molecular advancements in reproduction: from hair to calves collaborator s & dr. milt thomas
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Molecular Advancements in Reproduction: from Hair to Calves
Collaborators & Dr. Milt Thomas
Milt Thomas, Department of Animal and Range Sciences
Technology Implementation?
Milt Thomas, Department of Animal and Range Sciences
Molecular Advancements in Reproduction: From hair to calves.Outline:I.History II. Discovery: SNP from the GH-IGF axisIII. Discovery: QTL detectionIV. Discovery: transcriptome & proteomeV. Building research populations VI. Conclusions
Milt Thomas, Department of Animal and Range Sciences
Molecular Advancements in Reproduction: From hair to calves.
Participants: Past to Present
Konni Shirley, Pablo Luna, Sunday Peters, Kasey DeAtley, Gail Silver, Amelia Clayshulte, Tanner Schaub
Jim Reecy, Rohan Fernando, Kadir Kazilkaya, Dorian Garrick, Zhiliang Hu, John Pollak, Bob Weaber
Juan Medrano, Gonzalo Rincon, David Riley, Chad Chase
2006-35205-16651, 2008-35205-18751, 2009-35205-05100
Konni Shirley, Department of Animal and Range Sciences
Trait CC CT TT χ2
P-
value
First observed estrus 61.8 36.4 1.8 30.0 0.01
Estrus at synchronization 66.3 33.7 0 9.1 0.01
Pregnant during 1st breeding season 57.1 41.9 1.0 53.2 0.01
AI pregnancy 66.7 33.3 0 3.3 0.07
Calved by 2 yr of age 59.2 39.8 1.0 51.6 0.01
Percentages of IGF-I genotypes for categorical reproductive trait
Evolution of a Fertility ProjectNBCEC Large Herd Managers Symposium, KC -2005
– Spring born Brangus (n = 190) heifers
– Born 1997 to 2002
– Progeny of 14 Brangus sires
Milt Thomas, Department of Animal and Range Sciences
Evolution of a Fertility ProjectNBCEC Large Herd Managers Symposium, KC -2005
Initial Results from Rex Ranch Project Relative NMSU Angus Heifers
HerdPregnancy
StatusNumber
of Heifers
Genotypic Frequency, %
CC CT TT
1 non-pregnant 160 15 49.4 35.6
1 pregnant 166 19.2 41.6 39.2
3 non-pregnant 75 20 44 36
3 pregnant 85 20 40 40
NMSUPregnancy rate > 90% 110 10 51.8 38.2
Milt Thomas, Department of Animal and Range Sciences
Large Herd Managers Symposia (2005)
Identification of Molecular Markers to Improve Fertility of Beef Cattle; 200835205-18751.
Milt Thomas, Department of Animal and Range Sciences
QTL Detection
Validation of MAS tools in commercial
herds across environments and
production systems.
Technology Transfer
Fine Mapping
QTL Detection
Validation of MAS tools in commercial
herds across environments and
production systems.
Technology Transfer
Fine Mapping
Long-term goal: understand in molecular detail the genetic pathways regulating reproductive performance in beef cattle, with the intent of using this information to develop genetic improvement programs for fertility.
Milt Thomas, Department of Animal and Range Sciences
II. Discovery: SNP from the GH-IGF axis
IGF 1 38Mb
PMCH
STAT 6STAT 2
IGFBP6
SOCS2 15Mb
+ Pregnancy Associated
Plasma Protein(PAPPA)
orPappalysin
Chromosome 16
Bovine Resequencing Populationa. No common ancestors in 3 generation pedigree.b. Resequenced conserved regions of candidate genesc. 75 SNP discovered.
• Dairy breeds (n = 8/breed):
• Beef breeds (n = 6/breed):
Milt Thomas, Department of Animal and Range Sciences
Vista®
Assessment Population: Angus – Brahman – Romosinuano (n = 650)
Diallele = Mating system involving all breed-parent combinations to create hybrids.
Admixed Population = Group of animals with mixed ancestry because have inherited some fraction of their genome from different ancestry populations (e.g., populations stratification).
Ancestry-Informative Markers (AIM) = Set of polymorphisms with significant differences in allele frequencies between breed groups.
