Development of a 25-plex SNP assay for traceability in cattle
¨ B. Karniol*, A. Shirak*, E. Baruch*, C. Singrun†, A. Tal‡, A. Cahana‡, M. Kam‡, Y. Skalski‡, G. Brem†, J. I. Weller*, M. Ron* and E. Seroussi*
*Agricultural Research Organization, Institute of Animal Science, Bet-Dagan 50250, Israel. †Agrobiogen GmbH, Hilgertshausen-Tandern 86567, Germany. ‡Bactochem Ltd, Ness-Ziona 74031, Israel


Single nucleotide polymorphisms (SNPs) are amenable to automation and therefore have become the marker of choice for DNA profiling. SNaPshot, a primer extension-based method, was used to multiplex 25 SNPs that have been previously validated as useful for identity control. Detection of extended products was based on four different fluorochromes and extension primers with oligonucleotide tails of differing lengths, thus controlling the concise length of the entire chromatogram to 81 bases. Allele frequencies for Holstein, Simmental, Limousin, Angus, Charolais and Tux Cattle were estimated and significant positive Pearsoncorrelation coefficients were obtained among the analysed breeds. The probability that two randomly unrelated individuals would share identical genotypes for all 25 loci varied from 10)8 to 10)10 for these breeds. For parentage control, the exclusion power was found to be 99.9% when the genotypes of both putative parents are known. A traceability test of duplicated samples indicated a high genotyping precision of >0.998. This was further corroborated by analysis of 60 cases of parent–sib pairs and trio families. The 25-plex SNaPshot assay is adapted for low- and high-throughput capacity and thus presents an alternative for DNA-based traceability in the major commercial cattle breeds. Keywords ID power,

multiplex, parent exclusion, traceability.

DNA-based traceability provides the most reliable way to link the source of the carcass to meat products by matching genetic marker profiles (Dalvit et al. 2007). Microsatellites (STRs) have been widely used as genetic markers because of their high degree of polymorphism (Vignal et al. 2002; Dalvit et al. 2008). Nevertheless, single nucleotide polymorphisms (SNPs) have lower rates of genotyping errors (Weller et al. 2006), and are amenable to automation and high-throughput genotyping (Werner et al. 2004). Thus, SNPs have recently been multiplexed for human forensic purposes using SNaPshot system (Sanchez et al. 2006) and for individual identification and genetic traceability in pigs using SNPlex design (Ballester et al. 2007). Two SNP marker panels with 32 and 38 SNP loci have been proposed for traceability and parentage analysis for beef and dairy cattle respectively (Heaton et al. 2002; Werner et al. 2004). The SNPs in both panels were selected based on representation of the whole bovine genome with minimal linkage between the SNP loci, and allele frequency exceeding a threshold of 0.1. However,
Address for correspondence E. Seroussi, Institute of Animal Sciences, ARO, The Volcani Center, Bet-Dagan 50250, Israel. E-mail: Accepted for publication 18 November 2008

there was no attempt to multiplex the SNPs into a single assay. This study was aimed at the development of an economic high-throughput traceability system in cattle based on an effective set of 25-plex SNPs. In a previous study, we found that 25 informative SNPs provide statistical power equivalent to that of the commonly used panel of 11 microsatellites (Weller et al. 2006). Hence, we applied the IPLEX software (SEQUENOM, Latham et al. 2006) to select 25 SNPs using GenBank sequence files as the input (Werner et al. 2004). Variations other than the target SNPs were designated by the letter ÔNÕ. IPLEX software multiplexed 23 out of the 38 markers into a single-well reaction. Because of mass limitation for IPLEX (Ragoussis et al. 2006), nine other SNPs were multiplexed in a second reaction, and therefore remained relevant for potential use with the SNaPshot method. Ten individuals were genotyped using the 23-multiplex and we ruled out the gender-specific SNP (SNP1) due to a lack of polymorphism. To this 22primer design, we further added three additional SNPs based on the IPLEX design of the second reaction. Modification of mass was introduced by adding oligonucleotide-tails of differing lengths to the 5¢ end of the second PCR primer in a SNaPshot reaction (Sanchez et al. 2006). Monitoring of the results with GENEMAPPER (Applied Biosystems) indicated that minimal intervals of 1.0 base between two adjacent bins with the same colour are needed to prevent genotyping 353

Ó 2009 The Authors, Journal compilation Ó 2009 International Society for Animal Genetics, Animal Genetics, 40, 353–356

