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Genome Assembly Improvement and Mapping Convergently Evolved Skeletal Traits in Sticklebacks with Genotyping-by-Sequencing.

Glazer AM, Killingbeck EE, Mitros T, Rokhsar DS, Miller CT - G3 (Bethesda) (2015)

Bottom Line: In the revised genome assembly, 94.6% of the assembly was anchored to a chromosome.To assess linkage map quality, we mapped quantitative trait loci (QTL) controlling lateral plate number, which mapped as expected to a 200-kb genomic region containing Ectodysplasin, as well as a chromosome 7 QTL overlapping a previously identified modifier QTL.Finally, we mapped eight QTL controlling convergently evolved reductions in gill raker length in the two crosses, which revealed that this classic adaptive trait has a surprisingly modular and nonparallel genetic basis.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cell Biology Department, University of California-Berkeley, Berkeley, California 94720.

No MeSH data available.


Genotyping-by-sequencing (GBS) approach. (A) Flowchart of GBS. For each cross, the two grandparents were resequenced to determine homozygous SNP differences, which were filtered for high coverage levels and expected allele ratios in F2s (see Materials and Methods). (B) Sieve for high-coverage, segregating SNPs. For each SNP, the mean number of mapped reads supporting the marine and freshwater alleles, normalized for the number of millions of reads mapped per sample, is displayed. Data are shown for the FTC × LITC cross. Sieve is shown with red quadrilateral: freshwater allele frequency between 0.2 and 0.8 and total coverage between 0.2 and 3. (C) Diagram of binning approach. Low-coverage sequencing generated read pileup at a large number of SNPs. For each F2, SNPs were binned by counting the total number of marine and freshwater reads within the bin and determining a genotype from the pooled counts. Sample 2 illustrates a case in which a recombination breakpoint is near the boundary between two bins and Bin 1 containing the breakpoint is considered to have the FF genotype. Alternatively, bins containing recombination breakpoints also frequently were called with uncertain MF/FF or MM/MF genotypes (Figure S2). (D) Calling sex from sex chromosome (chromosome 19) coverage. Females (XX) have approximately equal sex chromosome and autosome coverage levels, whereas males (XY) have approximately half the coverage level on the sex chromosome compared to the autosomes. Data are shown for the FTC cross. Inset shows zoom-in of low-coverage samples showing that female and male fish can still be distinguished.
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fig1: Genotyping-by-sequencing (GBS) approach. (A) Flowchart of GBS. For each cross, the two grandparents were resequenced to determine homozygous SNP differences, which were filtered for high coverage levels and expected allele ratios in F2s (see Materials and Methods). (B) Sieve for high-coverage, segregating SNPs. For each SNP, the mean number of mapped reads supporting the marine and freshwater alleles, normalized for the number of millions of reads mapped per sample, is displayed. Data are shown for the FTC × LITC cross. Sieve is shown with red quadrilateral: freshwater allele frequency between 0.2 and 0.8 and total coverage between 0.2 and 3. (C) Diagram of binning approach. Low-coverage sequencing generated read pileup at a large number of SNPs. For each F2, SNPs were binned by counting the total number of marine and freshwater reads within the bin and determining a genotype from the pooled counts. Sample 2 illustrates a case in which a recombination breakpoint is near the boundary between two bins and Bin 1 containing the breakpoint is considered to have the FF genotype. Alternatively, bins containing recombination breakpoints also frequently were called with uncertain MF/FF or MM/MF genotypes (Figure S2). (D) Calling sex from sex chromosome (chromosome 19) coverage. Females (XX) have approximately equal sex chromosome and autosome coverage levels, whereas males (XY) have approximately half the coverage level on the sex chromosome compared to the autosomes. Data are shown for the FTC cross. Inset shows zoom-in of low-coverage samples showing that female and male fish can still be distinguished.

