Hidden diversity for abiotic and biotic stress tolerances in the primary gene pool of rice revealed by a large backcross breeding program

Low and unstable rice productivity in many areas of Asia is associated with many abiotic and biotic stresses such as drought, salinity, anaerobic conditions during germination, submergence, phosphorus and zinc deficiency, etc. To develop rice varieties with tolerance to these stresses, we undertook a large backcross (BC) breeding effort for the last 6 years, using three recurrent elite rice lines and 203 diverse donors, which represent a significant portion of the genetic diversity in the primary gene pool of rice. Significant progress has been made in the BC breeding program, which resulted in development of large numbers of introgression lines with improved tolerance to these stresses. Promising lines have been developed with excellent tolerances (extreme phenotypes) to salinity, submergence and zinc deficiency; resistance to brown plant hopper, ability to germinate under the anaerobic condition and low temperature. Our results indicated that there exist tremendous amounts of ‘hidden’ diversity for abiotic and biotic stress tolerances in the primary gene pool of rice. Furthermore, we demonstrated that despite the complex genetics and diverse physiological mechanisms underlying the abiotic stress tolerances, introgression of genes from a diverse source of donors into elite genetic backgrounds through BC breeding and efficient selection (careful screening under severe stress) is a powerful way to exploit this hidden diversity for improving abiotic stress tolerances of rice. We have developed three large sets of introgression lines, which not only provide an unique platform of breeding materials for developing new rice cultivars with superior yield and stability by trait/gene pyramiding, but also represent unique genetic stocks for a large-scale discovery of genes/alleles underlying the abiotic and biotic stress tolerances of rice using genomic tools.     

应用CRISPR/Cas9技术与分子标记辅助选择创制广东丝苗米新种质

【目的】创制优质香型丝苗米水稻新种质，探索水稻育种改良新途径，助力广东丝苗米品牌建设。【方法】以广东省农业主导品种粤农丝苗(YNSM)与优质稻粤王丝苗(YWSM)为材料，利用CRISPR/Cas9基因编辑技术定点编辑上述品种的香味基因Badh2，创制香稻新品系，随后通过分子标记辅助选择(MAS)技术定向导入GW7/GL7位点，系谱选育优质香型丝苗米水稻新种质。【结果】利用CRISPR/Cas9基因编辑技术成功创制两个香稻新品系yn-ko^badh2与yw-ko^badh2，其2-AP含量均极显著提高，达到239.39～440.79μg/kg，而有效穗数、每穗实粒数、糙米外观品质、千粒重与产量等主要农艺性状均未受到显著影响；MAS技术结合系谱选育的方法成功选育两个丰产性好、籽粒长宽比超过4.3的优质香型丝苗米水稻新品系NW^badh2GW7-1与NW^badh2GW7-2，达到广东丝苗米品种关于香味与外观粒型的认定标准。【结论】利用CRISPR/Cas9基因编辑与分子辅助选择技术相结合可精准、高效地创制新的优...     

利用CRISPR/Cas9技术创制Rc基因恢复红稻

【目的】将栽培稻品种恢复为米质优、抗逆性强的红稻具有较大的研究价值。利用CRISPR/Cas9基因编辑技术，编辑原花青素转录调节因子Rc基因，恢复红种皮特性，以改良水稻米质，提升抗逆性。【方法】利用CRISPR/Cas9技术，以Rc为靶基因，构建突变载体pYLCRISPR/Cas9-Rc-gRNA，以空育180、上育453为材料，转化获得转基因植株，通过测序手段和表型观察验证成果。【结果】分子水平检测获得Rc突变材料2种，其中KY-1在1414―1417 bp缺失4个碱基，终止子突变为苯丙氨酸；SY-1在1411 bp处缺失1个碱基，终止子突变为天冬氨酸。2种编辑材料均恢复为红米表型，且具有一定耐盐碱能力。【结论】利用CRISPR/Cas9基因编辑技术成功获得恢复红种皮表型的纯合株系，为红米改良提供基础材料。     

