Analysis of amplification of microsatellite loci in Iris pumila

Abstract

Utilization of microsatellite molecular markers was analyzed in Iris pumila and five other species belonging to the same genus: I. humilis, I. sibirica, I. pseudacorus, I. spuria and I. variegata, which inhabit the northern part of the Republic of Serbia. We selected 40 EST-SSR markers previously reported for the genus Iris. Sixteen markers that showed polymorphism were chosen for further research. To examine the potential of the selected microsatellite loci analysis in the detection of different I. pumila clones, we selected 18 individuals of this species that belonged to different genotypes. Optimization of amplification was performed for all 16 microsatellite loci: temperature program of the PCR protocol and number of amplification cycles were optimized as well as primer and DNA concentrations used in the reactions. Seven markers that were variable across the ana lyzed individuals were selected. The final analysis was performed using only 5 microsatellite loci since high-quality amplification for two loci was not possible. No greater deviations in the length of the fragments from the expected were observed, except for one primer. All microsatellite loci contained three-nucleotide repeats. I. pumila is tetraploid species and in several plants the presence of four alleles were observed. The observed number of alleles per locus ranged from 3 to 9. In the results obtained across 18 I. pumila individuals using 5 microsatellite markers, 17 multilocus genotypes were observed.