Genetic MarkersGenetic MarkersGenetic MarkersGenetic MarkersGenetic Markers Genetic Markers f Milk P d if Milk P d iGenetic Markers Genetic Markers 

f Milk P d if Milk P d ifor Milk Productionfor Milk Productionfor Milk Productionfor Milk Production

RalucaRaluca MateescuMateescu Oklahoma State UniversityOklahoma State UniversityRaluca Raluca MateescuMateescu, , Oklahoma State UniversityOklahoma State UniversityMichael Michael ThonneyThonney, , Cornell UniversityCornell University

Milk production & Sheep IndustryMilk production & Sheep Industry

Economically important trait 2007: $2 9 million for milk production (Shifl tt 2008) 2007: $2.9 million for milk production (Shiflett, 2008)

Increased demand: Increased demand: Sheep milk cheese

Decreased supply 23% decrease over 10 years 23% decrease over 10 years 

(USDA, 2009)

US Sheep Milk Cheese ImportsUS Sheep Milk Cheese Imports20062006

35,000

Import Quantity (tonnes)

2006200633 million kg33 million kg

30,000

20,000

25,000 125 ‐150 dairy sheep farmers ~ 4 mil. kg sheep milk ~ 800 000 k h

10 000

15,000

~ 800,000 kg cheese Need ~ 40x more sheep cheese   (Thomas)

10,000

1982 1985 1988 1991 1994 1997 2000 2003 2006

FAO, 2006

Benefits of Sheep MilkBenefits of Sheep Milk

Human Cow Sheep GoatSolids (%) 12.5 12.01 19.3 12.97Solids (%)Fat (%) 4.38 3.34 7.0 4.14

Protein 1.03 3.29 5.98 3.56Protein 1.03 3.29 5.98 3.56

Sheep milk is also more nutrient dense than cow milk

M di tibl th ilkMore digestible than cow milk

Higher cheese yield (18‐25%)Higher cheese yield (18 25%)

Milk production Milk production –– quantitative traitquantitative trait

Many genes  Difficult to see the effect of one gene Difficult to see the effect of one gene

Environment (frequency of milking, hi i i i )geographic region, nutrition)

Need to separate the genotype from the environment

Continuous phenotypicp yprange (250‐day milk yield)

Old Chatham Sheepherding Company

TraditionalTraditional SelectionSelection

Phenotypic evaluation

E h t h l t it Ewe has to reach sexual maturity

Sex limited trait (rams evaluated through daughters)

Repeated measurements (250‐day milk yield) Repeated measurements (250‐day milk yield)

Selection schemes would benefit from Detection of genes influencing milk production Implementation of MAS Implementation of MAS

ObjectiveObjective

Find molecular markers linked to milk productionproduction

Goal: marker assisted selection

Increase accuracy of selection

Reduce generation interval

Increase the rate of genetic progressgenetic progress

Quantitative Trait Locus (Quantitative Trait Locus (QTLQTL))

QTL = region of genome with one or more genes affecting a quantitative traitg g q

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 populationsp p p

Approach 1.Approach 1.

Candidate gene approach: Association between specific gene and the trait Association between specific gene and the trait

ProlactinProlactin

B l l b liBeta‐lactoglobulin

Kappa‐casein

ProlactinProlactin

Peptide hormone ‐ stimulates mammary growth & milk secretiongrowth & milk secretion

Endogenous rhythm of secretion modulated by photoperiod (highest and lowest conc. during summer and winter,lowest conc. during summer and winter, respectively.

Prolactin hormone shown to stimulate milk production in cattle

BetaBeta--lactoglobulinlactoglobulin

Major whey protein in milk

G hi hl & ifi ll d iGene highly & specifically expressed in bovine mammary gland during lactation

Major regulator: prolactin

Association found between beta‐lactoglobulin variants and milk protein g pyield/composition

CaseinsCaseins

αs1‐, αs2‐, β‐, Κ‐casein

Kappa‐casein shown to improve milk yield in cattlein cattle

Polymorphism discovered in kappa‐casein y p ppgene in sheep

i i i h kiKappa‐casein important in cheese making

PopulationPopulation

Old Chatham Sheepherding Company

676 East Friesian ewes

Ewes milked twice daily individual milkEwes milked twice daily, individual milk yields recorded once monthly.

