【作者】 程中平；

【导师】 黄宏文；

【作者基本信息】 中国科学院研究生院（武汉植物园） ， 植物学， 2007， 博士

【摘要】 桃亚属植物是核果类重要的果树植物资源，其中桃和扁桃是世界上重要的栽培果树。本研究通过现代分子生物学技术和方法，对桃亚属植物的分子系统学研究，揭示其内各种间关系和分类地位及与其近缘亚属的关系，以及对桃遗传多样性研究。为桃亚属植物在分类学中存在悬而未决问题的解决和桃资源的保存与利用提供分子生物学依据。采用核核糖体DNA内转录间隔区（ITS）对桃亚属的光核桃、甘肃桃、新疆桃、山桃、陕甘山桃、扁桃、野扁桃基因序列测序及来源于GenBank的李、杏、梅、樱的18个种的ITS区基因序列，以稠李和楼木、梅和杏分别作为对桃亚属及其近缘亚属植物、桃亚属各种类的外类群，进行系统发育树的建立。运用RAPD技术对223个桃、李、杏、梅、樱类植物进行分子标记，采用聚类方法对上述内容研究，综合两种方法研究结果，探讨桃亚属植物分子系统发育关系，并对引物在各类间的多态信息量分析。与此同时，利用RAPD标记对桃遗传多样性、类群的遗传结构、类群间的遗传差异等方面进行分析。主要研究结果如下：1．初步建立了桃种质资源数据库，该数据库包括1)223份材料（含近缘亚属20个种）的原产地、采集地、中文名称、英文名称、学名及果实性状；2)223份材料经22个引物扩出180个多态位点上的谱带，共计40140个数据；3)获得桃亚属主要种类的ITS区基因系列9个。2．桃亚属在核果类植物的系统发育经RAPD带进行聚类分析，可见杏类与梅类先聚在一起，再与李类植物相聚，进一步与桃类植物相聚，最后与樱类植物聚在一起。如果以0.533处作一接合线，可分为四大类第一类杏、梅，第二类李，第三类樱，第四类含郁李、GF₆₅₅（桃和李杂种）和桃及近缘种植物。基于ITs序列得到系统发育树为樱类位于系统发育树的基部，构成一单系分支，其内部支持率为68％。而桃、李、杏、梅聚在一起构成另一单系分支，内部支持率为100％，其分支内又分为两亚类，一亚类为桃类（含扁桃），内部支持率为100％，另一亚类则由杏亚属和李亚属的其他种类组成，内部支持率只有54％，而第二亚类中的杏与梅为一组的支持率达85％。3．桃亚属植物内的种间系统发育通过所记录的180个位点清晰带进行聚类分析，以0.87处作为结合线，可将它们分成内蒙古长柄扁桃；光核桃；山桃；甘肃桃；白花山桃、红花山桃、帚形山桃；白花山碧桃；陕西桃巴旦、桃巴旦；毛桃、新疆桃、喀什1号、喀什2号、喀什3号、喀什4号等8组。ITS全序列的简约性分析得到了5个相等的最短简约树，该树树长为80步，一致性指数（CI）和维持性指数（RI）分别为0.925，和0.941。每个树拓朴分析除山桃和陕甘山桃的位置有变化外，得到的结果是相同的。野扁桃和矮扁桃在桃亚属中其它种的基部，紧接着是扁桃，它单独占有一类位置。供试的8个种，分成一个四歧式的单系类群，第一类为光核桃和甘肃桃，第二类为桃和新疆桃，其它两类分别为山桃和陕甘山桃。经过严格一致树分析，构成姊妹群有光核桃和甘肃桃，桃和新疆桃，野扁桃和矮扁桃。4．针对桃亚属中种类分类地位存在异议的新疆桃、山桃、陕甘山桃进行分子生物学研究。基于ITS的系统树分析，自展支持率90％支持新疆桃和普通桃有较密切的亲缘关系。对203个以桃与李杂种为外类群的桃亚属种、变种、品种及类型的RAPD分析，得到的聚类图中，除新疆桃以外的其它种基本聚在普通桃的外围，而5个新疆桃并未聚到普通桃的外围，反而在许多普通桃的变种、品种及类型的内侧，分布于普通桃之间。采用ITS序列重建系统发育树，陕甘山桃和山桃为并行分支，各自成为独立一组，位于桃、新疆桃，光核桃、甘肃桃的基部。RAPD标记聚类分析，山桃为单独一组。5．从多态带、特殊位点对类群分析，提出了各类群核心种质，砧木类有列玛格哈露红及贝蕾、阿克拉娃；寿星桃有红重瓣、寿星桃（红花），寿粉，白单瓣；碧桃有人面桃，红碧桃、红花碧桃；垂枝桃有垂枝桃（江苏）、鸳鸯垂枝、垂枝桃（华农）；红叶桃有筑波3号、筑波6号和洛格红叶；硬肉桃有大红袍、五月鲜、秦岭冬桃、敦煌冬桃、一线红、阳泉肉桃和大甜桃；蜜桃有秋蜜、太原水蜜、深州水蜜和温州水蜜；水蜜桃有冈山白、Spring time、Rebin、白凤、霞晖2号、早艳、奉化玉露；蟠桃有玉露蟠桃、五月鲜扁干、苏联蟠桃、嘉庆、杨124；油桃有曙光、瑞光2号、五月火、艾米拉、红李光、阿姆肯；黄肉桃有爱保太，佛尔蒂尼莫蒂尼、金皇后、Red haven、西庄1号、金橙、橙香和锦锈。以上种质中，玉露蟠桃，秋蜜、爱保太和佛尔蒂尼莫蒂尼无论从带型还是在聚类图中的位置表现为更为特殊。6．利用特殊带型对各类群的品种、类型全部得到了识别，并依据特有带进行了分子检索表的编制。通过典型带对不同生态型、果实性状为类别进行分析，结果未发现有与生态型相吻合的特征带出现，而将供试所有品种按离核、粘核和溶质、不溶质划分归类，供试品种在Warburton等得到S₁₆₇-1050 bp处粘离核分子标记，出现既有离核（占供试离核41／56=73.2％），又有粘核（占供试粘核33／79=41.8％），可见此连锁标记连锁性不强，发生交换的机率较高，而S₁₆₇-850bp带和S₁₆₇-1050bp带同时出现时，该组包含6个离核，无粘核品种；6个溶质，而无不溶质。S₁₆₇-850bp和S₁₆₇-1400bp同时出现时，包含20个粘核品种和2个半离核品种。7．通过砧木、寿星桃、垂枝桃、红叶桃、碧桃、硬肉桃、蜜桃、水蜜桃、蟠桃、油桃、黄肉桃类群的多态标记、标记频率、扩增位点上的变异程度和各对应位点间相关性、类群多样性度及遗传分化和遗传结构分析。存在于桃类群间的变异为11.9％，类群内则为88.1％。各类群遗传多样度为黄肉桃＞蜜桃＞蟠桃＞红叶桃＞硬肉桃＞碧桃、水蜜桃＞油桃＞寿星桃＞垂枝桃。以类群进行聚类分析，在0.9586作为结合线，则有以食用为目的的栽培桃聚在一组（硬肉桃、水蜜桃、蜜桃、黄肉桃、油桃、蟠桃），其中最大的相似系数0.9806为硬肉桃和水蜜桃；而其它寿星桃、碧桃、红叶桃、垂枝桃各自成一组，其中最小相似系数0.9160为红叶桃和垂枝桃。依据以上研究结果，并结合前人对形态学、细胞学、孢粉学、酶学等方面的研究成果，认为核果类植物的属的分类为桃亚属、李亚属、杏梅亚属和樱亚属；新疆桃作为桃的一个变种；山桃为桃亚属中的一个种；陕甘山桃虽在本研究的系统树中单独为一类，对于其分类地位有待从多方面进一步研究。经对桃遗传多样性和遗传结构分析，证明特殊种质存在，且不同类群间存在不同的遗传变异，黄肉桃为其中遗传多样度最高的类群。类群内和类群间的遗传差异很大，类群内的变异是类群间变异近8倍。通过多态带和特殊带提出了桃核心种质，并利用聚类法、统计法、标记频率法、多态带数和特殊位点法、类群间和类群内的遗传差异法提出了种质保存策略；以及品种识别分子检索表的建立，为知识产权保护和正确选择品种提供了第一手资料。更多还原

【Abstract】 Plants in subgenus Amygdalus are an important resource of stone fruits, of which P. persica and P. communis are major fruit species in the world for fruit production. The study aims to disclose phylogeny among subgenera in stone fruit plants and species in P. persica, taxonomic rank, and genetic diversity of P. persica by molecular biological technology and methods. It will provide molecular evidences for solving problems in taxonomy of subgenus Amygdalus, conservation and utility of germplasm in P. persica.The internal transcribed spacer （ITS） regions of nuclear ribosomal DNA from 8 species of subgenus Amygdalus were sequenced, and analyzed together with other ITS data （from GenBank） of 18 species representing subgenus Cerasus, subgenus Armeniaca and subgenus Primus by using of Padus racemosa （Lam.） Gilib. and Padus buergeriana （Miq.） Yüet Ku as outgroup for studying phylogeny of subgenera in stone fruit plants. 223 accessions from subgenus Amygdalus, subgenus Prunus, subgenus Armeniaca, subgenus Cerasus were studied by RAPD technology and clustered according to mentioned phylogenic contents. Polymorphic information contents （PIC） were calculated among species in subgenera and demes in P. persica. Meanwhile, genetic diversity, genetic structure, genetic differences of demes were analyzed with RAPD markers. The main results as follow:1. A database of germplasm in subgenus Amygdalus （including 20 relative species in other subgenera） was established, which included （1） origin sites, collection places, common names, scientific names and fruit characters of 223 accessions; （2） 40140 data obtained from 180 loci and 22 primers; （3） 9 gene sequences of rDNA ITS in subgenus Amygdalus.2. Phylogeny of subgenus Amygdalus in stone fruit plants was analyzed by RAPD markers, indicating that apricot and mume in subgenus Armeniaca clustered firstly, and the two together with subgenus Prunus, further the three with subgenus Amygdalus, finally the four with subgenus Cerasus. If the cluster was made a joint line at the level of 0.533, there were four groups as above motioned subgenera. The consensus tree by ITS gene sequences indicated that subgenus Cerasus was basal to the other subgenera with a bootstrap value of 68% and occupied an isolated position （cladeⅠ）. Subgenus Prunus, subgenus Armeniaca and subgenus Amygdalus formed a monophyletic group （cladeⅡ） with a bootstrap value of 100%, which implied they had close relationships with each other and probably a common origin. CladeⅡwas divided into two subclades: One was subgenus Amygdalus with bootstrap values of 100%, the other was subgunus Prunus and subgenus Armeniaca with bootstrap values of 54%, but species in subgenus Armeniaca with bootstrap values of 85%.3. Phylogeny of species in subgenus Amygdalus was analyzed through markers in 180 loci and made a cluster, which inferred 8 groups: Neimengguchangbingbiantao（P. pedunculata）; Guanghetao（P. mira）; Shantao（P davidiana）, Ganshutao（P. kansuensis）; Badan（P. communis）; Xinjiangtao, Maoto （P. persica ） from joint line of 0.87. Treating gaps as missing, the parsimony analyses