Creating of novel Wx allelic variations significantly altering Wx expression and rice eating and cooking quality.

Granule-bound starch synthase I (GBSSI) encoding gene Waxy (Wx), which largely regulates the amylose content of rice grains, is a master module determining rice eating and cooking quality (ECQ). Fine-tuning amylose level of grains is an ideal strategy to improve rice quality. Through fine editing of Wx promoter and 5'UTR by CRISPR/Cas9 system, we created 14 types of novel Wx allelic variations, of which MT7 and MT13 were able to alter Wx expression and amylose content of grains. MT7 showed fragment deletion and base insertions in CAAT-boxes, hardly detectable expression levels of GBSSI mRNA and protein, and generated 5.87% amylose in grains. MT13 had fragment deletions in the A-box and the TATA-box, low expression levels of GBSSI mRNA and protein, and generated 9.61% amylose in grains. Besides of the amylose content, MT7 and MT13 significantly reduced protein content and increased lipid content of grains compared with Wx. A comparison of MT7, MT13 and other allelic lines demonstrated the importance of base insertion around the second CAAT-box and 31bp-deletion following the second TATA-box in modulating Wx expression. Thus, our study generated two novel Wx allelic variations which significantly alter Wx expression and amylose content of rice grains, providing not only new germplasms for soft rice breeding, but also insights into candidate cis elements of Wx.