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园艺学报 ›› 2025, Vol. 52 ›› Issue (12): 3202-3218.doi: 10.16420/j.issn.0513-353x.2024-0881

• 遗传育种·种质资源·分子生物学 • 上一篇    下一篇

番茄重要性状HRM分子标记开发及不同反应体系的分析效果评价

刘畅1, 魏凯1,2, 章力1,2, 路菲菲1, 余庆同1,3, 王梦雅1,4, 王孝宣1, 杜永臣1, 国艳梅1, 李君明1, 刘磊1, 李鑫1, 朱璨1, 黄泽军1,*()   

  1. 1 中国农业科学院蔬菜花卉研究所,蔬菜生物育种全国重点实验室,北京 100081
    2 中国农业大学园艺学院,北京 100193
    3 北京农学院植物科学技术学院,北京 102206
    4 浙江农林大学园艺科学学院,杭州 311300
  • 收稿日期:2025-08-06 修回日期:2025-10-17 出版日期:2025-12-25 发布日期:2025-12-20
  • 通讯作者:
    *(E-mail:
  • 基金资助:
    国家重点研发计划项目(2022YFD1200500); 国家自然科学基金项目(31872949); 现代农业产业技术体系建设专项资助(CARS-23-A06)

Development of HRM Molecular Markers for Important Traits in Tomato and Assessment of Analytical Effects Across Various Reaction Systems

LIU Chang1, WEI Kai1,2, ZHANG Li1,2, LU Feifei1, YU Qingtong1,3, WANG Mengya1,4, WANG Xiaoxuan1, DU Yongchen1, GUO Yanmei1, LI Junming1, LIU Lei1, LI Xin1, ZHU Can1, HUANG Zejun1,*()   

  1. 1 State Key Laboratory of Vegetable Biobreeding,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China
    2 College of Horticulture,China Agricultural University,Beijing 100193,China
    3 Institute of Plant Science and Technology,Beijing Agricultural University,Beijing102206,China
    4 College of Horticulture Science,Zhejiang A & F University,Hangzhou 311300,China
  • Received:2025-08-06 Revised:2025-10-17 Published:2025-12-25 Online:2025-12-20

摘要:

高分辨率熔解曲线(high-resolution melting,HRM)分型技术可以通过形成不同的熔解曲线鉴定样品的基因型,具有操作简单、速度快、通量高、分辨率高等优点。为开发番茄重要性状优异等位基因的HRM分子标记和评估不同因素对HRM分析效果的影响。基于23个优异等位基因或其附近的序列特征,成功开发了23个HRM分子标记,分别用于检测23个重要性状基因位点。包括抗病基因Bwr-12Frl、I-7PtoRx4、SmTy-1,耐非生物胁迫基因SlACR11ASlBBX31SlHAK20SlSOS2,果实性状相关基因Aft、LCGF、OVATEPT、R、SlALMT9、STP1Y,株型相关基因SD1、SPSP5G。另外,通过评估不同反应体积、模板DNA浓度和DNA提取方法对HRM分析效果的影响,发现5、7.5、10、12.5和15 µL的反应体积与试剂盒推荐的20 µL反应体积具有相同的分析效果;在5 µL反应体积下,DNA浓度在12.5 ~ 1 600 ng · µL-1的较宽范围内均具有良好的分析效果;NaOH法粗提的DNA也适用于HRM分析检测。本研究结果可应用于番茄优异等位基因鉴定及分子标记辅助育种中。

关键词: 番茄, 重要性状, 优异等位基因, HRM, 分子标记

Abstract:

High resolution melting(HRM)genotyping technology can identify the genotypes of different samples by forming different melting curves,be characterized by its simplicity,rapid processing,high throughput,and superior resolution. This study aims to develop HRM molecular markers for identifying superior alleles associated with important traits in tomato and to assess the impact of various factors on HRM analysis outcomes. A total of 23 HRM molecular markers were successfully developed based on the sequence characteristics of 23 superior alleles of specific genes or their adjacent regions. These genes include those associated with disease resistance,such as Bwr-12FrlI-7PtoRx4SmTy-1;genes conferring abiotic stress tolerance,including SlACR11ASlBBX31SlHAK20SlSOS2;genes related to fruit traits,such as AftLCGFOVATEPTRSlALMT9STP1Y;and genes associated with plant type,including SD1SPSP5G. Furthermore,an assessment of the impact of varying reaction volumes,template DNA concentrations,and DNA extraction methods on HRM analysis revealed that reaction volumes of 5,7.5,10,12.5 and 15 µL demonstrated comparable analytical performance to the recommended reaction volume of 20 µL specified in the kit. Notably,within the 5 µL reaction volume,DNA concentrations ranging from 12.5 ng · µL-1 to 1 600 ng · µL-1 exhibited robust analytical performance. Additionally,DNA extracted using the NaOH method was found to be suitable for HRM analysis. The findings of this study will facilitate the identification of superior alleles and marker-assisted breeding in tomato.

Key words: tomato, important trait, superior allele, HRM, molecular marker