Use of Microsatellites for the Identification of Potato Somatic Hybrids
Somatic hybrid plants were obtained through protoplast fusion of monoploid potato. Of three separate fusions, two were interspecific between Solanum phureja and S. chacoense, whereas one was intraspecific between two S. phureja clones. Microsatellites, or SSRs, were employed to distinguish true somatic hybrids from somaclones of unfused protoplasts. Primers flanking eight different SSR loci obtained from GenBank accessions for potato were developed for PCR amplification. Microsatellites consisted of di-, tri-, and tetra-nucleotide repeat units that varied from 4 to 20 repeats per locus. The majority of microsatellites were highly polymorphic between the S. phureja and S. chacoense clones and the presence of both parental alleles in fusion regenerants indicated their interspecific hybrid nature. One interspecifc somatic hybrid could be identified at three of the four examined loci (two tri- and one di-nucleotide repeat loci). The parents were monomorphic at the remaining di-nucleotide repeat, thereby rendering it unsuitable for hybrid identification. A similar result was obtained for another interspecific hybrid, with four of five loci appearing polymorphic between the parents and in the somatic hybrid. Less polymorphism was observed between the parents of an intraspecific S. phureja somatic hybrid, with only one locus (a tetra-nucleotide repeat) of five examined showing polymorphism. Results indicate that SSRs are a consistent and reliable means for somatic hybrid identification in potato. In order to reduce the cost and time of maintaining numerous calluses through a lengthy tissue culture regeneration phase, a technique was developed to screen calluses prior to regeneration. Using SSRs and a rapid DNA extraction method, hybrid and nonhybrid calluses could be distinguished rapidly without adversely affecting subsequent regeneration of shoots from the callus.