Milling quality (MQ) and grain shape (GS) of rice ( Oryza sativa L.) are correlated traits, both determine farmers’ final profit. More than one population under multiple environments may provide valuable information for breeding selection on these MQ-GS correlations. However, suitable analytical methods for reciprocal introgression lines with linkage map for this kind of correlation remains unclear. In this study, our major tasks were (1) to provide a set of reciprocal introgression lines (composed of two BC 2 RIL populations) suitable for mapping by linkage mapping using markers/bins with physical positions; (2) to test the mapping effects of different methods by using MQ-GS correlation dissection as sample case; (3) to perform genetic and breeding simulation on pyramiding favorite alleles of QTLs for representative MQ-GS traits. Finally, with four analysis methods and data collected under five environments, we identified about 28.4 loci on average for MQ-GS traits. Notably, 52.3% of these loci were commonly detected by different methods and eight loci were novel. There were also nine regions harboring loci for different MQ-GS traits which may be underlying the MQ-GS correlations. Background independent (BI) loci were also found for each MQ and GS trait. All these information may provide useful resources for rice molecular breeding.

>Rice heading date, or flowering time, is important for yield optimization and regional adaptation. While many genes controlling this trait in rice are known, breeders often need only slight adjustments. Our study used the CRISPR/Cas9 method to edit the Hd6 gene in the early-maturing, late-season japonica rice cultivar Nanjing 46 (NJ46), creating three mutants with reduced flowering times of 9-12 d under natural conditions in Nanjing city, Jiangsu Province, China. These mutants showed higher Hd3a and RFT1 expression levels without compromising yield or eating and cooking quality, demonstrating that Hd6 gene editing is an effective precision breeding tool for shortening heading date without laborious traditional methods.

Aleurone forms the outermost layer of the rice endosperm and plays a critical role in apoplastic nutrient uptake during endosperm development. Thickening the aleurone layer has been proposed to significantly increase the nutrient content of rice grains. In this study, we used a CRISPR/Cas9-mediated precise base editing method to target OsROS1,

In order to estimate the glycemic index（GI） of rice quickly and economically, the rice varieties Zijinnuo 2, Nanjing 04062 and Ningxiyou Y16 with different amylose contents were used as the research materials. The effects of sample pretreatment methods, the addition amount and method of α-amylase and amyloglucosidase on the hydrolysis characteristics of rice starch were analyzed. The suitable pretreatment method of rice starch in vitro digestion, the suitable addition amount and method of α-amylase and amyloglucosidase were determined. The GI value of rice was estimated according to the starch hydrolysis index and hydrolysis rate. The results showed that the suitable starch in vitro digestion test scheme for estimating the glycemic index of rice was as follows: 0.75 g of polished rice was boiled with water, then extruded 30 times by artificial stick, and the volume was adjusted to 15 ml with pH 6.8 phosphate buffer. Then, 1 ml 300 U/ml α-amylase and 1 ml 50 U/ml amyloglucosidase were added synchronously to hydrolyze for 60 min. By monitoring the dynamic change of glucose content in the hydrolysate, the GI value of rice could be estimated by the starch hydrolysis index within 1 h and the hydrolysis rate at 1 h. The GI estimation values of nine rice samples indicated that the experimental method established in this study could realize the preliminary screening of low GI rice resources and genetic materials.

Grain size, determined by grain length, grain width and grain thickness, is associated with grain yield and quality. Many genes controlling grain size were cloned and their related regulatory mechanisms were clearly clarified. However, whether these genes can be directly introduced into japonica rice for grain size improvement is unknown. We edited GS9,

氮素是促进水稻物质生产和产量形成的首要因素,其高效与合理的利用是农业可持续发展的重要保障.培育含有氮高效基因的水稻品种,充分发挥氮素高效吸收和利用遗传潜力是提高氮肥利用率、减少氮肥施用量的有效途径.本研究从氮素的吸收、转运、再分配和再利用等环节,选择了OsNR2、OsNPF6.1、OsTCP19、OsLHT1和OsGRF4共5个基因作为水稻氮高效遗传改良的基因组合,根据已报道的功能位点设计得到与目标基因共分离的基因功能标记,包括6对等位特异PCR标记和1对InDel标记,并对70份常规籼稻,34份常规粳稻和84份太湖资源水稻材料进行了鉴定.结果表明,OsNR2在籼稻中分布较广,OsNPF6.1、OsTCP19、OsGRF4在籼稻中分布较少,但是均未在常规粳稻中检出;常规粳稻中仅含有OsLHT1;同时我们还筛选出2份材料同时含有OsNR2、OsNPF6.1和OsGRF4高效单倍型.本研究开发的功能标记和筛选出的材料为通过分子标记辅助选择方法培育氮高效水稻新品种提供了技术支撑.

