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TECHNICAL NOTE
Identification of 104 rapidly-evolving nuclear protein-codingmarkers for amplification across scaled reptiles using genomicresources
Daniel M. Portik • Perry L. Wood Jr. •
Jesse L. Grismer • Edward L. Stanley •
Todd R. Jackman
Received: 30 November 2010 / Accepted: 31 May 2011 / Published online: 17 June 2011
� Springer Science+Business Media B.V. 2011
Abstract As the fields of molecular systematics and
phylogeography are advancing, it is necessary to incorpo-
rate multiple loci in both population and species-level
inference. Here, we present primer sets for 104 intronless
orthologus exons designed for amplification in all squa-
mates. When comparing the Anolis genome to the Gallus
genome, all the markers have less than 67.2% DNA
sequence identity, the percent identity of the first third of
the commonly used nuclear marker RAG-1. The rate of
evolution in these markers is therefore greater than nuclear
markers commonly used, and we demonstrate their use-
fulness for both phylogeographic and phylogenetic studies.
Keywords Nuclear markers � Squamates � Primers �Marker development � Intronless exons
A recent trend in both phylogenetics and phylogeography
is the development of new analytical techniques that
require the use of multiple nuclear loci (Brito and Edwards
2009; Edwards 2009; Heled and Drummond 2008; Hey
2010; Hey and Nielsen 2004; Pritchard et al. 2000), how-
ever, accurate estimates are only possible by using a large
number of loci (Leache and Rannala 2010). As the results
of these analyses have important implications for species
delimitation, identification of management units, and con-
servation policy, it is critical that a library of nuclear
markers is readily available to researchers. Anonymous
nuclear loci are not time or cost-effective for large-scale
studies, and typically exhibit low levels of variation and
informative sites (Jennings and Edwards 2005; Lee and
Edwards 2008). The importance of developing nuclear
markers with targeted levels of informative sites has long
been recognized (Graybeal 1994), and an attempt to
develop many informative nuclear protein-coding loci
(NPCL) has been made Townsend et al. (2008). However,
because these markers were developed based on distant
genomes (pufferfish and human), they are not ideal for
