[ Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use. 
?/dmtSO    Nwbar3    ?toch2009    3SN KU5-n03 
MOLECULAR 
PHYLOGENETICS 
DEVOLUTION        r^ //,A, 
EDITOIl-IN-CHIEF: 
f/ORRlS GOOOV^N 
This article appeared in a journal published by Elsevier. The attached 
copy is furnished to the author for internal non-commercial research 
and education use, including for instruction at the authors institution 
and sharing with colleagues. 
Other uses, including reproduction and distribution, or selling or 
licensing copies, or posting to personal, institutional or third party 
websites are prohibited. 
In most cases authors are permitted to post their version of the 
article (e.g. in Word or Tex form) to their personal website or 
institutional repository. Authors requiring further information 
regarding Elsevier's archiving and manuscript policies are 
encouraged to visit: 
http://www.elsevier.com/copyright 
Molecular Phylogenetics and Evolution 50 (2009) 654-660 
!^ 
<'???- ?<av- 
Contents lists available at ScienceDirect 
Molecular Phylogenetics and Evolution 
journal homepage: www.elsevier.com/locate/ympev 
PHYlOfeENETICS 
iEVOlUIION 
Short Communication 
A well-tested set of primers to amplify regions spread across the avian genome 
Rebecca T. Kimball ?*?*, Edward L Braun^ F. Keith Barker ^ Rauri C.K. Bowie''?''?^ Michael J. Braun^. 
Jena L Chojnowski'', Shannon J. Hackett^ Kin-Lan Han ^'^?^, John Harshman'^?'\ Victoria Heimer-Torres ^ 
Wallace Holznagel^. Christopher J. Huddleston^, Ben D. Marl<s', Kathleen J. MigliaJ, William S. Moore J. 
Sushma Reddy^ Frederick H. Sheldon', Jordan V. Smith'', Christopher C. Witt'?^ Tamaki Yuri'*'^ 
'Department of Zoology, University of Florida, P.O. Box 118525, Gainesville, FL 32611, USA 
^Bell Museum of Natural History, University of Minnesota, St. Paul, MN 55108, USA 
'^Zoology Department, Field Museum of Natural History, Chicago, IL 60605, USA 
'' Museum of Vertebrate Zoology & Department of Integrative Biology, University of California, Berkeley, CA 94720, USA 
'^DST/NRF Centre of Excellence at the Percy FitzPatrick Institute, Department of Botany and Zoology, Stellenbosch University, Matieland 7602, South Africa 
'Department of Vertebrate Zoology, Smithsonian Institution, Suitland, MD 20746, USA 
^Behavior, Ecology, Evolution, and Systematics Program, University of Maryland, College Park, MD 20742, USA 
''4869 Pepperwood Way, San Jose, CA 95124, USA 
' Museum of Natural Science, Louisiana State University, Baton Rouge, LA 70803, USA 
' Department of Biological Sciences, Wayne State University, Detroit, Ml 48202, USA 
''Department of Biology and Museum of Southwestern Biology, University of New Mexico, Albuquerque, NM 87131, USA 
ARTICLE     INFO 
Article history: 
Received 9 June 2008 
Revised 16 November 2008 
Accepted 18 November 2008 
Available online 6 December 2008 
1. Introduction 
DNA markers have been used to examine a broad range of 
biological problems, including those in phylogenetics, population 
genetics and the identification of individuals. Nuclear loci provide 
a rich source of information regarding evolutionary history and 
they allow researchers to examine many unlinked markers that 
have the potential to provide insight the coalescent process as well 
as introgression or hybridization events (e.g. Maddison, 1997; Nic- 
hols, 2001). Information about the nuclear genome can be collected 
by sequencing or by examining single nucleotide polymorphisms 
(SNPs) (Sunnucks, 2000; Avise, 2004). Unlike mitochondrial 
regions where virtually universal primers are available for verte- 
brates (e.g., Sorenson et al., 1999), extensively tested primer sets 
for nuclear regions that are likely to work on the majority of spe- 
cies within a group are not available. 
In phylogenetic studies, nuclear sequence data have provided 
additional insights into avian evolution (e.g., Avise, 2004; Hackett 
et al., 2008). The nuclear genome is heterogeneous, consisting of 
coding   regions,   introns,   untranslated   regions   (UTRs),   and 
* Corresponding author. Fax: +1 352 392 3704. 
E-mail address: rkimball@ufl.edu (R.T. Kimball). 
1055-7903/$ - see front matter ? 2008 Elsevier Inc. All rights reserved. 
doi:10.1016/j.ympev.2008.11.018 
intergenic regions. The markers exhibit varying patterns of molec- 
ular evolution (e.g., mutation rates, base composition, etc.), and 
thus can be used at a variety of different taxonomic levels for dif- 
ferent types of phylogenetic questions. In addition to substitutional 
variation, non-coding regions often exhibit length variation, pro- 
viding indel (insertion-deletion) characters that can be added to 
the region's information content (e.g.. Fain and Houde, 2004). 
Population-level questions can be examined using non-coding 
regions such as introns. Substitutional (and indel) variation of mul- 
tiple individuals of a single species or a set of closely related 
species provide insights into population divergence times and 
ancestral population sizes (e.g., Jennings and Edwards, 2005; 
Aitken et al., 2004). SNPs present in these rapidly evolving regions 
are increasingly being used as markers because they provide an 
opportunity to assess a large number of unlinked loci for a range 
of population questions (Brumfield et al., 2003; Morin et al., 
2004). In addition to population genetics, SNPs also have the 
potential to be used in parentage studies and for quantitative 
genetic studies in wild populations. 
In birds, there are several studies which have published sets of 
primers to amplify nuclear regions, particularly intron regions (e.g., 
Friesen et al., 1999, 1997; Primmer et al., 2002; Waltari and 
Edwards, 2002; Slade et al., 1993; Backstrom et al., 2008). How- 
ever, many of these primers have been tested on a limited set of 
R.T. Kimball et al. /Molecular Phylogenetics and Evolution 50 (2009) 654-660 655 
Table 1 
Characteristics of tiie loci included in tliis study. 