大米是典型的淀粉丰富的食物,是全世界大多数人口尤其是亚洲人口每日主要的卡路里来源.目前水稻品种大多数属高升糖指数(全称为血糖生成指数,glycemic index,GI)水稻,会诱发由于高热量摄入而致血糖失调的健康问题.已有大量研究者在探索降低大米血糖指数的方法.本文重点对稻米升糖指数的影响因素、筛选方法、遗传基础以及遗传改良等的相关研究进行总结,并对未来的研究方向提出了一些建议.

以籼稻高产品种扬稻6号、低血糖生成指数品种Basmati、粳稻香味品种R405为亲本,进行杂交和复交,历经多代系谱选择,获得多份丰产性、抗倒性等综合农艺性状优良的新品系;通过模拟精米人体外消化试验,获得消化速度慢的品系若干份;再测定直链淀粉含量以及抗性淀粉含量,同时进行食味品质评价,筛选到生育期适中、丰产性好的偏粳型新品系南粳丝苗.南粳丝苗具有优质、低血糖生成指数(GI)等特性,多次检测结果表明,其稻米主要品质指标达国标一级优质米标准,且适口性较好,血糖生成指数为55,明显低于普通稻米,是一个适合糖尿病、高血糖、肥胖等特殊人群的低升糖指数稻米品种.南粳丝苗已在江苏宜兴江南米道等企业进行规模化生产,具有较大的市场前景.下一步拟通过育种技术进一步提高低GI水稻品种的稻谷产量潜力,并强化低GI水稻配套栽培技术的研究.