use within any one particular vertebrate group. Here, we
describe a method for developing nuclear markers from
existing genomic resources and present 104 rapidly-evolving
NPCL designed primarily for squamates using the Anolis and
Gallus genomes.
Selection of candidate genes was the result of a multi-step
filtering process involving three databases. First, a database
of intronless genes was obtained from the SEGE Database
(Sakharkar and Kangueane, 2004), which contains amino
acid sequences of all intronless human genes. Genes
Electronic supplementary material The online version of thisarticle (doi:10.1007/s12686-011-9460-1) contains supplementarymaterial, which is available to authorized users.
D. M. Portik � P. L. Wood Jr. � J. L. Grismer �E. L. Stanley � T. R. Jackman
Department of Biology, Villanova University,
800 Lancaster Avenue, Villanova, PA 19085-1699, USA
D. M. Portik (&)
Museum of Vertebrate Zoology and Department of Integrative
Biology, University of California, 3101 Valley Life Sciences
Building, Berkeley, CA 94720-3160, USA
e-mail: [email protected]
Present Address:J. L. Grismer
Natural History Museum and Biodiversity Research Center
and Department of Ecology and Evolutionary Biology,
University of Kansas, 1345 Jayhawk Blvd, Dyche Hall,
Lawrence, KS 66045, USA
Present Address:E. L. Stanley
Department of Herpetology, American Museum of Natural
History, Central Park West at 79th Street, New York,
NY 10024, USA
123
Conservation Genet Resour (2012) 4:1–10
DOI 10.1007/s12686-011-9460-1
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2 Conservation Genet Resour (2012) 4:1–10
123
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Conservation Genet Resour (2012) 4:1–10 3
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4 Conservation Genet Resour (2012) 4:1–10
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R8
79
GA
TG
AT
GA
GG
AT
GA
GG
WA
GA
GG
ML
LP
LU
SF
1C
GC
AC
CG
TS
AA
GG
TS
AC
YC
TG
AC
TC
C6
2.0
F1
-R1
:1
10
4;
F2
-R1
:
70
6
SB
F2
MA
CA
GG
SA
AG
AA
GC
GW
GG
GA
AG
MG
GT
R1
TG
TC
YT
TG
CC
CC
GG
TT
GC
TR
MX
RA
5F
1Y
AT
TT
TG
GC
AA
AR
GT
CC
GT
GG
GA
AR
A4
6.2
F1
-R1
:2
31
7;
F2
-R2
:
10
20
AF
SB
SB
AF
SB
AF
F2
KG
CT
GA
GC
CT
KC
CT
GG
GT
GA
R1
WT
GT
GC
TG
CA
TA
TG
CT
GT
WA
TC
TC
WG
GT
R2
YC
TM
CG
GC
CY
TC
TG
CA
AC
AT
TK
MY
ST
3F
1R
CA
GA
AC
AT
GG
AG
AC
TA
GC
CC
50
.3F
1-R
1:
13
09
MB
R1
YC
CC
AT
CA
TY
CC
CA
TY
TG
CA
TC
TG
C
N4
BP
2F
1S
AA
