Baig M N R, Yu An, Guo Wenwu, Deng Xiuxin
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.
Genome. 2009 May;52(5):484-9. doi: 10.1139/g09-017.
Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in <0.5% chloroplast DNA contamination and no mitochondrial DNA contamination in both libraries. Eight and five positive clones harboring the gene encoding Phytoene synthase (Psy (EC 2.5.1.32)) were identified from the C. sinensis and C. reticulata libraries, respectively, using the filter hybridization procedure. These results suggest that the two BAC libraries are useful tools for the isolation of functional genes and advanced genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.
已构建并表征了两个深度覆盖的细菌人工染色体（BAC）文库，分别来自柑橘属两个最重要的物种——甜橙（Citrus sinensis (L.) Osbeck 'Cara Cara'脐橙）和宽皮柑橘（Citrus reticulata (L.) Blanco 'Egan No. 1'椪柑），以促进基因克隆并分析品种特异性基因组组成。甜橙BAC文库由36000个克隆组成，假阳性克隆可忽略不计，估计平均插入片段大小为126 kb，覆盖约4.5×10⁹ bp，因此提供了单倍体基因组等效物11.8倍的覆盖度；而宽皮柑橘文库由21000个克隆组成，同样假阳性克隆可忽略不计，估计平均为120 kb，覆盖约2.5×10⁹ bp，代表单倍体基因组等效物6.6倍的覆盖度。对两个文库进行了高拷贝载体、空pIndigoBAC536载体和细胞器DNA序列污染的评估。通过BAC滤膜的Southern杂交进行筛选，结果表明两个文库中叶绿体DNA污染均<0.5%，且均无线粒体DNA污染。使用滤膜杂交程序，分别从甜橙文库和宽皮柑橘文库中鉴定出8个和5个携带八氢番茄红素合酶（Psy (EC 2.5.1.32)）编码基因的阳性克隆。这些结果表明，这两个BAC文库是分离两个重要物种甜橙和宽皮柑橘功能基因及推进基因组学研究的有用工具。相关资源、高密度滤膜、单个克隆和整个文库均可公开分发，可在华中农业大学作物遗传改良国家重点实验室获取。