Locus Clir" Main regions'' bp in Gallus"^ % Coding" Exon rate*^ Intron rate"^ UTR rate'' Mix rate'' Effort' 
ACOl Z Intron 9 1060 3 ? 3.02 ? _ H 
ALDOB Z Introns 3- -7 2440 24 0.91 3.14 - 2.49 H 
ARNTL 5 Intron 12 550 19 ? 2.87 ? _ H 
BDNF 5 Exon 1 690 100 0.72 - ? - H 
CLOCK* 4 Intron 10 670 10 ? 2.97 ? - M 
CLOCK* 4 3'UTR 530 <1 ? - 1.14 - H 
CLTC 19 Introns 6- -7 1930 22 0.91 3.07 - 2.59 H 
CLTCLl 15 Intron 7 730 42 0.79 2.89 - 2.44 H 
CRYAA 1 Intron 1 1190 21 0.76 3.71 - 3.05 H 
CSDEl 26 Intron 5 470 27 0.30 3.14 ? 2.38 M 
CYPigAl" 10 5'UTR 530 22 1.47 - 2.34 1.79 H 
EEF2 28 Introns 5- -9 1700 33 0.85 3.79 ? 2.72 H 
EGRl 13 Exon 2, 3' UTR 1740 71 1.04 1.17 1.13 H 
FGB 4 Introns 4- -7 2580 22 0.92 2.86 ? 2.53 H 
GARS 2 Intron 11 303 19 - 2.69 - - M 
GHl 27 Introns 2- -3 1030 24 1.22 3.34 - 2.74 H 
HMGN2 23 Introns 2- -5 1600 13 0.69 3.88 - 3.47 H 
H0XA3 2 Intron 1 1460 6 ? 2.51 ? _ H 
IRFl 13 Intron 2 920 5 ? 3.49 ? - H 
IRF2 4 Intron 2 700 12 ? 2.06 ? - H 
NAT! 5 14 Intron 4 1010 12 ? 3.16 ? - M 
MB 1 Intron 2 950 3 - 1.96 - - H 
IMUSK Z Intron 3 700 28 0.94 3.16 - 2.72 H 
IMYC 2 Intron 2,1 ;xon 3, 3' UTR 1240 45 0.86 2.69 0.89 1.30 H 
NGF 26 Exon 4 750 95 1.67 - - - H 
NTF3 1 Exon 2 730 100 1.01 ? ? - H 
PARK7 21 Intron 2 700 14 ? 3.29 ? - M 
PAXIPl 2 Intron 20 480 <1 ? 2.53 ? - M 
PCBD 6 Introns 2- -3 1190 11 0.73 3.06 ? 2.68 H 
PER2 9 Intron 9 510 9 - 2.96 - - M 
RAPGEFl 17 Intron 18 1270 <1 - 2.83 - - M 
RHO 12 Introns 1- -3 1890 45 1.09 3.42 - 2.66 H 
SPINl Z Intron 2 940 14 ? 3.34 ? _ H 
TGFB2 3 Intron 5 560 3 ? 2.67 ? - H 
TPIMl 10 Intron 6 490 <1 ? 1.34 ? - H 
TXNDC12 8 Intron 6 479 5 ? 2.81 ? - M 
VIM 2 Intron 8 500 2 - 3.08 - - M 
" Chromosome in the chicken genome. 
'' Since exons in the 5' UTR of the chicken genome are not always annotated, our numbering system considered the first coding exon to be exon 1 (based on Build 2.1. 
assembly of the chicken genome released on May 2006 by the Genome Sequencing Center at Washington University in Saint Louis), and that the intron was numbered by the 
preceding exon. For previously published regions, published exon and intron designations were used [MYC (Harshman et al.. 2003); NGF (Bertaux et al., 2004). and TPMl 
(Primmer et al., 2002)]. For PAXIPl, the chicken genome lacked the 3' end, so numbering was based on the human genome. 
'^ Length in the chicken of the longest amplifiable region (excluding primer sequence). For MYC and GHl, this only includes regions amplified in the nested or semi-nested 
PCR. 
'' The percent of amplified product that is coding exon in the chicken. 
" The rate is the slope of the best fit line (constrained to go through the origin) of the p-distance regressed against the p-distances for all exons included in Hackett et al. 
(2008). For rates targeting a single region (e.g, a single intron) that have less than 20% exon, rates are only given for the intron. Mix is the rate of the combined region that 
contained introns, exons and/or UTRs. 
' High (H) effort are primers tested in ~200 species: Moderate (M) effort are tested in 42 species. 
* The two CLOCK segments are discontiguous and so are not included in Fig. 2. 
"* The amplified region includes the entire 5' UTR as well as upstream 5' non-coding sequence. 
taxa, and thus may not work in many groups. Here we present 
broadly tested primers for 36 nuclear loci (Table 1; including 
introns, exons, and UTRs) located on 21 chromosomes in the chick- 
en genome (Fig. 1). We have already collected a substantial dataset 
from these regions (e.g., Chojnowski et al., 2008; Yuri et al., 2008; 
Hackett et al., 2008), providing outgroups and a backbone for 
future studies that utilize the same regions. 
2. Loci and primers 
The primers presented here were tested either on 199 species 
(Table SI) representing all avian orders and most non-passerine 
families (High Effort in Table 1) or else on 42 selected species (Ta- 
ble SI) representing all major avian groups (Moderate Effort in 
Table 1). The primers presented here expand those published pre- 
viously (Harshman et al., 2003; Barker, 2004; Driskell and Chris- 
tidis, 2004; Zink et al., 2006; Cox et al., 2007; Barker et al., 2008; 
Chojnowski et al., 2008; Yuri et al., 2008; Hackett et al., 2008) in 
several major ways. First, we provide additional primers to ex- 
pand the utility of published loci, including alternative primers 
and internal primers that are anchored in highly conserved re- 
gions and can also be used both for nested PCR and to obtain 
shorter segments. Second, we provide well-tested primers that 
can be used to amplify segments of 16 additional loci. Finally, 
we provide detailed maps of all regions with multiple primers, 
indicating the locations of these primers. Based upon our tests, 
we include the primers (Table 2) that are most likely to success- 
fully amplify their target regions in the widest range of avian 
taxa; problems we have identified in the use of these primers 
are described below. 
