【作者】 陈姗姗；

【导师】 宋述尧；

【作者基本信息】 吉林农业大学 ， 作物栽培学与耕作学， 2015， 博士

【摘要】 茄子褐纹病每年给生产茄子的国家和地区造成巨大的经济损失,该病害不仅对茄子生长发育的危害非常严重,且主要侵染危害茄子果实,造成产量下降和品质降低,尤其是采种田更容易发病,严重影响茄子生产以及育种和制种。本研究以茄子褐纹病为研究对象,以植物病理学、植物育种学和分子生物学等理论和技术为依据,对不同抗性的茄子品种进行了褐纹病的抗病性鉴定及种质筛选,探讨茄子褐纹病的抗病机制和遗传规律,采用现代生物技术寻找与茄子抗褐纹病基因紧密连锁的分子标记,同时通过利用转录组测序技术研究茄子抗褐纹病的相关基因及转录组差异,以期为该抗病基因的鉴定、资源筛选、分子标记辅助育种及分子克隆奠定了良好的基础,从而加快茄子抗褐纹病育种进程。研究的主要结果如下:1.建立了茄子褐纹病苗期、结果期人工接种鉴定方法,提出了褐纹病病害分级和抗性分类标准。(1)苗期人工接种鉴定采用喷雾法,接种菌量为1×107cfu/m L,接种时期为5叶期,接种15d后调查病情。(2)活体果实接种鉴定用孢子浓度1×107cfu/m L浸湿的滤纸片放入茄子果蒂和果实交界处进行接种;离体果实接种鉴定以5 mm长“十”字伤口接种菌丝块法接种最好,接种12d后调查病情。2.结合苗期喷雾接种法和结果期离体菌丝块接种鉴定能明显区分供试的16个茄子品种的抗病类型,高抗品种(系)2份(83-02、成都墨茄王)、抗病品种2份(墨丽长茄、83-02×长茄1号F1)、中抗品种3份(朝研黑星、早熟黑茄王、83-02×长茄1号高世代自交系)、高感品种4份(长茄1号、黑又亮、早熟紫长茄、英七长茄)、中感品种3份(日本茄子、鹰嘴长茄、黑龙长茄)和感病品种2份(黑豹长茄、三月长茄)。3.不同抗、感品种的结构抗性和生化抗性不同。(1)用扫描电镜对不同抗病性的茄子品种果实表皮结构进行比较观察,结果发现:抗病品种表皮厚度在100μm左右,果实表皮细胞角质层厚,外果皮细胞层数多,细胞排列非常紧密;感病品种表皮厚度在50μm以内,果实表皮细胞角质层薄,外果皮细胞层数少,细胞排列疏松。经聚类分析发现,高抗病品种(系)聚为第一类,抗病品种聚为第二类,所有的感病品种被聚成第三类。(2)POD、SOD、PPO、PAL活性的高低可作为判断茄子品种抗病性的重要指标,即茄子叶片组织的酶活性越高,品种的抗褐纹病能力越强。POD、PAL、SOD和PPO活性在整个侵染期均呈现先升高后降低的趋势,CAT的活性变化在整个侵染期都呈现下降的趋势。(3)MDA含量和电导率变化与茄子品种的褐纹病抗性呈负相关关系;抗病品种体内含有的基础活性物质(可溶性糖、可溶性蛋白质及脯氨酸)的含量等均高于感病品种。4.高抗褐纹病品系‘83-02’的抗性为单基因显性遗传。茄子高抗褐纹病品系‘83-02’与高感品种杂交组合的正反交F1代,均表现抗病,F2代群体中的抗、感植株比例经卡方测验符合3:1,F1与感病亲本回交,其回交后代的抗、感植株分离比经卡方测验符合1:1。5.建立了一套清晰度高、重复性好的适合茄子褐纹病抗病基因的RAPD反应体系,采用BSA结合RAPD标记方法,在200个随机引物中共有157个引物获得了扩增产物,出现条带总数为624条,平均每个引物扩增的条带数为4.28条。在27条多态性引物中,筛选出来一条与茄子褐纹病抗病基因连锁的RAPD标记S60/400,标记与抗病基因之间的遗传距离约为5.4c M。6.抗病品系‘83-02’和感病品种‘长茄1号’的RNA样品通过转录组测序和组装得到了223,500条Unigene,总数据量约236,251,873bp。通过基因差异分析,发现抗病株与易感株的上调表达的基因为59,856个,下调表达的基因为51,976个。更多还原

