https://orcid.org/0000-0002-9781-841X
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Lingshang Lin, Zheng Li, Mengyuan Ning, Zihan Zou, Long Zhang, Qiaoquan Liu, Cunxu Wei, A mutant allele of the Wx gene encoding granule-bound starch synthase I results in extremely low amylose content in rice, Plant Physiology, Volume 196, Issue 4, December 2024, Pages 2296–2299, https://doi.org/10.1093/plphys/kiae507
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Dear Editor,
Amylose content (AC) plays critical roles in determining rice (Oryza sativa) qualities (Huang et al. 2020b). Amylose is synthesized by granule-bound starch synthase I (GBSSI) encoded by Wx. Eight Wxlv, Wxa, Wxin, Wxb, Wxmw/la, Wxop/hp, Wxmp, and wx alleles from rice natural germplasm are responsible for synthesizing amylose from high to low and glutinous and regulate rice qualities, especially eating and cooking quality (ECQ) (Zhang et al. 2019; Huang et al. 2020b; Zhou et al. 2021).
In recent years, CRISPR/Cas system has widely been used to edit Wx for fine-tuning AC in rice (Huang et al. 2020a, 2021; Xu et al. 2021; Zeng et al. 2020). Though the system is easy to use to create novel Wx alleles, it is more important and useful in practical production to mine novel Wx alleles from natural germplasm and mutation population to help breeders generate new rice cultivars with good ECQ. We identified a rice mutant M137 from EMS-induced mutation population of japonica rice cultivar Wuyungeng 8 (wild type, WT). The M137 grains exhibited translucent endosperm (Fig. 1A) and slightly altered grain shape and weight (Fig. 1B, 1 to 4; Supplementary Table S1). Some cavities were observed in the center of starch granule in M137, which might result in grain translucency (Supplementary Fig. S1). The agronomic traits of M137 were all similar to those of WT (Supplementary Table S2). Compared with WT, M137 brown grain markedly decreased AC from about 15.1 to 7.5%, but its starch, protein, and soluble sugar contents did not change (Fig. 1B, 5 to 8). The phenotype segregation ratio of F2 brown grains between M137 and its WT suggested that the causality is recessive, and that between M137 and glutinous rice cultivar Guanglingxiangnuo (GLXN) carrying wx indicated that Wx mutation might be the causal gene of M137 (Fig. 1C, 1; Supplementary Table S3). The Wx sequencing exhibited that the 20th cytosine (C) was substituted with thymine (T) in the 12th exon, resulting in an amino acid substitution of GBSSI, the 506th threonine (Thr/T) to isoleucine (Ile/I) (Fig. 1C, 2; Supplementary Fig. S2; Supplementary Table S4). Further, the Wx of japonica rice cultivar Zhonghua 11 (ZH11) was edited using CRISPR/Cas9 system, and 3 homozygous and independent lines with target C/T substitution were successfully obtained (Fig. 1C, 3; Supplementary Fig. S3). The transgene-free plants were screened from T1 plants, and their T2 brown grains exhibited translucent endosperm and extreme low AC from 2.9 to 4.1% (Fig. 1C, 3). The substantially lower AC in ZH11 edited lines than in M137 might be due to their different genetic backgrounds and growth environment, especially growth temperature (Huang et al. 2020b). The mutations in the 5 top-ranked potential off-target sites were not detected in T2-edited lines (Supplementary Fig. S4 and Table S5), and their T3 grains showed translucency and slightly altered grain shapes and weight (Supplementary Figs. S5 and S6).