Progress of CRISPR/Cas9 technology in breeding of Brassica napus

Gene editing technology provides important technical basics for the research in plant functional genes and crop genetic improvement. CRISPR/Cas9-mediated gene editing is an effective experimental tool for crop genome directed editing in recent years, which has been widely used in many crops as rice, wheat and other crops. CRISPR/Cas9 system was expected to be a powerful experimental tool in genetic improvement and molecular design breeding of rapeseed. This paper, which based on the development history and the latest research of CRISPR/Cas9-mediated gene editing technology in rapeseed, summarized the progress of CRISPR/Cas9 including plant type improvement, yield traits, quality improvement, disease and stress resistance improvement, yellow seed creation and other utilizes at present. The application scope, development direction and target analysis method of this technology in rape were focused. The problems of CRISPR/cas9 system in rapeseed breeding were analyzed and the improvement strategies were discussed. Finally, views on direction of rapeseed breeding by gene editing were emphasized.     

利用CRISPR/Cas9系统定向编辑水稻SD1基因

【目的】半矮秆水稻品种的选育和应用是水稻育种的最重大成果之一。半矮秆品种大多是半矮秆基因SD1(semi-dwarf1)功能缺失突变体,为了获得sd1突变体,本研究对SD1基因进行了定向编辑。【方法】利用CRISPR/Cas9系统,以SD1基因为靶基因,构建基因编辑载体CRISPR-SD1,用农杆菌介导的方法转化水稻恢复系申繁17和申繁24。【结果】在2个转化受体的T₀代均获得了纯合的sd1突变体,并且在T₁代株系中分离出了不含转基因序列的植株。2个品种的sd1突变体与各自的野生型相比,株高分别下降了25%左右。【结论】利用CRISPR/Cas9系统可以有效地对目的基因进行编辑,在水稻分子育种领域具有巨大的应用价值。     

利用CRISPR/Cas9技术编辑AFP1基因提高水稻耐逆性

目的 为鉴定水稻AFP1在非生物胁迫响应中的作用,创制非生物胁迫抗性的水稻新材料。方法 以优异籼稻恢复系华占为转化受体,利用CRISPR/Cas9技术创制afp1突变体,并对afp1突变体的耐逆性进行初步鉴定。结果 AFP1靶点1和靶点2的编辑效率分别为66.67%和75.00%。所有突变株系中,突变类型仅有插入和缺失突变,90%突变株系的突变长度为小片段突变（<5bp）。获得了6种无转基因成分的afp1纯合突变体。正常条件下,afp1突变体株高和结实率降低,有效分蘖增加,穗长显著升高,单株产量在-4.06%和11.75%之间变化。和野生型相比,afp1突变体的ABA敏感性和叶片水分散失率降低,耐干旱、热和渗透胁迫能力提高。结论 编辑AFP1基因可提高水稻多种非生物胁迫抗性。     

茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建

CRISPR/Cas9技术是一门新兴的基因组定点编辑技术,具有操作简单、高效的优点,可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术刚诞生,就受到了全球生命科学领域研究者的关注,不到3年的时间就已经成功应用于多种动、植物当中。然而CRISPR/Cas9技术在茶树中的应用面临载体构建问题,本文以茶树咖啡碱合成酶为例,联合采用常规PCR、Overlapping PCR和Golden Gate Cloning技术,构建了包含茶树咖啡碱合成酶双靶点的CRISPR/Cas9基因编辑载体,为CRISPR/Cas9介导的基因组编辑技术在茶树中的应用奠定了坚实基础。     

Improvement of Grain Shape and Fragrance by Using CRISPR/Cas9 System

【Objective】 CRISPR/Cas9-mediated genome editing has become an important way for molecular breeding in rice. To promote rice breeding, the non-fragrant japonica rice variety Longjing11 was used as the test material. This study aims to edit GS3, GS9 and Badh2 genes for obtaining valuable and stable long-grain fragrant rice materials. 【Method】 The target genes GS3, GS9 and BADH2 were selected to construct the vector pYLCRISPR/ Cas9-GS3/ GS9/Badh2-gRNA by using CRISPR/Cas9 gene editing technology. The Agrobacterium-mediated transformation was used to transform Longjing11. Specific mutations were introduced into the GS3, GS9 and Badh2 genes in Longjing 11. 【Result】 The grain length of the transgenic free homozygote gs3/gs9/badh2 increased by 26.43% to 27.01%, yield per plant by 10.82% to 12.11%, 1000-grain weight by 18.34% to 41.36%, rice became fragrant as compared with wild type of Longjing 11. This study efficiently transformed round-grain rice into long-grain fragrant rice. 【Conclusion】The homozygous mutant lines featured by stable inheritance and long-grain fragrant quality were obtained by using CRISPR/Cas9 system. This study provides a convenient and effective way of combining multiple quality traits together, which could significantly accelerate breeding process from a breeding perspective.     