Mentions: The two grandparents of the FTC cross and the two grandparents of the BEPA cross were resequenced to approximately 60× and 6× coverage, respectively. In each cross, sites where one grandparent was homozygous for one allele and the other grandparent was homozygous for a second allele were identified (“homozygous SNP positions”; see Supplemental Methods in File S8). GBS reads from F2s were sorted by barcode with a custom Perl script. Reads were mapped to the stickleback reference genome with BWA using default settings (www.bio-bwa.sourceforge.net), allowing up to a 4% difference between reads and the reference genome. We devised a method to identify high-quality, segregating SNPs. For each homozygous SNP position, F2 GBS reads overlapping the SNP were considered, and the number of reads supporting marine and freshwater alleles for each homozygous SNP position was determined with SAMtools (www.samtools.sourceforge.net). Genomic positions identified as not having a homozygous difference in the grandparent resequencing were not examined in the F2s. For each homozygous SNP position, a weighted average of these values was calculated across all F2s, normalized by the total number of mapped reads for each F2. We multiplied the marine and freshwater weighted averages by 106 to calculate reads per million mapped (RPMM). Properly segregating SNPs should have an approximately 1:1 ratio of marine:freshwater alleles, as was observed for most SNPs (Figure 1B). However, we observed some genomic regions that had a skewed allele ratio in the F2s, possibly due to meiotic drive and/or the lethality of particular genotypic classes. For example, a region of chromosome 2 centered at marker 0_32 in the FTC cross had a freshwater allele frequency of 0.61 (File S1). Therefore, a wider range of allele ratios was allowed for individual SNPs (a marine/freshwater RPMM ratio between 4:1 and 1:4). To include SNPs with true segregation bias, skewed markers adjacent to other similarly skewed markers were included, but skewed markers surrounded by nonskewed markers were removed (see Supplemental Methods in File S8). Additionally, SNPs were filtered for those with an average marine plus freshwater RPMM between 0.2 and 3.0 to have a set of SNPs with similar coverage levels. A separate set of sieving parameters was used to determine sex chromosome genotypes (see Supplemental Methods in File S8).


Genome Assembly Improvement and Mapping Convergently Evolved Skeletal Traits in Sticklebacks with Genotyping-by-Sequencing.

Glazer AM, Killingbeck EE, Mitros T, Rokhsar DS, Miller CT - G3 (Bethesda) (2015)

