柿属植物叶绿体测序及其系统发育和遗传变异研究

发布时间：2018-04-13 01:32

本文选题：柿属植物 + 叶绿体全基因组测序　；参考：《中南林业科技大学》2017年博士论文

【摘要】：柿属是柿树科最大的属,包括500余种,而柿(Diospyros kaki Thunb.)作为果树利用的代表种,具有显著的经济价值,是中国、韩国和日本重要的传统粮食资源。但是受其多倍性的限制,柿在分子生物学方面的研究进展缓慢,柿的起源及多倍化途径仍不明确。鉴定出与柿亲缘关系最近的二倍体近缘种,作为柿起源进化研究的材料具有重要的科学研究利用价值和重要的理论意义。因此,本课题采用高通量测序技术,开展了柿属5个物种的叶绿体全基因组测序、柿属植物叶绿体基因组图谱绘制、柿属植物的系统发育与进化地位、柿属植物分子标记开发和遗传变异等方面的研究。相关研究结果如下:1.利用 Illumina 测序技术,对柿(D.kaki Thunb.)、君迁子(D.lotus Linn.)、油柿(D.oleifera Cheng.)、浙江柿(D.glaucifolia Metc.)、金枣柿(D.'Jinzaoshi')等 5个柿属植物的叶绿体基因组进行全基因组测序,绘制了柿树科植物的第一份叶绿体基因组图谱。5个柿属植物的叶绿体基因组全长变化范围是157,300 bp～157,784 bp,呈典型的四分结构:2个反向重复区IRA和IRB、1个大的单拷贝序列LSC和1个小的单拷贝序列SSC。5个柿属植物的叶绿体基因组均注释到134个基因,其中包括80个蛋白编码基因,31个tRNA,和4个rRNA基因。此外,还鉴定出179个重复序列和283个简单序列重复;通过全序列比对分析,发现4个高度变异的区域,即trnQ_rps16,trnV_ndhC,和psbD_trnT等基因间隔区,以及ndhA的内含子区。2.通过与杜鹃花目下其他家族已发表的叶绿体基因组信息进行全序列比对分析,发现不同种间非编码区存在较大差异,尤其是蔓越莓与柿树科的差异最为显著;IRs区比SC区更为保守,4个区域的边界位置和长度在不同科的植物间也存在差异。此外,通过对柿和18个菊分支内已公布的叶绿体基因组的61个蛋白质编码基因进行系统进化分析,发现柿属于杜鹃花目,独立起源(单系类群),且杜鹃花目是菊分支中较为原始的一支。3.利用5个柿属植物叶绿体基因组间差异最大的trnQ_rps16序列对17种柿属资源进行了亲缘关系分析,序列对比后发现172个变异位点,80个信息位点;经MP和ML法构建系统进化树,初步筛选出柿的8个近缘种。随后,选取5个柿属植物叶绿体基因组高变区域和7种内切酶对柿及其近缘种进行PCR-RFLP试验,获得105个多态性的片段,聚类分析后表明:柿与金枣柿、油柿和云南野毛柿亲缘关系更近,而与浙江柿和君迁子亲缘关系较远。然后,为确定柿的最近近缘种,利用cpDNA ndh4和nrDNA ITS序列分别对柿及其近缘种进行亲缘关系分析,基于ndhA序列构建的ML进化树表明,柿与云南野毛柿聚为一支,油柿和金枣柿聚为一支,并且美洲柿和君迁子的雌雄资源被区分开,其中雌性资源与栽培柿亲缘关系比雄性资源近;而基于ITS构建的系统发育树中,柿和油柿聚为一支后与云南野毛柿聚为一支。最后,依据高通量测序结果,对柿及其4个近缘种的叶绿体全基因组序列、80个蛋白质编码基因序列、基因间隔区和内含子序列分别构建DNA矩阵后,利用ML算法和MP算法进行系统发育分析,结果均表明柿与油柿最近缘。4.根据柿属植物叶绿体全基因组测序结果,开发出4个柿属植物叶绿体基因组分子标记,并利用16份柿属材料对其在种间及柿种内的可行性进行了验证。4个标记在16份柿属植物中分别检测到31、27、7、25个变异位点,简约信息位点分别为14、5、2、13个。分别对4个标记进行聚类分析表明:CP1标记可以将11份柿品种与5个柿的近缘种完全分离,且柿与油柿距离最近,表明该标记适用于柿属植物种间资源鉴定和遗传变异分析,但不适用于柿种内;CP2、CP3、CP4标记均不能将柿品种与柿近缘种完全区分,但在11份柿品种间均检测到了多个碱基突变、插入和缺失位点,且多数柿品种发生变异的位点不同,只有'富有'和'中柿一号'两个品种未检测到特异的变异位点,故可以考虑用于柿种内的遗传变异研究。此外,本研究还借助ITS序列变异对栽培柿进行种质资源的遗传差异分析,共检测到4个变异位点,将21个栽培柿品种分成10个类型,表明ITS变异位点分析法可作为一种全新的栽培柿分子鉴定方法。
[Abstract]:Persimmon Diospyros is the largest genera, including 500 species, and persimmon (Diospyros kaki Thunb.) as a representative of fruit utilization, has significant economic value, is Chinese, South Korea and Japan are important traditional food resources. But by the polyploidy limit, persimmon in molecular biology research in slow the origin, and polyploidization of persimmon is not clear. Identify the closest relationship with persimmon diploid species, as persimmon phylogenetic study of material has important scientific research value and important theoretical significance. Therefore, this paper uses high throughput sequencing technology, carried out the 5 species of persimmon chloroplast genome sequencing, Diospyros plant chloroplast genome mapping, Diospyros phylogeny and evolutionary status of plants, research on plant molecular marker development and genetic variation etc. is related persimmon. The results were as follows: 1. by Illumina sequencing technology (D.kaki, Thunb.) of persimmon Diospyros lotus (D.lotus Linn.), persimmon (D.oleifera Cheng.), Zhejiang (D.glaucifolia Metc.), kumquat persimmon persimmon (D.'Jinzaoshi') 5 Diospyros chloroplast genome for whole genome sequencing, draw the Ebenaceae first chloroplast genome map.5 Diospyros chloroplast genome length in the range of 157300 BP to 157784 BP, with four typical structure: 2 inverted repeats of IRA and IRB, 1 large single copy sequence LSC and 1 small single copy of the chloroplast genome SSC.5 of Diospyros were annotated to 134 genes, among them including 80 protein encoding gene, 31 tRNA and 4 rRNA genes. In addition, we identified 179 repeat sequences and 283 simple sequence repeat; through sequence analysis, found 4 highly variable regions, namely trnQ_rps16, trnV_ndhC, And psbD_trnT and ndhA intergenic region, intron.2. through sequence analysis compared with the chloroplast genome information ericale other family published, that there are obvious differences between different kinds of non encoding