小麦BSR-Seq基因定位技术体系的建立和应用与粗山羊草3DS染色体臂序列分析

发布时间：2018-04-26 05:05

  本文选题：小麦 + 基因定位　； 参考：《中国农业大学》2016年博士论文

【摘要】：挖掘和克隆重要农艺性状相关基因是遗传解析和改良该性状的基础,但因小麦基因组复杂且缺乏参考序列,小麦基因的挖掘和克隆需要较多时间和人力。本研究试图在无参考基因组序列的情况下,利用下一代测序技术建立小麦中低成本和高效率的混池转录组测序(Bulked segregant RNA-Seq,BSR-Seq)基因定位技术体系,并利用该方法体系对多个小麦重要性状基因进行了定位；同时本研究也对小麦祖先种粗山羊草(Aegilops tauschii)基因组单个染色体臂进行了序列分析,以获得其参考序列,为小麦基因的克隆打下基础。获得以下主要结果：1.在利用比较基因组学对小麦抗白粉病基因Pm5e进行初定位的基础上,通过混池转录组测序建立了小麦BSR-Seq基因定位技术体系,该方法获得的候选SNP定位结果和初定位结果在同一个基因组区间,显示了其有效性。基于此,进一步对小麦BSR-Seq基因定位技术体系中的重要影响因素进行了方法学探讨,构建了更加有效和成熟的小麦BSR-Seq基因定位技术体系。通过对Pm5e的亲本和分离群体进行不同策略的混池转录组测序,明确了表型鉴定准确性对BSR-Seq基因定位结果影响较大,测序深度对其有一定程度的影响,亲本测序数据和生物学重复对其没有显著影响,为小麦BSR-Seq基因定位实验的设计提供了参考。2.利用建立的小麦BSR-Seq基因定位技术体系,对1个抗叶锈病基因(Lr42)、4个抗白粉病基因(Pm5e、PmTm4、MIHLT和MlH962)、5个抗条锈病基因(YrHuaiyangl、YrMengmai58、 YrZhengmai103、YrZhoumai22、YrZhongyul152)、1个抗叶枯病基因(Sb3)、1个矮杆基因(Rht-2BL)和1个蜡质合成基因(W1)进行了BSR-Seq分析,实现了对这些基因低成本高效的定位,将质量性状基因定位在7-36 Mb不等的物理区间内,遗传定位实验验证了BSR-Seq分析的准确性,定位区间内大量的候选SNP为这些基因的精细定位和图位克隆奠定了坚实的基础。同时这些应用验证了本研究建立的小麦BSR-Seq基因定位技术体系的有效性和高效性,显示其适用于不同类型的作图群体和性状。此外,还探讨了性状类型、多态性水平、作图群体类型和大小、混池策略和大小、测序读长和深度对BSR-Seq基因定位结果的影响,为小麦BSR-Seq基因定位实验的优化设计提供了参考。3.根据粗山羊草3DS染色体臂(At3DS)物理图谱对其MTP上3,337个BAC进行混池和下一代测序,组装得到135个super-scaffolds(N50值为4.3 Mb),并结合多个图谱数据得到了At3DS高质量参考序列247 Mb,覆盖度约为90%。At3DS序列中约81%是重复序列,含2,388个基因。At3DS含1,929个核心基因,较短柄草、水稻和高粱直系同源区域核心基因数多38%。高比例(约40%)的At3DS基因是非保守的,其中48%和21%分别来自于染色体间复制事件和染色体内复制事件。和Ta3BS相比,At3DS小100 Mb少879个基因,但基因密度无差别且着丝粒大小类似,染色体臂长度和基因数目的差异主要由非着丝粒区的差异引起。比较At3DS和Ta3DS发现,约0.36%的At3DS基因在Ta3DS中丢失,表明多倍体化后并未发生大量的基因丢失事件,且同源基因间存在大量SNP和Indel,表明多倍体化后3DS序列发生了显著分化。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S512.1
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本文编号：1804591