Combining QTL mapping and expression profile analysis to identify candidate genes of cold tolerance from Dongxiang common wild rice(Oryza rufipogon Griff.)

Rice(Oryza sativa L.), a tropical and subtropical crop, is susceptible to low temperature stress during seedling, booting, and flowering stages, which leads to lower grain quality levels and decreasing rice yields. Cold tolerance is affected by multiple genetic factors in rice, and the complex genetic mechanisms associated with chilling stress tolerance remain unclear. Here, we detected seven quantitative trait loci(QTLs) for cold tolerance at booting stage and identified one cold tolerant line, SIL157, in an introgression line population derived from a cross between the indica variety Guichao 2, as the recipient, and Dongxiang common wild rice, as the donor. When compared with Guichao 2, SIL157 showed a stronger cold tolerance during different growth stages. Through an integrated strategy that combined QTL-mapping with expression profile analysis, six candidate genes, which were up-regulated under chilling stress at the seedling and booting developmental stages, were studied. The results may help in understanding cold tolerance mechanisms and in using beneficial alleles from wild rice to improve the cold tolerance of rice cultivars through molecular marker-assisted selection.     

东乡野生稻苗期耐冷性的QTL定位

【目的】研究东乡野生稻苗期耐冷性的QTL和连锁标记,为水稻耐冷种质资源的利用及分子标记辅助选择育种提供理论和实践依据。【方法】以东乡野生稻作为非轮回亲本,南京11号为轮回亲本,构建144株BC2F1分离群体。通过SSR标记以根电导率作为耐冷性指标,以复合区间定位法对东乡野生稻苗期耐冷性进行QTL定位。【结果】检测到2个QTL qRC10-1和qRC10-2均位于第10染色体,对表型的贡献率分别为34.13%和37.02%,是两个主效的QTL。在与2个QTL的连锁标记RM171周围发展分子标记进一步定位,检测到3个QTL位于标记RM171附近。【结论】东乡野生稻第10染色体上的2个QTLqRC10-1,qRC10-2与苗期耐冷性有关,并位于SSR标记RM304-RM1108区间,可用于水稻耐冷性分子标记辅助选择育种。     

Genetic Diversity and Genetic Changes in the Introgression Lines Derived from Oryza sativa L. Mating with O. rufipogon Griff.

The objectives of the present study were to estimate genetic diversity and genetic changes of introgression lines (ILs) which derived from cultivated rice (Oryza sativa L. cv. Xieqingzao B, XB) mating with common wild rice (O. rufipogon Griff., CWR). The genetic data of 239 ILs were based on a total of 131 polymorphic microsatellite (SSR) markers distributed across the 12 chromosomes of rice. On average, these ILs possessed 77.1 and 14.31% homozygous bands from XB and CWR, respectively. Most of the ILs were clustered together with XB individual, which was revealed by principal coordinate analysis (PCA) and the program STRUCTURE analysis. The result from PCA demonstrated that some intermediate genotypes between XB and CWR were also found. Moreover, there were some genomic sequence changes including parental bands elimination and novel bands emergence in the ILs. The average Nei's gene diversity (He) was 0.296, which was higher than that of cultivated rice. It suggested that interspecific hybridization and gene introgression could broaden the base of genetic variation and lay an important foundation for rice genetic improvement. These different genotypic ILs would provide a better experimental system for understanding the evolution of rice species and the mechanism of alien gene introgression.     

利用SSR标记定位东乡野生稻苗期耐冷性基因

应用由 2 13个株系组成的协青早B/东乡野生稻的BC1F1群体 ,分析了东乡野生稻的耐冷基因与DNA标记的连锁关系。以苗期的死苗率为指标 ,对亲本和BC1F1群体各株系进行耐冷鉴定。结果表明 :死苗率在BC1F1群体中呈连续分布 ,表明耐冷性是由多基因控制的数量性状。应用单因素方差分析法 ,分别在第 4、第8染色体上发现有与耐冷性连锁的SSR标记RM 2 80、RM 337。     

东乡野生稻遗传资源的保护及其在育种上的利用

野生稻是水稻育种的重要遗传资源。东乡野生稻是普通野生稻的一种。由于人类的经济活动，导致东乡野生稻生境丧失，生境质量持续恶化，栖息地越来越少及外来种的入侵。对东乡野生稻的保护措施主要有就地保护（原地保护或原位保护）和迁地保护（易地保护或异位保护）。东乡野生稻具有许多优良特性，如特强的耐冷性，耐旱性，耐瘠性，抗病虫害，特强的再生性，广亲和性，胞质雄性不育基因及恢复性等。有些优良特性已被用于水稻常规育种和杂交育种中，并取得了巨大的社会效益和经济效益。有些优良特性还有待进一步的开发利用。     

Identification of a Resistance Gene bls1 to Bacterial Leaf Streak in Wild Rice Oryza rufipogon Griff.

