Expression and Functional Analysis of Candidate Genes Related to Aposporous Apomixis in the Grasses

PODIO M; MANCINI M; PERMINGEAT H; SIENA LA; DELGADO L; FELITTI S; SARTOR M; ESPINOZA F; ARRAIS-GUIMARAES L; DUSI D; CAMPOS CARNEIRO V; PUPILLI F; LEBLANC O; ORTIZ JPA; PESSINO SC

Oslo

Congreso; European Frontiers of Plant Reproduction Research; 2013

COST Action FA0903 ?Harnessing Plant Reproduction for Crop Improvement?

In the past decade, comparative transcriptome surveys carried out in Paspalum and Brachiaria sp. provided a vast list of genes displaying differential expression in reproductive organs of sexual and apomictic individuals. Here we picked a candidates?s subset to carry out further expression and functional analysis. Selection criteria consisted of: i) concurrent detection in comparative transcriptome surveys from both species; and/or ii) occurrence of experimental or in silico positional linkage to genomic regions associated with apospory control. Chosen transcripts were: Pnserk (somatic embryogenesis receptor kinase), Pnexs (exs domain-containing protein), Pn19 (unknown), Pn46 (MAP3K), Pn108 (far1 domain-containing protein), Pn69 (DNA methyltransferase) and PnMTA-70 like (RNA methyltransferase). Paspalum and Brachiaria candidates? full sequences were obtained by performing RACE (Rapid amplification of cDNA ends) or Illumina sequencing, respectively. Chronological quantitative expression was analysed by real time PCR in reproductive organs of sexual and apomictic genotypes at pre-meiosis, meiosis, post-meiosis and anthesis. Moreover, in situ hybridization was used to determine more accurately the cell types where expression was located. Tested candidates showed contrasting chronological and spatial expression patterns in sexual and apomictic plants, sometimes involving both coding and non-coding strands. Most of them displayed strong sense expression in the nucellus of aposporous plants, but not of sexual ones. A stable transformation platform is currently being used to modify the expression of these chosen genes, and to infer their potential functional role in reproductive development. Expression of candidate n46 (MAP3K) was down-regulated in a natural aposporous genotype via introduction of a hairpin construct. Transformed plants showed a diminished capacity to form aposporous embryo sacs with respect to wild-type and transformation controls. Our results suggest the existence of a molecular pathway ectopically activated in the nucellus of aposporous genotypes, which regulates the onset of apospory initials and their commitment to a gametophytic fate.