[Construction, characterization and screening of a transformation-competent artificial chromosome (TAC) library of wheat-Thinopyrum intermedium translocation line with resistance to barley yellow dwarf virus].
[Construction, characterization and screening of a transformation-competent artificial chromosome (TAC) library of wheat-Thinopyrum intermedium translocation line with resistance to barley yellow dwarf virus].
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发表时间：
2002
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通讯作者：
Xiao-Ping Wang;Zengyan Zhang;Qunyu Zhang;Yaoguang Liu;Z. Xin
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作者：
Xiao-Ping Wang;Zengyan Zhang;Qunyu Zhang;Yaoguang Liu;Z. Xin
以小麦Th.中间体易位系HW 642含有来源于大麦黄矮病毒（BYDV）的抗性基因。中场休息。该文库由2.3 × 10（6）个克隆组成，平均插入片段大小为22 kb，代表约2.5个单倍体基因组当量，并且能够给出大于95.77%的从该文库分离单拷贝DNA序列的概率。文库作为冷冻培养物储存在24个96孔格式中，每个孔含有大约1000个不同的克隆。用混合PCR技术可以鉴定出含有目的基因的TAC克隆。利用小麦微卫星标记wms 37和SSR标记wms 37中的一个与BYDV抗性基因共分离的SCAR标记筛选TAC文库。通过合并PCR方法成功地选择了12个克隆。PCR产物与Th.中场休息。在12个克隆中，有10个阳性克隆经Hind Ⅲ酶切后与Th.中场休息。这些结果表明，这10个克隆可作为分离黄矮病抗性及其相关基因的候选克隆，TAC文库是一个有用的基因分离资源。
A transformation-competent artificial chromosome (TAC) library was constructed from the genomic DNA of wheat-Th. intermidium translocation line HW642 that harbor the barley yellow dwarf virus (BYDV) resistance gene derived from Th. intermidium. The library consists of 2.3 x 10(6) clones with an average insert size of 22kb, representing approximately 2.5 haploid genome equivalents and is able to give a greater than 95.77% probability of isolating single-copy DNA sequences from this library. The library was stored as frozen cultures in 24 96-well formats, each well containing approximately 1000 different clones. TAC clones containing interest gene could be identified by the pooled PCR technique. A sequence characterized amplified region (SCAR) marker cosegregated with BYDV resistance gene, derived from a simple sequence repeat (SSR) or microsatellite marker wms37 of wheat, was applied to screen the TAC library. Twelve clones were successfully selected by the pooled PCR method. PCR products were identified by hybridizing with the SCAR marker band of Th. intermidium. Out of 12 clones, 10 positive clones restricted by Hind III were shown to hybridize with genomic DNA of Th. intermidium. These results showed evidences that the 10 clones could be used as candidate clones for isolation of BYDV resistance and its related genes, and the TAC library is a useful resource for isolating genes.