【摘要】：大量報(bào)道表明,馴化物種的受選擇基因組區(qū)域或者基因與遺傳育種程序高度相關(guān),能用于輔助產(chǎn)生新的或者期望的表型性狀,此外基因的甲基化水平和表達(dá)水平也與這些受選擇信號(hào)高度相關(guān)。恩施黑豬是一種典型的肉脂兼用型豬種,它具有肉質(zhì)好、適應(yīng)性強(qiáng)、蓄脂能力強(qiáng)以及抗病性強(qiáng)等優(yōu)良性狀。為了研究其馴化過程中這些優(yōu)良性狀的遺傳調(diào)控機(jī)制,本研究對(duì)來自三個(gè)不同地方的恩施黑豬群體的DNA分別進(jìn)行了混池測(cè)序,并獲得了約72倍豬參考基因組大小的重測(cè)序數(shù)據(jù)。此外,本研究還分別對(duì)90日齡的恩施黑豬、大白豬以及中國(guó)野豬進(jìn)行了全基因組甲基化測(cè)序、mRNA測(cè)序以及小RNA測(cè)序。最后對(duì)四種數(shù)據(jù)進(jìn)行了整合分析,主要獲得以下結(jié)果:(1)獲得了屬于三個(gè)不同亞群的5頭恩施黑豬的全線粒體DNA序列,長(zhǎng)度約為16.7kb,發(fā)現(xiàn)三種不同體型的恩施黑豬具有較近的遺傳關(guān)系。(2)三個(gè)亞群恩施黑豬以及野豬群體重測(cè)序共產(chǎn)生266.30Gb數(shù)據(jù),在四個(gè)群體中都發(fā)現(xiàn)了大約14M的SNP,恩施黑豬與中國(guó)野豬見共享了60.92%的SNP,而恩施黑豬群體之間則共享75.56%的SNP�；谌蚪MSNP信息位點(diǎn)的系統(tǒng)發(fā)育分析結(jié)果顯示,恩施黑豬遺傳距離較小,且大型豬和中型豬具有更近的遺傳距離。(3)在恩施黑豬基因組中鑒定出417個(gè)受到強(qiáng)烈選擇的基因,這些受選擇的基因主要與新陳代謝、免疫應(yīng)答以及生長(zhǎng)發(fā)育相關(guān)。其中RPS10、GSK3A、INSL6、TBX19以及VASN5個(gè)基因在恩施黑豬中呈現(xiàn)極強(qiáng)的正向選擇信號(hào),這些基因的功能涉及到免疫、肥胖、體型大小以及繁殖力等方面,與恩施黑豬的優(yōu)良性狀高度相關(guān)。(4)首次構(gòu)建了恩施黑豬肝臟組織的全基因組單堿基分辨率DNA甲基化圖譜,結(jié)果表明,絕大多數(shù)甲基化位點(diǎn)出現(xiàn)在CpG二核苷酸處,CHG和CHH三核苷酸中甲基化位點(diǎn)較少且甲基化水平較低。(5)恩施黑豬與大白豬以及中國(guó)野豬之間的差異甲基化基因主要富集于轉(zhuǎn)錄、細(xì)胞分化以及免疫相關(guān)通路。恩施黑豬與大白豬以及中國(guó)野豬之間的差異表達(dá)基因主要富集于能量代謝以及免疫相關(guān)通路。恩施黑豬與大白豬以及中國(guó)野豬之間的差異表達(dá)miRNA的靶基因主要富集于新陳代謝以及生長(zhǎng)發(fā)育相關(guān)通路。四種組學(xué)數(shù)據(jù)都能展示恩施黑豬優(yōu)良性狀相關(guān)的分子通路,且各組學(xué)的調(diào)控過程不是一個(gè)獨(dú)立的過程,而是以某種復(fù)雜的方式共同發(fā)揮作用的。(6)啟動(dòng)子區(qū)域高甲基化的基因表達(dá)水平較低,低甲基化水平的基因表達(dá)水平較高,受這種調(diào)控模式的差異表達(dá)基因占了總差異表達(dá)的基因的50%,說明這僅是甲基化對(duì)基因表達(dá)調(diào)控的主要模式之一。同理,啟動(dòng)子區(qū)域高甲基化的miRNA表達(dá)水平較低,低甲基化水平的miRNA表達(dá)水平較高,但只有極少數(shù)miRNA受到這種機(jī)制的調(diào)控,miRNA受甲基化調(diào)控的機(jī)制和mRNA具有較大的不同。此外,受選擇基因在以上三個(gè)不同豬種中都具有高表達(dá)低甲基化的特點(diǎn),說明這些基因可能對(duì)維持豬的生命功能發(fā)揮著重要的作用。綜上,本研究對(duì)恩施豬進(jìn)行線粒體DNA測(cè)序、重測(cè)序、DNA甲基化測(cè)序、mRNA測(cè)序、mi RNA測(cè)序等多層次的功能基因組學(xué)分析,鑒定出恩施黑豬基因組中受自然選擇的區(qū)域或基因,闡明了部分重要功能基因表達(dá)調(diào)控規(guī)律,揭示各組學(xué)的調(diào)控關(guān)系。這些發(fā)現(xiàn)有助于理解恩施黑豬重要優(yōu)良經(jīng)濟(jì)性狀的起源和發(fā)展,能夠?yàn)檫M(jìn)一步開發(fā)利用我國(guó)這一寶貴遺傳資源提供基礎(chǔ)理論。
[Abstract]:A large number of reports indicate that the selected genome area or gene of the domesticated species is highly related to the genetic breeding program, and can be used to assist in the production of new or expected phenotypic traits. In addition, the level of methylation and expression of the gene is highly correlated with these selected signals. The Enshi black pig is a typical type of meat fat concurrently type pig. In order to study the genetic regulation mechanism of these excellent characters in the course of domestication, the DNA of the Enshi black pig population from three different places was sequenced, and the retest number of about 72 times the size of the pig's reference genome was obtained. In addition, the whole genome methylation, mRNA sequencing and small RNA sequencing of 90 day old Enshi black pigs, big white pigs and Chinese wild boars were carried out. Finally, four data were integrated and analyzed, and the following results were obtained: (1) the whole mitochondrial DNA sequence of 5 Enshi black pigs belonging to three different subgroups was obtained. The length of the three Enshi black pigs of three different types had a close genetic relationship. (2) the weight sequencing of three subgroups of Enshi black pigs and the wild boar group produced a total of 266.30Gb data, the SNP of about 14M was found in the four population, and the Enshi black pig shared 60.92% SNP with the Chinese wild boar, while the Enshi black pig population shared 7. 