Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species
Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species
复制标题
DOI：
10.1186/1471-2164-11-122
复制
发表时间：
2010-02-19
期刊：
影响因子：
4.4
通讯作者：
Dvorak, Jan
中科院分区：
文献类型：
--
作者：
Luo, Ming-Cheng;Ma, Yaqin;Dvorak, Jan
背景资料：多倍体物种中密切相关的基因组的存在使得从由当前测序平台产生的鸟枪序列读数组装总基因组序列极其困难，如果不是不可能的话。利用细菌人工染色体（BAC）克隆的重叠群和基于BAC的物理图谱，可以按照有序克隆测序方法对多倍体物种的基因组进行测序。虽然BAC重叠群目前可以构建几乎任何二倍体生物体与SNaPshot高信息含量指纹（HICF）技术，目前还不知道这是否也适用于多倍体物种。来自同源染色体的正向区的BAC克隆可能共享许多限制性片段，因此被包括在共同的重叠群中。由于这一点和其他问题，利用SNaPshot HICF对多倍体物种的BAC文库进行物理作图尚未进行，也未评估这样做的可能性。唯一的例外是普通小麦，这是一种异源六倍体，其中可以从流式分选染色体的DNA中构建单染色体或单染色体臂BAC文库，并绕过多倍体造成的障碍。SNaPshot HICF技术用于利用全球BAC文库对多倍体植物进行物理作图的潜力通过在计算机合并的BAC文库中组装指纹克隆的重叠群来评估由小麦3AS和3DS两条同源染色体臂和完整的3B染色体组成的单染色体文库。由于每个克隆的染色体臂起源是已知的，因此可以估计重叠群组装的保真度。平均97.78%或更多的克隆（取决于文库）来自单个染色体臂。剩余克隆的大部分被证明是来自其他染色体的文库污染，这是在构建单染色体BAC文库期间不可避免的特征。在重叠群组装过程中，来自同源染色体臂的克隆掺入重叠群的水平低得可以忽略不计，这表明构建重叠群和物理图谱，使用小麦的全球BAC文库，几乎可以肯定也使用基因组大小与小麦相当的其他植物多倍体物种。由于所得组装的重叠群的高纯度，它们可以直接用于基因组测序。目前尚不清楚，但同样好的BAC重叠群也可以构建含有更小，更丰富的基因组的多倍体物种。
Background: The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information content finger-printing (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.Results: The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.Conclusions: The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.