8 U of shrimp alkaline phosphatase (USB) and then incubated at 37 °C for 60 min. Journal compilation Ó 2009 International Society for Animal Genetics. 2002). The PCR fragments were amplified from 7. red. 51 °C for 20 s and 60 °C for 30 s. Ó 2009 The Authors. Boxes denote each marker domain and allele-calls above and below the chromatogram respectively.). DNA was extracted from ear-tag sample (TypiFixTM) and was used as a template for simultaneous first PCR amplification using 25 primer pairs selected from a panel of 38 dairy cattle SNPs (Werner et al. 1. Sample sizes of the breeds were between 20 and 107 individuals as indicated in Table 1.5 ng of genomic DNA in a total volume of 10 ll with 10 lM of each dNTP. As an added value. Significant positive Pearson-correlation coefficients at the range of 0. A total of 289 individuals (see http://cowry.9%) cases of incorrect parenthood.11 · 10)8 (Limousin) to 2. A traceability assay performed on duplicated samples of 25 animals showed a perfect match of genotypes for all the 625 genotyped SNPs. and elongation at 72 °C for 1 min. 2004). Numbers on the left indicate fluorescence intensity. 2004). A high-throughput genotyping scheme was based on DNA extracted from ear-tag samples and further used as a template for simultaneous first PCR amplification with 25 primer pairs followed by the SNaPshot analysis. specific PCR primers in various concentrations (7–24 fmol/ll) and 0. 2 ll of SNaPshotTM ready reaction premix and specific PCR extension primers in various concentrations (3–150 fmol/ll). Table 1). Parental exclusion probabilities were calculated referring to two schemes: (i) when only one suspected parent was genotyped (one-parent exclusion. agri. The reaction mix was subjected to 30 cycles of 94 °C for 10 s. Samples were analysed by capillary electrophoresis using an ABI 3100 genetic analyzer (Applied Biosystems) and GENEMAPPER software V4.44–0. Inc. blue.01 significance level.3 · 10)10 (Holstein). Final extension was at 72 °C for 3 min. the traceability system based on the 25-plex assay may assist in herd management by better controlling the parenthood records. After enzyme inactivation at 75 °C for 15 min. The two parameters are presented in Table 1. 2 mM MgCl2. the 25-plex assay has statistical power to detect nearly all (above 99.0 (Applied Biosystems). assuming that Figure 1 A typical chromatogram depicting separation of SNP genotypes of the 25-plex using SNaPshot.pdf) were genotyped. 1). The chromatogram shows the allele peaks and bins using the following colour code: adenine. This representative sample indicates a genotyping precision reaching up to 100%. the PCR products were treated with 0. and Mmax ranged from 900 822 to 43 671 580 across breeds at the 0.huji. no family relationships were recorded among the animals. Apart from a few exceptions. Based on the allele frequencies shown in Table 1. thus affecting the power of genetic identification (Vignal et al. fluorescent nucleotide analogues and extension primers with oligonucleotide-tails. followed by 55 cycles of: denaturation at 94 °C for 30 s.5 U of HotStartTaq DNA polymerase (Qiagen. black (yellow for bins). SNaPshot analysis was based on a second PCR amplification using the first PCR products as a template. and (ii) when both suspected parents were genotyped (two-parent exclusion. Orange peaks represent fragments of DNA size standard (GS120LIZ). while still controlling the concise length of the entire chromatogram to 81 bases (Fig. ID power ranged from 1. green. To remove remaining single-stranded primers and dNTPs. SNP scoring was carried out by the single-base extension method using SNaPshotTM Kit (Applied Biosystems) according to the manufacturerÕs instructions.5 ll of the purified PCR product. primer annealing at 56 °C for 30 To remove unincorporated fluorescent ddNTPs. and guanine. 353–356 . Animal Genetics.5 ll. Allele frequencies of genetic markers may vary significantly between populations. thymine. Most SNPs were polymorphic in all the six cattle breeds and distributions of alleles did not deviate from Hardy– Weinberg equilibrium. Numbers on the scale above the chromatogram show size in bases. errors. In all breeds. An output of a GENEMAPPER plot demonstrating the allele separation and genotyping of an individual is presented. The cycling profile was: initial denaturation at 94 °C for 15 min.5 ll of the PCR products was treated with 4 U of Exonuclease I (USB) and 0. To evaluate the statistical power of the 25-plex assay for identity control and parentage testing. we estimated the allele frequencies in six cattle breeds.5 U of shrimp alkaline phosphatase (USB) at 37 °C for 60 min and then incubated at 75 °C for 15 min. 40. Table 1). we calculated for each population the theoretical probability that two random individuals would have identical genotypes for all markers by chance (ID power) and the maximum number of individuals that can be resolved (Mmax). Ready-made primer mixes for the first and second PCR are available from Bactochem Ltd.82 were obtained among all analysed breeds and between the distributions of allele frequency for Holstein that have been previously recorded (Werner et al.354 Karniol et al. Extension reactions were carried out in a final volume of 5. containing 1.

