【作者】 滕彩珠；

【导师】 韩永华； 胡中立；

【作者基本信息】 广西师范大学 ， 生态学， 2008， 硕士

【摘要】 本研究将DNA分子标记技术(ISSR标记和AFLP标记)用于魔芋的遗传研究,优化了有关的反应条件,完善了适用于魔芋遗传育种研究的DNA分子标记技术,建立了魔芋DNA分子标记技术体系。ISSR和AFLP标记首次在魔芋研究中应用。所用的材料分为两部分,30份魔芋材料和13份魔芋材料。1利用ISSR技术对30份魔芋种质资源进行DNA多态性分析。这30份资源是从云南省各地广泛收集来的,经过了初步的分类学鉴定,其中既有野生种材料(6份),也有大面积种植的5个种:花魔芋(Amorphophallus konjac) (16份),白魔芋(Amorphophallus albus) (5份),西盟魔芋(Amorphophallus krausei) (1份),甜魔芋(Amorphophallus dunnii) (1份),株芽魔芋(Amorphophallus guripingensis) (1份)。从65个随机引物中筛选出11个扩增基因组DNA,共获116条带,其中102条为多态性标记,每个引物平均提供10.55个标记信息。UPMGA法聚类分析结果表明:这30份魔芋种质资源间的Jaccard相似系数都比较大,各样品间的相似系数为0.65-0.95,各魔芋样品间的差异并不十分明显,甜魔芋和花魔芋以及西盟魔芋和株芽魔芋的关系比较近,野生魔芋分别聚在花魔芋、白魔芋和甜魔芋中,同一种的聚在一起,其中白魔芋的遗传多样性较为丰富。甜魔芋聚类于花魔芋中,西盟魔芋和株芽魔芋聚在一起,说明甜魔芋和花魔芋以及西盟魔芋和株芽魔芋的关系比较近。白魔芋聚为两大类,这两类之间差异较大,说明白魔芋的遗传多样性较为丰富,并且它与花魔芋的关系较近。野生的魔野24 (样品27)与甜魔芋聚在一起,所以野生的魔野24也应属于甜魔芋;野生魔芋30 (样品22)、野生魔芋1 (样品23)、野生魔芋2 (样品24)和野生魔芋4 (样品26)一起聚类于花魔芋中,应属于花魔芋。野生魔芋3 (样品25)聚类于白魔芋中,应属于白魔芋。这几个野生样品的分子聚类的分类结果与形态分类的结果是完全吻合的。2利用AFLP技术对13份魔芋种质资源进行DNA多态性分析。这13份品种包括花魔芋、白魔芋、西盟魔芋和珠芽魔芋。从10对引物中筛选出2对引物扩增基因组DNA,共获得112条带,其中64条为多态性标记,每个引物平均提供56个标记信息。这13份魔芋种质资源间的Jaccard相似系数都比较大,各样品间的相似系数为0.48-0.77。UPMGA法聚类分析结果表明:魔芋资源的遗传多态性比较丰富,西盟魔芋和株芽魔芋的关系比较近,平均相似系数为0.68,白魔芋和花魔芋的关系比较近,平均相似系数为0.64。花魔芋中除花魔芋1、花魔芋2和花魔芋9之外的聚为一类,白魔芋聚为第二类,珠芽魔芋聚为第三类,西盟魔芋聚为第四类。花魔芋1、花魔芋2和花魔芋9与西盟魔芋的相似系数分别高达0.77、0.72和0.68,所以这三个花魔芋资源和西盟魔芋的遗传关系应该很近,所以它们是否属于花魔芋值得进一步研究。在花魔芋中,来自巴东的花魔芋3和花魔芋8没有聚在一起,其它的材料也没有区域性分布。这说明魔芋资源的遗传多样性是很丰富的,主要原因是魔芋是以无性繁殖为主,不同地区之间基因交流是很困难的,所以就形成了各自的遗传多态性。本研究成果为魔芋优良品种选育、新品种保护以及种质资源保护利用奠定了分子标记研究的理论基础。本研究初步研究了魔芋种间和种内的亲缘关系,为进行分子标记辅助育种、重要基因的图位克隆等提供了先进有效的研究手段。此研究将对魔芋的分子遗传学研究产生重要影响。更多还原

【Abstract】 From optimizing the related reaction conditions and perfecting the technology of molecular markers for genetic studies, DNA molecular markers ISSR and AFLP were firstly used to study genetic relationships in Amorphophallus. The materials used in the research included two parts: thirty accessions and thirteen accessions.1 The ISSR analysis was conducted in the 30 germplasm resources from Yunnan Province in Amorphophallus. These materials included Amorphophallus konjac, Amorphophallus albus, Amorphophallus krausei, Amorphophallus dunnii and Amorphophallus guripingensis. Among 65 random primers, 11 were selected for amplification of the 30 genomic DNAs. Total 116 ISSR fragments were generated, 102 of them were polymorphic marks. Every primer offered 10.55 marker bands on average. The molecular phylogenetic tree obtained by UPMGA method showed that Jaccard similarity coefficient in the 30 germplasm resources was high from 0.65 to 0.95. Genetic diversity in Amorphophallus albus was richer than that of others, and Amorphophallus albus was close to Amorphophallus konjac. Amorphophallus dunnii was close to Amorphophallus konjac, while Amorphophallus krausei was close to Amorphophallus guripingensis. There were two big groups in the samples of Amorphophallus albus with low Jaccard similarity coefficient, which indicated there were rich genetic diversities in Amorphophallus albus. And Amorphophallus albus was close to Amorphophallus konjac. The wild Amorphophallus 24 (sample 27) clustered into the Amorphophallus dunnii, so it should belong to the Amorphophallus dunnii. The wild Amorphophallus 30 (sample 22), 1 (sample 23), 2 (sample 24) and 4 (sample 26) clustered into Amorphophallus konjac, which indicated they belonged to Amorphophallus konjac. The wild Amorphophallus 3 (sample 25) clustered into the Amorphophallus albus, thus it should belong to Amorphophallus albus. The molecular phylogenetic results of the wild Amorphophallus completely accorded with those of their morphology. 2 The AFLP analysis was conducted in the 13 germplasm resources in Amorphophallus. These materials included Amorphophallus konjac, Amorphophallus albus, Amorphophallus krausei and Amorphophallus guripingensis. Out of 10 pair primers, two pair primers were selected and then amplified the 13 genomic DNAs. Total 112 AFLP fragments were generated, 109 of them were polymorphic marks. Every primer offered 56 markers on average. The molecular phylogenetic tree obtained by UPMGA method showed that Jaccard similarity coefficient of the 13 germplasm resources was high from 0.48 to 0.77. The molecular phylogenetic tree showed genetic diversity was rich in Amorphophallus. Amorphophallus krausei was close to Amorphophallus guripingensis while Amorphophallus konjac was close to Amorphophallus albus. The samples of Amorphophallus konjac clustered into the first group with the exception of Amorphophallus konjac 1, 2 and 9. The samples of Amorphophallus albus clustered into the second group. The samples of Amorphophallus krausei clustered into the third group. And the samples of Amorphophallus guripingensis clustered into the fourth group. There were high genetic Jaccard similarity coefficients between Amorphophallus konjac 1, 2 and 9 and Amorphophallus krausei at the value of 0.77, 0.73 and 0.69, respectively. It needs further study whether the three samples belong to Amorphophallus konjac. Amorphophallus krausei and Amorphophallus guripingensis had close genetic relationships with the mean Jaccard similarity coefficient of 0.64. In Amorphophallus konjac, the Amorphophallus konjac 3 and 8 from the same region Badong did not cluster together, which suggested there were not region distribution of genetic diversity among Amorphophallus. In the mean time, it meant there were rich genetic diversities in the same region among Amorphophallus. The main reason is clonal propagation of Amorphophallus, which limits the gene flow in the same region. Therefore, it forms so many genetic polymorphisms.Our research based the molecular marker research theory for selecting excellent breeds, protecting of new breeds and utilizing of germplasm sources in Amorphophallus. The preliminary studies on genetic relationships among the species and in species in Amorphophallus provided advanced research means of molecular marker assistant breeding and map-based cloning of important genes and so on. It will make a deep impact on molecular genetics of Amorphophallus.更多还原