The genetic diversity and phenotypic variability of crop agronomic traits is valued by breeders for their benefits in crop breeding but are limited for most target traits. Genome editing has proved to be a powerful tool for quick and efficient creation of continuous beneficial genetic variation for crop breeding (Eshed and Lippman, 2019). The rice Waxy (Wx) gene (LOC_Os06g04200) encodes granule‐bound starch synthase I (GBSSI), which determines the amylose content (AC) of endosperm by controlling amylose synthesis. This is one of the major contributors for the eating and cooking quality (ECQ) of rice (Li et al., 2016), an attribute that is receiving increased attention in society because of the improvement in people’s living standards.

Rice AC ranges from 0 to ~30% depending on the presence of different Wx alleles, with Wx^a(relatively high AC of more than 20%) and Wx^b (intermediate AC of 14 to ~18%) being the major alleles found in the indica and japonica varieties, respectively (Teng et al., 2012). Amino acid changes in the Wx/GBSSI protein can affect the AC of rice grain, as in the well‐known 'soft rice' varieties (AC of 7%–10%) with genotypes Wx^op/^hp, Wx^mqorWx^mp (Zhu et al., 2015), which all have non‐synonymous mutations in the N‐terminal domain of Wx/GBSSI (Momma and Fujimoto, 2012). As rice varieties with moderately low AC (<12%), that is the 'soft rice' varieties, have become more popular commercially and for breeders (Li and Gilbert, 2018), both traditional and molecular breeding approaches including CRISPR/Cas9‐mediated gene knockout (Ma et al., 2015; Zhang et al., 2018) have been used to mutate Wx to reduce the AC of rice grain. However, only a limited number of Wx mutants have been generated, far fewer than needed to meet the diverse demands of ECQ. We hypothesized that the AC of rice grain could be fine‐turned by generating a series of novel amino acid substitution(s) close to the 'soft rice' allele responsible sites (such as the residues 158th inWx^mq or Wx^mp, 191th inWx^mq and 165th in Wx^op/^hp allele) in the N‐terminal domain of the Wx^b allele by state‐of‐the‐art base editing.

Based on the requirements of cytidine base editors (CBEs) (Zong et al., 2017), we designed three sgRNAs targeting the third (target site1, TS1), fourth (target site 2, TS2) or fifth (target site3, TS3) exons of Wx^b (Figure 1a), which were all close to the mentioned 'soft rice' allele responsible sites. The three sgRNAs were cloned into vector pH‐nCas9‐PBE to generate vectors PBE‐TS1, PBE‐TS2 and PBE‐TS3, respectively. The resulting plasmids were individually introduced into the japonica rice cultivar Nipponbare (NIP) by Agrobacterium‐mediated transformation. A total of 5, 10 and 7 independent T₀ transgenic lines, respectively, were generated, and 2, 5 and 2 representative edited lines (Figure 1b) were taken to the T₁ generation; only T‐DNA‐free homozygous individuals were then chosen and analysed in detail. We observed a variety of T₁ mutation types depending on the number and position of the base changes and substitutions within the editing window; these reflected the changes present in the parental lines, suggesting that the T₀alleles were faithfully transmitted to the next generation (Figure 1b). Using TS1, one line, Wx^m5 (from T₀ line B7‐2/6), carrying a C_{2, 3, 5}‐to‐T_{2, 3, 5} transition that led to P124F and R125W mutations was obtained; using TS2, four lines including Wx^m6 (from T₀ line B6‐29, a G_{6, 7}‐to‐A_{6, 7} transition leading to a G159K mutation), Wx^m7 (from T₀ line B2‐25, a G₆‐to‐C₆ transversion leading to a G159A mutation), Wx^m8 (from T₀ line B2‐25, with a G₁‐to‐A₁ transition and G₆‐to‐C₆ transversion, leading to G159A and D161N mutations) and Wx^m9 (from T₀ line B1‐68, a G₄‐to‐T₄ transversion and G₆‐to‐A₆ transition, leading to G159E and V160F mutations) were identified; in TS3, two lines including Wx^m10 (from T₀ line B2‐21, a C_{5, 6}‐to‐T_{5, 6} transition, leading to a T178I mutation) and Wx^m11 (from T₀ line B2‐21, a C₅‐to‐G₅ transversion and C₆‐to‐T₆ transition, leading to a T178S mutation) were obtained (Figure 1c). In addition, for all seven T₁ edited lines (Wx^m5‐Wx^m11), we failed to find any mutations in any of the potential off‐target sites (Figure 1d).

