【作者】 曾亮；

【导师】 李敏权； 杨晓明；

【作者基本信息】 甘肃农业大学 ， 草业科学， 2012， 博士

【摘要】 豌豆(Pisum sativum L.)是重要的粮菜饲兼用作物,具有耐寒、耐旱、耐瘠等特点,因其适应能力很强,在全世界范围内都有广泛的地理分布,广阔的分布范围和多样化的生境条件形成了其种质资源丰富的遗传多样性。由豌豆白粉菌(Erysiphe pisi)引起的豌豆白粉病是最严重的豌豆病害之一,抗病品种选育是防治其病害发生的最经济有效的措施,品种的抗病性鉴定是抗病育种的基础。本研究在对豌豆种质资源表型性状进行调查及测算的基础上,通过统计学分析手段,对其表型性状遗传多样性进行了研究,并进一步应用ISSR标记技术,从DNA水平上探讨了豌豆种质资源的遗传多样性与亲缘关系。同时,对豌豆材料白粉病田间自然发病情况做了详细调查,并对初步筛选出的抗病材料进一步进行了室内人工接种鉴定,在此基础上对豌豆白粉病不同抗性品种接种前后的生理生化变化进行了研究。主要的研究结果如下：1、通过对120份豌豆材料的20个表型性状分析,结果表明：豌豆属植物种质资源形态变异十分丰富,平均变异系数为32.68%,平均多样性指数为1.071。方差分析结果显示出供试材料种间存在显著或极显著差异,国内和国外群体内差异显著。主成分分析表明,前6个主成分的累积贡献率达到80.369%。120份材料在欧式遗传距离为56.59处聚为2大类,第1类主要为北方材料,第2类主要为南方材料,但是也有个别材料交叉出现,其聚类图位置依然较近,这说明系统聚类结果受材料的地理来源影响较大。2、建立了豌豆属ISSR分析的技术体系,对影响其ISSR-PCR反应体系中的5种主要因素进行了优化,最终确定了豌豆ISSR-PCR反应的最佳体系(20μL)为：15ng模板DNA,0.15mmol/LdNTP,0.5μmol/L ISSR引物,1U Taq DNA聚合酶,引物842号的最适退火温度为50℃。反应程序为：94℃充分变性3min,然后进行以下34个循环：94℃变性30s,50℃退火30s,72℃延伸45s,最后于72℃延伸10min,4℃终止反应保存。3、用ISSR标记技术对来自国内外的73份豌豆材料进行遗传多样性分析,结果表明：100个ISSR引物中共筛选出11个多态性明显、条带清晰、反应稳定的引物,73份材料DNA共扩增出91条条带,其中78条为多态性条带,平均每个引物扩增的条带数为8.2条,多态性比率为86.4%。Shannon多样性指数平均为0.4202,每个位点的有效等位基因数为1.4518,品种间遗传相似系数变幅范围为0.4065-0.9340,表现出丰富的遗传多样性。利用UPGMA聚类分析,以遗传相似系数0.52为界限,73份材料划分为5类,聚类基本符合地理来源相近的材料聚为一类,呈现出一定的地域性分布规律。4、通过2009-2011年连续3年对来自国内外的535份豌豆品种资源的白粉病田间自然鉴定,结果表明：田间表现高抗的有1个品种,占鉴定总数的0.19%；抗病品种2个,占总数的0.37%；中抗品种17个,占总数的3.18%；中感品种60个,占总数的11.12%；感病品种434个,占总数的81.12%；高感品种21个,占总数的3.93%。通过对田间表现出抗性的20个品种进行室内苗期接种鉴定表明,除了4个中抗品种的抗性发生了变化外,其余均与田间抗性表现一致,具有较好的抗性稳定性。5、通过对豌豆白粉病不同抗性品种接种前后的抗性相关酶活性变化及可溶性糖、可溶性蛋白和叶绿素含量变化的研究,结果表明：豌豆白粉病抗性反应相关防御酶中,苯丙氨酸解氨酶(PAL)、超氧化物歧化酶(SOD)及过氧化氢酶(CAT)的活性变化与豌豆白粉病抗性之间存在明显的正相关性,过氧化物酶(POD及多酚氧化酶(PPO)与豌豆白粉病抗性之间无明显的相关性。接种后,抗病品种中PAL、SOD和CAT3种酶活性明显大于感病品种。与抗性相关的物质中,可溶性蛋白和叶绿素的含量与品种抗病性之间存在明显的正相关,可溶性糖的含量与品种抗病性呈负相关。接种后,抗病品种中可溶性蛋白和叶绿素的含量明显高于感病品种。更多还原

