snp genotyping of bovine samples with kasp™ chemistry and

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SNP Genotyping of Bovine Samples with KASP™ Chemistry and Probe-Based Assays with a Variety of Master Mixes on the Nexar® System

ABSTRACTSingle Nucleotide Polymorphism (SNP) genotyping must be accurate, reliable, and cost-effective for agricultural marker-assisted selection and genomic selection programs to fully benefit from SNP analysis during the breeding process. To address these needs Douglas Scientific® developed the Nexar System, a flexible and automated high-capacity laboratory system for rapid processing of PCR-based SNP genotype analysis in miniaturized reaction volumes. This study evaluates the efficacy and economic advantages of the Nexar System for agricultural breeding programs. Twenty-nine beef samples were genotyped for four well known SNPs using KASP SNP genotyping chemistry and custom probe-based SNP genotyping assays with multiple master mixes. All conditions produced accurate, easily scorable results with matching genotypes for all replicates of each sample, and call rates ranging from 97% to 100%.

INTRODUCTIONPlant and animal breeding programs rely heavily on marker-assisted selection (MAS) and genomic selection (GS) techniques. Single Nucleotide Polymorphisms, or SNPs, are genetic markers found in plant and animal genomes. They are stable from generation to generation and prevalent throughout the genome making them ideal candidates for use in MAS and GS programs. The development of PCR-based SNP genotyping protocols has enabled researchers to analyze SNPs quickly and accurately. Additionally, improvements in laboratory automation have increased the capacity of MAS and GS laboratories, enabling high throughput sample screening and thus more rapid development of new agricultural products.

According to the USDA, the U.S. beef industry was valued at $88 billion in 2013, an increase of $14 billion since 2010. To keep up with demand, producers in the cattle industry are becoming increasingly reliant on genetic information for breeding programs. Of particular interest are SNPs which have been associated with economically relevant traits including animal weight gain and fat content, marbling, and meat tenderness. For example, polymorphisms in the calpain (CAPN1) and calpastatin (CAST) genes have been associated with meat tenderness as a result of postmortem proteolysis (Schenkel, 2006 and White, 2005). CAPN1-4751 is a C to T substitution. The CC and CT genotypes are associated with increased tenderness compared to the TT genotype (White et al., 2005). UoGCAST is a C to G substitution where the C allele is linked to overall increased tenderness (Schenkel et al., 2006). Leptin is a hormone related to feed intake, weight gain, and fat deposition. For one particular SNP in exon 2 of the leptin gene (C to T substitution) animals with the CC genotype have less fat and are less economically favorable than animals with the TT genotype (Lusk, 2007 and DeVuyst, 2008). Another SNP, located in the pro-melanin concentrating hormone (PMCH) gene, is associated

with fat content and overall meat quality (Walter et al., 2013). In addition to SNP genotyping for selecting favorable traits, SNP panels are also widely used for animal identification and tracking of beef products through the market.

In this study we genotyped beef samples using commercially-available PCR-based master mixes and custom SNP assays in miniaturized reactions on the Nexar System from Douglas Scientific. The beef samples were purchased from multiple local supermarkets in Minnesota. The four SNPs analyzed in this study were selected because of their association with several desirable traits related to overall meat quality. PCR-based SNP genotyping was completed using both KASP SNP genotyping chemistry and custom probe-based SNP genotyping assays with five different master mixes.

SNP Genotyping of Bovine Samples with KASP Chemistry and Probe-Based Assays with a Variety of Master Mixes on the Nexar System 1

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SNP Genotyping of Bovine Samples with KASP Chemistry and Probe-Based Assays with a Variety of Master Mixes on the Nexar System

MATERIALS AND METHODSDNA Extraction: Beef samples were obtained from local supermarkets in Minnesota. DNA was extracted using a Qiagen® DNeasy Blood and Tissue Kit according to the manufacturer’s instructions.

Assays and Reagents: Genomic information for the SNPs analyzed in this study can be found in Table 1. The PCR master mixes used in this study can be found in Table 2. Master mix was provided at 2X concentration and used according to the manufacturer’s instructions. BHQplus® probe-based SNP genotyping assays were designed using RealTimeDesign™ Software from LGC Biosearch Technologies. All primers and probes were obtained from LGC Biosearch Technologies. KASP SNP genotyping assays differ from traditional probe-based assays, as described in Figure 1. KASP primers were diluted according to the manufacturer’s instructions and were added at 2X concentration to the 2X KASP 1536 V4.0 Master Mix before use. For the probe-based assays, BHQplus probes and primers were added at 2X concentration to the master mix (400 nM and 1.8 µM, respectively) to achieve a final

concentration in the PCR reaction of 200 nM probes, 900 nM primers, and 1X master mix.

