Molecular Advancements in
Reproduction: from Hair to Calves
s & Dr. Milt Thomas
Collaborator
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 axis
III. Discovery: QTL detection
IV. Discovery: transcriptome & proteome
V. 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
Juan Medrano, Gonzalo Rincon, David Riley, Chad Chase
Jim Reecy, Rohan Fernando, Kadir Kazilkaya, Dorian
Garrick, Zhiliang Hu, John Pollak, Bob Weaber
2006-35205-16651, 2008-35205-18751, 2009-35205-05100
Milt Thomas, Department of Animal and Range Sciences
Evolution of a Fertility Project
NBCEC Large Herd Managers Symposium, KC -2005
Percentages of IGF-I genotypes for categorical reproductive trait
PTrait
CC CT TT χ2 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
– Spring born Brangus (n = 190) heifers
– Born 1997 to 2002
– Progeny of 14 Brangus sires
Konni Shirley, Department of Animal and Range Sciences
Evolution of a Fertility Project
NBCEC Large Herd Managers Symposium, KC -2005
Initial Results from Rex Ranch Project Relative NMSU Angus Heifers
Genotypic Frequency, %
Herd
Pregnancy
Status
Number
of Heifers
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
NMSU
Pregnancy
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
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.
QTL Detection
Fine Mapping
Validation of MAS
tools in commercial
herds across
environments and
production systems.
Technology Transfer
Milt Thomas, Department of Animal and Range Sciences
II. Discovery: SNP from the GH-IGF axis
+
SOCS2
15Mb
IGFBP6
STAT 2
STAT 6
PMCH
IGF 1
38Mb
Pregnancy
Associated
Plasma Protein
(PAPPA)
or
Pappalysin
Chromosome 16
Milt Thomas, Department of Animal and Range Sciences
Bovine Resequencing Population
a. No common ancestors in 3 generation pedigree.
b. Resequenced conserved regions of candidate genes
c. 75 SNP discovered.
Vista®
• Dairy breeds (n = 8/breed):
• Beef breeds (n = 6/breed):
Milt Thomas, Department of Animal and Range Sciences
Assessment Population: Angus – Brahman – Romosinuano (n = 650)
STARS – Brooksville, FL
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
Genotyping Results
Gene Name
SNP_assayed
Growth Hormone Receptor (GHR)
Glycosylation-Dependent Cell Adhesion Molecule 1 (GLCM1)
Insulin-like Growth Factor 1 (IGF1)
Insulin-like Growth Factor Binding Protein 6 (IGFBP6)
Pregnancy-Associated Plasma Protein (PAPPA)
Pro-Melanin Concentrating Hormone (PMCH)
Signal Transducer and Activator of Transcription 2 (STAT2)
Signal Transducer and Activator of Transcription 6(STAT6)
Suppressor of Cytokine Signaling 2 (SOCS2)
Total
SNP with
MAF >.10
1
9
6
5
9
5
6
6
12
----2
-1
---
59
3
3 Polymorphic Markers
56 Ancestry Informative Markers
Milt Thomas, Department of Animal and Range Sciences
Phylogenetic Tree
From AIMs
Ancestry proportion of Angus, Brahman and Romosinuano
(clusters outputted from Structure®; Bayesian-based)
Ancestry Clusters
Breed Group
Angus
Brahman
Romosinuano
Angus x Brahman
Angus x Romosinuano
Brahman x Romosinuano
N
1
2
3
66
59
129
116
136
143
0.012
0.878
0.040
0.462
0.039
0.474
0.694
0.029
0.461
0.334
0.497
0.234
0.294
0.082
0.498
0.204
0.464
0.292
Reproductive Traits:
Continuous: age of first calving, calving interval, days to calving
Categorical: pregnancy %, Perfect, %
Milt Thomas, Department of Animal and Range Sciences
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:σw2 = 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
11
Age at First Calving, d 551
Perfect, %
551
Calving Interval, d
441
Days to Calving, d
441
Pregnancy Rate, %
640
Trait
768.6 ± 36.8
53.8
500.1 ± 37.4a
448.8 ± 38.9a
77.8a
N
11
Age at First Calving, d
Perfect, %
Calving Interval, d
Days to Calving, d
Pregnancy Rate, %
a,bWithin
548
548
439
439
640
SNP 1 PAPPA
12
787.9 ± 9.6
56.3
450.4 ± 9.6b
387.6 ± 10.3b
85.8b
SNP 2 PAPPA
12
773.2 ± 13.7
52.7
403.4 ± 13.2a
344.9 ± 13.8a
90.6a
785.7 ± 9.7
59.2
439.9 ± 8.6b
379.4 ± 9.1b
87.9b
22
778.8 ± 11.2
53.7
421.2 ± 8.0b
360.9 ± 8.6b
89.6b
22
781.9 ± 13.3
52.2
445.1 ± 12.1b
384.4 ± 12.7b
86.3b
P-value
0.9999
0.8402
0.0595
0.0289
0.0136
P-value
0.9999
0.2823
0.0484
0.0288
0.0401
a row, means without a common superscript differ (P < 0.05)
Milt Thomas, Department of Animal and Range Sciences
Calving Interval, d
Interaction among STAT2 SNP genotypes and ancestral clusters
(ALL BREED GROUPS)
Brahman Cluster (1)
(Genotype*Cluster; P < 0.05)
Bos taurus Cluster (2 & 3)
Days to Calving, d
HH
Hh
Brahman Cluster (1)
hh
(Genotype*Cluster; P < 0.10)
Bos taurus Cluster (2 & 3)
HH
Hh
SNP Genotypes
hh
Milt Thomas, Department of Animal and Range Sciences
Calving Interval, d
Interaction among STAT2 SNP genotypes and ancestral clusters
(PUREBRED GROUPS)
Brahman Cluster (1)
Bos taurus Cluster (2 & 3)
HH
Days to Calving, d
(Genotype*Cluster; P < 0.05)
Brahman Cluster (1)
Hh
hh
(Genotype*Cluster; P < 0.10)
Bos taurus Cluster (2 & 3)
HH
Hh
SNP Genotypes
hh
Milt Thomas, Department of Animal and Range Sciences
Discovery: QTL detection + transcriptome & proteome
Identification of Molecular Markers to Improve Fertility of
Beef Cattle (USDA-NRI 200835205-18751)
Objective 1: Conduct a SNP-based whole-genome scan to
identify QTL associated with heifer pregnancy rate.
