Conservation genomic analyses of two Croatian autochthonous sheep breeds

Špehar M.1, Ferenčaković M.2, Curik I.2, Karapandža N.1, Barać Z.1, Sinković T. 1, Cubric-Curik V. 2

1Ministry of Agriculture, Zagreb, Croatia 2University of Zagreb, Faculty of Agriculture, Zagreb, Croatia About breeds – Istrian sheep (IS)

• Breeding began in 1771 • Native ewes x and Bergamo rams? • Southdown and Merinolandschaf rams • Breeding population – 1082 ewes, 86 rams • Dairy breed (lactation milk yield = 171 kg)

About breeds – Pag sheep (PS)

• Breeding began between 1805 and 1813 • Native ewes x merino Negretti, Southdown and Gentile di Puglia rams? • Sardinian White rams • Breeding population – 4292 ewes, 123 rams • Dairy breed (lactation milk yield = 137 kg)

Objective

• To define • Genomic position of IS and PS concerning Italian, Spanish, and some Merino and Merino-derived sheep breeds • To estimate • Linkage disequilibrium effective population size (NeLD)

• Genomic inbreeding levels (FROH) Material (animals) • 660 animals of 24 breeds • Italian, Spanish, and some Merino sheep breeds (15 – 20 animals per breed) • Available data from Dryad digital repository – Ciani et al., 2015 • 96 Istrian and 75 Pag sheep

Italian Spanish Merino • • Castellana • Arawapa • • Churra • Australian Merino • Gentile di Puglia • Ojalada • Australian Poll Merino • Laticauda • Rasa Aragonesa • Australian Industry Merino • • Chinese Merino • Croatian • Merinolandschaf • Massese • Istrian sheep • Rambouillet • Sardinian White • Pag sheep • Spanish Merino (Estremadura) • • Spanish Merino (Cordoba) Method • Illumina OvineSNP50 BeadChip • Quality control • Only autosomal SNPs with known position • GC score ≤ 0.7 • GenTrain score ≤ 0.4 • Gen Call > 0.9 • Animals with < 5% missing SNPs • 43,937 SNPs

• ROH detection and inbreeding levels estimate – Ferenčaković et al., 2013

• Min. 15 homozygous SNP in row • ROH > 2Mb F ≈ 25 gen ago ROH > 2Mb

• Min. density 1 SNP every 120kb • ROH > 4Mb FROH > 4Mb ≈ 12.5 gen ago

• Max. gap between 2 SNPs ≤ 1000kb • ROH > 8Mb FROH > 8Mb ≈ 6 gen ago

Method (cont)

• Principal Component Analysis

• Effective population size

• MAF < 0.05 • Barbato et al., 2015

NeEstimator (v2) • Do et al., 2014 Results Principal component analysis

0,05

Pag Sheep Gentile di Puglia

2

A 0,00

C

P Istrian Sheep Italian Z Appeninica Commisana Laticauda Leccesse * Massese x Sardinian White -0,05 -0,15 -0,10 -0,05 0,00 0,05 0,10 PCA 1 Effective population size IS PS Fit Plot for Ne Fit Plot for Ne 1000 2000

800 1500

Observations 13 Observations 13 Parameters 2 Parameters 2

Error DF 11 Error DF 11

e e

600 N N MSE 149.57 MSE 1336.8 R-Square 0.9973 R-Square 0.9939 1000 Adj R-Square 0.997 Adj R-Square 0.9934

400 푁푒푥 = 124 + 8.4*X 푁푒푥 = 173 + 16.7*X 500 푁푒0 = 124 푁푒0 = 173 200

20 40 60 80 100 20 40 60 80 100 GenAgo GenAgo

Fit 95% Confidence Limits 95% Prediction Limits Fit 95% Confidence Limits 95% Prediction Limits NeEstimator 101 (95% CI = 81 - 130) 222 (95% CI = 161 - 343)

Inbreeding levels

0.046 0.045 0.026 0.051 0.022 0.059 0.072 0.064 0.059 0.041 0.339 0,40

2

> 0,30

H

O 0,20

R

F 0,10 0,00 0.015 0.025 0.001 0.020 0.017 0.015 0.030 0.028 0.032 0.015 0.100

8

- 0,10

2

H

O 0,05

R

F

0,00 0.031 0.020 0.018 0.030 0.004 0.043 0.042 0.039 0.027 0.026 0.239 0,30

8

>

H 0,20

O

R

F 0,10

0,00 a o a a a a p e f p a c r li s a i in n r n g e h e b in r lla u a u e se c e o n e h is h s s h rd M te C P S d S o n l s m e a n e l a o il n M a g C p o C t ia l a s p P C n tr o P o A e s in n n G I r ri lia e e a M tr M s h u is A n a p S

Breed Conclusions

• The first genomic analysis of IS and PS • IS • Remote from the cluster of Mediterranean autochthonous breeds • Effective population size should be monitored • Higher inbreeding levels compared to PS and other breeds • PS • Closer to some Italian breeds • Effective population size is satisfied • Inbreeding was small compared to other breeds • Guidelines for the breeding program improvement

Acknowledgement • Rural Development Programme of the Republic of Croatia for the Period 2014 - 2020 • Measure 10.2. "Support for Conservation, Sustainable Use and Development of Genetic Resources in Agriculture"

• Support by the Croatian Science Foundation Anagrams 01.12. 2018 – 30.11.2022 (HRZZ-IP-2018-01-8708) “Application of NGS in assessment of genomic variability in ruminants”

Thank you for the attention!