Online Resource 1 Animals and tissue samples
Muscle samples were collected at an abattoir from adult pigs of the Pietrain and Czech
Large White breeds (m. biceps femoris, m.b.f.) and from 44-day-old fetuses (hind limb). Also,
muscle (m.b.f. and m. longissimus lumborum et thoracis, m.l.l.t.) and other tissue samples
(heart, tongue, diaphragm, liver, spleen, lung, kidney, ovary, adipose tissue, lymph node and
brain) were collected from an adult crossbred (Pietrain x Czech Large White x Czech
Landrace) for transcription profiling. Samples of m.l.l.t., m.b.f., heart, lung, kidney, brain and
liver tissues from Czech Landrace and commercial crosses with Czech Landrace in different
age categories (one-, seven- and 14-day-old piglets and adult animals) were collected to
isolate mRNA for real-time RT-PCR. Samples were collected within 30 min after slaughter,
immersed in RNAlater (QIAGEN, Hilden, Germany) and stored at -20 °C until used. All pigs
were slaughtered according to protocols for certified national slaughterhouses under the
supervision of an independent veterinarian. Principles of ethical standards were adhered to
during all sample collections.
For linkage mapping of EEF1A1 and EEF1A2 the Hohenheim three-generation pedigrees
Meishan x Pietrain (MxP), Wild Boar x Pietrain (WxP) and Wild Boar x Meishan (WxM)
[Geldermann et al., 2003] were used. Allele frequencies were estimated in eight pig breeds
(Czech Large White, Czech Landrace, Pietrain, Black Pied Prestice, Czech Meat Pig,
Hampshire, Duroc and Meishan).
Reference
Geldermann H, Müller E, Moser G, Reiner G, Bartenschlager H, Cepica S, Stratil A, Kuryl J,
Moran C, Davoli R, Brunsch C (2003) Genome-wide linkage and QTL mapping in porcine F2
families generated from Pietrain, Meishan and Wild Boar crosses. J Anim Breed Genet
120:363-393. doi: 10.1046/j.0931-2668.2003.00408.x
Online Resource 2 Strategy of sequencing of EEF1A1 cDNA and genomic DNA of EEF1A1
and EEF1A2
EEF1A1: cDNA sequence encompassing the whole coding sequence of EEF1A1 (1389
bp) was obtained from clones of a subtracted fetal hind limb muscle cDNA library [Bílek et
al., 2008; Stratil et al., 2008] and the sequence was deposited in the EMBL/GenBank database
under accession number FM995600 (submitted Feb. 9, 2009). By alignment with the human
EEF1A1 genomic sequence (ENSG00000156508, transcript EEF1A1-009; Ensembl,
http://www.ensembl.org/Homo_sapiens/Info/Index),
individual
exon
sequences
were
identified. The study of the porcine genomic sequence of EEF1A1 was initiated when the
genomic sequence was not available. On the basis of the cDNA sequence PCR primers
EEF1A1-2Aak, -2Bak; -Cf, -Br were designed (see Online Resource 3) and used to determine
the partial genomic sequence (from exon 2 to exon 8; Pietrain). For sequencing of the 5´ end
of the gene and the 5´ upstream and 3´ downstream sequences, BAC clones from the porcine
genomic BAC library were used [Rogel-Gaillard et al., 1999]. By using PCR primers
EEF1A1-D and -E (Online Resource 3), two positive BAC clones were identified (PigI153C5 and PigI-345G10). The clone PigI-345G10 was used to determine upstream and
downstream sequences of EEF1A1 by primer walking. From the obtained sequence, PCR
primers were designed to amplify fragments of genomic DNA and to sequence a Pietrain pig
(for the primers, see Online Resource 3; the primers for sequencing by primer walking are not
shown). The fragments with indels or repetitions (heterozygotes) were cloned in plasmids
using the QIAGEN PCR Cloningplus Kit (Qiagen). The sequence of the porcine EEF1A1,
including the 5´ UTR, 3´ UTR, 5´ upstream and 3´ downstream sequences, was deposited in
the EMBL/GenBank database under accession number FM995601 (submitted Feb. 9, 2009).
