Improving grain quality is one of the most important goals in rice (Oryza sativa L) breeding programs. Rice quality refers to the basic characteristics of rice in commodity circulation. It includes four main aspects, milling quality, appearance quality, cooking and eating quality and nutritional quality. Among them, appearance quality, cooking and taste quality (ECQ) are particularly important(Lau et al., 2015). ECQ is determined from amylose content (AC), gel consistency (GC), gelatinization temperature (GT), and viscosity (Phing Lau et al., 2016; Tian et al., 2009). Starch accounts up to 90 % of the dry weight in the rice grain(Zhou et al., 2002). Amylose, a constituent of starch, is a major indicator of ECQ in rice (Fitzgerald et al., 2009; Li et al., 2016; Tian et al., 2009). According to the content of amylose, rice was divided into four types, glutinous (AC < 2%), soft, intermediate to low and high (AC > 25%) (Pandey et al., 2012). Generally, the higher amylose content in rice grain is, the less sticky and harder the cooked rice is, resulting in poor taste (Jobling, 2004; Juliano, 1992). However, rice with too low amylose content is too sticky and soft. Thus, it is more popular with intermediate to low amylose content in rice.
The Waxy (Wx) gene encodes granule-bound starch synthase I(GBSSI), which was cloned by Wang et al in 1990(Sano, 1984; Sano et al., 1985; Wang et al., 1990). The Wx gene is a major gene controlling amylose content in rice endosperm,and plays a decisive role on rice ECQ(Tian et al., 2009). At present, there are great differences in amylose content among cultivated rice varieties. Such vast differences mainly come fromWx gene allele variation. At least 9 Wx natural alleles have been discovered and identified in rice. Those alleles include Wxa, Wxb, Wxin, Wxop /Wxhp, Wxmq, Wxmp, Wxlv, Wxla /Wxmw, and wx (Chen et al., 2008; Dobo et al., 2010; Mikami et al., 2008; Patrick D. Larkin 2003; Teng et al., 2012; Zhang et al., 2021; Zhang et al., 2019; Zhou et al., 2021).DNA sequence difference between Wx alleles, cause variation in Wx gene expression and enzymatic activity, which leads to the difference of amylose content and quality of rice. Researchers have designed different strategies to improved rice quality, including both transgenic methods and marker-assisted selection (MAS) breeding (Jin et al., 2010; Kimiko Itoh et al., 2003; Liu et al., 2005; Phing Lau et al., 2016; Rie Terada et al., 2000; Yu et al., 2009). Especially, introducing Wxb (ACཞ16%) and Wxin (ACཞ20%) alleles into rice germplasm with high amylose content via MAS or traditional breeding greatly expedites rice quality improvement.
With the development of rice consumption specialization, the demand for amylose content in rice is increasingly diversified, which requires new ways to modulate amylose content or create new Wx alleles. The third generation genome editing technology – CRISPR/Cas9 was developed in 2012(Jinek et al., 2012; Richter et al., 2012), which has been rapidly developed and widely used in the improvement of agronomic traits(Chen et al., 2019; Fiaz et al., 2019; Gao, 2021). Up to now, CRISPR/Cas9 system can realize gene regulation efficiently and conveniently, such as knock out, knockin, substitution, single base editing and epigenetic modification, and it also can manipulate gene translation and expression by editing gene uORF and promoter(Chen et al., 2019; Rodriguez-Leal et al., 2017; Zhang et al., 2018a; Zong et al., 2017). Most impotantly, the T-DNA insertion site and gene editing target sites are in different locations, and homozygous edited mutants free of transgenic components could be selected through progeny separation(Zhu et al., 2020).Compared with the traditional transgenic methods, the CRISPR/Cas9 technology is more prone to the changes of natural mutations and has more potential to be applied in production practice. Recently, CRISPR/Cas9 mediated rice Wx gene editing have been intensevly reported. These reporters can be grouped into 4 categories. First, researchers have created new glutinous rice by completely knocking out the Wx gene (Ma et al., 2015; Zhang et al., 2018b). Second,by editing the key cis-acting elements on the Wx gene promoter, researchers have achieved fine-tune of AC in the Wxb background (Huang et al., 2020a; Zeng et al., 2020). Third, researchers have tried to regulate rice AC by manipulating the splicing efficiency of Wx gene at the post-transcriptional level (Zeng et al., 2020). Fourth, researchers have used CRISPR/Cas9-mediated base editor to is modulate rice AC by modifying GBSSI enzyme activity(Xu et al., 2020). These studies have not only paved new avenues for rice AC improvement, but also generated a plenty of new Wx alleles and added precious new knowledge about the regulation of rice Wx gene expression and the modulation of GBSSI enzyme activity. In addition, a previous study shows that the first intron of the rice Wx gene greatly enhances foreign gene expression in rice protoplasts, but not in tobacco protoplasts. When it is inserted into the 5` UTR region between the CaMV 35S promoter and GUS (β-glucoronidase ) coding sequence, GUS expression is increased 15-fold in rice protoplasts(Li et al., 1995), strongly suggesting that the first intron of the Wx gene might play an important role in regulating the Wx gene expression, presumably at the transcriptional level.
In the study, we exploited the CRISPR/Cas9 gene editing technology to remove the entire first intron of the Wx gene. The intron removal created a totally noval Wx allele, and significantly increased the amylose content in rice inbreds with the Wxb allele, while the amylose content was not changed much in rice inbreds with the Wxa allele.