DONG Menghan,MA Menglu,WANG Ruikang,et al.Optimization of Trolliuschinensis Bunge ISSR-PCR Reaction System and Primer Screening[J].Northern Horticulture,2025,(17):10-18.[doi:10.11937/bfyy.20250074]
金莲花ISSR-PCR反应体系优化及引物筛选
- Title:
- Optimization of Trolliuschinensis Bunge ISSR-PCR Reaction System and Primer Screening
- 文章编号:
- 1001-0009(2025)17-0010-09
- Keywords:
- Trollius chinensis Bunge; ISSR-PCR; primer screening; reaction system
- 分类号:
- S 326
- 文献标志码:
- A
- 摘要:
- 以金莲花为试材，采用单因素水平筛选和正交实验方法，组合出最佳反应体系，系统分析了4个关键因素对体系的影响，包括DNA浓度、Mix酶用量、引物浓度、反应循环次数，并进行引物及其退火温度的确定，建立金莲花的ISSR-PCR反应体系，以期为金莲花种质资源创新和遗传多样性分析提供参考依据。结果表明：四因素对反应体系的影响依次为PCR循环次数>DNA浓度>Mix酶用量>引物浓度。最佳反应体系为DNA浓度35 ng·μL-1，引物浓度9 μmol·L-1，Mix酶用量9 μL。反应程序为94 ℃预变性5 min，94 ℃变性30 s，退火时间45 s，72 ℃延伸45 s，33个循环，72 ℃终延伸7 min，4 ℃保存。建立了金莲花ISSR分子标记最佳反应体系并筛选出26条ISSR引物，可应用于后续的种质资源研究工作。
- Abstract:
- Taking Trollius chinensis Bunge as experimental material，the optimal reaction system was combined by single factor horizontal screening and orthogonal experimental methods.The effects of four key factors on the system were systematically analyzed，included DNA concentration，Mix enzyme dosage，primer concentration，reaction cycle times，and the primers and their annealing temperature were determined.The aim was to establish the ISSR-PCR reaction system of Trollius chinensis，in order to provide reference for germplasm innovation and genetic diversity analysis of Trollius chinensis Bunge.The results showed that the influence of four factors on the reaction system was PCR cycles>DNA concentration>amount of Mix enzyme used>primer concentration.The best reaction system was,DNA concentration 35 ng·μL-1，primer concentration 9 μmol·L-1，and Mix enzyme amount 9 μL.Reaction procedure,94 ℃ pre-denaturation for 5 minutes，94 ℃ denaturation for 30 seconds，annealing time for 45 seconds，72 ℃ extension for 45 seconds，33 cycles，72 ℃ final extension for 7 minutes，4 ℃ preservation.The best reaction system of ISSR molecular markers was established and 26 ISSR primers were selected，which could be applied to the subsequent germplasm resources research work.
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备注/Memo
第一作者简介：董梦涵（1999-），女，硕士研究生，研究方向为园林植物种质资源。E-mail：1844839187@qq.com.责任作者：张芹（1972-），女，博士，教授，现主要从事园林植物资源及种质创新等研究工作。E-mail：zqin166@163.com.基金项目：河北省中央财政林草科技示范资助项目（冀TG［2024］014）；河北省现代农业产业体系建设专项资助项目（HBCT2024200203）。收稿日期：2025-01-07