Development and implementation of high-throughput SNP genotyping in barley

Abstract

Background: High density genetic maps of plants have, nearly without exception, made use of marker datasets containing missing or questionable genotype calls derived from a variety of genic and non-genic or anonymous markers, and been presented as a single linear order of genetic loci for each linkage group. The consequences of missing or erroneous data include falsely separated markers, expansion of cM distances and incorrect marker order. These imperfections are amplified in consensus maps and problematic when fine resolution is critical including comparative genome analyses and map-based cloning. Here we provide a new paradigm, a high-density consensus genetic map of barley based only on complete and error-free datasets and genic markers, represented accurately by graphs and approximately by a best-fit linear order, and supported by a readily available SNP genotyping resource.

Results: Approximately 22,000 SNPs were identified from barley ESTs and sequenced amplicons; 4,596 of them were tested for performance in three pilot phase Illumina GoldenGate assays. Data from three barley doubled haploid mapping populations supported the production of an initial consensus map. Over 200 germplasm selections, principally European and US breeding material, were used to estimate minor allele frequency (MAF) for each SNP. We selected 3,072 of these tested SNPs based on technical performance, map location, MAF and biological interest to fill two 1536-SNP "production" assays (BOPA1 and BOPA2), which were made available to the barley genetics community. Data were added using BOPA1 from a fourth mapping population to yield a consensus map containing 2,943 SNP loci in 975 marker bins covering a genetic distance of 1099 cM.

Conclusion: The unprecedented density of genic markers and marker bins enabled a high resolution comparison of the genomes of barley and rice. Low recombination in pericentric regions is evident from bins containing many more than the average number of markers, meaning that a large number of genes are recombinationally locked into the genetic centromeric regions of several barley chromosomes. Examination of US breeding germplasm illustrated the usefulness of BOPA1 and BOPA2 in that they provide excellent marker density and sensitivity for detection of minor alleles in this genetically narrow material.

Figure 1

Five 1536-plex GoldenGate assays. The numbers of SNPs selected from each Pilot OPA (POPA1, POPA2, POPA3) for the design of each production scale OPA (BOPA1, BOPA2) are indicted next to the arrows connecting the pilot and production OPAs. See Supplemental Text (Additional File 1) for complete details.

Figure 2

Examples of SNP data. A) Typical clustering of satisfactory data for POPA SNP 3_0004; red cluster area = homozygous AA, blue = homozygous BB, green dots within purple cluster area are 1:1 mixtures of parental DNA for three DH mapping populations. One germplasm sample (black dot) was outside of any call cluster and was thus scored "no call". B) Typical theta compressed data for POPA SNP 3_1104; although the polymorphism can be mapped in an individual population there are often wrong calls in such data and the cluster separation is problematic for general use in germplasm analyses or with multiple mapping populations; set to Gentrain 0.000, 100% "no call". C) Typical vertically separated clusters for POPA SNP 3_0070; generally polymorphic for a different locus than the source of the targeted SNP, which results in wrong annotation and degraded synteny; set to Gentrain 0.000, 100% "no call". D) Data for POPA SNP 1_1166 (ABC07305-1-4-322) from the OWB population; two DH samples behave as heterozygotes (purple cluster), far from the homozygotes (red = AA; blue = BB), instead with the 1:1 mixture of parental DNAs (green dot in purple cluster).

Figure 3

Venn diagram showing marker overlap. A four-way Venn diagram illustrates all unique, two-way, three-way and four-way sets of shared markers. The mapping populations are abbreviated as in the text: MxB = Morex × Barke, OWB = Oregon Wolfe Barley, SxM = Steptoe × Morex, HxO = Haruna Nijo × OHU602.

Figure 4

Segment of a consensus directed acyclic graph. A typical segment of a directed acyclic graph representing the consensus map of one barley linkage group is shown. Each oval represents one bin of SNP markers, using POPA names for SNPs. Where an oval contains more than one SNP, it means that there was no evidence of recombination in any mapping population between those markers. The observed recombination frequencies between marker bins are shown. The exact order of marker bins cannot be solved with certainty unless markers are shared between maps. Recombination frequencies are often not proportional to physical distance, nor consistent, when comparing two or more maps from different mapping populations. Therefore directed acyclic graphs provide a more exact description of the limit of knowledge of the marker order than does a linear map derived using approximations based on recombination values. See the text for further discussion.

Figure 5

Barley-rice synteny in detail for 5H. HarvEST screenshot showing barley-rice synteny for chromosome 5H. Colored lines connect each barley locus to the position of the best BLAST hit on the rice genome.

Figure 6

Barley-rice synteny summary. Seven barley linkage groups represented as rice synteny blocks. Numbers inside each barley chromosome indicate syntenic rice chromosome arm.