中国烟草科学2025，Vol.46Issue(3)：1-11,11.DOI:10.13496/j.issn.1007-5119.2025.03.001
基于Box-Behnken响应面法的烤后烟叶品种分子鉴定技术构建
Construction of Molecular Identification Technology for Cured Tobacco Varieties Based on Box-Behnken Response Surface Method
摘要
Abstract
To address the severe DNA degradation in cured tobacco leaves,which hinders subsequent molecular identification experiments,this study comprehensively considered factors such as DNA concentration,quality,extraction efficiency,cost,and safety for molecular marker applications.Based on the SLS method,we conducted single-factor experiments and employed a Box-Behnken response surface model to determine the optimal DNA extraction protocol for cured tobacco leaves.Sample amount,centrifugation time,and water bath duration were used as experimental variables,with DNA concentration and the A260/A280 ratio as response values.The results showed that the optimal conditions for DNA extraction from cured tobacco leaves were 20 mg of sample,10 minutes of centrifugation,and 15 minutes in water bath.Under these conditions,the DNA concentration was 588.16±27.09 ng/µL,and the A260/A280 ratio was 1.75±0.03.These results were consistent with the predictions of Design Expert 11 software,confirming the reliability of the optimization method.Molecular marker applicability tests revealed that DNA extracted using the optimized SLS method yielded clearer target fragment peaks in capillary electrophoresis after PCR amplification with SSR and KASP primers,and distinct genotyping plots were obtained by using KASP markers,demonstrating its applicability for SSR and KASP molecular marker detection in flue-cured tobacco leaves of different varieties.The established molecular identification system combining the optimized SLS method with 13 selected SSR primer pairs effectively distinguished the tested cured tobacco varieties,demonstrating its applicability for cured tobacco cultivar identification.关键词
烤后烟叶/基因组DNA提取方法/响应面分析法/SSR/KASP/分子鉴定技术Key words
cured tobacco leaf/genomic DNA extraction methods/response surface analysis methodology/simple sequence repeat/KASP/molecular identification分类
农业科技引用本文复制引用
田震,李媛,毛静静,任民,熊党安,张兴伟,殷瑜东,李少鹏,刘国祥..基于Box-Behnken响应面法的烤后烟叶品种分子鉴定技术构建[J].中国烟草科学,2025,46(3):1-11,11.基金项目
中国烟草总公司重大科技项目(110202201014) （110202201014）
江苏中烟工业有限责任公司科技项目(202208) （202208）
中国农业科学院科技创新工程(ASTIP-TRIC01) （ASTIP-TRIC01）
山东省自然科学基金面上项目(ZR2022MC145) （ZR2022MC145）