Bacterial leaf streak (BLS) of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a worldwide destructive disease. Development of resistant varieties is considered to be one of the most effective and eco-friendly ways to control the disease. However, only a few genes/QTLs having resistance to BLS have been identified in rice until now. In the present study, we have identified and primarily mapped a BLS-resistance gene, bls1, from a rice line DP3, derived from the wild rice species Oryza rufipogon Griff. A BC₂F₂ (9311/DP3//9311) population was constructed to map BLS-resistance gene in the rice line DP3. The segregation of the resistant and susceptible plants in BC₂F₂ in 1:3 ratio (χ²=0.009, χ²_{0.05, 1}=3.84, P>0.05), suggested that a recessive gene confers BLS resistance in DP3. In bulked segregant analysis (BSA), two SSR markers RM8116 and RM584 were identified to be polymorphic in resistant and susceptible DNA bulks. For further mapping the resistance gene, six polymorphic markers around the target region were applied to analyze the genotypes of the BC₂F₂ individuals. As a result, the BLS-resistant gene, designated as bls1, was mapped in a 4.0-cM region flanked by RM587 and RM510 on chromosome 6.     

水稻多逆境响应基因OsMsr13的克隆与功能分析

为了深入研究作物耐逆境胁迫的分子机制和发现新的水稻耐逆基因,采用Affymetrix基因芯片技术与水稻表达芯片(含51,279个转录本),分析了培矮64S不同生长发育时期、不同组织器官全基因组在低温、干旱、高温逆境胁迫下的表达水平变化,筛选出一批耐多逆境候选功能基因.OsMsr13是其中一个受低温、高温与干旱诱导,在各生长发育时期与组织器官均显著上调的基因.根据序列生物信息学分析,OsMsr13含有7个外显子和6个内含子,cDNA全长序列为1603bp,完整的ORF为1317bp,编码一个含438个氨基酸残基的蛋白质；在OsMsr13启动子区域发现多个与逆境应答相关的顺武作用元件；数据库查找和蛋白质多序列的比对表明Os Msr13的预测蛋白属于钙调素结合蛋白家族.为了进一步研究OsMsr13基因的功能,通过RT-PCR方法扩增到包含OsMsr13完整ORF的cDNA克隆,构建了含有OsMsr13重组基因的过量表达载体pCAM-JIT-OsMsr13,并经农杆菌介导法将重组基因导入到水稻中.转OsMsr13株系苗期的抗逆性试验表明OsMsr13过量表达能显著地提高水稻的耐寒性,但在抗旱和耐高温方面,转基因植株和野生型9311没有观察到多大差别.     

中菲水稻病毒病的比较研究Ⅴ．水稻种质对病毒及其介体的抗性

采用网箱集团接种结合玻管定苗定虫接种技术，测定比较了中菲栽培稻的８１个品种和野生稻的３个种，对两国水稻东格鲁病和水稻草状矮化病的病原病毒及其介体的抗性，筛选出了能同时抗水稻东格鲁球状病毒（ＲＴＳＶ）、水稻东格鲁杆状病毒（ＲＴＢＶ）和水稻草状矮化状病毒（ＲＧＳＶ）的３个株系以上的多抗性抗源─中山红无名种、Ｏｒｙｚａｎｉｖａｒａ和Ｏ．ｏｆｆｉｃｉｎａｌｉｓ，高抗这些病毒两个株系以上的多抗性品种─汕优桂３３、金早６号、ＨＢ４３７、Ｐａｎｋｈａｒｉ２０３、ＩＲ２８、ＩＲ５８和赤块矮选．高抗ＲＴＳＶ、ＲＴＢＶ介体黑尾叶蝉的品种─ＩＲ２８、ＧａｍＰａｉ３０１２－１５和Ｐａｎｋｈａｒｉ２０３，高抗ＲＧＳＶ介体褐飞虱的品种─ＩＲ２８和ＩＲ６４．     