Plant Biotechnology JournalVolume 19, Issue 1 p. 11-13 Brief CommunicationOpen Access Fine-tuning the amylose content of rice by precise base editing of the Wx gene Yang Xu, Yang Xu Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, China These authors contributed equally to this work.Search for more papers by this authorQiupeng Lin, Qiupeng Lin State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China These authors contributed equally to this work.Search for more papers by this authorXiufeng Li, Xiufeng Li Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin, ChinaSearch for more papers by this authorFangquan Wang, Fangquan Wang Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorZhihui Chen, Zhihui Chen Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorJun Wang, Jun Wang Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorWenqi Li, Wenqi Li Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorFangjun Fan, Fangjun Fan Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorYajun Tao, Yajun Tao Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorYanjie Jiang, Yanjie Jiang Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorXiaodong Wei, Xiaodong Wei Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, ChinaSearch for more papers by this authorRui Zhang, Rui Zhang State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, ChinaSearch for more papers by this authorQian-Hao Zhu, Qian-Hao Zhu CSIRO Agriculture and Food, Canberra, ACT, AustraliaSearch for more papers by this authorQingyun Bu, Corresponding Author Qingyun Bu buqingyun@iga.ac.cn orcid.org/0000-0002-5386-3577 Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin, China *Correspondence (Tel +86-10-64807727; fax +86-10-64807727; email: cxgao@genetics.ac.cn (CG); Tel +86-25-84390320; fax +86-25-84390319; email:yangjie168@aliyun.com (JY); Tel +86-451-86602723; fax +86-451-86603736; email:buqingyun@iga.ac.cn (QB)) † These authors contributed equally to this work.Search for more papers by this authorJie Yang, Corresponding Author Jie Yang yangjie168@aliyun.com orcid.org/0000-0003-2355-3022 Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, China *Correspondence (Tel +86-10-64807727; fax +86-10-64807727; email: cxgao@genetics.ac.cn (CG); Tel +86-25-84390320; fax +86-25-84390319; email:yangjie168@aliyun.com (JY); Tel +86-451-86602723; fax +86-451-86603736; email:buqingyun@iga.ac.cn (QB)) † These authors contributed equally to this work.Search for more papers by this authorCaixia Gao, Corresponding Author Caixia Gao cxgao@genetics.ac.cn orcid.org/0000-0003-3169-8248 State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China *Correspondence (Tel +86-10-64807727; fax +86-10-64807727; email: cxgao@genetics.ac.cn (CG); Tel +86-25-84390320; fax +86-25-84390319; email:yangjie168@aliyun.com (JY); Tel +86-451-86602723; fax +86-451-86603736; email:buqingyun@iga.ac.cn (QB)) † These authors contributed equally to this work.Search for more papers by this author Yang Xu, Yang Xu Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, China These authors contributed equally to this work.Search for more papers by this authorQiupeng Lin, Qiupeng Lin State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China These authors contributed equally to this work.Search for more papers by this authorXiufeng Li, Xiufeng Li Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin, ChinaSearch for more papers by this authorFangquan Wang, Fangquan Wang Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorZhihui Chen, Zhihui Chen Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorJun Wang, Jun Wang Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorWenqi Li, Wenqi Li Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorFangjun Fan, Fangjun Fan Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorYajun Tao, Yajun Tao Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorYanjie Jiang, Yanjie Jiang Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, ChinaSearch for more papers by this authorXiaodong Wei, Xiaodong Wei Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, ChinaSearch for more papers by this authorRui Zhang, Rui Zhang State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, ChinaSearch for more papers by this authorQian-Hao Zhu, Qian-Hao Zhu CSIRO Agriculture and Food, Canberra, ACT, AustraliaSearch for more papers by this authorQingyun Bu, Corresponding Author Qingyun Bu buqingyun@iga.ac.cn orcid.org/0000-0002-5386-3577 Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin, China *Correspondence (Tel +86-10-64807727; fax +86-10-64807727; email: cxgao@genetics.ac.cn (CG); Tel +86-25-84390320; fax +86-25-84390319; email:yangjie168@aliyun.com (JY); Tel +86-451-86602723; fax +86-451-86603736; email:buqingyun@iga.ac.cn (QB)) † These authors contributed equally to this work.Search for more papers by this authorJie Yang, Corresponding Author Jie Yang yangjie168@aliyun.com orcid.org/0000-0003-2355-3022 Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing, China Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, China *Correspondence (Tel +86-10-64807727; fax +86-10-64807727; email: cxgao@genetics.ac.cn (CG); Tel +86-25-84390320; fax +86-25-84390319; email:yangjie168@aliyun.com (JY); Tel +86-451-86602723; fax +86-451-86603736; email:buqingyun@iga.ac.cn (QB)) † These authors contributed equally to this work.Search for more papers by this authorCaixia Gao, Corresponding Author Caixia Gao cxgao@genetics.ac.cn orcid.org/0000-0003-3169-8248 State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China *Correspondence (Tel +86-10-64807727; fax +86-10-64807727; email: cxgao@genetics.ac.cn (CG); Tel +86-25-84390320; fax +86-25-84390319; email:yangjie168@aliyun.com (JY); Tel +86-451-86602723; fax +86-451-86603736; email:buqingyun@iga.ac.cn (QB)) † These authors contributed equally to this work.Search for more papers by this author First published: 17 June 2020 https://doi.org/10.1111/pbi.13433Citations: 30 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat 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 Wxa(relatively high AC of more than 20%) and Wxb (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 Wxop/hp, WxmqorWxmp (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 inWxmq or Wxmp, 191th inWxmq and 165th in Wxop/hp allele) in the N-terminal domain of