Identification of quantitative trait loci (QTLs) for grain length (GL) is important to rice breeding for increasing grain yield and appearance quality. Recent studies have identified a number of QTLs/genes as key grain length regulators by linkage mapping or genome‐wide association study (GWAS). These regulators are involved in G protein signalling, phytohormone signalling or transcriptional regulation, etc (Li et al., 2018). However, our current knowledge on GL is still fragmented in molecular mechanism and breeding utilization in rice. Here, we detected QTLs for GL by GWAS using 210 rice accessions from rice diversity panel 1 (RDP1) (McCouch et al., 2016) and linkage mapping using a recombinant inbred line (RIL) population with 116 lines derived from geng/japonica rice Suyunuo (long grain) and Bodao (short grain) (Figure 1a). Interestingly, one co‐localized locus was identified and LOC_Os06g15620 encoding a gibberellic acid‐stimulated regulator (GASR) protein included in this region was confirmed to control GL.

A GWAS performed for GL using the efficient mixed‐model association eXpedited (EMMAX) algorithm approach identified an associated locus over the threshold on chromosome 6 (−log₁₀(1/401 085) ≈ 5.6) (Figure 1b1). This locus was further narrowed down to a 62‐Kb region by linkage mapping (Figure 1b2). According to the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/), 10 annotated genes were located within the above‐described locus (Figure 1b3). Among them, a candidate gene OsGASR7 was also previously identified in a GWAS analysis for GL (Huang et al., 2012) and was found to be responsive to gibberellins and brassinosteroids in rice (Wang et al., 2009). Moreover, the wheat orthologous counterpart of OsGASR7, TaGASR7‐A1, was found to affect GL in common wheat under multiple cultivation conditions (Dong et al., 2014), and its CRISPR/Cas9‐induced aabbdd mutant significantly elevated thousand kernel weight (Zhang et al., 2016). Recently, OsGASR7/GW6 was found to regulate grain width and weight (Shi et al., 2020). Thus, OsGASR7 is likely a candidate for controlling GL. OsGASR7 contains a 437‐bp open reading frame (ORF) with two exons and one intron, and the sequence comparison of exons between Bodao and Suyunuo revealed three SNPs and one InDel (Suyunuo to Bodao: SNP1, 58G → A; SNP2, 70G → T; SNP3, 196A → G; InDel1, +AGCAGC between 209C and 210G). These variations lead to three amino acid substitutions and additional two serines (Figure 1b4).

To confirm the effect of OsGASR7 on grain length, the functional complementation test of OsGASR7 was performed. We introduced the entire genomic region of OsGASR7^SYN from Suyunuo (SYN) into Bodao (BD) and Nipponbare (Nip) which carries OsGASR7^BD, respectively, and generated two types of transgenic complemental rice, BD‐pOsGASR7^SYN::OsGASR7^SYN (B_CP) and Nip‐pOsGASR7^SYN::OsGASR7^SYN (N_CP). As expected, both BD‐pOsGASR7^SYN::OsGASR7^SYN and Nip‐pOsGASR7^SYN::OsGASR7^SYN exhibited increases in GL, confirming the role of OsGASR7 in controlling GL (Figure 1c1–c4). As grain elongation is related to cell division and/or cell expansion, the outer lemma surfaces of mature seeds were examined by scanning electron microscopy (SEM). The results showed that there was no significant difference in cell length between Bodao and BD‐pOsGASR7^SYN::OsGASR7^SYN (Figure 1c5, c6), indicating the involvement of OsGASR7 in cell division during spikelet hull development to control GL.

In order to further explore the variations in OsGASR7 in controlling GL, we conducted OsGASR7‐based candidate gene association analysis using 2004 accessions from RFGB (Wang et al., 2019) by general linear model (GLM) algorithm. The association analysis showed that the InDel1 of 6‐bp insertion (AGCAGC) was the most significantly associated with GL (Figure 1d1). Interestingly, InDel1 locates in the variable region containing a polyglycine tract of OsGASR7, which is specific in cereal species including common wheat (Dong et al., 2014). It indicates that InDel1 is critical for grain length regulation, and we thus divided OsGASR7 into six major genotypes based on the variable region where InDel1 locates (OsGASR7^BD, OsGASR7^SYN, OsGASR7^I, OsGASR7^II, OsGASR7^III and OsGASR7^Ⅳ) (Figure 1d2).

