Mapping molecular markers linked with two race specific loci conferring Colletotrichum trifolii resistance in Medicago sativa.

摘要

Estimates suggest about one-fourth of the U.S. alfalfa hay crop and one-tenth of the seed crop are lost annually to disease. Anthracnose caused by Colletotrichum trifolii Bain. & Essary is a major world-wide alfalfa foliar, stem and crown disease. It is controlled almost exclusively by genetic means relying on two race-specific genes ( An₁ and An₂) which have been shown to be unlinked and dominant in nature. The unique challenges inherent in breeding alfalfa (M. sativa) may be aided considerably by marker assisted selection. This study sought to produce molecular markers linked with C. trifolii resistance. For each resistance gene a single dose mapping population was created from a simplex by nulliplex cross and assessed phenotypically. Four methodologies consisting of covalent homology subtraction (CHS), resistance gene analog cleaved amplified polymorphic sequences (RGA-CAPS), a novel molecular technique (RTag), and amplified fragment length polymorphism (AFLP) were used. Application of the first three methodologies produced no linked polymorphisms. In using AFLP technology segregating subsets of the mapping populations were screened using 126 radiolabeled AFLP primer combinations. Polymorphisms were analyzed in a multipoint fashion for linkage. A few potentially linked markers were cloned, sequenced, and multiple PCR primers were designed which failed to maintain polymorphic capability even when single nucleotide polymorphisms were apparent. Therefore, all markers of interest were used as AFLPs with additional individuals in the mapping populations to estimate linkage. No markers in either population were considered linked. The overall results of this study suggest that a high degree of similarity is present between resistant and susceptible alleles making further molecular work challenging. Implications of this discovery are discussed.