Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis
Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis
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基于寡核苷酸多重PCR的柔性染色体涂色及其在黄瓜比较染色体分析中的应用
DOI：
10.1111/tpj.14600
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发表时间：
2019-12-08
期刊：
影响因子：
7.2
通讯作者：
Lou, Qunfeng
中科院分区：
文献类型：
--
作者：
Bi, Yunfei;Zhao, Qinzheng;Lou, Qunfeng
染色体涂色是染色体和基因组研究的一项强大技术。我们开发了一种基于黄瓜（Cucumis sativus L.，2n = 14）合成寡核苷酸（oligo）文库的多重 PCR 的灵活染色体涂色技术。文库中的每个寡核苷酸都与通用以及嵌套的特异性引物相关联，以进行扩增，从而可以从同一寡核苷酸文库生成不同的探针。通过使用荧光团缀合的引物对进行扩增，我们还能够生成双链标记寡核苷酸，其产生的信号比单链标记寡核苷酸强得多。在一个文库中合成了由八个片段组成的覆盖黄瓜 1 号染色体 (Chr1) 和 4 号染色体 (Chr4) 的寡核苷酸。从文库中生成的不同寡核苷酸探针明确地绘制了相应的染色体/片段，特别是在粗线期染色体上。然后，基于使用 Chr4 探针的跨物种染色体涂色，将该技术应用于研究黄瓜、C. hystrix 和 C. melo 染色体之间的同源关系。我们证明该探针可用于检测种间染色体同源关系和染色体重排事件。基于其优势和极大的便利性，我们预计这种灵活的寡核苷酸绘制技术对于研究任何具有已测序基因组的物种的染色体结构、组织和进化具有巨大的潜力。
Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double-stranded labelled oligos, which produced much stronger signals than single-stranded labelled oligos, by amplification using fluorophore-conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross-species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo-painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome.