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Development and preliminary evaluation of a 90 K Axiom® SNP array for the allo-octoploid cultivated strawberry Fragaria × ananassa.

Bassil NV, Davis TM, Zhang H, Ficklin S, Mittmann M, Webster T, Mahoney L, Wood D, Alperin ES, Rosyara UR, Koehorst-Vanc Putten H, Monfort A, Sargent DJ, Amaya I, Denoyes B, Bianco L, van Dijk T, Pirani A, Iezzoni A, Main D, Peace C, Yang Y, Whitaker V, Verma S, Bellon L, Brew F, Herrera R, van de Weg E - BMC Genomics (2015)

Bottom Line: Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing "haploSNPs" (11.7%).The array's high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies.This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS, NCGR, Corvallis, OR, USA. nahla.bassil@ars.usda.gov.

ABSTRACT

Background: A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca 'Hawaii 4' reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array.

Results: About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing "haploSNPs" (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative "codon-based" SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix's "SNPolisher" R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family 'Holiday' × 'Korona' with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM.

Conclusions: The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array's high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

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Six SNP quality classes (A) and four variance filters (B) applied to thePHRgenotype class. A. Default SNP quality classes produced by the Axiom Best Practices Genotyping Workflow. B. An example cluster plot for a SNP identified with each of the four variance filters used including: AB.varY identified large heterozygous cluster variance in the Y dimension; AA.varY for homozygous (AA) variance in the Y dimension; BB.varY, for homozygous (BB) variance in the Y dimension; and AB.varX, for heterozygous variance in the X dimension.
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Fig3: Six SNP quality classes (A) and four variance filters (B) applied to thePHRgenotype class. A. Default SNP quality classes produced by the Axiom Best Practices Genotyping Workflow. B. An example cluster plot for a SNP identified with each of the four variance filters used including: AB.varY identified large heterozygous cluster variance in the Y dimension; AA.varY for homozygous (AA) variance in the Y dimension; BB.varY, for homozygous (BB) variance in the Y dimension; and AB.varX, for heterozygous variance in the X dimension.

Mentions: DNA was isolated and quantified as described above for the octoploid strawberry validation set (Table 2). Up to 50 μL of DNA (≥20 ng/μL) from each sample was submitted to Affymetrix for genotyping. The Axiom assay was performed on four 96-sample Axiom arrays using the Affymetrix GeneTitan® system according to the procedure described by Affymetrix (http://media.affymetrix.com/support/downloads/manuals/axiom_2_assay_auto_workflow_user_guide.pdf). Next, cell intensity files (.CEL) generated by the GeneTitan instrument were converted to genotype calls using the Axiom Genotyping Algorithm version 1 (Axiom GT1) available through Affymetrix Power Tools or Genotyping Console™v4.1 software package. The procedure is documented by Affymetrix (http://media.affymetrix.com/support/downloads/manuals/axiom_genotyping_solution_analysis_guide.pdf). Executing the Axiom Best Practices Genotyping Workflow, SNPs sorted into six quality classes according to their clustering performance with respect to various quality-control measures (Figure 3A). These SNP classes were: (1) “Poly High Resolution” (PHR), which were polymorphic and passed all quality control (QC); (2) “No Minor Homozygote” (NMH), which passed all QC but only two clusters were observed; (3) “Off-Target Variant” (OTV), which had an additional low intensity cluster resulting from slight mismatches between the probe and the sequences for that group of individuals; (4) “Mono High Resolution” (MHR),which passed all QC but were monomorphic; (5) “CallRate Below Threshold” (CRBT), where genotype call rate was under 97%; and (6) “Other”, where the resultant SNP cluster pattern did not fall into any of the previous classes. SNPs that fell into the OTV class were further genotyped using OTV Caller, a statistical method developed by Affymetrix and included in the “SNPolisher” R package to identify samples that were homozygous for a -allele.Figure 3


Development and preliminary evaluation of a 90 K Axiom® SNP array for the allo-octoploid cultivated strawberry Fragaria × ananassa.