CA
AA
CW
AT
GG
GR
CA
GA
GR
GT
SA
AA
AR
A5
4.5
83
3S
B
R1
TC
AC
TT
TC
TT
CC
AC
AW
AT
GT
RC
TT
TT
NA
IPF
1A
AG
GA
GA
GG
CC
GG
CA
GT
GG
A5
5.2
80
9A
FA
F
R1
TG
GC
CA
AA
GA
CG
TT
GG
GC
TG
TG
NP
AT
F1
YA
AT
GC
TG
TT
TC
CA
GC
AT
CA
C5
2.7
F1
-R1
:4
55
MB
R1
GW
TC
TR
GG
AG
GA
GT
CC
GY
AA
TK
GC
TG
Conservation Genet Resour (2012) 4:1–10 5
123
Ta
ble
1co
nti
nu
ed
Gen
eP
rim
erS
equ
ence
50
to30
A.
A.
%ID
Ex
pec
ted
bas
ep
airs
TV
AL
CB
OM
GF
1R
YT
GC
CM
CA
CG
GA
CT
TC
A6
6.2
F1
-R1
F2
-R2
AF
MB
F2
CC
AG
GA
AY
AT
GG
TG
GA
AA
GR
G
R1
YK
GC
RG
CC
GC
TT
CG
TA
A
R2
YT
GG
AR
TA
GT
TT
GT
GG
TR
AT
R
PD
ZD
2F
1Y
TK
GA
GT
CA
GA
TG
AW
GA
RC
AA
AT
TG
AR
A4
7.0
F1
-R1
:1
53
5;
F2
-R2
:
70
4
MB
AF
MB
SB
F2
GG
AA
CA
AR
CC
AG
GR
CC
AA
AG
R1
GT
GC
GC
TG
AA
TT
TC
CT
GA
CC
R2
CT
CA
AT
GA
AW
ST
GC
GC
TG
AA
TT
PH
F3
F1
TG
CC
AG
TG
GA
TG
AC
AT
YC
TT
CA
RA
GC
C5
5.0
F1
-R1
:1
29
2;
F1
-R2
:
11
42
SB
AF
MB
R1
CC
AA
GG
RT
CA
CT
RT
GR
CG
CC
TY
R2
KA
CA
GG
KG
GC
CA
CG
GC
AT
CA
W
PIG
WF
1W
GT
GG
AY
TT
CC
CG
CA
GT
WT
CC
RC
GR
CG
61
.96
75
MB
R1
AG
GT
TS
GC
CA
TS
CG
GC
GG
GA
PK
DR
EJ
F1
36
0G
TA
GT
TT
CA
VC
AG
GG
TG
CA
AA
GG
GT
AT
CT
TG
T5
7.3
F1
36
0-R
24
80
:1
15
5;
F1
90
0-R
25
40
:
69
1;
GE
CK
OF
1-
GE
CK
OR
1:
60
4
SB
SB
AF
F1
90
0A
TT
AT
AT
TA
TA
TG
GT
TT
GA
CC
TA
TG
GC
TA
CA
CA
AC
GE
CK
OF
1M
AW
TT
TC
CA
TG
GT
GG
TG
SA
GA
TA
TG
T
GE
CK
OR
1T
CA
GT
GG
CA
CA
AA
GA
CA
TT
GC
R2
48
0T
TT
CA
GT
AT
CT
TT
DG
CC
CT
TA
TT
TG
CC
TC
AT
TC
R2
54
0G
AH
GG
CA
GT
GG
CT
TT
TA
CT
AA
TC
AC
AA
PR
DM
2F
1C
AY
CA
GC
GS
MG
GG
TY
CA
CG
AG
CG
56
.47
29
AF
R1
TC
GA
AG
WR
CC
GG
RC
TG
TG
CT
G
PR
LR
F2
AR
YG
AA
GA
CC
AG
CA
AC
TG
AT
GC
60
.37
70
SB
SB
R4
GG
CA
AG
GC
CT
CC
AY
TT
TG
Pse
ud
oZ
Ex
on
15
F1
SC
AG
CC
MC
TG
GA
YT
TC
TC
AG
G5
2.7
10
22
SB
R1
KG
GA
KC
CA
AA
SA
AC
GA
KG
AG
AT
G
Pse
ud
oZ
Ex
on
21
F1
YT
GA
TM
AA
RG
GC
TC
CG
TG
GA
TG
AA
G5
4.4
11
15
AF
SB
R1
RT
TC
AG
GT
CS
AT
YT
GG
AT
CA
TA
TC
TT
TC
RA
I4F
1W
CA
GA
KA
TY
TC
AG
AR
AA
TG
GC
TC
TG
AT
C6
5.5
43
7A
F
R1
YT
TK
TC
AR
YT
TG
TC
CT
GY
AG
CC
TT
TT
RA
T
RIF
1F
1C
CA
CG
AT
AC
CA
YA
CC
MG
RA
G5
0.0
F1
-R1
:6
19
;F
2-R
2:
89
8
MB
F2
MT
GY
CA
GC
AC
AA
RA
GA
AG
CA
R1
YT
GC
TT
CT
YT
TG
TG
CT
GR
CA
R2
GG
AW
GC
TG
AA
GG
AG
AC
CA
RIK
EN
F2
ST
TT
TA
TT
CR
WT
GC
CA
TC
YT
TA
TC
CT
TA
A5
3.1
70
2M
BS
B
R1
YK
CC
KT
TC
TA
GT
TC
TC
CA
GT
TA
TT
AC
AG
6 Conservation Genet Resour (2012) 4:1–10
123
Ta
ble
1co
nti
nu
ed
Gen
eP
rim
erS
equ
ence
50
to30
A.
A.