the Wxb 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 Wxb (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, Wxm5 (from T0 line B7-2/6), carrying a C2, 3, 5-to-T2, 3, 5 transition that led to P124F and R125W mutations was obtained; using TS2, four lines including Wxm6 (from T0 line B6-29, a G6, 7-to-A6, 7 transition leading to a G159K mutation), Wxm7 (from T0 line B2-25, a G6-to-C6 transversion leading to a G159A mutation), Wxm8 (from T0 line B2-25, with a G1-to-A1 transition and G6-to-C6 transversion, leading to G159A and D161N mutations) and Wxm9 (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 Wxm10 (from T0 line B2-21, a C5, 6-to-T5, 6 transition, leading to a T178I mutation) and Wxm11 (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 (Wxm5-Wxm11), we failed to find any mutations in any of the potential off-target sites (Figure 1d). Figure 1Open in figure viewerPowerPoint Fine-tuning amylose content by precise base editing of Wx in rice. (a) Diagram of the target Wxb gene. (b) Mutations in the edited T0 and T1 lines. The putative protospacer-adjacent motifs (PAMs) are shown in green. The number of altered bases in each line (coloured in red) is indicated by the letter S followed by a number. (c) A structural model of Wxb constructed using the PROTEIN DATA BANK server; mutated residues contributing to the changes of AC are shown as spheres and are coloured (P124 in apricot, R125 in blue, R158 in red violet, G159 in white, V160 in green, D161 in red, T178 in orange and Y191 in purple). (d) Analysis of potential off-target sites in the seven T1 edited lines. Red lower-case bases are mismatches to TS1-TS3; +, mutations detected; −, mutations not detected. (e) The AACs determined by iodine colorimetry of NIP, NG9108 and the edited lines. (f) GBSSI activities of the seeds of NIP and the edited lines. (g) SDS-PAGE analysis of starch granule-bound GBSSI (top) and total seed proteins (bottom) from mature seeds. M is a protein marker. (h) Comparisons of the appearance of different forms of milled rice. Scale bars, 1.75 cm. (i) The AACs of the two japonica varieties JG818, SJ18 and their corresponding Wx-edited lines. The different letters in (e), (f) and (i) indicate significant differences at P < 0.05 by Student's t-test. Data are means ± SD (n = 3). To determine the effect of these mutations on AC, we measured the apparent amylose contents (AACs) of grains from the seven mutant lines (Wxm5-Wxm11), NIP (Wxb) and a 'soft rice' control Nangeng9108 (NG9108) (Wxmp) (Figure 1e). Notably, Wxm5 had an AAC (1.4 ± 0.2%) as low as the glutinous rice. The AACs of Wxm6 (11.9 ± 0.1%), Wxm7 (11.3 ± 0.1%), Wxm10 (9.8 ± 0.2%) and Wxm11 (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 Wxm8 (5.8 ± 0.2%) and Wxm9 (4.2 ± 0.1%) lay between those of NG9108 and Wxm5. 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 Wxm5 and Wxm9 were opaque and glutinous rice-like, consistent with their low AAC. The milled grains of Wxm8, and Wxm11 were semi-translucent like those of NG9108. Interestingly, the appearance of the milled grain of Wxm6, Wxm7 andWxm10, 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 exampleWxm5, Wxm6, Wxm7 and Wxm10 (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. Wxa) 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. Acknowledgements This work was supported by grants from the National Transgenic Science and Technology Program (2019ZX08010-003), the National Natural Science Foundation of China (31701511), the National Science Foundation of Jiangsu Province (BK20170610), the National Key R&D Program of China (2018YFA0900600) and State Key Laboratory of Plant Cell and Chromosome Engineering (PCCE-KF-2020-01). Conflict of interest The authors have submitted a patent application based on the results reported in this paper. Author contributions J. Y., C. G. and Y. X. designed the research; Y. X., Q. L., X. L., F. W., Z. C., J. W., W. L., F. F., Y. T., Y. J., X. W. and R. Z. performed the research; Q. B. and C. G. contributed to the writing; and Y. X. and Q-H. Z. wrote the manuscript. References Eshed, Y. and Lippman, Z.B. (2019) Revolutions in agriculture chart a course for targeted breeding of old and new crops. Science, 366, x25. CrossrefWeb of Science®Google Scholar Li, H. and Gilbert, R.G. (2018) Starch molecular structure: The basis for an improved understanding of cooked rice texture. Carbohyd. Polym. 195, 9– 17. CrossrefCASPubMedWeb of Science®Google Scholar Li, H.Y., Prakash, S., Nicholson, T.M., Fitzgerald, M.A. and Gilbert, R.G. (2016) The importance of amylose and amylopectin fine structure for textural properties of cooked rice grains. Food Chem. 196, 702– 711. CrossrefCASPubMedWeb of Science®Google Scholar Li, Q., Huang, L., Chu, R., Li, J., Jiang, M., Zhang, C., Fan, X. et al. (2018) Down-regulation of SSSII-2 gene expression results in novel low-amylose rice with soft, transparent grains. J. Agric. Food Chem. 66, 9750– 9760. CrossrefCASPubMedWeb of Science®Google Scholar Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B. et al. (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant. 8, 1274– 1284. CrossrefCASPubMedWeb of Science®Google Scholar Momma, M. and Fujimoto, Z. (2012) Interdomain disulfide bridge in the rice granule bound starch synthase I catalytic domain as elucidated by X-Ray structure analysis. Biosci. Biotech. Bioch. 76, 1591– 1595. CrossrefCASPubMedWeb of Science®Google Scholar Teng, B., Zeng, R.Z., Wang, Y.C., Liu, Z.Q., Zhang, Z.M., Zhu, H.T. et al. (2012) Detection of allelic variation at the Wx locus with single-segment substitution lines in rice (Oryza sativa L.). Mol. Breed. 30, 583– 595. CrossrefWeb of Science®Google Scholar Zhang, J., Zhang, H., Botella, J.R. and Zhu, J. (2018) Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties. J. Integr. Plant Biol. 60, 369– 375. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Zhu, J., Zhang, C., Gu, M. and Liu, Q. (2015) Progress in the allelic variation of Wx gene and its application in rice breeding. Chinese J. Rice Sci. 29, 431– 438. CASGoogle Scholar Zong, Y., Wang, Y., Chao, L., Rui, Z., Chen, K., Ran, Y. et al. (2017) Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438– 440. CrossrefCASPubMedWeb of Science®Google Scholar Citing Literature Volume19, Issue1January 2021Pages 11-13 FiguresReferencesRelatedInformation

