Martin G B, Ganal M W, Tanksley S D
Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853.
Mol Gen Genet. 1992 May;233(1-2):25-32. doi: 10.1007/BF00587557.
We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22,000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11,000 YACs have been screened with RFLP markers that cosegregate with Tm-2a and Pto - loci conferring resistance to tobacco mosaic virus and Pseudomonas syringae pv. tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIII, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.
我们构建了一个用于染色体步移的番茄酵母人工染色体（YAC）文库，该文库包含相当于三个单倍体基因组的DNA（22,000个克隆）。高分子量DNA的来源是番茄品种VFNT樱桃和里奥格兰德 - PtoR的叶原生质体，它们共同包含编码对番茄六种病原体抗性的基因座。用与Tm - 2a和Pto共分离的RFLP标记筛选了约11,000个YAC，Tm - 2a和Pto基因座分别赋予对烟草花叶病毒和番茄丁香假单胞菌的抗性。鉴定出五个与这些标记杂交的YAC，因此它们是向这些基因进行染色体步移的起点。对文库的一个子集进行了各种重复序列的检测，鉴定出携带TGRI的YAC，TGRI是一种在大多数番茄染色体端粒附近成簇的重复序列，TGRII是一种散布重复序列，TGRIII是一种主要出现在着丝粒位点的重复序列。对文库中细胞器序列的评估表明，约10%的克隆含有叶绿体序列。许多这些YAC克隆似乎包含完整的155 kb番茄叶绿体基因组。用于构建文库的番茄品种，除了携带各种抗病基因外，还包含与通过经典连锁分析定位的大多数隐性突变相对应的野生型等位基因。因此，除了其在物理图谱绘制和基因组研究中的用途外，该文库对于向与几乎任何可在分离群体中评分的表型相对应的基因进行染色体步移也应是有用的。
