Assessment of genetic diversity in Citrus sinensis by ISSR marker and retrotransposon

Determining the classification, phylogenetic relationships, and genetic diversity in citrus is critical to determining genetic relationships, identifying germplasm, controlling genetic erosion, establishing breeding programs, and registering new cultivars. Researchers believe that genetic diversity is very important and is an essential part of any breeding program. The use of molecular techniques to assess DNA level, genetic diversity, and genetic distance is widely used in germplasm samples. Therefore, the aim of this study was to investigate the genetic diversity of orange genotypes using molecular markers.

In order to investigate the genetic diversity, 40 orange genotypes of Rudsar city in Guilan province were evaluated. DNA extraction from young leaf samples was performed using the Edward method with slight modifications. After DNA extraction of genotypes, polymerase chain reaction (PCR) was performed with 15 primers. Amplified genomic DNA images were analyzed and the data were entered into Excel software as a matrix. Polymorphic information content, marker index, effective multiple ratio, number of effective alleles, Shannon index and straw gene diversity were also examined.

The 15 primers used in this study were able to create a total of 140 bands, of which 101 were polymorphic bands, including TOS-1 and TOS-2 primers with 9 bands, UBC811 primer and UBC8821 + UBC826 hybrid primer with 4 Tape created the least number of polymorphic strips. Primers TOS-1, UBC813, UBC823 and TOS-2 and UBC811 with the highest PIC value, best markers and high values of effective allele, Shannon index, straw gene diversity as the top primers to study genetic diversity in this study, respectively were. Based on the results, the markers used were able to evaluate the genetic diversity of genotypes. The content of polymorphic information in this study ranged from 0.22 to 0.45. Principal coordinate analysis showed that the first three components were able to explain a total of 29.08% of the total variance. Cluster analysis by the farthest neighbor method divided 40 orange genotypes into five distinct groups based on similarities and differences. The grouping accuracy obtained from the cluster analysis was confirmed by the Fisher linear focal detection function 100%. The markers used in this study showed acceptable polymorphism. Also, the study of morphological traits and molecular markers used in this study showed the degree of similarity and differences of genotypes, although there were differences in the results of morphological and molecular studies that can be observed. Due to the high accuracy of molecular markers. However, using appearance and morphology, it was possible to distinguish orange genotypes from each other.

The results of this study showed that there is a high genetic diversity among orange genotypes. Due to the fact that the markers used in this study showed acceptable polymorphism, they can be used in future research on this plant and other citrus plants. In general, according to the results of this study, that ISSR and retrotransposon primers can be used as a simple molecular method based on PCR and relatively safe and reliable in determining the level of genetic diversity and phylogenetic relationships in citrus.