SA Na,WANG Pengyao,JU Fuzhu,et al.Optimization of SRAP Reaction System and Genetic Relationship in Paeonia[J].Northern Horticulture,2019,43(10):93-101.[doi:10.11937/bfyy.20183403]
芍药属SRAP反应体系优化与亲缘关系分析
- Title:
- Optimization of SRAP Reaction System and Genetic Relationship in Paeonia
- Keywords:
- Paeonia; SRAP; reaction system; optimization; genetic relationship
- 文献标志码:
- A
- 摘要:
- 以牡丹芍药杂交种‘东方金’和牡丹品种‘灰鹤’为试材，对SRAP-PCR反应体系和反应程序进行了优化，并利用优化的体系对芍药属65个品种进行了亲缘关系分析，以期建立稳定的芍药属植物SRAP分子标记技术体系。结果表明：芍药属植物的SRAP-PCR最佳程序为94 ℃预变性3 min；94 ℃变性30 s，37 ℃退火30 s，72 ℃延伸90 s，退火温度每个循环增加1 ℃，8个循环;94 ℃变性30 s，50 ℃退火30 s，72 ℃延伸90 s，32个循环；最后72 ℃延伸7 min。20 μL反应体系中各成分的最佳浓度依次为Mg2+ 2.5 mmol?L-1，dNTPs 0.2 mmol?L-1，Taq酶1 U、引物浓度0.3 μmol?L-1。16对引物对65份材料进行PCR扩增共扩增出了1 229个条带，均为多态性条带，多态性高达100%，芍药和杂种聚为一类，牡丹聚为一类，说明杂种与芍药亲缘关系更近。该研究建立的芍药属SRAP-PCR体系重复性好、稳定性强，适合芍药属植物SRAP的研究，为进一步研究芍药属植物种质资源遗传多样性、品种鉴定、分子标记辅助育种等方面奠定基础。
- Abstract:
- In order to establish stable and optimal SRAP-PCR protocol for plants of Paeonia，the factors of reaction system and procedure for PCR were optimized using cultivars of ‘Oriental Gold’ and ‘Hui He’ as materials．With the system,genetic relationship of 65 cultivars in Paeonia was analyzed.The results showed that most suitable protocol was an initial denaturation at 94 ℃ for 3 min，eight cycles of three steps,30 s of denaturing at 94 ℃，30 s of annealing at 37 ℃ and 90 s of elongation at 72 ℃，the temperature for annealing was increased 1 ℃ after each cycle．Followed by 32 cycles，at 94 ℃ for 30 s，50 ℃ for 30 s，72 ℃ for 90 s．With a final elongation at 72 ℃ for 7 min．The optimal concentration of Mg2+ was 2.5 mmol?L-1，0.2 mmol?L-1 of dNTPs，1 U of Taq DNA polymerase，each prime 0.3 μmol?L-1 for final 20 μL reaction system．A total of 1 229 polymorphic bands were amplified used 16 primer pairs,the polymorphism was as high as 100%,the herbaceous peony and the hybrids were clustered in a class,and the tree peony was clustered in a class,which proved that the hybrids were more closely related to the herbaceous peony.The newly established SRAP-PCR system of Paeonia was fully reproducible and stable,and suitable for the study of SRAP in Paeonia plants.It provided a foundation for further studies on genetic diversity of germplasm resources,cultivars identification and molecular marker assisted selection breeding of Paeonia．
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备注/Memo
第一作者简介：萨娜（1988-），女，硕士，助教，研究方向为园林植物种植与应用。E-mail:sana_qau@126.com.责任作者：郝青（1981-），女，博士，讲师，现主要从事观赏植物分子育种等研究工作。E-mail:haoqing5608@163.com.基金项目：山东省林业科技创新资助项目（LYCX05-2018-26）；青岛农业大学高层次人才科研基金资助项目（6631114306）。收稿日期：2019-01-15