水稻抗纹枯病QTLs元分析及候选基因鉴定

发布时间：2018-05-13 04:25

  本文选题：水稻 + 纹枯病　； 参考：《扬州大学》2017年硕士论文

【摘要】：由立枯丝核菌引起的水稻纹枯病是世界性水稻病害。水稻对该病的抗性为部分抗性,受多基因或数量性状基因座(quantitative trait loci/QTLs)控制,迄今未发现完全免疫的水稻种质和高抗的抗病基因。至目前,已先后有300多个抗纹枯病QTLs被报道分布于水稻全部12条染色体上。然而,由于采用的定位亲本、群体类型、遗传标记、鉴定方法以及环境条件等的不同,使得多数QTLs的位置不能直接比较,且置信区间较大,影响了育种应用。近年提出的QTLs元分析策略,其可利用已知QTLs的相关信息,通过数据整合再分析,推断出可靠性更高、位置区间更精确的一致性QTLs(Meta-QTLs)。本研究利用该方法,对抗纹枯病QTLs以及影响纹枯病的抗性的株高和生育期性状QTLs进行了元分析,并利用染色体片段代换系和高代回交群体对部分主效QTL进行验证及进一步定位;同时构建了高抗纹枯病水稻新种质YSBR1在江苏近年主推品种泰粳394(TJ394)背景下的全基因组染色体片段代换系。主要结果如下:1.收集前人有关水稻纹枯病抗性QTLs的研究报道,对符合元分析条件的188个抗纹枯病QTLs进行了元分析,获得34个抗纹枯病一致性QTLs,命名为MQSBR(Meta QTLs ofSheathblightresistance)。这些 MQSBRs 主要分布在水稻第 1、2、3、5、7、8、9、11和12染色体上,物理区间在0.2-4.2Mb范围内,其中近半数在1Mb区间内,明显小于前人报道的初级定位的置信区间。通过图谱比较,发现8个MQSBRs与MQHD(Meta QTLs of Heading Date)、MQPH(Meta QTLs of Plant Height)间连锁或区间重叠,余下 26 个MQSBRs可能与株高及生育期性状没有连锁关系。2.结合前人报道的可靠性较高的5个抗纹枯病QTLs(qSB-9TQ、qSB-11LE、qSB-11HJX74、qSB-11Tetep、qSB-12YSBR1、位置信息,发现MQSBR-25位于qSB-9TQ区间内,qSB-11LE位于MQSBR-27区间内,MQSBR-33和MQSBR-34位于qSB-12YSBR1区间内,这些结果证明元分析结果可靠性较好。本研究发现YSBR1/Lemont染色体片段代换系(Lemont背景)中有2个系的田间抗性显著强于对照Lemont,分别携带MQSBR-7、MQSBR-8和MQSBR-33、MQSBR-34,为了分析这2个系中是否还携带其它MQSBRs,通过236个分布于水稻12条染色体的多态性分子标记,检测发现这2个系的遗传背景中分别携带有7个和2个MQSBRs。3.为了发掘更多有利用价值的抗纹枯病主效QTLs,同时创建抗病育种中间材料,本研究构建了 YSBR1/TJ394全基因组染色体片段代换系。累计筛选到亲本间多态性分子标记266对,相对均匀的分布于水稻12条染色体上。累计获得了 111个以T394为背景的YSBR1染色体片段代换系,其中BC6F1世代代换系14份,BC5Fi世代代换系49份,BC4F1世代代换系37份,BC3F1世代代换系11份。代换系代换片段累计覆盖95%的YSBR1基因组信息。本研究利用覆盖qSB-12YSBR1的BC4F2回交分离群体(YSBR1/TJ394),通过复合区间作图法,发现一个抗纹枯病QTL,LOD值高达11.2,置信区间内包括MQSBR-34、MQPH-6和MQSBR-33的绝大部分区域。4.水稻第9染色体长臂中下端是不同研究学者多次检测到抗纹枯病QTLs的区间,在该区域共检测到5个MQSBRs,命名为MQSBR-22-MQSBR-26。结合纹枯病菌诱导差异表达基因信息,在MQSBR-24和MQSBR-25区间内各发现一个纹枯病菌显著诱导表达的基因 OsPIP(Plasma membrane intrinsic protein 2c)和 OsEXPR(Expansin-related protein 2 precursor)。通过RNA干扰,我们获得了 YSBR1背景下各基因转录表达显著下调的转基因株系。抗性鉴定结果显示,各基因的RNAi株系的病级数据与对照YSBR1间没有显著差异,表明它们可能不是MQSBR-24和MQSBR-25的候选基因。为了分析两个基因是否为qSB-9TQ的候选基因,我们利用各基因的RNA干扰系(OsPIP-Ri、OsEXPR-Ri/YSBR1背景)分别与Lemont背景下的qSB-9TQ导入系(LE-qSB-9TQ)杂交获得大量F1,同时配制对照F1(YSBR1×LE-qSB-9TQ)种子。对三套F1杂交种植株进行纹枯病菌接种鉴定,结果显示,不同材料间的病情差异不显著,暗示两个基因可能也不是qSB-9TQ的候选基因。以上结果为进一步克隆水稻抗纹枯病QTLs并开展育种应用奠定了材料基础,具有重要的理论和实际价值。
[Abstract]:Rice sheath blight caused by Rhizoctonia Rhizoctonia is a worldwide rice disease. The resistance of rice to this disease is partial resistance, controlled by multiple gene or quantitative trait loci (quantitative trait loci/QTLs). Up to now, no fully immune rice germplasm and high resistance genes have been found. Up to now, there have been more than 300 anti sheath blight QTLs. The reports are distributed on all 12 chromosomes of rice. However, the location of the parents, population types, genetic markers, identification methods and environmental conditions are different, which make the position of most QTLs can not be directly compared, and the confidence interval is large, which affects the breeding application. The QTLs meta analysis strategy proposed in recent years can make use of the known QTLs. Relevant information, through data integration and reanalysis, infers higher reliability and more accurate consistency QTLs (Meta-QTLs) in the position interval. This study uses this method to analyze the plant height of sheath blight and the resistance to sheath blight and the growth period character QTLs, and use the chromosome fragment substitution line and the high generation backcross population to the part of the QTLs. The main effect of QTL was verified and further located, and the genome segment substitution line of YSBR1, a new rice germplasm with high resistance to sheath blight, in Jiangsu, under the background of Thai japonica 394 (TJ394) in recent years, was constructed. The main results were as follows: 1. the study of the previous studies on the resistance of rice sheath blight to the rice sheath blight resistance was reported, and 188 of the conditions were conformed to the conditions of the analysis of the rice sheath blight. The resistance to