Several strategies were used for the development of novel loci. 
Work on some loci was initiated before the publication of the 
chicken {Callus gallus) genome, and these loci were selected from 
among the available chicken sequences that included intron and 
656 R.T. Kimball et al./Molecular Phylogenetics and Evolution 50 (2009) 654-660 
? 1 
-MB A ^GARS L.PAXIP1 
_ 
-NTF3 -VIM 
-H0XA3 r\ 
- -CRYAA 
-TGFB2 r\ 
-FGB 
^k_BDNF 
^ -IRF2 ARNTL 
  
-CLOCK ^VPCBDI u y -MYG U U        \J      \J 
1                         2                        3                    4                         5                  6 
O                         /'^PER2 ^.a^TPMI          ^^             ^-^ 
W-TXNDC12 U              ^CYP19A1 (J   RHOO^EGRI 0-NAT15 
8                             9                   10                          12               13                  14 
A 
QpCLTCLI   o_RApQEFi  O-CLTC ?-PARK7 
15                      17                         19                 21 -SPIN1 
^ALDOB 
? --HMGN2 ^NGF 
;i 
?-G HI ^EEF2 a ^MUSK ^ACOI 
Fig. 1. Karyotype of the chicken (Callus gallus) with loci included in this study 
designated using the HUGO (Human Genome Organisation) name (Table 1). 
Locations of specific loci were determined using MapViewer at the National Center 
for Biotechnology Information. 
exon structure (and introns of a suitable length). Primers were 
tlien developed by comparing sequences from chicken, human, 
and when available, reptiles or other avian species. Later loci 
were selected as they had regions (particularly introns) that were 
of a suitable length in the chicken genome, and additional se- 
quence data was available from at least one other avian species 
(typically Taenopygia guttata, Melopsittacus undulatus and Columba 
livia). Four introns were selected for development because they 
were identified as non-canonical (U12-dependent) introns in hu- 
man genes (Levine and Durbin, 2001); primers for these were 
developed by comparing human sequences with the chicken 
orthologs. All non-canonical introns used here (those in GARS, 
NAT!5, PAXIPl, and RAPGEF) have AT-AC terminal dinucleotides 
rather than the GT-AG terminal dinucleotides found in canonical 
introns. 
Basic chromosome structure appears largely conserved within 
birds (e.g. Griffin et al., 2007), suggesting that most loci that are 
unlinked in the chicken genome (Fig. 1) will remain unlinked in 
other avian lineages. The 36 loci represent a wide variety of genes 
(Table S2), and the amplified regions include differing amounts of 
coding and non-coding sequence (Table 1). Evolutionary rates vary 
(Table 1) though in almost all cases introns evolve at higher rates 
than either UTRs or coding regions. 
For many loci, the primers we present amplify a single region, 
while for other loci we present a suite of primers that allow ampli- 
fication of larger regions (or the targeting of only a subset of the re- 
gion we amplified) that can include combinations of introns, 
coding exons and UTRs (Fig. 2). Amplification of longer regions is 
advantageous when a large amount of data from a single locus is 
desired, for example to reduce variance in gene tree estimation 
(e.g., Chojnowski et al., 2008) or to obtain a large amount of data 
likely to have evolved under similar models of evolution to exam- 
ine patterns of sequence evolution (e.g.. Lake, 1997). Furthermore, 
larger regions also allow comparison of patterns of change across 
different types of data (e.g., coding vs. non-coding segments). We 
emphasize, however, that the regions selected are biased towards 
introns because we have found that avian introns are useful for 
phylogenetics at both deep and shallow level. 
Amplification of a single region (one intron) is relatively 
straightforward in most cases, generally requiring a single primer 
set. For regions with more than one primer pair (most of these span 
multiple introns and/or exons) the position of the different primers 
relative each other and to intron/exon structure are presented 
(Fig. 2). Most primers were used for PCR and sequencing but a 
few were used primarily for sequencing to allow double-stranded 
contigs to be obtained. The sequencing primers presented in Table 
2 include only those that are in highly conserved regions (primarily 
exons) that should also be useful for PCR across a broad range of 
taxa if amplification of smaller regions was desired. A few primers 
were used in nested PCR strategies (see below). To obtain complete 
double-stranded sequences for longer introns, it was often neces- 
sary to use primers that were not conserved across taxa (not in- 
cluded in Table 2). However, these primers can be readily 
developed for specific taxa once sequencing with the PCR primers 
has been completed. 
In general we used standard PCR conditions (annealing temper- 
atures and Mg** concentrations are noted in Table 2). However in a 
few cases, alternative PCR strategies (e.g., adjusting magnesium 
levels, use of specialized polymerases, nested PCR) were required 
to amplify regions for a small proportion of taxa. There were certain 
primer sets that generally required use of particular PCR strategies 
or that were not robust in certain taxonomic groups. There are two 
paralogs of ALDOB that occasionally amplify with exon-anchored 
ALDOB primers. To obtain the majority of the region, a semi-nested 
PCR strategy using Ald.3F and Ald.7intR followed by amplification 
using Ald.3F and Ald.7R increased specificity. The region amplified 
using primers Ald.6F and Ald.SR can be used without nesting, 
though amplification of a smaller product may occur under some 
conditions. Nested PCR was used to amplify the entire region of 
GHl (GH-F874 and GH-R3108, followed by GH-F897 and GH- 
R1925) while a semi-nested strategy was used for MYC (MYC- 
FOR-01 and MYC-REV-47 followed by MYC-FOR-02 and MYC- 
REV-47). 
