Rice is one of the most important crops. In fact, its greatest success has been achieved in the application of heterosis. For many years, researchers have devoted much attention to high-yield hybrid rice. However, in China, research on the quality of rice began fairly late, especially in the southern areas. With continued improvement in the standard of living, the quality of rice is now of greater concern than ever before. In the last 15 years, the quality traits of indica hybrid rice was improved to a certain extent; however, further improvements are still critical compared to the high-quality conventional rice, especially its eating quality (Chen et al. 2015; Tang et al. 2016). As many hybrid parents widely used from the same basic materials, the genetic polymorphism of hybrid rice may be relatively low (Li et al. 2000). Understanding the diversity and evolution of genes associated with eating quality is thus the foundation needed to fully utilize the rich genetic resources to achieve improvements in the quality of hybrid rice.
The quality of rice is a complex characteristic, with amylose content (AC) serving as a key determinant of its eating and cooking qualities (Tian et al. 2009; Qian et al. 2016). The Wx gene, encoding a granule bound starch synthase (GBSS), is a major gene in the control of AC. Its RNA expression and the activity of the OsGBSS1 protein positively correlate with AC (Liu et al. 2014). To date, at least eight Wx alleles have been identified in rice—Wxa, Wxb, Wxin, Wxop, Wxmp, Wxmw, Wxlv, and wx. The AC of the eight alleles decreases in sequence from Wxa (>25%), Wxlv (20–25%), Wxin (18–22%), Wxb (15–18%), Wxmw (10–14%), Wxmp (8–12%), and Wxop (5–10%) to wx (AC<2%) (Zhang et al. 2019; Yang et al. 2013; Liu et al. 2009; Sato et al. 1996; Mikami et al. 1999, 2000, 2008). Presently, eight Wx alleles have been identified. However, whether there are new functional alleles in the cultivated rice resources worldwide are yet to be elucidated. Nonetheless, we hope to develop a set of molecular markers for high-throughput genotyping in the Wx loci but whether the known alleles can cover most of the Wx allelic variations and explain the AC variances remain unknown. A complete understanding of the Wx allelic variations in cultivated rice worldwide is thus the premise for Wx genotyping and the efficiency of utilizing hybrid rice.
In 2015, a total of 3,000 germplasm accessions from 89 different countries/regions were selected for genome-wide resequencing, and a comprehensive SNP and InDel sub-database was established for the Rice Functional Genomics-based Breeding (RFGB) Database (Li et al. 2014). From the samples, 2,466 accessions were retrieved from a core collection established from more than 101,000 rice accessions in the IRGC while 534 accessions were selected from a core collection established from 61,470 rice accessions preserved in the CNCGB (Zhang et al., 2011). Thus, the 3000 germplasm accessions represent a panel with abundant genetic diversity in the global cultivation of rice. By utilizing the currently available genome information from the 3 K RGP sequencing data, different types of allelic variations in the Wx loci can be comprehensively analysed, and new allelic variants that are responsible for different AC classes can be discovered to improve future quality of hybrid rice.
A prior research revealed that a significant correlation exists between the number of (CT)n and AC (Larkin et al. 2003) and related microsatellite markers have also been developed. However, some of the AC variations cannot be accurately explained by these markers (Dobo et al. 2010). A series of Wx alleles has been recently identified in rice (Zhu et al. 2015), and numerous SNP genotyping methods have been used for polymorphism analysis of the Wx loci. Kompetitive Allele-Specific PCR (KASP) is a high throughput method that can type SNPs and InDels at specific sites. Based on terminal fluorescence reading, different genotypes at a single site can be detected by two-colour fluorescence (He et al. 2014) Therefore, owing to advantages such as efficiency, accuracy, and high throughput, establishing KASP to distinguish functional SNP would greatly accelerate the molecular design for breeding good quality rice.
Herein, to make better use of Wx allele resources for improving the eating-quality breeding of hybrid rice, we sought to understand the diversity of Wx alleles in cultivated rice and the evolution of the Wx genotype combination in hybrid rice. By using the 3K RGP re-sequencing data, we could identify not only the eight known alleles that widely existed in the germplasm resources, but also a potential novel allele. A set of KASP markers based on the Wx alleles was developed for high throughput genotyping of cultivated rice and the basic parent lines of hybrid rice obtained from 1976 to 2018 were selected for genotyping the Wx loci. As a result, only three Wx allelic variations were found in the main parents of the hybrids, and the allelic combination of hybrids changing from Wxlv/Wxb to Wxa/Wxbandthen to Wxb/Wxb with improvements in the quality of hybrids. Our results revealed the evolution of the eating quality of hybrid rice at the molecular level and provide a foundation for future breeding of hybrid rice with good eating quality. The present study also revealed new approaches for improving other quality traits in future hybrids.