Genetic Markers for Milk Production Raluca Mateescu, OklahomaStateUniversity Michael Thonney, Cornell University
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)
35,000 US Sheep Milk Cheese Imports Import Quantity (tonnes) 2006 33 million kg 30,000 25,000 20,000 15,000 10,000000 125 150 dairy sheep farmers ~ 4 mil. kg sheep milk ~ 800,000000 kg cheese Need ~ 40x more sheep cheese (Thomas) 1982 1985 1988 1991 1994 1997 2000 2003 2006 FAO, 2006
Benefits of Sheep Milk Human Cow Sheep Goat Solids (%) 12.5 12.01 19.3 12.97 Fat (%) 4.38 3.34 7.0 4.14 Protein 1.03 3.29 5.98 3.56 Sheep milk is also more nutrient dense than cow milk More digestible than cow milk Higher cheese yield (18 25%)
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
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
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
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 10 25 cm apart across the genome Experimental crosses vs. commercial populations p
Approach 1. Candidate gene approach: Association between specific gene and the trait Prolactin Beta lactoglobulin l li Kappa casein
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
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
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
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
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
Genotype determination: PRL Allele A 8% polyacrylamide gel 360 150 530 1400 1400 bp BB AB AA Allele B 360 150 510 20 1400 530 bp 510 bp 360 bp
Genotyping - BLG PCR amplification of 120 bp fragment (Dario et al., 2007) Digestion with RsaI (GT/AC) 1 2 3 4 5 6 7 RsaI RsaI Forward primer 2 Reverse primer
Genotype determination: BLG Allele A 8% polyacrylamide gel 17 66 BB AA AA AB 37 Allele B 103 bp 66 bp 37 bp 17 103
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
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 )
Lactation curves / day) Milk yield (g 2000 1800 1600 1400 1200 1000 800 600 400 200 Lactation 1 Lactation 2 Lactation >2 1 3 5 7 9 11 13 15 17 19 21 23 25 Days in milk (10 days intervals)
Monthly average production 1600 1400 Milk yi ield (g / day) 1200 1000 800 600 400 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Month
Prolactin Frequencies Gene frequency Genotypic frequency 0.13 087 0.87 001 0.01 0.23 0.76
Pairwise comparison of LSM Prolactin Milk Yield (g milk / day) Comparison Constant SE p value Adj. p value AA AB 35.15 139.69 0.80 0.96 AA BB 75.47 137.23 0.58 0.85 AB BB 110.62 36.92 0.002 0.008
Beta-Lactoglobulin Frequencies Gene frequency Genotypic frequency 069 0.69 0.31 0.43 0.52 0.05
Pairwise comparison of LSM Beta Lactoglobulin Milk Yield (g milk / day) Comparison Constant SE p value Adj. p value AA AB 34.91 31.3939 0.26 0.51 AA BB 26.79 71.45 0.71 0.92 AB BB 8.12 71.38 0.91 0.99
Kappa Casein frequencies Gene frequency Genotypic frequency 0.52 0.48 0.11 0.81 0.08 C allele T allele CC CT TT
Pairwise comparison of LSM Kappa Casein Milk Yield (g milk / day) Comparison Constant SE p value Adj. p value CC CT 11.73 48.85 0.81 0.97 CC TT 31.85 68.61 0.64 0.88 CT TT 43.58 54.94 0.43 0.71
Conclusions candidate gene approach Ewes with one A prolactin allele produce 110.6 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
Whole genome scan Approach 2. Experimental backcross pedigree Set of markers Interval mapping
Resource population Cornell Sheep Farm Dorset Non dairy sheep Selected for aseasonality and prolificacy OCSC East Friesian Dairy sheep Strong seasonal breeding
Breeding Design Dorset X East Friesian 72 F1 86 F1 ~ 140 BC ~ 100 BC Aseasonality Milk yield
East Friesian BC pedigree
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
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
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: 0 0.89) Sheep linkage map version 47 4.7 Genotyping GeneSeek Inc. 188 animals: 37 Dorset, 15 E. Friesian, 44 F1, 92 BC to E. Friesian
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.
Lactation curves 1 st lactation day) Milk yie eld (kg / 3 25 2.5 2 1.5 1 0.5 0 Lactation1 F1 Lactation1 BC 1 21 41 61 81 101 121 141 161 181 201 221 241 Days in milk (10 days intervals)
Lactation curves 2 nd lactation day) Milk yie eld (kg / 3 25 2.5 2 1.5 1 0.5 0 Lactation2 F1 Lactation2 BC 1 21 41 61 81 101 121 141 161 181 201 221 241 Days in milk (10 days intervals)
Lactation curves >2 lactation day) Milk yie eld (kg / 3 25 2.5 2 1.5 1 0.5 0 Lactation >2 F1 Lactation>2 BC 1 21 41 61 81 101 121 141 161 181 201 221 241 Days in milk (10 days intervals)
Summary statistics (BC ewes) Milk Milk Milk Peak Peak 50d 100d 250d DIM Yield (kg) (kg) (kg) (d) (kg) Min 26.34 50.5757 66.47 469 4.69 068 0.68 Max 154.28 280.69 549.29 79.07 336 3.36 Mean 70.73 143.01 265.46 38.88 1.66 StDev 26.08 47.42 91.58 18.21 0.55
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 +79.7.77 Min 371.1.52 Max +631.9.90 Lactation h 2 1 0.33 2 0.39 > 2 0.32 (92 backcrosses to EF) (entire OCSC population)
QTLs for milk production traits 5 chromosomes: OAR 2, 12, 18, 20, and 24 Trait OAR Pos (cm) F a MY50 (kg) 2 251.4 7.7878 26.98 (9.67) EBV (g) 12 59.4 5.12 172.87 (75.39) MY100 (kg) 18 56.1 5.02 33.83 (15.10) PMY (kg) 18 56.1 6.95 0.46 (0.17) EBV (g) 20 64.1 5.56 266.97 (113.20) EBV (g) 24 0 11.73 223.94 (65.39)
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) 2 251.4 7.78 78 26.98 (9.67) Prolactin EBV (g) 12 59.4 5.12 172.87 (75.39) QTL for fat % (Gutierrez Gil, 2009) MY100 (kg) 18 56.1 5.02 33.83 (15.10) QTL for MY, FY, PY (Barillet, 2005) PMY (kg) 18 56.1 6.95 0.46 (0.17) EBV (g) 20 64.1 5.56 266.97 (113.20) EBV (g) 24 0 11.73 223.94 (65.39)
Chromosome 24 F test t 14 12 10 8 6 4 2 0 EBV Peak Yield Milk50 Milk100 1% Milk250 5% 0 5 10 15 20 25 30 35 40 45 50 55 60 cm
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
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 # 2005-35205-17680 Erin Rogers OAES Hatch Project 2627 Connie Underwood NYAES Hatch Project 470
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