MOLECULAR CHARACTERIZATION AND EXPRESSION ANALYSIS OF THE APOMIXIS-LINKED EXS GENE IN SEXUAL AND APOMICTIC PASPALUM NOTATUM

PODIO M; DELGADO, L.; PESSINO SC; PUPILLI F; ORTIZ JPA

Bahia Blanca

Workshop; IV Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis en gramíneas forrajeras; 2014

Universidad Nacional del Sur/CERZOS-CONICET

A sequence homologous to the EXS (ERD/XPS/SPX) domain was found in the apomixis-linked BAC clone 346H10 of Paspalum simplex. A truncated form of this domain had been associated with proliferation in otherwise arrested cells in yeast, a function that resembles apospory to some extent. However the candidate?s function in plants is still unknown. The objective of this work was to isolate, sequence-characterize and analyze the expression patterns of EXS genes in apomictic and sexual genotypes of P. notatum. A conserved 2,290-bp gene fragment was amplified from genomic DNA of tetraploid sexual (Q4188) and apomictic (Q4117) plants using heterologous primers. Amplicons obtained from both genotypes were almost identical and highly similar to the EXS C-terminus of Zea mays and Oryza sativa, respectively. Southern-blot hybridization indicated the presence of at least four copies/alleles of EXS in the species. qRT-PCR analyses revealed that PnEXS was expressed in the four stages of the reproductive development tested (premeiosis, meiosis, postmeiosis and anthesis) and non-significant differences were detected between sexual and apomictic genotypes. However, in situ hybridization experiments revealed that PnEXS displayed a spatially- and chronologically altered expression pattern in reproductive organs of the apomictic and sexual flowers. At anthesis, PnEXS was strongly expressed in the nucellar tissue of the sexual genotype, but only in the megagametophyte border cells in the apomictic genotype. On the other hand, at meiosis, the sense probe hybridized in a region close to the megaspore mother cell in the apomictic genotype but no in the sexual one, suggesting the existence of an antisense-mediated mechanism leading to downregulate PnEXS expression in the apomict. Recently, RNAseq analysis allowed characterization of all splice-variants, alleles or paralogues expressed in P. notatum flowers. The sexual genotype showed two isotigs (splice variants/alleles/paralogs) while the apomictic one showed six. Interestingly, four of the six transcripts expressed in the apomictic genotype included one or two of the introns previously characterized in the PnEXS genomic sequences, suggesting the occurrence of variances in the splicing mechanism. The transcribed intron segments showed similarity with retrotransposons and introduced stop codons in the corresponding ORFs. Results presented in this work indicated that splice variants of PnEXS without protein-coding capacity are possibly associated with apomixis in P. notatum.