彩叶芋SCoT反应体系的建立与优化

Optimization and Establishment of SCoT Reaction System in Caladium bicolor

本试验通过单因子试验和正交试验的设计对‘红桃K’彩叶芋的SCoT-PCR反应体系进行研究及优化。研究结果表明,彩叶芋SCoT最佳反应体系为(20μL):引物浓度0.7μmol/L,d NTPs(10 mmol/L)浓度0.5 mmol/L,Taq DNA聚合酶1.25 U,DNA模板80 ng,Mg2+浓度1.5 mmol/L。PCR扩增程序为:94℃预变性4 min,94℃变性45 s,50.4℃条件下退火45 s,72℃延伸90 s,35个循环后,72℃再延伸7 min,最后16℃保存。此外,利用市场上观赏价值较高的9个彩叶芋品种为材料,对确立的‘红桃K’彩叶芋SCoT-PCR扩增体系进行再验证。结果表明,扩增条带清晰,重复后稳定性高,说明该反应体系为鉴定彩叶芋多样性合适的SCoT分子标记体系,同时为后期彩叶芋遗传多样性、分子辅助育种及遗传图谱的建立提供了依据。

In this study,the SCoT-PCR reaction system of Caladium bicolor ＇Hearts K＇ was studied and optimized by single factor experiment and orthogonal design.The results showed that final volume of the optimum reaction system for Caladium bicolor was 20 μL,including 0.7 μmol/L primer,0.5 mmol/L d NTPs,1.25 U Taq DNA polymerase,80 ng template DNA,and 1.5 mmol/L Mg^2＋.The PCR amplification profile was pre-denaturing at 94℃ for 4 min,denaturing at 94℃ for 45 s,annealing at 50.4℃ for 45 s and extending at 72℃ for 90 s.After 35 cycles,extending at 72℃ for 7 min and stored at 16℃ finally.In addition,the optimal reaction system of Caladium bicolor ＇Hearts K＇ was further verified by in 9 Caladium bicolor varieties with high ornamental value on the market.The results indicated that clear amplified bands and high stability after repetition suggesting that this reaction system was a suitable SCoT molecular marker system for the identification of the diversity of Caladium bicolor.Meanwhile,it provided reference for the genetic diversity,molecular marker assisted breeding and establishment of genetic map of Caladium bicolor in the later stage.