Novel genetic markers affect

rennet-induced milk coagulationrennet-induced milk coagulation

Vivi R. GregersenPostdoc

Molecular Biology and Genetics

Aarhus University, Denmark

12th International Symposium on Milk Genomics and Human Health

Milk Genomics

• Danish-Swedish milk genomics initiative

Milk phenotypes

Genotyping

620,000 SNPs~400 cows/breed

22

m/z200 400 600 800 1000 1200

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

[Abs. Int. * 1000]

a Yb P Y V R Y Ly V I V

185.250a 2

213.255b 2

310.284b 3

451.359y 3

473.328b 4

550.452y 4

572.546b 5

700.467a 6

728.456b 6

810.534y 6

863.598a 7

891.486b 7

923.583y 7

1004.991b 8

1022.621y 8

Cheese making

Modified from “Enzymes for cheese - CHY-MAX® M coagulant”, Chr. Hansen

Curd firming rate (CFR)

Fre

que

ncy 0 ,1 5

0 ,2 0

0 ,2 5

D a n is h H o ls te in

D a n is h J e rs e y

S w e d is h R e d

C F R (P a /m in )

2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 3 8 > 4 0

Fre

que

ncy

0 ,0 0

0 ,0 5

0 ,1 0

Poulsen et al., 2013, J. Dairy Sci. 96: 4830-4842

Milk coagulation

Danish Holstein

Coagulating

Poor coagulation

non-coagulation

Swedish Red

2%

16%

• High frequency of non-coagulating samples in

milk from Swedish Red

• Potentially, a severe problem for cheese

dairies

What causes variation among cows?

Minor environmental effect

Coagulation

Curd firming rate

H2 = 0.75

Poulsen et al., 2015, J. Dairy Sci. 98 :2079–2087

Naturmælk

http://obgyn.mcw.edu/

Large genetic effect

Objective

• Investigate the genetic contribution to the

phenomena non-coagulation

• To identify cause

Materials

Sampling

• Danish Holstein and Swedish Red– Milk and DNA samples

– Approx. 800 cows

– Sampling performed on 20 herds per breed– Sampling performed on 20 herds per breed

– Parity, lactation stage and milk yield in database

Genotyping

• Bovine HD SNP-chip

Methods

Milk rheological analysis

• Curd firming rate (CFR)[ΔG’/Δt]lin

G´= gel strength in PaG´= gel strength in Pa

t = time in minutes

(Poulsen et al., 2013)

Methods

Genome scan and haplotype analysis

• Simple GWAS

• Haplotype analysisAdditive genetic effects model

(Gregersen et al. 2015)

……

. . . …… . . . . . . …… . . .

SNP1

SNP2

SNPN

Allele 1

Allele 2

11

--1

11

--2

12

--1

21

--2

22

--1

22

--2

Leaf node

phase

# # # # # #

Haplotype analysis

• Highly associated SNPs only!

SNP1

SNP2

Allele 1

Allele 2

……

. . . …… . . . . . . …… . . . SNPN

11

--1

11

--2

12

--1

21

--2

22

--1

22

--2

Leaf node

phase

# # # # # #

CSN3, coding for κ-CN

• Genetic variants

Danish Holstein Danish Jersey Swedish Red

κ-CN

Poulsen et al., 2013, J. Dairy Sci. 96: 4830-4842

• Genetic B-variant is related to good coagulation properties

B

BB

A A

E EA

Methods

Genotyping of CSN3 genetic variants

• CSN3 (A,B and E) variants

• Custom TaqMan SNP assays(Poulsen et al. 2013)(Poulsen et al. 2013)

Methods

Sequencing of cows

• 4 cows were selected for (27x) whole

genome sequencing based on haplotype and

CSN3 genetic variantsCSN3 genetic variants

Results

• Milk data trimming

– Excluded samples with high somatic cell count

• Genotypes• Genotypes

– QC filtering

• SNPs (Call rate < 0.95) and individuals (Call freq. < 0.98)

for missingness

• Minor allele frequency (MAF < 0.02)

• Deviation from Hardy-Weinberg equilibrium (< 0.001)

Results

Major locus identified in relation to CSN3

Danish Holstein

Same three SNPs

Swedish Red

5% Bonferroni genome-wide significance

Position, bpPosition, bp

Swedish Red

Results

• SNPs selected for haplotype analysis

– Top three SNPs From GWAS

Non-coding

Located in relation to CSN3

– CSN3 B-variant SNPFrom Bovine HD SNPChip

In complete Linkage disequilibrium with the genetic B-variant

Results

4 haplotypes identified in Danish Holstein• CSN3 B-variant => good CFR

• CSN3 A-variant => good or poor CFR

HaplotypeDanish

Holstein n

Mean CFR

(Pa/min)Frequency Gene variant

hap1 416 7.28 57.46 A and E

hap2 177 10.78 24.45 B

hap3 72 9.46 9.94 A

hap4 59 11.83 8.15 A

Results

Same haplotype pattern identified in Swedish Red!

