Organelle DNA analysis of Solanum and Brassica somatic hybrids by PCR with ’universal primers’

Abstract 

In order to set up a quick and easy procedure for determining the cytoplasmic composition of somatic hybrids, we tested a set of ’universal primers’ for plastidial and mitochondrial DNA on 13 genotypes belonging to the following species: Nicotiana tabacum, Solanum commersonii, Solanum tuberosum, Solanum etuberosum, Solanum phureja, Brassica oleracea, Brassica rapa, ’Anand’ CMS B. rapa, ’Chiang’ CMS B. oleracea, and ’Ogura’ CMS B. oleracea. Such primers are homologous to conserved coding sequences and amplify polymorphic intergenic or intronic regions. cpDNA polymorphism within Solanum and Brassica spp. was found with two and four primer pairs, respectively. The primers for the intergenic region between the trnF and trnV genes gave polymorphism among several tested species and were used in S. commersonii (+) S. tuberosum somatic hybrids,and B. oleracea (+) ’Anand’ CMS B. rapacybrids. Two primer pairs for mtDNA revealed polymorphism between S. commersonii and S. tuberosum, and one showed intraspecific polymorphism in S. tuberosum. The primer pair for the intergenic region between the rps14 and cob genes (pumD) showed a fragment of about 1.5 kb in S. tuberosum and S. phureja. A shorter fragment and no amplification were found in S. etuberosum and S. commersonii, respectively, suggesting frequent intrageneric rearrangements in this genome region. All Brassicaceae evidenced a fragment about 150-bp longer than in S. tuberosum. The same primers were also used with interspecific Solanum spp. somatic hybrids. Both PCR with pumD primers and hybridization with rpl5/rps14 genes indicated lack of linkage between rpl5/rps14 and cob genes in S. commersonii. Compared to direct visualization of restricted organellar DNA or Southern analysis with labelled probes, amplification of cpDNA and mtDNA with universal primers, followed by electrophoresis of either entire or restricted amplified fragments, is a simpler, more rapid and less expensive method to determine the organelle genome composition of interspecific Solanum and Brassica somatic hybrids.