Supplementary Materials
Table S1: Sample list and Reduced Representation Bisulfite Sequencing (RRBS) results of
livers of fathers before and after heat treatment (F0LC and F0LH)
No. Animal Treatment Total
ID
group
reads
Mapped
reads
Mapping
ratios
Unique
CpGs
1
2
3
4
5
6
7
8
9
10
9,510,592
15,391,210
8,764,587
12,159,704
14,951,128
8,165,391
14,642,985
12,991,491
12,303,243
15,392,933
37.52%
38.19%
34.06%
35.95%
38.75%
38.58%
38.01%
37.89%
38.29%
37.14%
2,246,347
2,596,622
2,039,169
2,233,706
2,514,712
2,385,717
2,645,863
2,808,114
2,246,672
2,098,495
F0LC-F
F0LC-G
F0LC-H
F0LC-I
F0LC-J
F0LH-F
F0LH-G
F0LH-H
F0LH-I
F0LH-J
control
control
control
control
control
heat
heat
heat
heat
heat
25,347,599
40,301,574
25,730,278
33,821,458
38,585,298
21,167,448
38,527,683
34,284,688
32,127,702
41,447,873
CpG
Cov.
(X)
13
18
13
16
17
10
17
14
16
20
BS Conv.
rate
97.00%
98.23%
98.10%
97.79%
97.56%
98.28%
99.13%
98.78%
98.88%
95.26%
Sequencing and mapping results from male F0 Wild guinea pig liver samples (F0LC and
F0LH). Treatment groups are the five F0 fathers: either untreated (control: F0LC-F to F0LC-J)
or exposed for 2 months to 30°C ambient temperature (heat: F0LH-F to F0LH-J).
Table S2: Sample list and Reduced Representation Bisulfite Sequencing (RRBS) results of
livers of sons sired before and after heat treatment of their fathers (F1LC and F1LH)
No.
Animal
ID
Treatmen Total reads Mapped
t groups
reads
Mappin
g ratios
Unique
CpGs
CpG
Cov. ()
1
F1LC-F1
control
18,029,577
6,680,432
37.05%
3,309,580
7
BS
Conv.
rate
98.88%
2
F1LC-F2
control
16,059,059
6,066,076
37.77%
3,035,843
7
98.85%
3
F1LC-F3
control
22,521,636
8,711,906
38.68%
3,641,346
8
98.80%
4
F1LC-F4
control
24,850,183
9,780,109
39.36%
3,519,830
9
98.75%
5
F1LC-F5
control
21,141,617
7,974,523
37.72%
3,492,059
7
98.80%
6
F1LC-G1
control
29,401,669
11,042,394 37.56%
3,719,956
10
98.79%
7
F1LC-G2
control
22,183,595
8,798,513
39.66%
3,468,454
8
98.90%
8
F1LC-H1
control
20,205,777
7,907,817
39.14%
3,326,548
8
98.82%
9
F1LC-H2
control
24,959,451
9,423,385
37.75%
3,736,674
8
98.79%
10
F1LC-H3
control
15,427,493
6,206,456
40.23%
3,406,705
6
98.78%
11
F1LC-H4
control
20,939,405
8,425,873
40.24%
3,426,396
8
98.78%
12
F1LC-I1
control
22,053,277
8,336,943
37.80%
3,279,487
8
98.84%
13
F1LC-I2
control
16,424,032
6,771,022
41.23%
2,859,973
7
98.96%
14
F1LC-J1
control
17,776,958
7,315,599
41.15%
3,395,498
7
99.05%
15
F1LC-J2
control
19,264,973
7,702,700
39.98%
3,590,438
7
98.82%
16
F1LC-J3
control
24,919,184
9,606,720
38.55%
3,797,379
8
98.81%
17
F1LH-F1
heat
18,532,689
7,066,493
38.13%
3,516,644
6
98.82%
18
F1LH-F2
heat
28,644,120
10,804,520 37.72%
3,690,266
9
98.87%
19
F1LH-G1
heat
20,150,827
7,569,801
37.57%
3,389,117
7
98.84%
20
F1LH-G2
heat
19,321,403
6,701,990
34.69%
3,083,698
7
98.90%
21
F1LH-G3
heat
17,132,234
6,246,304
36.46%
3,300,790
6
98.93%
22
F1LH-G4
heat
19,276,868
6,637,384
34.43%
3,263,209
7
98.81%
23
F1LH-H1
heat
21,540,466
7,881,769
36.59%
3,425,142
7
98.98%
24
F1LH-H2
heat
18,390,731
7,099,149
38.60%
3,337,873
7
98.97%
25
F1LH-H3
heat
27,543,256
9,748,162
35.39%
3,632,603
9
98.99%
26
F1LH-H4
heat
33,786,783
11,335,437 33.55%
3,767,440
10
98.89%
27
F1LH-H5
heat
36,834,048
13,105,295 35.58%
4,015,123
10
98.92%
28
F1LH-I1
heat
35,310,760
11,730,786 33.22%
3,834,311
10
98.97%
29
F1LH-I2
heat
28,842,334
9,439,364
32.73%
3,778,893
8
98.80%
30
F1LH-I3
heat
35,306,161
12,372,854 35.04%
3,918,431
10
98.91%
31
F1LH-I4
heat
39,554,259
15,022,978 37.98%
3,814,662
13
98.84%
32
F1LH-I5
heat
16,821,180
6,590,335
39.18%
2,988,359
7
99.04%
33
F1LH-I6
heat
57,468,348
20,094,726 34.97%
3,101,848
21
99.02%
34
F1LH-J1
heat
37,827,572
12,759,894 33.73%
3,824,993
11
98.96%
Sequencing and mapping results from F1 Wild guinea pig liver samples (F1LC and F1LH).