• Same relation to genetic variants

• Same relation between mean CFR– Difference in mean CFR between breeds!

HaplotypeSwedish

Red n

Mean CFR

(Pa/min)Frequency Gene variant

hap1 400 5.76 60.1 A and E

hap2 108 8.88 16.2 B

hap3 29 8.58 4.4 A

hap4 117 9.86 19.3 A

Results

0,1

0,15

0,2

0,25

SR

DH

• 16% non-coagulation in SR

• 2% non-coagulation in DH

HaplotypeSwedish

Red n

Mean CFR

(Pa/min)Frequency Gene variant

hap1 400 5.76 60.1 A and E

hap2 108 8.88 16.2 B

hap3 29 8.58 4.4 A

hap4 117 9.86 19.3 A

0

0,05

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Results

Confirmation study in milk

• New sampling

Collection of milk from homozygous cows

Milk properties Milk properties Expression studies

Danish Holstein

Milk properties

(AU-FOOD)

MCP

Protein profile (LCMS)

Calcium distribution

Milk properties

(Arla Foods)

Micelle size

Expression studies

(AU-MBG)

Cvr nr Hap A Hap AE Hap B Total

DH1 2 3 2 7

DH2 2 9 6 17

DH3 3 8 7 18

DH4 3 8 6 17

DH5 2 5 5 12

Total 12 33 26 71

Animals carrying only hap 3 and/or hap 4 (A)

Homozygotes of hap1 (AE)

Homozygotes of hap 2 (B)

Protein content

• No significant difference between the

haplotypes

A AE B

4.5

4.0

3.5

3.0Pro

tein

(g

/10

0 g

)

Coagulation properties

30

20

CF

R (

Pa

/min

)

1800

1600

1400

1200

RC

T (

seco

nd

s)

Rennet coagulation time (RCT) is also affected by haplotype

Three out of 33 AE haplotype samples were non-coagulating!

A AE B A AE B

10CF

R (

Pa

/min

)

1000

800

RC

T (

Micelle size

Smaller micelles for good coagulation haplotypes

0,40,50,6

170

(nm

)

00,10,20,30,4

10

0-1

10

11

0-1

20

12

0-1

30

13

0-1

40

14

0-1

50

15

0-1

60

16

0-1

70

17

0-1

80

18

0-1

90

Micelle size (nm)

AE

A

B

A AE B

170

160

150

140

130

120

Mic

ell

esi

ze(n

m)

Micelle size relates to CSN3 expression levels

0.10

CN

/to

tal

pro

tein

A

AE

B

180

170

size

(nm

)A AE B

0.08

0.06

% KK KK

-CN

/to

tal

pro

tein

160

150

140

130

Mic

ell

esi

ze

% κ-CN/total protein

0.05 0.06 0.07 0.08 0.09 0.10 0.11

• Note, this is the quantitative expression level, NOT the exact amount of κ-CN

Confirmation study in milk

• Homozygotes of hap 1 (AE)

• Animals carrying only hap 3 and/or hap 4 (A)

• Homozygotes of hap 2 (B)

HAP AE

(n=33)

HAP A

(n=12)

HAP B

(n=26)

Protein% 3.59a 3.57a 3.45a

CFR (Pa/min) 6.24a 13.17b 11.82b

Micelle size (nm) 165.98a 150.41b 146.86b

a, b show significant difference within same row

Breeding potential

By lowering the frequency of the AE

haplotype, non- and poor coagulation

properties can be partly eliminated.Known variants

properties can be partly eliminated.

A B E

AE B A

0

0,05

0,1

0,15

0,2

0,25

4 6 8 10 12 14 16 18 20 22

CFR (Pa/min)

Today

future

Haplotypes

Conclusion

� Major locus affects curd firming rate

� Haplotype analysis reveals that not only the genetic variants of CSN3 affects CFRgenetic variants of CSN3 affects CFR

� The genetic A-variant of CSN3 is linked to both poor end good coagulation

� Obvious breeding potential

Further studies

• Pool-seq data of extreme phenotypes

CFR Danish Holstein

Bertelsen et al. 2015, J. Anim. Sci. (submitted)

Future Plans

• RNA-seq of mammary gland epithelia cells

from milk samples

� Two samples of each haplotype from four

different farmsdifferent farms

� Investigation:

� Which genes are expressed

� Expression level

� Genes in relation to associations

Acknowledgement

MBG, MGS Arla strategic Innovation Center ANIS, AU

Christian Bendixen Mikka S. Hansen Stig Purup

Lone Bruhn Madsen

Lars-Erik Holm FTEN, Lund University FOOD, AU

Frank Panitz Frida Gustavsson Nina A. Poulsen

Henriette P. Bertelsen Marie Paulsson Lotte B. Larsen

Bo Thomsen Maria GlantzBo Thomsen Maria Glantz

Rasmus O. Nielsen

Mette Jeppesen

Bente F. Majgren

Hanne Jørgensen

Bart Buitenhuis