Genetic diversity analysis and GWAS reveal the adaptive loci of milling and appearance quality of japonica rice (Oryza sativa L.) in Northeast China

Milling and appearance quality are important contributors to rice grain quality. Abundant genetic diversity and a suitable environment are crucial for rice improvement. In this study, we investigated the milling and appearance quality-related traits in a panel of 200 japonica rice cultivars selected from Liaoning, Jilin and Heilongjiang provinces in Northeast China. Pedigree assessment and genetic diversity analysis indicated that cultivars from Jilin harbored the highest genetic diversity among the three geographic regions. An evaluation of grain quality indicated that cultivars from Liaoning showed superior milling quality, whereas cultivars from Heilongjiang tended to exhibit superior appearance quality. Single- and multi-locus genome-wide association studies (GWAS) were conducted to identify loci associated with milling and appearance quality-related traits. Ninety-nine significant single-nucleotide polymorphisms (SNPs) were detected. Three common SNPs were detected using the mixed linear model (MLM), mrMLM, and FASTmrMLM methods. Linkage disequilibrium decay was estimated and indicated three candidate regions (qBRR-1, qBRR-9 and qDEC-3) for further candidate gene analysis. More than 300 genes were located in these candidate regions. Gene Ontology (GO) analysis was performed to discover the potential candidate genes. Genetic diversity analysis of the candidate regions revealed that qBRR-9 may have been subject to strong selection during breeding. These results provide information that will be valuable for the improvement of grain quality in rice breeding.     

栽培稻芽期耐低温全基因组关联分析

【目的】水稻是重要的粮食作物,芽期是水稻生长发育过程中最脆弱的时期,直播稻遭遇冷害时发芽率大幅降低,减产严重。深入了解耐冷性的遗传机制,为培育芽期强耐受性水稻品种奠定基础。【方法】以世界范围内14个国家代表性的238份水稻种质资源为试验材料,于2021和2022年在沈阳开展表型鉴定试验,统计不同水稻品种在人工气候培养箱15℃低温条件下第1—10天的发芽率和相对发芽率,利用R语言绘制5—10 d的频率直方图,通过表型丰富度Hill值选择宜作关联分析的天数,将发芽率和相对发芽率表型数据与重测序数据相结合,进行基于混合线性模型MLM（QK）的全基因组关联分析,并对所获得的SNP位点进行耐冷候选基因的预测。【结果】发芽率频数分布直方图和表型丰富度计算结果显示第8天发芽率多态性最好,其Hill值为0.84,高于其他几天发芽率（0.48—0.83）,可用于全基因组关联分析;主成分分析结果显示,这些水稻品种可以分为indica、aus、temperate japonica、tropical japonica和aromatic 5个亚群;2个指标进行的GWAS分析检测到3个相同的显著性SNP位点,均位于第4染色体,解释表型的11.9%—25.4%;在上下游各50 kb进行基因搜索,共发现24个相关候选基因,进一步开展LD和单倍型分析,发现LOC_Os04g24840和LOC_Os04g25140的不同单倍型耐冷性之间存在极显著差异。LOC_Os04g24840被编码区SNP分为5个单倍型,且Hap_3的耐冷性显著强于Hap_1;LOC_Os04g25140被编码区SNP分为18个单倍型,且77 bp处的氨基酸变异（S>L）存在籼粳差异。结果表明,编码糖基转移酶的基因LOC_Os04g24840和编码F-box蛋白基因LOC_Os04g25140可能与水稻芽期耐冷性密切相关。【结论】在238份水稻种质资源中共检测到3个与芽期耐冷性显著关联的SNP位点,筛选出2个与水稻芽期耐冷性相关的候选基因。     

野生稻对潜根线虫的抗性调查

在水稻分蘖期和乳熟期,调查了9份野生稻及4份栽培稻对潜根线虫的田间抗性,结果表明:根内线虫量栽培稻大于野生稻,普通野生稻大于其他类型野生稻;栽培稻和普通野生稻受多种潜根线虫侵染,药用野生稻、南方野生稻和尼瓦拉野生稻上仅发现水稻潜根线虫(Hirschnanniella oryzae),表明野生稻对潜根线虫种间存在选择抗性.     

