Optimization of SRAP-PCR System for Valeriana officinalis var. latifolia and Primer Screening
10.13422/j.cnki.syfjx.20211412
- VernacularTitle:宽叶缬草SRAP-PCR反应体系的优化及引物筛选
- Author:
Fang-yu LIANG
1
;
Mao-qiu HE
1
;
Chang-yan XU
1
;
Xiao-sheng YANG
2
;
Juan YANG
2
;
Zhong-sheng LUO
2
;
Rong-gui QIN
1
Author Information
1. School of Pharmaceutical Sciences，Guizhou Medical University，Guiyang 550025，China
2. The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences，Guiyang 550014，China
- Publication Type:Research Article
- Keywords:
Valeriana officinalis var. latifolia;
sequence-related amplified polymorphism （SRAP）;
system optimization;
primer screening;
orthogonal design
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2021;27(23):163-171
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish the sequence-related amplified polymorphism （SRAP）-polymerase chain reaction （PCR） system for Valeriana officinalis var. latifolia，so as to lay the theoretical and technical foundations for the breeding of V. officinalis var. latifolia. Method:Single factor test was applied to investigate the effects of Taq Mix dose，Mg2+ concentration，template DNA concentration，and Taq DNA polymerase content on SRAP-PCR amplification of V. officinalis var. latifolia，based on which the orthogonal experiments were performed to optimize the SRAP-PCR system for V. officinalis var. latifolia. The effective primers that could be used for genetic diversity studies of V. officinalis var. latifolia were selected under the optimal reaction condition. Result:The results of the single factor test showed that Taq Mix dose within the range of 8-11 μL resulted in better amplification. The addition of a low concentration of Mg2+，the medium to low concentrations of template DNA，or the low concentration of Taq DNA polymerase enhanced the amplification efficiency or richness. As demonstrated by the orthogonal experiments，the influencing degrees of related factors on SRAP-PCR amplification of V. officinalis var. latifolia were sorted in a descending order as follows： Taq Mix dose>Taq DNA polymerase content>Mg2+ concentration>template DNA concentration. The optimal reaction system for V. officinalis var. latifolia was determined to consist of 11 μL of Taq Mix，30 ng of template DNA，0.025 mmol·L-1 Mg2+，1.5 U of Taq DNA polymerase，5 μmol·L-1 forward primer，and 5 μmol·L-1 reverse primer，which was supplemented to 20 μL with ddH2O. The optimal annealing temperature was 36.8 ℃. A total of 17 pairs of effective primers with high band resolution and polymorphism were selected from 88 primer pairs for SRAP-PCR of V. officinalis var. latifolia. Conclusion:The established SRAP-PCR system for V. officinalis var. latifolia is stable， which can be used for genetic diversity studies of V. officinalis var. latifolia.
