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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

Five SNP linkage maps for linkage group 6D. Maps were derived from successive steps in map construction: I) 413 PHR SNPs on the full family (n = 79); II) using 75 progeny (four individuals removed due to poor performance based on graphical genotyping results or because they were duplicates; III) after scrutinizing genotype calls, some data points were replaced with missing values thus removing singletons and a pair; IV) adding 10 previously mapped SSR loci [38]; and V) a full data set totaling 10 SSRs and 667 SNPs consisting of 413 PHR, 247 NMH and 7 OTV SNPs. Black, blue and pink locus lines indicate PHR, NMH and OTV SNPs respectively, and green lines indicate SSR loci. The outer PHR SNPs of the maps are highlighted. PHR markers 1 to 3 refer to AX-89840764, AX-89799050 and AX-89799050 respectively. OTV markers 1 to 7 refer to the SNP (of the cross) AX-89814809 (∅∅ × A∅), AX-89869370 (∅∅ × A∅), AX-89866711 (A∅ × ∅∅), AX-89871559 (A∅ × ∅∅), AX-89896961 (AB × BB), AX-89897092 (AB × BB), and AX-89808404 (A∅ × B∅), respectively.
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Fig10: Five SNP linkage maps for linkage group 6D. Maps were derived from successive steps in map construction: I) 413 PHR SNPs on the full family (n = 79); II) using 75 progeny (four individuals removed due to poor performance based on graphical genotyping results or because they were duplicates; III) after scrutinizing genotype calls, some data points were replaced with missing values thus removing singletons and a pair; IV) adding 10 previously mapped SSR loci [38]; and V) a full data set totaling 10 SSRs and 667 SNPs consisting of 413 PHR, 247 NMH and 7 OTV SNPs. Black, blue and pink locus lines indicate PHR, NMH and OTV SNPs respectively, and green lines indicate SSR loci. The outer PHR SNPs of the maps are highlighted. PHR markers 1 to 3 refer to AX-89840764, AX-89799050 and AX-89799050 respectively. OTV markers 1 to 7 refer to the SNP (of the cross) AX-89814809 (∅∅ × A∅), AX-89869370 (∅∅ × A∅), AX-89866711 (A∅ × ∅∅), AX-89871559 (A∅ × ∅∅), AX-89896961 (AB × BB), AX-89897092 (AB × BB), and AX-89808404 (A∅ × B∅), respectively.

Mentions: Marker performance and map quality were further examined by a more in-depth analysis of LG6D through graphical genotyping of the single parent maps. Two offspring showed a high number of singletons (isolated double recombinants) that could not be reduced by alternative maps of at least equal quality. Two further offspring were represented twice (identical SNP profiles). These results from the four individuals indicated errors in labeling of samples after the SSR genotyping of HK [38]. Exclusion of data from these offspring resulted in a reduction in map size from 163 cM to 106 cM (Figure 10I and II). Graphical genotyping showed the ‘Holiday’ map to still be of poor quality with several blocks of multiple SNPs that had double recombination in multiple offspring (Additional file 13). The ‘Korona’ map had a block of SNPs with high degree of double recombination on LG6D. For both maps, these double recombinant regions could be resolved by manual adjustment of marker order, leaving five singletons for ‘Holiday’ (of which three were for the same marker) (Additional file 13) and one pair and three singletons for ‘Korona’. Genotype calling of these singletons and pair was validated through inspection of the relevant cluster plots. Due to their major negative impact on obtaining good quality high-density SNP maps, these data points were rescored as missing. The resulting integrated linkage map had a length of 90 cM (Figure 10III), and the JoinMap-derived single parental maps no longer contained suspicious double recombinations. We therefore concluded that the presence of even very few incongruent data points (<0.05%) had a major impact on the quality of a high marker density genetic linkage map. Once these were removed, previous marker data for ten SSR loci [38] could be easily integrated with the current SNP data (Figure 10IV). Graphical genotyping plots demonstrated lack of double recombination and the map size remained stable (Figure 10IV).Figure 10


