| 金庆敏 1,2，林毓娥 2，王 瑞 1，钟玉娟 2，吴廷全 1.基于 SSR 分子标记的‘粤秀 3 号’黄瓜杂交种子纯度及真实性鉴定[J].广东农业科学,2023,50(9):49-58 |
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基于 SSR 分子标记的‘粤秀 3 号’黄瓜杂交种子纯度及真实性鉴定 |
| Purity and Authenticity Identification of ‘Yuexiu No.3’ Cucumber Hybrid Seeds Based on SSR Molecular Markers |
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| DOI：10.16768/j.issn.1004-874X.2023.09.005 |
| 中文关键词: 黄瓜 ‘粤秀 3 号’ 种子纯度 SSR 分子标记 真实性鉴定 田间性状鉴定 |
| 英文关键词: Cucumber ‘Yuexiu No.3’ purity identification SSR molecular marker authenticity identification field trait identification |
| 基金项目:国家自然科学基金（32272713）；广东省重点领域研发计划项目（2020B02022001）；广东省农业科学院协同创新中心项目（XTXM202203） |
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| 中文摘要: |
| 【目的】‘粤秀 3 号’黄瓜具有生长势强、产量高、抗病及抗逆性强的特点，是华南地区推广面积较多的品种，本研究以期为其建立一套快速、准确、有效的种子纯度鉴定方法。【方法】利用 200 对 SSR 引物，根据杂种带为父母本互补带型的原则，对‘粤秀 3 号’黄瓜及其亲本进行特异性 SSR 标记物的筛选。将种子纯度鉴定结果与田间生物学鉴定结果进行比较，鉴定所筛选 SSR 引物的准确性。选取‘中农 108’‘园丰 6 号’‘津绿 5 号’‘中农 8 号’‘津研四号’5 个黄瓜品种种子及其他 49 份黄瓜种质材料，与‘粤秀 3 号’黄瓜品种同时检测，对所筛选的 SSR 引物特异性进行鉴定。【结果】从 200 对引物中筛选出符合父母带间成互补带要求的两对 SSR 引物（3B-10 和 2B-41）。3B-10 引物经 PCR 扩增后产生 160 bp 的母本特异条带（P1-A）及 190 bp 的父本特异条带（P2-B），测序比对这两条特异性条带，发现 P2-B 在 49~79 bp 处比 P1-A 多了 5 个 6 碱基的简单重复序列。2B-41 引物经 PCR 扩增后产生 218 bp 的母本特异条带（P1-C）和 206 bp 的父本特异条带（P2-D），经测序比对后发现，与 P2-D 相比，P1-C 在 120~131 bp 处有 12 个碱基的缺失。表明两对 SSR 引物均可有效区分‘粤秀 3 号’杂交种子及其父母本，可快速准确地鉴定‘粤秀 3 号’黄瓜种子纯度。此外，两对 SSR 引物的种子纯度鉴定结果均与田间生物学鉴定结果一致。两对引物均可有效区分‘粤秀 3 号’黄瓜种子与‘中农 108’‘园丰6 号’‘津绿 5 号’‘中农 8 号’‘津研四号’5 个黄瓜品种种子及其他 49 份黄瓜种质资源。但 3B-10 在 49 份种质资源中多态性较高，故 SSR 标记 2B-41 更适用于‘粤秀 3 号’的真实性鉴定。【结论】3B-10 和 2B-41 两对 SSR 引物可快速、准确、有效地对‘粤秀 3 号’杂交种子纯度进行鉴定，鉴定操作较简单、成本较低，可替代传统杂交种子纯度鉴定的方法，其商业应用价值极高。 |
| 英文摘要: |
| 【Objective】‘Yuexiu No.3’ cucumber is a variety that is widely promoted in the South China region due to its strong growth, high yield, disease resistance, and stress tolerance. This study aims to establishing a rapid, accurate and effective method for seed purity identification.【Method】200 pairs of primers were used to select SSR markers with specificity based on the principle of complementary band pattern between the parents and ‘Yuexiu No.3’. The seed purity identification results obtained using these primers were compared with the field biological identification results to verify their accuracy. The seeds of five cucumber varieties ‘Zhongnong 108’, ‘Yuanfeng No.6’, ‘Jinlv No.5’, ‘Zhongnong No.8’ and ‘Jinyan No.4’ and other 49 cucumber germplasm materials were selected and detected simultaneously with ' Yuexiu 3 ' cucumber varieties to identify the specificity of the selected SSR primers.【Result】Two pairs of SSR primers (3B-10 and 2B-41) were screened out from 200 pairs of primers that met the requirement of forming complementary bands between parental bands, PCR amplification using the 3B-10 primer produced a 160 bp maternal-specific band (P1-A) and a 190 bp paternal-specific band (P2-B). By comparing the sequencing results of the specific bands (P1-A, P2-B), it was found that the P2-B had an additional 6-base simple repeat sequence compared to the P1-A at positions 49-79 bp. PCR amplification using the 2B-41 primer produced a 218 bp maternal-specific band (P1-C) and a 206 bp paternal-specific band (P2-D). By comparing the sequencing results of the specific bands(P1-C, P2-D), it was found that the P1-C had a deletion of 12 bases compared to the P2-D at positions 120-131 bp. The results showed that these two pairs of SSR primers were able to effectively distinguish ‘Yuexiu No.3’ hybrid seeds from their parents and accurately identify the seed purity of ‘Yuexiu No.3’ cucumbers. In addition, the seed purity identification results of the two pairs of SSR primers were consistent with the field biological identification results. Both pairs of primers were capable of effectively distinguishing the ‘Yuexiu No.3’ cucumber seeds from the seeds of five other cucumber varieties, namely ‘Zhongnong 108’, ‘Yuanfeng No.6’, ‘Jinlv No.5’, ‘Zhongnong No.8’and ‘Jinyan No.4’, as well as 49 other germplasm resources. However, among these 49 germplasm resources, 3B-10 displayed a higher level of polymorphism. Therefore, SSR marker 2B-41 was more suitable for confirming the authenticity of the ‘Yuexiu No.3’ cucumber variety.【Conclusion】The 3B-10 and 2B-41 pairs of SSR primers can rapidly, accurately and efficiently identify the purity of ‘Yuexiu No.3’ hybrid cucumber seeds. The identification is simple and low cost, which can replace the traditional method of hybrid seed purity identification, and has high commercial application value. |
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