Milt Thomas, Department of Animal and Range Sciences
STARS – Brooksville, FL
Gene Name SNP_assayed SNP with MAF >.10
Growth Hormone Receptor (GHR) 1 --Glycosylation-Dependent Cell Adhesion Molecule 1 (GLCM1) 9 --Insulin-like Growth Factor 1 (IGF1) 6 --Insulin-like Growth Factor Binding Protein 6 (IGFBP6) 5 --Pregnancy-Associated Plasma Protein (PAPPA) 9 2Pro-Melanin Concentrating Hormone (PMCH) 5 --Signal Transducer and Activator of Transcription 2 (STAT2) 6 1Signal Transducer and Activator of Transcription 6(STAT6) 6 --Suppressor of Cytokine Signaling 2 (SOCS2) 12 --
Total 59 3
3 Polymorphic Markers56 Ancestry Informative Markers
Genotyping Results
Milt Thomas, Department of Animal and Range Sciences
Phylogenetic TreeFrom AIMs
Ancestry proportion of Angus, Brahman and Romosinuano
(clusters outputted from Structure®; Bayesian-based) Ancestry Clusters
Breed Group N 1 2 3
Angus 66 0.012 0.694 0.294Brahman 59 0.878 0.029 0.082Romosinuano 129 0.040 0.461 0.498Angus x Brahman 116 0.462 0.334 0.204Angus x Romosinuano 136 0.039 0.497 0.464Brahman x Romosinuano 143 0.474 0.234 0.292
Milt Thomas, Department of Animal and Range Sciences
Reproductive Traits:Continuous: age of first calving, calving interval, days to calving Categorical: pregnancy %, Perfect, %
Mixed Model yijklmn = µ + Ai + Bj + Ck + Dl + Em + Fn + eijklmn, Proc Mixed,
Proc Glimmix
• yijkl= phenotypic value of traits,• µ = population mean or frequency,• Ai = fixed effect of SNP genotype or haplotype,• Bj = fixed effect of year of birth,• Ck = fixed effect of age of dam (BIF, 2006), • Dl = covariate of coefficient of ancestry,• Em = covariate of Julian birth date, • Fn = random effect of sire using the Z statistic to test if Ho:σw
2 = 0 (Littell et al., 1996),
• eijklmn = random residual error.
Milt Thomas, Department of Animal and Range Sciences
Least squares means ± SE and levels of significance for fertility traits among SNP genotypes of PAPPA
Trait N SNP 1 PAPPA 11 12 22 P-value
Age at First Calving, d 551 768.6 ± 36.8 787.9 ± 9.6 778.8 ± 11.2 0.9999Perfect, % 551 53.8 56.3 53.7 0.8402Calving Interval, d 441 500.1 ± 37.4a 450.4 ± 9.6b 421.2 ± 8.0b 0.0595Days to Calving, d 441 448.8 ± 38.9a 387.6 ± 10.3b 360.9 ± 8.6b 0.0289Pregnancy Rate, % 640 77.8a 85.8b 89.6b 0.0136
Trait N SNP 2 PAPPA 11 12 22 P-value
Age at First Calving, d 548 773.2 ± 13.7 785.7 ± 9.7 781.9 ± 13.3 0.9999Perfect, % 548 52.7 59.2 52.2 0.2823Calving Interval, d 439 403.4 ± 13.2a 439.9 ± 8.6b 445.1 ± 12.1b 0.0484Days to Calving, d 439 344.9 ± 13.8a 379.4 ± 9.1b 384.4 ± 12.7b 0.0288Pregnancy Rate, % 640 90.6a 87.9b 86.3b 0.0401
a,bWithin a row, means without a common superscript differ (P < 0.05)
Milt Thomas, Department of Animal and Range Sciences
HH Hh hh
HH Hh hh
SNP Genotypes
D
ays
to C
alvi
ng,
d
Cal
ving
In
terv
al, d
Interaction among STAT2 SNP genotypes and ancestral clusters (ALL BREED GROUPS)
Bos taurus Cluster (2 & 3)
Bos taurus Cluster (2 & 3)
Brahman Cluster (1) (Genotype*Cluster; P < 0.05)
Brahman Cluster (1) (Genotype*Cluster; P < 0.10)
Milt Thomas, Department of Animal and Range Sciences
HH Hh hh
HH Hh hh
SNP Genotypes
Day
s to
Cal
vin
g, d
C
alvi
ng I
nte
rval
, d
Interaction among STAT2 SNP genotypes and ancestral clusters (PUREBRED GROUPS)
Brahman Cluster (1) (Genotype*Cluster; P < 0.05)
Brahman Cluster (1) (Genotype*Cluster; P < 0.10)
Bos taurus Cluster (2 & 3)
Bos taurus Cluster (2 & 3)
Milt Thomas, Department of Animal and Range Sciences
Milt Thomas, Department of Animal and Range Sciences
Objective 1: Conduct a SNP-based whole-genome scan to identify QTL associated with heifer pregnancy rate.
Identification of Molecular Markers to Improve Fertility of Beef Cattle (USDA-NRI 200835205-18751)
Discovery: QTL detection + transcriptome & proteome
Relationship fostered from efforts of IBBA –BIC.
