VALIDATION OF COMMERCIAL DNA TESTS FOR BEEF QUALITY TRAITS

Gene mapping and discovery programs have resulted in the detection of numerous DNA ‘markers’ for various beef cattle production traits. Prior to commercializing genetic markers, it is important to validate their purported effects on the traits of interest in different breeds and environments, and assess them for correlated responses in associated traits. One of the biggest challenges in achieving this objective is the availability of cattle populations with sufficient phenotypic data to assess the association between various traits and newly discovered genetic markers. Results from such validation studies to date have not been widely published and genetic marker tests sometimes may be commercialized prior to the collection of field validation data. In addition, conflicting reports about some commercially available markers, as well as the recognized occurrence of well-proven bulls with a high EPD for a given trait but carrying two copies of the “wrong” (unfavorable) marker for that trait, have made some producers wary of investing in DNA-based testing. Producers want to know whether DNA-based tests perform in accordance with the claims of the marketing company and are interested in third-party, independent validation of these tests. The objective of this study was to validate three commercially-available genetic tests (GeneSTAR Quality Grade, GeneSTAR Tenderness, and Igenity TenderGENE).


Introduction
Gene mapping and discovery programs have resulted in the detection of numerous DNA 'markers' for various beef cattle production traits.Prior to commercializing genetic markers, it is important to validate their purported effects on the traits of interest in different breeds and environments, and assess them for correlated responses in associated traits.One of the biggest challenges in achieving this objective is the availability of cattle populations with sufficient phenotypic data to assess the association between various traits and newly discovered genetic markers.Results from such validation studies to date have not been widely published and genetic marker tests sometimes may be commercialized prior to the collection of field validation data.In addition, conflicting reports about some commercially available markers, as well as the recognized occurrence of well-proven bulls with a high EPD for a given trait but carrying two copies of the "wrong" (unfavorable) marker for that trait, have made some producers wary of investing in DNA-based testing.
Producers want to know whether DNA-based tests perform in accordance with the claims of the marketing company and are interested in third-party, independent validation of these tests.The objective of this study was to validate three commercially-available genetic tests (GeneSTAR Quality Grade 8 , GeneSTAR Tenderness 8 , and Igenity TenderGENE 9 ).

Experimental Procedures
Validation Process.The National Beef Cattle Evaluation Consortium (NBCEC, www.NBCEC.org)conducts independent validations of commercially-available genetic tests for beef cattle production traits.This process is a collaboration of owners of the DNA and phenotypes (e.g., breed associations) and commercial testing companies, facilitated by the NBCEC.

DNA Testing Companies and Sample
Populations.Phenotypic data and DNA were mostly collected as part of the Carcass Merit Project funded by the Cattlemen's Beef Board and cooperating breed associations.Each commercial testing company selected the breed groups to be used for the validation.

Genetic Tests.
Genotyping for the GeneSTAR Quality Grade and GeneSTAR Tenderness marker panels (Bovigen, LLC) and the TenderGENE marker panel (Merial) was done by the respective companies.The 2 tenderness panels share two common μcalpain and similar but different calpastatin markers.The µ-calpain enzyme system is primarily responsible for tenderization that occurs during aging.Calpastatin is an inhibitor of the calpain enzyme system, so high levels of calpastatin are undesirable.
Phenotypes.Traits analyzed were longissimus lumborum (loineye) Warner-Bratzler shear force (WBSF) and subjectively recorded marbling score.Muscle sections were harvested 24 to 48 hours postmortem from numerous processing plants, with nearly all using relatively high-voltage electrical stimulation.Steaks (1 inch thick) were vacuum packaged and aged at 1° to 2°C until 14 days post-mortem.Steaks were cooked to an internal temperature of 71°C at 163°C.After reaching the endpoint temperature, steaks were cooled at 1° to 2°C for 24 hours, and eight 1/2-inchdiameter cores were removed parallel to the muscle fibers and sheared with a WBSF Vblade attached to an Instron Universal Testing Machine.A numeric score was used to record marbling, with 400 corresponding to Slight 00 , 500 corresponding to Small 00 , and 600 corresponding to Modest 00 , etc. Quality grade was analyzed as percentage qualifying as USDA Choice or Prime based entirely on marbling score ≥ 500 (all carcasses were A maturity).
Statistical Analyses.The basic model was y = CG + marker effect + sire + e, where CG denotes a fixed contemporary group and sire was a random effect.For GeneSTAR Tenderness and Igenity TenderGENE marker panels, there were two linked markers, so the regression was on the expected number of copies of each of the four haplotypes (one of which was rare).Genotype frequencies were estimated and analyses carried out with SAS Proc HAPLOTYPE and Proc Mixed, respectively (SAS 9.1.3,SAS Inst.Inc., Cary, NC).

