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湖南生态科学学报

花匠小妙招
2025-06-17 16:36

摘要：

【目的】系统挖掘钟花樱InDel位点，筛选具有多态性的InDel标记，并应用于钟花樱核心种质资源的脱氧核糖核酸(DNA)指纹图谱构建。【方法】以‘湘妍’全基因组序列为参考，利用Illumina平台对18份钟花樱种质资源进行重测序，利用Trimmomatic软件对原始数据进行质量控制，去除低质量数据，应用BWA软件与参考基因组进行对比，然后应用GATK软件检测InDel位点。利用Primer-BLAST设计候选InDel位点的聚合酶链式反应(PCR)引物，分别采用琼脂糖凝胶电泳和凝胶成像系统进行PCR产物检测和分析。【结果】18份不同种源地钟花樱重测序数据经过质检过滤后，数据质控参数Q30平均值达到了91.10%,平均碱基数为11 124 455 100 bp,平均测序深度达到30×以上。共挖掘到InDel位点1 048 575个，其中插入位点共有583 382个，缺失位点共有465 193个，100 bp以上的共有2 640个。随机筛选了40个InDel位点进行引物设计及验证，结果表明31对引物具有多态性，多态率为77.5%。选用5对多态性好、分辨率高的引物构建了7份钟花樱核心种质资源的DNA指纹图谱，得到汝城1号的DNA指纹图谱代码为1-1-1-1-2,莽山1号为1-2-2-2-1、新宁1号为2-1-2-1-1、东安1号为2-1-1-1-2、龙山1号为2-1-2-1-2、福建1号为2-2-1-2-1、阳明山1号为2-1-1-2-2。【结论】基于基因组重测序技术挖掘钟花樱InDel标记，具有数量多、稳定性强和多态性高等优势，本研究为其他樱亚属植物InDel标记的开发提供了参考依据，促进了樱亚属植物分子标记辅助育种和种质资源保护的发展。

Abstract：

【Objective】The objective of this study was to identify InDel loci systematically, then detected InDel markers with polymorphism, and apply them to construct DNA fingerprinting of core germplasm resources of Prunus campanulata.【Method】Using the whole genome sequence of Prunus campanulata ‘Xiangyan' as reference, 18 germplasm resources were resequenced by Illumina platform. The quality control of the original data was carried out by Trimmomatic software, the low quality data was removed, and the BWA software was used to compare with the reference genome. Then GATK software was used to detect InDel loci. Prime-BLAST was used to design PCR primers, and then agarose gel electrophoresis and gel imaging systems were used to detect and analyze PCR products, respectively.【Result】After quality inspection and filtering, the average Q30 of data quality control parameters was 91.10%, the average base number was 11 124 455 100 bp, and the average sequencing depth was more than 30×. A total of 1 048 575 InDel loci were identified, including 583 382 insertion loci, 465 193 deletion loci. There were 2 640 InDel loci with a size larger than 100 bp. Then, 40 InDel loci were randomly selected for primer design and validation. The results showed that 31 pairs of primers were polymorphic, and the polymorphism rate was 77.5%. Five pairs of primers with high polymorphism and resolution were selected to construct the DNA fingerprint of seven core germplasm resources of Prunus campanulata as follows: The DNA fingerprint code of Rucheng No.1 was 1-1-1-1-2, Mangshan No.1 was 1-2-2-2-1, Xinning No.1 was 2-1-2-1-1, Dongan No.1 was 2-1-1-1-2, Longshan No.1 was 2-1-2-1-2, Fujian No.1 was 2-2-1-2-1, Yangmingshan No.1 was 2-1-1-2-2.【Conclusion】The InDel markers identified based on genome resequencing technology in Prunus campanulata has the advantages of large data volume, strong stability, and high polymorphism in Prunus campanulata. This study provides a reference for the development of InDel markers in other subg. Cerasus plants, and promotes the development of marker-assisted breeding and germplasm resource protection of subg. Cerasus plants.

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