【作者】 李小孟；

【导师】 周志钦；

【作者基本信息】 西南大学 ， 果树学， 2010， 博士

【摘要】 柑橘是世界上最重要的经济作物之一,广泛栽培在热带和亚热带地区。柑橘属(Citrus L.)属于芸香科(Rutaceae)柑橘亚科(Aurantioideae)植物。自林奈1753年确立柑橘属以来,在过去250余年的时间里,国内外学者从形态到分子对柑橘属植物的分类和进化问题开展了一系列研究。然而,柑橘及其近缘属植物间的系统进化关系仍未解决,柑橘属植物中真正原始野生类型的种类及数目仍无定论,主要原始野生种类间的系统进化关系仍模糊不清,柑橘属植物系统进化的基本框架仍有待建立。由于种属间易于杂交、高频率芽变和栽培历史悠久等因素,柑橘属植物的分类、进化和重要栽培(杂)种的起源等问题仍待解决。本研究以柑橘属及其近缘属植物为材料,采用AFLP(Amplified Fragment Length Polymorphism)分子标记、叶绿体片段序列和核基因ITS(Internal transcribed spacer)序列分析方法,研究了柑桔属及其近缘属植物间的系统进化关系、柑橘属植物原始野生类型间系统进化的基本框架以及重要栽培柑橘类型的起源。有关重要研究结果如下：1.以九里香、蚝壳刺和酒饼簕为外类群,分别分析柑橘属及其近缘属59份材料和25份非杂种材料的叶绿体片段(psbH-petB,trnL-trnF和trnS-trnG)序列、AFLP分子标记和核基因ITS序列,重建真正柑橘果树类(the true citrus fruit trees group)六属植物间的系统进化关系。综合我们的研究,结果表明：真正柑橘果树类是个单系类群,枳属首先从真正柑橘果树类六属中分离出来,澳指檬属(Microcitrus Swingle)、澳沙檬属(Eremocitrus Swingle)和多蕊橘属(Clymenia Swingle)可能来自一个共同的祖先,其中澳指檬属和澳沙檬属关系更近,金柑属(Fortunella Swingle)与柑橘属关系紧密。另外,cpDNA数据显示澳指檬属、澳沙檬属、多蕊橘属可能与柑橘属中的枸橼(Citrus medica L.)有较近的亲缘关系。AFLP聚类分析显示澳指檬属、澳沙檬属、多蕊橘属远离柑橘属。ITS聚类分析表明金柑属与柑橘属中的宽皮柑橘关系更近。cpDNA和ITS数据的聚类分析表明澳指檬属中的澳指檬(M. australasica)可能是杂种起源,大翼橙可能是其亲本之一。2.以枳属为外类群,分别分析柑橘属植物47份材料、17份非杂种材料[包含宜昌橙(C.ichangensis Swingle)]和15份非杂种材料(不包含宜昌橙)的叶绿体片段序列、AFLP分子标记和核基因ITS序列,构建柑橘属植物原始野生类型间系统进化的基本框架。结果表明：所有数据都支持柑橘属是个单系类群,柑橘属植物中的四个基本类型枸橼(C. medica)、柚(C.grandis)、宽皮柑橘(C. reticulata)和大翼橙(Papeda)均为单系起源。另外,cpDNA数据显示大翼橙和柚关系比较紧密,枸橼、宜昌橙和宽皮柑橘关系较近。AFLP聚类分析表明宜昌橙与宽皮柑橘和柚关系最近,宽皮柑橘与柚关系紧密,大翼橙与宽皮柑橘关系较远,枸橼与宜昌橙、宽皮柑橘、柚、大翼橙四个类型所聚分支形成姊妹关系。ITS数据及合并数据均显示枸橼、柚和大翼橙关系较近,它们一起与宽皮柑橘形成姊妹关系,其中柚与大翼橙的关系最近。3.以枳属为外类群,分析柑橘属植物27份材料的叶绿体片段序列、AFLP分子标记和核基因ITS序列,确定重要栽培柑橘类型的起源。cpDNA数据表明甜橙(C. sinensis)、酸橙(C.aurantium)和葡萄柚(C. paradisi)的母本是柚,大翼橙是墨西哥莱檬(C. aurantifolia’Mexican’)的母本,粗柠檬(C. jambhiri)、兰卜莱檬(C. limonia ’Rangpur’)、广西土藜檬(C. limonia ’Guangxi’)的母本可能是宽皮柑橘,费米耐劳(C.limon ’Femminello’)、尤力克(C. limon ’Eureka’)和巴柑檬(C. bergamia)的母本可能是酸橙代代(C. aurantium ’Daidai’)。AFLP聚类分析显示宽皮柑橘是葡萄柚、甜橙和酸橙的一个亲本,粗柠檬可能是兰卜莱檬和广西土藜檬的亲本,枸橼可能是墨西哥莱檬、巴柑檬和柠檬(C. limon)的一个亲本。ITS数据表明甜橙和酸橙是由柚和宽皮柑橘杂交而来,甜橙是葡萄柚的一个亲本,酸橙是尤力克的一个亲本,枸橼是费米耐劳、巴柑檬、粗柠檬、兰卜莱檬和广西土藜檬的一个亲本,对于墨西哥莱檬我们的分子数据显示大翼橙和枸橼分别是它的两个亲本,橘可能参与了粗柠檬、巴柑檬、柠檬和莱檬的起源。更多还原