Treatment groups are the sons (Fn-Jn) of fathers either untreated (control: F1LC-F1 - F1LH-J3)
or exposed for 2 months to 30°C ambient temperature (heat: F1LH-F1 - F1LH-J1).
Table S3: Sample list and Reduced Representation Bisulfite Sequencing (RRBS) results of
testis of sons sired before and after heat treatment of their fathers (F0TC and F0TH)
No. Label
1
2
3
4
5
6
7
8
9
10
F0TC-F
F0TC-G
F0TC-H
F0TC-I
F0TC-J
F0TH-F
F0TH-G
F0TH-H
F0TH-I
F0TH-J
Treatment
group
Total reads
Mapped
reads
Mapping
ratio
Unique
CpGs
control
control
control
control
control
heat
heat
heat
heat
heat
34,941,270
32,377,904
30,188,067
30,078,031
31,940,052
35,502,770
40,249,815
33,981,470
40,151,093
30,637,704
12,661,449
12,016,680
11,317,550
11,325,352
12,143,485
13,115,294
14,883,650
12,863,185
14,686,904
11,240,908
36.24%
37.11%
37.49%
37.65%
38.02%
36.94%
36.98%
37.85%
36.58%
36.69%
4,252,743
3,885,977
3,994,599
4,223,878
4,266,646
4,427,390
4,378,046
4,177,212
4,367,847
4,128,242
CpG
Cov.
()
9
9
9
8
9
9
10
10
11
8
BS
Conv.
rate
99.29%
98.84%
99.33%
99.26%
99.28%
99.21%
98.95%
99.25%
99.22%
99.23%
Sequencing and mapping results from F1 Wild guinea pig testis samples (F1TC and F1TH).
Treatment groups are the sons of fathers that were either untreated (control: F0TC-F to F0TC-J)
or exposed for 2 months to 30°C ambient temperature (heat: F0TH-F to F0TH-J). Testis DNA
was pooled of sons from the same father resulting in 10 RRBS libraries.
Table S4: Number of annotated regions with DMRs per father group
4/5 fathers
promoter 11
5/5 fathers
0
CDS
33
12
CGI
45
15
The number of annotated regions including DMRs (annotated DMRs), which occurred in at
least four fathers or in offspring of at least four out of five fathers (F-J), as well as in all
comparison groups (F0LC vs. F0LH, F1LC vs. F1LH and F1TC vs. F1TH; Fig. 2) were listed
according their annotation (promoter, CDS and CGI).
Table S5: Annotated regions with DMRs per father group after comparing F0LC vs. F0LH,
F1LC vs. F1LH and F1TC vs. F1TH
ID
Gene name
F0LC
vs.
F0LH
F1LC
vs.
F1LH
F1TC
vs.