5.56% SNP. based on the phylogenetic analysis of the whole genome SNP information sites showed that the genetic distance of Enshi black pigs was smaller, and the large and medium pigs had closer genetic distance. (3) 417 strongly selected genes were identified in the Enshi black pig genome, and these selected genes were mainly associated with metabolism, immune response and Growth and development related. Among them, RPS10, GSK3A, INSL6, TBX19 and VASN5 genes are very strong positive selection signals in Enshi black pigs. These genes are related to immunity, obesity, size and fecundity, and are closely related to the excellent characters of Enshi black pigs. (4) the whole gene of the liver tissue of Enshi black pig was first constructed. The single base resolution DNA methylation map showed that most methylation sites appeared at CpG dinucleotide, CHG and CHH trinucleotides were less methylation sites and lower methylation levels. (5) the difference methylation genes between Enshi black pig and big white pig and Chinese wild boar were mainly enriched in transcription, cell differentiation and immunity. The differential expression genes between the Enshi black pig and the big white pig and the Chinese wild boar are mainly enriched in energy metabolism and immune related pathways. The differential expression of miRNA between Enshi black pig and big white pig and Chinese wild boar is mainly enriched in metabolism and growth related pathways. The four types of data can be displayed. The molecular pathways related to the excellent characters of Enshi black pigs are shown, and the regulation process of each group is not an independent process, but in a complex way. (6) the gene expression level of hypermethylation in the promoter region is low, the level of the low methylation level is higher, and the differential expression base of this regulation mode is different. 50% of the genes expressed in the total difference show that this is only one of the main modes of regulation of gene expression by methylation. Similarly, the miRNA expression level of hypermethylation in promoter region is low, and the level of miRNA expression at low methylation level is higher, but only a few miRNA are regulated by this mechanism, and the mechanism of miRNA regulation by methylation is the mechanism and the regulation of methylation. MRNA has a large difference. In addition, the selected genes have high expression of low methylation in all three different pigs. It shows that these genes may play an important role in maintaining the life function of pigs. In this study, the mitochondrial DNA sequencing, sequenced, DNA sequencing, mRNA sequencing, MI RNA sequencing of Enshi pigs were carried out in this study. In order to identify the regions or genes selected naturally in the genome of Enshi black pig, the regulation regularity of some important functional gene expression is clarified and the regulation relationship of each group is revealed. These discoveries help to understand the origin and development of the important and excellent economic traits in Enshi black pigs, and can be further developed for the benefit of further development. The basic theory of this precious genetic resource in China is provided.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

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