Polymorphism of polygalacturonase-inhibiting protein genes by fluorescence-based PCR single-strand conformation profiling and sequencing in autotetraploid alfalfa (Medicago sativa) populations

Abstract

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins involved in plant defence. The aim of the current study was to isolate the genomic fragment of PGIP genes in alfalfa (Medicago sativa) and to analyse their sequence variation. Four PGIP genes, namely MsPGIP1, MsPGIP2, MsPGIP3 and MsPGIP4, were discovered using the homology cloning method, and sequence variation among these four PGIP genes was higher than those among PGIP genes reported in non-Medicago leguminous plants. Fluorescence-based PCR single-strand conformation polymorphism (FPCR–SSCP) analysis, which was further improved by adding primers to PCR products, combined with sequencing revealed that MsPGIP4 had the most alleles (17) and most amino acid variation sites (12) while MsPGIP1 had the least (8 and 3, respectively). Furthermore, the extents of allele frequency divergence in MsPGIP1 and MsPGIP4 were obviously greater than those of MsPGIP2 and MsPGIP3 among the studied populations. Three populations, namely ‘Rambler’, ‘C/W5’ and ‘PG sutter’, showed higher frequency divergence for three of the four PGIP genes than other studied populations. In conclusion, the four alfalfa PGIP genes found in this study possessed high intergene and intragene sequence variation, and showed different extents of allele frequency divergence among the studied eight alfalfa populations. Overall, this study lays a foundation for studying their biological and molecular functions. In addition, the improved SSCP analysis technique described here provides a useful tool for molecular biology research in autopolyploids.