發(fā)布時間：2018-04-13 01:32

  本文選題：柿屬植物 + 葉綠體全基因組測序��； 參考：《中南林業(yè)科技大學(xué)》2017年博士論文

【摘要】：柿屬是柿樹科最大的屬,包括500余種,而柿(Diospyros kaki Thunb.)作為果樹利用的代表種,具有顯著的經(jīng)濟(jì)價值,是中國、韓國和日本重要的傳統(tǒng)糧食資源。但是受其多倍性的限制,柿在分子生物學(xué)方面的研究進(jìn)展緩慢,柿的起源及多倍化途徑仍不明確。鑒定出與柿親緣關(guān)系最近的二倍體近緣種,作為柿起源進(jìn)化研究的材料具有重要的科學(xué)研究利用價值和重要的理論意義。因此,本課題采用高通量測序技術(shù),開展了柿屬5個物種的葉綠體全基因組測序、柿屬植物葉綠體基因組圖譜繪制、柿屬植物的系統(tǒng)發(fā)育與進(jìn)化地位、柿屬植物分子標(biāo)記開發(fā)和遺傳變異等方面的研究。相關(guān)研究結(jié)果如下:1.利用 Illumina 測序技術(shù),對柿(D.kaki Thunb.)、君遷子(D.lotus Linn.)、油柿(D.oleifera Cheng.)、浙江柿(D.glaucifolia Metc.)、金棗柿(D.'Jinzaoshi')等 5個柿屬植物的葉綠體基因組進(jìn)行全基因組測序,繪制了柿樹科植物的第一份葉綠體基因組圖譜。5個柿屬植物的葉綠體基因組全長變化范圍是157,300 bp～157,784 bp,呈典型的四分結(jié)構(gòu):2個反向重復(fù)區(qū)IRA和IRB、1個大的單拷貝序列LSC和1個小的單拷貝序列SSC。5個柿屬植物的葉綠體基因組均注釋到134個基因,其中包括80個蛋白編碼基因,31個tRNA,和4個rRNA基因。此外,還鑒定出179個重復(fù)序列和283個簡單序列重復(fù);通過全序列比對分析,發(fā)現(xiàn)4個高度變異的區(qū)域,即trnQ_rps16,trnV_ndhC,和psbD_trnT等基因間隔區(qū),以及ndhA的內(nèi)含子區(qū)。2.通過與杜鵑花目下其他家族已發(fā)表的葉綠體基因組信息進(jìn)行全序列比對分析,發(fā)現(xiàn)不同種間非編碼區(qū)存在較大差異,尤其是蔓越莓與柿樹科的差異最為顯著;IRs區(qū)比SC區(qū)更為保守,4個區(qū)域的邊界位置和長度在不同科的植物間也存在差異。此外,通過對柿和18個菊分支內(nèi)已公布的葉綠體基因組的61個蛋白質(zhì)編碼基因進(jìn)行系統(tǒng)進(jìn)化分析,發(fā)現(xiàn)柿屬于杜鵑花目,獨(dú)立起源(單系類群),且杜鵑花目是菊分支中較為原始的一支。3.利用5個柿屬植物葉綠體基因組間差異最大的trnQ_rps16序列對17種柿屬資源進(jìn)行了親緣關(guān)系分析,序列對比后發(fā)現(xiàn)172個變異位點(diǎn),80個信息位點(diǎn);經(jīng)MP和ML法構(gòu)建系統(tǒng)進(jìn)化樹,初步篩選出柿的8個近緣種。隨后,選取5個柿屬植物葉綠體基因組高變區(qū)域和7種內(nèi)切酶對柿及其近緣種進(jìn)行PCR-RFLP試驗(yàn),獲得105個多態(tài)性的片段,聚類分析后表明:柿與金棗柿、油柿和云南野毛柿親緣關(guān)系更近,而與浙江柿和君遷子親緣關(guān)系較遠(yuǎn)。然后,為確定柿的最近近緣種,利用cpDNA ndh4和nrDNA ITS序列分別對柿及其近緣種進(jìn)行親緣關(guān)系分析,基于ndhA序列構(gòu)建的ML進(jìn)化樹表明,柿與云南野毛柿聚為一支,油柿和金棗柿聚為一支,并且美洲柿和君遷子的雌雄資源被區(qū)分開,其中雌性資源與栽培柿親緣關(guān)系比雄性資源近;而基于ITS構(gòu)建的系統(tǒng)發(fā)育樹中,柿和油柿聚為一支后與云南野毛柿聚為一支。最后,依據(jù)高通量測序結(jié)果,對柿及其4個近緣種的葉綠體全基因組序列、80個蛋白質(zhì)編碼基因序列、基因間隔區(qū)和內(nèi)含子序列分別構(gòu)建DNA矩陣后,利用ML算法和MP算法進(jìn)行系統(tǒng)發(fā)育分析,結(jié)果均表明柿與油柿最近緣。4.根據(jù)柿屬植物葉綠體全基因組測序結(jié)果,開發(fā)出4個柿屬植物葉綠體基因組分子標(biāo)記,并利用16份柿屬材料對其在種間及柿種內(nèi)的可行性進(jìn)行了驗(yàn)證。4個標(biāo)記在16份柿屬植物中分別檢測到31、27、7、25個變異位點(diǎn),簡約信息位點(diǎn)分別為14、5、2、13個。分別對4個標(biāo)記進(jìn)行聚類分析表明:CP1標(biāo)記可以將11份柿品種與5個柿的近緣種完全分離,且柿與油柿距離最近,表明該標(biāo)記適用于柿屬植物種間資源鑒定和遺傳變異分析,但不適用于柿種內(nèi);CP2、CP3、CP4標(biāo)記均不能將柿品種與柿近緣種完全區(qū)分,但在11份柿品種間均檢測到了多個堿基突變、插入和缺失位點(diǎn),且多數(shù)柿品種發(fā)生變異的位點(diǎn)不同,只有'富有'和'中柿一號'兩個品種未檢測到特異的變異位點(diǎn),故可以考慮用于柿種內(nèi)的遺傳變異研究。此外,本研究還借助ITS序列變異對栽培柿進(jìn)行種質(zhì)資源的遺傳差異分析,共檢測到4個變異位點(diǎn),將21個栽培柿品種分成10個類型,表明ITS變異位點(diǎn)分析法可作為一種全新的栽培柿分子鑒定方法。
[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.

【學(xué)位授予單位】：中南林業(yè)科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S665.2

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