This document summarizes a study on the genetic architecture of developmental traits in gypsy moth populations. The study established 7 gypsy moth populations in common gardens and sequenced 188 individuals to identify 11,021 SNPs. Three phenotypes - pupal duration, mass, and total development time - were measured. Population structure was corrected using PCA. Several SNPs were significantly associated with each trait, though effect sizes were small. Multilocus models explained over 50% of trait variation. Future work could involve refining the genome assembly and studying additional populations to detect smaller genetic effects.

1. Genetic architecture of
developmental traits in
populations of male gypsy moths
Christopher J. Friedline, Ph.D.!
Virginia Commonwealth University
@noituloveand @cfriedline
Evolution 2014!
Raleigh, NC!
6.21.2014

2. Lymantria dispar
http://bugguide.net/node/view/553307Adapted from: http://www.fs.fed.us/ne/morgantown/4557/gmoth/atlas/quar1a.gif

3. http://green.blogs.nytimes.com/2011/09/13/the-toll-from-tree-boring-pests/?_php=true&_type=blogs&_r=0

4. Can we gain insight into the
genetic architecture of invasion
using L. dispar as a model?

5. Experimental design
• 7 populations established in
replicated common gardens
(NY, VA)
• NC (1), VA (2), NY (1),
Quebec (2) + 1 lab strain
(CPHST Otis Lab)
• Single reference assembly
(HiSeq 2500 PE)
• 188 barcoded individuals
• 3 phenotypes (developmental)
http://www.fs.fed.us/ne/morgantown/4557/gmoth/

8. Parry and Grayson, EntSoc 2013

9. Reference Assembly
• MaSuRCA Zimin et al. (2013), Bioinformatics, 29 (21),
2669-2677
• 591,450 scaffolds (90M QC PE reads)
• 588,164,124 Mb (~1 Kb/contig, max=28 Kb)
• 268 contigs > 10 Kb
• N50 = 1.3 Kb
• 35.3% GC
• 32%/56% CEGMA complete/partial

10. ddRADseq
• 316 M reads
(41.5 GB)
• 188 individuals
• 690 Kb/individual
[10, 1400] Kb (QC)
Peterson et al. (2012) Plos One 7.5 (2012): e37135-e37135

11. SNP Calling/Filtering
• Called with Freebayes (n = 30,791)
• Kept only biallelic SNPs
• Removed > 50% missing
• Removed FIS outliers (> |0.5|)
• Removed MAF < 0.01
• n = 11,021

12. Phenotypes
NC NY OTIS QC32 QC93 VA1 VA2
7
8
9
10
11
12
13
Pupual Duration
NC NY OTIS QC32 QC93 VA1 VA2
0.2
0.3
0.4
0.5
0.6
0.7
Mass
NC NY OTIS QC32 QC93 VA1 VA2
65
70
75
80
85
90
95
Total Dev Time

13. 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
FST
0
1000
2000
3000
4000
5000
6000
7000
8000
Multilocus FIS = 0.0486, FST = 0.0224, FIT = 0.0700
n=11021 [0.00, 0.34]
FST
MAF Bin F F F
50 0.1542 0.0234 0.174
40 0.1007 0.0303 0.128
30 0.0757 0.0295 0.103
20 0.0093 0.0169 0.0261
10 -0.0673 0.0088 -0.0579

14. Population Structure
Correction by PCA
• Price et al. (2006) ”Principal Components Analysis
Corrects for Stratification in Genome-wide Association
Studies." Nat Genet 38.8
• First principal component approximates FST
• Number of axes chosen using a Tracy-Widom test,
described in Eckert et al. (2010). Genetics 185:
969-982.
• Correlation of genotype vs. phenotype residuals to X2
to p-values

15. Population Structure
Correction by PCA
• Price et al. (2006) ”Principal Components Analysis
Corrects for Stratification in Genome-wide Association
Studies." Nat Genet 38.8
• First principal component approximates FST
• Number of axes chosen using a Tracy-Widom test,
described in Eckert et al. (2010). Genetics 185:
969-982.
• Correlation of genotype vs. phenotype residuals to X2
to p-values
40 20 0 20 40 60 80
PC1 (0.021%)
60
50
40
30
20
10
0
10
20
30
PC2(0.015%)
PCA of n=7 populations on 11021 loci

