Rice Genomics and Genetics 2025, Vol.16, No.4, 219-236 http://cropscipublisher.com/index.php/rgg 231 6 Strategies for Grain Quality Improvement Based on Starch Biosynthesis Regulation 6.1 Application of marker-assisted selection (MAS) and gene editing technologies The major genes related to the starch synthesis pathway, such as Wx, Alk, BADH2, etc., provide clear selective targets in the improvement of rice quality. Molecular marker-assisted selection (MAS) has been widely used to track the superior alleles of these genes. In breeding practice, by developing molecular markers closely linked to target genes, breeders can identify the genotype of plants at the seedling stage, thereby significantly improving the selection efficiency. For instance, in response to different alleles of the Wx gene (such as distinguishing Wxa from Wxb, wx, etc.), researchers have established functional InDel and SSR markers to achieve precise selection of amylose content traits. In a high-yield indica rice background, by using MAS to select the low straight-chain alleles of Wxb and eliminate Wxa, an improved variety with reduced amylose content and softer rice was successfully cultivated while maintaining the yield of the original variety (Yang et al., 2019). For instance, for the Alk gene in japonica rice that determines the gel consistency, the designed CAPS marker can effectively distinguish between high gel consistency (soft rice) and low gel consistency (hard rice) alleles, thereby guiding the introduction of soft rice alleles into the target variety (Gao et al., 2011). In conclusion, MAS makes the selection of complex quality traits efficient and reliable, especially in the utilization of heterosis and multi-gene aggregation breeding. Molecular markers can simultaneously track multiple quality genes, increasing the probability of aggregation. In the past decade, the rise of gene editing technologies (such as CRISPR/Cas9) has opened up new paths for quality improvement. Compared with traditional mutagenesis, gene editing can precisely alter the gene loci of interest, thereby directly obtaining the desired allelic variations. For the major genes in rice quality, gene editing can instantaneously "knock out" or modify the target genes in one variety without introducing additional exogenous DNA. For instance, as mentioned earlier, by knocking out the Wx gene with CRISPR, a common rice can be quickly transformed into glutinous rice. Knocking out the BADH2 gene can endow rice with a fragrant aroma. In terms of enhancing the taste of steamed and cooked rice, Japanese scholars have utilized gene editing to destroy two negative effect genes in large-grain indica rice with poor taste, significantly improving the texture without losing yield. Gene editing can also be used to precisely regulate the expression level or activity of quality genes without complete knockout. For instance, by editing the repetitive sequences of the Wx promoter, different degrees of expression inhibition were achieved, and a series of materials with gradually changing amylose content were obtained. This makes it possible to develop rice of various textures. It can be foreseen that in the future quality breeding of rice, the organic combination of conventional MAS and emerging gene editing will significantly enhance the efficiency and accuracy of breeding. For clear major effect loci, superior alleles can be screened from germplasm resources through MAS. For ideal alleles that do not exist in germplasm, they can be directly created through gene editing and introduced into breeding materials. The combined application of these two methods is expected to cultivate new rice materials that meet the target quality indicators in a targeted manner within one or two generations, thereby accelerating the breeding process of high-quality rice varieties. 6.2 Strategies for multi-gene coordinated improvement of starch quality The cooked taste and nutritional quality of rice are often the result of the combined effect of multiple starch-related traits. Therefore, multi-gene collaborative improvement is needed to achieve an overall enhancement. For instance, merely reducing the content of amylose might make the rice soft but overly sticky, which is not accepted by some consumers. If the proportion of long-chain amylopectin is appropriately increased while reducing the linear chain (increasing the viscosity of the gel), a balance can be struck between soft stickiness and elasticity. This requires the simultaneous regulation of multiple genes such as Wx and SSIIa. The strategy of aggregating multiple genes through conventional hybridization has been achieved to a certain extent, such as the combination of high-yield genes and high-quality genes in the cultivation of "super rice". However, more efficient multi-gene improvement can be achieved by using gene-edited components or stacks. In 2019, researchers from IRRI used CRISPR/Cas9 to simultaneously edit the quality genes of two restorer lines - Wx from an sterile line material and BADH2 from a restorer line. Eventually, they combined stickiness and aroma to cultivate the world's first hybrid rice with both fragrant and glutinous lines. This achievement proves that through
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