Background: Rye (Secale cereale L.) is the most frost tolerant cereal species. As an outcrossing species, rye exhibits high levels of intraspecific diversity, which makes it well-suited for allele mining in genes involved in the frost responsive network. For investigating genetic diversity and the extent of linkage disequilibrium (LD) we analyzed eleven candidate genes and 37 microsatellite markers in 201 lines from five Eastern and Middle European rye populations. Results: A total of 147 single nucleotide polymorphisms (SNPs) and nine insertion-deletion polymorphisms were found within 7,639 bp of DNA sequence from eleven candidate genes, resulting in an average SNP frequency of 1 SNP/52 bp. Nucleotide and haplotype diversity of candidate genes were high with average values π = 5.6 × 10^-3 and Hd = 0.59, respectively. According to an analysis of molecular variance (AMOVA), most of the genetic variation was found between individuals within populations. Haplotype frequencies varied markedly between the candidate genes. ScCbf14, ScVrn1, and ScDhn1 were dominated by a single haplotype, while the other 8 genes (ScCbf2, ScCbf6, ScCbf9b, ScCbf11, ScCbf12, ScCbf15, ScIce2, and ScDhn3) had a more balanced haplotype frequency distribution. Intra-genic LD decayed rapidly, within approximately 520 bp on average. Genome-wide LD based on microsatellites was low. Conclusions: The Middle European population did not differ substantially from the four Eastern European populations in terms of haplotype frequencies or in the level of nucleotide diversity. The low LD in rye compared to self-pollinating species promises a high resolution in genome-wide association mapping. SNPs discovered in the promoters or coding regions, which attribute to non-synonymous substitutions, are suitable candidates for association mapping.

Background: Frost is an important abiotic stress that limits cereal production in the temperate zone. As the most frost tolerant small grain cereal, rye (Secale cereale L.) is an ideal cereal model for investigating the genetic basis of frost tolerance (FT), a complex trait with polygenic inheritance. Using 201 genotypes from five Eastern and Middle European winter rye populations, this study reports a multi-platform candidate gene-based association analysis in rye using 161 single nucleotide polymorphisms (SNPs) and nine insertion-deletion (Indel) polymorphisms previously identified from twelve candidate genes with a putative role in the frost responsive network. Results: Phenotypic data analyses of FT in three different phenotyping platforms, controlled, semi-controlled and field, revealed significant genetic variations in the plant material under study. Statistically significant (P < 0.05) associations between FT and SNPs/haplotypes of candidate genes were identified. Two SNPs in ScCbf15 and one in ScCbf12, all leading to amino acid exchanges, were significantly associated with FT over all three phenotyping platforms. Distribution of SNP effect sizes expressed as percentage of the genetic variance explained by individual SNPs was highly skewed towards zero with a few SNPs obtaining large effects. Two-way epistasis was found between 14 pairs of candidate genes. Relatively low to medium empirical correlations of SNP-FT associations were observed across the three platforms underlining the need for multi-level experimentation for dissecting complex associations between genotypes and FT in rye. Conclusions: Candidate gene based-association studies are a powerful tool for investigating the genetic basis of FT in rye. Results of this study support the findings of bi-parental linkage mapping and expression studies that the Cbf gene family plays an essential role in FT.