1. Effects of  -Calpain DNA Tests on Tenderness R. Mark Thallman U.S. Meat Animal Research Center Clay Center, NE

2.  -Calpain Quantitative Trait Locus (QTL) for WB Shear Force on Bovine Chromosome 29  -calpain gene located in the region of the QTL  -calpain is a proteolytic enzyme that plays a key role in postmortem tenderization of meat

3. MARC  -Calpain Team Tim Smith Eduardo Casas Roger Stone Brent Page Stephen White Mohammad Koohmaraie Tommy Wheeler Steven Shackelford Renee Godtel Kevin Tennill Steve Simcox Linda Flathman

4. Tests for  -Calpain are Marketed By: Frontier Beef Systems - TenderGENE Genetic Solutions – GeneStar Tenderness II MMI Genomics – plan to market with their parentage products

5. QTL on Bovine Chromosome 29 L×J P×AP×A BTA29 ToughTenderToughTender

6. What is an SNP? (Single Nucleotide Polymorphism) Functional Genotype Test Result +/-+/- Functional Mutation +/-+/- Test Location AGAATTTGAAT  TGTGATGAACACTCCAC AGAATATGAAT  TGTGATGAACAGTCCAC +/++/++/++/+ AGAATTTGAAT  TGTGATGAACACTCCAC Association between test result and functional mutation Or, the test polymorphism may actually be the functional polymorphism, but that is difficult to prove and not really necessary.

7. What is an SNP? (Single Nucleotide Polymorphism) Functional Genotype Test Result +/-+/- Functional Mutation +/-+/- Test Location AGAATTTGAAT . .  TGTGATGAACAC TCCAC AGAATATGAAT ..  TGTGATGAACAG TCCAC +/++/++/++/+ AGAATTTGAAT . .  TGTGATGAACAC TCCAC AGAATTTGAAT ..  TGTGATGAACAC TCCAC Association between test result and functional mutation

8. SNP Association L×J P×AP×A G|AG|A ToughTenderToughTender C|GC|G 316 530 G|AG|A C|GC|G 316 530

9. Distribution of  –Calpain Genotypes GGAGAA CC4.00.40.0 CG22.38.80.8 GG27.028.48.2 SNP 316 SNP 530 GPE Cycle VII steers

10. Possible Test Results Can be Viewed from Two Perspectives GGAGAA CCCC GG CC AG CC AA CGCG GG CG AG CG AA GGGG GG AG GG AA SNP 316 SNP 530 CGCGG GAGA C A CGCG CC GG CG GG CG GA CC GA G GC GGGG GG GA GC GA GAGA GC AG GG AG GG AA GC AA C ACC AG CG AG CG AA CC AA Haplotype

11. Reporting Results with Multiple SNP GGAGAA CC+/+ 4.0 +/? 0.4 ?/? 0.0 CG+/? 22.3 +/- 8.8 -/? 0.8 GG?/? 27.0 -/? 28.4 -/- 8.2 SNP 316 SNP 530 CGCGG GAGA C A CGCG +/+ 4.0 +/? 11.2 +/- 4.0 +/? 0.2 G +/? 11.2 ?/? 27.0 -/? 14.2 ?/? 0.4 GAGA +/- 4.0 -/? 14.2 -/- 8.2 -/? 0.4 C A+/? 0.2 ?/? 0.4 -/? 0.4 ?/? 0.0 Haplotype Genotype frequencies

12. Genotypic Effects on WB Shear Force (lb) (higher is tougher) GGAGAA CC-1.35 ± 0.79 2% -0.58 ± 1.09 1% N/A 0% CG-0.36 ± 0.35 12% -0.29 ± 0.33 16% -0.99 ± 0.93 1% GG0 24% 0.0 ± 0.27 33% 0.82 ± 0.37 10% SNP 316 SNP 530 ASA 362 hd

13. Two Possible Models for Observed Intermediate Haplotype Effect Avg. Effect of Haplotype Tender Intermediate Tough Too Rare to Estimate Well CGCG 316 530 GAGA 316 530 GGGG 316 530 CACA 316 530 CGCG 316 530 GAGA 316 530 GGGG 316 530 CACA 316 530 GGGG Three Functional Alleles Mixture of Two Functional Alleles Effect -.22 .07 0.09 .06 -.10 .34 Freq..193.527.268.011 GPE Cycle VII-2 557 hd

14. A Challenge and … B×H ToughTender G|GG|G G|GG|G 316 530 Heterozygous for QTL, but homozygous at test loci. Red vs blue alleles in progeny are determined by flanking microsatellites

15. … an Opportunity B×H ToughTender G|G|TG|G|T G|G|CG|G|C 316 530 This situation suggests rather strongly that the G-G haplotype is actually associated with a mixture of functional alleles. Provides opportunity to make the test more powerful by adding SNP SNP X?

16. Why Did We Discover this Opportunity to Improve the Test? Because we looked Because we had an appropriate population in which to find it

17. How Should You Use the  -Calpain Tests? To augment other selection criteria Certainly not as an all-or-none criteria We are confident that 316-C (*) is associated with a tender form of  -calpain and that 530-A is associated with a tough form of  -calpain. However, it appears that 316-G (no-*) and 530-G could be associated with either tough or tender alleles. Therefore, I would give preference to animals that are 316-C (*), but would not cull animals that are otherwise desirable, but are 316-G (no-*).

18. Use of  -calpain in Bos indicus-influenced cattle Less data to support the association Effect of SNP 316 seems consistent with Bos taurus, but SNP 530 is not so consistent Our recommendation is to use only the results for SNP 316 in Bos indicus-influenced cattle This is the SNP that GS offers and the one that FBS recommends for Brahman-influenced animals. We hope to have additional SNP to augment the use of the test in Brahman-influenced animals

19. We Are Learning a Great Deal From the  -Calpain DNA Test Multiple SNP present some challenges. The test works very well as it is. I am confident that it will work even better when we add a few more SNP to it.

20. Conclusions DNA testing is not as simple as it first appears. DNA tests should be viewed as very fluid systems. The potential benefits are enormous. It will eventually become widespread in cattle breeding. It is important that producers have realistic expectations