利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状

【目的】基因组定点编辑技术已成为分子育种的重要手段。本研究拟对GS3和Gn1a功能缺失突变对目标性状的改良效应进行分析,以期为培育高产水稻提供理论基础。【方法】利用CRISPR/Cas9系统,以控制粒型基因GS3和控制每穗粒数基因Gn1a为编辑对象,构建了共敲除载体p C1300-2×35S::Cas9-g~(GS3)-g~(Gn1a)a,用农杆菌介导法转化4个优质水稻品种,分析了基因突变的特征和相应农艺性状。【结果】构建的敲除载体成功地实现了对GS3和Gn1a基因的定点编辑。在4个转化受体的T_0代均分别获得了gs3和gs3gn1a的移码突变体。对T_1 代中无选择标记突变体的农艺性状分析表明,突变体gs3和gs3gn1a与野生型相比粒长变长,千粒重增加;突变体gs3gn1a与突变体gs3相比,每穗粒数显著增加。【结论】利用CRISPR/Cas9系统进行水稻基因编辑可以快速改良品种的目标性状,在水稻品种的定向改良方面具有巨大的潜力。     

Defensive Role of Plant Hormones in Advancing Abiotic Stress-Resistant Rice Plants

Consistent climatic perturbations have increased global environmental concerns, especially the impacts of abiotic stresses on crop productivity. Rice is a staple food crop for the majority of the world’s population. Abiotic stresses, including salt, drought, heat, cold and heavy metals, are potential inhibitors of rice growth and yield. Abiotic stresses elicit various acclimation responses that facilitate in stress mitigation. Plant hormones play an important role in mediating the growth and development of rice plants under optimal and stressful environments by activating a multitude of signalling cascades to elicit the rice plant’s adaptive responses. The current review describes the role of plant hormone-mediated abiotic stress tolerance in rice, potential crosstalk between plant hormones involved in rice abiotic stress tolerance and significant advancements in biotechnological initiatives including genetic engineering approach to provide a step forward in making rice resistance to abiotic stress.     

Improvement of Rice Production under Drought Conditions in West Africa: Application of QTLs in Breeding for Drought Resistance

Rice plays a paramount role in food and nutrition security in many West African countries. Despite the doubling of production during the last decade, rice consumption has grown faster, creating a deficit between the demand and supply. Although the West African sub-region remains the main rice-producing centre on the continent, production is severely hampered by biotic and abiotic stresses. Drought is one of the factors that most severely reduce grain yields of rice. Systems of production need to be established in order to mitigate yield loss as a result of drought. This review discusses the effects of drought on rice production in West Africa and its mitigation with an emphasis on the improvement of tolerance to drought stress. Yield stability can be achieved by developing drought-tolerant varieties through several processes encompassing profiling of known QTLs and identification of new ones, marker-assisted selection, genomic selection, and extensive multi-locational yield trials. We suggest a comprehensive strategy for breeding drought-tolerant rice varieties in West Africa.     

CRISPR/Cas9系统在植物育种中的应用

人工核酸酶系统是在特定的基因组位点,进行切割进而利用生物内源的修复系统对目标基因进行编辑,创造新基因型的基因编辑技术。CRISPR/Cas9系统是原核生物抵御噬菌体侵染的天然适应性免疫系统。经过优化的CRISPR/Cas9编辑系统凭借操作简单,突变效率高,成本低等特点优越于其他核酸酶系统,比如锌指核酸酶系统和TALE核酸酶系统。目前,优化改造后的CRISPR/Cas9编辑系统已经在植物功能基因研究和新材料创制中得到了广泛地应用。本研究简述了CRISPR/Cas9编辑系统的结构和作用机理,归纳并论述了CRISPR/Cas9系统在植物中的编辑效率与脱靶效应和在改良作物农艺性状中的应用。最后,总结了CRISPR/Cas9系统在功能基因组学研究中应用扩展,以及展望了该系统在作物育种中的发展前景及应用价值,期望为高效利用CRISPR/Cas9系统进行新材料创制、作物品种改良提供参考。      

