A Novel Allele in the Promoter of Wx Decreases Gene Expression and Confers Lower Apparent Amylose Contents in Japonica Rice ( Oryza sativa L.).

Wx is the key gene that controls amylose content (AC), and various alleles have been found in rice populations. Wx b is the major allele in japonica and produces moderate AC (15~18%). It was recently found that editing the promoter of Wx could produce a series of alleles that have different Wx activities. Although some studies have edited the promoter, few studies have focused on the natural variations in Wx . Here, we used the Rice3K database to investigate variations in the Wx promoter and found that the allele Wx1764178 (A/G) has a higher LD (linkage disequilibrium) with the two key SNPs (1765751, T/G; 1768006, A/C), which could produce different Wx alleles and influence AC, as reported previously. Further study showed that the Wx1764178 allele (A/G) is functional and influences the expression of Wx positively. Editing the A allele using CRISPR‒Cas9 produced 36 and 3 bp deletions and caused a decrease in the expression of Wx . The apparent amylose content (AAC) in the edited lines was decreased by 7.09% and 11.50% compared with that of the wild type, which was the japonica variety Nipponbare with Wx b and the A allele at 1764178, while a complementary line with the G allele showed a lower AAC than the A allele with no effect on other agronomic traits. The AAC of the edited lines showed a higher increase than that of the wild type (Nipponbare, Wx b ) in low-nitrogen conditions relative to high-nitrogen conditions. We also developed a dCAPS marker to identify the allele and found that the G allele has widely been used (82.95%) in japonica -bred varieties from Jiangsu Province, China. Overall, we found a functional allele (Wx1764178, A/G) in the Wx promoter that could affect AAC in japonica cultivars and be developed as markers for quality improvement in rice breeding programs.