For some regions, one primer set worked much of the time, but an 
alternative set was often necessary to achieve amplifications across 
all taxa. This included amplification of FGB intron 5 (Fib5 and Fib6 
worked much of the time, but Fib.5F2 and Fib.6R2 often worked 
when those primers failed), MUSK (MUSK-13F2 and MUSK-I3R2 
were most robust), ACOl (AC01-I9F and AC01-I9R were used most 
commonly), and IRFl (about half of the taxa amplified with IRF1.2F 
and IRFl.3R, while the remainder were amplified with IRF1.2F2 
and IRFl .3R2). For MB, the use of MY02 and MYOintR avoided ampli- 
fication of a homopolymer run in many taxa, decreasing the need to 
clone products due to length polymorphisms. In addition, for some 
taxa, amplification of two smaller regions (MY02 and MYOintR plus 
MYOintF and MY03F) was necessary. For these regions, the specific 
primer combination that was most successful depended strongly 
upon phylogeny so it may be necessary to try multiple combinations. 
Alternatively, some primer sets amplified less exon relative to in- 
tron, such as in CLTCLl, where both CLTCLl.eSRnew and 
CLTCLl.e8Ralt amplified robustly when paired with CLTCLl .e7F, 
but use of CLTCLl .e8Rnew gave almost 150 bp more of exon 
sequence. 
Some primer sets failed to work, or amplified poorly, in specific 
groups. For example, in CLTC, CLTC.e7Falt and CLTC.e8Ralt amplifies 
intron 7 from the paralog CLTCLl in passerines; for passerines, prim- 
ers CLTC.e7Fpass2 and CLTC.e8Rpass amplified the correct paralog, 
but were not very robust. The CLK UTR primers (CLK.21F and 
CLI<.3'UTR) amplified poorly in Coraciiformes and Psittaciformes, 
while Ald.6F and Ald.SR amplified weakly in Psittaciiformes. The 3' 
end of RHO (covering all primers amplifying exons 2-4) did not am- 
plify from ratites (but did amplify from Tinamiformes). CYPl 9A1 and 
TPMl amplified robustly in only 75% of the taxa (150 species), 
though there was little phylogenetic pattern to which taxa amplified 
and which did not. The published primers for the SPINZ (Z-linked 
copy of spindling) occasionally amplify SPINW instead. 
R.T. Kimball et al. /Molecular Phylogenetics and Evolution 50 (2009) 654-660 657 
Table 2 
Primers used to amplify regions from Table 1. For regions with more than two primers (except CLOCK), see Fig. 2 for primer locations. 
Locus Primer name Sequence (5'-3') Use' Annealing T? (Mg** cone.) Authors'" or references 
ACOl AC01-I9F CTGTGGGAATGCTGAGAGATTT 55? (2.0 mM) FKB 
AC01-I9R2 CAACITTGICCIGGGGICITT FKB 
AC01-I9F2 CTCCTCTCAGGATCCAGACTT 55? (2.0 mM) FKB 
AC01-I9R CTGCAGCAAGGCACAACAGT FKB 
ALDOB AldB.3F GCCATTTCCAGCTCTCATCAAAG 55?(1.5mM) RTK & ELB 
AldBJintR CAYAGGAATGAATCRRGAATCAC 3 RTK & ELB 
AldBJR AGCAGTGTCCCTTCCAGGTASAC 58?(1.5mM) RTK & ELB 
AldB.6F GAGCCAGAAGTCTTACCTGAYGG 50?(1.5mM)" RTK & ELB 
AldB.SR GCTCKCCCGTATGAGAAGGTCAGYTT RTK & ELB 
AldB.4F GCAGGAACAAATGGAGAAACSAC 2 RTK & ELB 
AldB.SF GAGCGCTGTGCCCAGTACAAGAA 2 RTK & ELB 
AldBJF G11CTGGCTGCTGTCTACAAGGC 2 RTK & ELB 
AldB.4R GTSG111CTCCATTTGTTCCTGC 2 RTK & ELB 
AldB.SR ACTTGTTGGCAGATGCTGGCGTA 2 RTK & ELB 
AldB.SR AGCGCTGGAGRTCATGGTCT 2 RTK & ELB 
ARNTL ARNTL.12F TGGTTCAGTTTCATGAACCCTTG 55? (2.0 mM) RTK & ELB 
ARNTL.13R CCTGAAGCACRCTGTCCATGCT RTK & ELB 
BDNF ChickBDNF-5' ATGACCATCL1111CCTTACTATG 55? (2.0 mM) Sehgal and Lovette (2003) 
ChickBDNF-3' TCTTCCCLI11 lAATGGTTAATGTAC Sehgal and Lovette (2003) 