EEF1A2: Porcine sequence TC348097 (http://compbio.dfci.harvard.edu/tgi/; an updated
sequence is currently listed under TC603294 and it does not differ in the region of interest),
which was highly identical (93% of 1155 nt) with the human EEF1A2 coding sequence
(Ensembl, ENST00000217182), was used to design four pairs of PCR primers (EEF1A2-1A,
-1B; -2A, -2B; -3A, -3B; -4A, -4B) to sequence the porcine EEF1A2 gene, from exon 4 to
exon 8 in genomic DNA. Information on the primers is presented in Online Resource 3. A
complete genomic sequence of the porcine EEF1A2 was obtained by sequencing the PCR
fragments amplified on a PAC clone with EEF1A2 using the above-mentioned primers, as
well as other primer pairs (see Online Resource 3) and primers for primer walking (designed
on the basis
of the obtained sequences;
data not
shown). The PAC
clone
(TAIGP714G09049Q) was identified by screening PAC library (PAC-TAIGP714; breed
German Landrace) [Al-Bayati et al., 1999] with PCR primers 9A and 9B (Online Resource 3).
Exon/intron organization of EEF1A2 was determined on the basis of comparison with human
EEF1A2 (Ensembl, release 52; ENSG00000101210; transcript EEF1A2-201). The obtained
sequence has been deposited in EMBL/GenBank database under accession number
FM992107.
References
Bílek K, Knoll A, Stratil A, Svobodová K, Horák P, Bechyňová R, Van Poucke M, Peelman
LJ (2008) Analysis of mRNA expression of CNN3, DCN, FBN2, POSTN, SPARC and
YWHAQ genes in porcine foetal and adult skeletal muscles. Czech J Anim Sci 53:181-186.
Stratil A, Knoll A, Horák P, Bílek K, Bechyňová R, Bartenschlager H, Van Poucke M,
Peelman LJ, Svobodová K, Geldermann H (2008). Mapping of the porcine FBN2, YWHAQ,
CNN3, DCN, POSTN, SPARC, RBM39 and GNAS genes, expressed in foetal skeletal muscles.
Anim Genet 39:204-205. doi: 10.1111/j.1365-2052.2007.01678.x
Rogel-Gaillard C, Bourgeaux N, Billault A, Vaiman M, Chardon P (1999) Construction of a
swine BAC library: application to the characterization and mapping of porcine type C
endoviral elements. Cytogenet Cell Genet 85:205-211. doi: 10.1159/000015294
Al-Bayati HK., Duscher S, Kollers S, Rettenberger G, Fries R, Brenig B (1999) Construction
and characterization of a porcine P1-derived artificial chromosome (PAC) library covering
3.2 genome equivalents and cytogenetical assignment of six type I and type II loci. Mamm
Genome 10:569-572. doi: 10.1007/s003359901046
Online Resource 3 PCR primers for sequencing, SNP analysis, SCH and RH mapping, and amplification conditions of EEF1A1 and EEF1A2
Gene
Accession no.
Primer name
Primer sequences (5´-3´)
EEF1A1-Zf
EEF1A1-Zr
EEF1A1-U
EEF1A1-V
EEF1A1-Q
ccgcgttgctgtggctct
ttggttggcaatcaggcagatat
catatgcttcggagccactcttag
cgcgtcctccatcttgagac
ctgggaaagtggtgtcgtgtg
Location in
genomic sequence
5´ upstream
5´ upstream
5´ upstream
intron 1
5´ upstream
EEF1A1-R
ccgatgacgacgatgttgat
exon 2
EEF1A1-2Aak
EEF1A1-2Bak
EEF1A1-Cf
EEF1A1-Br
EEF1A1-3f
EEF1A1-3r
acgtcgattcgggcaagtc
ccagaaattggcacaaatgcta
ttggctacaaccctgatacag
cgttcttccaccactgattaag
ggtggaagaacggtctcagaact
ccctttttgagccattttcagat
exon 2
exon 4
exon 4
exon 8; 3´ UTR
exon 8; 3´ UTR
