植物单染色体单倍型构建的技术体系

The difference between homologous chromosomes is one of the hot topics that geneticists concerned about. Every single chromosome harbors its unique haplotype, and the combination of different genetic loci, located on a pair of homologous chromosomes, has an important effect on the phenotypes. Up to now, it is difficult to directly distinguish the sequence differences between homologous chromosomes by high-throughput sequencing. However the haplotype analysis technique of single chromosome can be used to provide important information for the comparative analysis of homologous chromosomes, personal relationship tracking, and the analysis of allele differentiation. Recently, haplotype based on chromosome microdissection has been developed in human. However there has been no efficient method to construct haplotype of single chromosome in plants. In this study, the single chromosome haplotype analysis system will be developed using wheat 7DL ditelomeric additional line as the material. Firstly, two 7DL homologous chromosomes will be separately microdissected using the chromosome microdissection technique developed in our lab, then amplified using the multiple displacement amplification (MDA) system. Secondly, the quantified and qualified single chromosome DNA will be sequenced by the high-throughput sequencing technique and assembled using De Novo Magic assembly method. Thirdly, the haplotype of two 7DL homologous chromosomes will be constructed and comparatively analyzed. Taken together the technique system of plant single chromosome haplotype construction will be developed in this study. This study will be significant in providing a new opportunity for useful alleles digging, genetic recombination studying and heterosis theory investigation.

同源染色体差异一直是遗传学家关注的热点之一，每一条染色体都有自己独特的单倍型，同源染色体不同位点的组合对表型有重要影响。目前高通量测序技术尚不能直接区分同源染色体的差异。而利用单染色体单倍型分析技术，可为同源染色体序列比较、个体亲缘关系追踪、等位基因差异等分析提供重要信息。目前已经在人类建立了基于染色体微分离的单倍型分析技术，然而在植物中还没有有效的单染色体单倍型构建技术。本课题拟以六倍体小麦7DL端体附加系为材料，利用本实验室建立的染色体显微分离体系，分别分离7DL两条同源染色体，通过MDA扩增单染色体获得其大量DNA，并在此基础上进行高通量测序，同时利用De Novo Magic 序列拼接新技术进行组装，以此构建7DL１对同源染色体单倍型,并对其单倍型进行比较分析,从而获得构建植物单染色体单倍型的技术体系。该研究对未来规模挖掘植物优异等位基因、遗传重组理论及杂种优势理论研究有重要意义。

同源染色体差异一直是生物学科的研究热点课题之一。目前高通量测序技术尚不能直接区分同源染色体差异。本研究以普通小麦7DL双端体附加系为材料，用成熟的单染色体显微分离技术成功地将同一分裂相中分散较好的两条7DL同源染色体分离出来，分别用MDA和MALBAC技术对其进行基因组扩增。利用HiFi、stLFR和NGS等技术构建了基于多种不同测序策略的单倍型测序技术体系。采用多种组装策略获得小麦7DL两个单倍型的基因组序列，约占小麦7DL全长（648Mb）的30%左右, 构建了基于单倍体扩增测序的单倍型基因组组装技术体系。共线性分析发现两个单倍型之间、两个单倍型与7DL参考基因组之间具有较好的共线性。通过单倍型间的比较分析，鉴定了不同种类的序列变异，包括SNP、Indel和SV。其中有害变异可能为抗病相关研究提供基础。综上，该技术对同源染色体比较、等位基因差异分析、作物中优异等位基因的发掘、植物杂交优势理论、遗传重组理论等研究具有重要意义。

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