Relationship fostered from
efforts of IBBA –BIC.
Milt Thomas, Department of Animal and Range Sciences
Population Description
1.
2.
3.
4.
Brangus heifer pregnancy records collected since 1993.
DNA and phenotypes on >800 heifers from > sires born 2005, 2006, 2007.
Trait, pregnant as a yearling, yes (80.6%) or no (19.4%).
Simple statistics for growth and carcass traits:
Birth weight
77.2 ± 0.4 lbs
205-day weight
532.2 ± 2.2 lbs
365-day weight
799.0 ± 3.3 lbs
Ribeye area
9.8 ± 0.05 in2
Historic Data
Fat thickness
0.24 ± 0.002 in
provided by
intramuscular fat %
4.24 ± 0.032 %
Matt Jones
100
80
Not Pregnant
Heifer
Pregnancy
Percent
Pregnant
70
80
Number
60
Spring
Fall
60
50
40
of Sires
40
30
20
10
20
0
0
03
20 1
0
20 9
9
19 7
9
19 5
9
19 3
9
19
03
20
01
20
99
19
97
19
95
19
93
19
Milt Thomas, Department of Animal and Range Sciences
Discovery: QTL detection + transcriptome & proteome
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17
18 19 20 21 22 23 24 25 26
Birth Weight ,
365-day Weight
Intramuscular Fat %
Ribeye Area
Rib Fat
27 28 29 x/y
Pregnant as yearling:
(12 to 15 mo) of age to calve by 24 mo.
BIGS, Bayes C
http://bigs.ansci.iastate.edu/bigsgui/login.html
Milt Thomas, Department of Animal and Range Sciences
Pipeline for Marker Discovery: Example from Chromosome 2
QTL detection
BIGS, Bayes C
http://bigs.ansci.iastate.edu/bigsgui/login.html
SNPlotZ
GBrowse
http://www.animalgenome.org/
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation.
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
GBrowse
Assessment and Validation
Milt Thomas, Department of Animal and Range Sciences
Cattle Genome Tracks
http://www.animalgenome.org/cgi-bin/gbrowse/cattle/#search
Cattle Genome Tracks
Pipeline for Marker Discovery: Example from Chromosome 2
QTL detection
SNPlotZ
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation.
Milt Thomas, Department of Animal and Range Sciences
QTL detection
SNPlotZ
RNA-Seq-Alpheus
GBrowse
Reads/
million
Transcriptome
Transcript
Matches
Proteome
Functional SNP ID
Gene
Matches
Assessment and Validation.
Milt Thomas, Department of Animal and Range Sciences
Chemical Analysis and Instrumentation
Lab
Cattle Genome Tracks
Potassium inwardly-rectifying channel, subfamily J, member 3, G protein-activated inward rectifier potassium channel 1
Calcium binding, maganese ion binding, sugar binding, transferace activity, protein amino acid O-linked
Transcriptomic
Expression
Gene Name
Pre
pubertal
Pubertal
17.7
14.03
Genomic
Expression
Pre
pubertal
Axon-axon contacts: link neurons with supporting schwann cells, adhesion, proliferation,apoptosis
Pubertal
LOC100138296
KCNJ3
Cell cycle arrest, regulation of mitotic cell cycle
LOC10013916
Endonuclease reverse transcriptase
GALNT13
LOC526988
RPRM
1.38
23.67
LOC100139440
10.48
10.31
1.03
2.21
0.99
17.57
QTL detection
SNPlotZ
Chemical Analysis &
Instrumentation Lab
Quantitative Proteome Profiling
FT-ICR Mass Spectrometry
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation
Milt Thomas, Department of Animal and Range Sciences
QTL detection
SNPlotZ
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.
GBrowse
Transcriptome
Proteome
Functional SNP ID
Assessment and Validation
Milt Thomas, Department of Animal and Range Sciences
Locations of Cooperators (numbers 1-23) and Research Scientists (letters A-D)
7
8
16
5
12B
9
14
18
19
22
13
4
D
20
11
A 1
C
15
21
10
3
172
6
Milt Thomas, Department of Animal and Range Sciences
What about Angus?
~ 20 SNP explain
32.1% of the
variance in heifer
pregnancy EPD.
BovineSNP50
results from a deep
Angus pedigree.
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.
Conclusions
Molecular Advancements in Reproduction: from Hair to Calves
1. 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
Milt Thomas, Department of Animal and Range Sciences