<i>Pho1a </i>GENE FRAGMENT VARIABILITY IN TUBER-BEARING AND NONTUBER-BEARING POTATO SPECIES (<i>Solanum </i>subgenus <i>Potatoe</i>) AND<i> S. tuberosum </i>L. CULTIVARS
Starch is the main metabolite in potato tubers. Therefore structure and functional analysis of starch metabolism genes are of fundamental and applied interest. The final starch amount in sink organs (fruits, seeds and tubers) depends not only on the amylose and amylopectin synthesis, but also on the catabolic enzymes activity. Proteins that participate in starch biosynthesis are rather well studied, while the starch degradation reactions are not fully understood. To date, more data on the crucial role of starch-phosphorylases in this process have been reporting. Starch phosphorylases are widespread among plant species, but the coding genes structure and genetic diversity remain unclear. In potato tubers starch is cleaved by L-form of starch phosphorylase encoded by the Pho1a ( STP23 ) gene. In the current work Pho1a gene fragment (exon II-exon IV) variability was analyzed for the first time in 15 wild and 81 cultivated potato accessions. The chosen gene fragment corresponds to the regulatory part of the glycosyltransferase domain and comprises glucose-6-P binding site, pyridoxal phosphate cofactor binding site and active site (glucose binding). The nucleotide and amino acid polymorphism is determined. A total of 96 potato accessions were used for allelic diversity analysis: 15 wild species from Potatoe and Estolonifera subsections (where S. etuberosum is a nontuber-bearing species), 67 cultivated potato varieties and 14 breeding lines of S. tuberosum. Nuclear DNA was isolated from young leaves using potassium-acetate method with phenol-chloroform additional purification. Primer combination Pho2F (5´-CTGAACATGAAGCAAGCGTA-3´)-Pho4R (5´-GGCTA-TGGACTTAGGTACA-3´) was designed for chosen fragment amplification. The sequences of all varieties and breeding lines of S. tuberosum had a length of 670 bp. The length of the obtained Pho1a sequences in species ranged from 666 bp ( S. vernei, S. lignicaule ) up to 672 bp ( S. pinnatisectum ). Totally 59 SNPs were detected, 15 of them localized in exons. It allowed us to identify 11 allelic variants, moreover 9 of them were found in wild species. Cultivated potato S. tuberosum has two allelic variants. The Pho1a_A2 allelic variant was observed in the majority of analyzed potato cultivars and all the breeding lines. Interestingly, the same variant was detected in some wild potato species, belonging not only to superseries Rotata, but also to Stellata that is considered to be more ancient. The Pho1a_A10 allelic variant was found in 9 cultivars (Bintje, Red Scarlett, Ushkonir, Karasaiskii, Aurora, Aladin, Chernskii, Plamya, Udacha). The Pho1a gene fragment translation revealed that 3 out of 15 exonic SNPs led to amino acid substitutions. In potato cultivars with the Pho1a_A10 allelic variant neutral M139I substitution was detected. The other neutral substitutions M139L and T157S were identified in S. circaefolium and S. vernei, correspondingly. The only radical substitution R212S was detected in nontuber-bearing S. etuberosum. The potential role of the found amino acid substitutions in the functional protein domain requires the further investigation. Further search for the allelic variants associated with starch content in tubers can be used in potato breeding programs