Development of a dense SNP-based linkage map of an apple rootstock progeny using the Malus Infinium whole genome genotyping array

Abstract

Background: A whole-genome genotyping array has previously been developed for Malus using SNP data from 28 Malus genotypes. This array offers the prospect of high throughput genotyping and linkage map development for any given Malus progeny. To test the applicability of the array for mapping in diverse Malus genotypes, we applied the array to the construction of a SNP-based linkage map of an apple rootstock progeny.

Results: Of the 7,867 Malus SNP markers on the array, 1,823 (23.2%) were heterozygous in one of the two parents of the progeny, 1,007 (12.8%) were heterozygous in both parental genotypes, whilst just 2.8% of the 921 Pyrus SNPs were heterozygous. A linkage map spanning 1,282.2 cM was produced comprising 2,272 SNP markers, 306 SSR markers and the S-locus. The length of the M432 linkage map was increased by 52.7 cM with the addition of the SNP markers, whilst marker density increased from 3.8 cM/marker to 0.5 cM/marker. Just three regions in excess of 10 cM remain where no markers were mapped. We compared the positions of the mapped SNP markers on the M432 map with their predicted positions on the 'Golden Delicious' genome sequence. A total of 311 markers (13.7% of all mapped markers) mapped to positions that conflicted with their predicted positions on the 'Golden Delicious' pseudo-chromosomes, indicating the presence of paralogous genomic regions or mis-assignments of genome sequence contigs during the assembly and anchoring of the genome sequence.

Conclusions: We incorporated data for the 2,272 SNP markers onto the map of the M432 progeny and have presented the most complete and saturated map of the full 17 linkage groups of M. pumila to date. The data were generated rapidly in a high-throughput semi-automated pipeline, permitting significant savings in time and cost over linkage map construction using microsatellites. The application of the array will permit linkage maps to be developed for QTL analyses in a cost-effective manner, and the identification of SNPs that have been assigned erroneous positions on the 'Golden Delicious' reference sequence will assist in the continued improvement of the genome sequence assembly for that variety.

Figure 1

SNP genotype clusters revealed following analysis by GenomeStudio. Examples of SNP genotype clusters revealed following analysis using GenomeStudio generated from the M432 mapping progeny using the IRCS genoptying array. (a). Expected patterns of genotype clusters for markers with the parental genotype conformation i) AA × AB, ii) AB × AB and iii) AB × BB. (b). Genotype clusters displaying evidence of sub-clusters possibly as a result of hybridisation to paralogous loci. (c). Clusters not locating to the expected region of graph space, leading to possible mis-assignation of genotype in the i) AB × BB, and ii) AA × AB marker types.

Figure 2

SNP-based linkage map of the M432 progeny. An consensus genetic linkage map of the M432 Malus mapping population composed of 2,579 molecular markers, including 2,272 SNPs generated with the IRSC array, 306 SSRs and the S-locus, spanning 1,282.2 cM over 17 LGs. The scale in centi-Morgans is given at the edge of the figure.

Figure 3

Comparison of SSR positions on the M432 linkage map. A comparison of the genetic positions of 306 SSR markers and the S-locus in the M432 mapping progeny. Marker positions on the left (A) were determined following mapping of only SSR markers by Fernández-Fernández et al. [9], whilst those on the right (B) were determined following mapping of an additional 2,269 SNP markers in this investigation. Marker order was generally conserved except for the positions of two loci (Ch05d03; LG12 and NH014a.z; LG17) given in red which were removed from subsequent analyses.

Figure 4

Comparison of genetic and physical positions of the mapped IRSC SNPs on the M432 genetic and ‘Golden Delicious’ physical maps. Plots for each of the SNPs mapped in the M432 mapping progeny as a function of their physical positions on the ‘Golden Delicious’ genome sequence. Each plot (LG1-LG17) represents one of the 17 LGs of the M432 map and one of the pseudo-chromosomes (1–17) of the ‘Golden Delicious’ genome sequence.