Milt Thomas, Department of Animal and Range Sciences
Population Description1.Brangus heifer pregnancy records collected since 1993. 2.DNA and phenotypes on >800 heifers from > sires born 2005, 2006, 2007.3.Trait, pregnant as a yearling, yes (80.6%) or no (19.4%).4.Simple statistics for growth and carcass traits:
Birth weight 77.2 ± 0.4 lbs205-day weight 532.2 ± 2.2 lbs365-day weight 799.0 ± 3.3 lbsRibeye area 9.8 ± 0.05 in2
Fat thickness 0.24 ± 0.002 inintramuscular fat % 4.24 ± 0.032 %
0
20
40
60
80
100
Not Pregnant Pregnant
0
10
20
30
40
50
60
70
80
Spring Fall
Number
of Sires
Heifer Pregnancy Percent
0
20
40
60
80
100
Not Pregnant Pregnant
0
10
20
30
40
50
60
70
80
Spring Fall
Number
of Sires
Heifer Pregnancy Percent
Historic Data provided by Matt Jones
Milt Thomas, Department of Animal and Range Sciences
Discovery: QTL detection + transcriptome & proteome
1 2 3 5 6 7 8 94
29
11 12 14 15 16 17
18
13
19 20 21 23 24 25 26 2722
10
28
Birth Weight , 365-day WeightIntramuscular Fat %Ribeye AreaRib Fat
Pregnant as yearling: (12 to 15 mo) of age to calve by 24 mo.
http://bigs.ansci.iastate.edu/bigsgui/login.htmlBIGS, Bayes C
x/y
Milt Thomas, Department of Animal and Range Sciences
Pipeline for Marker Discovery: Example from Chromosome 2
QTL detection
SNPlotZ
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation.
http://bigs.ansci.iastate.edu/bigsgui/login.htmlBIGS, Bayes C
http://www.animalgenome.org/
Milt Thomas, Department of Animal and Range Sciences
Pipeline for Marker Discovery: Example from Chromosome 2
QTL detection
SNPlotZ
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation
GBrowse
Cattle Genome Trackshttp://www.animalgenome.org/cgi-bin/gbrowse/cattle/#search
Cattle Genome Tracks
Milt Thomas, Department of Animal and Range Sciences
Pipeline for Marker Discovery: Example from Chromosome 2QTL detection
SNPlotZ
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation.
Milt Thomas, Department of Animal and Range SciencesChemical Analysis and Instrumentation Lab
QTL detection
SNPlotZ
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation.
RNA-Seq-Alpheus
Reads/million
TranscriptMatches
GeneMatches
Calcium binding, maganese ion binding, sugar binding, transferace activity, protein amino acid O-linked
Axon-axon contacts: link neurons with supporting schwann cells, adhesion, proliferation,apoptosis
Cell cycle arrest, regulation of mitotic cell cycle
Endonuclease reverse transcriptase
Potassium inwardly-rectifying channel, subfamily J, member 3, G protein-activated inward rectifier potassium channel 1
Cattle Genome Tracks
Transcriptomic Expression
Genomic Expression
Gene NamePre
pubertalPubertal
Prepubertal
Pubertal
LOC100138296
KCNJ3 17.7 14.03
LOC10013916
GALNT13
LOC526988 1.03 2.21
RPRM 1.38 23.67 0.99 17.57
LOC100139440 10.48 10.31
Milt Thomas, Department of Animal and Range Sciences
Chemical Analysis & Instrumentation LabQTL detection
SNPlotZ
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation
Quantitative Proteome Profiling FT-ICR Mass Spectrometry
Milt Thomas, Department of Animal and Range Sciences
QTL detection
SNPlotZ
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation
Objective 2: Develop data and DNA resources! a. Different environments and production systems.
AES = 60%; seedstock = 25%; commercial = 15%Influence of Angus = 95%; Continental = 25%; Brahman = 40%
b. Opportunity for multiple traits.
Milt Thomas, Department of Animal and Range Sciences
1
23
45
6
78
9
10
11
12
1314
15
16
17
18
19
A
B
C
D
Locations of Cooperators (numbers 1-23) and Research Scientists (letters A-D)
2021
22
What about Angus?
Triennial Reproduction Symposium, ASAS, Montreal.
J. F. Taylor, S. D. McKay, J. E. Decker, D. Vasco, M. C. McClure, J. W. Kim, M. A. Rolf, T. Taxis, and R. D. Schnabel. 2009. Application of genome based technologies for identifying genes and their expression that are important for livestock reproduction. J. Anim. Sci. 87 (E-Suppl. 1): 2.
~ 20 SNP explain 32.1% of the variance in heifer pregnancy EPD.
BovineSNP50 results from a deep Angus pedigree.
Milt Thomas, Department of Animal and Range Sciences
ConclusionsMolecular Advancements in Reproduction: from Hair to Calves1.Collaborations needed to move research forward. 2.Resequencing candidate genes can yield effective SNP for genotype to phenotype associations. 3.Genomic approaches help isolate functional mutations. 4.Admixture???
Much work to do!
Milt Thomas, Department of Animal and Range Sciences
Discussion
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