Economically important trait. Increased demand: Decreased supply. Sheep milk cheese. 2007: $2.9 million for milk production (Shiflett, 2008)

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1 Genetic Markers for Milk Production Raluca Mateescu, OklahomaStateUniversity Michael Thonney, Cornell University

2 Milk production & Sheep Industry Economically important trait 2007: $2.9 million for milk production (Shiflett, 2008) Increased demand: Sheep milk cheese Decreased supply 23% decrease over 10 years (USDA, 2009)

3 35,000 US Sheep Milk Cheese Imports Import Quantity (tonnes) million kg 30,000 25,000 20,000 15,000 10, dairy sheep farmers ~ 4 mil. kg sheep milk ~ 800, kg cheese Need ~ 40x more sheep cheese (Thomas) FAO, 2006

4 Benefits of Sheep Milk Human Cow Sheep Goat Solids (%) Fat (%) Protein Sheep milk is also more nutrient dense than cow milk More digestible than cow milk Higher cheese yield (18 25%)

5 Milk production quantitative trait Many genes Difficult to see the effect of one gene Environment (frequency of milking, geographic region, nutrition) ii Need to separate the genotype from the environment Continuous phenotypic range (250 day milk yield) Old Chatham Sheepherding Company

6 Traditional Selection Phenotypic evaluation Ewe has to reach sexual maturity Sex limited trait (rams evaluated through daughters) Repeatedmeasurements (250 day milkyield) Selection schemes would benefit from Detection of genes influencing milk production Implementation of MAS

7 Objective Find molecular markers linked to milk production Goal: marker assisted selection Increase accuracy of selection Reduce generation interval Increase the rate of genetic progress

8 Quantitative Trait Locus (QTL) QTL = region of genome with one or more genes affecting a quantitative trait Detecting QTL for milk production: Candidate gene approach Use a known trait/gene in one species to see if similar in a different species Genome wide scans Markers cm apart across the genome Experimental crosses vs. commercial populations p

9 Approach 1. Candidate gene approach: Association between specific gene and the trait Prolactin Beta lactoglobulin l li Kappa casein

10 Prolactin Peptide hormone stimulates mammary growth & milk secretion Endogenous rhythm of secretion modulated by photoperiod (highest and lowest conc. during summer and winter, respectively. Prolactin hormone shown to stimulate milk production in cattle

11 Beta-lactoglobulin Major whey protein in milk Gene highly hl & specifically expressed in bovine mammary gland during lactation Major regulator: prolactin Association found between betalactoglobulin variants and milk protein yield/composition

12 Caseins αs1, αs2, β, Κ casein Kappa casein shown to improve milk yield in cattle Polymorphism discovered in kappa casein gene in sheep Kappa casein important in cheese making

13 Population Old Chatham Sheepherding Company 676 East Friesian ewes Ewes milked twice daily, individual milk yields recorded once monthly. Milk production records (1/1/97 7/31/07) + pedigree information Old Chatham Sheepherding Company

14 Genotyping -PRL DNA extracted from blood PCR amplification i of a 25Kb 2.5 fragment (Vincent & Rothschild, 1997) Digested with HaeIII (GG/CC) 2500 bp 8620 bp HaeIII HaeIII HaeIII HaeIII Forward primer Reverse primer

15 Genotype determination: PRL Allele A 8% polyacrylamide gel bp BB AB AA Allele B bp 510 bp 360 bp

16 Genotyping - BLG PCR amplification of 120 bp fragment (Dario et al., 2007) Digestion with RsaI (GT/AC) RsaI RsaI Forward primer 2 Reverse primer

17 Genotype determination: BLG Allele A 8% polyacrylamide gel BB AA AA AB 37 Allele B 103 bp 66 bp 37 bp

18 Genotyping CSN3 DNA extracted from blood PCR amplification i of a 87/97 bp fragment (Feligini, 2005) Forward primer 87 bp T CC CT CT TT Forward primer 97 bp C

19 Statistical Analysis Calculate gene and genotypic frequencies Eti Estimate t association iti of each gene polymorphism with milk production: Y = YRMO + GENE + DIM(LACT) + A + E Y = amount of milk in test day sample from an individual ewe YRMO = year and month; GENE = gene polym. fixed effect DIM(LACT) = days in milk within lactation random effect A = animal random effects; E = random residuals Differences between LSM (Tukey Kramer multiple comparison adjustment )

20 Lactation curves / day) Milk yield (g Lactation 1 Lactation 2 Lactation > Days in milk (10 days intervals)

21 Monthly average production Milk yi ield (g / day) Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Month

22 Prolactin Frequencies Gene frequency Genotypic frequency

23 Pairwise comparison of LSM Prolactin Milk Yield (g milk / day) Comparison Constant SE p value Adj. p value AA AB AA BB AB BB

