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Optimization of SRAP-PCR System for Valeriana officinalis var. latifolia and Primer Screening / 中国实验方剂学杂志
Article de Zh | WPRIM | ID: wpr-905877
Bibliothèque responsable: WPRO
ABSTRACT
Objective:To establish the sequence-related amplified polymorphism (SRAP)-polymerase chain reaction (PCR) system for <italic>Valeriana officinalis</italic> var. <italic>latifolia</italic>,so as to lay the theoretical and technical foundations for the breeding of<italic> V. officinalis </italic>var. <italic>latifolia</italic>. Method:Single factor test was applied to investigate the effects of <italic>Taq</italic> Mix dose,Mg<sup>2+ </sup>concentration,template DNA concentration,and <italic>Taq </italic>DNA polymerase content on SRAP-PCR amplification of <italic>V. officinalis </italic>var. <italic>latifolia</italic>,based on which the orthogonal experiments were performed to optimize the SRAP-PCR system for <italic>V. officinalis </italic>var. <italic>latifolia</italic>. The effective primers that could be used for genetic diversity studies of <italic>V. officinalis</italic> var. <italic>latifolia </italic>were selected under the optimal reaction condition. Result:The results of the single factor test showed that <italic>Taq </italic>Mix dose within the range of 8-11 μL resulted in better amplification. The addition of a low concentration of Mg<sup>2+</sup>,the medium to low concentrations of template DNA,or the low concentration of <italic>Taq</italic> 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 <italic>V. officinalis</italic> var. <italic>latifolia </italic>were sorted in a descending order as follows: <italic>Taq</italic> Mix dose><italic>Taq</italic> DNA polymerase content>Mg<sup>2+</sup> concentration>template DNA concentration. The optimal reaction system for <italic>V. officinalis</italic> var. <italic>latifolia </italic>was determined to consist of 11 μL of <italic>Taq</italic> Mix,30 ng of template DNA,0.025 mmol·L<sup>-1 </sup>Mg<sup>2+</sup>,1.5 U<italic> </italic>of<italic> Taq </italic>DNA polymerase,5 μmol·L<sup>-1</sup> forward primer,and 5 μmol·L<sup>-1</sup> reverse primer,which was supplemented to 20 μL with ddH<sub>2</sub>O. 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 <italic>V. officinalis</italic> var. <italic>latifolia</italic>. Conclusion:The established SRAP-PCR system for <italic>V. officinalis</italic> var. <italic>latifolia</italic> is stable, which can be used for genetic diversity studies of <italic>V. officinalis</italic> var. <italic>latifolia</italic>.
Mots clés
Texte intégral: 1 Indice: WPRIM Type d'étude: Diagnostic_studies / Screening_studies langue: Zh Texte intégral: Chinese Journal of Experimental Traditional Medical Formulae Année: 2021 Type: Article
Texte intégral: 1 Indice: WPRIM Type d'étude: Diagnostic_studies / Screening_studies langue: Zh Texte intégral: Chinese Journal of Experimental Traditional Medical Formulae Année: 2021 Type: Article