Dispensing, Thermal Cycling, and Analysis: Douglas Scientific instruments including the Nexar, Soellex®, and Araya® were used for all sample processing and SNP genotyping reactions in Array Tape® and are described in Figure 2. DNA samples (800 nL) were dispensed into Array Tape with the multi-channel, 384-tip pipette head from CyBi® Product Line. KASP 1536 V4.0 Master Mix containing 2X SNP genotyping assay (800 nL) was dispensed with the non-contact Dispense Jet to create 1.6 µL total reaction volume in Array Tape. PCR amplification and thermal cycling were performed in the Soellex using a touchdown PCR protocol with a total of 41 cycles according to the standard protocol for KASP genotyping chemistry. Alternatively, master mix containing 2X BHQplus probe-based assay (800 nL) was dispensed with the non-contact Dispense Jet and PCR amplification was performed in the Soellex using the manufacturer’s instructions for a total of 45 cycles. End-point fluorescence values were determined by scanning the Array Tape in the Araya. Cluster plot analysis was completed with Douglas Scientific’s Intellics® Software Suite. All SNP genotyping reactions were performed in duplicate. Data were managed and analysed using IntelliScore® software.

SNP ID Genbank Accession Number and Base Position Chromosome Substitution SNP Description

Leptin AY138588 - 305 4 C/T TT genotype associated with fatter, more economically favorable animals

CAPN1-4751 AF248054 - 6545 29 C/T CC and CT genotypes associated with increased meat tenderness

PMCH DQ499531 - 38 5 A/T AA genotype associated with high body fat and more tender meat

UoGCAST AY008267 - 282 7 C/G CC genotype linked to increased meat tenderness

Table 1: SNP information

Reagent Source

KASP 1536 V4.0 Master Mix LGC Genomics

TaqMan® GTXpress™ Master Mix Life Technologies

TaqMan Genotyping Master Mix Life Technologies

PerfeCTa® qPCR ToughMix® Quanta BioSciences

SensiFAST™ Lo-ROX Genotyping Kit Bioline

SsoAdvanced™ Universal Probes Supermix Bio-Rad Laboratories

Unlabeled primers and BHQplus probes Biosearch Technologies

Table 2: Reagents and assays

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Figure 1: Assay design for probe and KASP SNP genotyping reactions. A) Probe-based SNP genotyping assays utilize forward and reverse primers to amplify a segment of genomic DNA surrounding a SNP. Two fluorogenic probes differentially bind to their allele-specific complement. Fluorescent signal is produced during each PCR cycle by separation of the dye and quencher, as probes are hydrolyzed through Taq exonuclease activity. B) KASP SNP genotyping assays utilize a common reverse primer and two allele-specific forward primers, ending at the SNP site. Each forward primer contains an allelic discrimination nucleotide at the 3’ end and a tag that is specific to one of the FRET cassettes at the 5’ end. Tag sequences are identical to dye-labeled strands of the FRET cassettes, which are supplied in the master mix. The tag sequence is incorporated into the PCR product along with the forward primer as PCR amplification occurs. During subsequent PCR cycles, the dye-labeled strand of the FRET cassette acts as a forward primer. Fluorescent signal is produced by irreversible separation of the FRET cassette dye and quencher.

ARRAY TAPE NEXAR SOELLEX ARAYA

• Flexible microplate replacement • Liquid handler optimized for Array Tape • High capacity water bath PCR • End-point fluorescence scanner

• Reduced reaction volumes • 800 nL DNA, 384-channel dispense • Optimized for Array Tape • Optimized for Array Tape

• Total well volume of 2 µL • 800 nL master mix, 384-well dispense in 38 seconds • Three tanks for PCR optimization • Scan 384 wells in 28 seconds

• Optically clear cover seal • Seal Array Tape for thermal cycling • Touchdown or traditional PCR • Data ready for analysis in Intellics

Figure 2: Nexar System Overview

FAM™ Dye

FAM Dye

VIC®/HEX™ Dye

HEX Dye

Quencher

Quencher

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RESULTSAll SNP genotyping reactions in this experiment produced clusters that were easily scored in IntelliScore data analysis software. Cluster plot images for all assays and master mixes are shown in Figure 3. A summary of the calls using each master mix is provided in Table 3. Three samples were not scored because of poor amplification. Genotyping results for all four probe-based SNP assays studied in this experiment matched across all master mixes used and were concordant with the results achieved using the corresponding KASP assays. The genotypes for each sample are shown in Table 4. The 29 samples were sorted and ranked based on favorable genotypes.

