【作者】 罗楠；

【导师】 王永清；

【作者基本信息】 四川农业大学 ， 果树学， 2011， 博士

【摘要】 本研究对枇杷属植物亲缘关系和遗传多样性进行了研究,取得以下主要研究结果：1.在采用ISSR标记对41份枇杷属植物材料进行亲缘关系研究和遗传多样性分析方面：(1)进行了枇杷属植物DNA提取方法的优化。采用改良的蛋白酶-K法,在裂解细胞核之前加入不含CTAB的提取介质Ⅰ,经低速离心后可有效将多糖、多酚等物质与细胞核分离。蛋白酶K的加入能有效降解与DNA结合的蛋白质,避免蛋白质与DNA共沉淀。0.3mmol/L的NaAc可以使多糖和小分子rRNA不沉淀而只沉淀DNA。所提DNA纯度较高,能满足ISSR扩增要求。(2)优化并建立了枇杷属植物ISSR反应体系和扩增程序。优化后的枇杷属植ISSR反应体系为：PCR扩增总体积为25μL,10×buffer2.5μL, Mg2+浓度2.0mmol/L,Taq酶1.5U,引物0.3μmol/L,模板60ng, dNTPs0.15mmol/L。反应程序为94℃预变性5mim；94℃变性1mim,退火70s,72℃延伸1.5mim,40次循环；72℃延伸7mim,4℃保存。(3)在41份供试材料中,20条ISSR引物分别产生436条带,多态性带分别为392条,多态性为89.9%。(4)根据ISSR数据分析,各供试材料间的相似系数分别为0.6216—0.9106之间。聚类结果首先将41份供试材料分为非栽培类群和栽培类群,但栽培类群分类存在一定差异,并且与传统的枇杷分类有所不同。说明枇杷品种分类是由多因素共同决定的,其表现型也受多因素共同影响。(5)研究结果并未发现枇杷属植物春季开花型或秋冬开花型的特异谱带,并且聚类结果也不支持将枇杷属植物按花期分为春季开花和秋冬开花两大类,说明开花时期可能为多基因控制性状,将枇杷属植物依开花时期分类是不可靠的。2.在对普通枇杷、栎叶枇杷及大渡河枇杷叶、果实、种子等形态特征观测及RAPD和ISSR两种分子标记综合分析,探讨栎叶枇杷、大渡河枇杷和普通枇杷的亲缘关系方面：(1)栎叶枇杷、大渡河枇杷和普通枇杷间存在着极显著的差异性,大渡河枇杷的各种性状(叶、果实、种子)都处于栎叶枇杷和普通枇杷之间；(2)大渡河枇杷不同单株的许多性状也有不同程度的分离,有些偏向于栎叶枇杷,有些偏向于普通枇杷；(3) RAPD和ISSR相似系数分析,大渡河枇杷总是处于栎叶枇杷和普通枇杷之间,偏向于栎叶枇杷,是连接枇杷属其他植物与普通枇杷之间的纽带。以大渡河枇杷作为待定杂种获得了最高的叠加性(44.9%和45.8%)。通过DNA指纹图谱分析发现,栎叶枇杷和普通枇杷的特异性谱带在大渡河枇杷上均有不同程度的表现。形态特征聚类分析及RAPD和ISSR相似系数分析均支持大渡河枇杷为栎叶枇杷和普通枇杷种间杂种的观点。3.在采用RAPD技术对四川枇杷产区的8个枇杷品种(系)进行品种(系)鉴定和系谱分析方面：(1)采用RAPD分析,利用选出的能稳定扩增的6个引物对‘大五星’、‘龙泉1号’、‘川农1号’等8个枇杷品种(系)进行了扩增,共扩增出54条带,其中26条具有多态性,多态性比例为48.15%。(2)利用NTSYSpc 2.1软件进行统计分析,得到8个品种(系)间的遗传距离。其中,‘早钟6号’与‘解放钟’的遗传距离最小,只有0.042,相似系数达到95.8%；‘川农1号’与‘早钟6号’的遗传距离最大为0.217,相似系数为78.3%。(3)初步建立了大五星、龙泉1号、川农1号等品种(系)的DNA指纹图谱,并找到部分特异谱带,利用DNA指纹图谱和特异谱带成功地对参试品种(系)进行了鉴别。(4)对供试品种(系)进行了聚类分析。4.在采用ISSR分子标记技术对‘大五星’、‘龙泉1号’、‘龙泉5号’及其小种子株系进行遗传多样性分析方面：18条ISSR引物在62份供试材料中共扩增出324条带,其中310条为多态性带,多态性比率为95.7%,18条引物在62份枇杷材料的平均有效等位基因数、平均Nei’s基因多样性指数、平均Shannon信息指数分别为1.5654,0.3249,0.5283,表明枇杷小种子株系具有丰富的遗传多样性,是极好的育种材料。更多还原

