杨树同源重组特点及其遗传变异研究

Recombination between non-sister chromatin of homologous chromosomes is the genetic basis of of heterosis, closely related with the formation of the excellent characters of the forest tree. Studies on characteristics of homologous recombination and genetic variation in forest tree have great significance in theoretical study. Due to the lack of direct method for detecting homologous recombination in plant, the applicant had issued a patent named "a method to directly detect DNA homologous recombination in higher plant (Patent Application NO: 201110363048.5)". Using this method, chromosome doubling is induced by physical and chemical mutagens during the first mitosis of embryo sac development to inhibit the post-meiotic segregation of heteroduplex DNA resulting from homologous recombination, and then formed a 2n egg cell containing two sister chromosomes. Subsequently, hybridization with a sperm cell could fix two sister chromosomes into a triploid zygote with hDNA information. Finally, using codominant SSR markers, this type of triploid population could be used to directly detect heteroduplex DNA resulting from homologous recombination during megasporogenesis and then assess features of homologous recombination in female parent. Using the above patent, and based on the triploids resulting from embryo sac chromosome doubling in Sect.Tacamahaca and Sect.Leuce, the present project was designed to detect the occurring times, frequencies and hot spots of homologous recombination in different poplar species, different individual trees within the same species, and different linkage groups in a certain plant, and then reveal the characteristic of homologous recombination during meiosis in Populus. The research results will provide a theoretical and methodological foundation for further researches of the formation mechanism of the excellent characters in forest tree.

同源重组是杂种优势形成的基础，与林木优良性状形成密切相关，开展林木同源重组特点及其遗传变异研究意义重大。针对缺乏直接检测植物DNA同源重组方法的问题，申请人提出了"一种直接鉴定高等植物DNA同源重组的方法"，通过诱导杨树胚囊染色体加倍阻止减数后分离，使异源双链DNA经过复制形成的两条姊妹染色体不分离而共存于同一个2n配子中，并通过与某一父本杂交固定于三倍体后代内，然后采用SSR等共显性分子标记鉴定异源双链DNA产生与否，从而实现母本同源重组直接检测。本项目采用上述发明，在获得胚囊染色体加倍来源的PMR型青杨、白杨杂种三倍体群体基础上，利用SSR毛细管电泳技术，检测不同杨树种、同一杨树种的不同个体植株，以及同一植株的不同连锁群的同源重组发生次数、发生频率、热点区域等，揭示杨树DNA同源重组特点以及种的差异性，为杨树等林木优良性状形成机制研究奠定理论和方法学基础。

采用 “一种直接鉴定高等植物DNA 同源重组的方法”，以 ‘哲引3号杨’（Populus pseudo-simonii × P.nigra ‘Zheyin3#’）ב北京杨’（P. pyramidalis × P.cathayana cv. ‘Beijingensis’）PMR型青杨杂种三倍体群体，以及毛白杨3119×银腺杨(P.alba×P. glandulosa)、毛白杨3532×银腺杨(P.alba×P. glandulosa) PMR型白杨杂种三倍体群体为对象，利用筛选出的106对适宜于青杨和98对适宜于毛白杨的母本处于杂合状态且与父本有差异的多态性SSR引物开展研究，完成了预期研究目标，包括：揭示了杨树同源重组发生次数、频率以及变动范围，绝大多数的染色体发生了1-3次同源重组事件，青杨和白杨染色体上不同SSR标记位点处的hDNA发生频率分别介于5.3%-76.6%和8.5-82.2%之间；同源重组位点存在偏好性，染色体两端的SSR位点较中部位点更易发生重组，基因越密集的区域越容易发生同源重组，表明SDR和PMR型2n配子在育种中的利用价值不一定比与FDR型2n配子低；杨树不同种，以及同种不同植株、同一植株的不同连锁群的同源重组发生次数、发生频率、发生部位等存在一定差异；并基于同源重组发生与不同2n配子形成的关系，证明只有挑选位于低重组率区域SSR位点才可能准确鉴定2n配子来源，而居于染色体臂的中段的位点最代表整条染色体杂合性等。有关研究为杨树等被子植物的同源重组分析和以及林木优良性状形成机制研究奠定理论与方法学基础。