61 0. (2004). We compared the 25-plex system to a commercial set of 11 STRs (bovine StockMarksÒ Animal Parentage Typing System) for cases of parent–sib pairs and trio families from various breeds (Holstein.63 0.79 0.932 0.79 0. Using IPLEX software.10 0.23 0.92 0.46 0.7 · 106 Sim.34 0.60 0.9 0.0 · 106 Ang.51 0. SNPlex and IPLEX. In the remaining two cases.56 0.34 0..38 0. 3 The probability of exclusion.4 0.72 0.30 0.9985 1.25 0. 1 SNP numbers follow Werner et al.76 0.14 0. Simmental.63 0.45 0.73 0.53 0.59 0. 2002).67 0.58 0.9995 2. i.72 0.35 0.78 0.42 0. and was also a more reliable assay than IPLEX for the subset of 22 markers tested. Weller et al. Tux Cattle..14 0.49 0.40 0.75 0.87 0.23 0. 5 Mmax is the maximum number of individuals that can be resolved (equation 3.50 0..8 · 106 Lim.34 0.6 0. 40.892 0.69 0.5–93.38 0.63 0.46 0.72 0. The main feature of the 25-plex SNaPshot assay is its scalable flexibility for low.2%.65 0.89 0. while a microsatellite panel for traceability would quickly reveal the mixed samples by observing the presence of more than two alleles per marker.42 0. of which 38 and 22 were confirmed and excluded respectively.50 0. 2006).26 0.49 0. Tux.74 0.9 · 106 Char.69 0.90 0.37 0.61 0.46 0. Jamieson & Taylor 1997).15 0.42 0. this subset was the largest multiplex we could pick out from the panel of the 38 SNPs published by Werner et al. 353–356 .58 0.37 0. Holstein.9988 3.67 0. the SNaPshot was the most economical. Char. 2 The probability of detecting a falsely reported parent with offspring.37 0.4 · 10)9 4.25 0.75 0.00 0.41 0.45 0.38 0.47 0.03 1. Simmental.79 0.26 0.71 0.1 · 106 355 SNP no. in the range of 84.33 0.56 0. However.68 0.08 0.16 0. 2006).37 0.14 0.66 0.61 0.47 0.34 0.39 0.61 0.5 · 10)9 4.60 0.8 0.19 0.9990 2.893 0.6 · 106 Tux.49 0.45 0. For 20 of the 22 cases.50 0.58 0.79 0.53 0.66 0.78 0. Brown Swiss and Pinzgauer). the exclusion was based on at least two conflicts.89 0. (N = 40) 0.16 0.10 0.81 0. Weller et al. AF440368 AF440372 AF440381 AF440377 AF440369 AF440365 AJ505155 AJ505160 AJ505159 AJ496639 AJ496641 AJ496635 AJ496636 AJ496762 AJ496763 AJ496765 AJ496773 AJ496774 AJ506786 AJ496776 AJ496782 AJ496785 AJ496786 AF440378 AJ505157 Hol.892 0.51 0. Angus.54 0.9 · 10)9 2.1 2 5 6 7 8 9 11 13 14 15 16 17 18 19 20 21 25 26 27 28 30 31 32 35 38 Allele 1 G C C C G C C G C G C C G G C G C G C G C C G G G Allele 2 C T T T A T T A T A T T A A T A T T T A T T A C T Exclusion probability ID power4 Mmax5 One-parent2 Two-parent3 Hol.53 0.42 0.11 0. Jamieson & Taylor 1997).11 0.39 0. the exclusion probabilities are significantly reduced if only one parent is available. identity control and exclusion probabilities in six cattle breeds.e.3 · 10)10 43. Journal compilation Ó 2009 International Society for Animal Genetics.24 0. (2004).61 0.27 0.72 0.892 0... the one observed conflict was further validated by STR analysis.59 0. It should be noted that compared with STRs. Limousin.39 0.A 25-plex SNP assay for traceability in cattle Table 1 SNP-allele frequencies.38 0.59 0. We compared the three commercially available platforms of SNaPshot.844 0.92 0.91 0.41 0.44 0. Sim.78 0.44 0.79 0. Numerous methodologies for SNP genotyping have been reported (Vignal et al..63 0.29 0.24 0.15 0. where both parents are falsely recorded (equation 3a.17 0.37 0. Ang. Ó 2009 The Authors.45 0.65 0.06 0.1 · 10)8 0. Animal Genetics.53 0. Charolais. (N = 31) 0.1 0.38 0. Frequency of allele 1 GenBank accession no. where the other parent is missing (equation 2a. We analysed 60 cases of family relationships.25 0. and found that in our hands. (N = 107) 0.5 · 10)9 6.19 0. (N = 20) 0. Lim.24 0.42 0.9990 2. (N = 39) 0.76 0. both parentsÕ genotypes are available.28 0.45 0.66 0. 4 The probability that two individuals would have identical genotypes by chance (equation 1.12 0.54 0. Such mixtures would not be easily detected by a panel of SNPs.62 0. SNPs have also drawbacks in scenarios where samples contain DNA from multiple individuals.84 0.9992 1.and high-throughput capacity to address individual cases of fraud along with a population-wide traceability.78 0. (N = 52) 0.