Milk production records (1/1/97 –records (1/1/97 7/31/07)  + pedigree informationinformation Old Chatham Sheepherding Company

Genotyping Genotyping -- PRLPRL

DNA extracted from blood

PCR lifi i f 2 5 KbPCR amplification of a 2.5 Kb fragment (Vincent & Rothschild, 1997)

Digested with HaeIII (GG/CC)

2500 bp

HaeIII HaeIII HaeIII

8620 bp

HaeIII

Reverse primerForward primer

HaeIII HaeIII HaeIIIHaeIII

p

Genotype determination: PRLGenotype determination: PRL

8% polyacrylamide gelAllele A

1400 bp360 150

530 1400BB AB AA

530 bp530 bp510 bp

360 bp360 150

Allele B

p

510 20 1400

Genotyping Genotyping -- BLGBLG

PCR amplification of 120 bp fragment (Dario et al., 2007)

Digestion with RsaI (GT/AC)

1 2 53 4 76

R I R IRsaI RsaI2

Reverse primerForward primer

Genotype determination: BLGGenotype determination: BLG

8% polyacrylamide gelAllele A

17 66

37

BB ABAAAA

37

103 bp

66 bp

37 bp

17

Allele B

103

Genotyping Genotyping –– CSN3CSN3

DNA extracted from blood

PCR lifi i f 87/97 bPCR amplification of a 87/97 bpfragment (Feligini, 2005)

CC CT CT TT

Forward primer

87 bp TCC CT CT TT

Forward primer

97 bp C

Statistical Analysis

Calculate gene and genotypic frequencies

E ti t i ti f hEstimate association 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 eweYRMO d th GENE l fi d ff tYRMO = year and month; GENE = gene polym. fixed effect DIM(LACT) = days in milk within lactation random effectA = animal random effects; E = random residuals

Differences between LSM (Tukey‐Kramer multiple comparison adjustment )multiple comparison adjustment )

Lactation curves

18002000

Lactation 1L i 2

120014001600

/ day

) Lactation 2Lactation >2

80010001200

yiel

d (g

200400600

Milk

y

2001 3 5 7 9 11 13 15 17 19 21 23 25

Days in milk (10 days intervals)Days in milk (10 days intervals)

Monthly average production

1400

1600

1200

1400

/ day

)

800

1000

ield

(g /

600

800

Milk

yi

400Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Month

ProlactinProlactin FrequenciesFrequencies

Genotypic frequency Genotypic frequency Gene frequency Gene frequency

0 87 0.76

0.13

0.87

0 010.23

0.01

Pairwise comparison of LSM

ProlactinMilk Yi ld ( ilk / d )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

BetaBeta--LactoglobulinLactoglobulin FrequenciesFrequencies

Genotypic frequency Genotypic frequency Gene frequency Gene frequency Genotypic frequency Genotypic frequency Gene frequency Gene frequency

0 690.69

0.31

0.430.52

0.05

Pairwise comparison of LSM

Beta‐LactoglobulinMilk Yi ld ( ilk / d )Milk Yield (g milk / day)

Comparison Constant SE p‐value Adj. p‐value

AA ‐ AB ‐34 91 31 39 0.26 0.51AA AB 34.91 31.39 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

0.520.52 0.480.480.080.08

0.810.810.110.11

C allele T allele CC CT TT

Pairwise comparison of LSM

Kappa‐CaseinMilk Yi ld ( ilk / d )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 A110.6 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 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 yieldsignificant effect on milk yield

Approach 2.Approach 2.

Whole genome scan

Experimental backcross pedigree

Set of markers

Interval mapping

Resource population

Cornell Sheep Farm OCSC Dorset Non‐dairy sheep

OCSC East Friesian  Dairy sheep

Selected for aseasonality and 

lifi

Strong seasonal breeding

prolificacy

Breeding Design

Dorset♀

East Friesian♂X♀ ♂

86 F172 F1 86 F1♀

72 F1 ♂

~ 140 BC ~ 100 BC

Milk yieldAseasonality

East Friesian BC pedigree

Phenotypes

Ewes milked twice daily, individual milk i ld d d hl (OCSC)yields recorded once monthly (OCSC)

Milking records and pedigree informationMilking records and pedigree information through the end of July 2007

Genetic evaluation => EBV

Lactation curves fitted to each BC ewe Lactation curves fitted to each BC ewe

Phenotypes

EBV (estimated breeding value)

PMY – peak milk yield

0 l i ilk i ld 0 dMY50 – cumulative milk yield to 50 days

MY100 – cumulative milk yield to 100 daysMY100  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 26 autosomes; average marker interval ~ 29cM

Average # alleles = 7.03 (range: 1 ‐ 21)

Average PIC = 0.63 (range: 0 ‐ 0.89)

Sh li k i 4 7Sheep linkage map version 4.7

Genotyping – GeneSeek Inc.Genotyping  GeneSeek Inc.

