【作者】 李映志；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2006， 博士

【摘要】 分布在我国南岭山脉的野橘种质是我国重要的宽皮柑橘种质资源，不仅对研究我国宽皮柑橘的起源和演化，而且对引进野橘抗性性状进行宽皮柑橘育种来说，都具有重要的意义。宽皮柑橘地方品种是人们在柑橘栽培实践中，长期驯化和选择产生的一些栽培历史悠久的柑橘品种，这些种质中不仅蕴涵着优良的抗性和品质性状基因，而且同样是研究柑橘起源与演化的重要材料。为便于这些种质的有效收集和保存，并分析他们与我国宽皮柑橘起源的关系，本文以宽皮柑橘地方品种和野橘为试材，对我国宽皮柑橘和野橘的遗传多样性及系统发育关系进行了研究。获得的主要结果有：1．在对33份宽皮柑橘地方品种和19份野橘种质的遗传多样性研究中，共筛选出具有多态性的10对核基因组SSR引物和8对叶绿体基因组SSR引物，这些引物共扩增出60个核基因组SSR多态性条带和19个叶绿体基因组多态性条带。除分布于3组由7份种质所形成的组内种质外，核基因组SSR标记可将所有的种质区分开，包括2个不同的砂糖橘品系。2．位点多态性信息含量(Locus polymorphic information content，PIC)的计算结果表明，在叶绿体基因组和核基因组水平上，野橘种质的遗传多样性都要高于宽皮柑橘地方品种，在核基因组SSR水平上，二者的PIC值分别为0.6138和0.5091；在叶绿体基因组SSR水平上，二者的PIC值分别为0.4921和0.3769。3．利用Jaccard距离，通过UPGMA(The unweighted pair group method based on arithmetic averages，非加权对等平均法)法的聚类分析结果表明，在核基因组SSR水平上，宽皮柑橘地方品种聚成5组，野橘种质聚成2组；在叶绿体基因组水平上，宽皮柑橘地方品种和野橘种质都分别聚成2组：但叶绿体SSR和核SSR的聚类模式并不完全一致。通过分析叶绿体SSR和核SSR聚类模式上的差别，对一些地方品种的杂种起源进行了讨论。从聚类分析的结果还可看出，野橘种质中，崇义野橘与宽皮柑橘地方品种的关系最近，莽山野橘A类的种质距宽皮柑橘地方品种最远；宽皮柑橘地方品种中，茶枝柑与野橘种质聚在一起。4．系统发育分析所使用的3个序列中，trnL-trnF的序列长度为1009bp-1083bp，比对后序列长1157bp，其中，变异位点94个(0.081％)，简约信息位点17个(0.015％)。matK区域的5’端3’端分别测序后合在一起用于进化树分析。5’端长733bp-748bp，3’端长779bp-789bp，合起来比对后的序列长1556bp，其中，变异位点136个(0.087％)，简约信息位点26个(0.017％)。FLint2的序列长度为766bp-783bp，比对后的序列长795bp，其中变异位点91个(0.1145％)，简约信息位点36个(0.0452％)。5．对3个DNA数据集进行的数据集间相容性分析结果表明，三个数据集间差异较大，不相容性较高。因此，分别构建了最大简约树(排除gaps和将gaps处理为新性状)、最大似然树、邻接距离树和trnL-trnF与matK联合数据集的最大简约树。根据建树的结果，对核DNA序列与叶绿体DNA序列间的不相容性与相关种质的杂种起源进行了讨论。6．构树的结果表明，尽管3个序列数据集构建出的进化树在拓扑结构上有些差异，但他们之间存在很多共同点。其中，大多数宽皮柑橘地方品种与大多数野橘种质能形成一个单系分支，即宽皮柑橘分支。宽皮柑橘地方品种中，滑皮柑、臭皮柑、夔柑、本地广橘、瓯柑5份种质为宽皮柑橘作父本，柚类或橙类作母本的杂种。宽皮柑橘地方品种中的青皮应与香橙为同一类种质。野橘种质中，莽山野橘A1和道县大果野橘5与宽皮柑橘分支成姐妹系。位于宽皮柑橘分支中的莽山野橘B0和江永野橘4与宽皮柑橘地方品种成并列分支，而道县小果野橘1、崇义野橘A1、江永粗皮野橘1和崇义野橘A2则在宽皮柑橘分支中形成一个进一步分化的单系分支。7．根据序列数据分析结果对与宽皮柑橘有关的一些生物型的起源进行了讨论。大多数结果与前人研究结果一致，但通过叶绿体基因组证据和核基因组证据的结合可进一步明确他们的父母本来源。在这些结果中，值得注意的有：1)大翼橙类的3份种质中，大种橙、宜昌橙与红河大翼橙在叶绿体基因组和核基因组上都有区别，并且能分别与其他种质形成分支，他们可能分别与宽皮柑橘、柚类或枸橼类的起源有关；2)金柑类种质宁波金柑和四季橘在叶绿体DNA数据集和核DNA数据集中都能形成一个分支，因此本研究中的3个DNA数据集不支持四季橘的起源与宽皮柑橘有关的假说。此外，本文还对人工合成柑橘嵌合体红江橙的几种分离类型进行了分子标记鉴定。研究中，使用了5种红江橙嵌合体材料，和叶绿体SSR、核SSR及RAPD等DNA分子标记。结果表明，1对叶绿体SSR引物和1个RAPD引物分别在5种红江橙材料中表现出多态性，谱带模式与嵌合体的结构有一定的联系。对RAPD特异引物片段回收测序后，根据序列丌发出相应的SCAR标记，可用于进一步的嵌合机理研究。更多还原

【Abstract】 Wild mandarins played important roles both in caltivar breeding and citrus phylogenetic relationship analysis. In the first chapter of this thesis, genetic diversity of mandarin accessions and phylogenetic relationships among them and related citrus species were studied. Hongjiangcheng is a valuable cutivar in China, and as a citrus synthetic chimera, in the second chapter of this thesis, its genetic background were studied. The main results were as follows:1. In the genetic diversity study of 33 loose skin mandarin landraces and 19 wild mandarin accessions, ten nuclear simple sequence repeat(nSSR) and eight chloroplast simple sequence repeat(cpSSR) markers were seleted as polymorphic markers. These primers amplified 60 nuclear SSR polymorphic bands and 19 choloroplast SSR polymorphic bands. Except for accessions in 3 groups formed by 7 accessions, nuclear SSR primers could discriminated all accessions studied, even two differently distributed’Shatangju’ accessions.2. Our analysis of nuclear