gldss 2009 mateescu - university of wisconsinansci.wisc.edu/extension-new...
Post on 01-Feb-2018
216 Views
Preview:
TRANSCRIPT
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
top related