To determine the effect of these mutations on AC, we measured the apparent amylose contents (AACs) of grains from the seven mutant lines (Wx^m5‐Wx^m11), NIP (Wx^b) and a 'soft rice' control Nangeng9108 (NG9108) (Wx^mp) (Figure 1e). Notably, Wx^m5 had an AAC (1.4 ± 0.2%) as low as the glutinous rice. The AACs of Wx^m6 (11.9 ± 0.1%), Wx^m7 (11.3 ± 0.1%), Wx^m10 (9.8 ± 0.2%) and Wx^m11 (7.9 ± 0.1%) were all moderately but significantly lower than that of NIP (14.4 ± 0.2%), but comparable with that of NG9108 (9.6 ± 0.2%). The AACs of Wx^m8 (5.8 ± 0.2%) and Wx^m9 (4.2 ± 0.1%) lay between those of NG9108 and Wx^m5. The GBSSI activities in developing seeds of the Wx‐edited lines 10 days after flowering ranged from 231.5 ± 16.5 to 712.1 ± 54.1 nmol/g/min (Figure 1f), all lower than in NIP. The reduced GBSSI activities are likely due to the lower total amount of GBSSI protein (Figure 1g). These results demonstrate that amino acid substitutions in TS1‐TS3 indeed can reduce the total GBSSI abundance and activity and decrease the AC of seeds.

In general, the quality of the appearance of the milled rice (especially the transparency of the grain) is negatively correlated with AAC (Li et al., 2018). The milled rice grains of the 'soft rice' varieties with 7%–10% AAC (e.g. NG9108) are semi‐translucent while the glutinous rice grains with AAC < 2% are opaque. We compared the appearance of the milled rice grains (10% moisture) of the seven Wx‐edited lines (T₂ generation) with those of NIP and NG9108. As indicated in Figure 1h, the milled grains of Wx^m5 and Wx^m9 were opaque and glutinous rice‐like, consistent with their low AAC. The milled grains of Wx^m8, and Wx^m11 were semi‐translucent like those of NG9108. Interestingly, the appearance of the milled grain of Wx^m6, Wx^m7 andWx^m10, with AACs of 9.8%–11.9%, tended to be like that of NIP rather than NG9108, being almost transparent rather than semi‐translucent, indicating that we successfully generated novel germ plasms with moderately reduced AC (~10%) but without affecting the quality of the appearance of the milled rice.

The results achieved in NIP were confirmed in two other japonica varieties, Jingeng818 (JG818) and Suijing18 (SJ18), by generating T‐DNA‐free and homozygous T₁ mutants like those observed in NIP, for exampleWx^m5, Wx^m6, Wx^m7 and Wx^m10 (Figure 1i), indicating that the strategy used in this study is reliable and can be used to fine‐tune AC in elite japonica varieties.

In summary, we have used a base‐editing system to create a series of mutants with AACs of 1.4%–11.9% and have achieved the goal of fine‐tune rice AC over the range of 0%–12% to enrich the range of breeding materials available to breeders. Furthermore, we speculated that base‐editing other sites (e.g. the C‐terminal domain) and/or base editing of the varieties with other Wx alleles (e.g. Wx^a) could be available to further extend the range of AC.

This study shows that it is possible to obtain a range of mutations by substituting many individual amino acids in the critical domains of genes controlling economically important traits. This provides an important new strategy for crop breeding.

育种家对作物农艺性状的遗传多样性和表型变异性的评价是育种者对作物育种的益处，但对于大多数目标性状却受到限制。事实证明，基因组编辑是快速高效创建作物育种连续有益遗传变异的强大工具（Eshed and Lippman，2019）。水稻Waxy（Wx）基因（LOC_Os06g04200）编码与颗粒结合的淀粉合酶I（GBSSI），该酶通过控制直链淀粉的合成来确定胚乳的直链淀粉含量（AC）。这是大米的饮食质量（ECQ）的主要贡献者之一（Li等，2016），由于人们生活水平的提高，该属性正日益受到社会的关注。

根据不同的Wx等位基因的存在，水稻AC的范围为0至〜30％，其中Wx ^a（相对较高的AC超过20％）和Wx ^b（中等AC的14至〜18％）是水稻中发现的主要等位基因。分别是in稻和粳稻品种（Teng等，2012）。Wx / GBSSI蛋白中的氨基酸变化会影响稻米的AC，就像在著名的“软米”品种（AC为7％–10％）中，基因型为Wx ^op / ^hp，Wx ^mq或Wx ^mp（朱等人，2015），它们在Wx / GBSSI的N末端域均具有非同义突变（Momma和Fujimoto，2012年）。随着AC适度较低（<12％）的水稻变种（即``软米''变种）在商业上和育种者中变得越来越流行（Li and Gilbert，2018），传统和分子育种方法都包括CRISPR / Cas9介导基因敲除（Ma et al。，2015 ; Zhang et al。，2018）已用于突变Wx以降低稻米的AC。但是，只有有限数量的Wx已经产生了突变体，远远少于满足ECQ多样化需求所需的突变体。我们假设可以通过在“软水稻”等位基因负责位点附近（例如Wx ^mq或Wx ^mp的第158位，在Wx ^mp的第191位）产生一系列新的氨基酸取代来精细调节稻米的AC。通过最新的基础编辑，在Wx ^b等位基因N末端域的Wx ^op / ^hp等位基因中的Wx ^mq和165th ）。