and chloroplast genome polymorphism showed that the genetic diversity in wild accessions was slightly higher than that found in landraces, with polymorphic information content values of 0.6138 and 0.5091 at nSSR loci, and 0.4921 and 0.3769 at cpSSR loci, respectively.3. Five groups of landrace and two groups of wild mandarin were identified based on the nSSR data and two groups each of landraces and wild mandarins were identified at the cpSSR loci. Different distribution patterns of landraces and wild mandarins were observed between dendrograms constructed using these two marker systems, and hybrid origins of several landraces were suggested by these results. Combined analysis also suggested that, among wild mandarin,’Chongyi’ showed the closest relationship to landraces, and that’Chazhigan’ landrace was the primary genotype among landraces.4. Of the 1157 aligned positions in the trnL-trnF regions, 94 sites were variable, pairwise sequence divergences ranged from 0 to 2.8%. Of the 1556 aligned positions in the matK regions, 136 sites were variable, pairwise sequence divergences ranged from 0 to 2.6%. Of the 795 aligned positions in the Flint2 region, 91 were variable, pairwise divergence ranged from 0 to 3.4%. 5. Compatibility analysis of three DNA sequence dataset showed difference among three dataset. Therefore, maximum parsimony, maximum likelihood and neighbor join distance phylogenetic trees were constructed using three dataset sperately and using combinated dataset of trnL-trnF and matK sequence dataset. Incompatiablity between nuclear DNA sequence dataset and chloroplast DNA sequence dataset were used to analysize hybrid origination of related accessions.6. Sequence data showed that most of landraces and most of wild accessions form a manophyletic clade. Among other landraces,’Choupigan’,’Huapigan’,’Kuigan’,’Bendiguangju’ and’Ougan’ were of hybrid orign with mandrin as male parent and pummelo or orange as female parent; Qingpi is a variety of Yuzu(Citrus junos Sieb. Ex Tanaka). Two other wild accessions,’Mangshan wild mandarin Al’ and’Daoxian wild mandarin No. 5’ were sister to loose skin mandarin clade.’Mangshan wild mandarin B0’,’Jangyong wild mandarin No. 4’ was sister to landraces in loose skin mandarin clade.’Daoxian wild mandarin No. 1’,’Chongyi wild mandarin Al’,’Jiangyong wild mandarin No. 1’ and’Chongyi wild mandarin A2’ formed a clade in loose skin mandarin clade.7. Several genotypes related to mandarin were also discussed in this thesis, and their possible paternal and mathernal origins were also proposed. Two kinds of pepada were identified according their phylogenetic relationships to species of genera Citrus. No phylogenetic relationships were found between calamadin and loose skin mandarin.8. In addition, SSR and RAPD molecular markers were employed to study chimerism of five’Hongjiangcheng’ chimeras. One pair of cpSSR primers and one RAPD primer amplified polymorhic bands in five materials, and the distribution of polymorhic bands could be explained by chimera itselves. One polymorhic RAPDs was sequenced, and results showed that it was 61.9% homologous to Poncirus trifoliata citrus tristeza virus resistance gene locus.更多还原