GENETICS
Diptera-Specific Polymerase Chain Reaction Amplification Primers of
Use in Molecular Phylogenetic Research
JOEL F. GIBSON,1,2,3 SCOTT KELSO,1 MORGAN D. JACKSON,4 JOEL H. KITS,4
GIL F. G. MIRANDA,4 AND JEFFREY H. SKEVINGTON1,2
Ann. Entomol. Soc. Am. 104(5): 976Ð997 (2011); DOI: 10.1603/AN10153
ABSTRACT DNA sequence data from a variety of mitochondrial and nuclear gene regions are
signiÞcant components of phylogenetic research in entomology. Polymerase chain reaction (PCR)
ampliÞcation primers for many gene regions have been developed that are speciÞc to a range of
dipteran groups. Here, we review the existing Diptera-speciÞc PCR ampliÞcation primers that have
been published for 11 mitochondrial and nuclear gene regions: 12S small ribosomal subunit, cyto-
chromeb, cytochrome oxidase c subunit I, 28S ribosomal RNA, alanyl-tRNA synthetase, the carbamoyl
phosphate synthase region of CAD, elongation factor-1
, 6-phosphogluconate dehydrogenase, triose
phosphate isomerase, white, and wingless. We also have designed in total 94 new PCR ampliÞcation
primers for use in these same gene regions. Our new primers have been developed and tested using
our DNA sequence database of
1,600 specimens representing 40 families of Diptera. All of the past
and newly developed primer sequences are presented in tables, and their locations are shown on gene
maps. This combined data will facilitate future molecular phylogenetic research within Diptera.
KEY WORDS gene maps, phylogenetics, mitochondrial DNA, nuclear DNA, ribosomal DNA
The most recent review of published, DNA sequence-
based, phylogenetic studies of insects (Caterino et al.
2000) lists a large number of studies using a broad
range of target taxa and gene regions. In the time since
the publication of this review, many more papers have
been published that use many different gene regions
to study relationships between a variety of taxa. Al-
though someof these studies include thedevelopment
of new, taxon-speciÞc polymerase chain reaction
(PCR) ampliÞcation primers, many rely on existing,
published primers. These existing primers, however,
may not be appropriate for the taxa being investigated
and may lead to inefÞciency or sequencing failure.
Generally, the primers used to generate DNA se-
quence data were developed for use in groups of
insects other than those that are the focus of the new
study. For these primers to be of use in sequencing
taxa that have never before been sequenced, universal
primers are a necessity. Universality takes the form of
oligonucleotide degeneracy or an acceptable level of
oligonucleotide mismatch. Both situations can make
the ampliÞcation and sequencing of target gene re-
gions extremely difÞcult, if not impossible. Degener-
ate primers can produce nonspeciÞc ampliÞcation,
multiplex PCR product, and the necessity of isolating
thedesiredPCRproductbefore sequencing.Although
new techniques exist (Ma and DiFazio 2008, Gibson et
al. 2010a) to facilitate the isolation of desired PCR
products frommultiplexPCRproducts, thesemethods
require additional time and money. Also, when de-
generacy is included in a primer sequence, each dif-
ferent possible version of the primer sequence is pro-
ducedand included in themanufacturedproduct.This
can lead to an exponential increase in the number of
primer sequences present in the PCR reaction. For
example, a manufactured primer oligonucleotide se-
quence containing four Ns and three YÕs would ac-
tually consist of 2,048 different nucleotide sequences,
only one of which would match the genomic template
DNA sequence. This leads not only to a greatly re-
duced concentration of the correctly matching primer
but also a disruption in reaction kinetics as genomic
template primer locations are blocked by poorly
matching versions of the primer. Primers with less
degeneracy, but developed for distantly related
groups, often lead to sufÞcient nucleotide mismatch to
result in ampliÞcation failure.
Another consideration is the condition of the spec-
imen from which DNA is being extracted. In molec-
ular phylogenetic research, the specimens being used
may not have been prepared or stored under optimal
conditions. These conditions may have lead to degra-
dation and fragmentation of genomic DNA. In these
instances, a number of alternate primer pairs may be
necessary to amplify and sequence the target gene
region in smaller segments.
1 Agriculture and Agri-Food Canada, Canadian National Collection
of Insects, Arachnids and Nematodes, 960 Carling Ave., Ottawa, ON
K1A 0C6, Canada.
2 Department of Biology, 209 Nesbitt Bldg., Carleton University,
1125 Colonel By Dr., Ottawa, ON K1S 5B6, Canada.
3 Corresponding author, e-mail: joel.gibson@agr.gc.ca.
4 School of Environmental Sciences, 1216 Edmund C. Bovey Bldg.,
University of Guelph, Guelph, ON N1G 2W1, Canada.

Due to the potential problems introduced by overly
degenerate or mismatched primers and the necessity
for primers located throughout the length of target
gene regions, a variety of taxon-speciÞc primers are
essential. In the case of Diptera, many primers are
used time and time again despite the fact that they
were designed originally for use in nondipteran taxa.
