【作者】 杨朝东；

【导师】 包满珠；

【作者基本信息】 华中农业大学 ， 植物学， 2005， 博士

【摘要】 1 梅花AFLP体系构建和引物筛选 梅花（Prunus mume,2n=16）属于蔷薇科（Rosaceae）,Prunoidese亚科,李属（Prunus L.）植物,在中国、朝鲜和日本是著名的早春开花的观赏植物。AFLP是目前研究物种遗传变异常用的分子标记之一,它首次应用于梅花种质资源的遗传多样性和亲缘关系的研究。本研究采用EcoR Ⅰ/Pst Ⅰ限制性内切酶组合酶切梅花基因组DNA的AFLP实验流程。从256对AFLP引物中筛选出10对多态性位点最高的引物,这些引物扩增出高的多态性位点在‘雪梅’等14个梅花品种中具有可靠的重复性,共扩增出清晰可记录524个位点,其中233个为多态性位点,多态性位点的百分率从35.71%到59.67%之间不等,平均百分率为44.46%,可用来分析更多梅花品种之间的遗传关系。AFLP是一种效率高、花费适中、节约时间、但操作比较复杂的研究梅花遗传分化的技术。 2 应用AFLP初步探讨梅花品种间的亲缘关系 应用AFLP技术初步研究了1个半野生种,1个杂交子代和18个梅花品种,代表梅花主要类、型,共计20株梅花的亲缘关系,19对选择性引物扩增出清晰的总位点数为918个,其中多态性位点324个,多态性位点百分率为35.29%。采用SAS类平均法聚类分析AFLP数据,λ值为0.9759时,将20株梅花分为四个类群。半野生‘曲梗梅’的多态位点数最高,‘多萼朱砂’、‘复瓣小宫粉’等梅花品种次之。在类群Ⅳ中,具有明显亲缘关系的两组杂交亲本及其子代各自聚为一组,表明AFLP技术和聚类分析方法可以用来研究梅花品种间的亲缘关系。‘小宫粉’、‘江南朱砂’和它们的子代亲缘关系密切,聚类分析结果仍能区别其较小差异,这有助于解决梅花中不同产地相同品种异名和同名而品种不同的情况。垂枝类梅花品种间有较近的亲缘关系或者遗传来源,支持枝姿作为梅花分类的重要形态特征,垂枝类品种属于梅花中比较进化的类群与其历史演化记录相吻合。本研究中所采用的DNA限制性内切酶酶切组合及所筛选的AFLP引物对适合研究梅花品种间的亲缘关系和遗传多样性。 3 AFLP技术对梅花杂交种的快速鉴定 应用银染法AFLP鉴定以父本‘多萼朱砂’与母本‘雪梅’杂交所得12株F₁子代。以E-AT/P-AT选择性引物对扩增,父本180bp左右的特异带在12株F₁代中都呈阳性带;母本170bp左右的两条特异带在12株F₁代中也都呈阳性带,双亲基因组DNA中这3个特异位点在F₁子代中稳定遗传。结果表明这12株均为‘多萼朱更多还原

【Abstract】 1. Establishment of AFLP technique and Assessment of Primer Combinations for Mei FlowerMei flower is one of the most famous ornamental flowers in eastern Asia for its blossoming in early spring. The AFLP technique is one of the most frequently used techniques for analysis of genetic variation and used herein for the first time in Prunus mume. This research provides a detailed and modified AFLP protocol for Mei genomic DNA digested by EcoR I/Pst I restriction endonuclease combinations. The best ten primer pairs of high polymorphism were screened from 256 primer combinations that could reliably and repetitively distinguish 14 Mei samples and would be suitable for genetic analysis of more cultivars. Ten primer pairs produced total AFLP loci up to 524 and polymorphic loci up to 233. The ratio of polymorphic loci scoped from 35.71% to 59.67% and the average ratio was 44.46% in the ten primers. AFLP is an effective, inexpensive and time saving technique for the genetic differentiation of the Mei cultivars as evidenced in this study.2. Preliminary analyzed of genetic relationships for Mei cultivars by AFLP MarkersThe genetic relationships among a semi-wild species, a hybrid and 18 Mei flower cultivars were preliminary investigated using AFLP. 19 selected primer combinations revealed 918 legible loci, 324 of which were polymorphic loci that accounting for 35.29% of the total. Based on the SAS average similarity cluster analysis, 20 Mei samples were classified into four groups as λ =0.9759. P. mume var. cernua showed the highest polymorphic loci in these Mei samples. ’Duo-e Zhusha’ and ’Fuban Xiao Gongfen’ showed less polymorphism than P. mume var. cernua. In the group IV, AFLP and cluster analysis results are as follows: Parentages of some Mei flower cultivars were successfully identified. The results show difference between ’Xiao Gongfen’, ’Jiangnan Zhusha’ and their hybrids which will be useful to clear the confusions of homonym and synonymous.There are close genetic relationships among the cultivars of pendulous which support traditional classification using branch gesture as the important morphological character in Mei flowers. The pendulous cultivars belong to the more evolutionary type and in accordance with the records in its development history. These results demonstrate that AFLP technique is an efficient method for evaluating the genetic relationships and diversity among Mei flower cultivars.3. Rapid Identification of Parentage in Prunus mume (Sieb. et Zucc.) by AFLP AssayParentage determination of 12 individuals offsprings from the cross Prunus mume ’Xue Mei’ xPrunus mume ’Duo-e Zhusha’ was conducted using silver staining AFLP method. The result of amplification in Fi population with the selective primers combination of ￡-AT / F-AT showed as follows: Specific band of 180bp of the male parent was present in all the 12 progenies of Fi population and two specific bands of 170bp and 171bp of the female parent were present in all the 12 progenies too. And these three bands could inherit from parents to progenies of Fi population constantly. It is concluded these 12 individuals are all hybrids. Therefore, AFLP is a rapid method for parentage determination in Prunus mume. It will be useful for early selection of hybrids in cross breeding of Prunus mume.4. The analysis of genetic diversity and genetic relationships of Mei germplasm 4.1 The cluster analysis of accessions in this studyThe line A divided the 125 accessions into 7 branches as the SM coefficient was 0.846. The branch I were the true mei flower, branch II was the Prunus armeniaca, branch III were the hybrid of mei and Prunus armeniaca, branch IV was the hybrid of mei and Prunus cerasifera, branch V was Prunus salicina, branch VI was Prunus persica (L.) Batsch f. camelliae- flora and branch VD was Prunus pseudocerasus, respectively.The line B divided the true mei of Branch I into 8 groups as the SM coefficient was 0.846. The group (D was the feral mei; group ? were consisted with single flowered form, green calyx form, part of pink double form, cinnabar purple form, albop-lena form, flavescens form, versicolor form, pendulous mei and tortuous dragon; group (3) were part of pink double form, such as ’Hongfen Taige’ et al; group ? were the cultivars of ’Liu Bao’,et al; group (D were the cultivars of ’Fushou Mei’, et al; group (6)were the cultivars of Tu Fen’,et al; group ? was the cultivar of ’Guhong Chuizhi’ and group (D was the cultivar of ’LijiangHongmei’.There were 9 forms under line B and line C. The form (1) were single flower form; form (2) were green calyx form; form (3) were the large part cultivars of pink double form; form (4) were alboplena form; form (5) were part of cinnabar purple form; form (6) were the other part of cinnabar purple form; form (7) were the flavescens form and versicolor form; form (8) were the pendulous mei and form (9) were the tortuous dragon mei in turn.4.2 The summary of genetic relationships and Phylogeny revealed by AFLP markers in MeiPrunus mume was close to Prunus armeniaca, and close to Prunus salicina and Prunus persica (L.) Batsch f. camelliaeflora secondly between species; it was far away from Prunuspseudocerasus. ’Dan Fenghou’, ’Feng Hou’ and ’Song Chun’ were belonged to inter-species hybrids of Mei and Prunus armeniaca, but they are more close to Prunus armeniaca. ’Meiren Mei’ was belonged to inter-species hybrids of Mei and Prunus cerasifera. ’Dan Fenghou’ was more close to ’Feng Hou’ than ’Song Chun’in the dendrogram. These results were consistent with morphological classification by Chen (1996).In Prunus mume, these cultivars with typical branchs, leaves and flowers of Mei were formed its originality group. But the cultivars of ’Xue Mei’ et al were showed subtle differentiation in the form (1) of group (2). The cultivars collected from Japan showed more primitively characters in group ?. These cultivars sexported from China in history and maybe fail to gene flow introgression with the origin group, thus it created reproductive isolation.Form (3) cultivars in group (2); ’Hongfen Taige’ et al cultivars in group (§); ’Fu Fen’ and ’Bianban Ddahong’ in group ?; ’Lijiang Hongmei’ in group ? were divided from pink double form for its complicated derivation. They existed different evolution classes and maybe the hybrids in Mei. ’Lijiang Hongmei’ was close to the varieties of Mei. Thus, we postulate the double flower and pink color of Mei maybe derivated early in its evolutionary history.Cinnabar purple form was divided into (5) and (6) in these studies. But the similarity was highly between them. Green calyx form were classified in form (2) of group (2) and group (6), we postulate its origin maybe early in mei evolutionary history. Alboplena form in form (4) agree to Chen (2004) classification system.Pendulous Mei group were scattered in group (D, group (7) , (8) and (6) of group d) in the dendrogram. They evolved in different degree for their located in unlike groups. Pendulous group and tortuous dragon Mei represented the most evolutionary in branch gesture of Mei. Flavescens form and versicolor form represented the most evolutionary in petal color, type of flower and fragrance of Mei in the dendrogram. So we considered the two forms as one. The classification of Mei is not relation to their geographical distrubition.43 The analysis of genetic diversity of Mei flower germplasm resourcesAccording to morphological and molecular markers evidences in these studies, we classified 117 accessions of Mei into 12 groups. Molecular variance of AFLP markers was caculated by AMOVA program. The results showed the molecular variance among groups was 31.01% and the molecular variance within groups was 68.99%. The molecular variance among groups was lower than the variance within groups. It showed that the genetic diversity was low in Mei germplasm resources.更多还原