& Eggen A.J. 353–356 . Mercade A. (2007) Genetic traceability of livestock products: a review. & Cassandro M. Dalvit C. Animal Genetics 37.... & Poulton K... Gervaso M. time-of-flight mass spectrometry in genomics research. Bennett G. No 533/08.. Harhay G. Weller J. Bounce M.. Dalvit C. Elvidge G. Bet-Dagan. contribution from the Agricultural Research Organization.A. & Taylor S.P. (2008) Genetic traceability of meat using microsatellite markers. De Marchi M. 663–5.W. 920–9. et al.356 Karniol et al. References Ballester M. (2004) Detection and characterization of SNPs useful for ID control and parentage testing in major European dairy breeds. Moreover.. 1713–24. in parent exclusions that are based on a single SNP conflict. Seroussi E. Latham K..G..P.. The reliability of the new 25-plex assay in determining genetic profiles was high and hence a single conflict was sufficient for parentage exclusion.. Targhetta C. Werner F. & Colella S. PLoS Genetics 2. Habermann F.P. Ó 2009 The Authors.S... (2006) Matrixassisted laser desorption/ionisation. Phillips C. (2007) Individual identification and genetic traceability in the pig using the SNPlexTM genotyping system. Israel. 40. 275–305. beef cattle. when only one parent is available or when considering multi-sire pasture situations or situations where sires are related. Animal Genetics 38. Animal Genetics 28. Additional incentive to use the developed traceability system is the possibilities it raises for parentage control capabilities. (2006) Estimation of the number of genetic markers required for individual animal identification accounting for genotyping errors. Journal compilation Ó 2009 International Society for Animal Genetics. Stone R. Food Research International 41. Ollier W..W.L. Animal Genetics 35. SanCristobal M. Institute of Animal Science. Crosby I. 2006). Borsting C. De Marchi M....I. 44–9.. Milan D.. Kaur K..T. Durstewitz G.P. it is advisable to supplement the 25-plex with additional markers.A. Jamieson A. 437–49. Sanchez J. Genetics Selection Evolution 34. Acknowledgements This work was supported by the Chief Scientist of Israel Industry and Trade Ministry as part of the German–Israeli binational BioDisc programme. (1997) Comparisons of three probability formulae for parentage exclusion.. Vignal A. Electrophoresis 27. (2006) The SEQUENOM MassARRAY technology as a high through-put SNP genotyping system.. 387–9. Santamartina J. Stephens R. 272–81.S. 397–400.... 301–7. International Journal of Immunogenetics 33. Heaton M. Van Haandel B.. & Sanchez A. & Laegreid W. Chitko-McKown C.. (2002) Selection and use of SNP markers for animal identification and paternity analysis in U. 321. it is advisable to perform additional tests to confirm the result (Weller et al. (2002) A review on SNP and other types of molecular markers and their use in animal genetics. Keele J. Ragoussis J.M. & Cassandro M. & Ron M. Meat Science 77. Mammalian Genome 13. Casas E. However. Grosse W. (2006) A multiplex assay with 52 single nucleotide polymorphisms for human identification. Smith T. et al. Animal Genetics.

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