Identification and characterization of sex related genes in Actinidia arguta by suppression subtractive hybridization

Abstract

Abstract Actinidia arguta, with sweet flavour, high nutritional value and health promoting role, is a typical dioecious plant. But dioecy represents an important constraint in kiwifruit breeding programmes and also requires identification of male and female genotypes before planting an orchard. Therefore, understanding molecular basis of sex determination in A. arguta is important for molecular identification of seedling gender at early stage. PCR-based suppressive subtractive hybridization (SSH) was performed to screen genes related to sex determination. A total of 533 and 508 positive clones, from forward and reverse libraries respectively, were selected for sequencing. The clean ESTs were assembled into 309 (forwad) and 369 (reverse) unigenes, and 228 and 299 of them hit known homologous sequences of the NCBI nr database. Based on Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, four unigenes that might be involved in sex determination were selected for validation by qRT-PCR. The expression of PME was very strong in male flower at late development stage, with 72 times higher than that of female flower. Reversely, male flower exhibited extremely low expression level of ACO, MAN1 and MYC2 at late development stage as compared with female flower. The variable expression patterns of these genes between male and female flowers suggested their importance in controlling sex expression. These results advanced our understanding of the molecular mechanism of sex determination in A. arguta.