regions, especially between Cranberry with Diospyros most significantly; IRs district area more than SC conservative, boundary location and the length of the 4 regions also exist in different families of plants in different groups. In addition, phylogenetic analysis by 61 protein encoding gene of chloroplast genome of persimmon and 18 chrysanthemum branch published, found that persimmon belongs to the Ericales, independent origin (monophyletic), and Rhododendron take orders is a.3. primitive utilizedju branch in 5 Diospyros plants among the biggest differences in chloroplast genome trnQ_rps16 sequence of 17 species of Diospyros resources by phylogenetic analysis and sequence comparison found 172 variation Site 80 information sites; by MP and ML methods to construct the phylogenetic tree. The results showed that 8 species of persimmon. Subsequently, the persimmon and its sibling species PCR-RFLP test hypervariable region 7 restriction endonucleases and selected 5 Diospyros chloroplast genome, 105 polymorphic fragments, clustering the analysis showed that persimmon persimmon and kumquat, persimmon persimmon and Yunnan wild hair a closer relationship with Zhejiang, dateplum persimmon and distantly related. Then, in order to determine the nearest relatives of persimmon, persimmon and its relatives of the genetic relationship was analyzed by cpDNA ndh4 and nrDNA ITS sequence, ML the phylogenetic tree constructed by ndhA sequences showed that based on the persimmon and Yunnan wild hair Shiju a persimmon and kumquat, persimmon were clustered into one clade, and America and dateplum persimmon female resources are separated, the relationship between female and male than the genetic resources of persimmon cultivation resources; and constructing the system based on ITS With the tree, persimmon persimmon and clustered into one with Yunnan as a wild hair Shiju. Finally, on the basis of high-throughput sequencing results of persimmon and its 4 species of chloroplast genome sequences, sequences of 80 protein encoding gene, intergenic region and intron sequences were constructed for the DNA matrix. The phylogenetic analysis was carried out using ML algorithm and MP algorithm. The results show that with the recent.4. according to the edge of persimmon persimmon Diospyros chloroplast genome sequencing results, the development of molecular plant chloroplast genome of 4 persimmon markers, and the use of 16 persimmon materials on the interspecific and intraspecific persimmon in the feasibility was verified by.4 a marker in 16 Diospyros spp. were detected 31,27,7,25 mutation sites, parsimony informative sites are 14,5,2,13 respectively. Each of the 4 markers cluster analysis shows that CP1 markers can be relatives of 11 persimmon varieties and 5 persimmon completely From, and persimmon persimmon and the nearest, show that the marker for analysis of Diospyros species resource identification and genetic variation, but not suitable for persimmon in; CP2, CP3, CP4 mark could not be persimmon varieties and persimmon species completely distinguish, but in 11 copies among persimmon cultivars were detected a plurality of mutation, insertion and deletion sites, and most of the variation of persimmon varieties in different sites, only the 'rich' and 'one' two persimmon varieties did not detect specific mutation sites, it can be considered for the study of genetic variation within species of persimmon. In addition, this study also by ITS sequence analysis the variation of Germplasm Resources of persimmon cultivation the genetic difference of 4 mutation sites were detected, 21 cultivated persimmon varieties are divided into 10 types, showed that ITS mutation analysis can be used as a new method for molecular identification of persimmon cultivation.

【学位授予单位】：中南林业科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S665.2

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