In the 2004 rice accessions, 55.3%, 38.8%, 2.0%, 0.3%, 0.5% and 3.1% of them carry OsGASR7^BD, OsGASR7^SYN, OsGASR7I, OsGASR7II, OsGASR7III and OsGASR7^Ⅳ, respectively, and OsGASR7^SYN has the strongest effect on GL no matter in xian/indica (XI), geng/japonica (GJ) or total population (Figure 1e1). Especially in GJ, the grains of rice with OsGASR7^SYN are significantly longer than that without OsGASR7^SYN (Figure 1e1), and this is consistent with the results of complementation test. Although containing the InDel1, rice accessions with OsGASR7^I and OsGASR7^III exhibit short grains. However, these rare alleles also show variations in flanking sequence of InDel1 compared to OsGASR7^SYN, indicating that the flanking sequence or the position of InDel1 may also affect the grain length regulation. To find out whether OsGASR7^SYN has been utilized in breeding, we analysed OsGASR7 sequences of 42 cultivars. Compared with the distribution of OsGASR7^SYN in XI of 2004 accessions, that in XI cultivars has increased (57% versus 74%) (Figure 1e). What is more, the grains of cultivars with OsGASR7^SYN are still longer than those without OsGASR7^SYN (non‐OsGASR7^SYN) (Figure 1e2). These results show that OsGASR7^SYN has been likely selected in many XI cultivars.

At last, we examined the subcellular localization of OsGASR7^SYN and OsGASR7^BD protein carrying green fluorescent protein (GFP) in rice protoplast expressing a nuclear marker OsD53‐mCherry. Both fluorescence of OsGASR7^SYN‐GFP and OsGASR7^BD‐GFP were mainly observed in cytoplasm (Figure 1f). Through in silico gene expression analysis, it was found that OsGASR7 was regulated by brassinolide treatment and accumulated in panicles, suggesting that OsGASR7 may involve brassinosteroid (BR) pathway to regulate grain length in rice.

Functions of some GASR genes have been studied in rice. OsGASR1 controls seedling growth and amylase production (Lee et al., 2017), and OsGASR9 plays a positive role in the response to GA and grain development (Li et al., 2019). In this work, we confirmed that a GASR protein OsGASR7 is responsible for grain length regulation in rice by GWAS, linkage analysis and transgenic complemental study. More importantly, a natural variation of 6‐bp insertion (AGCAGC) in OsGASR7 was found to be significantly associated with GL and could be potentially used as a molecular marker for rice breeding. The allele variations in different rice cultivars may lead to the functional diversity of OsGASR7 observed in the studies of GW6 (Shi et al., 2020) and this work. Further studies will be conducted to clarify how OsGASR7 modulates grain length and the role of additions of two serines in OsGASR7 function and regulation.

鉴定籽粒长度（GL）的数量性状基因座（QTL）对于提高水稻产量和外观质量至关重要。最近的研究已经通过连锁作图或全基因组关联研究（GWAS）将许多QTL /基因鉴定为关键的粒长调节剂。这些调节剂参与G蛋白信号转导，植物激素信号转导或转录调节等（Li等，2018）。但是，我们目前对GL的了解仍然在水稻的分子机制和育种利用方面尚不完善。在这里，我们使用了水稻多样性研究小组1（RDP1）的210份水稻材料，通过GWAS检测到了GL的QTL（McCouch et al。，2016）和使用重组自交系（RIL）群体的连锁图谱，该群体具有来自耿/粳稻Suyunuo（长粒）和Bodao（短粒）的116个品系（图1a）。有趣的是，鉴定出一个共定位的基因座，并证实了编码该区域内包含赤霉素刺激的调节蛋白（GASR）的LOC_Os06g15620可控制GL。

使用有效的混合模型关联证明（EMMAX）算法为GL执行的GWAS确定了在6号染色体上的阈值之上的相关基因座（-log ₁₀（1/401 085）≈5.6）（图1b1）。通过连锁作图，该基因座进一步缩小到62-Kb区域（图1b2）。根据水稻基因组注释项目（http://rice.plantbiology.msu.edu/），在上述基因座内有10个注释基因（图1b3）。其中，先前还在GLAS的GWAS分析中鉴定了候选基因OsGASR7（Huang等，2012），发现该基因对水稻中的赤霉素和油菜素甾体有反应（Wang等，2009）。）。此外，小麦直系同源配对OsGASR7，TaGASR7 - A1，被发现影响GL在多个培养条件下普通小麦（冬等人，。2014，和它的CRISPR / Cas9诱导）AABBDD突变显著升高的千粒重（张等人，2016）。最近，发现OsGASR7 / GW6调节晶粒的宽度和重量（Shi等，2020）。因此，OsGASR7可能是控制GL的候选对象。OsGASR7含有一个437bp的开放阅读框（ORF），带有两个外显子和一个内含子，Bodao和Suyunuo之间的外显子序列比较显示了三个SNP和一个InDel（Suyunuo与Bodao：SNP1，58G→A; SNP2，70G→T ； SNP3，196A→G； InDel1，+ AGCAGC（介于209C和210G之间）。这些变异导致三个氨基酸取代和另外两个丝氨酸（图1b4）。

