Zeng Qingdong, Yuan Fengping, Xu Xin, Shi Xue, Nie Xiaojun, Zhuang Hua, Chen Xianming, Wang Zhonghua, Wang Xiaojie, Huang Lili, Han Dejun, Kang Zhensheng
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China.
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Agronomy, Northwest A&F University, Yangling, Shaanxi 712100, China.
Biomed Res Int. 2014;2014:845806. doi: 10.1155/2014/845806. Epub 2014 May 5.
For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sublibraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was composed of total 765,696 clones, of which 390,144 were from the HindIII digestion and 375,552 from the BamHI digestion. Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selected from the HindIII sublibrary and 573 clones from the BamHI sublibrary, the average insert sizes were estimated as 129 and 113 kb, respectively. Thus, the HindIII sublibrary was estimated to have a 3.01-fold coverage and the BamHI sublibrary a 2.53-fold coverage based on the estimated hexaploid wheat genome size of 16,700 Mb. The 765,696 clones were arrayed in 1,994 384-well plates. All clones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence (such as gene Yr26 for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5D pools with Xwe173, a marker tightly linked to Yr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning of Yr26 and other genes of interest.
为了对六倍体小麦品系92R137中赋予重要性状的基因进行基于图谱的克隆，使用经限制性内切酶HindIII和BamHI消化的92R137基因组DNA构建了一个细菌人工染色体（BAC）文库，该文库包括两个子文库。BAC文库总共由765,696个克隆组成，其中390,144个来自HindIII消化产物，375,552个来自BamHI消化产物。通过对从HindIII子文库中随机选择的453个克隆和BamHI子文库中573个克隆进行脉冲场凝胶电泳（PFGE）分析，估计平均插入片段大小分别为129 kb和113 kb。因此，基于估计的16,700 Mb六倍体小麦基因组大小，估计HindIII子文库有3.01倍的覆盖率，BamHI子文库有2.53倍的覆盖率。这765,696个克隆排列在1,994个384孔板中。所有克隆还被排列成板池，并进一步排列成五维（5D）池。从该文库中鉴定出与任何小麦DNA序列（如抗条锈病基因Yr26）相对应的克隆的概率估计超过99.6%。通过用与Yr26紧密连锁的标记Xwe173对5D池进行聚合酶链反应筛选，成功获得了6个BAC克隆。这些结果表明，该BAC文库是用于Yr26和其他感兴趣基因的定位克隆的宝贵基因组资源。
