基于性染色体特异反转座子分析的石刁柏性染色体演化研究

It is well known that plant sex chromosomes originated from a pair of autosomes, however, it is still unclear how did this pair of autosomes trigger and evolve to become sex chromosomes. As the mobile elements of genome, retrotransposon was one of the important elements causing the instability of chromosomes, so it may play essential roles in the origin and evolution of plant sex chromosome. In our previous work, male genome and female genome of Asparagus officinalis were sequenced separately by Solexa high-throughput sequencing technique, and 35 male-specific and 60 female-specific retrotransposons were obtained. On the basis of these results, in this project, we would like to use A. officinalis (2n=20, dioecious) and A. setaceus (2n=20, bisexual flower) as materials, detect and locate these sex-specific retransposons using PCR and FISH, and then, by using combined techniques of metaphase chromosome FISH, meiosis (mainly pachytene and diakinesis stages) FISH, RT-PCR and Southern blotting to systematically compare and analyze the distribution patterns on metaphase chromosomes, dynamic changes during main meiosis stages and the change of activities and copy numbers of the identified sex chromosome-specific retrotransposons. Based on the above studies, we wish to speculate the function of these sex chromosome-specific retrotrnaposons in the early stage of origination and evolution of asparagus sex chromosomes. The findings in this project will provide new evidences for further discovery of the key evolution events of plant sex chromosome.

植物性染色体起源于一对常染色体，但这对常染色体如何启动向性染色体演化的开关至今仍是一个未解之谜。反转座子作为可移动的基因组成分是造成染色体不稳定的重要因素之一，因此有可能是植物性染色体演化的启动器和推进器。在前期工作中，我们分别对石刁柏雌雄株基因组进行Solexa高通量测序，数据分析获得35个雄性特异和60个雌性特异的反转座子序列。在此基础上，本项目拟以石刁柏（2n=20，雌雄异株）及其近缘种文竹（2n=20，花两性）为材料，采用PCR和FISH对这些序列进行鉴定和染色体定位，进一步利用有丝分裂FISH、减数分裂粗线期和终变期FISH、RT-PCR及Southern等技术比较分析性染色体特异反转座子在石刁柏和文竹相应染色体上的分布特点、减数分裂过程中的动态变化、基因组内转座活性和拷贝数的变化，推测反转座子在石刁柏性染色体早期演化过程中的作用，为揭示植物性染色体进化早期关键步骤提供新的证据。

本研究以雌雄异株植物石刁柏为主要实验材料，采用高通量测序、生物信息分析、消减杂交、斑点杂交及荧光原位杂交等手段，对石刁柏雌雄基因组的转座子等重复序列进行了研究。主要研究结果如下：(1) 转座子序列约为石刁柏组装序列的53%。在总的转座子序列中LTR反转录转座子比例最高(28%)。与LTR反转录转座子性比，非LTR反转录转座子和DNA转座子较为少见。雄性和雌性基因组之间几乎所有的转座子组成高度相似的。(2) 在石刁柏基因组中共鉴定出87,576个SSR位点，基于22对多态性SSR引物的10个石刁柏品种的聚类分析表明石刁柏品种间遗传相似度很高，性别是影响品种间聚类的重要因素。(3) 石刁柏基因组消减杂交结合斑点杂交共鉴定出98个雌雄差异序列。进一步的基因组半定量PCR 验证表明其中9个序列为稳定的雄性偏向序列，且大多为(6/9, 67%)多拷贝的重复序列。FISH表明这些序列多定位在石刁柏染色体的着丝粒附近。这些研究结果为石刁柏性别分化及性染色体起源演化的研究奠定了良好的基础。