根据胞苷碱基编辑器（CBE）的要求（Zong等，2017），我们设计了三种靶向第三个（靶位点1，TS1），第四个（靶位点2，TS2）或第五个（靶位点3，TS3）的sgRNA。Wx ^b的外显子（图1a），均与提到的“软水稻”等位基因负责位点接近。将这三个sgRNA克隆到载体pH‐nCas9‐PBE中以分别生成载体PBE‐TS1，PBE‐TS2和PBE‐TS3。通过农杆菌介导的转化将所得质粒分别导入粳稻品种日本晴（NIP）。共有5、10和7个独立的T ₀分别产生了转基因品系，并将2、5和2个代表性编辑品系（图1b）用于T ₁代；然后仅选择无T-DNA的纯合子个体并进行详细分析。我们观察到了多种T ₁突变类型，具体取决于编辑窗口中碱基变化和取代的数量和位置。这些反映了亲本系中存在的变化，表明T ₀等位基因如实地传递给了下一代（图1b）。使用TS1，一条线，蜡质^M5（在T ₀线B7-2 / 6），携带Ç _2，3，5 -to-T _2，3，5获得了导致P124F和R125W突变的转变；使用TS2，四行包括Wx ^m6（从T ₀行B6-29，由G _6、7到A _6、7过渡导致G159K突变），Wx ^m7（从T ₀行B2-25，从G 0K ₆到C _6转换导致G159A突变），Wx ^m8（来自T ₀线B2-25，具有G ₁到A ₁转换和G ₆到C _6转换，导致G159A和D161N突变）和Wx ^m9（来自T₀行B1-68，G ₄到T _4转换和G ₆到A ₆转换，导致了G159E和V160F突变。在TS3中，两条线包括Wx ^m10（从T ₀线B2-21，从C _5、6到T _5、6过渡，导致T178I突变）和Wx ^m11（从T ₀线B2-21，a获得了C ₅到G _5的转化和C ₆到T _6的转化，从而导致T178S突变（图1c）。此外，对于所有7条T ₁编辑的行（Wx ^m5 - Wx ^m11），我们未能在任何潜在的脱靶位点中发现任何突变（图1d）。

为了确定这些突变对AC的影响，我们测量了七个突变株（Wx ^m5 - Wx ^m11），NIP（Wx ^b）和“软稻”对照Nangeng9108（NG9108）的谷物的表观直链淀粉含量（AAC） （Wx ^mp）（图1e）。值得注意的是，Wx ^m5的AAC（1.4±0.2％）低至糯米。的气冷蜡质^M6（11.9±0.1％），蜡质^M7（11.3±0.1％），蜡质^M10（9.8±0.2％）和蜡质^M11（7.9±0.1％）均中等，但明显低于NIP（14.4±0.2％），但与NG9108（9.6±0.2％）相当。的气冷蜡质^M8（5.8±0.2％）和蜡质^M9（4.2±0.1％）的那些NG9108和之间铺设蜡质^M5。花后10天，Wx编辑品系的发育种子中的GBSSI活性范围为231.5±16.5至712.1±54.1nmol / g / min（图1f），均低于NIP。GBSSI活性降低可能是由于GBSSI蛋白总量较低（图1g）。这些结果表明，TS1-TS3中的氨基酸取代确实可以降低GBSSI的总丰度和活性，并降低种子的AC。

通常，碾米的外观质量（特别是谷物的透明度）与AAC呈负相关（Li等，2018）。AAC为7％–10％的“软米”品种的碾米米粒（例如NG9108）是半透明的，而AAC <2％的糯米米粉是不透明的。我们比较了Wx编辑的7条品系（T ₂代）与NIP和NG9108的碾米粒（10％水分）的外观。如图1h所示，Wx ^m5和Wx ^m9的磨粒不透明且呈糯米状，与低AAC一致。Wx ^m8的磨碎颗粒，以及Wx ^m11像NG9108一样是半透明的。有趣的是，Wx ^m6，Wx ^m7和Wx ^m10的铣削颗粒的外观，AAC为9.8％–11.9％，倾向于像NIP而不是NG9108，几乎是透明的而不是半透明的，这表明我们成功产生了具有适度降低的AC（〜10％）的新型种质，但又不影响碾米的外观质量。

在NIP中获得的结果在另外两个粳稻品种Jingeng818（JG818）和Suijing18（SJ18）中得到了证实，方法是产生无T-DNA和纯合的T ₁突变体，如在NIP中观察到的突变体，例如Wx ^m5，Wx ^m6，Wx ^m7和Wx ^m10（图1i），表明本研究中使用的策略可靠，可用于微调优良粳稻品种的AC。

总之，我们已经使用碱基编辑系统创建了一系列AAC为1.4％–11.9％的突变体，并实现了在0％–12％范围内对水稻AC进行微调的目标，以丰富AAC的范围。育种材料可供育种者使用。此外，我们推测可以对其他位点进行碱基编辑（例如C末端域）和/或与其他Wx等位基因对品种进行碱基编辑（例如Wx ^a）来进一步扩大AC的范围。

这项研究表明，有可能通过在控制经济重要性状的基因的关键域中取代许多单个氨基酸来获得一系列突变。这为作物育种提供了重要的新策略。