3´ downstream
PWa on BAC
PigI-345G10
PWa on BAC
PigI-345G10
FM995601
EEF1A1-X
EEF1A1-Y
EEF1A1-Y1
EEF1A1-Y2
EEF1A1-D
EEF1A1-E
aaagaagcattttgaggaactatg
tggcacttcctaaaatttgtagta
tctggaccaagagggagatagat
gctgctacatcaaacctcgtact
aattatgctgtgctgtgaatac
gcatttagcacattttagacat
3´ downstream
3´ downstream
3´ downstream
3´ downstream
intron 5
intron 6
CR904955
EEF1A1-5F
EEF1A1-5R
ggccttgctcagtgggttag
cccctcattcagcaatcaca
BAC end of
PigI-345G10
EEF1A1 PWa on BAC
PigI-345G10
PWa on BAC
PigI-345G10
PWa on BAC
PigI-345G10
FM995600
FM995600
FM995600
FM995600
PWa on BAC
PigI-345G10
Amplicon MgCl2
Size (bp) (mM)
~600
HotStarb
Ta (°C) Comments
1810
1.0
50
882
1.0
60
857
2.25
60
1258
1.5
57
925
1.0
57
1480
2.0
52
379
2.0
55
356
2.5
57
394
1.0
55
62
HotStarTaq polymeraseb;
sequencing
HotStarTaq polymerase;
sequencing
LA DNA polymeraseb;
sequencing;
SNP analysis
(FM995601:g.2111A>G)
RsaI PCR-RFLP:
allele G: 754 + 128 bp
allele A: 581 + 173 + 128 bp
LA DNA polymerase;
sequencing
LA DNA polymerase;
sequencing
LA DNA polymerase; cloning;
sequencing
LA DNA polymerase;
sequencing
LA DNA polymerase;
sequencing
LA DNA polymerase;
BAC screening; SNP analysis
(FM995601:g.3535C>T)
HpaII PCR-RFLP:
allele T: 356 bp
allele C: 295 + 61 bp
LA DNA polymerase; SCH
mapping
Online Resource 3 PCR primers for sequencing, SNP analysis, SCH and RH mapping, and amplification conditions of EEF1A1 and EEF1A2 (continued)
EEF1A2 TC348097
FM992107
EEF1A2-1A
EEF1A2-1B
EEF1A2-2A
EEF1A2-2B
EEF1A2-3A
EEF1A2-3B
EEF1A2-4A
EEF1A2-4B
EEF1A2-27A
EEF1A2-16B
EEF1A2-22A
EEF1A2-15B
EEF1A2-9A
EEF1A2-9B
cgacaacatgctggagccct
gagcctccagcagggacacg
ggttcaagggctggaaagtgga
ggcctcgctcagagcctcg
accttcgcgcccgtgaaca
gggccgtgtggcagtcaatg
gcagatcagcgccggctact
gccgctcttcttctccacgttc
gtgggcaccgcacagacatt
agaaaggggaggtcagccagtg
attttgcagacagtgggggtgc
gggacaccctgacctcaaccaa
tgagcaagtatgaggcccagga
ctggtctccctggcggtgac
exon 4
exon 5
exon 5
exon 6
exon 6
exon 7
exon 7
exon 8
intron 1
intron 2
intron 2
intron 2
intron 5
intron 5
EEF1A2-5A
EEF1A2-2B
EEF1A2-11A
EEF1A2-11B
acgcaggcattggcactgtg
see above
ctgaggtgaagtcggtggagatg
cgctgcctgggaagacgg
intron 5/exon 6
exon 6
exon 6
intron 6
745
1.5
65
1224
2.0
62
1447
2.0
62
878
2.0
62
969
HotStarb
60
646
HotStar
60
169
2.25
60
140
2.0
60
LA DNA polymerase;
sequencing
LA DNA polymerase;
sequencing
LA DNA polymerase;
sequencing
LA DNA polymerase;
sequencing
HotStarTaq polymerase;
comparative sequencing
HotStarTaq polymerase;
comparative sequencing
LA DNA polymerase, without
DMSO;
PAC screening; RH mapping
LA DNA polymerase;
comparative sequencing
212
HotStar
58
HotStarTaq polymerase;
SNP analysis
(FM992107:g.6609C>G)
Hin6I PCR-RFLP
allele G: 212 bp
allele C: 173 + 39 bp
a
Primers were designed on the sequence that was obtained by sequencing (primer walking, PW) of BAC clone PigI-345G10 (Jouy-en-Josas, France).
b
HotStarTaq Master Mix Kit (QIAGEN, Valencia, CA, USA); LA DNA Polymerases Mix (Top-Bio, Prague, Czech Republic).
Online Resource 4 PCR primers for transcription profiling and analysis of expression of EEF1A1
and EEF1A2 in various tissues, and for housekeeping genes GAPDH and HPRT1
Gene
Accession no.
