大白菜基于单拷贝基因的染色体涂染及结球甘蓝易位片段的鉴定

Chromosome painting (CP) based on fluorescence in situ hybridization (FISH) has been playing an important role in the research of chromosome identification, rearrangement, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. Recently, a chromosome painting method with single copy gene pools in Cucumis sativus was successfully developed, which has provided the reference for carrying out chromosome painting in the sequenced crops..In this study, our aims are to screen single copy genes free of repetitive sequences from B. rapa genome database using the RepeatMaker software online (http://www.brassicadb.org), and to develop a chromosome painting method with single copy gene pools in Chinese cabbage Chinese cabbage (Brassica rapa L. ssp. pekinensis) through the procedures such as primer design, long-PCR amplification, purification, pooling, labeling and hybridizing onto chromosome spreads. Further, the chromosome painting method will be applied to develop comparative chromosome painting between Chinese cabbage A3 chromosome and head cabbage（B. oleracea ssp. capitata） chromosomes, and to identify the translocation fragment with head cabbage chromosome under Chinese cabbage genetic background. .The results will not only offer the technical support for constructing a high-resolution integrated cytogenetic map of Chinese cabbage genome, and carrying out the comparative chromosome painting between Chinese cabbage and head cabbage at the whole genome level, but also settle the foundation for plant breeders to study chromosome addition, substitution and introgression of B. rapa or B. oleracea, and to track the lineage of chromosomes carrying significant agronomic genes in Brassica.

基于FISH的染色体涂染在人类和动物染色体鉴定、重排、畸形诊断和进化研究中扮演了重要角色。由于植物染色体存在大量重复序列，染色体涂染并没有广泛应用。近来，基于单拷贝基因池的染色体涂染方法在黄瓜上被建立，为已测序作物开展染色体涂染提供了借鉴。.本项目拟基于白菜基因组测序数据资料，利用软件RepeatMaker查找无重复序列单拷贝基因，通过引物设计、长片段PCR扩增、纯化、池化、标记以及与染色体制片杂交等程序，建立大白菜基于单拷贝基因池的染色体涂染技术，在此基础上开展大白菜A3染色体和结球甘蓝染色体比较涂染以及大白菜遗传背景下结球甘蓝易位片段鉴定等研究。.该结果将为大白菜高密度整合分子细胞遗传图谱的构建、大白菜和结球甘蓝整个基因组的比较染色体涂染等研究提供技术支持，也将为育种学家研究白菜或甘蓝染色体的附加、代换和渐渗，追踪携带特异农艺性状基因的染色体世系奠定基础。

荧光原位杂交（FISH）是一项重要的分子细胞遗传学工具，随着不同物种基因组测序的进展，以基于单低拷贝基因组或基因序列的长片段PCR产物为探针的FISH技术在验证基因组序列组装准确性、进行全基因组染色体核型分析以及跨物种比较研究方面表现出很大的优越性。本项目基于白菜基因组测序数据资料，通过单拷贝基因组/基因序列分析、引物设计、长片段PCR扩增技术体系优化以及染色体制片、原位杂交等程序的研究，建立了大白菜基于单低拷贝序列长片段PCR-FISH技术体系，利用长片段PCR-FISH技术进行了白菜基因探针在甘蓝染色体上的杂交和大白菜—甘蓝易位系的鉴定。主要研究结果如下：.（1）从基因组 DNA 模板质量、dNTPs 用量、退火温度和延伸时间等方面对PCR反应体系和反应程序进行优化，确定了适合 5~15 kb 长片段 PCR 的技术体系。.（2）通过对高质量的粗线期制片的酶液浓度、酶解时间、标记探针大小、用量、以及探针和染色体变性方式和时间等因素摸索，建立了长片段 PCR-粗线期 FISH 技术体系。.（3）利用在线软件 Repeatmarker 对大白菜A03基因组DNA序列和基因序列进行分析设计引物，获得97个3~15kb的PCR产物探针，FISH结果表明72个探针在近着丝粒异染色质区检测出了类似着丝粒或rDNA重复的杂交信号。.（4）利用在线Giri repbase对供试探针DNA序列进行分析，揭示了白菜基因组和基因序列中含有大量的转座子或反转录转座子元件片段，推测其可能是FISH产生类重复杂交信号的原因。基于Giri repbase软件对大白菜大于5 kb的基因序列分析，获得了31个3~7 kb的PCR产物探针，FISH结果表明14个探针检测到类重复杂交信号，8个探针检测到特异杂交信号。.（5）针对白菜、甘蓝共线性基因差异位点设计引物开发基因标记，对大白菜—甘蓝易位系‘AT1-41’和‘AT1-47’外源片段位置进行鉴定，初步确定2个易位系的外源甘蓝外源片段与大白菜A03染色体顶端物理位置包含422, 779 bp~1,720, 999 bp一段片段发生了代换。.研究结果为白菜作物开展单低拷贝长片段PCR-FISH技术以及进行易位系外源片段鉴定提供了借鉴。.