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.

Genotyping‐by‐sequencing‐based genome‐wide association studies on Verticillium wilt resistance in autotetraploid alfalfa (Medicago sativaL.)

“…In the present study, two were identified on chromosome 6 and one on chromosome 7, suggesting similar locations of loci for VW resistance in M. sativa and M. truncatula . However, additional resistance loci to VW have also been identified in M. sativa by both our previous (Zhang et al ., ) and present studies, whereas these loci were not found in M. truncatula . For instance, several VW resistance loci were identified on chromosome 8 by both our previous (Zhang et al ., ) and present studies, whereas no VW resistance locus has been reported on chromosome 8 in M. truncatula .…”

“…They include a major QTL on chromosome 7, and two minor QTLs on chromosomes 2 and 6 (Ben et al ., ). In our previous study, three resistance loci associated with VW resistance were identified on chromosome 7 and one on chromosome 2 (Zhang et al ., ). In the present study, two were identified on chromosome 6 and one on chromosome 7, suggesting similar locations of loci for VW resistance in M. sativa and M. truncatula .…”

“…Miller and Christie () reported that general combining ability (GCA) significantly influenced the expression of resistance to VW in ‘Vertus’ alfalfa, and suggested that additive genetic variance is the primary source of VW resistance. Our previous report (Zhang et al ., ) identified multiple loci (i.e. quantitative resistance) associated with VW resistance in two alfalfa mapping populations.…”

“…Sequence alignment to the M. truncatula genome revealed multiple resistance loci, several of which are linked to known resistance genes. These putative resistance loci identified here had similar chromosomal locations as those previously reported in tetraploid alfalfa (Zhang et al ., ) and in its diploid relative M. truncatula . We plan to validate these putative QTLs in a broader range of alfalfa germplasm.…”

“…() developed a real‐time polymerase chain reaction (PCR) procedure to quantify the amount of V. alfalfae in plant materials, and found significant quantitative differences in the pathogen load between VW‐resistant and VW‐susceptible cultivars. Our previous investigation applied single nucleotide polymorphism (SNP)‐derived markers to identify multiple loci significantly associated with VW resistance in alfalfa by bulked segregant analysis in susceptible or resistant pools constructed from 13 synthetic populations, and by high‐resolution melting in two F1 populations consisting of 352 individuals (Zhang et al ., ). Multiple loci at different chromosomal positions of the associated markers suggested that VW resistance is most likely under the control of multiple genes in these alfalfa populations.…”