Consumers were asked to swab the inside of their cheeks for approximately 1 min on each cheek using Isohelix DNA buccal swabs. These were then stored at room temperature until DNA extraction and kept dry through the use of Isohelix Dri-Capsules (Cell Projects Ltd., Kent, UK). The swabs were sent to IDna Genetics Ltd. (Norwich, UK) for extraction and genotyping, with 10% of the swabs sent as blinded replicates to ensure accuracy. DNA was extracted using Isohelix Buccalyse DNA Extraction Kit (Cell Projects, Kent, UK) according to the manufacturer’s instructions, and then diluted 1:8 with water prior to analysis. TAS2R38 polymorphisms (rs713598, rs1726866 and rs10246939) were analysed using the KASP genotyping chemistry (LGC Group, Middlesex, UK). Diluted DNA was dried into 384-well PCR plates (Life Technologies, UK), and then 5 μL of KASP Master mix (LGC Group, Middlesex, UK) and primers were added. PCR amplification was performed as follows: 94 °C for 15 min, 94 °C for 15 s, 65 °C for 20 s, 94 °C for 15 s, 57 °C for 20 s (Life Technologies, UK). The fluorescent products were detected in an Applied Biosystems instrument (Life Technologies, UK).
Kasp genotyping chemistry
Manufactured by LGC
Sourced in United Kingdom
KASP genotyping chemistry is a flexible and reliable technology for single nucleotide polymorphism (SNP) genotyping. It utilizes a competitive allele-specific PCR and fluorescence-based detection system to accurately identify different allelic variants.
Automatically generated - may contain errors
6 protocols using kasp genotyping chemistry
1
Buccal Swab DNA Extraction and Genotyping
2
SLC26A9 SNP Genotyping with KASP
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The genotyping of SLC26A9 SNPs (rs7512462, rs4077468, rs7419153, rs12047830, rs4077469, rs12741299, and rs1874361) was carried out using Kompetitive Allele Specific PCR (KASP) genotyping chemistry (LGC, Teddington, United Kingdom).
3
Validating QTL Region on Chromosome A02
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To validate the QTL region identified on chromosome A02, allele‐specific KASP (Kompetitive allele‐specific PCR) primers (LGC Genomics, https://www.biosearchtech.com/services/genotyping-services ) were designed to target five SNPs within a 6Mbp region located between base pair positions 136 553 and 6 375 504 on chromosome A02 (Table S2 ). The genomic positions for each SNP were according to the Darmor‐bzh reference genome and included SNPs within orthologues of the flowering time genes AtFLC (SNP FLC‐136553 within BnaFLC.A02) and AtFT (SNP FT‐6375504 within BnaFT.A02). DNA was extracted from the 94 earliest and 94 latest F2 lines under both NVERN and VERN treatments, in addition to the parental lines Cabriolet and Darmor, and assayed for SNP genotype using the KASP genotyping chemistry according to the manufacturer's instructions (LGC Genomics, Hoddesdon, UK).
4
Genotyping Pigmentation-Related Genes
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Genomic DNA was isolated from saliva samples using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol.
We selected genes previously associated with skin sensitivity to sunlight and tanning ability (4, 5, 16, 18, 19) . Nine SNPs located in 8 pigmentation-related genes were finally genotyped: rs4911442 (located in the ASIP gene), rs2153271 (BNC2 gene), rs12913832 (HERC2 gene), rs1800407 (OCA2 gene), rs12896399 (SLC24A4 gene), rs16891982 (SLC45A2 gene), rs1393350 and rs1042602 (TYR gene), and rs12203592 (IRF4 gene).
Genotyping assays were performed by using KASP Genotyping Chemistry (LGC, Hoddesdon, United Kingdom) or TaqMan technology (Applied Biosystems, Foster City, USA). PCR conditions varied depending on the requirements of each probe, always according to the manufacturer's recommendations. For quality control, we Unsuccessful genotyping rate was lower than 5% in all SNPs analysed.