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)

Five SNP linkage maps for linkage group 6D. Maps were derived from successive steps in map construction: I) 413 PHR SNPs on the full family (n = 79); II) using 75 progeny (four individuals removed due to poor performance based on graphical genotyping results or because they were duplicates; III) after scrutinizing genotype calls, some data points were replaced with missing values thus removing singletons and a pair; IV) adding 10 previously mapped SSR loci [38]; and V) a full data set totaling 10 SSRs and 667 SNPs consisting of 413 PHR, 247 NMH and 7 OTV SNPs. Black, blue and pink locus lines indicate PHR, NMH and OTV SNPs respectively, and green lines indicate SSR loci. The outer PHR SNPs of the maps are highlighted. PHR markers 1 to 3 refer to AX-89840764, AX-89799050 and AX-89799050 respectively. OTV markers 1 to 7 refer to the SNP (of the cross) AX-89814809 (∅∅ × A∅), AX-89869370 (∅∅ × A∅), AX-89866711 (A∅ × ∅∅), AX-89871559 (A∅ × ∅∅), AX-89896961 (AB × BB), AX-89897092 (AB × BB), and AX-89808404 (A∅ × B∅), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig10: Five SNP linkage maps for linkage group 6D. Maps were derived from successive steps in map construction: I) 413 PHR SNPs on the full family (n = 79); II) using 75 progeny (four individuals removed due to poor performance based on graphical genotyping results or because they were duplicates; III) after scrutinizing genotype calls, some data points were replaced with missing values thus removing singletons and a pair; IV) adding 10 previously mapped SSR loci [38]; and V) a full data set totaling 10 SSRs and 667 SNPs consisting of 413 PHR, 247 NMH and 7 OTV SNPs. Black, blue and pink locus lines indicate PHR, NMH and OTV SNPs respectively, and green lines indicate SSR loci. The outer PHR SNPs of the maps are highlighted. PHR markers 1 to 3 refer to AX-89840764, AX-89799050 and AX-89799050 respectively. OTV markers 1 to 7 refer to the SNP (of the cross) AX-89814809 (∅∅ × A∅), AX-89869370 (∅∅ × A∅), AX-89866711 (A∅ × ∅∅), AX-89871559 (A∅ × ∅∅), AX-89896961 (AB × BB), AX-89897092 (AB × BB), and AX-89808404 (A∅ × B∅), respectively.
Mentions: Marker performance and map quality were further examined by a more in-depth analysis of LG6D through graphical genotyping of the single parent maps. Two offspring showed a high number of singletons (isolated double recombinants) that could not be reduced by alternative maps of at least equal quality. Two further offspring were represented twice (identical SNP profiles). These results from the four individuals indicated errors in labeling of samples after the SSR genotyping of HK [38]. Exclusion of data from these offspring resulted in a reduction in map size from 163 cM to 106 cM (Figure 10I and II). Graphical genotyping showed the ‘Holiday’ map to still be of poor quality with several blocks of multiple SNPs that had double recombination in multiple offspring (Additional file 13). The ‘Korona’ map had a block of SNPs with high degree of double recombination on LG6D. For both maps, these double recombinant regions could be resolved by manual adjustment of marker order, leaving five singletons for ‘Holiday’ (of which three were for the same marker) (Additional file 13) and one pair and three singletons for ‘Korona’. Genotype calling of these singletons and pair was validated through inspection of the relevant cluster plots. Due to their major negative impact on obtaining good quality high-density SNP maps, these data points were rescored as missing. The resulting integrated linkage map had a length of 90 cM (Figure 10III), and the JoinMap-derived single parental maps no longer contained suspicious double recombinations. We therefore concluded that the presence of even very few incongruent data points (<0.05%) had a major impact on the quality of a high marker density genetic linkage map. Once these were removed, previous marker data for ten SSR loci [38] could be easily integrated with the current SNP data (Figure 10IV). Graphical genotyping plots demonstrated lack of double recombination and the map size remained stable (Figure 10IV).Figure 10

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