CRISPR-induced miRNA156-recognition element mutations in TaSPL13 improve multiple agronomic traits in wheat

Increase in grain yield is always a major objective of wheat genetic improvement. The SQUAMOSA promoter-binding protein-like (SPL) genes, coding for a small family of diverse plant-specific transcription factors, represent important targets for improving grain yield and other major agronomic traits in rice. The function of the SPL genes in wheat remains to be investigated in this respect. In this study, we identified 56 wheat orthologues of rice SPL genes belonging to 19 homoeologous groups. Like in rice, nine orthologous TaSPL genes harbour the microRNA156 recognition elements (MRE) in their last exons except for TaSPL13, which harbour the MRE in its 3'-untranslated region (3'UTR). We modified the MRE of TaSPL13 using CRISPR-Cas9 and generated 12 mutations in the three homoeologous genes. As expected, the MRE mutations led to an approximately two-fold increase in the TaSPL13 mutant transcripts. The phenotypic evaluation showed that the MRE mutations in TaSPL13 resulted in a decrease in flowering time, tiller number, and plant height, and a concomitantly increase in grain size and number. The results show that the TaSPL13 mutants exhibit a combination of different phenotypes observed in Arabidopsis AtSPL3/4/5 mutants and rice OsSPL13/14/16 mutants and hold great potential in improving wheat yield by simultaneously increasing grain size and number and by refining plant architecture. The novel TaSPL13 mutations generated can be utilized in wheat breeding programmes to improve these agronomic traits.