Development and validation of allele-specific PCR-based SNP typing in a gene on chromosome D03 conferring resistance to Fusarium wilt race 4 in Upland cotton (Gossypium hirsutum).

Abstract

Upland cotton (Gossypium hirsutum) is the most important fiber crop for the global textile industry. Fusarium oxysporum f. sp. vasinfectum (FOV) is one of the most destructive soil-borne fungal pathogens in cotton. Among eight pathogenic races and other strains, FOV race 4 (FOV4) is the most virulent race in US cotton production. A single nucleotide polymorphism (SNP) in a glutamate receptor-like gene (GhGLR4.8) on chromosome D03 was previously identified and validated to confer resistance to FOV race 7, and targeted genome sequencing demonstrated that it was also associated with resistance to FOV4. The objective of this study was to develop an easy and convenient PCR-based marker assay. To target the resistance SNP, a forward primer for the SNP with a mismatch in the 3rd position was designed for both the resistance (R) and susceptibility (S) alleles, respectively, with addition of 20-mer T7 promoter primer to the 5' end of the forward primer for the R allele. The two forward primers, in combination with each of five common reverse primers, were targeted to amplify amplicons of 50-260bp in size with R and S alleles differing in 20bp. Results showed that each of three common reverse primers in combination with the two forward primers produced polymorphic markers between R and S plants that were consistent with the targeted genome sequencing results. The polymorphism was distinctly resolved using both polyacrylamide and agarose gel electrophoreses. In addition, a sequence comparative analysis between the resistance gene and homologous sequences in sequenced tetraploid and diploid A and D genome species showed that none of the species possessed the resistance gene allele, suggesting its recent origin from a natural point mutation. The allele-specific PCR-based SNP typing method based on a three-primer combination provides a fast and convenient marker-assisted selection method to search and select for FOV4-resistant Upland cotton.