generated five equally shortest trees with a length of 80 steps, a consistency index （CI） of 0.925, a retention index （RI） of 0.941, a rescaled consistency index （RC） of 0.871. The topology of each tree was identical except for placement of Shantao （P. davidiana） and Shanganshantao（P. potanini）. In every case, P. ledebouriana and P. tenella were basal to the other species in subgenus Amygdalus, followed by P. communis which occupied an isolated position. Above these species, a tetrachotomy separated four monophyletic groups, the first one consisting of P. mira and P. kansuensis, the second one consisting of P. persica and P. persica var.ferganensis, and the rest two including P. potanini and P. davidiana, respectively. A strict consensus tree depicted sister groups of （P. mira, P. kansuensis）, （P. persica, P. persica var. ferganensis）, and （P. ledebouriana, P. tenella）.4. In allusion to dispute on taxonomic ranks about Xingjiangtao, Shantao, Shanganshantao, they were analyzed with ITS gene sequences and RAPD markers. A bootstrap value of 90% supported extreme relative relationship between Xingjiangtao and common peach. 203 species, varieties, cultivars and forms clustered with RAPD markers, inferring that all species except Xingjiangtao in subgenus Amygdalus displayed in outer of the dendrogram, while 5 accessions of Xingjiangtao did not cluster out of common peach and mixed with accessions in common peach. Systematic tree was constructed by ITS sequences, indicating that Clades of Shanganshantao and Shantao existed parallel, turning into own group at the base of the other species in subgenus Amygdalus. The dendrogram from RAPD markers supported that Shantao was in an independent group. 5. Core germplasm in each deme was put forward and listed as Liemage, Haluhong, Bailey, Akelawa in rootstock group; Hongchongban, Shouxintao（Red）, etc. in shouxingtao group; Hongbitao, Honghuabitao, Renmiantao, etc. in bitao group; Zhubo 3, Zhubo 6, Luogehongye etc. in red leaf peach group; Dahongpao, Wuyuexian, Qinglingdongtao, Yixianhong, Yangquanroutao, Datiantao, etc. in crisp peach group; Qiumi, Taiyuanshuimi, Shenzhouhongmi, Wenzhoushuimi, etc. in honey peach group; Okayamahaku, Spring time, Rebin, Hakuho, Xiahui No2, Zhaoyian, Fenghuayulu, etc. in juicy peach group; Yulupantao, Wuyuexianbiangan, Sulianpantao, Jiaqing, Yangzhou 124, etc. in flat peach group; Shuguang, Ruiguang No.2, Mayfire, Aimila, Hongliguang, Armking, etc. in nectarine group; Fertinimoroteini, Xizhuang No.1, Nong 1-2-4, Gold queen, Red haven, Jinxiu, Chengxiang, Elberta, Jingcheng, etc. in yellow peach group. Among above mentioned germplasm, Yulupantao, Qiumi, Elberta, Fertinimoroteini appeared very special because of both RAPD markers and clustering place in dendrogram.6. Molecular checking indexes which identified the experimented accessions were edited according to special markers. Typical bands were used to analyze relation with ecotypes and characters of fruits, indicating that there was no relation