The MC1R gene was studied by direct genetic sequencing with the Sanger method, as previously described (29) . A sample with known MC1R genotype per 96-well plate was added for quality control. Sixteen samples (3.49%) were discarded because of unsuccessful MC1R sequencing.
We selected genes previously associated with skin sensitivity to sunlight and tanning ability (4, 5, 16, 18, 19) . Nine SNPs located in 8 pigmentation-related genes were finally genotyped: rs4911442 (located in the ASIP gene), rs2153271 (BNC2 gene), rs12913832 (HERC2 gene), rs1800407 (OCA2 gene), rs12896399 (SLC24A4 gene), rs16891982 (SLC45A2 gene), rs1393350 and rs1042602 (TYR gene), and rs12203592 (IRF4 gene).
Genotyping assays were performed by using KASP Genotyping Chemistry (LGC, Hoddesdon, United Kingdom) or TaqMan technology (Applied Biosystems, Foster City, USA). PCR conditions varied depending on the requirements of each probe, always according to the manufacturer's recommendations. For quality control, we Unsuccessful genotyping rate was lower than 5% in all SNPs analysed.
The MC1R gene was studied by direct genetic sequencing with the Sanger method, as previously described (29) . A sample with known MC1R genotype per 96-well plate was added for quality control. Sixteen samples (3.49%) were discarded because of unsuccessful MC1R sequencing.
5
Genetic Mapping of BLR38 Resistance Loci
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Ten leaf discs (Ø = 6 mm) were punched per plant and placed in a 96‐well tray. Leaf discs were dried by placing a silica bag on top of the plate and applying a vacuum to the sealed package. DNA was isolated using the sbeadex maxi plant kit (LGC Genomics, Berlin, Germany) and verified on a NanoDrop‐8000 spectrophotometer (ThermoFisher Scientific, Wilmington, DE, USA). Genotyping was carried out using KASP genotyping chemistry (LGC Genomics, Berlin, Germany) on Fluidigm chips. Data were analysed using Fluidigm SNP genotyping software. A genetic linkage map was constructed using JoinMap 4.0 software (Van Ooijen, 2006 ), and QTLs were detected using MapQTL 5.0 software (Van Ooijen, 2004 ).
To determine the smallest mapping interval on chr 4, BLR38‐unresponsive plants homozygous for L. serriola LS102 alleles at the QTL on chr 8 were selected. In these plants, BLR38 unresponsiveness was caused by homozygous L. sativa GreenTowers alleles on chr 4 at marker positions closely linked to BLR38 recognition. After delineating the interval on chr 4, the smallest mapping interval on chr 8 was determined with BLR38‐unresponsive plants that were homozygous for L. serriola LS102 on chr 4. For the markers defining the smallest mapping intervals on chr 4 and chr 8, the SNP with its flanking sequence is listed in TableS5 .
To determine the smallest mapping interval on chr 4, BLR38‐unresponsive plants homozygous for L. serriola LS102 alleles at the QTL on chr 8 were selected. In these plants, BLR38 unresponsiveness was caused by homozygous L. sativa GreenTowers alleles on chr 4 at marker positions closely linked to BLR38 recognition. After delineating the interval on chr 4, the smallest mapping interval on chr 8 was determined with BLR38‐unresponsive plants that were homozygous for L. serriola LS102 on chr 4. For the markers defining the smallest mapping intervals on chr 4 and chr 8, the SNP with its flanking sequence is listed in Table
6
Genotyping HABP2 G534E Variant
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Genomic DNA was isolated from whole blood samples using Promega Maxwell 16 system (Promega). HABP2 G534E variant was genotyped using competitive allele-specific KASP genotyping chemistry (LGC Genomics, Teddington, UK) as described previously (11 (link)). The genotyping call rates were >98%, with controls derived from replicates with verified genotypes used in all assays. Four additional markers, two upstream of G534E (rs10787491, rs932650) and two downstream of G534E (rs10885478, rs1885434), were genotyped in five G534E heterozygous individuals for haplotype analysis.
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