Bassil NV, Davis TM, Zhang H, Ficklin S, Mittmann M, Webster T, Mahoney L, Wood D, Alperin ES, Rosyara UR, Koehorst-Vanc Putten H, Monfort A, Sargent DJ, Amaya I, Denoyes B, Bianco L, van Dijk T, Pirani A, Iezzoni A, Main D, Peace C, Yang Y, Whitaker V, Verma S, Bellon L, Brew F, Herrera R, van de Weg E - BMC Genomics (2015)

Six SNP quality classes (A) and four variance filters (B) applied to thePHRgenotype class. A. Default SNP quality classes produced by the Axiom Best Practices Genotyping Workflow. B. An example cluster plot for a SNP identified with each of the four variance filters used including: AB.varY identified large heterozygous cluster variance in the Y dimension; AA.varY for homozygous (AA) variance in the Y dimension; BB.varY, for homozygous (BB) variance in the Y dimension; and AB.varX, for heterozygous variance in the X dimension.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374422&req=5

Fig3: Six SNP quality classes (A) and four variance filters (B) applied to thePHRgenotype class. A. Default SNP quality classes produced by the Axiom Best Practices Genotyping Workflow. B. An example cluster plot for a SNP identified with each of the four variance filters used including: AB.varY identified large heterozygous cluster variance in the Y dimension; AA.varY for homozygous (AA) variance in the Y dimension; BB.varY, for homozygous (BB) variance in the Y dimension; and AB.varX, for heterozygous variance in the X dimension.
Mentions: DNA was isolated and quantified as described above for the octoploid strawberry validation set (Table 2). Up to 50 μL of DNA (≥20 ng/μL) from each sample was submitted to Affymetrix for genotyping. The Axiom assay was performed on four 96-sample Axiom arrays using the Affymetrix GeneTitan® system according to the procedure described by Affymetrix (http://media.affymetrix.com/support/downloads/manuals/axiom_2_assay_auto_workflow_user_guide.pdf). Next, cell intensity files (.CEL) generated by the GeneTitan instrument were converted to genotype calls using the Axiom Genotyping Algorithm version 1 (Axiom GT1) available through Affymetrix Power Tools or Genotyping Console™v4.1 software package. The procedure is documented by Affymetrix (http://media.affymetrix.com/support/downloads/manuals/axiom_genotyping_solution_analysis_guide.pdf). Executing the Axiom Best Practices Genotyping Workflow, SNPs sorted into six quality classes according to their clustering performance with respect to various quality-control measures (Figure 3A). These SNP classes were: (1) “Poly High Resolution” (PHR), which were polymorphic and passed all quality control (QC); (2) “No Minor Homozygote” (NMH), which passed all QC but only two clusters were observed; (3) “Off-Target Variant” (OTV), which had an additional low intensity cluster resulting from slight mismatches between the probe and the sequences for that group of individuals; (4) “Mono High Resolution” (MHR),which passed all QC but were monomorphic; (5) “CallRate Below Threshold” (CRBT), where genotype call rate was under 97%; and (6) “Other”, where the resultant SNP cluster pattern did not fall into any of the previous classes. SNPs that fell into the OTV class were further genotyped using OTV Caller, a statistical method developed by Affymetrix and included in the “SNPolisher” R package to identify samples that were homozygous for a -allele.Figure 3

Bottom Line: Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing "haploSNPs" (11.7%).The array's high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies.This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS, NCGR, Corvallis, OR, USA. nahla.bassil@ars.usda.gov.

ABSTRACT

Background: A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca 'Hawaii 4' reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array.

Results: About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing "haploSNPs" (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative "codon-based" SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix's "SNPolisher" R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family 'Holiday' × 'Korona' with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM.

Conclusions: The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array's high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

Show MeSH
Related in: MedlinePlus