The Florida Cracker Sheep (FCS) is one of the oldest sheep breeds in the United States. This heritage breed from Florida, naturally adapted to humid and hot climate conditions, is one of the most parasite resistant breeds from the Southern US. However, approximately 1,000 individuals remain alive in the world. Therefore, more research and conservational efforts are required to support all the FCS producers from Florida and rescue FCS from extinction. Advancements in NGS technologies and reduction in genotyping costs have allowed the utilization of these tools in animal genetics and genomics. We followed up a FCS population (n = 350) from a commercial farm to evaluate parasite resistance traits (FEC, FAMACHA score, hematocrit) using a longitudinal study and genotyped 300 sheep using the GGP Ovine 50k array. Analysis with Golden Helix SVS software identified 15 SNPs with additive and non-additive effects associated with parasite resistance in chromosome 1, 2, 3, 6, 8, 10, 11,12, 13 and 21. Also, a deletion CNV was associated with parasite resistance (FEC) in chromosome 21. Some of these DNA variants were located in STAT5B, NRIPI, TRPM3, WC1, GPC5, CELF2 and RAB3IL genes which control immune response mechanisms in sheep. Validation of these results and implementation of genomic selection utilizing information from NGS, SNP genotyping and WGS can be easily performed by Golden Helix SVS software. This will allow the implementation of breeding and conservational programs in FCS farms and will improve the profitability of farms over the long term by incorporating the use of genetically parasite resistant sheep and promote local sheep meat production in Florida.

2. Outline
• Introduction
• FL Cracker sheep
• Parasite Resistance
• SNPs
• Results
• CNVs
• Future Work

3. Parasite Resistance: ability to limit the establishment
and fecundity of the parasite
Objectives: identify DNA variants controlling parasite
resistance in Florida Cracker sheep and develop tools
for selective breeding
What is parasite resistance?

4. Gastrointestinal
nematode
infections
Dead
50,331 sheep
86,701 goats
Anthelmintic
resistance
Southern US:
high disease
incidence
Gastrointestinal Nematodes: major threat to small
ruminant operations in the Southeast
• Haemonchus contortus:
the most pathogenic parasite

5. Potential Solution: Selection for parasite resistance
(Artificial selective breeding)
Resistance
Limits the
establishment
(reduce burden)
and fecundity of
parasite
- Innate immune
response
- Acquired
immunity
Tolerance/Resilience increase infectivity of environment and
determination of status requires accurate data on the level of
parasite infection
Potential Solution: selection for parasite resistance

6. Selection for improved parasite
resistance will result in a general
reduction in parasite burden which in
turn, may lead to reduced losses in
production and meat quality in FCS
vs
Potential Solution: selection for parasite resistance
RR SS

7. Phenotypic markers used to detect parasite
resistance
Parasitological
measures
FEC
FAMACHA
Hematocrit
IgA
Eosinophilia
IgE
IgG
Immunological
measures

8. Florida Cracker Sheep: a heritage sheep breed
from Florida

9. Florida Cracker Sheep: naturally adapted to hot
and humid conditions
• The Florida Cracker is one of the oldest
breeds of sheep in North America
• Native and heritage breed from Florida
• Adapted to hot and humid conditions like
that in Florida
• It is an endangered sheep breed under
critical conservation priority (< 1,000)

10. Spain introduced 400 sheep
(Churra) during St.
Augustine foundation
1565 1945
Florida Native sheep
re-domestication
Sheep remained feral
2019
Great health and
reproductive behavior
Breed domesticated and naturally adapted to tropical
environmental conditions of Florida
Re-domestication
Florida Cracker Sheep: a heritage sheep breed
from the Southeast

11. Florida Cracker sheep population
FAIRMEADOW SHEEP
FARM
Location: Ocala, FL
• Florida Cracker sheep from commercial farm
located in Ocala, FL (n = 380; 3 and 5 months old)
• Phenotypes collected from 2018 -2020
• Animals were dewormed before the study. After
confirmation of FEC reduction, animals were
naturally infected with H. contortus.