F1TH
ENSCPOG00000001921_promoter
ENSCPOG00000006486_promoter
ENSCPOG00000012928_promoter
ENSCPOG00000020508_promoter
ENSCPOG00000020731_promoter
ENSCPOG00000021486_promoter
ENSCPOG00000023463_promoter
ENSCPOG00000024113_promoter
ENSCPOG00000024501_promoter
ENSCPOG00000025237_promoter
ENSCPOG00000025665_promoter
ENSCPOG00000000591_CDS
ENSCPOG00000000999_CDS
ENSCPOG00000001618_CDS
ENSCPOG00000002223_CDS
ENSCPOG00000003802_CDS
ENSCPOG00000007730_CDS
ENSCPOG00000009376_CDS
ENSCPOG00000010080_CDS
ENSCPOG00000012359_CDS
ENSCPOG00000013220_CDS
ENSCPOG00000021078_CDS
ENSCPOG00000026620_CDS
ENSCPOG00000000387_CDS
ENSCPOG00000000682_CDS
ENSCPOG00000000777_CDS
ENSCPOG00000001125_CDS
ENSCPOG00000001215_CDS
ENSCPOG00000002024_CDS
ENSCPOG00000003039_CDS
ENSCPOG00000003239_CDS
ENSCPOG00000003371_CDS
ENSCPOG00000006604_CDS
Topaz1
Ticam1
A9lpa2
C5orf45
Kiaa1609
Zc3h18
Sncg
C19orf52
Ak4
Svp
Krtap17-1
Notch4
Icam5
Kcnh6
Myh14
Eps8l2
Rara
Pclo
Plekhh3
Map3k6
Hmcn2
Ntf4
Dock6
Phkg1
Pde11a
Mast1
Palm3
Fgd2
Cmbl
Rasal3
Apba1
Stab2
Sox13
4
5
5
4
4
5
4
4
5
5
4
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
4
5
4
4
5
4
4
5
4
4
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
4
4
4
4
4
4
4
4
5
5
4
4
4
4
5
5
5
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
5
5
5
4
4
No.
of
fathe
rs
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
4
4
4
4
4
4
4
4
4
4
ENSCPOG00000006927_CDS
ENSCPOG00000007786_CDS
ENSCPOG00000009248_CDS
ENSCPOG00000009593_CDS
ENSCPOG00000009735_CDS
ENSCPOG00000009742_CDS
ENSCPOG00000009747_CDS
ENSCPOG00000010411_CDS
ENSCPOG00000011364_CDS
ENSCPOG00000011963_CDS
ENSCPOG00000012253_CDS
ENSCPOG00000012929_CDS
ENSCPOG00000012950_CDS
ENSCPOG00000013646_CDS
ENSCPOG00000013669_CDS
ENSCPOG00000015562_CDS
ENSCPOG00000019861_CDS
ENSCPOG00000021309_CDS
ENSCPOG00000021486_CDS
ENSCPOG00000023599_CDS
ENSCPOG00000024030_CDS
ENSCPOG00000024356_CDS
ENSCPOG00000025737_CDS
CGI_scaffold137_2593957_2594458
CGI_scaffold24_26911431_26912365
CGI_scaffold25_8916300_8917298
CpG_Island_scaffold27_7015314_7016000
CpG_Island_scaffold347_182392_182891
CGI_scaffold4_14096129_14096696
CGI_scaffold53_10797058_10798420
CGI_scaffold53_12338161_12339640
CGI_scaffold65_4665560_4666457
CGI_scaffold70_8308344_8309287
CGI_scaffold8_8119090_8119649
CGI_scaffold912_7518_8043
CGI_scaffold927_16_3441
CGI_scaffold927_3618_16028
CGI_scaffold96_4454758_4455442
CGI_scaffold10_22854406_22855263
CGI_scaffold10_49324092_49324882
CGI_scaffold115_4041100_4041661
Bambam1
Ush2a
Gnas
Lama5
Wiz
Pcdha5
Slc27a1
Fcho1
Inpp5d
Cacna1i
Ptpn21
Hspb2
Pik3cd
Kcns1
Ehbp1l1
Gpt
C9orf142
Tmem44
Zc3h18
Hgfac
Atcay
Ppp1r18
Fbll1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
4
4
5
5
5
5
5
4
4
4
4
4
4
5
4
4
5
4
4
4
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
4
4
4
4
4
4
4
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
4
4
5
5
5
5
4
5
4
4
4
5
5
4
5
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
4
4
CGI_scaffold117_1774019_1774529
CGI_scaffold15_34244619_34245449
CGI_scaffold156_1121544_1122341
CGI_scaffold16_36817085_36817599
CGI_scaffold17_14716398_14717393
CGI_scaffold18_38301297_38301912
CGI_scaffold19_27074656_27075589
CGI_scaffold2_67477859_67478928
CGI_scaffold20_31185302_31186764
CGI_scaffold226_426882_427711
CGI_scaffold226_632579_633632
CGI_scaffold25_13488570_13489078
CGI_scaffold27_2619059_2619665
CGI_scaffold30_10384183_10384700
CGI_scaffold3088_1_2831
CGI_scaffold314_110299_111344
CGI_scaffold32_299187_300192
CGI_scaffold322_240862_241395
