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, 3 (10), e3376

Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

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Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

Nathan A Baird et al. PLoS One.

Abstract

Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F(2) individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms.

Conflict of interest statement

Competing Interests: EAJ has patents filed on the RAD marker, and partial interest in a company commercializing the system. TSA is a paid employee of that company.

Figures

Figure 1
Figure 1. RAD marker generation.
(A) Genomic DNA was digested with a restriction enzyme and the P1 adapter was ligated to the fragments. The P1 adapter contains a forward amplification primer site, an Illumina sequencing primer site, and a barcode (colored boxes represent P1 adapters with different barcodes). (B) Adapter-ligated fragments were combined (if multiplexing), sheared and (C) ligated to a second adapter (P2, white boxes). The P2 adapter is a divergent “Y” adapter, containing the reverse complement of the reverse amplification primer site preventing amplification of genomic fragments lacking a P1 adapter. (D) RAD tags, which have a P1 adapter, will be selectively and robustly enriched.
Figure 2
Figure 2. Sequenced RAD marker mapping.
(A) A native saltwater stickleback population, Rabbit Slough (RS), have complete lateral plate armor (brackets) while these structures are absent in the derived, freshwater Bear Paw (BP) population. The freshwater fish also have a reduction in pelvic structure (arrow) compared to the oceanic population. These two phenotypes segregate independently in an F2 mapping cross. Using SbfI (B) or EcoRI (C), we mapped polymorphic RAD markers from RS (red) and BP (green) parental fish along the 21 stickleback linkage groups. The apparent size differences of the linkage groups between (B) and (C) reflect the fact that the EcoRI recognition sequence occurs more frequently than SbfI. Red and green bars above the linkage groups are measures of lateral plate linkage in the F2 progeny, indicating the number of tightly linked markers in the local region. (D) Sequence reads per barcoded F2 individual used to create (C). Variable numbers of reads were obtained from each of the 96 individuals used in our analysis, reflecting different concentrations of starting DNA template. 68% of individuals had between 50 K and 150 K RAD tags sequenced (∼0.4–1.0× coverage of the ∼150 K tags present in the genome). Only 2 individuals had less than 10,000 reads (red). (E) A close-up of the boxed region from (C) showing recombination breakpoints in six informative low plate F2 fish on LGIV. Black tick marks are 1 Mb apart in physical distance. (F) F2 individuals were repooled in silico based on the pelvic structure phenotype (A, arrow). Linkage was determined as in (B, C), mapping the locus for a reduction in pelvic structure to the end of LGVII.
Figure 3
Figure 3. Mapping a novel induced mutation.
(A) Southern blot of digested N. crassa genomic DNA with methylation-sensitive AvaII, showing loss of methylation in the AX7 mutant strain, as compared to the parental mutagenized N2977 strain. (B) Polymorphic RAD markers from N32 (red) and N2977 (green) parental strains were mapped along the seven N. crassa linkage groups. Red and green bars above the linkage groups are measures of linkage in the recombinant progeny, indicating the number of tightly linked markers in the local region. (C) A close-up view of linkage group II, showing the locations of confirmative RFLP markers (arrows). (D) RFLP marker confirmation. RFLP markers were designed using polymorphic RAD markers at 2.1 Mb and 3.1 Mb on LGII for N. crassa. The marker at 2.1 Mb confirmed the lack of recombinants in the wild-type pool, while a portion of individuals in the mutant pool have undergone recombination at this location. RFLP analysis at 3.1 Mb showed complete linkage in both wild-type and mutant pools to the methylation-deficient phenotype.

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