sheath blight QTLs was analyzed by meta analysis, and 34 conformance QTLs of resistance to sheath blight were obtained, named MQSBR (Meta QTLs ofSheathblightresistance). These MQSBRs were mainly distributed on the 1,2,3,5,7,8,9,11 and 12 chromosomes of rice. The physical interval was within the 0.2-4.2Mb range, and nearly half of them were within the 1Mb interval, obviously smaller than those of the previous reports. By comparison, 8 MQSBRs and MQHD (Meta QTLs of Heading Date), MQPH (Meta QTLs of Plant) are linked or overlapped, and the remaining 26 may have no linkage to plant height and growth stage traits. 11HJX74, qSB-11Tetep, qSB-12YSBR1, location information, found that MQSBR-25 is located in the qSB-9TQ interval, qSB-11LE is located in the MQSBR-27 interval, MQSBR-33 and MQSBR-34 are in the qSB-12YSBR1 interval. These results show that the results of the meta analysis are reliable. This study found that there are 2 fields in the YSBR1/Lemont chromophore segment substitution system (Lemont background). Compared with control Lemont, MQSBR-7, MQSBR-8 and MQSBR-33, MQSBR-34 were carried respectively. In order to analyze the other MQSBRs in the 2 lines and 236 polymorphic markers distributed on the 12 chromosomes of rice, we found that 7 and 2 MQSBRs.3. were carried in the genetic background of the 2 lines to explore more utilization. The main effect of anti sheath blight was QTLs, and the intermediate material of disease resistance breeding was created at the same time. The YSBR1/TJ394 genome fragment substitution line was constructed in this study. A total of 266 pairs of polymorphic molecular markers were screened and distributed on 12 chromosomes of rice. 111 YSBR1 chromosome fragments with T394 as the background were obtained. Substitution lines, of which 14 generations of BC6F1 generation, 49 generations of substitution lines for generation BC5Fi, 37 generations of substitution lines in BC4F1 generation and 11 generations of substitution lines for generation BC3F1. The substitution line substitution fragment covers 95% of YSBR1 genome information. This study uses BC4F2 backcross segregated populations (YSBR1/ TJ394) covering qSB-12YSBR1, and discovers an anti grain by composite interval mapping method. The value of QTL, LOD, is up to 11.2. In the confidence interval, the middle and lower end of the ninth chromosome ninth chromosome long arm of.4. rice, including MQSBR-34, MQPH-6 and MQSBR-33, is the interval between different researchers on the anti sheath blight QTLs, and 5 MQSBRs are detected in the region, and the differential expression gene is induced by the MQSBR-22-MQSBR-26. combined with the Rhizoctonia solani. We found a gene OsPIP (Plasma membrane intrinsic protein 2C) and OsEXPR (Expansin-related protein 2 precursor) in the MQSBR-24 and MQSBR-25 intervals. The results showed that there was no significant difference between the disease level data of the RNAi strains of each gene and the control YSBR1, indicating that they may not be candidate genes for MQSBR-24 and MQSBR-25. In order to analyze whether the two genes are candidate genes for qSB-9TQ, we use the RNA interference lines of each gene (OsPIP-Ri, OsEXPR-Ri/YSBR1 background) with qSB-9T in the Lemont background, respectively. A large number of F1 were obtained by Q Introduction (LE-qSB-9TQ) hybridization and control of F1 (YSBR1 * LE-qSB-9TQ) seeds. Three sets of F1 hybrid plants were inoculated with Rhizoctonia solanae. The results showed that the difference between different materials was not significant, suggesting that two genes might not be the candidate genes of qSB-9TQ. The above results were the further cloning of rice anti grain. The QTLs and the application of breeding have laid the foundation for the disease and have important theoretical and practical value.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.111.42
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本文编号：1881659