Genetic Diversity of Superior Walnut Genotypes Revealed by ISSR Markers in Shahrood, Iran

Iran is known as the origin of Persian walnut in the world, and so the study of genetic diversity, identifying and introducing superior walnut genotypes from the main walnut production areas in Iran is very important. From the beginning of the year 2001, seeds of selected walnut genotypes from important walnut production area of Iran were collected and cultivated in a walnut collection in Semnan Research Center (Shahrood), Iran. From this collection, promising walnut genotypes were selected based on climate adaptability to Shahrood area and pomological characteristics. In this research, in addition to introducing fruit characters of these walnut selected genotypes, genetic diversity and their genotypes relationship in the genome level have been also investigated with the ISSR markers. 40 selected genotypes of important walnut regions of Iran (Tuyserkan, Orumieh, Karaj and Khorasan province) were cultivated in a walnut collection at the Shahrood Research Center in 2001. From these seedlings, 21 genotypes were selected on the base of pomological characteristics and they were grafted onto Persian walnut seedlings. Pomological traits of the fruits of these selected genotypes including the average of nut weight, kernel weight, kernel percentage, shell attachment to the kernel, kernel color, shell thickness and nut size and shape were measured. DNA was extracted from young leaves of walnut genotype by CTAB method and its quality and quantity evaluated on the agarose gel as well as the Nano drop, and then genomic DNA was amplified with the 10 ISSR primers in PCR. The amplified bands were separated by Metaphor agarose gel and stained with Ethidium bromide. For each primer, the amplified band range, the total number of bands, the number of polymorphic bands, the polymorphic percentage, Average informative band (AvIb) and resolving power (Rp) was determined. UPGMA dendrogram based on Jaccard similarity matrix was the performance by Ntsys 2.0 software. Principle Coordinate analysis was performed based on the genetic distance matrix with GenAlex 6.2 software. The mean of fruit and kernel weight in selected walnut genotypes were 14.34 and 7.33 g, respectively. The highest nut and kernel weight was found to be 17.5 and 10.3 g, respectively in OR23. The Kernel weight was more than kernel weight reported by Shamlu et al. (2015), Yarilgak et al. (2001) and Tasmuris et al. (2002) (9.40, 8.88 and 6.32 g). The average of kernel percentage in selected walnuts was 51.5%. The highest percentage of the kernel (62.7%) was observed in T12 genotype. from the ten ISSR primers in 21 walnut genotypes, 112 DNA fragments were amplified and 102 DNA bonds of them were polymorphic and they were used for genetic variation analysis. The highest number of amplified DNA bands was observed in UBC826 and UBC888 primers with 14 bands. The highest average band informative (AvIb), 0.62, was observed in UBC887 and UBC886 primers. The highest resolving power (Rp) was found to be 7.71 in UBC826 primer and then in UBC.887 primer. The Jaccard' similarity coefficient of genotypes varies from 0.51 to 0.88. The results showed that the genetic distance of selected genotypes of walnut is medium. The dendrogram analysis of 21 genotypes of walnuts was divided genotypes into two main groups and three subgroups in the first group, which greatly matched the results of the PCoA. The genotypes were collected from Shahrood were inserted on the first subgroup of GI in dendrogram analysis, except of KH4 and OR37 genotypes, and the highest genetic similarity was observed between some of these genotypes (R1G2 with R2G1 and R1G7). The second group includes genotypes KH34, OR26, and SH1. Subgroup 3 of the first group contains R2G8, K26, OR23, T12, and K28. The second group has high genetic distances including R2G4, KH31, T1, OR4, and T9. The ISSR marker technology is an inexpensive, easy and satisfactory way to evaluate genetic relationships and genetic variation among walnut cultivars. The results of this research showed that these markers (ISSR) are suitable for study of variation in walnut genotypes. The genotypes collected from Shahrood had a lower genetic diversity, and the observed diversity is probably related to human interactions. The Tuyserkan and Urmia genotypes showed more genetic variation. The results of clustering based on molecular markers were largely matched with the grouping based on qualitative traits. The results showed that there is a moderate genetic distance between selected walnuts genotypes. The genotypes collected from Urmia and Tuyserkan regions have a higher genetic distance than other genotypes and which according to their superior characteristics, they can be used as parent along with selected walnut genotypes from the Shahrood area in a breeding program.