%ID
Ex
pec
ted
bas
ep
airs
TV
AL
CB
RL
FF
1S
AA
AC
AC
CT
YC
GM
AG
GG
CT
CA
TC
C5
6.9
98
1A
F
R1
CT
CC
CC
TC
TC
KT
GC
TT
CY
AG
TY
RM
I1F
1G
AG
TG
GA
AA
CC
TG
GC
TA
TC
AT
CT
AC
AT
56
.89
41
MB
R1
AA
AC
AA
AT
CG
TC
AT
CT
AA
TG
GA
AA
GT
C
RN
AS
EL
F1
GG
GA
AG
GA
GG
AA
GC
CC
TG
AG
GT
T6
3.0
F1
-R1
:5
01
;F
2-R
1:
73
4;
F3
-R2
:7
25
;
F4
-R2
:5
19
MB
F2
WT
GG
WT
GG
AC
RC
CS
CT
TC
AY
AG
TG
F3
GA
CR
CC
SC
TT
CA
YA
GT
GC
TG
F4
TC
AT
GG
AR
GC
TG
CT
TG
GT
AT
R1
YC
TT
GC
WC
CY
TT
TT
CA
CA
CA
RC
R2
CT
TG
CW
CC
YT
TT
TY
AC
AC
A
SE
C1
6A
F1
WC
AG
AA
CC
AA
GA
RG
TK
YT
GC
CM
AG
YG
AG
CC
50
.61
13
7A
F
R1
GG
CT
GR
GC
CA
AG
YT
RT
AR
CT
YT
GR
TT
MG
GC
TG
SN
X1
9F
1R
CC
TG
CA
AC
GT
GC
TG
YT
GC
C5
3.8
91
6A
F
R1
TG
TG
CT
CY
CG
GG
CW
GT
GA
TG
GT
SO
X1
0R
1A
TG
TG
CT
AC
TT
GC
AT
AA
AT
AA
GG
47
.81
79
6M
BM
B
F1
YA
TW
GG
CC
TT
CT
AG
AT
GA
GG
A
SP
EN
F1
YA
GC
GC
MA
AG
AT
CA
GY
CA
GA
TC
CC
61
.4F
1-R
1:
77
3;
F1
-R2
:
10
03
MB
R1
GS
GT
GA
CG
CT
GT
GC
GG
GG
GC
AT
R2
SA
CG
TC
GG
RC
TG
SA
CG
GG
GG
C
TL
R3
F1
TG
AT
TG
CA
CY
TG
TG
AM
AG
YA
TW
GC
TT
GG
TT
TG
66
.35
10
MB
R1
WA
TA
AT
CT
TC
CT
GC
TC
CT
TY
TT
AT
GC
TL
R4
F1
RA
GA
GT
GC
TY
CG
KA
TY
AC
CA
AG
A5
9.6
13
27
AF
R1
KC
GG
RA
CA
GK
CC
CA
GY
YT
YT
GC
C
TL
R5
F1
TG
GC
TR
AA
TG
AA
AC
CA
AT
GT
MA
CY
YT
AG
CT
GG
62
.05
30
AF
R1
AC
AC
AC
CA
SC
CA
TC
YT
TG
AG
AA
AC
TG
C
TL
R7
F1
WG
GC
CC
AG
GG
RC
AY
RS
AR
AG
GG
A6
6.0
45
3A
F
R1
KT
GC
CA
CT
TK
YA
AT
RT
AC
TT
GT
TK
GT
TR
AN
K1
F1
SA
AG
TT
CA
TT
GY
WG
GC
TT
GA
AS
TG
TG
AG
G6
4.4
11
35
SB
R1
WA
CA
GT
WC
GY
TC
AG
CC
TC
TC
CT
GA
UB
N1
F2
CC
TC
CC
TS
GA
AG
CM
GT
CT
CT
AA
GG
AA
CT
67
.29
66
MB
SB
SB
R2
YM
AC
AG
CW
GG
CT
TY
AG
GG
AG
GA
GG
TC
AG
US
PL
1F
1W
TG
GC
TT
GA
RT
GT
GA
TG
AY
T4
8.2
12
76
MB
R1
YT
TY
TC
CT
TT
TT
AG
CW
TT
AA
G
WD
R8
1F
1W
TG
GG
GT
YG
TS
CA
GC
TC
TT
TG
AC
CA
G6
1.3
11
09
SB
MB
AF
SB
AF
R1
CT
GG
GC
CA
CR
AA
GC
AG
TC
TG
TG
TA
SA
GG
TA
GA
A
Conservation Genet Resour (2012) 4:1–10 7
123
containing over 1,500 base pairs (bp) were selected. Second,
we obtained exons consisting of over 1,500 bp from the
Gallus gallus database by screening the three databases
(ILD, ULD, and EID) at the University of Toledo (Shepelev
and Fedorov 2006). Last, we used Biomart (Smedley et al.
