ISSR and chloroplast DNA analyses indicate frequent hybridization of alien Medicago sativa subsp sativa and native M-sativa subsp falcata

Alfalfa (Medicago sativa subsp. sativa) was introduced to Lithuania as a fodder crop and now grows as a weed in natural ecosystems adjacent to its wild relative, sickle medic (M. sativa subsp. falcata). The hybrid form of these taxa, variegated alfalfa (M. sativa subsp. varia), is considered an invasive taxon due to its intensive spread. Here, we use inter-simple sequence repeat (ISSR) markers and haplotype analyses of three regions of chloroplast DNA (psbA-trnH, trnS-trn2GS and trnL-trnF) and the nuclear ribosomal DNA internal transcribed spacer region to study the spontaneous hybridization of alfalfa and sickle medic in natural subpopulations. Plants of the three taxa were collected from 19 subpopulations located at seven sites. ISSR analysis revealed similar values of genetic diversity (expected heterozygosity, band richness, polymorphic band proportion) in subpopulations of the three studied subspecies. Our study revealed a lack of genetic differentiation among these taxa and high differentiation among subpopulations (I broken vertical bar (ST) = 0.389, P = 0.001). Analysis of the chloroplast DNA regions provides direct evidence for the hybrid origin of variegated alfalfa. Taxon-characteristic haplotypes were identified for subsp. sativa and subsp. falcata in local subpopulations, and two haplotypes specific to the parental taxa were detected in subpopulations of M. sativa subsp. varia. Our study demonstrates the extensive genetic impact of alfalfa on adjacent native subpopulations of sickle medic.