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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

Effect of excluding 51 “non-ananassa” octoploid accessions from genotyping co-cluster runs for two SNPs. The cluster plots in the left column are for 310 strawberry samples. The cluster plots in the right column are for 284 strawberry samples, excluding the non-ananassa samples (F. chiloensis and F. virginiana). SNP A is a robust diploid-like SNP site that produces only three genotype clusters in the presence as well in the absence of the wild progenitor samples, and thus is genotyped correctly in both circumstances. SNP B is a more challenging SNP site at which the genomes of the divergent non-ananassa form a 4th genotype cluster (arrow). This 4th cluster causes the samples in the BB homozygous genotype cluster to be incorrectly called as heterozygous (colored gold). When the non-ananassa samples are excluded (bottom right), the software correctly calls the samples in the homozygous BB cluster as “BB” (colored blue).
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Fig5: Effect of excluding 51 “non-ananassa” octoploid accessions from genotyping co-cluster runs for two SNPs. The cluster plots in the left column are for 310 strawberry samples. The cluster plots in the right column are for 284 strawberry samples, excluding the non-ananassa samples (F. chiloensis and F. virginiana). SNP A is a robust diploid-like SNP site that produces only three genotype clusters in the presence as well in the absence of the wild progenitor samples, and thus is genotyped correctly in both circumstances. SNP B is a more challenging SNP site at which the genomes of the divergent non-ananassa form a 4th genotype cluster (arrow). This 4th cluster causes the samples in the BB homozygous genotype cluster to be incorrectly called as heterozygous (colored gold). When the non-ananassa samples are excluded (bottom right), the software correctly calls the samples in the homozygous BB cluster as “BB” (colored blue).

Mentions: The first attempt at genotyping the polyploid samples included the 306 F. ×ananassa samples and 51 octoploid “non-ananassa” accessions (Table 2, Additional file 1). For some SNPs, adequate genotyping was hampered due to the inclusion of different octoploid species, which caused samples to form more than three intensity clusters (Figure 5). SNPs with such complex cluster groups resulted in a higher error rate of automated assignments of genotypes to clusters. In addition, samples whose intensities fell between the three prototypical clusters were more likely to have miscalled genotypes. While procedures and filter thresholds proposed by Affymetrix adequately identified SNPs with complex cluster patterns in diploid species, they needed to be extended for use with allo-polyploid species. Therefore, a second attempt was performed using advanced approaches to identify such SNPs and samples and filter them appropriately.Figure 5


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)

Effect of excluding 51 “non-ananassa” octoploid accessions from genotyping co-cluster runs for two SNPs. The cluster plots in the left column are for 310 strawberry samples. The cluster plots in the right column are for 284 strawberry samples, excluding the non-ananassa samples (F. chiloensis and F. virginiana). SNP A is a robust diploid-like SNP site that produces only three genotype clusters in the presence as well in the absence of the wild progenitor samples, and thus is genotyped correctly in both circumstances. SNP B is a more challenging SNP site at which the genomes of the divergent non-ananassa form a 4th genotype cluster (arrow). This 4th cluster causes the samples in the BB homozygous genotype cluster to be incorrectly called as heterozygous (colored gold). When the non-ananassa samples are excluded (bottom right), the software correctly calls the samples in the homozygous BB cluster as “BB” (colored blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Effect of excluding 51 “non-ananassa” octoploid accessions from genotyping co-cluster runs for two SNPs. The cluster plots in the left column are for 310 strawberry samples. The cluster plots in the right column are for 284 strawberry samples, excluding the non-ananassa samples (F. chiloensis and F. virginiana). SNP A is a robust diploid-like SNP site that produces only three genotype clusters in the presence as well in the absence of the wild progenitor samples, and thus is genotyped correctly in both circumstances. SNP B is a more challenging SNP site at which the genomes of the divergent non-ananassa form a 4th genotype cluster (arrow). This 4th cluster causes the samples in the BB homozygous genotype cluster to be incorrectly called as heterozygous (colored gold). When the non-ananassa samples are excluded (bottom right), the software correctly calls the samples in the homozygous BB cluster as “BB” (colored blue).
Mentions: The first attempt at genotyping the polyploid samples included the 306 F. ×ananassa samples and 51 octoploid “non-ananassa” accessions (Table 2, Additional file 1). For some SNPs, adequate genotyping was hampered due to the inclusion of different octoploid species, which caused samples to form more than three intensity clusters (Figure 5). SNPs with such complex cluster groups resulted in a higher error rate of automated assignments of genotypes to clusters. In addition, samples whose intensities fell between the three prototypical clusters were more likely to have miscalled genotypes. While procedures and filter thresholds proposed by Affymetrix adequately identified SNPs with complex cluster patterns in diploid species, they needed to be extended for use with allo-polyploid species. Therefore, a second attempt was performed using advanced approaches to identify such SNPs and samples and filter them appropriately.Figure 5

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