杨树高分辨率核型图构建及第19号染色体结构分化研究

The chromosomes of poplar are so small that it is difficult to accurately identify each chromosome on the basis of mitotic metaphase chromosome morphology, which leads to the lag in its molecular cytogenetic study. The developing of the molecular cytogenetic techniques and the poplar genome information offer us a unique opportunity to conduct molecular cytogenetic research on poplar. By establishing the high quality protocol of pachytene chromosome preparation and the efficient and stable technology about pachytene-FISH, the project aims to mark the 19 pachytene chromosomes of poplar with the chromosome-specific single-copy gene selected from the poplar genome, developing the high-resolution karyotype of poplar, which lays the foundation for improving the genetic linkage map and constructing the cytogenetic map of poplar. Sex differentiation is the focus in the field of developmental biology, however the mechanism of sex differentiation in different poplars still exists obvious divergence. The study is to construct the single-copy gene probe pools and oligo libraries for the chromosome 19 and do comparative mapping among different poplars to find the interspecific structure variation of chromosome 19 based on pachytene chromosome painting. It is hopefully to provide molecular cytogenetics basis for the mechanism of sex differentiation, besides it lays the foundation for the further development in comparative mapping among poplar genome, the study on the chromosome structure differentiation in poplars and related species.

杨树由于其染色体较小，依据有丝分裂中期染色体的形态难以准确辨别每条染色体，导致分子细胞遗传学研究滞后。快速发展的分子细胞遗传学技术及杨树的全基因组信息，为开展杨树的分子细胞遗传学研究提供了新机遇。本项目拟通过探讨杨树高质量减数分裂粗线期染色体制片及高效稳定的FISH技术体系，充分利用杨树的全基因组信息，筛选染色体特异的单拷贝基因来标记杨树粗线期19条染色体，构建高分辨率细胞学图谱，为完善杨树的遗传连锁图谱、构建细胞遗传图谱奠定基础；性别分化是发育生物学的热点研究领域，但不同杨树种的性别分化机制尚存在明显分歧。本研究拟构建杨树19号染色体单拷贝基因探针池和Oligo库，通过粗线期染色体涂染技术，对不同杨树种的19号染色体比较作图，分析19号染色体的种间结构分化。为揭示杨树的性别分化机制提供分子细胞遗传学依据，也为进一步开展杨树全基因组比较作图，研究杨属及近缘种的染色体结构分化奠定基础。

杨树由于其自身的生物学特性及其重要地位而成为林木研究的模式树种，然而与其它模式物种相比，杨树的细胞遗传学研究明显滞后，这主要是由于杨树染色体较小，依据中期染色体的形态难以准确辨别每条染色体。快速发展的分子细胞遗传学技术及杨树的全基因组信息，为开展杨树的分子细胞遗传学研究提供了新机遇。本项目依据毛果杨基因组信息，开发并合成了19对染色体特异的Oligo库，通过Oligo-FISH技术可以准确识别杨属树种的每条染色体；基于多轮Oligo-FISH技术构建了杨树中期染色体核型图，比较分析了5大派杨树代表种的核型特点，发现杨属树种表现出十分保守的核型；通过对不同杨树19号染色体粗线期配对行为观察及单基因比较作图，发现19号染色体在不同杨树中保存着很好的共线性，短臂末端出现未被涂染的序列缺失区域，且短臂末端在22-24％减数分裂粗线期细胞中没有配对，说明杨树的性别决定区域出现了染色体水平的结构分化，推断毛白杨和美洲黑杨的性别决定区均位于19 号染色体短臂末端，性别决定机制均为 XY 型。综上可见，本项目的研究结果为进一步开展杨树全基因组比较作图、杨属及近缘种的染色体结构分化研究奠定了扎实的分子细胞遗传学工作基础。