【作者】 王芳；

【导师】 李灿辉；

【作者基本信息】 云南师范大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 马铃薯是一大类起源、分类地位和系统演化关系尚存争议的茄属植物。马铃薯种质资源的准确鉴定不仅在研究其起源、分类和系统演化方面有重要的理论意义;同时,它对于种质资源保存、育种和育种者权益保护,及种质资源交流等也具有实际的应用价值。本文利用过氧化物酶(EC 1.11.1.7,POD)同工酶、酯酶(EC 3.1.1.1,EST)同工酶和SSR(Simple sequence repeat)分子标记技术对云南师范大学薯类研究所新引进或选育的100多份马铃薯种质资源(品种/系)进行了鉴定研究,并主要基于SSR标记初步建立了109份马铃薯种质资源的分子指纹数据库。主要研究结果包括以下几个方面:1、马铃薯过氧化物酶同工酶电泳(POD-PAGE)结果对可供马铃薯种质资源鉴定的适用材料(根、茎、叶、块茎和无病毒试管苗)筛选实验证实,同一品种(系)不同组织器官的POD同工酶谱存在明显的差异;但处于休眠期的成熟块茎和不同培养时期无病毒试管苗(2～6周)的POD同工酶谱均稳定,品种间差异明显,且结果重复性好。通过比较70份马铃薯种质资源休眠块茎和试管苗POD同工酶标记的鉴别率发现,休眠块茎POD标记的鉴别率(88.7%)略高于试管苗的鉴别率(85.0%)。利用休眠块茎POD同工酶标记对94份不同的马铃薯种质资源进行鉴定,聚类分析结果可区分出62份品种(系),鉴别率为66.0%。结果表明,马铃薯POD同工酶标记具有较高水平的多态性,可用于种质资源鉴定;但是,仅利用该标记尚不足以区辩所有供试的马铃薯种质资源。2、马铃薯块茎和试管苗酯酶同工酶电泳(EST-PAGE)结果以马铃薯成熟块茎和/或试管苗为材料,对112份品种(系)的EST同工酶进行电泳分析,结果发现:1)只有块茎样品的EST同工酶谱清晰可辩,结果重现性好;而不同品种(系)试管苗材料的EST同工酶谱成带效果差,实验结果难以统计分析,且可重复性差。2)所有参试品种(系)成熟块茎样品的EST谱带主要集中在相对迁移率为0.483～4.648区域,并表现出一定水平的多态性。3)利用块茎EST同工酶为标记,对94份不同来源的种质资源进行聚类分析(UPGMA),结果仅能区别27份不同的品种资源,鉴别率为28.7%;由此说明,仅通过成熟块茎EST同工酶标记不能对供试马铃薯种质资源进行准确的鉴别。3、马铃薯核DNA的SSR标记分析结果选用分别分布于马铃薯单倍体染色体组(n=12)上的12对SSR引物,对109份马铃薯品种(系)进行标记鉴定研究。发现所用引物的扩增产物均超出文献报道的范围;不同引物对扩增到的等位基因数不同,最少的扩增到4个等位基因(STM0037),最多达19个等位基因(STM1104),平均为12.7个等位基因;在供试的马铃薯品种资源中,12对引物的多态信息含量(PIC)在0.709(STM0037)～0.902(STM0030)之间,平均为0.833。排除相同和可能混杂的马铃薯品种资源,利用上述12个SSR标记可以将剩余的89品种(系)准确区分,鉴别率达100%。但是,SSR标记聚类分析结果未能准确反映供试种质资源间的亲缘关系。在对每个SSR标记位点鉴别率分析的基础上,筛选出了4组引物组合。它们都能对109份供试马铃薯种质资源进行较准确的鉴别,可大幅度节省鉴定相关的成本。4、分子指纹数据库的构建主要根据12个SSR标记结果,初步构建了109份供试马铃薯种质资源的分子指纹数据库;为马铃薯品种鉴定、育种和育种者权益保护等工作奠定了很好的基础。综合分析上述结果表明,不同分子标记方法和不同标记位点对四倍体栽培种马铃薯种质资源的鉴别率有明显差异;本文所采用的分子标记尚不足以揭示品种资源间的亲缘关系。开发与常规植物学和农艺性状紧密连锁的分子标记,将进一步促进分子标记技术在种质资源鉴定等方面的研究和应用工作。更多还原

