SNP Genotyping For Fine mapping Eggshell Quality

Traits in ChickenTerhi Iso-Touru

MTT Agrifood Research Finland

MTT Agrifood Research Finland

• leading research institute in the agriculture, food and environment sectors in Finland• operates in 14 different locations around the country• operates under the Ministry of Agriculture and Forestry• 800 professionals, 300 of whom work in research

SNP Genotyping For Fine mapping Eggshell Quality Traits in ChickenBACKGROUND

• EU project: SABRE (http://www.sabre-eu.eu/)• SABRE = Cutting Edge Genomics for Sustainable Animal Breeding • WP7 product safety

• task 7.1 Fine mapping of QTL in a resource population

• Partners• MTT Agrifood Research Finland• Lohmann Tierzucht GmbH, Germany• The Roslin Institute, Scotland• INRA, France

BACKGROUND

• egg quality traits are poorly studied

• only some QTLs for eggshell quality

• poor eggshell quality→cracked or broken eggs→a route for pathogen contamination→ losses may be up to 10 % of total production

GENOME SCAN WITH MICROSATELLITES

• mapping population (1791 individuals)

• 162 microsatellites, 26 chromosomes

• Parent generation• reciprocal line cross• RIR x White Rock• phenotypes for egg quality

• F1 generation• phenotypes for egg quality• for producing F2 generation

• F2 generation• phenotypes for egg quality

X

F1

F2

P

QTL REGIONS FROM GENOME SCAN

• QTL (Quantitative Trait Loci) analyses were done using line cross model• five interesting QTL regions from five different chromosomes

• 230 genes specifically expressed in the shell gland • 28 are located within the QTL regions found in this study

MCW02130,0LEI02515,0

GGA13

ADL02630,0

MCW012335,0

ADL020072,0

GGA14

ADL02020,0MCW01518,0

ADL029339,0

GGA17

LEI02580,0MCW03121,0

GGA16

ADL02060,0LEI00831,0MCW023127,0

GGA15

HUJ00100,0ADL03043,0MCW021718,0MCW021940,0

GGA18

ADL03760,0ROS03156,0

GGA27

MCW02660,0MCW009412,0

MCW034945,0

GGA19

MCW01190,0ADL03248,0ADL003413,0

GGA20

ROS00730,0SCW025623,0

GGA22

ADL02620,0MCW01651,0

MCW024987,0

GGA23

ROS01130,0

MCW030139,0

GGA24

MCW03550,0MCW028513,0

ADL028548,0

GGA26

ADL03720,0

MCW033271,0

GGA12

LEI01350,0

MCW034735,0

MCW022777,0

GGA28ADL01170,0

MCW033122,0MCW005534,0 MCW025840,0

LEI017168,0 ADL020172,0 MCW024178,0 LEI022983,0 MCW015484,0 MCW024685,0 LEI025490,0 MCW029491,0 ROS001794,0 LEI011197,0 LEI014498,0 LEI012199,0

LEI0075127,0 MCW0269132,0

GGA0Z

FINE MAPPING OF QTL REGIONS

• 1599 F2 hens • from 16 different families• parents included to the fine mapping population→ totally 1791 individuals, three generations

X

F1

F2

SNP GENOTYPING

• BeadXpress Reader• Veracode application: genotyping with Goldengate assay kit • GoldenGate oligo pools (OPAs)• 48-, 96-, 144-, 192-, and 384- SNP plex• one OPA set → 480 individuals (5x96 plates)

• fine mapping in two different laboratories with sameindividuals

• MTT: one OPA, two QTL regions (chromosomes 14 and Z)• The Roslin institute: one OPA, three QTL regions

DESINGING THE OPA

• validated SNPs were available from public databases (www.ncbi.nlm.nih.gov/sites/entrez)

• totally 449 SNPs available for the two QTL regions (chr14; 9Mb and Z; 43Mb)

• OPA was designed with online Assay Design Tool (www.illumina.com/)

DESINGING THE OPA

• Chr 14• 202 SNPs, 181 SNPs passed the quality restriction (SNP score > 0.7)

• Chr Z• 247 SNPs, 203 SNPs passed the quality restriction (SNP score > 0.7)

→Custom OPA for 384 chicken SNPs• 5 OPA sets, totally 2400 individuals

GENOTYPING

• DNA was extracted from the blood samples using salting out procedure (phenol – chloroform)

• 250 ng of DNA needed per individual

• 2 – 3 plates per week• about 1,5 months to get the system work correctly• remote sessions with Illumina’s experts has been useful

• 19 plates genotyped, ~1800 individuals

• genotypes analysed with BeadStudio Genotyping Module • manual checking • remote sessions with Illumina’s experts has been useful

RESULTS

• 331 successfully genotyped SNPs

out of them

• 120 were monomorphic• 33 from chr 14• 87 from chr Z

• hens: ZW, roosters:ZZ

RESULTS

• 53 SNPs failed • multiple clusters (12), heritability errors (10), poor signals (31)

• 5 individuals were in wrong families

RESULTS

• MAF < 0.05• 24 SNPs

• 13 from Chr14• 11 from ChrZ

• In summary: out of 384 SNPs• 211 were in good quality• 120 were monomorphic• 53 failed

ISSUES

• 1 OPA set was from different synthesizing lot• created genotyping problems• can not be clustered with genotypes from other 4 OPA sets

• 1 plate worked poorly

→ 3 different genotyping project• One with genotypes from 4 OPA sets, one with genotypes from 1 OPA set and

one plate separately

• family structure helps genotyping and SNP clustering• replicates are useful

• 3 to 5 per plate• costly

• technical reasons caused some errors• BeadXpress reader stopped during the run

NEXT

• association analyses using different models

• genotype commercial chicken population using the same OPA we have already used and test the marker effects • 500 individuals• DNA extraction from feather sheaths using lysis

• DNA quality• preliminary results show that DNA lysis works

Thank You for Your Attention!