【Abstract】 Citrus is one of the most important fruit crops in the world. It is widely grown in the tropical and subtropical areas of the world. The genus Citrus established by Carl Linneaus in 1753 belongs to the subfamily Aurantioideae of the family Rutaceae. Since the establishment of the genus, Citrus has attracted a lot of research interest for more than 250 years. However, until now, the phylogenetic relationships between Citrus and its closely related genera have remained unresolved, and the number of "basic species" types in Citrus is still in controversy, and the phylogenetic relationship between the key wild species of Citrus still needs to be clarified. Mainly due to sexual compatibility between Citrus and related genera, high frequency of bud mutations, long history of cultivation, the questions concerning the taxonomy and evolution of the genus Citrus, and the genetic relationship between the cultivated species and their putative wild progenitors have remained unresolved.In the present study, the amplified fragment length polymorphisms (AFLP) fingerprints, nuclear internal transcribed spacer (ITS), and three chloroplast DNA regions (psbH-petB, trnL-trnF and trnS-trnG) of the genus Citrus and its five closely related genera were analyzed in attempt to reconstruct the molecular phylogenetic relationship in the true citrus fruit trees group, establish the phylogenetic framework of the genus Citrus and identify the origin of the cultivated citrus. The main results obtained are as follows:1. Using Murraya paniculata, Atalantia buxifolia, and Severinia buxifolia as outgroups, AFLP fingerprints, ITS sequence, and three chloroplast DNA regions of 59 accessions of the genus Citrus and its five closely related genera, and 25 accessions of the key "non-hybrid" wild species were analyzed respectively in an attempt to reconstruct the molecular phylogenetic relationship of the true citrus fruit trees group (Citrinae, Citreae, Aurantioideae, Rutaceae). Our results showed that the true citrus fruit trees group was monophyletic, and Poncirus was possibly the first genus separated from the other five genera in the group.The genera Microcitrus, Eremocitrus and Clymenia, may be of the same origin, and the genus Microcitrus was more closely related to Eremocitrus, Fortunella might have a close relationship with Citrus. In addition, our cpDNA data indicated that Microcitrus, Eremocitrus and Clymenia together formed a sister relationship to C. medica. Our AFLP dendrogram showed that Microcitrus, Eremocitrus and Clymenia were distant from the genus Citrus. Our ITS data demonstrated that Fortunella had more close relationship with C. reticulata. Our cpDNA and ITS data suggested that some species of M. australasica might have been hybrid origin and papeda might be one of its parents.2. Using Poncirus as outgroup, AFLP fingerprints, ITS sequence, and three chloroplast DNA regions of 47 accessions of genus Citrus, and 17 accessions of "non-hybrid" wild species of the genus Citrus, and 15 accessions of "non-hybrid" wild species of the genus Citrus were analyzed respectively in an attempt to establish the phylogenetic framework of the genus Citrus. All of our molecular data supported that the genus Citrus was monophyletic and the four basic biotypes of Citrus, i.e., Papeda, pummelo, citron and tangerine were of monophyletic origin. Our cpDNA data showed that C. grandis had close relationship with the Papeda species, C. medica, Ichang papeda and C. reticulata were more closely related to each other than they were to other types in the genus. Our AFLP dendrogram showed that Ichang papeda, C. reticulata and C. grandis were more closely related to each other than they were to other types in the genus, and papeda was distant from C. reticulata. Our ITS sequence and combined data analysis demonstrated that C. grandis was most closely related with the Papeda species, C. medica was more closely related to C. grandis and Papeda species than to C. reticulata, and that C. reticulata most likely have a sister relationship to the common ancestor of C. grandis, C. medica, and Papeda species.3. Using Poncirus as outgroup, AFLP fingerprints, ITS sequence, and three chloroplast DNA regions of 27 accessions of the cultivated citrus and their putative wild ancestors were analyzed in an attempt to identify both their paternal and maternal origins. Our cpDNA tree clearly indicated that pummelo (C. grndis) was female parent of sweet orange, sour orange and grapefruit; Papeda was the female parent of mexican lime (C. aurantifolia); The female parent of rough lemon (C. limon), rangpur lime (C. limonia) and guangxi local lemon (C. limonia) was C. reticulata. The chloroplast genome of bergamot (C. aurantifolia) and lemon was derived from sour orange. Our AFLP tree suggested that C. reticulata was probable a parent of, sweet orange and sour orange; Sweet orange was probable a parent of grapefruit; Rough lemon was most likely a parent of rangpur lime and guangxi local lemon; C. medica could be a parent of mexican lime, bergamot and lemon. Our ITS data demonstrated that sweet orange and sour orange were hybrids of mandarin (C. reticulata) and pummelo (C. grandis); Mexican lime was a hybrid of Papeda and C. medica; Sweet orange was a parent of grapefruit; C. medica could be a parent of C. limon’Femminello’, bergamot, rough lemon, rangpur lime and guangxi local lemon; Mandarin could take part in the origin of rough lemon, bergamot, lemon and lime.更多还原