CONCLUSIONSThis study demonstrates that the Nexar System can be successfully used to produce repeatable and easily scored SNP genotyping results with bovine samples in miniaturized reaction volumes. BHQplus probe-based SNP assays with multiple master mixes and KASP chemistry produced accurate, matching results, demonstrating the flexibility of the Nexar System for use with a variety of chemistry options. One interesting finding from the genotyping results was that meat samples within a single package were not necessarily from the same animal. For example, sample 3-A and 3-B were two steaks grouped in the same pack-age. However, when samples were ranked based on favorable geno-types, 3-A came out on top and 3-B was at the bottom. Even with the small panel of SNPs used in this study, these results demonstrate the usefulness of SNP genotyping for both trait selection as well as animal identification.

Figure 3: SNP genotyping cluster plots. Endpoint fluorescence values are plotted with Cal Fluor Orange or Hex signal on the y-axis and FAM signal on the x-axis. All values are normalized with ROX.

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CONCLUSIONS CONT'D.In addition to automating the SNP genotyping process for MAS and GS programs, the Nexar System provides significant cost savings in the form of reduced PCR reaction volumes. The results shown in this study were generated with 1.6 µL reactions in Array Tape, a significant volume reduction com-pared to standard plate-based systems. Taken together, the results of this experiment demonstrate that the Nexar System is a solution for agricultural SNP genotyping applications.

REFERENCESDeVuyst, E. A. The economics of gene testing cattle. Okla-homa Cooperative Extension Service (2008) AGEC-255.

Lusk, J. L. Association of single nucleotide polymorphisms in the leptin gene with body weight and backfat growth curve parameters for beef cattle. J Anim Sci (2007) 85:1865-1872.

Schenkel, F.S., Miller, S.P., Jiang, Z., Mandell, I.B., Ye, X., Li, H., Wilton, J. W. Association of a single nucleotide polymorphism in the calpastatin gene with carcass and meat quality traits of beef cattle. J Anim Sci (2006) 84:291-299.

USDA Economic Research Service(http://www.ers.usda.gov/topics/animal-products/cattle-beef/statistics-information.aspx visited July 20, 2014).

Walter, L. J., Gasch, C. A., McEvers, T. J., Hutcheson, J. P., De-Foor, P., Marquess, F. L. S., Lawrence, T. E. Association of pro-melanin concentrating hormone genotype with beef carcass quality and yield. J Anim Sci (2014) 92:325-331.

White S. N., Casas E., Wheeler T. L., Shackelford S. D., Koohma-raie M., Riley D. G, Chase C.C. Jr, Johnson D.D., Keele J.W.,

Smith T.P.L. A new single nucleotide polymorphism in CAPN1 extends the current tenderness marker test to include cattle of Bos indicus, Bos taurus, and crossbred descent. J Anim Sci (2005) 83:2001-2008.

SNP KASP 1536 V4.0

TaqMan GTXpress

TaqMan Genotyping

PerfeCTa ToughMix

SensiFAST Genotyping

Bio-Rad SsoAdvanced

Leptin(CC/CT/TT) 2/19/8 2/19/8 2/19/8 2/19/8 2/19/8 2/19/8

CAPN1-4751 (CC/CT/TT) 8/10/11 8/10/11 8/9*/11 8/10/11 8/10/11 8/10/11

PMCH (AA/AT/TT) 13/14/2 13/13*/2 13/14/2 13/14/2 13/14/2 13/14/2

UoGCAST (CC/CG/GG) 8/10/11 8/10/10* 8/10/11 8/10/11 8/10/11 8/10/11

Table 3: Summary of SNP genotyping calls *One sample did not fall within a cluster

Table 4: Bovine sample genotypes for each SNP. The samples are sorted and ranked based on favorable combination of the four SNPs. Green indicates the favorable genotype, yellow indicates intermediate, and orange indicates the less favorable genotype.

NEXAPP-5-1

*For research use only. The products of Douglas Scientific, LLC are not FDA-approved for use in human diagnostic procedures.

snp genotyping of bovine samples with kasp™ chemistry and

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