Genotyping-by-sequencing (GBS) approach. (A) Flowchart of GBS. For each cross, the two grandparents were resequenced to determine homozygous SNP differences, which were filtered for high coverage levels and expected allele ratios in F2s (see Materials and Methods). (B) Sieve for high-coverage, segregating SNPs. For each SNP, the mean number of mapped reads supporting the marine and freshwater alleles, normalized for the number of millions of reads mapped per sample, is displayed. Data are shown for the FTC × LITC cross. Sieve is shown with red quadrilateral: freshwater allele frequency between 0.2 and 0.8 and total coverage between 0.2 and 3. (C) Diagram of binning approach. Low-coverage sequencing generated read pileup at a large number of SNPs. For each F2, SNPs were binned by counting the total number of marine and freshwater reads within the bin and determining a genotype from the pooled counts. Sample 2 illustrates a case in which a recombination breakpoint is near the boundary between two bins and Bin 1 containing the breakpoint is considered to have the FF genotype. Alternatively, bins containing recombination breakpoints also frequently were called with uncertain MF/FF or MM/MF genotypes (Figure S2). (D) Calling sex from sex chromosome (chromosome 19) coverage. Females (XX) have approximately equal sex chromosome and autosome coverage levels, whereas males (XY) have approximately half the coverage level on the sex chromosome compared to the autosomes. Data are shown for the FTC cross. Inset shows zoom-in of low-coverage samples showing that female and male fish can still be distinguished.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Genotyping-by-sequencing (GBS) approach. (A) Flowchart of GBS. For each cross, the two grandparents were resequenced to determine homozygous SNP differences, which were filtered for high coverage levels and expected allele ratios in F2s (see Materials and Methods). (B) Sieve for high-coverage, segregating SNPs. For each SNP, the mean number of mapped reads supporting the marine and freshwater alleles, normalized for the number of millions of reads mapped per sample, is displayed. Data are shown for the FTC × LITC cross. Sieve is shown with red quadrilateral: freshwater allele frequency between 0.2 and 0.8 and total coverage between 0.2 and 3. (C) Diagram of binning approach. Low-coverage sequencing generated read pileup at a large number of SNPs. For each F2, SNPs were binned by counting the total number of marine and freshwater reads within the bin and determining a genotype from the pooled counts. Sample 2 illustrates a case in which a recombination breakpoint is near the boundary between two bins and Bin 1 containing the breakpoint is considered to have the FF genotype. Alternatively, bins containing recombination breakpoints also frequently were called with uncertain MF/FF or MM/MF genotypes (Figure S2). (D) Calling sex from sex chromosome (chromosome 19) coverage. Females (XX) have approximately equal sex chromosome and autosome coverage levels, whereas males (XY) have approximately half the coverage level on the sex chromosome compared to the autosomes. Data are shown for the FTC cross. Inset shows zoom-in of low-coverage samples showing that female and male fish can still be distinguished.
Mentions: The two grandparents of the FTC cross and the two grandparents of the BEPA cross were resequenced to approximately 60× and 6× coverage, respectively. In each cross, sites where one grandparent was homozygous for one allele and the other grandparent was homozygous for a second allele were identified (“homozygous SNP positions”; see Supplemental Methods in File S8). GBS reads from F2s were sorted by barcode with a custom Perl script. Reads were mapped to the stickleback reference genome with BWA using default settings (www.bio-bwa.sourceforge.net), allowing up to a 4% difference between reads and the reference genome. We devised a method to identify high-quality, segregating SNPs. For each homozygous SNP position, F2 GBS reads overlapping the SNP were considered, and the number of reads supporting marine and freshwater alleles for each homozygous SNP position was determined with SAMtools (www.samtools.sourceforge.net). Genomic positions identified as not having a homozygous difference in the grandparent resequencing were not examined in the F2s. For each homozygous SNP position, a weighted average of these values was calculated across all F2s, normalized by the total number of mapped reads for each F2. We multiplied the marine and freshwater weighted averages by 106 to calculate reads per million mapped (RPMM). Properly segregating SNPs should have an approximately 1:1 ratio of marine:freshwater alleles, as was observed for most SNPs (Figure 1B). However, we observed some genomic regions that had a skewed allele ratio in the F2s, possibly due to meiotic drive and/or the lethality of particular genotypic classes. For example, a region of chromosome 2 centered at marker 0_32 in the FTC cross had a freshwater allele frequency of 0.61 (File S1). Therefore, a wider range of allele ratios was allowed for individual SNPs (a marine/freshwater RPMM ratio between 4:1 and 1:4). To include SNPs with true segregation bias, skewed markers adjacent to other similarly skewed markers were included, but skewed markers surrounded by nonskewed markers were removed (see Supplemental Methods in File S8). Additionally, SNPs were filtered for those with an average marine plus freshwater RPMM between 0.2 and 3.0 to have a set of SNPs with similar coverage levels. A separate set of sieving parameters was used to determine sex chromosome genotypes (see Supplemental Methods in File S8).

Bottom Line: In the revised genome assembly, 94.6% of the assembly was anchored to a chromosome.To assess linkage map quality, we mapped quantitative trait loci (QTL) controlling lateral plate number, which mapped as expected to a 200-kb genomic region containing Ectodysplasin, as well as a chromosome 7 QTL overlapping a previously identified modifier QTL.Finally, we mapped eight QTL controlling convergently evolved reductions in gill raker length in the two crosses, which revealed that this classic adaptive trait has a surprisingly modular and nonparallel genetic basis.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cell Biology Department, University of California-Berkeley, Berkeley, California 94720.

No MeSH data available.