水稻低温应答基因OsLCL3的分离与功能鉴定

为探明水稻响应低温的分子机制,改良水稻对低温的抗性,从水稻低温诱导表达谱中挑选了1个受低温诱导的基因OsLCL3,通过实时定量PCR和启动子驱动GUS报告基因转化水稻的方法验证其功能。对OsLCL3进行同源蛋白搜索,发现它属于DUF761超家族,并对这个超家族中的部分蛋白序列进行了系统树分析;通过水稻原生质体瞬时表达系统,初步确定OsLCL3蛋白定位于细胞核和细胞质中。在水稻中超量表达OsLCL3基因,并对转基因植株进行低温胁迫处理,发现超量表达OsLCL3的植株在低温胁迫下的电导率显著低于野生型对照植株,胁迫后超量表达植株的存活率显著高于野生型对照植株。结果表明,OsLCL3可能在水稻对低温的应答中起着重要作用。     

Effects of Phosphate Fertilizer on Cold Tolerance and Its Related Physiological Parameters in Rice Under Low Temperature Stress

The study was designated to explore the physiological mechanism of cold tolerance enhanced by phosphate in rice. An experiment was conducted to investigate the effects of different levels of phosphate ...     

云南粳亚种生态群间孕穗期耐冷性遗传研究

用热带粳、光壳稻和普通粳群中7个不同耐冷级别品种及其1／2P(P—1)配制的21个组合进行云南粳亚种生态群间穗期耐冷性遗传研究。结果表明：(1)云南高原冷凉稻区鉴定出孕穗期耐冷性同属极弱级热带粳、光壳稻与普通粳型所携的耐冷基因不同与冷驯化有关,粳亚种的孕穗期耐冷性划分为热带粳、光壳稻和普通粳3大类型。(2)耐冷性弱的不同生态群品种为背景,推断昆明小白谷、丽粳2号和半节芒穗期耐冷基因的显隐关系、耐冷性的受控基因对数及其基因效应存在明显的差别。     

水稻芽期耐寒性综合评价及耐寒指标筛选

为筛选水稻芽期耐寒指标并建立可靠的水稻芽期耐寒性数学评价模型,采用49个水稻种质资源,测定发芽率(GR)、相对发芽率(RGR)、发芽势(GP)、相对发芽势(RGP)、发芽指数(GI)、种子萌发指数(PI)、平均发芽时间(MGT)、成苗率(SR)和萌发系数(GC)9个指标并进行主成分及隶属函数等分析,对水稻芽期耐寒性进行综合评价。结果表明,根据水稻芽期耐寒性综合评价值(D)可以将49个水稻品种分为5个等级,分别是耐寒性极强品种(IRIS 313-11965、IRIS 313-8956和IRIS 313-11038,D为0.790～0.830)、耐寒性强品种(IRIS 313-11944等17个品种,D为0.609～0.745)、耐寒性中等品种(IRIS 313-11049等7个品种,D为0.503～0.576)、耐寒性弱品种(IRIS313-11986等13个品种,D为0.364～0.482)和耐寒性极弱品种(IRIS 313-10891等9个品种,D为0.187～0.282)。采用逐步回归分析法建立水稻芽期耐寒性的最优回归方程:D=0.326+0.020GI+0.021SR-0.028MGT+0.025PI+0.012GR+0.137RGP,筛选出发芽率、相对发芽势、发芽指数、种子萌发指数、平均发芽时间和成苗率6个与水稻芽期耐寒性显著相关的指标。低温胁迫条件下,可通过测定这6个指标对水稻品种的芽期耐寒性进行快速准确的鉴定及评价。     

Dissecting the genetic basis of maize deep-sowing tolerance by combining association mapping and gene expression analysis

Deep-sowing is an important method for avoiding drought stress in crop species, including maize. Identifying candidate genes is the groundwork for investigating the molecular mechanism underlying maize deep-sowing tolerance. This study evaluated four traits (mesocotyl length at 10 and 20 cm planting depths and seedling emergence rate on days 6 and 12) related to deep-sowing tolerance using a large maize population containing 386 inbred lines genotyped with 0.5 million high-quality single nucleotide polymorphisms (SNPs). The genome-wide association study detected that 273 SNPs were in linkage disequilibrium (LD) with the genetic basis of maize deep-sowing tolerance. The RNA-sequencing analysis identified 1 944 and 2 098 differentially expressed genes (DEGs) in two comparisons, which shared 281 DEGs. By comparing the genomic locations of the 273 SNPs with those of the 281 DEGs, we identified seven candidate genes, of which GRMZM2G119769 encoded a sucrose non-fermenting 1 kinase interactor-like protein. GRMZM2G119769 was selected as the candidate gene because its homologs in other plants were related to organ length, auxin, or light response. Candidate gene association mapping revealed that natural variations in GRMZM2G119769 were related to phenotypic variations in maize mesocotyl length. Gene expression of GRMZM2G119769 was higher in deep-sowing tolerant inbred lines. These results suggest that GRMZM2G119769 is the most likely candidate gene. This study provides information on the deep-sowing tolerance of maize germplasms and identifies candidate genes, which would be useful for further research on maize deep-sowing tolerance.     