between typical bands and ecotypes, but some extent relation between bands and characters of fruits existed. If all cultivars were divided into freestone and clingstone, melting and nonmelting, both freestone （ratio shared total survey, 41/56=73.2%） and clingstone （ratio shared total survey, 33/79=41.8%） appeared in the loci of S₁₆₇-1050 bp linked marker raised by Warburton et al. It was apparently not strong linkage between the marker and the character, and exchange rate of genes was high. If combinations of S₁₆₇-850bp and S₁₆₇-1050bp or S₁₆₇-850bp and S₁₆₇-1400bp were used to distinguish characters of fruits, 6 freestone accessions without clingstone ones and 6 melting accessions without nonmelting ones displayed in the first combination of markers, 20 clingstone accessions and 2 semi-freestone ones without freestone ones appeared in the second combination of markers.7. Shouxintao group, weeping peach group, red leaf peach group and bitao group, crisp peach group, mitao group, honey peach group, flat peach group, nectarine group, yellow peach group were studied from aspects of polymorphic markers, frequency of markers, variation degree of amplified loci, genetic diversity, genetic differentiation and genetic structure. There were 11.9% and 88.1% of genetic variation existing among and within demes, respectively. Genetic diversity of the demes was expressed as follow: yellow peach group＞honey peach group＞flat peach group＞red leaf peach group＞crisp peach group＞bitao group and juicy peach group＞nectarine group＞shouxingtao group＞weeping peach group. Demes clustered to be divided into five sections at joint line of 0.9586, the first one was cultivated cultivars including crisp peach, honey peach, juicy peach, flat peach, nectarine, yellow peach, of which crisp peach and juicy peach had 0.9806 of highest similarity; the others were shouxingtao, bitao, red leaf peach, weeping peach, of which red leaf peach and weeping peach had 0.9160 of lowest similarity.According to above results and former research achievements of morphology, cytology, pollen, isozyme, plants of stone fruits should be classified as subgenus Amygdalus, subgenus Prunus, subgenus Armeniaca, subgenus Cerasus; Xingjiangtao is a variant and suggested scientific name as Prunus persica var. ferganensis; Shantao as Prunus davidiana. Although shanganshantao clustered as independent group in the experiment, it needs studying further about taxonomical rank. There were special germplasm by means of genetic diversity. Different demes had various genetic variations, which of yellow peach was highest. Genetic variation in demes appeared 8 times higher than among demes. The paper also gave a few methods such as dendrogram, statistic, frequency of markers, polymorphic bands and special loci, genetic variations within and among demes for conservation and utility, molecular checking indexes for cultivars can provide basic information for protection of intellectual property rights and selection of wanted cultivars.更多还原