EEF1A1 FM995600
Primer name
EEF1A1_11A
EEF1A1_11B
EEF1A2 FM992107
EEF1A2_7A
EEF1A2_7B
GAPDH AF141959
GAPDH_3A
GAPDH_3B
HPRT1 NM_001032376.2 HPRT_1AF
HPRT_1BR
Primer sequences (5´- 3´)
Location
ttgcattctaccaccaactcgt
aacattgactggagcaaaggtg
aacgtgtcagtcaaggacatcc
cgggtggttcagaatgatga
aaaggccatcaccatcttcc
gccccacccttcaagtgagcc
aaggacccctcgaagtgttg
cacaaacatgattcaagtccctg
exon 5
exon 6
exon 6
exon 7
exon 4
exons 5-6
exon 7
exon 8
Amplicon
size (bp)
157
111
135
122
Online Resource 5 Allele frequencies at SNPs FM995601:g.2111A>G and FM995601:g.3535C>T
(EEF1A1), and FM992107:g.6609C>G (EEF1A2) in eight pig breeds
Czech Large White
FM995601:g.2111A>Ga
(EEF1A1)
n
G
A
14
0.68
0.32
FM992107:g.6609C>G
(EEF1A2)
n
G
C
14
0.14
0.86
Czech Landrace
22
0.77
0.23
25
0.00
1.00
Czech Meat Pig
15
0.93
0.07
15
0.30
0.70
Pietrain
26
0.54
0.46
23
0.04
0.96
Black Pied Prestice
7
0.21
0.79
7
0.29
0.71
Hampshire
6
0.92
0.08
6
0.83
0.17
Duroc
11
1.00
0.00
12
0.54
0.46
Meishan
22
0.80
0.20
8
1.00
0.00
Breed
a
The same animals were tested for SNP FM995601:g.3535C>T; all pigs were homozygotes CC,
except two Meishan and one Czech Meat Pig, who were heterozygotes CT.
Online resource 6 Potential pseudogenes and pseudogene fragments of porcine EEF1A1 in the pig genome sequence (Sscrofa10.2)a
Scrofa10.2 sequence (GCA_000003025.4) was blasted with the EEF1A1 cDNA sequence derived from FM995601.1. The sequences were adjusted
manually for maximum alignment lengths and analysed by ClustalW2.
Chromosome
Position
Strand Identities
Gaps
SSC1
119675490 - 119677217
Minus 1493/1751 (85%)
32/1751 (2%) Processed pseudogene (from exon 1 to exon 8)
139609140 - 139610422
Plus
1005/1329 (76%)
51/1329 (4%) Processed pseudogene (from exon 1 to exon 7)
11959737 - 11961058
Plus
1104/1348 (82%)
27/1348 (2%) Processed pseudogene (from exon 3 to exon 8)
133456562 - 133458318
Minus 723/916 (79%)
225/292 (77%)
(without 601-nt
insertion)
44/916 (5%)
9/292 (77%)
Processed pseudogene (from exon 1 to exon 7;
insertion is in exon 3)
98804674 - 98805016
91658469 - 91659113
Plus
Plus
270/344 (78%)
541/673 (80%)
20/344 (6%)
29/673 (4%)
Fragment homologous to exon 8
Processed pseudogene (from exon 5 to exon 8)
97802672 - 97804350
Plus
1505/1721 (87%)
52/1721 (3%) Processed pseudogene (from exon 1 to exon 8)
119617258 - 119619084
Minus 1135/1376 (82%)
(without 2 insertions
of 204 and 278 nt)
78/1376 (6%) Fragmented and truncated processed
Within
pseudogene (3 fragments from exon 3 to exon 8 LOC100156563
are within the sequence)
75507884 - 75517394
145475919 - 145476277
Minus cds 1050/1391
(75%)
Plus
302/360 (84%)
cds 7/1391
(1%)
1/360 (0%)
13578954 - 13579494
Minus 450/554 (81%)
19/554 (3%)
SSC2
SSC3
SSC4
SSC6
SSC7
Comments
Exon-intron structure (exons 2 – 8). Closed
reading frame (potential pseudogene)b
Fragment of processed pseudogene (from exon
1 to exon 3)
Fragment of processed pseudogene (from exon
7 to exon 8)
NCBI gene
LOC100517693
(partial)
Within
LOC100157010
Within
LOC102160176
Within
LOC100512950
Within
LOC102165177
LOC100511506
LOC100620900
-
Online resource 6 Potential pseudogenes and pseudogene fragments of porcine EEF1A1 in the pig genome sequence (Sscrofa10.2)a (continued)
