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Detection of the genetic variation of polygalacturonase-inhibiting protein gene 2 in autotetraploid alfalfa (Medicago sativa) using an improved SSCP technique

文献类型： 外文期刊

作者： Gui, Z.;Liu, H. Q.;Xin, N.;Zhang, X.;Pi, Y. S.;Gao, J. M.;Wang, Y.;Yuan, Q. H.;Li, X. L.

作者机构：

关键词： Lucerne;Autopolyploid;Primer concentration;Single-strand conformation polymorphism;Fluorescent labeling;Sequence variation

期刊名称： GENETICS AND MOLECULAR RESEARCH

ISSN： 1676-5680

年卷期： 2014 年 13 卷 4 期

页码：

收录情况： SCI

摘要： In this study, 2 approaches were adopted to obtain good single-strand conformation polymorphism (SSCP) data for autotetraploid alfalfa; primers were added to PCR products, and fluorescent-labeled primers were utilized. PCR-SSCP conditions for a 331-bp fragment in the coding region of poly galacturonase-inhibiting protein gene 2 in alfalfa (MsPGIP2) were optimized, and the results showed that the best SSCP gel pattern could be obtained when the loading mixture was made by mixing 1 mu L PCR products, 0.2 to 0.8 mu L unlabeled primers (50 mu M) and 4 to 16 mu L loading buffer. Furthermore, the use of the fluorescent-labeled primers resulted in 2 separated electrophoresis images from 2 complementary single DNA strands, thus making the determination of alleles and idiotypes a relatively easy task. In addition, the results of sequencing prove that the determination of alleles and idiotypcs were accurate based on SSCP analysis. Finally, a total of 9 alleles with 18 SNP sites were identified for MsPGIP 2 in the alfalfa variety 'Algonquin'. In conclusion, MsPGIP2 possessed great genetic variation, and the addition of primers to the PCR products in combination with the fluorescent labeling of primers could significantly improve the sensitivity and resolution of SSCP analysis. This technique could be used for genetic diversity detection and marker-assisted breeding of useful genes in autopolyploid species such as alfalfa.

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