利用CRISPR/Cas9技术高效创制长粒香型水稻

【目的】CRISPR/Cas9基因编辑技术已成为水稻分子育种的重要手段。为了促进水稻育种的发展,本研究以非香型粳稻品种龙粳11为试验材料,对GS3、GS9和Badh2基因进行编辑,以期获得能稳定遗传的长粒香水稻材料。【方法】利用CRISPR/Cas9技术,以GS3、GS9和Badh2为靶基因,构建敲除载体pYLCRISPR/Cas9-GS3/ GS9/Badh2-gRNA,通过农杆菌介导法,在龙粳11的GS3、GS9和Badh2基因中引入了特定的突变。【结果】T₂代无转基因的gs3/gs9/badh2纯合突变体与野生型龙粳11相比,粒长增加26.43%~27.01%,单株产量增加10.82%~12.11%,千粒重增加18.34%~41.36%,稻米变香,高效地将圆粒水稻变成长粒香型水稻。【结论】利用CRISPR/Cas9技术获得能够稳定遗传并具有长粒香品质的纯合突变株系,为组合多个品质性状提供了一种方便有效的方法,从育种角度加快了新品系创制过程。     

Introgressive Hybridization in Potato Revealed by Novel Cytogenetic and Genomic Technologies

Potato is the third most important food crop in the world and is crucial to ensure food security. However, increasing biotic and abiotic stresses jeopardize its stable production. Fortunately, breeders count on a rich pool of wild relatives that provide sources for disease resistance and tolerance to environmental stresses. To use such traits effectively, breeders require tools that facilitate exploration and exploitation of the genetic diversity of potato wild relatives. Introgression programs to incorporate such alien chromatin into the crop have so far relied on cytogenetic and genetic studies to tap desired traits from these wild resources. The available genetic and cytogenetic tools, supplemented with more recent genomic technologies, can assist in the use of potato relatives in pre-breeding. This information can also facilitate cisgenesis and genome editing to improve potato cultivars. Despite the abundant and rapidly growing genomic information of potato, that of its wild relatives is still limited.     

CRISPR/Cas技术及其在作物遗传育种中的应用

简要概述了ZFN、TALEN和CRISPR/Cas系统3种基因组编辑技术的工作原理,介绍了CRISPR/Cas技术在功能基因组学研究、候选基因功能分析(验证)、分子育种方面的应用及其在作物产量、品质、抗病性及水稻雄性不育系创制上的研究进展,提出该项技术在植物遗传育种应用中的新思路及安全性等研究方向。     

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.     

利用CRISPR/Cas9技术创制高效抗除草剂水稻

【目的】培育抗除草剂品种在水稻育种中具有重要意义。利用CRISPR/Cas9基因编辑技术,以黑龙江优质粳稻品种为材料,编辑乙酰乳酸合酶ALS基因,创制具有抗除草剂特性的水稻材料。【方法】利用CRISPR/Cas9技术,以乙酰乳酸合酶ALS为靶基因,构建单碱基突变载体pH-nCas9-PBE-ALS,以松粳22、龙粳46和绥粳18为转化材料,利用农杆菌介导转化获得转基因植株,通过对转基因植株的突变位点进行测序结合除草剂喷施试验,鉴定基因型及表型。【结果】经分子水平检测验证,获得ALS^S627N突变植株10株,ALS^S627N且¹⁸⁸⁴G-A但第628位氨基酸未改变突变植株1株,ALS^S627N/G628E突变植株1株。相较于野生型,以上三类突变植株均具有较强抗除草剂特性。【结论】利用CRISPR/Cas9基因编辑技术获得具有抗除草剂特性,能够稳定遗传,不含转基因标记的纯合株系,可为抗除草剂水稻育种提供基础材料。