【作者】 王步云；

【导师】 徐世昌；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2014， 博士

【摘要】 小麦条锈病是当今我国小麦生产上最为严重的病害之一，严重影响小麦的品质和产量。小麦条锈菌是专性寄生菌，目前利用鉴别寄主的抗病基因仍是鉴定条锈菌小种致病基因的唯一有效方法。因此，明确小麦条锈菌鉴别寄主抗条锈病基因及遗传特点和抗性特点，进行分子标记定位，并通过转录组测序进行分析，利于在基因水平和转录水平上进行小麦条锈菌生理专化研究和抗病性分析。尤皮Ⅱ号是小麦条锈菌鉴别寄主，对我国小麦条锈菌具有特别的鉴别能力，对其所携带的抗条锈病基因进行遗传分析和标记定位，并予以命名，具有重要的理论和实际意义。本研究通过常规杂交分析，以轮回亲本铭贤169为母本与近等基因系铭贤169*6/YrJu4（N9）杂交构建F2代作图群体。采用SSR和EST-SSR技术，利用已有的引物，并通过共线性分析设计引物，用高通量测序技术对铭贤169和铭贤169*6/YrJu4的不同时间点样品进行转录组测序并开发SSR引物，对基因供体尤皮Ⅱ号、轮回亲本铭贤169及抗条锈病近等基因系铭贤169*6/YrJu4基因组DNA进行PCR扩增和变性聚丙烯酰胺凝胶电泳分析，通过近等基因系铭贤169*6/YrJu4，对其基因供体尤皮Ⅱ号中抗条锈病基因YrJu4进行了分子标记筛选和精确定位；以轮回亲本铭贤169和铭贤169*6/YrJu4的N9株系为试材，接种CYR17②，选取时间点为0h、6h、12h、24h和48h，分别提取叶片总RNA用于转录组测序，筛选候选基因。研究结果如下：（1）通过4种方法，筛选出31对引物，即pTtksuG5、CFD6、CFD168、WMC702、WMS445、CFA2058、GPW2111、GPW2125、PSP3153、GPW7379、BE407000、BE423182、BE444378、BE497393、BE606324、BG606824、R2-10-2、R3-7、R6-1-2、R8-1-2、R11-6-2、R12-3、6a、12b、18a、19b、25a、40a、47a、49b和54b在近等基因系铭贤169*6/YrJu4与轮回亲本铭贤169间能稳定扩增出差异DNA片段，经40株抗感单株和354株F2代作图群体的遗传连锁性检测，发现有7对引物（GPW7379、49b、GPW2125、R2-10-2、BE423182、WMC702、WMS445）的扩增位点与目的基因YrJu4具有遗传连锁性，遗传距离依次为17.1cM、14.6cM、14.1cM、13.5cM、4.1cM、11.7cM和15.2cM。Xbe423182与Xwmc170（王冬梅，2013）可作为YrJu4的两个侧翼标记，将YrJu4标定在小麦染色体4.6cM范围内，可用于目的基因的分子标记与定位。尤皮Ⅱ号抗条锈病基因YrJu4在小麦2A染色体上的位置为：Xgpw7379—X49b—Xgpw2125—XR2-10-2—YrJu4—Xbe423182—Xwmc702—Xgwm445。鉴于尤皮Ⅱ号是具有特别鉴别力的小麦条锈病中国鉴别寄主，其中所含YrJu4基因是不同于其它抗条锈病基因的未知新基因，建议将YrJu4按国际命名为Yr60。构建了用于目的基因YrJu4精细定位的4253株F2代作图群体，并采用常规杂交分析方法，接种条锈菌系CYR17②，对轮回亲本铭贤169、基因供体尤皮Ⅱ号和近等基因系铭贤169*6/YrJu4及其杂交后代进行苗期抗性鉴定及统计分析，结果显示，近等基因系铭贤169*6/YrJu4对CYR17②的抗病性是由1对隐性基因控制。（2）转录组测序结果显示，测序结果理想，质量较高，共获得241947条N50值为1021的unigenes序列，平均长度为640nt，丰富了小麦的转录组信息。经过blast比对，在Nr库中比对上unigenes数目最多的前3个物种依次是大麦、二穗短柄草和粳稻；19404个unigene被注释到KOG数据库中24种类别中，包含样本基因最多的3个类别分别为信号转导机制、翻译后修饰和一般功能预测；有21496个unigene被注释到KEGG数据库，在KEGG中注释最多的3个代通路分别是核糖体通路（ko03010）、植物－病原菌互作通路（ko04626）和淀粉和蔗糖代谢通路（ko00500）；有49019个unigene被注释到3大类65亚类的GO分类上，与抗真菌病害有关的unigenes共有90个。分析了接种条锈菌CYR17②生理小种6h和12h后近等基因系铭贤169*6/YrJu4与铭贤169的转录组表达差异，发现上调表达unigenes共有46个，下调表达unigenes共有39个；以接种12h后为起点，以铭贤169作为对照，发现近等基因系铭贤169*6/YrJu4中21个unigenes表达在12h、24h和48h呈现持续上调，有31个unigenes在12h、24h和48h呈现持续下调表达。以上分析结果为进一步研究抗病基因YrJu4调控途径提供重要信息。更多还原

