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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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Related in: MedlinePlus

Allelic configurations of SNP (di-allelic and multi-allelic) and indel markers in an octoploid. Panel A) Di-allelic SNPs: To qualify as di-allelic, only two alleles can be detected at the site. The “marker allele” is present only in one subgenome (the marker subgenome), within which it can be homozygous present, heterozygous, or homozygous absent. In case 1 a single probe can be used to interrogate the marker because the indicated polymorphism is neither A/T nor G/C. In case 2 two probes must be used because the indicated polymorphism is an A/T (also true for a G/C polymorphism). Panels B and C) Multi-allelic SNPs: More than two alleles are represented at the site. Three distinctive cases are shown for tri-allelic (Panel B) and for tetra-allelic sites (Panel C). In tri-allelic case 1 the marker polymorphism is G/T, while there is a C at the same site in the background subgenomes. Genotyping of this marker would require two probes. In case 2 the marker polymorphism is G/T, with a background G in one subgenome and a background C in the others. Genotyping of this marker would require two probes. In case 3 there are two marker polymorphisms, a G/T in one subgenome and a G/C in another, while there is a C at the site in the background subgenomes. Three probes and a non-standard analysis algorithm are needed for this polymorphism. Genotyping of case 3 tri-allelic markers, and of tetra-allelic markers (Panel C) is currently not possible. Panel D) Di-allelic indels: Only two alleles are represented at the site. Although they are genomic insertions and deletions, the indel polymorphisms are genotyped as SNPs, and various probing strategies may be employed depending upon the sequence characteristics within and immediately adjacent to the indel.
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Fig1: Allelic configurations of SNP (di-allelic and multi-allelic) and indel markers in an octoploid. Panel A) Di-allelic SNPs: To qualify as di-allelic, only two alleles can be detected at the site. The “marker allele” is present only in one subgenome (the marker subgenome), within which it can be homozygous present, heterozygous, or homozygous absent. In case 1 a single probe can be used to interrogate the marker because the indicated polymorphism is neither A/T nor G/C. In case 2 two probes must be used because the indicated polymorphism is an A/T (also true for a G/C polymorphism). Panels B and C) Multi-allelic SNPs: More than two alleles are represented at the site. Three distinctive cases are shown for tri-allelic (Panel B) and for tetra-allelic sites (Panel C). In tri-allelic case 1 the marker polymorphism is G/T, while there is a C at the same site in the background subgenomes. Genotyping of this marker would require two probes. In case 2 the marker polymorphism is G/T, with a background G in one subgenome and a background C in the others. Genotyping of this marker would require two probes. In case 3 there are two marker polymorphisms, a G/T in one subgenome and a G/C in another, while there is a C at the site in the background subgenomes. Three probes and a non-standard analysis algorithm are needed for this polymorphism. Genotyping of case 3 tri-allelic markers, and of tetra-allelic markers (Panel C) is currently not possible. Panel D) Di-allelic indels: Only two alleles are represented at the site. Although they are genomic insertions and deletions, the indel polymorphisms are genotyped as SNPs, and various probing strategies may be employed depending upon the sequence characteristics within and immediately adjacent to the indel.

Mentions: The GDP VCF files were entered into various filtering pipelines (Additional files 3, 4, 5, 6, 7 and 8) aimed at discovering marker candidates of several types: di-allelic SNPs (Figure 1A; Additional file 3), multi-allelic SNPs (mSNPs) (Figure 1B-C; Additional file 4), di-allelic indels (Figure 1D, Additional file 5), and three categories of haploSNPs (Figure 2A-C; Additional files 6, 7 and 8). The term “haploSNP” denotes the coupling of two variants: (1) a marker SNP and (2) a closely adjacent HSV SNP or indel that provides a critical “destabilization site”, which is intended to confer subgenomic exclusivity and thereby achieve technical ploidy reduction at the respective site of probe hybridization.Figure 1


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)

Allelic configurations of SNP (di-allelic and multi-allelic) and indel markers in an octoploid. Panel A) Di-allelic SNPs: To qualify as di-allelic, only two alleles can be detected at the site. The “marker allele” is present only in one subgenome (the marker subgenome), within which it can be homozygous present, heterozygous, or homozygous absent. In case 1 a single probe can be used to interrogate the marker because the indicated polymorphism is neither A/T nor G/C. In case 2 two probes must be used because the indicated polymorphism is an A/T (also true for a G/C polymorphism). Panels B and C) Multi-allelic SNPs: More than two alleles are represented at the site. Three distinctive cases are shown for tri-allelic (Panel B) and for tetra-allelic sites (Panel C). In tri-allelic case 1 the marker polymorphism is G/T, while there is a C at the same site in the background subgenomes. Genotyping of this marker would require two probes. In case 2 the marker polymorphism is G/T, with a background G in one subgenome and a background C in the others. Genotyping of this marker would require two probes. In case 3 there are two marker polymorphisms, a G/T in one subgenome and a G/C in another, while there is a C at the site in the background subgenomes. Three probes and a non-standard analysis algorithm are needed for this polymorphism. Genotyping of case 3 tri-allelic markers, and of tetra-allelic markers (Panel C) is currently not possible. Panel D) Di-allelic indels: Only two alleles are represented at the site. Although they are genomic insertions and deletions, the indel polymorphisms are genotyped as SNPs, and various probing strategies may be employed depending upon the sequence characteristics within and immediately adjacent to the indel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Allelic configurations of SNP (di-allelic and multi-allelic) and indel markers in an octoploid. Panel A) Di-allelic SNPs: To qualify as di-allelic, only two alleles can be detected at the site. The “marker allele” is present only in one subgenome (the marker subgenome), within which it can be homozygous present, heterozygous, or homozygous absent. In case 1 a single probe can be used to interrogate the marker because the indicated polymorphism is neither A/T nor G/C. In case 2 two probes must be used because the indicated polymorphism is an A/T (also true for a G/C polymorphism). Panels B and C) Multi-allelic SNPs: More than two alleles are represented at the site. Three distinctive cases are shown for tri-allelic (Panel B) and for tetra-allelic sites (Panel C). In tri-allelic case 1 the marker polymorphism is G/T, while there is a C at the same site in the background subgenomes. Genotyping of this marker would require two probes. In case 2 the marker polymorphism is G/T, with a background G in one subgenome and a background C in the others. Genotyping of this marker would require two probes. In case 3 there are two marker polymorphisms, a G/T in one subgenome and a G/C in another, while there is a C at the site in the background subgenomes. Three probes and a non-standard analysis algorithm are needed for this polymorphism. Genotyping of case 3 tri-allelic markers, and of tetra-allelic markers (Panel C) is currently not possible. Panel D) Di-allelic indels: Only two alleles are represented at the site. Although they are genomic insertions and deletions, the indel polymorphisms are genotyped as SNPs, and various probing strategies may be employed depending upon the sequence characteristics within and immediately adjacent to the indel.
Mentions: The GDP VCF files were entered into various filtering pipelines (Additional files 3, 4, 5, 6, 7 and 8) aimed at discovering marker candidates of several types: di-allelic SNPs (Figure 1A; Additional file 3), multi-allelic SNPs (mSNPs) (Figure 1B-C; Additional file 4), di-allelic indels (Figure 1D, Additional file 5), and three categories of haploSNPs (Figure 2A-C; Additional files 6, 7 and 8). The term “haploSNP” denotes the coupling of two variants: (1) a marker SNP and (2) a closely adjacent HSV SNP or indel that provides a critical “destabilization site”, which is intended to confer subgenomic exclusivity and thereby achieve technical ploidy reduction at the respective site of probe hybridization.Figure 1

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