Molecular characterization and expression analysis of the apomixis-linked EXS genes in sexual and apomictic Paspalum notatum.

PODIO, MARICEL; DELGADO LUCIANA; PESSINO, SILIVINA CLAUDIA; PUPILLI, FULVIO; ORTIZ, JUAN PABLO AMELIO

Bahía Blanca

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

Cerzos-CONICET

A sequence homologous to the EXS (ERD/XPS/SPX)domain was found in the apomixis-linked BAC clone346H10 of Paspalum simplex. A truncated form of thisdomain had been associated with proliferation inotherwise arrested cells in yeast, a function thatresembles apospory to some extent. However thecandidate's function in plants is still unknown. Theobjective of this work was to isolate, sequencecharacterizeand analyze the expression patterns of EXSgenes in apomictic and sexual genotypes of P. notatum. Aconserved 2,290-bp gene fragment was amplified fromgenomic DNA of tetraploid sexual (Q4188) and apomictic(Q4117) plants using heterologous primers. Ampliconsobtained from both genotypes were almost identical andhighly similar to the EXS C-terminus of Zea mays andOryza sativa, respectively. Southern-blot hybridizationindicated the presence of at least four copies/alleles ofEXS in the species. qRT-PCR analyses revealed thatPnEXS was expressed in the four stages of thereproductive development tested (premeiosis, meiosis,postmeiosis and anthesis) and nonsignificant differenceswere detected between sexual and apomictic genotypes.However, in situ hybridization experiments revealed thatPnEXS displayed a spatially- and chronologically alteredexpression pattern in reproductive organs of the apomicticand sexual flowers. At anthesis, PnEXS was stronglyexpressed in the nucellar tissue of the sexual genotype,but only in the megagametophyte border cells in theapomictic genotype. On the other hand, at meiosis, thesense probe hybridized in a region close to the megasporemother cell in the apomictic genotype but no in thesexual one, suggesting the existence of an antisensemediatedmechanism leading to downregulate PnEXSexpression in the apomict. Recently, RNAseq analysisallowed characterization of all splice-variants, alleles orparalogs expressed in P. notatum flowers. The sexualgenotype showed two isotigs (splice variants/alleles/paralogs) while the apomictic one showed six.Interestingly, four of the six transcripts expressed in theapomictic genotype included one or two of the intronspreviously characterized in the PnEXS genomicsequences, suggesting the occurrence of variances in thesplicing mechanism. The transcribed intron segmentsshowed similarity with retrotransposons and introducedstop codons in the corresponding ORFs. Results presentedin this work indicated that splice variants of PnEXSwithout protein-coding capacity are possibly associatedwith apomixis in P. notatum.