【Abstract】 Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), has been one of the mostdestructive diseases of wheat in China. It could cause great wheat yield losses and decrease wheatproduct quality. The pathogen is an obligate fungus. The effective identification of the virulence gene(s)in races of Pst is achieved with the aid of resistance gene(s) of a set of wheat differentials. Therefore, itwas important to find out the genetic traits and defense characteristics of the resistance genes ofdifferential varieties. Through resistance gene mapping and transcriptome sequencing, results would beused in the characterization of race-specific resistance of differential varieties. The differential varietyJubilejina Ⅱ has special identification ability for the races ofPst found in China. It had importanttheoretical significance and application value to genetically analyze and map the resistance gene(s) anddesignate it in Jubilejina Ⅱ.In this study, we used classic hybridization analysis and constructed F2mapping population byhybridization of Mingxian169as recurrent female parent and N9line of the near-isogenic line (NIL)Mingxian169*6/YrJu4as male parent. SSR and EST-SSR methods were used and primers weredesigned on the base of colinearity analysis between wheat and rice. Transcriptome sequencing analysisof Mingxian169and N9line of Mingxian169*6/YrJu4was conducted with de novo sequencingtechnology. The PCR products with the DNA templates of gene donor Jubilejina Ⅱ, recurrent parentMingxian169and N9line was analyzed with polyacrylamide gel electrophoresis for fine mapping ofthe resistance gene YrJu4. Mingxian169and N9line of Mingxian169*6/YrJu4were inoculated with thePst race CYR17. The total RNA of Mingxian169and N9line of Mingxian169*6/YrJu4after beinginoculated with CYR17for0h,6h,12h,24h and48h was isolated for the transcriptome sequencinganalysis. The results were shown as follows:1.There were31pairs of primers selected could stably amplify special DNA bands betweenMingxian169and Mingxian169*6/YrJu4, such as pTtksuG5, CFD6, CFD168, WMC702, WMS445,CFA2058, GPW2111, GPW2125, PSP3153, GPW7379, BE407000, BE423182, BE444378,BE497393, BE606324, BG606824, R2-10-2, R3-7, R6-1-2, R8-1-2, R11-6-2, R12-3,6a,12b,18a,19b,25a,40a,47a,49b and54b. Seven primers (GPW7379,49b, GPW2125, R2-10-2, BE423182,WMC702,WMS445) were found to be genetically linked with YrJu4after detection with20resistantand20susceptible plants and354F2plants. The genetic distance between the7markers above andYrJu4was17.1cM,14.6cM,14.1cM,13.5cM,4.1cM,11.7cM,15.2cM, respectively. Xbe423182andXwmc170(Wang Dongmei,2013) were the flanking markers of YrJu4in the region of4.6cM. Thelocation of YrJu4on wheat chromosome2A was Xgpw7379—X49b—Xgpw2125—XR2-10-2—YrJu4—Xbe423182—Xwmc702—Xgwm445. YrJu4in Jubilejina Ⅱwas different from other wheat stripe rustresistance genes located on chromosome2A. We suggested that it should be designated as Yr60.A fine mapping population of4253F2plants was constructed. Using classic hybridization analysismethod, Mingxian169, Jubilejina II, N9line of Mingxian169*6/YrJu4and the F2plants wereinoculated with CYR17and phenotype scoring was conducted.The results showed that the resistance of Mingxian169*6/YrJu4to CYR17②was controlled by a pair of recessive genes.2.The transcriptome sequencing results revealed that the isolated mRNA was of high quality and241947unigenes of which N50value was above1021were found totally, and their average length was640nt. The transcriptome results would enrich the wheat transcription data. After being blasted with Nrdatabase, the most annotated unigenes were hitted in the databases of Hordeum vulgare, Brachypodiumdistachyon and Oryza sativa japonica.19404unigenes were annotated and classified into24categoriesin KOG database, and the first three categories were related to signal transduction mechanisms,posttranslational modification and protein turnover, chaperones, and general function prediction only.21496unigenes were annotated in KEGG database. The first three pathways were ribosome (ko03010),plant-pathogen interaction (ko04626) and starch and sucrose metabolism (ko00500). Besides,49019unigenes were annotated in3categories and65subclasses of GO database.The expression pattern differences between Mingxian169and N9line of Mingxian169*6/YrJu4at6h and12h after being inoculated with CYR17were analyzed and46up-regulated unigenes and39down-regulated unigenes were found. We took12h after inoculation as the start point and Mingxian169as control, and found21unigenes continuously up-regulated and31unigenes continuouslydown-regulated from12h to48h post inoculation. These results could provide important information forfurther investigation of the regulatory pathways of resistance gene YrJu4.更多还原