SNP discovery and chromosome anchoring provide the first physically-anchored hexaploid oat map and reveal synteny with model species

Abstract

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Figure 1. In silico SNP discovery approaches.

Both methods started with a set of quality-trimmed 454 sequence reads identified by source germplasm from either the cDNA libraries or from DArT-based genomic complexity reductions. A. In the single template approach (STA), the reads were assembled by MIRA software to generate a consensus sequence for each contig. Four reference genomes were selected based on membership in four different quadrants of a principle component analysis that had been conducted previously using DArT markers. Consensus sequences from all varieties were assembled against each of the four reference genomes, and candidate SNPs were called using Roche GSMapper. SNPs were filtered based on several criteria, as described in the methods. Redundant SNPs were identified using BLASTN. B. In the composite template approach (CTA), reads were assembled within varieties at a high stringency using DNAstar Seqman Software. Then the consensus reads were filtered and truncated to include only those parts having perfect alignment with greater than two reads. The consensus reads were then concatenated and subjected to a single composite assembly at lower stringency. The consensus from this assembly was used as a composite reference genome to call SNPs. Although the SNP calling and filtering process was similar to CTA, this pipeline was automated using in-house software called “Ace-of-Base”.

Figure 2. Marker density and positions for individual linkage maps (Otana/PI 260616, A; Provena/94197A1-9-2-2-2-5, B; Ogle/TAM O-301, C; Hurdal/Z-959-1, D; SolFi/HiFi, E; and Kanota/Ogle, F) in relationship to the consensus.

Marker positions in the consensus map are indicated by the scales on the left axes; positions of each corresponding marker in the component maps are indicated by a color gradient described in the key.

Figure 3. Chromosome anchoring of consensus linkage map.

Grey dots indicate positions of non-anchored markers on each linkage group, while colored dots indicate positions of physically anchored SNPs (blue dots), DArTs (red up arrows), and RFLPs (green down arrows).

Figure 4. Regions of sequence similarity between SNP markers on 21 chromosomes from an oat consensus map (8A to 21D) and chromosomes from the sequenced genomes of Brachypodium distachyon (Bd) and Oryza sativa (Os).

Regions of substantial colinearity have been interpolated using chromosome-specific colors.