Development and application of universal ND-FISH probes for detecting P-genome chromosomes based on Agropyron cristatum transposable elements
Development and application of universal ND-FISH probes for detecting P-genome chromosomes based on Agropyron cristatum transposable elements
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DOI：
10.1007/s11032-022-01320-w
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发表时间：
2022-08-01
影响因子：
3.1
通讯作者：
Li，Lihui
中科院分区：
文献类型：
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作者：
Sun，Yangyang;Han，Haiming;Li，Lihui
荧光原位杂交(FISH)是广泛应用于细胞遗传学研究的一种基本工具。常规鱼类的检测效率由于其耗时的性质而受到限制。带有荧光标记的寡核苷酸探针已应用于非变性鱼类(ND-FISH)的分析，极大地简化了实验过程，节省了成本和时间。冰草含有一个基本基因组P，是小麦改良的重要野生近缘种。然而，基于ND-FISH技术检测P-基因组染色体的寡核苷酸探针尚未见报道。本研究根据转座元件(TES)在小麦基因组中的分布，设计了94个基于三种类型转座元件的寡核苷酸探针。CristatumSequence。ND-FISH验证表明，12个单条寡核苷酸探针在小麦背景的全P染色体上产生了稳定而明显的杂交信号。为了提高信号强度，用成功的12个探针制备了混合探针，并在二倍体材料A中进行了验证。CristatumZ1842，一个小的节段性易位系和6个含有P基因组的异源多倍体野生近缘。寡核苷酸的信号覆盖了OFA的整个染色体。与单探针法相比，其强度更高。结果表明，寡聚酶链式反应可以取代传统的基因组原位杂交(GISH)探针来鉴定非P基因组背景下的P染色体或片段。最后，我们提供了一种快速、有效的检测小麦背景中P染色体的方法，它可以取代传统的GISH/FISH方法，将寡核苷酸探针与寡聚-pSc119.2-1和寡聚-pTa535-1探针结合起来。综上所述，我们开发了一套基于ND-FISH技术的寡核苷酸探针来鉴定P-基因组染色体，从而促进了对FA的利用。小麦改良计划中的克氏杆菌。
Fluorescence in situ hybridization (FISH) is a basic tool that is widely used in cytogenetic research. The detection efficiency of conventional FISH is limited due to its time-consuming nature. Oligonucleotide (oligo) probes with fluorescent labels have been applied in non-denaturing FISH (ND-FISH) assays, which greatly streamline experimental processes and save costs and time.Agropyron cristatum, which contains one basic genome, “P,” is a vital wild relative for wheat improvement. However, oligo probes for detecting P-genome chromosomes based on ND-FISH assays have not been reported. In this study, according to the distribution of transposable elements (TEs) in Triticeae genomes, 94 oligo probes were designed based on three types ofA. cristatumsequences. ND-FISH validation showed that 12 single oligo probes generated a stable and obvious hybridization signal on whole P chromosomes in the wheat background. To improve signal intensity, mixed probes (Oligo-pAc) were prepared by using the 12 successful probes and validated in the diploid accessionA. cristatumZ1842, a small segmental translocation line and six allopolyploid wild relatives containing the P genome. The signals of Oligo-pAc covered the entire chromosomes ofA. cristatumand were more intense than those of single probes. The results indicate that Oligo-pAc can replace conventional genomic in situ hybridization (GISH) probes to identify P chromosomes or segments in non-P-genome backgrounds. Finally, we provide a rapid and efficient method specifically for detecting P chromosomes in wheat backgrounds by combining the Oligo-pAc probe with the Oligo-pSc119.2–1 and Oligo-pTa535-1 probes, which can replace conventional sequential GISH/FISH assays. Altogether, we developed a set of oligo probes based on the ND-FISH assays to identify P-genome chromosomes, which can promote utilization ofA. cristatumin wheat improvement programs.