Wu Xiaolei, Zhong Guohua, Findley Seth D, Cregan Perry, Stacey Gary, Nguyen Henry T
Division of Plant Sciences and National Center for Soybean Biotechnology, University of Missouri-Columbia, Columbia, MO 65211, USA.
BMC Genomics. 2008 Jan 22;9:28. doi: 10.1186/1471-2164-9-28.
Integrated genetic and physical maps are extremely valuable for genomic studies and as important references for assembling whole genome shotgun sequences. Screening of a BAC library using molecular markers is an indispensable procedure for integration of both physical and genetic maps of a genome. Molecular markers provide anchor points for integration of genetic and physical maps and also validate BAC contigs assembled based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy and an in silico approach to anchor molecular markers onto the soybean physical map.
A total of 1,470 markers (580 SSRs and 890 STSs) were anchored by PCR on a subset of a Williams 82 BstY I BAC library pooled into 208 pools in six dimensions. This resulted in 7,463 clones (approximately 1x genome equivalent) associated with 1470 markers, of which the majority of clones (6,157, 82.5%) were anchored by one marker and 1106 (17.5%) individual clones contained two or more markers. This contributed to 1184 contigs having anchor points through this 6-D pool screening effort. In parallel, the 21,700 soybean Unigene set from NCBI was used to perform in silico mapping on 80,700 Williams 82 BAC end sequences (BES). This in silico analysis yielded 9,835 positive results anchored by 4152 unigenes that contributed to 1305 contigs and 1624 singletons. Among the 1305 contigs, 305 have not been previously anchored by PCR. Therefore, 1489 (78.8%) of 1893 contigs are anchored with molecular markers. These results are being integrated with BAC fingerprints to assemble the BAC contigs. Ultimately, these efforts will lead to an integrated physical and genetic map resource.
We demonstrated that the six-dimensional soybean BAC pools can be efficiently used to anchor markers to soybean BACs despite the complexity of the soybean genome. In addition to anchoring markers, the 6-D pooling method was also effective for targeting BAC clones for investigating gene families and duplicated regions in the genome, as well as for extending physical map contigs.
整合的遗传图谱和物理图谱对于基因组研究极为重要，是组装全基因组鸟枪法测序序列的重要参考。利用分子标记筛选BAC文库是整合基因组物理图谱和遗传图谱不可或缺的步骤。分子标记为遗传图谱和物理图谱的整合提供锚定位点，同时也验证仅基于BAC指纹组装的BAC重叠群。我们采用了六维BAC混合策略和计算机模拟方法将分子标记定位到大豆物理图谱上。
通过PCR将总共1470个标记（580个SSR和890个STS）定位到威廉姆斯82 BstY I BAC文库的一个子集中，该文库被混合成208个六维池。这产生了与1470个标记相关的7463个克隆（约1倍基因组当量），其中大多数克隆（6157个，82.5%）由一个标记定位，1106个（17.5%）单个克隆包含两个或更多标记。通过这种六维池筛选工作，这有助于1184个重叠群有锚定位点。同时，利用来自NCBI的21700个大豆单基因集对80700个威廉姆斯82 BAC末端序列（BES）进行计算机模拟定位。这种计算机模拟分析产生了9835个阳性结果，由4152个单基因定位，这些单基因有助于1305个重叠群和1624个单拷贝序列。在1305个重叠群中，305个以前未通过PCR定位。因此，1893个重叠群中的1489个（78.8%）被分子标记定位。这些结果正在与BAC指纹整合以组装BAC重叠群。最终，这些努力将产生一个整合的物理图谱和遗传图谱资源。
我们证明，尽管大豆基因组复杂，但六维大豆BAC池可有效地用于将标记定位到大豆BAC上。除了定位标记外，六维混合方法对于靶向BAC克隆以研究基因组中的基因家族和重复区域以及扩展物理图谱重叠群也有效。