【Abstract】 The potatoes are common name for a number of species belong to Solamum genus, of which origin, taxonomic status and phylogenetic relationships are still being argued. The proper method of potato germplasm identification not only has theory significance on studying for its origin, classification and phylogenesis, but also has applicable values for the germplasm conservation, breeding and breeders’ rights protection, germplasm exchange and so on. Using peroxidase isozyme, esterase isozyme and SSR marker methods, over 100 potato accessions, including that introduced or locally selected by the Root and Tuber Crop Research Institute of Yunnan Normal University, were identified. A molecular fingerprinting database of 109 accessions was finally constructed mainly based on the results of SSR marker analyses.The main results were summarized as follows:1. The results of peroxidase isozyme analysis of potato accessionsThe results of experiments with available materials, including roots, stems, leaves, mature tubers and in vitro plantlets, for peroxidase (POD) isozyme analysis of potato accessions showed the profile of POD isozymes were evidently different among materials employed even from the same accessions, but it was stable and repeatable with mature tubers and in vitro plantlets at different growth stages (2-6 week), and showed acceptable diversity among potato accessions used. By comparing the identification efficiency revealed by POD marker of mature tubers with that of in vitro plantlets of 70 accessions, it was found that the former (88.7%) was slightly higher than that of the latter (85.0%). Cluster analysis based on POD marker of mature tubers of 94 different potato germplasms showed that 62 advanced clones/cultivars could be distinguished with identification efficiency of 66.0%. The above results demonstrated that the potato POD marker had higher level of polymorphism among potato accessions and thus could be applied to potato germplasm identification, but it was not enough to discriminate all accessions by POD marker only in our experiments. 2. The results of esterase isozyme analysis of mature tuber and in vitro plantlet of potato accessionsThe mature tubers and/or in vitro plantlets were employed as materials for the analysis of esterase (EST) isozymes of 112 advanced potato clones/cultivars, we found that: 1) Only the mature tubers’ electrophoretic profiles were clearly distinguishable and repeatable, while that of in vitro plantlets could not be well resolved and repeated. 2) The profiles of EST isozymes of mature tubers from all potato accessions tested were concentrated in the range of Rm 0.483-0.648 and showed lower level of polymorphism among accessions. 3) Cluster analysis based on EST marker could only discriminate 27 cultivars which accounted for 28.7% of 94 different accessions tested. Thus, it was concluded that EST marker of mature tuber was not enough to discriminate the potato germplasms used in the experiments.3. The results of SSR marker analysis of potato accessionsTwelve SSR primer pairs, each dispersed on one of the chromosomes of potato haploid genome (2n=12), were employed to identify 109 advanced potato clones/cultivars. From the results we observed that the size of amplified products (base pair) of selected primer pairs were out of the ranges reported in other references, and the allele numbers amplified by these primer pairs were also quite different from that reported, which ranged from the lowest 4 (STM0037) to the highest 19 (STM1104), with an average of 12.7 alleles, among the accessions. PIC for primer pairs in all tested accessions ranged from 0.709 (STM007) to 0.902 (STM0030), with an average of 0.833. Excluding the same and possible mixed accessions, the remainder 89 advanced clones/cultivars could be discriminated exactly with 100% of identification efficiency. But the results of clustering couldn’t reflect exactly the relative relationships among tested accessions.In order to save the cost of SSR marker based identification of potatoes, we have selected out 4 groups of primer pairs based on its identification efficiency of each SSR marker. All of these 4 groups of primer pairs were successful in distinguishing the accessions used in our experiments with higher efficiency.4. Constructing the molecular fingerprinting database of potato accessionsWe have constructed a molecular fingerprinting database of 109 accessions mainly based on SSR marker results. This database would provide a common ground for potato accessions identification, breeding and breeders’ rights protection and so on.From the above results, it was concluded that there was evident difference in identification efficiency of potato accessions between different molecular marker method and different marker locus adopted, and the molecular markers used in present paper were not powerful enough to reveal exactly relative relationships among tested accessions. To develop new molecular markers closely linked with traditional taxonomic and/or agronomic characters will further promote its application in the germplasm identification.更多还原