Rice Genomics and Genetics 2025, Vol.16, No.4, 219-236 http://cropscipublisher.com/index.php/rgg 227 5 Case Studies of Representative Genes and Mutants 5.1 Wx gene and regulation of amylose content TheWaxy (Wx) gene is a core gene that controls the synthesis of amylose in rice and plays a decisive role in the formation of grain quality. The Wx gene encodes particle-binding starch synthase (GBSS), and its function is directly related to the level of amylose content. There are various Wx allelic variations in rice. Among them, the loss-in-function (wx) allele reduces the amylose content within the grains to less than 2%, endowing the grains with a sticky texture (such as in glutinous rice and glutinous corn). Another important variation is the aforementioned Wxa andWxb: Indicarice carrying Wxa has 25% amylose, and the rice is drier and harder. Japonica rice with 15% straight chains carrying Wxb has soft and sticky rice. Therefore, in hybrid breeding, the selection of the Wx allele can effectively alter the rice quality of the offspring. For instance, introducing Wxa into japonica rice can enhance amylose content and improve the overly soft and sticky texture. Conversely, introducing Wxb into indica rice can reduce amylose and make the rice softer. In addition to natural alleles, modern technology has also achieved targeted modification of the Wx gene. CRISPR/Cas9 gene editing was used to knock out the Wx gene, resulting in a series of artificial glutinous rice materials. For instance, research teams from Japan and China reported almost simultaneously that CRISPR was used to cause the deletion of exon 1 of Wx or the generation of an early stop codon, obtaining phenotypically stable glutinous rice strains. These edited glutinous rice varieties contain nearly zero amylose, have a soft and sticky texture, and are no different from natural glutinous rice. Moreover, they do not require years of hybridization and homozygism, which greatly accelerates the targeted breeding of glutinous rice varieties. In terms of increasing amylose, researchers have also attempted to enhance Wx expression or alter regulatory elements through gene editing. For instance, a team edited a microsatellite polymorphism ((CT)n repeat) on the Wx promoter, obtaining a series of repeat variations of different lengths, which led to gradient changes in Wx expression and amylose content, achieving fine regulation of the softness and hardness of rice (Figure 2) (Huang et al., 2020). In addition, after introducing the wild rice Wx ancestor allele Wxlv into cultivated rice, it was found that the amylose content could be reduced by approximately 5 percentage points, while the viscoelasticity of the rice was also enhanced. This indicates that some excellent Wx alleles have potential in improving the quality of rice. In breeding practice, molecular markers have been utilized to efficiently screen Wx alleles. For instance, in hybrid offspring, through functional markers of the Wx gene, individual plants carrying waxy alleles, low linear alleles and high linear alleles can be accurately distinguished, accelerating the aggregation of quality traits (Yao et al., 2010). In conclusion, whether it is the utilization of traditional variations or the creation of modern gene editing, the mechanism by which Wx genes regulate amylose content has been deeply mastered and successfully applied to rice quality breeding. From the cultivation of pure japonica glutinous rice to the development of soft rice with moderate straight-chain in hybrid indica rice, the Wx gene is a key regulatory target. It can be expected that with the enrichment of understanding of the Wx allelic variation effect and the application of new technologies, people will be able to cultivate more rice varieties that meet different consumer demands. 5.2 Functional characterization of SBE and SS gene families In addition to Wx, functional variations of other enzyme-coding genes in the starch synthesis pathway also have a significant impact on rice quality. The research on the starch branched enzyme (SBE) and soluble starch synthase (SS) families has provided us with multiple gene targets for improving the structure and properties of starch. The classic "high amylose corn" is achieved by mutating the SBEIIb gene (amylomaize, ae mutation) in corn, which increases the amylose content to over 50% (Han et al., 2022). Similarly, knocking out the homologous SBEIIb gene in rice can significantly increase the content of amylose and resistant starch in grains. A research team from NIAS in Japan silenced rice SBEIIb using RNAi technology, obtaining high-amylose rice with an amylose content of over 30%. At the same time, the starch digestion rate was significantly reduced, which is conducive to the development of low-glycemic index varieties.
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