High levels of nucleotide diversity and fast decline of linkage disequilibrium in rye (Secale cereale L.) genes involved in frost response

Abstract

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.

Figure 1

Haplotype frequencies of eleven candidate genes in five rye populations (PR, EKO, SMH, ROM, Petkus). The different haplotypes occurring within each gene are represented by different coloured bars (see legend). Haplotypes occurring at a frequency < 0.05 are pooled and shown as black bars. The number of investigated lines in each population is shown in brackets.

Figure 2

Principal co-ordinate analysis of 201 rye lines from five populations (PR, EKO, SMH, ROM, Petkus) based on candidate gene haplotypes. Analysis was based on a similarity matrix of candidate gene haplotypes. PCo1 and PCo2 are the first and second principal co-ordinates and percentages indicate percent variation explained.

Figure 3

Principal co-ordinate analysis of 201 rye lines from five populations (PR, EKO, SMH, ROM, Petkus) based on genome-wide SSR markers. Analysis was based on a similarity matrix from 37 SSR loci. PCo1 and PCo2 are the first and second principal co-ordinates and percentages indicate percent variation explained.

Figure 4

LD heat plots of ten candidate genes. Analysed sequences, including the promoter and complete coding sequences of ScCbf6 and ScCbf9b, and partial coding sequences of ScCbf12, ScCbf14, and ScCbf15; ScVrn1 was not included due to a lack of pairwise comparisons, since only one Indel was observed. Exons, and 5'- or 3'-flanking regions are represented by grey cylinders and black lines, respectively. White cylinders with dashed lines indicate non-amplified exons. Black triangles represent polymorphic sites starting from "SNP1" on the top of each graph. Each grid represents the strength of LD estimated by r² for each pairwise comparison between polymorphic sites with a minor allele frequency (MAF) > 0.05. The colour legend for r² values is given on the right side.

Figure 5

Scatterplots of pairwise distances and LD. LD based on r² between all SNPs (MAF > 5%) in eleven candidate genes within five rye populations (PR, EKO, SMH, ROM, Petkus) and across populations (over all), with non-linear fitting curve from the mutation-recombination-drift model (see methods). Thresholds for LD (see methods) are indicated by a horizontal solid line.