氮是水稻生产发育过程中所必须的大量营养元素，但是大量的氮肥投入在提高产量的同时对环境造成了严重危害。选用含有氮高效基因的水稻品种提高水稻自身的氮肥利用效率是减少氮肥使用量降低环境氮污染的最有效途径之一。 NRT1.1B 是一个影响水稻籼、粳亚种间氮肥利用效率的关键基因，主要分布在籼稻品种中。为了筛选携带 NRT1.1B 基因的粳稻资源，根据氮高效基因 NRT1.1B 与其等位基因 nrt1.1b 在功能区域存在的单核苷酸变异，设计和筛选出 NRT1.1B 的等位基因特异 PCR 功能标记 1nrt / 1NRT 。结合测序分析验证， 1nrt / 1NRT 可以准确快速鉴定出 NRT1.1B 的不同基因型。利用 1nrt / 1NRT 对 71 份籼稻品种和 134 份粳稻品种进行 NRT1.1B 基因型检测，结果表明 71 份籼稻品种均携带 NRT1.1B 基因， 134 份粳稻品种均携带 nrt1.1b 基因。进一步对 172 太湖流域地方粳稻资源和 99 份粳稻育种中间品系进行 NRT1.1B 基因型检测，结果发现粳稻品系‘常粳 144 ’携带 NRT1.1B 基因，测序分析也进一步证实了该结果。本研究为利用 NRT1.1B 改良粳稻氮高效育种提供了科学依据。

