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

Abstract

ABSTRACT. 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 polygalacturonase-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 µL PCR products, 0.2 to 0.8 µL unlabeled primers (50 µM) and 4 to 16 µL 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 10185 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (4): 10184-10193 (2014) Detecting the alleles of MsPGIP2 by improved SSCP technique of alleles and idiotypes were accurate based on SSCP analysis. Finally, a total of 9 alleles with 18 SNP sites were identified for MsPGIP2 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.

Sensitivity and applications of the PCR Single-Strand Conformation Polymorphism method

“…PCR-SSCP could be used for the authentication of snapper species by SSCP [48], the population genetic structure of zeugodacus tau species complex [49], for the genetic diversity of citrus trees [50], identification of phytophthora genes polymorphism of potato [51], identification of genetic diversity of lemon and mandarin varieties [52], detection of the genetic variation of alfalfa gene [53], prediction of the chloroplast gene polymorphism in barley varieties [54] and bacteria identification in wood tick [55].…”

Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review

“…• Stained bands are easy to be read and interpret in almost all cases   However, both techniques have been employed to study several mechanisms in plants with a variety of genetic polymorphism purposes that ranged from splicing alterations to fingerprinting and diagnostic markers. [62][63][64] With respect to fish and amphibians, species identification and the detection of environmental samples and closely related organisms were recently conducted using PCR-RFLP. [56][57][58][59][60][61][62][63][64][65][66][67][68] The PCR-SSCP was used in the detection of parasite species.…”