Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting

Abstract

Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.

Figure 1

Distribution of disease severity scores in the Pioneer (A) and Alforex populations (B). 1 =  resistant with no or minimal chlorosis of lower leaves; 2 =  moderately resistant with chlorosis of lower and middle leaves but no chlorosis of terminal leaves; 3 =  susceptible with well developed symptoms of chlorosis, and necrotic and twisted terminal leaflets on at least one, but not all main stems, 4 =  susceptible with severe symptoms of chlorosis, necrosis, and twisting of all leaflets on all main stems; and 5 =  dead plant.

Figure 2

High resolution melting profiles of SNP markers MSCWSNP1843 in the Pioneer (PIO) (A, B) and Alforex (AFX) (C, D) populations. The real time PCR was performed by LightCycler 480) and the HRM profiles were analyzed using the gene scanning module software. Normalized melting curves (A, C) and temperature shift difference plots (B, D) are presented. Three groups of genotypes were distinguished by three colors (red, blue and green). Melting curves for parents in each population are indicated by arrows. Resistant parents: 58(R)  =  55V50-58(R) (Pioneer) and 3.13.2 (R)  =  CW065006-3-13-2 (R) (Alforex), susceptible parents: 118(S)  =  55V50-118(S) (Pioneer) and 4.01.1(S)  =  CW065006-4-01-1(S) (Alforex).

Figure 3

High resolution melting profiles of SNP markers MSCWSNP1873 in the Pioneer (PIO) (A, B) and Alforex (AFX) (C, D) populations. The real time PCR was performed by LightCycler 480) and the HRM profiles were analyzed using the gene scanning module software. Normalized melting curves (A, C) and temperature shift difference plots (B, D) are presented. Five colors (red, blue, green, pink and grey) represent five distinct groups of genotypes. Melting curves for parents in each population are indicated by arrows. Resistant parents: 58(R)  =  55V50-58(R) (Pioneer) and 3.13.2 (R)  =  CW065006-3-13-2 (R) (Alforex), susceptible parents: 118(S)  =  55V50-118(S) (Pioneer) and 4.01.1(S)  =  CW065006-4-01-1(S) (Alforex).

Figure 4

Principal components analysis of marker data in the Pioneer (A) and Alforex populations (B). The first and second principal components were used for graphing. Each data point represents a genotype. No subpopulation was detected in the Pioneer population (A), while two clusters were distinguishable in the Alforex population (B).

Figure 5

Comparison of allele effects of six significant SNP markers associated with Verticillium wilt resistance between the pioneer and Alforex populations. Estimated values were represented by bars and different allele types (up to 5 alleles per locus) were distinguished by different colors. Star “*” indicates the population in which the significant marker was identified.