Lijavetzky D, Muzzi G, Wicker T, Keller B, Wing R, Dubcovsky J
Department of Agronomy & Range Science, University of California, Davis 95616-8515, USA.
Genome. 1999 Dec;42(6):1176-82.
A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276,480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720,384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.
已构建了小麦A基因组的基因组细菌人工染色体（BAC）文库。为此选择了一粒小麦品种DV92，因为它是一种栽培二倍体小麦，也是构建饱和遗传图谱所用的亲本系之一。用该品种的叶片从细胞核中分离高分子量DNA。该DNA用限制性内切酶Hind III进行部分消化，经过两次大小选择，电洗脱并克隆到pINDIGO451 BAC载体中。该文库由276,480个克隆组成，平均插入片段大小为115 kb。排除1.33%的空克隆和0.14%含有叶绿体DNA的克隆，该文库的覆盖率为5.6个基因组当量。有了这样的基因组覆盖率，该文库中出现任何DNA序列的概率高于99.6%。克隆被分选到720,384孔板中，并印迹到15个高密度滤膜上。用几个单拷贝或低拷贝克隆筛选高密度滤膜，选择了5个阳性BAC克隆进行进一步分析。由于大多数一粒小麦BAC末端包含重复序列，因此对经典的末端分离程序进行了改进，以选择用于染色体步移的低拷贝序列。