Results
Allele Frequencies.The sample genotypes and allele frequencies for each of the markers included in the commercial tests in this validation study are shown in Table 1.Some alleles were extremely rare (< 0.5%) in certain populations.Specific haplotype frequencies are reported in Table 2. Haplotypes are combinations of genes affecting different traits.
GeneSTAR Quality Grade.One of the Quality Grade (QG) alleles was almost fixed in the Hereford-sired sample population, so the analysis included only the 387 Charolaissired × Angus cattle (Table 1).The GeneSTAR QG test was not associated with marbling score; however, an increase in the percentage of Choice plus Prime approached significance (P≤ 0.06; Table 3).The association of the test with quality grade was primarily attributable to the effect of the favorable allele of the QG marker.Bovigen, LLC categorizes the different genotypes into categories of 0, 1, or 2 "stars".In this sample population, the average effect of each "star" (0, 1, or 2) of the GeneSTAR QG test was associated with a 6.2% increase in the percentage of Choice plus Prime.
GeneSTAR Tenderness.Improved tenderness was associated with substituting a T allele at Calpastatin-T1 and a C allele at both μ-calpain loci.The GeneSTAR Tenderness analysis included 1302 cattle (372 Charolais × Angus, 260 Hereford, and 670 Brahman).The association of calpastatin-T1 and the μ-calpain haplotypes with WBSF were each highly significant (P<0.01), as was the combination of these markers (P<0.0001).Each calpastatin T was associated with a decrease of 0.33 lb. in WBSF, and substituting the Calpain T2-T3 C-C haplotype for the Calpain T2-T3 G-T haplotype was associated with a decrease of 0.75 lb in WBSF (Table 4).
Igenity TenderGENE.Improved tenderness was associated (P<0.001) with substituting a C allele at calpastatin and a C allele at both μ-calpain loci.The association of calpastatin and the μ-calpain alleles with WBSF were each highly significant (P<0.001), and the combination of all three even more so (P<0.0001).Table 5 shows the improvement in WBSF for each of the possible haplotypes contrasted to the least tender genotype (calpastatin GG, μ-calpain TT, μ-calpain GG) calculated from a combined analysis of 1209 cattle (181 Brangus, 400 Charolais × Angus crosses, 310 Red Angus and 318 Brahman).In this sample population, each calpastatin C was associated with a decrease of 0.42 lb in WBSF and substituting C-C for G-T at CAPN1 was associated with a decrease of 0.73 lb of WBSF.Combined genotypic effects for GeneSTAR Tenderness and Igenity Tender-GENE are presented in Table 6.There was a 2.2 lb difference in WBSF between the most and least tender genotypes in both panels.

Discussion
Our study did not show a significant association of the GeneSTAR Quality Grade marker with marbling score, but there was a definite trend (P = 0.06) toward increased quality grade associated with substituting the favorable allele in Charolais × Angus crossbred cattle that had been fed for less than 250 days.However, the binary trait of percentage of Choice plus Prime represents a considerable loss of information compared to the continuous trait of marbling score.The association of quality grade with the results from the GeneSTAR Quality Grade test in the absence of a significant association with marbling score was probably the result of a high proportion of animals on the borderline of the USDA Select/Choice grade.The absolute improvement in quality grade associated with any marker will always be dependent upon marbling endpoint, which emphasizes the importance of environment and management on results derived from validation studies.
The genotype effects of the two tenderness panels, GeneSTAR Tenderness and Igenity TenderGENE, were very similar to each other (Table 6), suggesting that the two calpastatin alleles are marking the same tendernessassociated region of the genome.The magnitude of the WBSF reduction associated with the most favorable genotypes compared to the least favorable genotypes is distinctly greater than the difference in tenderness that has been recorded between Select and low Choice quality grades, as well as being greater than the tenderness difference between Select and "premium" Choice (upper two-thirds of Choice) beef.From the perspective of genetic improvement, it is interesting to observe that the frequency of the μ-calpain G-T haplotype is relatively high (Table 2).This suggests that the beef industry may have the opportunity to improve tenderness by increasing the frequency of the μ-calpain C-C haplotype.
Failure to achieve statistical significance should never be interpreted as evidence that an effect is zero.In this case, the major allele frequency in one or more validation populations may be so high that there is no real opportunity to evaluate the effect of the test.This should not be considered a negative result, but rather a 'no result' (e.g. the GeneSTAR QG test in the Hereford population in this study).Given these considerations, it is perhaps not surprising that few marker validation studies in cattle have been published.However, validation of the effects of genetic markers in independent populations is likely to be vital to the success of genetic testing technology, as producers are likely to be reluctant to invest in unproven markers.
Validation studies can also serve to generate information that is essential for the process of incorporating DNA tests into cattle evaluation.Although there is a tendency to label DNA tests as being associated with one particular trait, markers with a large effect on any one trait are also likely to have correlated effects on other traits because most genes influence a variety of traits.The widespread adoption of marker-assisted selection in the industry will likely depend upon the successful integration of marker information into cattle evaluation schemes to enable eventual development of "DNA marker-assisted EPDs."

Implications
Tenderness could be markedly improved by selecting for the favorable calpastatin and μ-calpain genotypes included in GeneSTAR Tenderness and Igenity TenderGENE marker panels.Using the GeneSTAR Quality Grade marker panel could result in an increased percentage of USDA Choice plus Prime carcasses.

Table 3 . Effects of GeneSTAR Quality Grade Panel Results on Marbling Score and % of Animals Grading Choice and Prime Phenotypes from 387 Charolais-sired × Angus Cattle
Average effects of Quality Grade favorable alleles.

Table 4 . Effects of GeneSTAR Tenderness Panel Results on Warner-Bratzler Shear Force (lb) Phenotypes from 372 Charolais-sired ×Angus, 260 Hereford, and 670 Brahman Cattle
The low number of animals with the C-T haplotype in this study made it difficult to accurately estimate their effects.

Table 5 . Effects of Igenity TenderGENE panel results on Warner-Bratzler Shear Force (lb) Phenotypes from 181 Brangus, 400 Charolais-sired × Angus Cross, 310 Red Angus and 318 Brahman Cattle
The low number of animals with the C-T haplotype in this study made it difficult to accurately estimate its effect.