Summary Moderately rolled leaf is one of the target traits of the ideal plant architecture in rice breeding. Many genes, including homeodomain leucine zipper IV transcription factors ROC5 and ROC8 , regulating rice leaf rolling have been cloned and functionally analysed. However, the molecular mechanism by which these genes modulate leaf‐rolling remains largely elusive. In this study, we demonstrated the transcription activation activity of both ROC8 and ROC5. Overexpressing ROC8 caused adaxially rolled leaves due to decreased number and size of bulliform cells, whereas knockout of ROC8 induced abaxially rolled leaves due to increased number and size of bulliform cells. ROC8 and ROC5 each could form homodimer, but ROC8 interacted preferably with ROC5 to forms a heterodimer. Importantly, we showed that the ROC8‐ROC5 heterodimer rather than the homodimer of ROC8 or ROC5 was functional as neither overexpressing ROC8 in the ROC5 mutant nor overexpressing ROC5 in the ROC8 ‐knockout line could rescue the mutant phenotype. This was further partially supported by the identification of a large number of common differentially expressed genes in single and double mutants of roc8 and roc5 . ROC8 and ROC5 were functionally additive as the phenotype of abaxially rolled leaves was stronger in the roc5roc8 double mutant than in their single mutants. Our results provide evidence for the role of dimerization of ROC members in regulating leaf rolling of rice.

As the overuse of nitrogen fertilizer has caused series of environmental problems, improving the nitrogen use efficiency of rice crop has been drawing much attention. Since developing nitrogen-efficient rice genotypes is a feasible solution, illustrating the microbiological characteristics of the nitrogen-efficient rice genotypes is of great importance. We performed a field experiment, and planted 16 genotypes of rice. The nitrogen utilization efficiencies and yields of rice were examined. Real-time PCR and Illumina sequencing were employed to investigate the abundance of the nitrogen-cycle related genes and the bacterial community structure of the rhizosphere. The results revealed that rice genotypes enriched specific bacterial communities to inhabit in the rhizosphere at both the taxonomic and functional levels. The enriched bacteria were mainly involved in the functions of nitrogen transformation, ferric iron reduction, organic compound biodegradation, and sulfur transformation. We observed a strong correlation between the yield and the abundance of rhizospheric functional bacteria, especially the groups of Proteobacteria and Chloroflexi. The analysis of nitrite reductase and denitrifying genes showed that the denitrification activity in the rhizosphere inversely correlate with the efficiency of nitrogen utilization. We reason that a smaller denitrifying bacteria population leads to less the nitrogen loss in the rhizosphere, thus an increase of available nitrogen and a higher rice yield. This research revealed how functional bacteria assembled in the rhizosphere of rice genotypes with varied nitrogen use efficiencies, which may provide valuable information with regard to the improvement of nitrogen use efficiency and reduction of fertilizer application.

