1. Effectiveness of SNPs genotyping assay as a tool for genetic traceability of cattle production chain
Anna Pozzi
36° International Society for Animal Genetics Conference
16th – 21st July 2017
Animal Forensic Genetics Workshop

2. FOOD SAFETY & CONSUMERSEU
greater control over processing and distribution channels through TRACEABILITY
FOOD SAFETY & CONSUMERS
1. ingredients and finished products
2.ACCURATE/ SAFE ANIMAL IDENTIFICATION
3. Genetic traceability
as a way to further enhance conventional traceability
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

3. Spallanzani Institute
Rivolta d’Adda (CR)
Since 1996 involved in Official Semen Control
1997 start in Italy “IDENTITY TEST” (IT)
To verify correct identity of bulls producing frozen semen
for Artificial Insemination Centre (AI)
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

4. Genetic traceability experience
From Services…
biological reference
semen sample
Genetic profile of DNA extracted from sample batch
Genetic profile of DNA extracted from biological reference (blood or semen)
AI centres send when young broodstock begin to produce
Identity test OK
Identity test NO
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

5. Genetic traceability experience
…to research
Genetic traceability in typical Italian products (PDO) chain
(Salame VARZI)
TRACCIA Project
Development of small fingerprinting protocol by SNPS for traceability in food supply chain
RAIZ Project
development of a RealTime PCR assay for Myostatin point mutation in Piedmontese breed
COGECA
genetic traceability to support the conventional paper-based traceability in beef production chain
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

6. Genetic traceability evolution
From Microsatellites… to SNPs
gacctaatcacacacataccata
gacctaatcacacacacacacacacacacacacacatacca
gacctaatcacacacacacacacacataccata
International standard for parental verification, genetic id, traceability
+ high degree of polymorphism
- higher cost
- error rate
- turnaround time
- not comparable
+ abundance
+ cost efficiency
+ potential for automation
- lower informativity due bi allelic nature
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

7. Constructed new 32 SNPs panel MAF 0.5
Previous study
11 microsatellite markers
StockMarks for Cattle Bovine Genotyping Kit
128 SNPs
88 ISAG Bovine Comp. Test
40 selected from NCBI database
353 bulls, 15 breeds
Constructed
new
32 SNPs panel
MAF 0.5
Allele frequencies for SNPs and STRs: direct counting .
Genetic Variability PIC, He, Ho, Fis
PI , MAF
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

8. Previous study Informativeness of the 32 SNPs panel at the breed level
Cattle breed Ho He
Fis PI
MAF
n (MAF<0.3)
Belgian Blue
0.40
0.42
0.04
1.02 E-12
0,357 9
Italian Brown
0.41
0.03
1.29 E-12
0,342 10
Castana
0.45
0.43
-0.03
4.01 E-13
0,370 8
Chianina
0.39
0.00
5.32 E-12
0,315 11
Italian Holstein
7.13 E-13
0,345
Grigio Alpina
0.44
1.35 E-13
0,396 4
Limousin
0.46
0.06
9.24 E-14
0,408 1
Marchigiana
-0.06
1.20 E-12
0,341
Italian Red Pied
0.48
-0.04
7.80 E-14
0,410
Piedmontese
9.81 E-14
0,420
Pinzgau
-0.09
3.65 E-13
0,377 7
Reggiana
1.15 E-13
0,403 3
Rendena
2.95 E-13
0,372
Romagnola
3.06 E-12
0,336 12
Aosta Red Pied
0,339
All samples (353)
0.09
2.53 E-14
0,475
PI (11 MS)= 1.95 X
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

9. Goals
Verify the amount information of 32 SNPs finger printing protocol applied in the food supply chain
Test 32 SNPs fingerprinting protocol on different matrices
Milk instead of Blood
Protocol for DNA extraction from milk 1° 2°
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

10. 32 SNPs Methods & Materials 140 samples 50 semen batches
Traceability on food supply chain on several matrices
140 samples
50 semen batches
30 meat samples
30 hair samples
30 MILK SAMPLES
OPEN ARRAY platform
SNP Genotyping sw
TaqMan Genotyper 1.0 sw
DNA extraction
Bio Sprint 96 DNA Blood Kit- specific modifications
Mag Attract technology
developed protocol for milk
32 SNPs
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