【Abstract】 Phomopsis vexans is one of the most important diseases of eggplant that caused huge economic loss to produce eggplant countries and regions each year. It not only produced serious hazard for growth and development of eggplant, but also infected fruits,caused poor yield and decreased quality, especially the field harvested seed incidented easily, affected the eggplant production and breeding and seed production seriously.The Phomopsis vexans of eggplant was used as the research object in this research, used the theory and technologyof plant pathology、plant breeding and microbiology as the basis,different cultivars of eggplants were screened for resistant identification and germplasm screening of Phomopsis vexans, discussed the resistant mechanism of Phomopsis vexans, adopt modern biological technology search for molecular marker close linkage to disease resistant gene, meanwhile by using the transcriptome sequencing technology research eggplant brown grain disease resistant genes and the difference between the transcriptome. In order to establish solid foundation for the cloning of resistant genes and molecular marker assisted breeding, thus accelerated the progress of eggplant resistan breeding.The main results as follows:1.Set up the artificial inoculation appraisal method during seedling stage and fruit period in Phomopsis vexans of eggplant, first proposed the standards of disease grading and resistance classification in Phomopsis vexans.(1) Used spray method of artificial inoculation identification during seedling, the amount of inoculant was 1 x 107 cfu/m L, inoculation period is 5 leaves stage, after inoculating 15 days was suitable for investigated the disease resistant.(2) Set up inoculation methods in vivo fruit and in vitro fruit :in vivo fruit inoculation identification put the spore concentration in 1 x 107 cfu/m L wet filter paper pieces into the junction of pedicel and fruit of eggplant; in vitro fruit inoculation identification with 5 mm " 十 " wound inoculated sclertium was the best inoculation. after inoculating 12 days were suitable for investigated the disease resistant of each cultivar.2. Used spray method during seedling stage and in vitro fruit inoculated sclertium could identify the disease resistant of sixteen eggplant cultivars obviously, two high resistant cultivars(83-02,Chengdumoqie), two resistant cultivars(Molichangqie, 83-02×Changqie NO.1F1) 、three middle disease-resistant cultivars(Chaoyanheixing 、Zaoshuheiqiewang、83-02×Changqie NO.1 high generation inbred lines) 、 four high susceptible cultivars(Changqie NO.1 、 Heiyouliang 、Zaoshuzichangqie、 Yingqichangqie)、three middle susceptible cultivars(Japanese eggplant、Yingzhuichangqie 、 Heilongchangqie) 、 two susceptible cultivars( Heibaochangqie 、Sanyuechangqie).3. The resistant cultivars and susceptible cultivars had different structural resistance and biochemical resistance.(1) The pericarps of different resistant eggplants were observed and compared with SEM. The results showed that the thickness of epidermal cells was about 100μm in the resistant cultivars, had thickness stratum corneum of epidermal cells,more layers of pericarp cells and the cells were arranged closely; the thickness of epidermal cells was less than50μm in the susceptible cultivars,had thinness stratum corneum of epidermal cells、less layers of pericarp cells and the cells were arranged loosen. The cluster analysis showed, the high resistant cultivars(lines) were gathered into the first kind, the resistant cultivars(lines) were gathered into the second kind, all the susceptible cultivars were gathered into the third kind.(2)The activities of POD、SOD、PPO and PAL higher, the ability resistance Phomopsis vexans stronger, the enzymes activities could use to judge disease resistance to different cultivars of eggplant. The activities of POD、 SOD、PPO and PAL presented a trend of increased and decreased, only the activity of CAT presented different degrees of decline during the whole infection period.(3) The contents of MDA and changes of EC had a negative correlation; The content of base active substances(soluble sugar、soluble protein and free praline) in the resistant cultivars were more than the susceptible cultivars.4. The resistance in Phomopsis vexans of high resistant line ‘83-02’ was single gene dominant inheritance. The combinations(F1) being reciprocal crossed between high resistant line ’83-02’and high susceptible cultivar all presented disease-resistant, the ratio of resistant plants and susceptible plants in F2 generation group was 3:1 which tested with chi square test, the ratio of backcross generation was 1:1 which tested with chi square test.5. A highly stable and optimized RAPD response system which was suitable for eggplant was established. BSA(Bulked Segregant Analysis) was used to search for RAPD markers,in the 200 random primers there had 157 primers obtained amplicons, appeared with a total of 624 stripes,the average number of primer amplification bands is 4.28. In the 27 polymorphism primers,screening out one RAPD markers S60/400 was were screen out 5.4 c M away from resistant gene.6. The RNA samples of resistant line‘83-02’ and susceptible cultivar ‘Changqie NO1’ cultivars obtained 223,500 Unigene by transcriptoming sequencing and assembling, the total amount of data size were about 236,251,873 bp. Through genetic variance analysis, found that the resistant plant and susceptible plant had 59,856 higher expression of genes, 51,976 lower expression of genes.更多还原