【作者】 谢一青；

【导师】 姚小华；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2013， 博士

【摘要】 小果油茶（Camellia meiocarpa Hu.）是我国栽培面积和年产量仅次于普通油茶的油料树种，但对其遗传改良研究仍处于初级阶段。小果油茶为异花授粉植物，种内变异类型极为丰富。为充分挖掘和利用现有的遗传变异，本文在前期充分调查的基础上，对小果油茶种内自然变异类型进行了划分，同时针对小果油茶分类地位仍存在异议的问题，从孢粉学、表型、基因组DNA和基因转录水平上对典型小果油茶类型的亲缘关系进行了研究。主要结论如下：1．根据小果油茶生物学特性及种群遗传特点，结合前期的调查资料，初步对小果油茶种内变异类型进行了划分。结果表明：小果油茶树体、果实、叶片、花等表型性状变异十分丰富，其中树体性状的变异系数最大（平均CV=43.79%），其次是果实性状（平均CV为26.09%），叶和花性状的变异系数相对较小，平均为16.84%和16.53%。综合R型聚类和主成分分析结果及成熟期等生态表现，提出以果实横径、果皮厚度和成熟期作为类型划分的指标，将小果油茶初步划分为10个类型。2．比较了部分典型小果油茶类型和普通油茶的花粉形态，并分析其与山茶属物种间的亲缘关系，结果表明：不同类型小果油茶的花粉均具有山茶属植物花粉的种属特征，即：花粉粒为长形球、近球形，赤道面观长椭圆形，极面观三裂圆形，具三孔沟，但花粉粒的外观表面纹饰特征因不同类型而异。相关分析表明，不同小果油茶类型的单果质量、鲜出籽率与花粉粒极轴长（P值）呈显著负相关，与沟间距离呈显著正相关，因此可以通过花粉形态特征来鉴别小果油茶类型。通过比较小果油茶和36个山茶物种间的花粉形态特征，发现小果油茶花粉大小与油茶组、红山茶组的大部分种相似，而与连蕊茶组、短柱茶组、糙果茶组等大部分种相差甚远，初步说明，小果油茶与油茶组和红山茶组的大部分种有较近的亲缘关系。3．对部分典型小果油茶类型和42个山茶属油用类物种间的亲缘关系进行分析。结果表明：46个物种（类型）的表型性状变异极为丰富，叶片、花、果实及其经济性状等39个性状的变异系数达4.95%~159.41%，尤其是单果质量和单果籽质量的变异系数更是高达159.41%和134.72%；通过聚类分析将46个物种（类型）分为2大类群，其中小果油茶大果薄皮立冬籽（龙眼茶）、中果薄皮霜降籽（羊屎茶）、小果薄皮寒露籽（珍珠茶）和中果薄皮寒露籽（宜春白皮中籽）先与钝叶短柱茶聚在一起，再与短柱茶、粉红短柱茶、小果短柱茶、普通油茶等12个物种聚为一个亚类，说明小果油茶的表型性状和这些物种的表型性状相似性较高。4、采用AFLP标记对部分典型小果油茶类型和42个山茶属油用类物种间的亲缘关系进行了分析。结果表明：18对AFLP引物组合共检测到642条多态性条带，多态性条带占95.69%；小果油茶羊屎茶与珍珠茶遗传相似系数最大，为0.9539，越南油茶与滇北红山茶遗传相似系数最小（0.5272）。通过聚类分析将46个物种（类型）划分为5个类群：越南油茶和小果短柱茶分别单独为一类；糙果茶组聚为一类；14个红山茶属物种和短柱茶组中果实性状较为类似的陕西短柱茶、攸县油茶和闽鄂山茶聚为一大类；小果油茶与除小果短柱茶外的油茶组、短柱茶组部分种聚为一类。与形态学标记相比较，发现两种标记的聚类结果并不完全一致，但在一定程度上可以互补。5．以小果油茶和普通油茶的嫩叶及种仁为材料，建立了适合油茶的cDNA-AFLP反应体系。结果表明：总RNA中分离的mRNA经合成双链cDNA后，用限制性内切酶EcoRⅠ和MseⅠ在37℃下5h内完全酶切，经5U T4连接酶16℃连接的产物稀释10倍后直接用于预扩增；在20μL选择性扩增体系中，以预扩增产物稀释30倍为模版、dNTP (10mM)1.5μL、引物(10μM)1.0μL、Taq酶用量1.25U时的扩增效果较好。利用优化体系成功筛选出61对可以获得带型丰富且重复性好的引物组合。6．为进一步分析典型小果油茶类型间差异的遗传学基础，本文尝试用cDNA-AFLP技术对小果油茶基因表达差异进行分析。138对引物组合共扩增出3649条转录衍生片段（TDFs），其中差异显示的TDFs有3557个，多态率高达97.48%。研究发现在大果薄皮立冬籽龙眼茶和普通油茶中特异表达的TDFs占总差异带的4.27%，在龙眼茶和宜春白皮中籽中特异表达的TDFs占4.53%，在龙眼茶和羊屎茶中特异表达的TDFs有1.57%，在龙眼茶、羊屎茶和宜春白皮中籽中均特异表达的TDFs占1.88%，而在龙眼茶、宜春白皮中籽和普通油茶中均特异表达的TDFs占3.54%，说明龙眼茶与普通油茶和宜春白皮中籽的亲缘关系可能较近，而与羊屎茶的亲缘关系可能相对较远。基于TDFs表达模式差异的多态性将5个参试材料分为3类，其中龙眼茶和宜春白皮中籽聚为一类，羊屎茶和珍珠茶归为一类，普通油茶独立为一类，这与基因组DNA的AFLP分析结果一致。差异TDFs经回收及测序，获得了789个差异基因片段。经BlastX/N同源性比对，有716个在GeneBank中找到了同源序列，其功能主要涉及信号转导、转录因子、蛋白质合成、代谢、运输、细胞防御等方面。以上研究结果为小果油茶的分类、遗传改良、种质资源保护及进一步开发利用提供了理论依据。更多还原

【Abstract】 Camellia meiocarpa is a kind of native oil plant species, whose cultivation area andannual output were only less than that of Camellia oleifera. While the genetic improvement,breeding and application of C. meiocarpa was just on its germinal stage. C. meiocarpa is across-pollination plant, which is rich in genetic variation. In order to genetic improve, theintraspecific type of C. meiocarpa was primarily divided based on previous work, and thegenetic relationships of four typical types that were studied on phenotypic, morphological,DNA and transcription level. The main conclusions were drawn as follows:1. The variation types of C. meiocarpa were classified