Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides
Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides
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DOI：
10.1534/genetics.115.177642
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发表时间：
2015-07-01
期刊：
影响因子：
3.3
通讯作者：
Jiang, Jiming
中科院分区：
文献类型：
--
作者：
Han, Yonghua;Zhang, Tao;Jiang, Jiming
染色体特异性涂染是分子细胞遗传学和基因组学研究中的一项重要技术。我们开发了一种基于寡核苷酸（oligo）的黄瓜（Cucumis sativus）染色体彩绘技术，该技术将适用于任何具有测序基因组的植物物种。利用新开发的生物信息学管道鉴定黄瓜单个染色体特异性寡核苷酸，然后并行地从头大量合成。将合成的寡聚物进行扩增并用生物素或洋地黄毒苷标记，用于荧光原位杂交（FISH）。我们开发了三种不同的探针，每种探针含有23，000 - 27，000个寡核苷酸。这些探针在靶黄瓜染色体上跨越8.3-17 Mb的DNA，并且具有1.5-3.2个寡核苷酸/酶的密度。这些探针在单个黄瓜染色体上产生FISH信号，并用于绘制其他黄瓜物种中的同源染色体，这些黄瓜物种与黄瓜分化长达1200万年。大量的寡核苷酸探针使我们能够在减数分裂的早期阶段跟踪单个染色体。我们能够精确地定位黄瓜7号染色体和Cucumis hystrix的1号染色体在F-1杂种中的配对。这两个homeopathy染色体配对在71%的前期I细胞，但只有25%的中期I细胞，这可能提供了一个解释较高的重组率相比，在植物杂交报告homeopathy染色体之间的交叉频率。
Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000-27,000 oligos. These probes spanned 8.3-17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5-3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F-1 hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids.