發(fā)布時間：2018-04-26 05:05

  本文選題：小麥 + 基因定位　； 參考：《中國農業(yè)大學》2016年博士論文

【摘要】：挖掘和克隆重要農藝性狀相關基因是遺傳解析和改良該性狀的基礎,但因小麥基因組復雜且缺乏參考序列,小麥基因的挖掘和克隆需要較多時間和人力。本研究試圖在無參考基因組序列的情況下,利用下一代測序技術建立小麥中低成本和高效率的混池轉錄組測序(Bulked segregant RNA-Seq,BSR-Seq)基因定位技術體系,并利用該方法體系對多個小麥重要性狀基因進行了定位；同時本研究也對小麥祖先種粗山羊草(Aegilops tauschii)基因組單個染色體臂進行了序列分析,以獲得其參考序列,為小麥基因的克隆打下基礎。獲得以下主要結果：1.在利用比較基因組學對小麥抗白粉病基因Pm5e進行初定位的基礎上,通過混池轉錄組測序建立了小麥BSR-Seq基因定位技術體系,該方法獲得的候選SNP定位結果和初定位結果在同一個基因組區(qū)間,顯示了其有效性�；�于此,進一步對小麥BSR-Seq基因定位技術體系中的重要影響因素進行了方法學探討,構建了更加有效和成熟的小麥BSR-Seq基因定位技術體系。通過對Pm5e的親本和分離群體進行不同策略的混池轉錄組測序,明確了表型鑒定準確性對BSR-Seq基因定位結果影響較大,測序深度對其有一定程度的影響,親本測序數(shù)據(jù)和生物學重復對其沒有顯著影響,為小麥BSR-Seq基因定位實驗的設計提供了參考。2.利用建立的小麥BSR-Seq基因定位技術體系,對1個抗葉銹病基因(Lr42)、4個抗白粉病基因(Pm5e、PmTm4、MIHLT和MlH962)、5個抗條銹病基因(YrHuaiyangl、YrMengmai58、 YrZhengmai103、YrZhoumai22、YrZhongyul152)、1個抗葉枯病基因(Sb3)、1個矮桿基因(Rht-2BL)和1個蠟質合成基因(W1)進行了BSR-Seq分析,實現(xiàn)了對這些基因低成本高效的定位,將質量性狀基因定位在7-36 Mb不等的物理區(qū)間內,遺傳定位實驗驗證了BSR-Seq分析的準確性,定位區(qū)間內大量的候選SNP為這些基因的精細定位和圖位克隆奠定了堅實的基礎。同時這些應用驗證了本研究建立的小麥BSR-Seq基因定位技術體系的有效性和高效性,顯示其適用于不同類型的作圖群體和性狀。此外,還探討了性狀類型、多態(tài)性水平、作圖群體類型和大小、混池策略和大小、測序讀長和深度對BSR-Seq基因定位結果的影響,為小麥BSR-Seq基因定位實驗的優(yōu)化設計提供了參考。3.根據(jù)粗山羊草3DS染色體臂(At3DS)物理圖譜對其MTP上3,337個BAC進行混池和下一代測序,組裝得到135個super-scaffolds(N50值為4.3 Mb),并結合多個圖譜數(shù)據(jù)得到了At3DS高質量參考序列247 Mb,覆蓋度約為90%。At3DS序列中約81%是重復序列,含2,388個基因。At3DS含1,929個核心基因,較短柄草、水稻和高粱直系同源區(qū)域核心基因數(shù)多38%。高比例(約40%)的At3DS基因是非保守的,其中48%和21%分別來自于染色體間復制事件和染色體內復制事件。和Ta3BS相比,At3DS小100 Mb少879個基因,但基因密度無差別且著絲粒大小類似,染色體臂長度和基因數(shù)目的差異主要由非著絲粒區(qū)的差異引起。比較At3DS和Ta3DS發(fā)現(xiàn),約0.36%的At3DS基因在Ta3DS中丟失,表明多倍體化后并未發(fā)生大量的基因丟失事件,且同源基因間存在大量SNP和Indel,表明多倍體化后3DS序列發(fā)生了顯著分化。
[Abstract]:Mining and cloning important agronomic traits are the basis for genetic analysis and improvement of this trait. However, because of the complexity of the wheat genome and lack of reference sequences, the mining and cloning of wheat genes need more time and manpower. This study attempts to establish the middle and low yield of Wheat by the next generation sequencing technology without the reference genome sequence. The gene mapping system of Bulked segregant RNA-Seq (BSR-Seq) was used to locate the genes of several important traits of wheat, and the single chromosome arm of the Aegilops tauschii group of wheat ancestral species (Aegilops tauschii) was sequenced. The reference sequence was used to lay the foundation for the cloning of wheat gene. The following main results were obtained: 1. on the basis of comparative genomics for wheat resistance to powdery mildew gene Pm5e, the gene mapping system of wheat BSR-Seq was established by the sequencing of mixed pool transcriptional group. The results of the candidate SNP location and the initial positioning results obtained by this method were obtained. In the same genomic region, its effectiveness was shown. Based on this, the important influencing factors in the wheat BSR-Seq gene mapping system were further studied, and a more effective and mature BSR-Seq gene mapping system for wheat was constructed. The mixed pool transcription of different strategies was carried out by the parent and separated population of Pm5e. The results showed that the accuracy of phenotypic identification had a great influence on the BSR-Seq gene location results, and the sequencing depth had a certain influence on it. The sequencing data and biological repetition had no significant influence on it. It provided a reference for wheat BSR-Seq gene mapping system based on the reference.2. for the design of Wheat BSR-Seq gene localization experiment. 1 anti leaf rust genes (Lr42), 4 powdery mildew resistance genes (Pm5e, PmTm4, MIHLT and MlH962), 5 stripe rust genes (YrHuaiyangl, YrMengmai58, YrZhengmai103, YrZhoumai22, YrZhongyul152), 1 anti leaf blight genes (Sb3), 1 dwarf genes (Rht-2BL) and 1 wax synthetic genes were analyzed to realize these bases. The quality trait genes were located in the physical interval of 7-36 Mb for low cost and high efficiency, and the accuracy of BSR-Seq analysis was verified by genetic localization experiments. A large number of candidate SNP in the positioning interval laid a solid foundation for the fine localization and mapping of these genes. These applications verified the wheat BS established by this study at the same time. The effectiveness and efficiency of the R-Seq gene mapping system showed that it was suitable for different types of mapping populations and traits. In addition, the types of traits, polymorphisms, population types and sizes, mixing pool strategy and size, the effect of sequence reading length and depth on the localization results of BSR-Seq gene, and the location of BSR-Seq genes in wheat were also discussed. The test optimization design provides reference.3. for the mixed pool and next generation of 3337 BAC on its MTP based on the physical map of the 3DS chromosome arm (At3DS) of the Leymus chinensis. The assembly gets 135 super-scaffolds (N50 value 4.3 Mb), and the At3DS high quality reference sequence 247 Mb is obtained by combining multiple Atlas data. The coverage is about 8 in the 90%.At3DS sequence. 1% is a repeat sequence, containing 2388 genes.At3DS containing 1929 core genes. The At3DS gene with high ratio of more than 38%. (about 40%) of the core genes of rice and sorghum homologous region (about 40%) is not conservative, and 48% and 21% are derived from the inter chromosome and dyed replication events respectively. Compared with Ta3BS, the At3DS small 100 Mb is 879 less than 879. But the gene density is not different and the size of the centromere is similar. The difference in the length of the chromosome arm and the number of genes is mainly caused by the difference in the non centromere region. Compared with At3DS and Ta3DS, about 0.36% of the At3DS gene is lost in Ta3DS, indicating that there is no large number of genetic loss events after polyploidy, and a large number of S exists between the homologous genes. NP and Indel showed that 3DS sequences were significantly differentiated after polyploidy.

【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S512.1
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本文編號：1804591