CGI_scaffold33_18212468_18213158
CGI_scaffold347_333299_334244
CGI_scaffold368_180343_181204
CGI_scaffold4_33132022_33132631
CGI_scaffold41_873326_873825
CGI_scaffold42_13041239_13041738
CGI_scaffold42_13324813_13325557
CGI_scaffold429_95158_95955
CGI_scaffold45_11118285_11118918
CGI_scaffold509_141021_141523
CGI_scaffold53_11638212_11638734
CGI_scaffold6_3164487_3165970
CGI_scaffold6_598782_599924
CGI_scaffold61_9054899_9055727
CGI_scaffold687_5368_6432
CGI_scaffold7_45860487_45861026
CGI_scaffold70_8735848_8736348
CGI_scaffold72_7980612_7981332
CGI_scaffold72_8633449_8633948
CGI_scaffold72_8651524_8652515
CGI_scaffold81_1145048_1146070
CGI_scaffold86_6254900_6255830
CGI_scaffold86_6260228_6261562
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
4
4
5
5
5
5
5
5
4
4
5
4
4
5
4
5
5
4
5
4
4
4
4
4
4
4
5
5
4
5
4
4
5
4
4
5
4
4
5
4
5
5
4
4
4
4
4
5
4
4
4
4
5
4
4
5
4
4
4
4
4
4
4
4
4
4
4
5
4
4
4
5
5
4
5
5
4
4
4
4
5
4
5
5
5
4
5
5
4
5
5
5
5
4
5
5
5
5
4
5
5
4
5
4
5
5
5
4
4
4
5
5
4
5
5
5
5
5
4
5
5
4
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
CGI_scaffold90_5370669_5371337
NA
4
5
5
4
All annotated regions with DMRs among at least four father groups are listed. The ID includes
the Ensembl-ID as well as regulatory regions followed by gene name. To localize CGIs also
the scaffold and position were included. The table was sorted by regulatory regions, starting
with promoters, followed by CDS and CGIs and by their occurrence in five and four father
groups, for F0LC vs. F0LH, F1LC vs. F1LH and F1TC vs. F1TH separately, as well as for all
three combined (No. of fathers) as shown in Fig. 2.
Table S6: Number of genes in CpGs and DMRs
F0LC vs. F0LH F1LC vs. F1LH F1TC vs. F1TH
Genes incl. mCpGs
Genes incl. mCpGs sign.
Genes incl. mCpGs insign.
Genes incl. DMRs
10,860
13,900
12,522
8,235
7,311
6,795
10,589
13,845
12,461
304
103
353
The number of genes are listed including the methylated CpGs (mCpGs) in the fathers` livers
before and after heat exposure (F0LC vs. F0LH) and sons sired before and after the fathers`
exposure in liver (F1LC vs. F1LH) and testis (F1TC vs. F1TH). Most of the genes had several
CpGs, which were partly significantly (mCpGs sign.) and partly insignificantly (mCpGs
insign.) changed in their methylation levels. In addition, the table gives the number of genes
that were located in DMRs.
Fig. S1: DMRs located in the Seminal vesicle polypeptide (Svp) gene
Hypermethylated (red bars) and hypomethylated (blue bars) DMRs detected in the Seminal
vesicle polypeptide (Svp) gene in the heat-treated father groups. This male-specific gene
indicated 1) a tissue-specific pattern and 2) a composition of DMRs for regulatory control.
Whereas Svp was hypomethylated in livers samples of fathers (F0LH), it showed
hypermethylation in the son’s testis (F1TH) and an additional DMR located further upstream
of the DMRs detected in livers.
Fig. S2: Thermoregulation pathway genes showing single mCpG changes detected in heat
group compare to control
Thermoregulation genes with significantly changed methylation ratio (CpGn) in the heat
exposed father livers (F0LC vs. F0LH) and their sons` livers and testes (F1LC vs. F1LH and
F1TC vs. F1TH F0LH). The genomic location, such as coding sequence (CDS) illustrated as
circles and promoter illustrated as triangle, as well as gene methylation states (blue
hypomethylated, red hypermethylated) are given. GO terms describe the thermoregulatory
pathway the genes are involved in.