2009) to obtain all Anolis exons consisting of over 1,500 bp.
We focused on intronless genes because they allow a greater
chance for specific lengths of target sequence to be obtained,
and conservation of a reading frame allows better align-
ments. Human intronless genes were searched for in the
Gallus genome using HomoloGene (NCBI), keeping only
genes with less than 70% amino acid identity. As a pre-
caution, the Human-Gallus genes were compared to the
Anolis genome using the Gallus version of the correspond-
ing human ortholog to check for the presence of paralogs in
the Anolis genome. The Anolis and Gallus exons obtained
were first compared to Anolis or Gallus genomes using a
discontiguous megablast in Geneious v5.3.6 (Drummond
et al. 2011) to discover and remove paralogs and remove
genes with DNA identities of over 75%. The tBlastN func-
tion was then used for whole exon comparisons to screen the
Anolis-Gallus genes for our ultimate cutoff of 67.2% DNA
identity (faster than the first third of RAG-1). Across all
databases, only orthologous genes over 1,500 bp with less
than 67.2% DNA identity between Anolis and Gallus were
selected. Although use of the three databases may appear
redundant, there were cases in which useful genes were
revealed through the use of one database that were absent
from the other two databases.
Primers were designed from exon alignments between
Gallus and Anolis using Primer3 (Rozen and Skaletsky
2000). Amplifications occurred in 25lL volume reactions
initiated at 95�C for 2 min followed by 35 cycles of
95�C for 35 s, 50�C for 35 s and 72�C for 1 min 35 s (with
extension increasing 4 s per cycle) as in Portik et al.
(2011).
Initial sorting of exons over 1,500 bases produced over
500 exons from the three databases. After the filtering
process, 104 genes were selected for primer design in
squamates. From these 104 genes, primer sets were
developed for 170 gene fragments ranging in size from 407
to 2,492 base pairs, with the average fragment containing
897 base pairs. We present results from 70 genes tested
in at least one squamate group in Table 1, and 34 untested
genes are presented in Online Resource 1. Resulting
genetic diversity indices are presented in Table 2.
We have developed 104 rapidly-evolving orthologous
NPCL useful for both interspecific and intraspecific studies
within squamates. We have tested a subset of these markers
using skinks, varanids, gekkonids, cordylids, and agamids.
Several markers have proven useful for diagnosing intra-
specific populations in skinks (EXPH5, KIF24, Table 2;
Portik et al. 2010; Portik et al. 2011) as well as delimitingTa
ble
1co
nti
nu
ed
Gen
eP
rim
erS
equ
ence
50
to30
A.
A.
%ID
Ex
pec
ted
bas
ep
airs
TV
AL
CB
XIR
P1
F1
YG
GW
GA
TG
TC
AR
AA
CA
GC
CA
AG
TG
G6
3.5
F1
-R1
:6
97
;F
2-R
2:
12
47
;F
3-R
3:
11
16
SB
F2
RA
AC
AG
CC
AA
GT
GG
CT
CT
TT
GA
AA
CK
CA
AC
CY
A
F3
YG
AG
AA
GG
GA
GA
TC
TG
GA
CT
AY
CT
GA
AG
R1
RA
AC
CT
TT
TG
CC
YC
CA
AC
AT
CT
CC
R2
YR
GG
CT
GG
TT
TT
CA
AA
AA
GC
CA
GG
TR
GA
T
R3
AA
AT
CC
CC
TT
TG
GA
GA
CA
TT
AA
CG
TT
AR
AC
TT
T
ZH
X3
F1
YC
GG
AA
RT
GG
TT
YA
GC
GA
YA
GG
A6
0.6
85
6A
F
R1
SC
GA
CT
RT
CM
CC
AA
AC
CA
GC
G
ZN
F4
51
F1
WC
GT
TG
TC
GT
AA
TK
CT
GG
CC
C6
1.3
56
9M
B
R1
YC
CT
CC
AT
GR
AA
YC
GG
CT
CA
TR
TG
CA
Inca
ses
of
mu
ltip
lep
rim
erse
ts,
spec
ific
com
bin
atio
ns
of
pri
mer
sp
airs
and
exp
ecte
dp
rod
uct
size
are
giv
en.