according to the biologicalcharacteristics and population genetic characteristics of C. meiocarpa. The results showed thatthe phenotypic variation of C. meiocarpa plant type, fruit, leaves and flowers were very high.The variations of plant type (43.79%) were the highest among four phenotypic traits, thesecond was fruit traits (26.09%), and variations of leaf (16.84%) and flower (16.53%) were theleast. Based on the results the main principle for classifying the variation types of C.meiocarpa were fruit diameter, pericarp thickness and maturation stage. The intraspecific typeof C. meiocarpa was primarily divided into ten different types.2. Pollen morphology of Camellia meiocarpa and C. oleifera were analysted and thegenetic relationships of C. meiocarpa with other Camellia species were also studied. Underelectron microscope, the pollen morphology, polar view and equatorial view of different typesof C. meiocarpa were similar with Camellia pollen. The pollen gains were prolate orsubsphaeroidal, and its equatorial view was long ellipe, while the polar view was3-labedrounded with3narrow operculums. But there were some distinctive differences in pollen exineornament. The correlation analysis showed that the fruit weight and fresh seed ratio of C.meiocarpa was significant negatively related to pollen polar axis length (P) and positivelyrelated to the distance between colporates, respectively. Compared with the36species pollen morphology of Camellia, the pollen grain size of different types in C. meiocarpa were similarto that of Oleifera and Camellia, which indicated that C. meiocarpa had a close geneticrelationship with Oleifera and Camellia species.3. Morphological makers were used to evaluate the genetic relationships of someCamellia meiocarpa variation types and42Camellia species that seeds were used to beproduced oil. The results showed that the variation of phenotypic traits between46sampleswere very high, the coefficient of variation (CV) of39traits from leaf, flower and fruit wasfrom4.95%to159.41%, and the cv of fruit and seed weight reached up to159.41%and134.72%, respectively. The results showed that46samples were divided into2categories. C.meiocarpa cv.‘Longyan’,‘Yangshi’,‘Zhenzhu’and ‘Yichunbaipi’ were classificated togetherwith C. obtusifolia, then C. brevistyla, C. puniceiflora, C. confusa, C. oleifera, C. weiningensis,C. sasanqua, C. lanceoleosa, C. fluviatilis, C. yuhsienensis, C.grijsii, C. parafurfuracea, C.shensiensis were classificated together.4. AFLP makers were also used to evaluate the genetic relationships of four variationtypes of Camellia meiocarpa and42Camellia species. The results showed that642bands wereobtained using18primer combinations and95.69%of them were polymorphic loci. Thegenetic distance between ‘Yangshi’ and ‘Zhenzhu’ was maximum (0.9539), when that of C.Vietnamensis and C. boreali-yunnanica was minimum (0.5272). The results of clusteringanalysis