筛选和转化药用野生稻TAC克隆获得耐旱水稻

【目的】利用分子生物学技术建立一种可有效转移药用野生稻有利基因的方法,为进一步大量开展利用药用野生稻有利基因提供技术基础。【方法】以植物抗性相关转录因子AP2/EREBP和bZIP家族基因保守序列为探针,采用同位素α-32P对探针进行标记,通过Southern 杂交对药用野生稻TAC文库进行第一轮筛选;所获得阳性克隆用地高辛标记的探针进行第二轮筛选;最后利用PCR方法对第二轮筛选所获得的阳性克隆进行验证。通过农杆菌介导法将阳性克隆转入栽培稻品种日本晴,利用TAC载体插入片段两端的抗性标记Hpt和SacB,通过PCR扩增检测和和Southern杂交对转化植株进行鉴定。采用PEG胁迫法和生理指标变化对转基因T2植株的芽期、苗期耐旱性进行鉴定。【结果】第一轮TAC文库筛选共获得1 073个阳性克隆,其中,以AP2/EREBP的保守序列为探针筛选出606个、bZIP为探针筛选出467个。对这些阳性克隆进行第二轮筛选,从中共获得147个阳性克隆,其中,AP2/EREBP为探针筛选出95个、bZIP为探针筛选出52个。PCR验证有103个阳性克隆能够扩增出目的片段,分别是利用AP2/EREBP设计的引物筛选出63个克隆和bZIP的40个克隆,阳性克隆得率分别为66.32%和76.92%。通过农杆菌转化,以编号为52D-M16和22D-P2（与AP2/EREBP相关）以及55R-A17、49R-O14和8-A24（与bZIP相关）的克隆转化水稻日本晴均获得转化植株,转化试验结果发现外源插入片段越大则克隆转化的成苗率越低。抗性鉴定、PCR和Southern杂交结果均证明外源片段已转入受体水稻品种日本晴中。芽期抗旱性鉴定表明R12-23、R15-41和R1-8 3份材料的耐旱性优于受体日本晴,苗期鉴定显示M63-9和R12-23的耐旱性较强。综合认为,R12-23具有较好的耐旱性。【结论】通过构建药用野生稻TAC文库并结合特异克隆筛选和遗传转化方法,可以进行有利基因的转移,结合进一步的育种,有望实现药用野生稻在育种上的利用。     

Study on Difference in Cold Tolerance Between Weedy Rice and Cultivated Rice in Heilongjiang Province of China

A long-red awn weedy rice and rice cultivars named Ken99004（ZCI）, Crossing-503（ZC2）, Ken Sticky Rice（ZC3）, Shashani（ZC4）, Long-Grain Aromatic Rice（ZC5） were used to study typical wild traits of weedy rice and to identify the cold resistance of all genotypes. The results showed that the stem of long-red awn weedy rice was the weakest. No difference was found in the early stage of rice growth, e.g. the vegetable growth stage, between long-red awn weedy rice and other rice cultivars in the stem rigidity, but in the later stage, or reproductive growth stage, their leaves and stems were senescent rapidly because of the speedy supply of nutrients for panicle growth, meanwhile the rigidity of stem was reduced sharply just as withered weeds. The germination rate of long- red awn weedy rice was the highest in cold condition, and in turn were ZCI, ZC2, ZC3, ZC4, ZC5, respectively. The performances of genotypes in cold tolerance were identical, those with a high ability of germination in low temperature also showed a strong cold tolerance in main traits in whole growth period, the order from strong to weak in the extent of cold tolerance were long-red awn weedy rice, ZCl, ZC2, ZC3, ZC4, ZC5, respectively