Investigation on genetic diversity of some unknown genotypes of citrus in Iran according to morphological and molecular characteristics based on ISSR and PCR-RFLP markers

Citrusgermplasm has an extensive diversity in Iran which is due to cross pollination, long history of seed propagation and abundance of genetic variations. For determining the classification status, the phylogenetic relationships and the genetic distance between members of this valuable genetic reserve, it is necessary to use of morphological characters along with DNA-based molecular markers. In the present study, to obtain information on the degree of genetic affinity between 79 unknown citrus biotypes in Kotra Citrus Research Station and determining their relative distance from 18 commercial cultivars, three types of markers (morphological, ISSR and PCR-RFLP) were used.

The present study was carried out asa three-year research toobtain passport data for 79 biotypes and 18 commercial cultivars (check) of citrus trees, recognition of phylogenetic relationships and determination of genetic distance between these genotypes. This study was conducted on the basis of a comparative study of 19 vegetative and 40 reproductive traits and the analysis of chloroplast DNA based on ISSR and PCR-RFLP markers of leaf samples.The three-year average of morphological traits according to standard citrus descriptor were recorded.In order to perform molecular investigations, DNA extraction from the leaf samples of each genotype was performed and DNA was quantified using UV-visible spectrophotometer (nano-drop) at 260 nm. Cluster analysis of morphological and molecular data was performed based on the unpaid pairs (UPGMA) and Jaccard's similarity coefficient anddifferences between genotypes were determined by coding and ranking of them.

Obtained results from cluster analysis of morphological and molecular data with NTSYS-pc and POPGENE software, using the UPGMA method with Jaccard's coefficient of similarity showed that all genotypes can be categorized according to morphological traits, ISSR and PCR-RFLP markers into 12, 9 and 5 main groups respectively in the 40% , 53% and 60% similarity coefficient respectively. According to morphological traits, the first cluster (A) can be divided into two subgroups which there are three lemon varieties in one of them. Another group (B) contains all sweet orange and mandarin cultivars and the third group (C) was incorporates three genotypes including Citron, Pomelo and Duncan grapefruit.The use of ISSR marker showed that polymorphism of examined genotypes was varied from 92% to 53% for N10 and N1 markers, respectively and a significant number of them had close affinity to sweet orange.The results of this study also showed that kumquat, based on molecular characteristics and apparent traits, is a distinct genus of Citrus family and should be placed in separate group.On the other hand, sweet oranges, grapefruits and pomelos were all in the same group which is consistent with the fact that grapefruits are hybrids of sweet oranges and pomelo. In this way, the degree of similarity of the local unknown genotypes was determined to each other and to the commercial varieties (checksamples(.Also, the differentiation of three species (C. reticulata, C. medica and C. maxima) was possible.

The data obtained from the measurement of morphological and molecular characteristics of unknown citrus genotypes could obtain a passport data for each biotypes and determine the phylogenetic relationships of biotypes with each other and with commercial cultivars. Obtained results could determine the genetic distance of biotypes from each other as well as from commercial varieties. In this way, in the future, parents can be efficiently chosen to perform corrective breeding programs for creating of new varieties.