CAPS与熔解曲线技术相结合的菜薹SNP基因分型体系的建立与验证

园艺学报 ›› 2013, Vol. 40 ›› Issue (10): 1916-1927.

CAPS与熔解曲线技术相结合的菜薹SNP基因分型体系的建立与验证

李平霞1,2，黄英金1，刘博2，梁建丽2，程锋2，武剑2，王晓武2,*

（1江西农业大学农学院，南昌 330045；2中国农业科学院蔬菜花卉研究所，北京 100081）

收稿日期:2013-05-11 出版日期:2013-10-25 发布日期:2013-10-25

Establishment and Validation of SNP Genotyping System by CAPS Combining Melting Curve in Brassica campestris

LI Ping-xia1,2，HUANG Ying-jin1，LIU Bo2，LIANG Jian-li2，CHENG Feng2，WU Jian2，and WANG Xiao-wu2,*

（1College of Agronomy，Jiangxi Agricultural University，Nanchang 330045，China；2Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 100081，China）

Received:2013-05-11 Online:2013-10-25 Published:2013-10-25

摘要/Abstract

摘要： 根据不同长度或不同碱基序列的DNA片段的熔解温度（T_m）不同的原理，将酶切扩增多态性序列（cleaved amplified polymorphic sequence，CAPS）与熔解曲线（melting curve，MC）技术相结合，建立了CAPS-MC技术，从而实现SNP分子标记基因分型。通过菜薹‘L58’和紫菜薹‘ZCT095’的基因组重测序数据与白菜基因组的参考序列（‘Chiifu-401-42’的基因组序列）的比对，预测了719个HindⅢ内切酶识别碱基序列的SNP位点，并将其设计成CAPS-MC分子标记，从中随机选择10对标记进行体系建立与优化，结果显示本体系有灵敏度强、精准性高、快速、高通量、经济实用等优势。

关键词: 菜薹, 酶切扩增多态性序列, 熔解曲线, 单个核苷酸多态性, 高通量基因分型

Abstract: Melting temperature（T_m）was different according to the different lengths or nucleotide sequences of DNA fragments. In this report，we established a novel single nucleotide polymorphism（SNP）genotyping system refered as CAPS-MC approach. The CAPS-MC approach combined cleaved amplified polymorphic sequence（CAPS）with melting curve（MC）analysis. By aligning whole genome re-sequencing data from two Brassica campestris accessions，‘L58’and‘ZCT095’，to the reference genome sequence of‘Chiifu-401-42’，a total of 719 HindⅢ endonuclease recognition nucleotide sequence SNP polymorphisms were converted to CAPS-MC markers. We established and optimized CAPS-MC system by randomly selecting 10 pairs of these markers. Our results clearly demonstrated that CAPS-MC approach was sensitive，accurate，rapid and inexpensive. Because the melting curve of 96 samples can be measured in 22 mins once，it is well suited for high-throughput genotyping.

Key words: Brassica campestris, cleaved amplified polymorphic sequence（CAPS）, melting curve, font-size: 10.5pt, mso-bidi-font-weight: bold, mso-bidi-font-size: 12.0pt, mso-fareast-font-family: 宋体, mso-font-kerning: 1.0pt, mso-ansi-language: EN-US, mso-fareast-language: ZH-CN, mso-bidi-language: AR-SA" lang="EN-US">single nucleotide polymorphismfont-size: 10.5pt, mso-bidi-font-weight: bold, mso-ascii-font-family: 'Times New Roman', mso-hansi-font-family: 'Times New Roman', mso-bidi-font-size: 12.0pt, mso-font-kerning: 1.0pt, mso-ansi-language: EN-US, mso-fareast-language: ZH-CN, mso-bidi-language: AR-SA, mso-bidi-font-family: 'Times New Roman'">（font-size: 10.5pt, mso-bidi-font-weight: bold, mso-bidi-font-size: 12.0pt, mso-fareast-font-family: 宋体, mso-font-kerning: 1.0pt, mso-ansi-language: EN-US, mso-fareast-language: ZH-CN, mso-bidi-language: AR-SA" lang="EN-US">SNPfont-size: 10.5pt, mso-bidi-font-weight: bold, mso-ascii-font-family: 'Times New Roman', mso-hansi-font-family: 'Times New Roman', mso-bidi-font-size: 12.0pt, mso-font-kerning: 1.0pt, mso-ansi-language: EN-US, mso-fareast-language: ZH-CN, mso-bidi-language: AR-SA, mso-bidi-font-family: 'Times New Roman'">）, font-size: 10.5pt, mso-bidi-font-size: 12.0pt, mso-fareast-font-family: 宋体, mso-font-kerning: 1.0pt, mso-ansi-language: EN-US, mso-fareast-language: ZH-CN, mso-bidi-language: AR-SA" lang="EN-US">high-throughput genotyping

中图分类号:

S 634

李平霞1,2，黄英金1，刘博2，梁建丽2，程锋2，武剑2，王晓武2,*. CAPS与熔解曲线技术相结合的菜薹SNP基因分型体系的建立与验证[J]. 园艺学报, 2013, 40(10): 1916-1927.

LI Ping-xia1,2，HUANG Ying-jin1，LIU Bo2，LIANG Jian-li2，CHENG Feng2，WU Jian2，and WANG Xiao-wu2,* . Establishment and Validation of SNP Genotyping System by CAPS Combining Melting Curve in Brassica campestris[J]. ACTA HORTICULTURAE SINICA, 2013, 40(10): 1916-1927.

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