indicated that46samples were divided into5groups. C. Vietnamensis and C. confusawere respectively clustered group Ⅰand Ⅱ, group Ⅲ were Section Furfuracea, group Ⅳwere Section Camellia and a subset of C.grijsii, C. shensiensis, C. yuhsienensis, which hadsimilar fruit traits. Group Ⅴ were C. meiocarpa, Section Oleifera and most of SectionParacamellia.5. A suitable cDNA-AFLP system for C. meiocarpa and C. oleifera was established afteroptimizing several key factors, including enzyme digestion system, pre-amplification andselective amplification. The results showed that total RNA of the tender leaf and kernel wasextracted by quick extraction kit of EASYspin Plus plant RNA and Trizol method. mRNA was synthesized into double stranded cDNA after it was isolated from total RNA, then it wasdigested completely by EcoRⅠand MseⅠat37℃for5hours. The digested product wasconnected with5U T4ligase at16℃for an overnight. Then reducible pre-amplification wasobtained when ligation product were diluted by10times for pre-amplification. And the optimalconditions for a20μl volume of selective amplification were obtained when the template ofpre-expanded product diluted by30times and with dNTP (10mM)1.5μL, primer (10μM)1.0μL, Taq enzyme1.25U. Moreover,61out of210primer pairs were successfully selected bythe improved method.6. In order to understand genetics bases of differentation of C. meiocarpa variation types,transcripts profile was analysted by cDNA-AFLP.3649transcript-derived fragments (TDFs)were obtained with138primer pairs, a3557TDFs appeared to be polymorphic loci (97.48%).Among them, the specific expression TDFs in C. meiocarpa cv.‘Longyan’ and C. oleifera upto4.27%of the total, the specific expression TDFs in C. meiocarpa cv.‘Longyan’ and‘Yichunbaipi’ was about4.53%, the specific expression TDFs in C. meiocarpa cv.‘Longyan’and ‘Yangshi’ were1.57%, and that of C. meiocarpa cv.‘Longyan’,‘Yangshi’ and‘Yichunbaipi’ were only1.88%, while the specific expression TDFs in C. meiocarpa cv.‘Longyan’,‘Yichunbaipi’ and C. oleifera occupied3.54%, which explained that C. meiocarpacv.‘Longyan’ may have closer relationship with C. meiocarpa cv.‘Yichunbaipi’ and C. oleifera,five materials were divided into3classese, cluster Ⅰ includes C. meiocarpa cv.‘Yangshi’ and‘Zhenzhu’, cluster Ⅱ includes ‘Longyan’ and ‘Yichunbaipi’, while C. oleifera separatecategorization. The result based on cDNA-AFLP was consistent with the AFLP taxa. A subsetof1176differential TDFs were sequenced, which789sequences could be obtained.716ofthem were annotated for their function with GeneBank database by BlastX/N, and functionalontology showed genes related to signal transduction, transcription factor, metabolism,transportation, cell defence mechanisms and protein sythesis.Theses results provided a theory basis for the further study of classificaton, geneticimprovement, resource protection and utilization of C. meiocarpa.更多还原