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首页> 外文期刊>Theoretical and Applied Genetics >Construction of two BAC libraries from the wild Mexican diploid potato, Solanum pinnatisectum, and the identification of clones near the late blight and Colorado potato beetle resistance loci
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Construction of two BAC libraries from the wild Mexican diploid potato, Solanum pinnatisectum, and the identification of clones near the late blight and Colorado potato beetle resistance loci

机译：从墨西哥野生二倍体马铃薯Solanum pinnatisectum构建两个BAC文库，并鉴定晚疫病和科罗拉多马铃薯甲虫抗性基因座附近的克隆

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摘要

To facilitate isolation and characterization of disease and insect resistance genes important to potato, two bacterial artificial chromosome (BAC) libraries were constructed from genomic DNA of the Mexican wild diploid species, Solanum pinnatisectum, which carries high levels of resistance to the most important potato pathogen and pest, the late blight and the Colorado potato beetle (CPB). One of the libraries was constructed from the DNA, partially digested with BamHI, and it consists of 40,328 clones with an average insert size of 125 kb. The other library was constructed from the DNA partially digested with EcoRI, and it consists of 17,280 clones with an average insert size of 135 kb. The two libraries, together, represent approximately six equivalents of the wild potato haploid genome. Both libraries were evaluated for contamination with organellar DNA sequences and were shown to have a very low percentage (0.65–0.91%) of clones derived from the chloroplast genome. High-density filters, prepared from the two libraries, were screened with ten restriction fragment length polymorphism (RFLP) markers linked to the resistance genes for late blight, CPB, Verticillium wilt and potato cyst nematodes, and the gene Sr1 for the self-incompatibility S-locus. Thirty nine positive clones were identified and at least two positive BAC clones were detected for each RFLP marker. Four markers that are linked to the late blight resistance gene Rpi1 hybridized to 14 BAC clones. Fifteen BAC clones were shown to harbor the PPO (polyphenol oxidase) locus for the CPB resistance by three RFLP probes. Two RFLP markers detected five BAC clones that were linked to the Sr1 gene for self-incompatibility. These results agree with the library’s predicted extent of coverage of the potato genome, and indicated that the libraries are useful resources for the molecular isolation of disease and insect resistance genes, as well as other economically important genes in the wild potato species. The development of the two potato BAC libraries provides a starting point, and landmarks for BAC contig construction and chromosome walking towards the map-based cloning of agronomically important target genes in the species.
机译：为了促进对马铃薯重要的疾病和昆虫抗性基因的分离和鉴定，从墨西哥野生二倍体物种茄茄（Solanum pinnatisectum）的基因组DNA构建了两个细菌人工染色体（BAC）文库，该文库对最重要的马铃薯病原体具有高水平的抗性和害虫，晚疫病和科罗拉多马铃薯甲虫（CPB）。其中一个文库是根据DNA构建的，部分被BamHI消化，它由40,328个克隆组成，平均插入片段大小为125 kb。另一个文库是由用EcoRI部分消化的DNA构建的，它由17,280个克隆组成，平均插入片段大小为135 kb。这两个文库一起代表了野生马铃薯单倍体基因组的大约六个当量。对这两个文库均进行了细胞器DNA序列污染的评估，结果表明它们具有非常低的百分比（0.65-0.91％）的叶绿体基因组克隆。从这两个文库中制备的高密度滤膜，用十个限制性片段长度多态性（RFLP）标记进行筛选，这些标记与晚疫病抗性基因，CPB，黄萎病和马铃薯囊肿线虫的抗性基因以及自交配的基因Sr1 S座鉴定了39个阳性克隆，每个RFLP标记至少检测到2个阳性BAC克隆。与晚疫病抗性基因Rpi1连锁的四个标记与14个BAC克隆杂交。通过三个RFLP探针显示，十五个BAC克隆具有CPB抗性的PPO（多酚氧化酶）基因座。两个RFLP标记检测到5个BAC克隆，这些克隆与Sr1基因连锁以实现自身不相容性。这些结果与该文库预测的马铃薯基因组覆盖范围相符，并表明该文库是分子分离疾病和昆虫抗性基因以及野生马铃薯物种中其他重要经济基因的有用资源。两个马铃薯BAC文库的开发提供了一个起点，并为BAC重叠群构建和染色体走向该物种中农学上重要的目标基因的图基克隆提供了里程碑。

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  • 来源
    《Theoretical and Applied Genetics》 |2004年第6期|1002-1009|共8页
  • 作者

    Q. Chen; S. Sun; Q. Ye; S. McCuine; E. Huff; H.-B. Zhang;

  • 作者单位

    Agriculture and Agri-Food Canada Lethbridge Research Centre;

    Department of Soil and Crop Sciences and Institute for Plant Genomics and Biotechnology Texas AM University;

    Department of Soil and Crop Sciences and Institute for Plant Genomics and Biotechnology Texas AM University;

    Department of Soil and Crop Sciences and Institute for Plant Genomics and Biotechnology Texas AM University;

    Department of Soil and Crop Sciences and Institute for Plant Genomics and Biotechnology Texas AM University;

    Department of Soil and Crop Sciences and Institute for Plant Genomics and Biotechnology Texas AM University;

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