12. Phenotype collection
H. contortus
FEC
Hematocrit
Body condition score & weight
FAMACHA
score

13. Objective
• Identify DNA variants (SNPs, CNVs) associated with FEC,
FAMACHA, hematocrit level (PCV), and average daily gain

14. Genotyping and Quality control of SNP data
• DNA extracted from blood samples
• Genotyped with GGP Ovine 50k
• Call rate < 95%, MAF ≤ 0.05, LD pruning
• Initial number of SNPs before quality control: 45,205 SNPs
• Final number of SNPs after quality control: 32,500 SNPs

15. Genome Wide Association Analysis with SNP data

16. Results: SNPs with additive and non-additive effects
associated with FEC

17. Results: SNPs with additive effects associated with
FEC
• Antigen WC1.1 gene encodes a receptor
that is uniquely expressed on γδ- T cells
• Previous studies with resistant Canaria
Hair sheep infected with H. contortus, have
suggested that immune response
modulated by WC1+ γδ- T cells is the
primary mechanism used for regulation of
parasite growth and fecundity
(González et al., 2011; Guo et al., 2016; Hernández et al., 2017)

18. Results: SNPs with additive and non-additive effects
associated with FAMACHA and ADG

19. Results: SNPs with additive and non-additive effects
associated with PCV

20. Results: SNPs with additive and non-additive effects
associated with PCV
• For PCV, the SNP with additive genetic
effects was located close to STC1 gene
whereas the SNP with recessive genetic
effects was located downstream of TGFB2
gene
• STC1 controls calcium transport in intestinal
epithelia and negatively regulates TRP
channels
• Calcium entries TRP channels for agonist
induction of NFKB activation which leads to
IL1B production and activation of
inflammatory response

21. Quality Control for CNV detection
• Intensity values from 45,205 SNPs were available for quality control procedures
using SVS Golden Helix
• Principal component analysis (PCA) was applied to detect and correct for the
presence of batch effects and to correct the LRR values

22. CNV Segmentation
• The copy number analysis module (CNAM) for optimal segmenting was used to
identify CNVs using the univariate method (default options)
• This analysis considers only one sample at a time and detects rare or large
CNVs
• Univariate outlier removal, maximum number of 10 segments per 20,000
markers, a minimum of 1 marker per segment, and 2,000 permutations per pair
with a p-value cutoff of 0.05 were used
• Individuals with a waviness factor (WF) - 0.05>WF>0.05 were also excluded

23. Genome Wide Association Analysis with CNVs

24. Results: CNVs associated with FEC

25. Results: CNVs associated with FEC
• A significant deletion CNV in chromosome 21 was associated with FEC
• This deletion was located in intron 2 of RAB3IL gene and overlapped a QTL
associated with changes in eosinophil number
• This deletion was also located close to a QTL associated with FEC in sheep
• Intronic polymorphisms in RABIL3 are associated with Chron’s disease
(affects gastrointestinal tract)
• Symptoms: diarrhea, anemia, weight loss, abdominal pain, blood in feces, etc

26. Results: CNVs associated with FEC
• A significant deletion CNV in chromosome 21 was associated with FEC
• This deletion was located in intron 2 of RAB3IL gene and overlapped a QTL
associated with changes in eosinophil number
• This deletion was also located close to a QTL associated with FEC in sheep
• Intronic polymorphisms in RABIL3 are associated with Chron’s disease
(affects gastrointestinal tract)
• Symptoms: diarrhea, anemia, weight loss, abdominal pain, blood in feces, etc

27. Conclusions
• Significant SNPs in chromosome 1, 2, 3, 6, 8, 10, 11,12, 13 and
21 and a deletion CNV in chromosome 21 may contribute to
parasite resistance

28. Future Work
Utilize SVS to:
• Validate identified SNPs and CNVs in FCS
• Estimate Runs of Homozygosity to identify
the genomic footprint of inbreeding in FCS
• Perform Genomic Selection for parasite
resistance in FCS

29. Acknowledgments
• Dr. Owen Rae (UF)
• Brittany Diehl (UF)
• Dr. Ibukun Ogunade (WVU)
• Dr. Thomas Terrill (FVSU)
• Dr. Ignacy Misztal (UGA)
• Dr. Andres Pech-Cervantes (FVSU)

30. Acknowledgments
- This research was partially supported by
Sustainable Agriculture Research and
Education, U.S. Department of
Agriculture, under award No. GS17-173.
- We thank Neogene for free genotyping.

31. QUESTIONS?