CLOCK CLK.10F CATGTGGATGATCTAGATAATCTGGC 55? (2.0 mM) RTK & ELB 
CLK.10R GYAATGTGTTTGCAGCCAAATCCA RTK & ELB 
CLK.21F CCTTCCAAAGCTCAGCCACAGTA 57? (2.0 mM) RTK & ELB 
CLICS'UTR GCCTACAGATAACAGATTACGTTTCATGC RTK & ELB 
CLTC CLTCeSFnew CTACATGAACAGAATCAGTGGAGAGAC 64?(1.5mM) RTK, JLC, & ELB 
CLTC.eVRnew GCTGCCACITTIGCTGCCTCTGAATA RTK, JLC, & ELB 
CLTC.eVFalt CAGAATCCTGATCTAGLI1 lACGAATGGC 53?(1.5mM) RTK, JLC, & ELB 
CLTC.e8Ralt CATTTCTCCAGAAGTTGI1IGCGTCC RTK, JLC, & ELB 
CLTC.e7Fpass2 CAGGTGCTCTCAGTGTG1G1GGAAGA 52? (2.5 mM) RTK, JLC, & ELB 
CLTC.eSRpass TGWGCTGGAACACTCTGGAACCG RTK, JLC, & ELB 
CLTCLl CLTCLl .e7F CACCAATGTTCTGCAGAATCCTGA 55? (2.5 mM) RTK, JLC, & ELB 
CLTCLl .eSRnew CCAGCTTATCTTCCTTNAGCCATTTCTC RTK, JLC, & ELB 
CLTCLl .eSRalt GGCTGAGCTGGTACACTCTGGAACTTCC 58? (2.0 mM) RTK & ELB 
CRYAA CRY.IF TTACTATYCAGCACCCCTGGTTCAA 63?(1.5mM) RTK & ELB 
CRY.2R CTGTL111CACTGTGCTTGCCRTGRAT RTK & ELB 
CSDEl CSDE.5F CTGGTGCTGTAAGTGCTCGTAAC 59? (2.5 mM) RTK, JVS, & ELB 
CSDE.6R CCAGGCTGTAAGGI1ICTAGGTCAC RTK, JVS, & ELB 
CYP19A1 CYP.5UTRF GAACTCATTCAATGCTGCCATRTGTG 52? (2.0 mM) RTK & ELB 
CYP.IR GGCCCTGGTATTGATGATGITTCnCAT RTK & ELB 
EEF2 EEF2.5F GAAACAGITTGCTGAGATGTATGTTGC 60?(1.5mM) RTK & ELB 
EEF2.7R GGITTGCCCICCIT GTCCTTATC RTK & ELB 
EEF2.6F CCTTGAYCCCATCTTYAAGGT 58?(1.5mM) RTK & ELB 
EEF2.9R CCATGATYCTGAL111CARGCCAGT RTK & ELB 
EEF2.7F GACGCGATCATGACCTTCAAGAAAGA 2 RTK & ELB 
EEF2.8F ACCTGCCTTCTCCTGTCACAG 2 RTK & ELB 
EEF2.6R CACCTTRAAGATGGGRTCAAG 2 RTK & ELB 
EEF2.8R TATGGCRGCCTCATCATCAGG 2 RTK & ELB 
EGRl" ZIF AGAAACCAGCTATCCCAAYCAA 1 60?(2.0mM) Chubb (2004) 
Z9R CTCAATTGTCCTTGGAGAAAAGG 1 Chubb (2004) 
Z5F CCl 11ICTCCAAGGACAATTGA 1 45? (2.0 mM) Chubb (2004) 
ZIOR AAAACAAAACTTCTGCCAC 1 Chubb (2004) 
Z3F CCCTATGCCTGCCCAGTGGAGTCC 2 Chubb (2004) 
Z7R CGTGAAAACCTCCGGTCACAG 2 Chubb(2004) 
FGB Fib3 CTGTAATATCCCGGTGGITTCAGG 1 55? (2.0 mM) FKB 
Fib4 ATTTCAGATGrn CACCICCCITTC 1 FKB 
Fib5 CGCCATACAGAGTATACTGTGACAT 1 54? (2.0 mM) FKB 
Fib6 GCCATCCTGGCGATTCTGAA 1 FKB 
Fib.5F2 GTACCTCATCCAGCCAGATCCT 1 54? (2.0 mM) SH, SR, RCKB, & JH 
Fib.6R2 TTCTGAATCAAAGTCCAGCC 1 SH, SR, RCKB, & JH 
Fib.6F TTGCAAAGAGTGGAGGGAAG 1 53.8? (2.0 mM) KJM 
Fib.8R CCATCCACCACCATCTTCTT 1 KJM 
Fib.7R TTGGCTGAriTTGTCATrrCC 2 KJM 
Fib.7F TGATGGAAGGAGCTTCACAG 2 KJM 
Fib.7intF CCTACTCAGAAGACAGGAGCTCA 2 KJM 
Fib.7intR TGAGCTCCTGTCTTCTGAGTAGG 2 KJM 
GARS GARS.llF G11CCTCTYATAGCTGAGAAGC 1 62? (2.5 mM) RTK, VHT. & ELB 
GARS.12R CGTCCTTCTTRTAAGLI1IGCC 1 RTK, VHT. & ELB 
GHl GH-F874 CCTTCCCWGCCATGCCCCriT CCAACC 3 70-60? (2.0 mM)"^ TY&MJB 
GH-R3108 CCGTAGTTCnCAGCAGGGCSTCCTCG 3 TY&MJB 
GH-F897 TGLIT GCCAACGCTGTGCTGAGG 1 60?(1.5mM) TY&MJB 
GH-R1925 TCCCTTCTTCCAGGTCCriTART 1 TY&MJB 
GH-F1391 GATGTCTCCACAGGAACGYA 2 TY&MJB 
GH-R1476 GATTTCTGCTGGGCATCATCCTTCC 2 TY&MJB 
{continued on next page) 
658 R.T. Kimball et al./Molecular Phylogenetics and Evolution 50 (2009) 654-660 
Table 2 (continued) 
Locus Primer name Sequence (5'-3') Use" Annealing T? (Mg** cone.) Authors'" or references 
HMGN2 HMG17.2F GCTGAAGGAGATACCAARGGCGA 62?(1.5mM) RTK & ELB 
HMG17.4R ClTlGGAGCIGCCl-ll-nAGG RTK & ELB 
HMG17.3F AACGGAGATCGGCGAGGTTATC 58?(1.5mM) RTK & ELB 
HMG17.6R2 AGCACCTTCGGLIIICTG TY 
HMG17.6R3 TAGAGTCACCAGAAGTACACAGTTATC TY 
HMG17.4F AAACCTGCYCCTCCRAAGCCAGAGCC RTK & ELB 
HMG17.5F AAAACGGAGATGCCAAAACAGAC TY 
HMG17.3R GATAACCTCGCCGATCTCCGTT RTK & ELB 
HMG17.5R CTGGTCTGTnTGGCATCTCC RTK & ELB 
H0XA3 H0XA3.1F GTCTCGGCAAAACACAAAGC 55? (2.0 mM) KJM 
