MiR396 regulatory network and its expression during grain development in wheat.

Wheat contains the largest number of miR396 family with 17 miR396 in Poaceae. MiR396 regulatory network underlying wheat grain development has not comprehensively been explored. Our results showed that precursor miR396 family in Poaceae exhibited not only conservativeness but also diversification especially in wheat. Five haplotypes were detected in Poaceae species, while 4 haplotypes in wheat with Hap-4 (miR396a) and Hap-5 (miR396n) unique to wheat. GO enrichment analysis of target genes showed that the first 20 enrichment functions of miR396a and miR396n are completely different from each other, and also completely different from miR396(b-g), miR396(h-m), and miR396(o-q). Functional annotation on the 18 target genes shared by miR396(b-g), miR396(h-m), and miR396(o-q) found that 11 of the 18 target genes are growth-regulating factor (GRF) genes. Our results indicated that, during the grain filling stage of wheat, miR396 is involved in the development of grains by regulating the expression of GRF genes (GRF1, GRF6, and GRF9). Although the enrichment function of miR396(b-g), miR396(h-m), and miR396(o-q) is the same, the gene functional networks they formed differ greatly. Our results indicated that polyploidization enriches not only the diversity of miR396 family and its target genes but also gene functional networks in wheat. These results laid foundation for further elucidating function of miR396 gene family underlying wheat grain development.