188 animals: 37 Dorset, 15 E. Friesian, 44 F1, 92 BC to E FriesianBC to E. Friesian

QTL mapping

GridQTL – regression‐based, interval i (H l d K )mapping (Haley and Knott)

F‐test = RSS (full model) / RSS (reducedF test = RSS (full model) / RSS (reduced model)

Permutation tests (10,000 iterations) 

Chromosome wide significance levelsChromosome‐wide significance levels. 

Lactation curves – 1st lactation

2 5

3 Lactation1 F1

2

2.5

day)

Lactation1 BC

1

1.5

eld (kg / 

0.5

1

Milk yie

01 21 41 61 81 101 121 141 161 181 201 221 241

Days in milk (10 days intervals)Days in milk (10 days intervals)

Lactation curves – 2nd lactation

2 5

3Lactation2 F1

2

2.5

day)

Lactation2 BC

1

1.5

eld (kg / 

0.5

1

Milk yie

01 21 41 61 81 101 121 141 161 181 201 221 241

Days in milk (10 days intervals)Days in milk (10 days intervals)

Lactation curves – >2 lactation

2 5

3 Lactation >2 F1

2

2.5

day)

Lactation>2 BC

1

1.5

eld (kg / 

0.5

1

Milk yie

01 21 41 61 81 101 121 141 161 181 201 221 241

Days in milk (10 days intervals)Days in milk (10 days intervals)

Summary statistics (BC ewes)

Milk  50d 

Milk 100d 

Milk 250d 

Peak DIM 

Peak Yield 

(kg) (kg) (kg) (d) (kg)

Mi 26 34 50 57 66 47 4 69 0 68Min 26.34 50.57 66.47 4.69 0.68

Max 154 28 280 69 549 29 79 07 3 36Max 154.28 280.69 549.29 79.07 3.36

Mean 70.73 143.01 265.46 38.88 1.66Mean 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)

Lactation h2EBV Acc

1 0.33

2 0.39

Mean +79.7 .77

Min ‐371.1 .52

> 2 0.32Max +631.9 .90(entire OCSC population)(92 backcrosses to EF) (entire OCSC population)(92 backcrosses to EF)

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 78 26 98 (9 67)MY50 (kg) 2 251.4 7.78 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)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

MY50 (kg) 2 251 4 7 78 26 98 (9 67)OAR 20 (close to position 20cM):P l iMY50 (kg) 2 251.4 7.78 26.98 (9.67)

EBV (g) 12 59.4 5.12 ‐172.87 (75.39)‐ Prolactin‐ QTL for fat % (Gutierrez‐Gil, 2009)

MY100 (kg) 18 56.1 5.02 ‐33.83 (15.10)

PMY (kg) 18 56.1 6.95 ‐0.46 (0.17)‐ QTL for MY, FY, PY (Barillet, 2005)

EBV (g) 20 64.1 5.56 266.97 (113.20)

EBV (g) 24 0 11 73 223 94 (65 39)EBV (g) 24 0 11.73 223.94 (65.39)

Chromosome 24

12

14EBV

P k Yi ld

8

10

t

Peak Yield

Milk50

Milk1001%

6

8

F te

st Milk100

Milk250 5%

2

4

00 5 10 15 20 25 30 35 40 45 50 55 60

cMcM

Conclusions

Dairy sheep industry – growing

High demand for sheep milk cheeses –growth opportunitygrowth opportunity

Need selection programs, MAS could play an important role

Prolactin gene marker for OCSC popProlactin gene – marker for OCSC pop.

Fine mapping OAR 2, 12, 18, 20, 24pp g , , , ,

Acknowledgements

Department of Animal Science OSU

Old Chatham SheepherdingScience, OSU

Toni OltenacuAndrea Sexten

Sheepherding Company

Ann StaigerKarista HudelsonJ ti B h

Financial supportUSDA CSREES NRI GrantJustin Buchanan

Erin RogersConnie Underwood

USDA-CSREES NRI Grant # 2005-35205-17680

OAES Hatch Project 2627Connie Underwood NYAES Hatch Project 470

QUESTIONSQ