HOXA3.2R AGCTGGGCACTTG1G1AAGC KJM 
IRFl IRF1.2F GCATGAGACCCTGGTTGG 52.5? (2.0 mM) KJM 
IRF1.3R ATGCTTAGCTGCATGI1 ICC KJM 
IRF1.2F2 TGCAGATCAATTCCAATCAAATAC 51.5? (2.0 mM) KJM 
IRF1.3R2 CAGGCGICl-llCTCCATGTC KJM 
IRF2 IRF2.2F ATGlCl-l-IGGGTCGGGTITA 55.5? (2.0 mM) KJM 
IRF2.3R GAAACTGGGCAATTCACACA KJM 
NAT! 5 NAT.4F ATCAGAGGGGTTCTCAAAGATGG 62? (2.0 mM) RTK, VHT, & ELB 
NAT.5R AGAGAAGGCTCTGGGCTTGTCGGTA RTK, VHT, & ELB 
MB MY02 GCCACCAAGCACAAGATCCC 52? (2.0 mM) Sladeetal.(1993) 
MY03F TTCAGCAAGGACCTTGATAATGACIT Heslewoodetal. (1998) 
MYOintF ATAAACCAGCCCATGCAGCCT 52? (2.0 mM) SH, SRRCKB.&JH 
MYOintR CCAGACTAAGAAATAGGTTGC SH, SR, RCKB.&JH 
MUSK MUSK-I3F CTTCCATGCACTACAATGGGAAA 50?(2.0mM) FKB 
MUSK-I3R CTCTGAACATTGTGGATCCTCAA FKB 
MUSK-I3F2 AAATAACCCGACCACCTGTAAA 50?(2.0mM) FKB 
MUSK-I3R2 TAGGCACTGCCCAGACTGTT FKB 
MYC MYC-FOR-01 TAATTAAGGGCAGCTTGAGTC 53? (1.75 mM) MJB, CH, & W/H 
MYC-REV-47 CTATAAAGACl 1 lATTAAAGGTATTTACAT MJB, CH, & W/H 
MYC-FOR-02 TGAGTCTGGGAGCl-llATTG 55?(1.7mM) MJB, CH, & W/H 
MYC-FOR-03 AGAAGAAGAACAAGAGGAAG 2 MJB, CH, & W/H 
MYC-FOR-05 CACAAACTYGAGCAGCTAAG 2 MJB, CH, & W/H 
MYC-REV-04 GGCTTACTGTGa C11CT 2 MJB, CH, & W/H 
MYC-REV-06 TTAGCTGCTCAAG111GTG 2 MJB, CH, & W/H 
NGF A1INGF5' GGTGCATAGCGTAATGTCCATG 52?(2.0mM) Sehgal and Lovette (2003) 
AIINGF3' ATAATTTACAGGCTGAGGTAG Sehgal and Lovette (2003) 
NTF3 ChickNT3-5' ATGTCCATCn GITl-l ATGTG 50? (2.0 mM) Sehgal and Lovette (2003) 
ChickNT3-3' Gl-IClTCClAlTl-l-ICnGAC Sehgal and Lovette (2003) 
PARK7 PARK.2F GCAGGCCTRRCTGGAAAAGARCC 56?(1.5mM) RTK & ELB 
PARK.3R TTCTGAGCTCCWAGRTTACC RTK & ELB 
PAXIPl PAX.20F CCCTCAGACACTGGATTAYGAATCAT 60? (2.5 mM) RTK, VHT, & ELB 
PAX.21R CCAAGGATTCCGAAGCAGTAAG RTK, VHT, & ELB 
PCBDl PCBD.2F AGAGCTGTGGGGTGGAACGAGGTGGA 64?(1.5mM) RTK & ELB 
PCBD.4R TCRTGGGTGCTCAAGGTGATGTGAAC RTK & ELB 
PCBD.3F CYAGAGTGGCTCTACARGCAGAA RTK & ELB 
PCBD.3R CCTTRirGTACACRTTGAACC RTK & ELB 
PER2 PER.9F CATCTTCAYCCAAATGACAGACC 55? (2.0 mM) RTK & ELB 
PER. 1 OR CCTGATTGGTGAATAGTCAAAAGG RTK & ELB 
RAPGEFl RAP.18F GCCCATCAAGAAGCTRCARTACAGAT 62? (2.5 mM) RTK, VHT, & ELB 
RAP.19R CTGGGRAGTGGCAAAAdTCTCAT RTK, VHT, & ELB 
RHO Rhod.S'F CACCTCRCAARCCGCAGCCAT 57?(1.5mM) RTK & ELB 
Rhod.exlR GTAGCAAAGAAGCCITCRAYGTAGC RTK & ELB 
Rhod.lF GAACGGGTACl-n Gl Cl'll GGAGTAAC 64?(1.5mM) RTK & ELB 
Rhod.lR CCCATGATGGCGTGGTTCTCCCC RTK & ELB 
Rhod.2F GAAATTGCTCTCTGGTCRCTGGTYGT 60?(1.5mM) RTK & ELB 
Rhod.4R AAAGAANGCYGGGATGGTCATGAAGA RTK & ELB 
Rhod.3F CTGAAGCCAGAGRTCAACAACGAAT RTK & ELB 
Rhod.3R ATCCCRCACGAGCACTGCAT RTK & ELB 
SPINl Spin319F TATGGACTAGAACTGCACAAAG 60? (2.0 mM) Handley et al. (2004) 
Spin472R AGACCATCCCCCTCCATTCATC Handley et al. (2004) 
TGFB2 TGFB2.5F GAAGCGTGCTCTAGATGCTG 58? (2.0 mM) Primmer et al. (2002) 
TGFB2.6R AGGCAGCAATTATCCTGCAC Primmer et al. (2002) 
TPMl Trop.SaF AATGGCTGCAGAGGATAA 60? (4.0 mM) Primmer et al. (2002) 
Trop.ebR TCCTCTTCAAGCTCAGCACA Primmer et al. (2002) 
TXNDC12 TXN.6F GGAAACCCCAGCTACAAGTATTTC 57?(1.5mM) RTK, JVS, & ELB 
TXN.7R GGCCTCCTTCATCCCITGG RTK, JVS, & ELB 
VIM VIM.8F GACCGTGGAAACTAGAGATGGAC 57?(1.5mM) RTK, JVS, & ELB 
VIM.9R GTCATCGTGATGCTGGGAAGnrC RTK, JVS, & ELB 
' Use refers to the way in which we used primer: 1 is a primer we used for both PCR and sequencing, 2 indicates primers used primarily for sequencing but likely to work 
for PCR as well; type 3 primers were used for PCR to produce an initial product used as template in a nested or semi-nested PCR strategy. 
