JIAO Li,LIU Xiang-guo,YANG Chun-ming,et al.Application of High Resolution Melting Analysis in Soybean Ecotilling[J].Soybean Science,2011,30(05):843-846,850.[doi:10.11861/j.issn.1000-9841.2011.05.0843]
高分辨率溶解曲线分析技术在大豆Ecotilling中的应用
- Title:
- Application of High Resolution Melting Analysis in Soybean Ecotilling
- 文章编号:
- 1000-9841（2011）05-0843-04
- 关键词:
- 大豆; 高分辨率溶解曲线分析技术; Ecotilling; SNP
- Keywords:
- Soybean; High resolution melting; Ecotilling; SNP
- 分类号:
- S565.1
- 文献标志码:
- A
- 摘要:
- 将高分辨率溶解曲线（High Resolution Melting，HRM）技术运用在大豆Ecotilling中，对256份栽培大豆的大豆异黄酮合酶基因（IFS1）的外显子区域进行SNP筛查，并采用直接测序的方法对HRM检测结果进行验证。结果表明：模板均一化后的检测效果较未均一化的检测效果差异显著，野生型样本与待测样本按1∶1及1∶4比例混样溶解曲线阈值较高，其中1∶4混样适合高通量检测。HRM对IFS1基因外显子区域的检测结果与PCR产物测序的结果比对显示，对IFS1第5段外显子和第7段外显子检测的准确率分别达到91.2％和100％，表明该研究利用HRM技术建立的栽培大豆的Ecotilling技术体系是一种高灵敏度、高通量和低成本检测SNP的新方法。
- Abstract:
- Identification of allelic variants in natural populations by Ecotilling has became a rapid Single nucleotide polymorphisms(SNP) discovery method,but it is not convenient for detection.High resolution melting(HRM)analysis is a rapid,sensitive and high-throughput method that can be used in the examination of SNP.In this paper,HRM techniques was used in Ecotilling to detect the SNP of IFS1 exons in 256 soybean cultivars.The results of HRM was verified by direct sequencing.The effect of homogenization and non-homogenization of the template on the testing was discussed and the result showed that the former had more remarkable effect.The testing effect of different proportions of mixed samples was compared and found that 1∶1 and 1∶4 mixed samples were optimal.The comparison between the testing results of exon regions of IFS1 by HRM and results of PCR products sequencing showed the accuracy of fifth and seventh sections of exons were 91.2% and 100%,respectively.It demonstrates that the soybean-cultivating Ecotilling established by the use of HRM is a new method for SNP detection with high sensitivity, throughput and low cost.
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备注/Memo
基金项目：吉林省科技发展计划重大资助项目（20086029）。