These primers are often not adequate to amplify and
sequence target gene regions in dipteran taxa, leading
to considerable frustration andwaste.Diptera-speciÞc
primers for a variety of gene regions are needed to
reduce time and effort spent and to increase sequenc-
ing success in molecular phylogenetic research.
Our purpose is to review unique PCR ampliÞcation
primers that have been developed and published as
part of research on Diptera phylogenetics. We have
chosen eleven gene regions that include the most
commonly sequenced gene regions as well as some
that have only recently been developed. These gene
regions are small ribosomal subunit (12S), cyto-
chrome b (Cytb), cytochrome oxidase c subunit I
(COI), 28S ribosomal RNA (28S), alanyl-tRNA syn-
thetase (AATS), the carbamoyl phosphate synthase
region of CAD (CAD), elongation factor-1
(EF-1
),
6-phosphogluconate dehydrogenase (PGD), triose
phosphate isomerase (TPI), white, and wingless. To
the list of existing primers, we seek to add our own
newly designed primers that will provide further op-
tions to future researchers that wish to amplify a given
gene region for their own dipteran target taxa. We
intend to provide sufÞcient primer alternatives, both
old and new, such that any of these eleven gene re-
gions could be ampliÞed and sequenced in any future
molecular phylogenetic research involving Diptera.
Materials and Methods
We surveyed the scientiÞc literature to identify
unique PCR ampliÞcation primers developed as a part
of phylogenetic research involving Diptera. Primers
that were different from past primers by at least one
nucleotide were included in both primer tables and
Table 1. Number of specimens of each Diptera family included in data set used to create new PCR amplification primers for each
gene region
Family
Gene region
12S Cytb COI 28S AATS CAD EF-1a PGD TPI white wingless
Atelestidae 1 0 1 1 0 1 0 0 0 0 0
Bombyliidae 1 0 1 1 0 1 0 0 0 0 0
Brachystomatidae 1 0 1 1 1 1 1 1 1 1 1
Conopidae 71 65 69 71 66 11 6 8 8 9 10
Curtonotidae 1 0 1 1 0 1 0 0 0 0 0
Cypselosomatidae 1 0 1 1 0 1 0 0 0 0 0
Diopsidae 4 0 3 4 3 4 4 3 4 2 4
Dolichopodidae 1 0 1 1 0 1 0 0 0 0 0
Drosophilidae 1 1 1 1 1 1 1 1 1 1 1
Empididae 1 0 1 1 0 1 0 0 0 0 0
Heleomyzidae 1 1 1 1 1 0 0 0 0 0 0
Hybotidae 1 0 1 1 0 1 0 0 0 0 0
Ironomyiidae 1 0 1 1 0 1 0 0 0 0 0
Lauxaniidae 2 2 3 2 2 2 2 1 1 2 2
Lonchopteridae 2 1 2 2 1 2 1 1 1 1 1
Marginidae 1 0 1 1 0 1 0 0 0 0 0
Megamerinidae 1 0 1 1 1 1 1 1 1 1 1
Micropezidae 46 1 38 13 5 42 11 5 5 4 35
Muscidae 2 1 2 2 1 2 0 0 1 1 1
Neriidae 1 0 1 1 1 1 1 1 1 1 1
Opetiidae 1 0 1 1 0 1 0 0 0 0 0
Pallopteridae 1 1 1 1 1 1 1 1 0 1 1
Phoridae 3 1 3 3 1 3 1 1 1 0 1
Pipunculidae 88 42 80 2 44 78 1 1 1 1 1
Platypezidae 2 1 2 2 1 2 1 1 1 0 1
Platystomatidae 1 1 1 1 1 1 1 1 1 1 1
Psilidae 2 0 2 2 2 2 2 1 1 2 2
Pyrgotidae 1 1 1 1 0 1 1 1 0 1 1
Richardiidae 1 0 1 1 0 1 0 0 0 0 0
Sciadoceridae 1 0 1 1 0 1 0 0 0 0 0
Sciomyzidae 1 0 1 1 0 1 0 0 0 0 0
Somatiidae 1 0 1 1 1 1 1 1 1 1 1
Sphaeroceridae 26 22 24 23 18 2 1 0 1 1 1
Strongylophthalmyiidae 2 1 2 2 2 2 2 1 2 2 2
Syringogastridae 1 0 0 1 1 1 1 1 1 1 1
Syrphidae 44 39 44 40 32 39 1 1 1 1 1
Tachinidae 1 0 1 1 0 1 0 0 0 0 0
Tanypezidae 1 0 1 1 1 1 1 1 1 1 1
Tephritidae 1 1 1 1 1 1 1 1 0 0 0
Therevidae 1 0 1 1 0 1 0 0 0 0 0
Total no. specimens included 319 182 298 193 189 215 44 35 36 36 72
Total no. families included 40 17 39 40 24 39 23 22 21 21 23
September 2011 GIBSON ET AL.: DIPTERA-SPECIFIC PRIMERS 977