'' For primers published by our group, primer authors are provided if there are questions about the primers or locus. 
?^ For this set of ALDOB primers we used a 10 s annealing time to limit amplification of a paralog. 
'' A gene map has been published in Chubb, 2004 and so is not shown in Fig. 1. 
?^ A touchdown PCR was used for this locus, stepping down from 70? to 60?, see also Yuri et al. (2008). 
R.T. Kimball et al. /Molecular Phylogenetics and Evolution 50 (2009) 654-660 659 
AC01 
??AC01-I9F 
?AC01-I9F2 
cu- -Tiol 
-AC0M9R 
-AC0M9R; 
CLTCL1 
-?CLTCLI .eBRnew 
rCLTCLLeSRalt 
ALDOB 
AldB.SF AldB.4F     AldB.SF AldB.6F 
?0> 
AldB.BR AldB.TR AldB.7intR AldB.SR 
CLTC 
CLTC.e6Fnew CLTC.e7Fpass2 -^ 
CLTC.e7Falt- 
CLTC.e7Rnew 
? CLTC.eaRalt 
hCLTCeBRpass 
EEF2 
EEF2.5F EEF2.6F 
-CD- 
EEF2.7F EEF2.8F 
 CZI DZh 
I EEF2.7R EEF: 
MB 
MY02 
MYOintR MY03F 
FGB 
Fib3 Fib.5F2?I 
Fib5- 
C^D- -cn- 
Fib4 
Fib.6F Fib.7F 
?m?nnz 
Fib6, Fib.6R2     Fib.7R 
BFib.7intF 
 ETUI 
BFib.7intR Fib.SR 
GH1 
-?GH-F874      QH-F1391 
-m i~4~i? 
?^QH-R1476      -^?C 
HMGN2 
HMG17.2F HMG17.3F     HMG17.4F 
m 0? -i^- Q^- 1^ 
HMG17.6R2 
?HMG17.6R3 
IRF1 
? IRF1.2F 
^IRFI .2F2 [T]  
^. ?JRF1.3R 
-?IRF1.3R 
PCBD1 
PCBD.2F PCBD.3F 
CO n}- -m 
MUSK 
- MUSK-I3F2 
?MUSK-I3F n> 
-MUSK-I3R 
^-MUSK-I3R2 
RHO 
Rhod.SF      Rhod.lF 
?^  D- 
Rhod.lR    Rhod.3R 
MYC MYC-FOR-01 ?I 
MYC-FOR-02- 
MYC-REV-06    MYC-REV-47 
H 250 bp 
Fig. 2. Primer locations for regions wliere we include more tlian two primers, scaled to the same length. Exons are boxes, with the exon number inside. Primer arrows are not 
proportional to primer length though the arrow tip indicates the 3' end of the primer. A single arrow was used when the 3' end of two primers differed by less than 10 bp. For 
some longer introns, additional non-conserved primers (not shown) were used for sequencing. 
3. Conclusions Acknowledgments 
Given the importance of comparing unlinl<ed loci, resources 
sucli as tiie one we have developed here should have broad useful- 
ness and a high probability of being used successfully. We hope fu- 
ture studies can build upon the sequences we have already 
collected in many avian taxa, and can contribute to a better under- 
standing of avian evolution, ecology and conservation. In addition, 
we hope to stimulate the development of additional well-tested loci 
that can be added to the resource that we have begun. Most impor- 
tantly, we hope that this resource stimulates other groups to com- 
pile similar easy to use resources in a single location for other 
taxonomic groups. 
We thank two anonymous reviewers for providing helpful sug- 
gestions on an earlier version of this manuscript. This research 
was funded by the National Science Foundation (DEB-0228682 to 
R.T.K., E.L.B. and D.W. Steadman; DEB-0228675 to SJH; DEB- 
0228688 to FHS and DEB-0228617 to WSM). We thank the following 
collections for tissue samples: American Museum of Natural His- 
tory, Auckland Museum, Australian National Wildlife Collection, 
Burke Museum of Natural History and Culture (University of Wash- 
ington), Field Museum of Natural History, University of Kansas Nat- 
ural History Museum & Biodiversity Center, L Densmore private 
collection, Louisiana State University Museum of Natural Science, 
660 R.T. Kimball et al./Molecular Phylogenetics and Evolution 50 (2009) 654-660 
Marjorie Barrick Museum (University of Nevada, Las Vegas), Mu- 
seum of Southwestern Biology (University of New Mexico), Museum 
of Vertebrate Zoology (University of California, Berkeley), Museum 
Victoria, National Museum of Natural History, San Francisco Zoolog- 
ical Garden, and Zoological Museum University of Copenhagen. 
Appendix A. Supplementary data 
Supplementary data associated with this article can be found, in 
the online version, at doi:10.1016/j.ympev.2008.11.018. 
References 
Aitken, N., Smith, S., Schwarz, C, Morin, P.A., 2004. Single nucleotide polymorphism 
(SNP) discovery in mammals: a targeted-gene approach. Mol. Ecol. 13, 1423- 
1431. 
Avise, J.C, 2004. Molecular Markers, Natural History, and Evolution. Sinauer 
Associates, Inc., Sunderland, MA. 
Backstrom.  N.,  Fagerberg,  S.,  Ellegren,  H.,  2008.  Genomics  of natural  bird 
populations: a gene-based set of reference markers evenly spread across the 
avian genome. Mol. Ecol. 17, 964-980. 
Barker. F.K., 2004. Monophyly and relationships of wrens (Aves: Troglodytidae): a 
congruence   analysis   of  heterogeneous   mitochondrial   and   nuclear   DNA 
sequence data. Mol. Phylogenet. Evol. 31. 486-504. 
Barker, F.K., Vandergon. A.J., Lanyon, S.M.. 2008. Assessment of species limits among 
yellow-breasted meadowlarks (Stume/to) using mitochondrial and sex-linked 
markers. Auk 125, 869-879. 
Bertaux, 0., Toselli-Mollereau, E.. Auffray. C. Devignes, M.D., 2004. Alternative 
usage   of  5'   exons   in  the   chicken   nerve  growth  factor  gene:   refined 
characterization of a weakly expressed gene. Gene 334, 83-97. 