【Abstract】 The pea(Pisum sativum L.) was an important food, vegetable and feeding mixture-purposed crop, with cold and drought and barren tolerant or other characteristics, it had the wide distribution in the world because of its strong ability to adapt, the broad geographical distribution and diversification of habitat conditions forming rich genetic diversity. The powdery mildew of pea caused by Erysiphe pisi was the one of the most serious diseases of pea, the most economic and effective measures of prevention and control the disease was selecting resistant varieties, the varieties resistance identification was the basic of selecting resistant varieties. The study on the basic of investigation and measure the pea germplasm resources phenotypic character, through the statistical analysis method, research the genetic diversity of traits, and further application ISSR markers, discuss the pea germplasm resources of genetic diversity and paternity. At the same time, make a detailed investigation of pea’s powdery mildew in the field on natural, and take the further indoor inoculating artificially identification to the resistance materials, on this basis, research the physiological changes of different varieties of powdery mildew resistance pea before and after inoculated. The main research results were as follows:1. Through analysis to20phenotypes of120pea materials, the results showed that:the pea germplasm resources variation form was very rich, the average coefficient of variation was32.68%, and the average diversity index was1.071. The results of variance analysis showed that the material had significant or very significant difference, the domestic and foreign community significantly different. Principal component analysis showed that, the first six main composition accumulation contribution rate reached80.369%.120material place for2categories in genetic distance of56.59, the clustering results divided south and north materials into2kinds, one kind of main materials for the north, the second type of main materials for the south, but also have individual materials cross appear, its clustering figure position remains relatively close, it showed that system clustering results from the geographical source material had big effect.2. Set up the technical analysis of pea ISSR system, optimized ISSR-PCR amplification system on pea in five factors. The most suitable ISSR-PCR system for pea was established, namely20μL reaction system containing15ng DNA template,0.15mmol/L dNTP,0.5μmol/L ISSR primer,1U Taq DNA Polymerase, the suitable annealing temperature of primer842is50℃. Amplificative thermal reaction program suitable for the ISSR-PCR analysis in the Pisum sativum was devised as followings:one preliminary denaturation at94℃for3min;34cycles each involved at94℃for30s, anneal at50℃for30s, extended at72℃for45s; and a final extension at72℃for10min, and keeping final products at4℃3. The genetic diversity of the73materials of Pisum sativum from both at home and abroad were analyed by ISSR marker, the results showed that:11primers were selected from100primers which have the clearly and polymorphic bands, a total of91bands were obtained by73materials, including78polymorphism bands.On average, each primer amplification site for8.2and the polymorphism percentage was86.4%. Shannon’s index was0.4202in average and the number of effective alleles was1.4518, the genetic similarity coefficient ranged from0.4065to0.9340and showed rich genetic diversity. Use groups by UPGMA cluster analysis to the genetic similarity coefficient0.52for boundaries,73materials was divided into five types, clustering basic accord with geographical origin of close materials for a class together, has a certain regional distribution.4. In order to make full use of disease-resistant peas germplasm resources, the study tested the resistance of pea to powdery mildew in field natural of535pea resources from international and domestic in2009-2011.The result showed that one variety was high resistant, accounting for0.19%of the total number of appraisal; two varieties showed resistance, accounting for0.37%of the total;17varieties showed middle resistance, accounting for3.18%;60varieties showed middle susceptible, accounting for11.12%;434varieties showed susceptible, accounting for81.12%;21varieties showed high susceptible, accounting for3.93%. The resistance indoor was consistent with it in field except4middle resistance varieties through the indoor inoculation identification of20varieties which have showed resistance in field.5. Through to research the resistance related enzyme activity change and soluble sugar, soluble protein and chlorophyll content change of different resistant varieties of pea powdery mildew before and after inoculation, the results showed that:in pea’s powdery mildew resisitance response enzyme which related defense, PAL, SOD and CAT activities change had obviously positive correlation with pea’s powdery mildew resistance, POD and PPO had no obvious correlation with pea’s powdery mildew. After inoculation, the PAL, SOD and CAT in disease-resistant varieties had significant enzyme activity greater than susceptible varieties. In related resistance material, soluble protein and chlorophyll content had obviously positive correlation with variety resistance, soluble sugar content had negatively correlated with variety resistance. After inoculation, disease-resistant varieties of soluble protein and the chlorophyll content was significantly higher than the susceptible varieties.更多还原