Weeds and weedy rice plague commercial rice fields in many countries. Developing herbicide-tolerance rice is the most efficient strategy to control weed proliferation. CRISPR/Cas9-mediated gene editing, which generates small InDels and nucleotide substitutions at and around target sites using error-prone non-homologous end joining DNA repairing, has been widely adopted for generation of novel crop germplasm with a wide range of genetic variation in important agronomic traits. We created a novel herbicide-tolerance allele in rice by targeting the acetolactate synthase (OsALS) gene using CRISPR/Cas9-mediated gene editing. The novel allele (G628W) arose from a G-to-T transversion at position 1882 of OsALS and conferred a high level of herbicide tolerance. Transgene-free progeny carrying homozygous G628W allele were identified and showed agronomic performance similar to that of wild-type plants, suggesting that the G628W allele is a valuable resource for developing elite rice varieties with strong herbicide tolerance. To promote use of the G628W allele and to accelerate introgression and/or pyramiding of the G628W allele with other elite alleles, we developed a DNA marker for the G628W allele that accurately and robustly distinguished homozygous from heterozygous segregants. Our result further demonstrates the feasibility of CRISPR/Cas9-mediated gene editing in creating novel genetic variation for crop breeding.

Rice yield and sustainable production are important issues for global food safety (Itoh et al., 2005). Sterility mutants are appropriate materials for understanding the molecular mechanisms underlying fertility regulation in rice, and are potential germplasm for production of hybrid seeds. The availability of the rice whole genome sequence enabled fine mapping and cloning of the key genes underlying the sterility trait and shed insights into the development of male and female gametophytes in the past decade, but our understanding on the genetic and molecular mechanisms underlying fertility remains limited. REPRESSOR OF SILENCING 1 (ROS1) encodes a bi-functional DNA demethylase that removes 5-methylcytosine and nicks double-stranded DNA (Gong et al., 2002; Tang et al., 2016). It was found that OsROS1a, a rice homolog of Arabidopsis ROS1, is indispensable for the development of gametophytes (Ono et al., 2012). A recent study reported that accumulation of an alternatively spliced ROS1 transcript reduced seed-setting by 8.2% and produced seeds with multiple layers of aleurone (Liu et al., 2018). CRISPR/Cas9 gene editing has been proved to be a powerful tool to generate knockout mutants for characterization of gene function in plants (Chen et al., 2019). In this study, we create a number of OsROS1 knockout mutants using CRISPR/Cas9 and further explored the role of OsROS1 in regulation of rice fertility. Two 20-bp guide RNAs specific to OsROS1 (LOC_Os01g11900) targeting the first (targeted site 1, TS1) and the fifteenth exons (targeted site 2, TS2) (Figure 1a) were designed, and used to generate the knockout vectors pOsCas9-TS1 and pOsCas9-TS2. Each vector was individually introduced into the rice cultivar Zhennuo19 by Agrobacterium transformation. For each target site, we identified seven mutated transgenics. Four lines were homozygous mutants; the remaining lines were bi-allelic mutants (Figure 1b). Most of the mutations caused frameshifts in the coding region, giving rise to prematurely terminated proteins, and two edited lines (line 1-3-3 and 2-4-7) contained amino acid deletions and/or substitutions. Furthermore, we failed to find any mutations in any of the potential off-target sites (Figure 1c). During the vegetative growth stage, the growth state and appearance of all the mutant lines were indistinguishable from the wild type (WT); however, during the reproductive growth stage, most of the mutants were sterile. Of the 14 T0 mutants, only two produced seeds with a seed-setting rate of 7.3 ± 2.2% (line 1-3-3) and 5.8 ± 2.9% (line 2-4-7), while the remaining 12 T0 frameshift mutants failed to produce any seed (Figure 1d). We analysed the nature of mutations by sequencing in the T1 generation of the two seed-producing T0 mutants. We found that all the 11 T1 progeny of line 1-3-3 were homozygous mutants with the same 9-bp deletion observed in the T0 plant, and that all the 9 T1 progeny of line 2-4-7 were homozygous mutants with the same 6-bp deletion found in the T0 plant. The seed-setting rates of these T1 mutants were comparable to that of the WT (Figure 1d). These results suggest that the transmission efficiencies of the mutant alleles were different. The in-frame mutant alleles could be efficiently transmitted; while all of the editing events causing frameshift could not be passed on to the next generation. These results also showed that all the frameshifted mutants of TS1 and TS2 have the same effect on rice sterility determination. Combining the results of previous studies (Ono et al., 2012), we speculated that the predominant function domain should be located in the C-terminal of OsROS1. To determine the underlying cause of the fertility defect of the gene editing mutant, we conducted hand-pollinated reciprocal crosses between the ratoon plant of the homozygous mutant line 1-1-2 and the WT. When line 1-1-2 was used as the pollen receiver, the seed-setting rate was zero; when the WT stigmas were sprinkled with the mutant pollen grains, the seed-setting rate was zero as well. Only when WT stigmas were sprinkled with its own pollen grains, the seed-setting rate was normal (Figure 1e). This finding indicates that the fertility defect of the OsROS1 knockout mutants (e.g. line 1-1-2) is due to defects in both male and female gametophytes. To dissect the cellular defects responsible for the sterility of the mutant, we compared development of the floret structure and gametophytes between line 1-1-2 and WT. The lemma, palea, glume, stamen, stigma, style and ovary of the mutant appeared to be normal, and after glume opening, the anther shape and dehiscence of the mutant were similar to that of the WT (Figure 1f). However, we observed difference in pollen fertility between the mutant and WT. I2-KI staining showed that the pollens of the WT were regularly round and deeply stained; in contrast, the mutant pollens were irregular and slightly or not stained (Figure 1g). In addition, we also examined the tricellular pollens stained with DAPI and found that the WT pollen contained one dispersed vegetative nucleus and two smaller generative nuclei; however, in the mutant, about half of the pollen grains exhibited shaded and a bicellular feature, and the other half aborted showing irregularly shaped (Figure 1h). To further characterize the pollen difference between the WT and the mutant line 1-1-2, we examined the pollen grains using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Mature WT pollen grains were spherical and plump, containing large numbers of starch granules, and the wall of which composed of exine and intine; while in the mutant, the surface of about half of the pollen grains was wrinkled, and the other half had an irregular and shrunken appearance (Figure 1i). Furthermore, the pollen grains of both abnormal types accumulated little and abnormal starch granules and their wall lacked intine (Figure 1i). These findings suggest that the pollens of the OsROS1 knockout mutants (e.g. line 1-1-2) are defective and aborted. We further examined cytologically the embryo sacs. In the WT, the mature embryo sacs were plump, and the normal-size sac cavities contained eight nuclei that could be recognized clearly, showing that antipodal cells located at the chalazal end, one egg cell and two synergid cells constituted the egg apparatus and located at the micropylar end, and two polar nuclei horizontally arranged above the egg apparatus; however, the embryo sacs were degenerated in the mutant, in which neither cavities nor nuclei could be observed (Figure 1j). At 24 h after pollination, when the double fertilization is completed in rice, almost all of the WT embryo sacs were normally fertilized each with a multi-celled globular embryo and a layer of free endosperm nuclei; while in the mutant, although the degenerated sac cavities enlarged slightly, no fertilized egg was observed at all (Figure 1j). These findings suggest that the embryo sacs of the OsROS1 knockout mutants (e.g. line1-1-2) are defective and aborted. In summary, our results indicate that OsROs1 is essential for normal development of both male and female gametophytes and provide clues for further elucidating the biological mechanisms related to OsROS1-mediated fertility regulation in rice. Moreover, we believe that the CRISPR/Cas9-mediated genome editing technology would not like to be limited to OsROS1 but applicable to investigating other genes with lethal effect on plant development. This work was supported by grants from the National Transgenic Science and Technology Program (2018ZX08001-02B), the National Natural Science Foundation of China (31701511) and the Science Foundation of Jiangsu Province (BK20170610). The authors have declared no conflict of interest. Y. X. and J. Y. designed the research; Y. X., F. W., Z. C., J. W., W. L., F. F., Y. T. and Y. J. performed the research; and Y. X. and Q-H. Z. wrote the paper.