24 Beta-Lactoglobulin Frequencies Gene frequency Genotypic frequency

25 Pairwise comparison of LSM Beta Lactoglobulin Milk Yield (g milk / day) Comparison Constant SE p value Adj. p value AA AB AA BB AB BB

26 Kappa Casein frequencies Gene frequency Genotypic frequency C allele T allele CC CT TT

27 Pairwise comparison of LSM Kappa Casein Milk Yield (g milk / day) Comparison Constant SE p value Adj. p value CC CT CC TT CT TT

28 Conclusions candidate gene approach Ewes with one A prolactin allele produce g more milk per day than ewes no A allele. No difference between AA and AB ewes 110 g/ day ~ 7% of the average TD milk Requires validation before use in other pop. Beta lactoglobulin and kappa casein no significant effect on milk yield

29 Whole genome scan Approach 2. Experimental backcross pedigree Set of markers Interval mapping

30 Resource population Cornell Sheep Farm Dorset Non dairy sheep Selected for aseasonality and prolificacy OCSC East Friesian Dairy sheep Strong seasonal breeding

31 Breeding Design Dorset X East Friesian 72 F1 86 F1 ~ 140 BC ~ 100 BC Aseasonality Milk yield

32 East Friesian BC pedigree

33 Phenotypes Ewes milked twice daily, individual milk yields recorded donce monthly (OCSC) Milking records and pedigree information through the end of July 2007 Genetic evaluation => EBV Lactation curves fitted to each BC ewe

34 Phenotypes EBV (estimated breeding value) PMY peak milk yield MY50 cumulative milk yield to 50 days MY100 cumulative milk yield to 100 days MY250 cumulative milk yield to 250 days

35 Markers and Linkage maps 120 microsatellite markers 26 autosomes; average marker interval ~ 29cM Average # alleles = 7.03 (range: 1 21) Average PIC = 0.63 (range: ) Sheep linkage map version Genotyping GeneSeek Inc. 188 animals: 37 Dorset, 15 E. Friesian, 44 F1, 92 BC to E. Friesian

36 QTL mapping GridQTL regression based, interval mapping (Haley and Knott) F test = RSS (full model) / RSS (reduced model) Permutation tests (10,000 iterations) Chromosome wide significance levels.

37 Lactation curves 1 st lactation day) Milk yie eld (kg / Lactation1 F1 Lactation1 BC Days in milk (10 days intervals)

38 Lactation curves 2 nd lactation day) Milk yie eld (kg / Lactation2 F1 Lactation2 BC Days in milk (10 days intervals)

39 Lactation curves >2 lactation day) Milk yie eld (kg / Lactation >2 F1 Lactation>2 BC Days in milk (10 days intervals)

40 Summary statistics (BC ewes) Milk Milk Milk Peak Peak 50d 100d 250d DIM Yield (kg) (kg) (kg) (d) (kg) Min Max Mean StDev

41 EBVs and heritability 365 rams and 3,219 ewes (Jan 97 Jul 07) Average production: 1,171 g milk / day (entire OCSC population) EBV Acc Mean Min Max Lactation h > (92 backcrosses to EF) (entire OCSC population)

42 QTLs for milk production traits 5 chromosomes: OAR 2, 12, 18, 20, and 24 Trait OAR Pos (cm) F a MY50 (kg) (9.67) EBV (g) (75.39) MY100 (kg) (15.10) PMY (kg) (0.17) EBV (g) (113.20) EBV (g) (65.39)

43 QTLs for milk production traits 5 chromosomes: OAR 2, 12, 18, 20, and 24 Trait OAR Pos (cm) F a OAR 20 (close to position 20cM): MY50 (kg) (9.67) Prolactin EBV (g) (75.39) QTL for fat % (Gutierrez Gil, 2009) MY100 (kg) (15.10) QTL for MY, FY, PY (Barillet, 2005) PMY (kg) (0.17) EBV (g) (113.20) EBV (g) (65.39)

44 Chromosome 24 F test t EBV Peak Yield Milk50 Milk100 1% Milk250 5% cm

45 Conclusions Dairy sheep industry growing High demand for sheep milk cheeses growth opportunity Need selection programs, MAS could play an important role Prolactin gene marker for OCSC pop. Fine mapping OAR 2, 12, 18, 20, 24

46 Acknowledgements Department of Animal Old Chatham Science, OSU Sheepherding Toni Oltenacu Company Andrea Sexten Ann Staiger Karista Hudelson Financial support Justin Buchanan USDA-CSREES NRI Grant # Erin Rogers OAES Hatch Project 2627 Connie Underwood NYAES Hatch Project 470

47 QUESTIONS