Leibniz Institute of Plant Genetics and Crop Plant Research, Corrensstr, 3, 06466 Gatersleben, Germany.
BMC Genomics. 2011 May 19;12:247. doi: 10.1186/1471-2164-12-247.
BACKGROUND: Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. RESULT: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. CONCLUSION: BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.
背景:尽管第二代测序(2GS)技术允许对以前经过金标准测序的基因组进行重新测序,但对大型和复杂真核生物基因组进行全基因组鸟枪法测序和从头组装仍然具有挑战性。因此,对于大麦等基因组而言,获得全基因组物理图谱仍然是全基因组测序的前提条件。为了开始进行此类工作,需要准备好能够代表单倍体基因组深度覆盖的大片段基因组文库,即细菌人工染色体(BAC)文库。
结果:为大麦(Hordeum vulgare L.)品种 Morex 构建了 5 个新的 BAC 文库。这些文库构建在各自载体的不同克隆位点(HindIII、EcoRI、MboI 和 BstXI)上。为了增强基因组代表性的无偏性并最大程度地减少 BAC contigs 之间的缺口数量,这些缺口通常是由于限制性位点分布不均匀所致,还生成了一个机械剪切的文库。通过仔细检查主要质量参数,如平均插入大小、污染程度(平板宽、相邻和叶绿体)、空孔和超出范围的克隆(<30 或>250 个片段的克隆),对新的 BAC 文库进行了全面深入的特征描述。此外,一组基于基因的探针与高密度 BAC 过滤器杂交,表明每个文库的基因组覆盖度在 2.4 到 6.6 X 之间。
结论:代表>20 个单倍体基因组的 BAC 文库已作为大麦研究界的新资源提供。系统地利用这些文库进行高通量 BAC 指纹图谱分析,应该能够为大麦基因组构建全基因组物理图谱,这对于基于图谱的基因分离和基因组测序将是非常重要的。
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