为了确认效果OsGASR7上粒长，的功能互补试验OsGASR7进行。我们将来自Suyunuo（SYN）的OsGASR7 ^SYN的整个基因组区域分别引入了携带OsGASR7 ^BD的Bodao（BD）和Nipponbare（Nip），并生成了两种类型的转基因互补水稻，BD-p OsGASR7 ^SYN :: OsGASR7 ^SYN（ B_CP）和Nip-p OsGASR7 ^SYN :: OsGASR7 ^SYN（N_CP）。如预期的那样，BD-p OsGASR7 ^SYN :: OsGASR7 ^SYN和Nip-p OsGASR7 ^SYN:: OsGASR7 ^SYN的GL升高，证实了OsGASR7在控制GL中的作用（图1c1-c4）。由于籽粒伸长与细胞分裂和/或细胞扩增有关，因此通过扫描电子显微镜（SEM）检查了成熟种子的外表面。结果表明，Bodao和BD-p OsGASR7 ^SYN :: OsGASR7 ^SYN之间的细胞长度没有显着差异（图1c5，c6），表明OsGASR7参与了小穗壳发育以控制GL的细胞分裂。

为了进一步探索OsGASR7在控制GL中的变异，我们使用2004年RFGB（Wang等人，2019）的材料通过通用线性模型（GLM）算法进行了基于OsGASR7的候选基因关联分析。关联分析表明，插入6 bp的InDel1（AGCAGC）与GL的关联最明显（图1d1）。有趣的是，InDel1位于包含OsGASR7的聚甘氨酸束的可变区，该序列在包括普通小麦在内的谷物品种中具有特异性（Dong等，2014）。这表明InDel1对晶粒长度调节至关重要，因此我们划分了OsGASR7成基于所述可变区6种主要的基因型，其中InDel1所处（OsGASR7 ^BD，OsGASR7 ^SYN，OsGASR7^我，OsGASR7 ^II，OsGASR7 ^III和OsGASR7 ^Ⅳ）（图1D2）。

在2004年的大米加入，55.3％，38.8％，2.0％，0.3％，0.5％和它们的3.1％携带OsGASR7 ^BD，OsGASR7 ^SYN，OsGASR7 I，OsGASR7 II，OsGASR7 III和OsGASR7 ^Ⅳ，分别和OsGASR7 ^SYN具有无论在西安/印度（XI），耿/粳稻（GJ）还是总人口中，对GL的影响最强（图1e1）。尤其是在GJ中，含有OsGASR7 ^SYN的水稻的籽粒明显比没有水稻的更长。OsGASR7 ^SYN（图1e1），这与互补测试的结果一致。尽管包含InDel1，但带有OsGASR7 ^I和OsGASR7 ^III的水稻种质却表现出短粒状。然而，与OsGASR7 ^SYN相比，这些稀有等位基因还显示InDel1的侧翼序列变异，表明侧翼序列或InDel1的位置也可能影响晶粒长度调节。为了确定OsGASR7 ^SYN是否已用于育种，我们分析了42个品种的OsGASR7序列。与分布比较OsGASR7 ^SYN在XI在2004年的收成中，XI品种的收成有所增加（57％对74％）（图1e）。而且，具有OsGASR7 ^SYN的品种的籽粒仍比没有OsGASR7 ^SYN的品种（非OsGASR7 ^SYN）更长（图1e2）。这些结果表明，在许多XI品种中都可能选择了OsGASR7 ^SYN。

最后，我们在表达核标记OsD53-mCherry的水稻原生质体中检测了带有绿色荧光蛋白（GFP）的OsGASR7 ^SYN和OsGASR7 ^BD蛋白的亚细胞定位。OsGASR7 ^SYN- GFP和OsGASR7 ^BD- GFP的荧光都主要在细胞质中观察到（图1f）。通过计算机基因表达分析，发现OsGASR7受到油菜素内酯处理的调控并积聚在穗中，提示OsGASR7可能参与油菜素类固醇（BR）途径调控水稻的籽粒长度。

在水稻中已经研究了一些GASR基因的功能。OsGASR1控制幼苗的生长和淀粉酶的产生（Lee等人，2017），而OsGASR9在对GA和谷物发育的响应中起积极作用（Li等人，2019）。在这项工作中，我们证实了GASR蛋白OsGASR7负责通过GWAS，连锁分析和转基因互补研究对水稻的籽粒长度进行调控。更重要的是，6-bp的插入在自然变化（AGCAGC）OsGASR7被发现与GL显着相关，并有可能被用作水稻育种的分子标记。在GW6的研究（Shi等人，2020）和这项工作中，不同水稻品种的等位基因变异可能导致OsGASR7的功能多样性。将进行进一步的研究以阐明OsGASR7如何调节晶粒长度以及添加两个丝氨酸对OsGASR7功能和调节的作用。