title: Detection of the genetic variation of polygalacturonase-inhibiting protein gene 2 in autotetraploid alfalfa (Medicago sativa) using an improved SSCP technique.
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 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.
authors.affiliation: Key Laboratory of Crop Genetics and Breeding, Department of Agronomy, Tianjin Agricultural University, Tianjin, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Gui
authors.affiliation: Key Laboratory of Crop Genetics and Breeding, Department of Agronomy, Tianjin Agricultural University, Tianjin, China.
authors.forename: H Q
authors.initials: HQ
authors.lastname: Liu
authors.affiliation: Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing, China.
authors.forename: Q H
authors.initials: QH
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Crop Genetics and Breeding, Department of Agronomy, Tianjin Agricultural University, Tianjin, China.
authors.forename: N
authors.initials: N
authors.lastname: Xin
authors.affiliation: Key Laboratory of Crop Genetics and Breeding, Department of Agronomy, Tianjin Agricultural University, Tianjin, China.
authors.forename: X
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing, China.
authors.forename: X L
authors.initials: XL
authors.lastname: Li
authors.affiliation: Key Laboratory of Crop Genetics and Breeding, Department of Agronomy, Tianjin Agricultural University, Tianjin, China.
authors.forename: Y S
authors.initials: YS
authors.lastname: Pi
authors.affiliation: Key Laboratory of Crop Genetics and Breeding, Department of Agronomy, Tianjin Agricultural University, Tianjin, China gao11799713@163.com.
authors.forename: J M
authors.initials: JM
authors.lastname: Gao
mesh_terms: D000483:Alleles; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D005453:Fluorescence; D000455:Medicago sativa / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D018807:Polymorphism, Single-Stranded Conformational
chemical_list: D017931:DNA Primers; C081479:PGIP protein, plant; D010940:Plant Proteins