【Abstract】 In the present study, the genetic relationship and diversity in Eriobotrya were investigated using morphology and molecular markers. The main results were as follows.1 In the study on genetic relationship and diversity of Eriobotrya accessions using ISSR markers:(1) Methods for genomic DNA extraction in loquat were optimized. The improved CTAB-protease K method was suitable to remove polysaccharides and polyphenols by low-speed centrifugal because the solvents I, which did not contain CTAB, could not dissolve karyon but membrane. The protein, which was combined with DNA could be degraded by protease K, and avoided precipitating with DNA. Only DNA but not polysaccharides and small rRAN could be precipitated by 0.3mmol/LNaAc.(2) Amplification system of ISSR for loquat was established and optimized systematically. The optimal amplification system of ISSR was as follows. The total 25μL contained 10×buffer2.5μL,2.0mmol/LMg2+,1.5U Taq Polymerase,0.3μmol/L primer, 60ng template DNA, and 0.15mmol/L dNTPs. The temperature profile used for PCR was: 94℃for 5min, followed by 40 cycles of 94℃for70s, annealing temperature for 70s,72℃for1.5mim, and was terminated with a 7min DNA extension step at 72℃. Amplification products were conserved at 4℃.(3) 436 bands were amplified by 20 ISSR primers, among which 392 bands were polymorphic, and the percentage of polymorphism was 89.9%.(4) Similarity coefficients and dendrograms were obtained based on ISSR data. The similarity coefficients were 0.6216—0.9106. The 41 loquat accessions were divided into two groups based on both dendrograms. One group was non-cultivated type while the other was cultivated type. But the classification of sub-groups of cultivated type was somewhat different between two dendrograms, and also a little different from traditional classification.(5) No specific bands were gained between spring florescence and autumn-winter florescence according to ISSR markers. And also the accessions could not be divided into spring florescence and autumn-winter florescence according to the dendrogram.2 In the study on the genetic relationships between E. prinoides, E. prinoides var. dadunensis and E. japonica using phenotypic characters, RAPD and ISSR markers:(1) The results of variance analysis, multiple comparisons, correlation coefficients, cluster analysis on the phenotypic character data showed that E. prinoides, E. prinoides var. dadunensis and E. japonica displayed very significant differences. E. prinoides var. dadunensis was between E. prinoides and E. japonica.(2) Different individuals of E. prinoides var. dadunensis also had varying degrees of separation:some were close to E. prinoides, and some were close to E. japonica.(3) The results from RAPD and ISSR showed that the similarity coefficient between oakleaf loquat and common loquat was minimal (0.6996 and 0.7219, respectively) and the similarity coefficient between oakleaf loquat and Daduhe loquat was maximal (0.8403 and 0.8211, respectively), while the similarity coefficient between Daduhe loquat and common loquat was intermediate (0.7195 and 0.7219, respectively). The highest additivity was obtained when Daduhe loquat was regarded as the undetermined hybrid (44.9% and 45.8%, respectively). The specific bands of oakleaf loquat and common loquat were present in Daduhe loquat. Thus, it could be concluded that Daduhe loquat was a hybrid of oakleaf loquat and common loquat.3 In the RAPD analysis on 8 cultivars or lines of Loquat cultivated in Sichuan:(1) 6 primers producing polymorphic RAPD products were selected from 80 random primers and successfully used for amplifying the DNAs.54 bands were obtained, among which 26 bands showed polymorphism, amounting to 48.15%.(2) The genetic distances and similarities among the cultivars or lines were estimated By using NTSYSpc 2.1 software.’Zaozhong 6’and’Jiefangzhong’showed the smallest genetic distance (0.042) and a greatest genetic similarity (95.8%), while’Chuannong 1’ and’Zaozhong 6’showed the greatest genetic distance (0.217) and the smallest genetic similarity (78.3%).(3) The DNA fingerprints of the cultivars or lines were established. The specific bands of some cultivars or lines were found out. All the cultivars or lines could be identified by DNA fingerprints and the specific bands.(4)Genetic distances and cluster analysis were also performed for the cultivars or lines.4 In the analysis on the genetic diversity of maternal plants (’Dawuxing’,’Longquan No.1’and’Longquan No.5’) and their 59 offsprings derived from miniature seeds by ISSR markers:18 ISSR primers were applied to the amplification, producing 324 bands, in which 310 bands (95.7%) were polymorphic. The average value of effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index were 1.5654,0.3249 and 0.5283, respectively. The genetic difference between these cultivars and miniature seed derived plants was revealed based on similarity coefficients and cluster analysis. The results showed that the miniature seed derived plants had an abandant genetic diversity which could provide excellent germplasm resources for breeding in loquat.更多还原