11. Methods & Materials 32 SNPs panel Animal Forensic Genetics Workshop
SNP ID
Chromosome
RefSNP
Allele 1
Allele 2
ISAG_1.3 1 rs A C
ISAG_1.6 rs T
ISAG_3.2 3 rs
ISAG_3.5 rs G
ISAG_4.2 4 rs
ISAG_5.4 5
DQ500958
ISAG_6.2 6 rs
ISAG_7.1 7 rs
ISAG_8.2 8 rs
ISAG_8.3 rs
ISILS_8.1 rs
ISILS_9.5 9 rs
ISAG_10.4 10 rs
ISAG_10.5 rs
ISAG_12.1 12 rs
ISILS_13.2 13 rs
ISAG_14.4 14 rs
ISAG_15.1 15
DQ866817
ISAG_15.3
EF042090
ISILS_16.6 16 rs
ISAG_17.2 17
AY860426
ISAG_18.4 18 rs
ISAG_20.2 20
DQ888313
ISILS_20.5 rs
ISAG_21.1 21
DQ995976
ISILS_23.1 23 rs
ISILS_23.4 rs
ISAG_24.3 24 rs
ISAG_27.3 27 rs
ISAG_28.4 28 rs
ISAG_29.2 29
DQ404153
ISILS_29.2 rs
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

12. Viability of 32 SNPS panel
Results
Informativeness 32 SNPs
SNP ID Ho He FI
PE(1)
PE(2) PI
ISAG_1.3
0.46
0.50
0.08
1,87E-1
1,88E0
3,75E-1
ISAG_1.6
0.52
-0.04
1.87E-1
1.87E0
3.75E-1
ISAG_10.4
0.60
-0.20
ISAG_10.5
0.55
-0.09
ISAG_12.1
0.43
0.14
1.88E0
3.76E-1
ISAG_14.4
0.07
ISAG_15.1
0.51
-0.02
ISAG_15_3
0.48
0.04
ISAG_17.2
0.39
0.47
0.17
1.80E-1
3.91E-1
ISAG_18.4
0.49
0.03
1.86E-1
3.77-E1
ISAG_20.2
3.77E-1
ISAG_21.1
0.01
1.85E-1
3.78E-1
ISAG_24.3
0.06
3.79E-1
ISAG_27.3
0.13
ISAG_28.4
-0.01
1.84E-1
3.81E-1
ISAG_29.2
0.41
0.16
ISAG_3.2
-0.05
ISAG_3.5
0.54
-0.07
ISAG_4.2
-0.13
1.82E-1
3.86E-1
ISAG_5.4
0.22
ISAG_6.2
0.44
3.89E-1
ISAG_7.1
ISAG_8.2
ISAG_8.3
0.09
3.82E-1
ISILS_13.2
ISILS_16.6
0.53
-0.06
ISILS_20.5
0.38
0.23
ISILS_23.1
ISILS_23.4
-0.03
ISILS_29.2
ISILS_8.1
-0.12
3.90E-1
ISILS_9.5
All
8.40E-14
Results about 1st goal
Viability of 32 SNPS panel
PI value confirms:
the results obtained in our previous work
data found in bibliography
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

13. Blood Milk Methods & Materials Sampling No veterinary Easy sampling
No stress
Easy sampling No
veterinary
stress
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

14. Milk : alternative DNA source
Methods & Materials
Milk : alternative DNA source
Several protocol for DNA isolation published: showed different difficulties
Milk
Blood
Quantity /quality for different applications
Standardised approaches
Small Volumes
Low DNA quantity/quality not for large scale genotyping
Large volumes
are necessary
Develop an innovative protocol
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

15. Methods & Materials Milk protocol
30 milk samples from Italian Holstein
Somatic cells count
(ranging from 16 x 103 to 97 x 104 )
3 groups
SCC < cells/mL
SCC < cells/mL
SCC > cells/mL
Additive
Sodium Azide
No Additive
1 day
3 days
5 days
Storage condition:
ambient, refrigerated and frozen
quantity and quality DNA were investigated
samples refrigerated and frozen: no significative differences
we excluded ambient conservation
(Results not presented here)
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

16. 32 SNPs panel Conclusion can be a in addition
valid fingerprinting tool for …
32 SNPs panel
breeding plans
disease monitoring
pedigree analysis
Agri-Food traceability
in addition
can be an important tool
Animal
Milk
Meat
Tannery
Reproduction
not only as a control ( to avoid fraud) but especially for :
Producers, to implement the value of their products
Consumers, to guarantee the real product origin, expecially for products as PDO and PGI, of which Italy is rich.
in the agri-food supply chain (milk, meat, tannery, reproduction)..
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop

17. THANK YOU ! anna.pozzi@istitutospallanzani.it Molecular Genetic Lab
Graziella Bongioni – lab head
Anna Pozzi
Cristina Previtali
Rossana Capoferri
Caterina Cambuli
Sabina Arabi
Silvia Cenadelli
Dublin, 17 July 2017
A. Pozzi
Animal Forensic Genetics Workshop