<i>Tae-miR396b</i> regulates <i>TaGRFs</i> in spikes of three wheat spike mutants

Tillering and spike differentiation are key agronomic traits for wheat (<i>Triticum aestivum</i> L.) production. Numerous studies have shown that miR396 and growth-regulating factor genes (<i>GRFs</i>) are involved in growth and development of different plant organs. Previously, we have reported that wheat miR396b (<i>tae-miR396b</i>) and their targets <i>TaGRFs</i> (<i>T. aestivum GRFs</i>) play important roles in regulating wheat tillering. This study was to investigate the regulatory roles of <i>tae-miR396b</i> and <i>TaGRFs</i> played during wheat spike development. Wheat cultivar Guomai 301 (wild type, WT) and its three sipke mutants dwarf round spike mutant (<i>drs</i>), apical spikelet sterility mutant (<i>ass</i>) and prematurely terminated spike differentiation mutant (<i>ptsd1</i>) were studied. Three homeologous genes of <i>tae-miR396b</i> on the long arms of chromosomes 6A, 6B, and 6D were identified, and they encoded the same mature miRNA. Complementary sequences of mature <i>tae</i>-<i>miR396b</i> were identified in 23 <i>TaGRFs</i>, indicating they were the target genes of <i>tae</i>-<i>miR396b</i>. <i>Tae-miR396b</i> had different regulatory effects on <i>TaGRFs</i> between Guomai 301 and its mutants. <i>TaGRF2-7A</i> was confirmed to be the target gene of <i>tae-miR396b</i> by molecular interaction assay. The expression levels of <i>tae-miR396b</i> and <i>TaGRFs</i> were different between WT and mutants <i>drs</i>, <i>ass</i> and <i>ptsd1</i> at the floret primordium visible (S1), the two awns/spikelet reaching apical meristem of the spikelet (S2), and the green anther stage (S3). The expression level of <i>tae-miR396b</i> in WT was significantly higher than that in mutants <i>drs</i> and <i>ass</i>. The most <i>TaGRFs</i> were negatively regulated by <i>tae-miR396b</i>. The abnormal expressions of <i>TaGRF1</i> (<i>6A</i>, <i>6D</i>), <i>TaGRF2</i> (<i>7A</i>, <i>7B</i>, <i>7D</i>), <i>TaGRF4</i> (<i>6A</i>, <i>6B</i>), <i>TaGRF5</i> (<i>4A</i>, <i>7A</i>, <i>7D</i>), and <i>TaGRF10</i> (<i>6A</i>, <i>6B</i>, <i>6D</i>) were important causes for abnormal spike development in the three mutants. This study laid foundation for further elucidating functions of <i>tae-miR396b</i> and <i>TaGRFs</i> underlying wheat spike development. Regulating <i>tae-miR396b</i> and <i>TaGRFs</i> will be a new approach for wheat high yield breeding.