Brumfield. R.T., Beerli, P., Nickerson, D.A., Edwards, S.V., 2003. The utility of single 
nucleotide polymorphisms in inferences of population history. Trends Ecol. 
Evol. 18, 249-256. 
Chojnowski. J.L., Kimball. R.T., Braun. E.L., 2008. Introns outperform exons in analyses 
of basal avian phylogeny using clathrin heavy chain genes. Gene 410,89-96. 
Chubb, A.L., 2004. New nuclear evidence for the oldest divergence among neognath 
birds: the phylogenetic utility of ZENK (i). Mol. Phylogenet. Evol. 30,140-151. 
Cox, W.A., Kimball, R.T., Braun, E.L.. 2007. Phylogenetic position of the New World 
quail (Odontophoridae): eight nuclear loci and three mitochondrial regions 
contradict morphology and the Sibley-Ahlquist tapestry. Auk 124. 71-84. 
Driskell, A.C.. Christidis, L., 2004. Phylogeny and evolution of the Australo-Papuan 
honeyeaters (Passeriformes. Meliphagidae). Mol. Phylogenet. Evol. 31,943-960. 
Fain, M.G., Houde, P., 2004. Parallel radiations in the primary clades of birds. 
Evolution 58. 2558-2573. 
Friesen, V.L., Congdon. B.C., Kidd. M.G., Birt. T.P.. 1999. Polymerase chain reaction 
(PCR) primers for the amplification of five nuclear introns in vertebrates. Mol. 
Ecol. 8, 2141-2152. 
Friesen. V.L, Congdon, B.C., Walsh, H.E., Birt, T.P., 1997. Intron variation in marbled 
murrelets   detected   using   analyses   of   single-stranded   conformational 
polymorphisms. Mol. Ecol. 6,1047-1058. 
Griffin. D.K., Robertson, L.B.W.. Tempest, H.G.. Skinner, B.M., 2007. The evolution of 
the  avian  genome  as   revealed   by  comparative  molecular  cytogenetics. 
Cytogenet. Genome Res. 117. 64-77. 
Hackett, S.J., Kimball, R.T.,  Reddy, S.,  Bowie,  R.C.K.. Braun, E.L.,  Braun,  M.J.. 
Chojnowski, J.L., Cox, W.A., Han, K.-L., Harshman, J., Huddleston, C.J., Marks, 
B.D., Miglia, K.J.. Moore. W.S., Sheldon, F.H., Steadman, D.W., Witt, C.C, Yuri, T., 
2008. A phylogenomic study of birds reveals their evolutionary history. Science 
320, 1763-1768. 
Handley, L.J.L., Ceplitis. H., Ellegren, H.. 2004. Evolutionary strata on the chicken Z 
chromosome: Implications for sex chromosome evolution. Genetics 167, 367- 
376. 
Harshman,]., Huddleston, C.J., Bollback.J.P.. Parsons.T.J.. Braun. M.J.. 2003. True and 
false gharials: A nuclear gene phylogeny of Crocodylia. Syst. Biol. 52.386-402. 
Heslewood,  M.M.,  Elphinstone,  M.S.,  Tidemann,  S.C,  Baverstock,  P.R.,   1998. 
Myoglobin   intron   variation   in   the   Gouldian   Finch   Erythrura   gouldiae 
assessed by temperature gradient gel electrophoresis. Electrophoresis  19, 
142-151. 
Jennings. W.B., Edwards, S.V., 2005. Speciational history of Australian grass finches 
(Poepltila) inferred from thirty gene trees. Evolution 59, 2033-2047. 
Lake, J.A.,  1997.  Phylogenetic  inference.  How  much  evolutionary  history  is 
knowable? Mol. Biol. Evol. 14. 213-219. 
Levine, A., Durbin, R., 2001. A computational scan for U12-dependent introns in the 
human genome sequence. Nucleic Acids Res. 29. 4006-4013. 
Maddison, W.P.. 1997. Gene trees in species trees. Syst. Biol. 46. 523-536. 
Morin, P.A., Luikart. G., Wayne, R.K., Grp, S.W., 2004. SNPs in ecology, evolution and 
conservation. Trends Ecol. Evol. 19, 208-216. 
Nichols, R., 2001. Gene trees and species trees are not the same. Trends Ecol. Evol. 
16,358-364. 
Primmer,   C.R.,   Borge,   T..   Lindell.  J.,   Saetre,   G.P.,   2002.   Single-nucleotide 
polymorphism characterization in species with limited available sequence 
information: high nucleotide diversity revealed in the avian genome. Mol. Ecol. 
11,603-612. 
Sehgal. R.N.M.. Lovette. I.J., 2003. Molecular evolution of three avian neurotrophin 
genes: implications for proregion functional constraints.]. Mol. Evol. 57.335-342. 
Slade, R.W., Moritz, C, Heideman, A., Hale, P.T., 1993. Rapid assessment of single 
copy nuclear DNA variation in diverse species. Mol. Ecol. 2, 359-373. 
Sorenson. M.D., Ast, J.C, Dimcheff, D.E., Yuri, T, Mindell, D.P., 1999. Primers for a 
PCR-based approach to mitochondrial genome sequencing in birds and other 
vertebrates. Mol. Phylogenet. Evol. 12, 105-114. 
Sunnucks, P.. 2000. Efficient genetic markers for population biology. Trends Ecol. 
Evol. 15, 199-203. 
Waltari, E., Edwards, S.V.. 2002. Evolutionary dynamics of intron size, genome size, 
and physiological correlates in archosaurs. Am. Nat. 160. 539-552. 
Yuri, T., Kimball, R.T., Braun, E.L., Braun, M.J., 2008. Duplication and accelerated 
evolution of growth hormone gene in passerine birds. Mol. Biol. Evol. 25. 352- 
361. 
Zink, R.T., Pavlova, A., Rohwer, S., Drovetski, S.V., 2006. Barn swallows before barns: 
population histories and intercontinental colonization. Proc. R. Soc. Lond. B 273, 
1245-1251.