16. Population Structure
Correction by PCA
• Price et al. (2006) ”Principal Components Analysis
Corrects for Stratification in Genome-wide Association
Studies." Nat Genet 38.8
• First principal component approximates FST
• Number of axes chosen using a Tracy-Widom test,
described in Eckert et al. (2010). Genetics 185:
969-982.
• Correlation of genotype vs. phenotype residuals to X2
to p-values

17. Top SNP
C/C C/T T/T
Locus 14103 (ctg7180001511349/152)
0.2
0.3
0.4
0.5
0.6
0.7
Mass (p = 0.000004, FST = 0.008543)
T/T T/C C/C
Locus 14908 (ctg7180001527347/31)
65
70
75
80
85
90
95
Total Dev Time (p = 0.000111, FST = 0.016849)
A/A A/T T/T
Locus 10529 (ctg7180001452692/364)
7
8
9
10
11
12
13
Pupual Duration (p = 0.000022, FST = 0.024810)

18. 468
444
44
444
73 29
9
Total Dev Time Pupual Duration
Mass
• Corrected for population structure (Price et al. 2006), binned by MAF
• By p value (p < 0.05):
• Mass: n = 555
• Pupual duration: n = 526
• Total development time: n = 524
• By q value (Storey and Tibshirani, 2003)
• Mass: n = 3 (14103
(*,10)
, 27843
(40)
, 9023
(40)
)
• Pupual duration: n = 1 (S10529
(40)
)
• Total development time: n = 0
Significant SNPs

19. Multilocus effectS27843
S5330
S13360
S23045
S23046
S14681
S14682
S27841
S30469
S25851
S10170
S23791
S12194
S25917
S7263
S7870
S1134
S14103
S8633
S13228
S14762
S2609
S26153
S17855
S9023
Top 25 (p-value) loci
0.005
0.000
0.005
0.010
mult.reg.coeff
Mass regression coefficients
(R2
= 0.556, R2
adj = 0.494)
S10056
S20804
S30588
S21213
S11490
S23793
S29819
S18637
S14030
S21212
S12563
S24930
S26120
S23756
S23892
S12194
S14908
S15702
S12218
S15492
S30168
S29721
S28752
S19836
S10057
Top 25 (p-value) loci
1.0
0.5
0.0
0.5
1.0
mult.reg.coeff
Total Dev Time regression coefficients
(R2
= 0.639, R2
adj = 0.584)
S16063
S6315
S10193
S18784
S21858
S5008
S2156
S18532
S18465
S12721
S12012
S28904
S2571
S20272
S6963
S30060
S3196
S3195
S16560
S17662
S10529
S20271
S18530
S6316
S16062
Top 25 (p-value) loci
1.0
0.5
0.0
0.5
1.0
1.5
mult.reg.coeff
Pupual Duration regression coefficients
(R2
= 0.541, R2
adj = 0.473)

20. Blast results
Mass
reverse transcriptase*
non-LTR
retrotransposon
predicted craniofacial
development protein
sulfotransferase
(amine, estrogen)
Pupual duration
endonuclease-reverse
transcriptase
phosphatidylinositol 3-
kinase
Development time
reverse transcriptase
endonuclease-reverse
transcriptase
transcription initiation factor
TFIID subunit 2-like protein
chosen by p+q*!
chosen by q+

21. Conclusions/Future Work
• Assembly curation is likely necessary for more
robust biological conclusion
• Small effect sizes difficult to detect with small
sample size and populations
• High degree of multilocus effects
• Additional replicate gardens with related material
• Probabilistic genotype calling with full set

22. Acknowledgments
Johnson Lab!
Derek Johnson
Kristine Grayson
Trevor Faske
NPGI: NSF Postdoctoral Fellowship in Biology FY 2013
Rodney Dyer, VCU
Dylan Parry, SUNY-ESF
Eckert Lab!
Andrew Eckert
Brandon Lind
Erin Hobson
Ethan Harwood
VCU NARF
VCU CHiPC