The Use of Microsatellites Markers in Molecular Characteristics of Hybrids between Pulsatilla Species ^†

Abstract

Pulsatilla × hackelii Pohl. is the most common hybrid between Pulsatilla patens and Pulsatilla pratensis. It occurs in the natural environment, most often in sites together with parental forms. Pulsatilla × hackelii is morphologically intermediate between the pure Pulsatilla species. Separation of hybrid individuals from pure Pulsatilla species requires a whole set of morphological features. Completed plastid genome provides molecular evidence for the hybrid origin of Pulsatilla × hackelii. In our study, we present the—genotypic characteristics of Pulsatilla × hackelii based on microsatellite markers developed for Pulsatilla vulgaris. A specimen of the hybrid was found in the Świętokrzyskie Mountains, near Bocheniec village, in the vicinity of a forest road in a fresh mixed coniferous forest (Querco roboris-Pinetum (W. Mat. 1981) J. Mat. 1988). We amplified a set of 10 microsatellites in two multiplex PCR reactions. From the 10 analyzed loci, we successfully amplified eight. The analyzed microsatellites in population studies of three Pulsatilla species showed some species-specific alleles. In the case of the Pulsatilla × hackelii sample, we obtained PCR products in the case of 5 loci developed for P. vulgaris but successfully crossamplified in P. patens and P. pratensis. Additionally, we obtained results for two loci, which crossamplified only for Pulsatilla patens and one only for Pulsatilla pratensis. Among the investigated markers, two were monomorphic and the remaining six were polymorphic, which makes them highly informative in population studies for pasque-flower species as Pulsatilla patens, P. pratensis, and P. vernalis. The obtained results indicated that analyzed microsatellite markers could be used for hybrids identification, although there is a need for further research on a larger number of hybridized individuals.

1. Introduction

Hybridization is widespread in plants. The genus Pulsatilla contains more than 30 species of herbaceous perennials. It is characterized by a relatively high hybridization rate. Spontaneous hybridization between P. patens and P. pratensis occurs in the natural habitats, most often in sites together with parental forms. Completed plastid genome provides molecular evidence for the hybrid origin of Pulsatilla × hackelii [1]. P. × hackelii is morphologically intermediate between the pure P. patens and P. pratensis. The distinction between hybrid individuals and pure Pulsatilla species requires a whole set of morphological features. In our study, we present the genotypic characteristics of P. × hackelii based on microsatellite markers developed for P. vulgaris [2]. Some of the loci can crossamplified only for one species from P. patens and P. pratensis.

2. Materials and Results

A hybrid specimen was found in the Świętokrzyskie Mountains, near Bocheniec village (southern Poland), in the vicinity of a forest road in a fresh mixed coniferous forest (Querco roboris-Pinetum). We amplified a set of ten microsatellites in two multiplex PCR reactions. From the ten analyzed loci, we successfully amplified eight. The analyzed microsatellites in the population of three Pulsatilla species showed some species-specific alleles. In the case of the P. × hackelii sample, we obtained PCR products in the case of five loci developed for P. vulgaris, but successfully crossamplified in P. patens and P. pratensis. Additionally, we obtained results for two loci, which crossamplified only for P. patens and only one for P. pratensis (Figure 1). Two of the analyzed loci were polymorphic (PV32 and PV65).

3. Discussion and Conclusions

Among the investigated markers, two were monomorphic (PV44 and PV48), and the remaining six were polymorphic. Markers PV32, PV65, and PV9 are highly polymorphic, which makes them highly informative in population studies of pasque-flower species such as P. patens, P. pratensis, and P. vernalis. The obtained results indicated that analyzed microsatellites markers can be used for hybrids identification, although there is a need for further research on a larger number of hybridized individuals.