Microsatellite-based identification of doubled haploid plants by androgenesis in Anemone coronaria L

Anemone coronaria L. (2n=2x=16) is a perennial, allogamous, and highly heterozygous plant belonging to the Ranunculaceae family, marketed both as cut flower and garden plant. The production of F1 hybrids obtained by crossing highly homozygous inbred lines is hampered by the marked inbreeding depression characterizing the species. In order to overcome this limit, protocols have been developed to obtain doubled haploid homozygous plants following diploidization of haploid plants obtained through in vitro androgenesis. However, during the androgenesis process a high frequency of spontaneous diploidization has been observed. This makes it difficult to discriminate between doubled haploid plants (DH) and plants originated from somatic anther tissues, as both have the same number of chromosomes (2x), and are thus not discriminable on the basis of their DNA content through the flow-cytometric technique or following chromosome count. Following a genome-wide survey, we previously developed a microsatellite markers database (AnCorDB) in the species. By selecting microsatellites showing two alleles (heterozygous) in the anthers mother plant, we developed a fast and easy to apply technique suitable for selecting the DH plants in an early stage of plant development. DHs are characterized by the presence of one allele, whereas plants originated by somatic embryogenesis are characterized by the presence of two alleles.