and planted in a completely randomized design with three replications.Genetic material was extracted using CTAB method (Rovers & Bendich, ۱۹۸۵). The quality and quantity of extracted DNA were measured using agarose gel electrophoresis and nano-drop spectrophotometer.The extracted DNA was diluted to reach ۱۵ ng / μg.۹ primers were used in the PCR reaction.PCR amplification was planned by performing ۴۰ cycles after an initial cascade cycle. The initial flushing cycle was performed at ۹۴ ° C for ۳ min. Each cycle of the main cycle consists of a thawing step for ۳۰ seconds (۹۴ ° C), a slow cooling step at the primer binding temperature for ۱ minute (۵۲ to ۵۸ ° C) and a ۷۲ ° elongation step. Celsius was for ۱ minute. The final primer extension step was performed for ۷ min at ۷۲ ° C in a final cycle.The AFLP procedure was slightly modified according to the method of Wuce (۱۹۹۵) .۲۰۰ ng of genomic DNA with ۵ units of EcoR , Taq restriction enzymes for ۳ h at ۳۷ ° C, ۱ h at ۶۵ ° C and ۲۴ h at complementary stage. The ۲۵ cuttings were then attached to the end of the cuttings for ۴ hours at ۲۵ ° C and ۲۴ hours at ۳۷ ° C.Duplication of adapter.Results: When the first two or three components account for about ۱۰-۲۰% of the marker-related variations, genetically expressed markers are favorably sampled from the whole genome. Comparing the results of the principal components analysis revealed that the AFLP marker performed better than the ISSR marker and the AFLP primers had a good distribution across the rye plant genome. Also their grouping results were in perfect agreement with the cluster analysis grouping.Inter- and intra-species diversity was calculated with ISSR and AFLP primers using GenAlex software. Molecular variance analysis was used to calculate it. The results of this analysis showed that in both ISSR and AFLP markers the intra-species diversity was higher than the inter-species diversity and ۹۷% of the diversity was related to intra-species diversity and ۳%. There was no difference between the results of molecular variance analysis of the two markers.Conclusion: Overall, the AFLP marker was better able to group the species in terms of genetic similarity

Keywords:

Genetic diversity , rye , molecular marker , ISSR , AFLP