氮是水稻生产发育过程中所必须的大量营养元素，但是大量的氮肥投入在提高产量的同时对环境造成了严重危害。选用含有氮高效基因的水稻品种提高水稻自身的氮肥利用效率是减少氮肥使用量降低环境氮污染的最有效途径之一。 NRT1.1B 是一个影响水稻籼、粳亚种间氮肥利用效率的关键基因，主要分布在籼稻品种中。为了筛选携带 NRT1.1B 基因的粳稻资源，根据氮高效基因 NRT1.1B 与其等位基因 nrt1.1b 在功能区域存在的单核苷酸变异，设计和筛选出 NRT1.1B 的等位基因特异 PCR 功能标记 1nrt / 1NRT 。结合测序分析验证， 1nrt / 1NRT 可以准确快速鉴定出 NRT1.1B 的不同基因型。利用 1nrt / 1NRT 对 71 份籼稻品种和 134 份粳稻品种进行 NRT1.1B 基因型检测，结果表明 71 份籼稻品种均携带 NRT1.1B 基因， 134 份粳稻品种均携带 nrt1.1b 基因。进一步对 172 太湖流域地方粳稻资源和 99 份粳稻育种中间品系进行 NRT1.1B 基因型检测，结果发现粳稻品系‘常粳 144 ’携带 NRT1.1B 基因，测序分析也进一步证实了该结果。本研究为利用 NRT1.1B 改良粳稻氮高效育种提供了科学依据。

水稻籽粒长宽比是影响水稻品质和产量的重要农艺性状之一,是由多基因控制的数量性状.染色体片段代换系由于可以减少分离群体中个体间遗传背景的干扰,已成为定位和克隆复杂性状QTL的重要材料.本研究利用以籼稻品种9311为背景、以粳稻品种日本晴为代换片段构建的128个经过2代重测序的染色体片段代换系群体作为试验材料,利用多元回归,结合Bin-map图谱,定位到了4个控制水稻籽粒长宽比的QTL.其中,qLWR2.1被定位在第2染色体上的812145 bp区间内,加性效应值为-0.04,加性效应百分率为-1.12％;qLWR2.2被定位在第2染色体上的324166 bp区间内,加性效应值为0.17,加性效应百分率为4.14％;qLWR3.1被定位在第3染色体上的17825 bp区间内,加性效应值为-0.25,加性效应百分率为-7.73％;qLWR11.1被定位在第11染色体上的945168 bp区间内,加性效应值为0.21,加性效应百分率为5.15％.本研究结果为精细定位并克隆相应QTL,进而探明水稻籽粒长宽比QTL的分子调控机制奠定了基础.

为了筛选江苏苏北地区稻麦周年生产力优势品种组合,选用16个主栽水稻品种和12个小麦品种为材料,设置6月15日、6月22日2个水稻播期处理;11月10日、11月30日2个小麦播期处理,探讨早播、晚播条件对水稻和小麦各产量要素及稻麦周年生产力的影响.结果表明,随着播期推迟,各水稻及小麦品种产量都有不同程度下降,其中武运粳21号、淮稻12号、连麦7号受播期影响最小.不同水稻品种构成的稻麦组合在迟播条件下周年产量损失波动较大,稻麦周年产量受水稻影响较大.其中,以华粳8号、武运粳21号、盐粳16、中稻1号和淮稻12等为代表的水稻与各小麦品种构成的周年产量受迟播影响较小.中稻1号和徐麦35的组合无论是在早播还是迟播条件下,周年产量均最高,可作为优势组合.

多胺是一类广泛存在于生物体内的小分子含氮碱,参与了植物生长发育的整个过程,并与抗逆性密切相关.目前,植物中的多胺合成代谢途径已基本揭示,多个代谢途径中编码相关酶的基因也已克隆.本文综述了植物多胺代谢途径的异同点及演化过程,重点阐述了多胺的生理功能、多胺与激素的关系以及多胺在抗性中的作用,希望给未来的生物合成和基因组育种提供帮助.

Intron-targeted gene insertion strategy using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated Cas9) has been shown to be a potential tool for crop genetic improvement by targeted mutagenesis or gene replacement of an elite allele into widely cultivated rice varieties. The rice blast resistant protein Pi-ta, differs from its susceptible counterpart, pi-ta, by a single amino acid in exon 2. To create new materials resistant to the rice blast disease, we inserted a genomic fragment containing the exon 2 and 3′ untranslated region (3′ UTR) of Pi-ta into intron 1 of pi-ta in rice materials susceptible to rice blast using the intron-targeted insertion strategy. The gene insertion frequency was 3.8%. Several novel transgene-free progeny with stably inherited homozygous insert were identified in the T1 generation, which have been crossed to rice germplasm bearing other resistance gene (R gene) for pyramiding of R genes. This work verified the feasibility of using the genome editing technology in improvement of qualitative agronomic trait in crops.