SSC7
81509056 -81510833
Minus
425/503 (84%)
148/176 (84%)
333/395 (84%)
17/503 (3%)
3/176 (2%)
1/395 (0%)
Fragmented and truncated processed
Within
pseudogene (3 fragments from exon 4 to exon 8 LOC102160893
are within the sequence; two insertions are
SINE (PRE-1))
SSC8
SSC9
28631144 - 28632821
28754240 - 28756842
Plus
Minus
LOC100521383
LOC100514912
Plus
Exon-intron structure (exons 2 – 8)b. ORF
LOC100737797
29641242 -29642530
Plus
99/1760 (6%)
cds 3/1389
(0%)
cds 3/1389
(0%)
0/180 (0%)
1/258 (0%)
4/218 (2%)
3/95 (3%)
Processed pseudogene, complete (exons 1 – 8)
Exon-intron structure (exons 2 – 8)b. ORF
28922157 - 28924779
1336/1760 (76%)
cds 1075/1389
(77%)
cds 1078/1389
(78%)
152/180 (84%)
197/258 (76%)
164/218 (75%)
67/95 (71%)
Fragmented and truncated pseudogeneb (4
fragments, exons 3, 6, 7 and 8 are within the
sequence); exon – intron structure
LOC100512298
7533723 -7535904
Minus
1459/1756 (83%)
(two insertions
excluded)
80/1756 (5%) Processed pseudogene, complete (exons 1 – 8). LOC100524033
Insertion (235 nt incl. one direct repeat) in exon (partial)
6, and SINE (PRE-1) (258 nt incl. one direct
repeat) in exon 8
SSC10
29315076 - 29315786
Plus
698/713 (98%)
2/713 (0%)
SSC11
SSC12
58620529 - 58620889
48458853 - 48460381
Plus
Minus
336/403(83%)
1283/1563 (82%)
SSC13
804499 – 805617
Minus
923/1193 (77%)
11467914 – 11469648
Minus
26413088 - 26413397
Minus
1371/1749 (78%)
(without 53-nt
insertion)
260/323 (80%)
Fragment of processed pseudogene (from exon
6 to exon 8)
43/403 (11%) Fragment homologous to exon 8
36/1563 (2%) Processed pseudogene (from exon 1 to exon 8)
80/1193 (7%) Fragment of processed pseudogene (from exon
3 to exon 8)b
72/1749 (4%) Processed pseudogene, complete (exons 1 – 8)
13/323 (4%)
Fragment of processed pseudogene (from exon
5 to exon 7)b
LOC100516935
(partial)
LOC100153897
LOC100152300
-
Online resource 6 Potential pseudogenes and pseudogene fragments of porcine EEF1A1 in the pig genome sequence (Sscrofa10.2)a (continued)
SSC14
95159960- 95160956
Minus
SSC15
28341082 - 28342983
Minus
SSC16
75371586 - 75372415
Plus
708/851 (83%)
(without 162-nt
insertion)
1474/1682 (88%)
(without 226-nt
insertion, PRE-1)
779/838 (93%)
SSC17
2488137 - 2488560
Minus
372/468
SSCX
67392142 - 67393219
Plus
10301240 - 103013277
Plus
177/209 (85%)
519/608 (85%)
(284-nt insertion
and 40-nt deletion
excluded)
748/881 (85%)
a
31/851 (4%)
Fragment of processed pseudogene (from exon
6 to exon 8)
-
26/1682 (2%) Processed pseudogene (exons 1– 8)
LOC100738918
(partial)
10/838 (1%)
Fragment of processed pseudogene (from exon
6 to exon 8)
45/468 (10%) Fragment of processed pseudogene (exons 7 –
8)
3/209 (1%)
Fragmented and truncated processed
26/608 (4%) pseudogene (two fragments; from exon 5 to
exon 8)
-
16/881 (2%)
Within
LOC102162801
Fragment of processed pseudogene (from exon
5 to exon 8)
-
The specificity of the EEF1A1 pseudogene status was ascertained on the basis of homologies of 5´ UTR and/or 3´ UTR of EEF1A1 gene and the query
sequences.
b
It could not be unequivocally proved whether this is EEF1A1 or EEF1A2 pseudogene.