COMPARAÇÃO DOS MARCADORES MICROSSATÉLITES E ISSR NA AVALIAÇÃO DA DIVERSIDADE GENÉTICA DE Psidium guajava L. E DETERMINAÇÃO DO TAMANHO IDEAL DE AMOSTRAGEM PARA ESTUDOS DE GENÉTICA DE POPULAÇÕES

Abstract

To determine reliable genetic diversity and population structure data the correct choice of molecular markers to be used and the number of individuals to be sampled in each population are limiting in obtaining reliable results. The objective of this study was the comparison of microsatellite (SSR) and ISSR markers in the evaluation of the genetic diversity of Psidium guajava and to determine the ideal size sample for population genetic studies in plants. For comparison of microsatellite and ISSR markers, DNA from 90 individuals of three populations of P. guajava was collected in Goiatuba (GO), Lagarto (SE) and Ceará Mirim (RN) and were amplified separately using 12 microsatellite pairs primers and seven ISSR primers. The data obtained for each marker were used for obtaining genetic parameters that were comparatively evaluated. To determine the optimal sample size, data obtained with microsatellite markers from P. guajava and Campomanesia xanthocarpa species and a set of simulated data were used. For P. guajava and C. xanthocarpa were collected 60 individuals and they are evaluated using 12 microsatellite loci. For each species, the original population of 60 individuals were used to randomly generated subpopulations with sample sizes of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 59 individuals. Each subpopulation was randomly generated 10 times to obtain 100 subpopulations of each species to be used in statistical analysis. For the simulated data, the original population was generated by the program GenAlEx considering 12 microsatellite loci. After obtaining the simulated original population, subpopulations were generated as described for the species. The effective number of alleles (Ne), number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He) and genetic differentiation index (FST) were obtained for each repetition of each subpopulation and used in the statistical analysis. The comparison between the markers showed that the Shannon index and the genetic diversity of Nei were higher with microsatellite markers and the population of Ceará Mirim (RN) show greater diversity, while with ISSR markers population with greater diversity was from Goiatuba (GO ). Already, gene flow was three times higher with ISSR (Nm = 4.01). The Fst, the variation between and within populations and the number of genetic groups (K) were similar with the two markers. Although the same number of the genetic groups, the distribution of these groups in the population was different for each marker, with ISSR presenting greater agreement with the geographical distribution of the populations. The results of this study show that the ISSR markers were more informative to Psidium guajava and to obtain better results with microsatellite loci number should be high. This low efficiency of microsatellite markers in P. guajava is probably due to the high rate of homozygous of the species due to your mixed reproductive system with self-pollination of around 65%. In determining the optimal sample size, the results showed significant correlation between the number of alleles (Na), effective number of alleles (Ne), expected heterozygosity (He), FST and the sample size. Only the observed heterozygosity (Ho) showed no significant correlation with sample size. The set of simulated data showed different results from the other sets, which was expected because they do not represent what occurs in nature. For data sets of P. guajava and C. xanthocarpa the genetic index evaluated showed similar results for each sample size. In these sets was also observed that increasing the sample size results in a decrease of the significant difference of the estimates of the effective number of alleles (Ne), the number of alleles (Na), the expected heterozygosity (He) and FST, but these decreases were minimal when compared samples with more than 20 individuals. The results obtained allow us to conclude that a sample of 20 to 30 individuals per population is sufficient for population genetic studies with microsatellite markers in plants.