Res
ult
sar
eab
bre
via
ted
asfo
llo
ws:
SB
rep
rese
nt
sin
gle
-ban
dP
CR
pro
du
cts
of
exp
ecte
dsi
ze,
MB
mu
ltip
leb
and
s,A
Ffa
ilu
reto
amp
lify
.B
lan
kn
ot
test
ed.
Th
efo
llo
win
gta
xo
no
mic
abb
rev
iati
on
sar
eu
sed
:T
,T
rach
ylep
is(S
cin
cid
ae);
V,
Va
ran
us
(Var
anid
ae);
A,
Aca
nth
osa
ura
(Ag
amid
ae);
L,
Lei
ole
pis
(Ag
amid
ae);
C,
Co
rdyl
us,
Ch
am
aes
au
ra,
Pla
tysa
uru
s,P
seu
do
cord
ylu
s(C
ord
yli
dae
);B
,B
ava
yia
(Gek
ko
nid
ae).
Pri
mer
sw
ith
aste
risk
sp
ub
lish
edin
Po
rtik
etal
.(2
01
0)
8 Conservation Genet Resour (2012) 4:1–10
123
species boundaries in cordylid lizards (KIF24, PRLR,
Table 2; Stanley et al. 2011). Several markers have amplified
broadly across five diverse squamate families (Agamidae,
Cordylidae, Gekkonidae, Scincidae, Varanidae) and have
potential for resolving higher-level squamate relationships
(Table 1).
Our identified NCPL have great potential for squamate
conservation efforts, as they can be used at a variety of
levels. A critical step to the protection of evolutionary
lineages is their initial identification, which is often com-
pleted using molecular evidence. The NCPL in this study
can be used individually or in a multilocus framework to
accomplish this task and allow evolutionary lineages to
be targeted for conservation efforts or protective status.
Alternatively, within particular species multiple NCPL can
be used to determine the genetic cohesiveness of popula-
tions in a metapopulation system and allow researchers to
define management units. These management units can be
assessed for genetic variation and informed decisions can
be made to protect the overall genetic diversity within a
species.
Acknowledgments We would like to thank Nicole Rocha, Andrew
Feiter, Arianna Kuhn, Maria Tempera, Lauren Adderly, and Stuart
Love Nielsen for contributions in laboratory work. We thank Aaron
Bauer for providing many tissue samples used in this study. Funding for
this project was provided by a National Science Foundation grant (DEB
0515909) and by the Department of Biology at Villanova University.
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Table 2 Nucleotide diversity of markers for sequenced squamate species
Gene Acanthosaura (6 sp.) Cordylus (14 sp.) Leiolepis (5 sp.) Trachylepis (2 sp.) Varanus (8 sp.)
Intra Inter Intra Inter Intra Inter Intra Inter Intra Inter
RAG-1 0.0014–0.0145 0.1461 0.0019–0.0089 0.0112 0.0024–0.0130 0.0298
C10orf71 0.0015–0.0058 0.0067
EXPH5 0.0013–0.0098 0.0217
KIAA1217 0.0043–0.0056 0.0311
KIAA1549 0.0092–0.0786 0.0406 0.0016–0.0092 0.0228
KIAA2018 0.0023–0.0072 0.0105 0.0015–0.0058 0.0067
KIF24 0.0095–0.0220 0.0317 0.0063–0.0191 0.0235
MXRA5 0.0034–0.0064 0.0179 0.0023–0.0133 0.0302
PKDREJ 0.0016–0.0056 0.0115 0.0012–0.0184 0.0714
PRLR 0.0016–0.0501 0.0547 0.0015–0.0189 0.0220
Diversity calculations were conducted at the interspecific (all sequenced species within the genus) and intraspecific (within each species,
presented as a range) levels
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