【作者】 刘翔；

【导师】 孟金陵；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2005， 硕士

【摘要】 油菜菌核病是造成油菜严重减产的主要病害之一。尽管很多科学家对菌核病进行了广泛而深入的研究,但有关该方面的进展却差强人意。以本研究室现有的资源为基础,探讨油菜菌核病的研究方法,发掘抗病资源,将为后期抗菌核病育种利用和相关基因的分离、克隆奠定基础。 本研究包括三个方面的内容,即通过PCR、Southern blotting分子鉴定和抗生素筛选三种方法对转基因甘蓝型油菜的后代株系进行了鉴定;通过草酸毒素法和离体叶片接种核盘菌法鉴定几个甘蓝型油菜品种（系）的抗病特性;利用TN群体（由Tapidor×宁油7号杂种一代建立的DH群体,共包含202个株系）,进行油菜抗菌核病OTLs定位。 本研究的主要结果如下: 1) 转基因材料分子鉴定 转AP₂₄-Glu基因株系的PCR检测结果为:NPTⅡ引物特异性扩增后代株系,两年的结果均显示阳性;而以AP₂₄、35S、35S-AP₂₄特异引物筛选转AP₂₄-Glu基因的后代株系,结果均显示阴性。Southern blotting检测结果为:利用NPTⅡ,AP₂₄和35S序列作为探针没有筛选到阳性株系。 转Chi-Oxo基因株系的PCR检测结果:NPTⅡ引物扩增后代株系DNA混合样,两年的结果均为阳性;Southern blotting检测结果:利用NPTⅡ基因序列作为探针,只有一个转基因株系为阳性。 利用卡那霉素筛选转基因后代种子,两种转基因材料均不抗卡那霉素。 2) 常规品种的草酸抗性鉴定和核盘菌抗性分析 品种间的草酸抗性和核盘菌抗性差异是显著的,其中表现为高度抗草酸毒素的品种有Tapidor、恢5200、中油821、6-203;表现高感草酸的材料为湘油15、宁油7号、宁RS-1、华双3号。抗菌核病较好的品种有Tapidor和6-203;Westar表现最为感病。不同品种对核盘菌抗性和对草酸抗性的相关性不同。 3) 利用TN群体进行抗病相关QTLs定位 在已有的高密度遗传图谱的基础上,利用WinQTLCart 2.0作图软件进行QTLs扫描,共检测到19个抗菌核病相关QTLs,分布在9个连锁群中,单个位点解释的表型变异为更多还原

【Abstract】 Sclerotinia stem rot caused by S. sderotiorum is a kind of stubborn disease of rapeseed, which results in serious yield loss. The scientists in the world have made a wide spread and profound research on the disease, but the development is not satisfying. Based on the resource of our lab, explorating technique of studying on Sderotinia stem rot, investigating the resistant resource, and cloning the gene resistant to S. sderotiorum is our main aim.This research includes three aspects, verifing transgenic breeds with technique of PCR、 southern blotting and antibiotic screening, testing the resistance to S. Sderotiorum of 12 breeds with detached leaves inoculation and ethane diacid toxin at the seedling stage and mapping QTLs relating to resistance to S. sderotiorum in a DH population （derived from cross combination of Tapidorxningyou 7, and including 202 lines）. The research results are as follows:1) Transgenic materials testifyingPCR results of transgenic descendant lines with AP24-Glu gene indicated: all the transgenic plants were positive to NPTII primer amplification and negative to AP₂₄、 35S、 35S-AP₂₄ primer amplification; Southern blotting results indicated: all plants were negative to NPTII、 AP₂₄ and 35S probe hybridization.PCR results of transgenic descendant lines with Chi-Oxo gene indicated: all the transgenic plants were positive to the NPTII primer amplification; Southern blotting results indicated: there is one plant was blotting result indicated: there only one line was positive to NPTII probe hybridization and all the lines were negative to AP₂₄ and 35S probe hybridization.Seeds of the the two transgenic descendant line were sensitive to kanamydn.2) Resistance to ethanedioic acid and S. sderotiorum analysisThe results indicated that the difference of the two resistances among breeds is significant. Tapidor、 Hui 5200、 Zhongyou 821 and 6-203 show strong resistant to ethanedioic acid, and Xiangyou 15、 Ningyou 7、 Ning RS-1 and Huashuang 3 are strong sensitive to ethanedioic acid, Tapidor and 6-203 show better on resistance to Sderotiniasclerotiorum, and Westar is prone to sensitive to Sclerotinla sclerotiorum. Variant breeds for resistance to S. sclerotiorum and ethanedioic acid were different. 3) QTLs mapping in TN populationBased on the genetic map construct, 19 QTLs were detected in all with WinQTLCart 2.0, and covered 9 linkages, accounted for 3.53%-11.24% of the total phenotypic variation respectively. Among which, the peak LOD of rsO4j is O.OlcM away from the nearest marker, the peak LOD of rsO6g is 0.009 cM away from the left and 0.914cM from the right nearest marker, the peak LOD of rsO4h is 0.01 cM away from the left and 1.04 cM from the right nearest marker, the peak LOD of rsO7c is only 0.007cM away from the left marker更多还原