Bonacum, J., R. DeSalle, P. O’Grady, D. Olivera, J. Wintermute, and M. Zilversmit. 2001. New nuclear and mitochondrial primers for systematics and comparative genomics in Drosophilidae. Dros. Inf. Serv. 84: 201-204. |
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New nuclear and mitochondrial primers for systematics and comparative genomics in Drosophilidae.
Bonacum,
J., R. DeSalle, P. O’Grady, D. Olivera, J. Wintermute, and M. Zilversmit.
Division of Invertebrate Zoology, American Museum of
Natural History, New York, NY 10024, USA.
Introduction
Several reviews
of both mitochondrial (Simon et al.,
1994) and nuclear (Brower and DeSalle, 1994) primers useful for molecular
systematics and molecular evolution have recently been published. Our laboratory has been developing a battery
of primers capable of amplifying a wide range of Drosophilid species.
Here we report on a number of primer pairs useful for examining a wide
range of divergences (from the population to genus level).
Primer design and amplification protocols for high throughput applications
can be found in Zilversmit et al.
(2002). These primers should
prove useful to a researchers studying population genetics, molecular evolution
and phylogenetic systematics in the family Drosophilidae.
Mitochondrial Primers
We have developed
a series of primers that will amplify an entire Drosophila mitochondrion.
Below are a number of primer pairs that work well in a large range
of species and constitute about 1/4 of the mitochondrial sequence.
N2-J-1006
TAGGTGGACTACCTCCATTTTYAGG
C1-N-1560
TGTTCCTACTATTCCGGCTCA
C1-J-1718
GGAGGATTTGGAAATTGATTAGTTCC
C1-N-2191
CCCGGTAAAATTAAAATATAAACTTC
C1-J-2183
CAACATTTATTTTGATTTTTTGG
C1-N-2659
GCTAATCCAGTGAATAATGG
C2-J-3696
GAAATTTGYGGRGCWAATCATAG
A8-N-4102
AARTTTGTTATCATTTTC
C2-J-3696
GAAATTTGYGGRGCWAATCATAG
A8-N-4478
GTTGTGTATGATTAATTCAACC
C3-J-5014
TTATTTATTKTWTCWGAAGT
C3-N-5460
TCAACAAAGTGTCAGTATCA
C3-J-5041
TTATTTATTKTWTCWGAAGT
C3-N-5460
as above
C3-J-5778
TGAATGYGGRTTTGAYCC
N5-N-6708
GGTTCWATATGATTTATACC
Nuclear Primers
Nuclear primers have recently become used in an effort to examine a variety of phylogenetic questions. The complete genome sequence of Drosophila melanogaster (Adams et al., 2000) has made design of nuclear primers much more tractable. Below we list several that we have developed in our laboratory and are useful at a variety of levels.
Several primer pairs flank non-coding or highly variable regions in the species we have surveyed. CG3869, an unnamed gene of unknown function, has a large intron of up to 400 base pairs in some taxa. The bride of sevenless (boss) gene also contains an intron in some species. Short non-coding regions can also be found in sans fille (snf) and lethal (2) neighbor of tid (tumorous imaginal discs). The glass gene also has some interesting variation in some groups. Two other genes we have examined, seven in absentia (sia) and forkhead (fkh), show little variation, but amplify in a wide range of taxa, including vertebrates.
A number of other nuclear primers are also being explored in our laboratory. These include wee, extra sex combs (esc), and wingless (wg). Other
primers have been designed to genes discovered by the Drosophila
melanogaster genome project, but not associated
with any phenotype or function. This latter class of primers is assigned only a “CG”
number below. Finally, many of
our primers have been engineered to contain the T7 and T3 universal priming
sites. This facilitates rapid
sequencing by high throughput methodology (Zilversmit et al.,
2002). Some sequences we have had positive results with include fkh, glass, amylase (amy), esc,
mago nashi (mago), ntid, boss,
snf, and sia. All primers
are listed 5’— 3’.
CG3869F CCCAACATCTTCATCCTGAACAAYMGNTGGGA
CG3869R GCGGACTGGGAGATGCAYTCYTCRAA
BossF1 ACCAGATGCCCTGGGGNGARAA
BossR1 TGGACAGGGAGCCGCKNARCCARTT
T3/BossF1 ATTAACCCTCACTAAAGACCAGATGCCCTGGGGNGARAA
T7/BossR1 AATACGACTCACTATAGTGGACAGGGAGCCGCKNARCCARTT
snfL GAAGATGCGGGGCCARGCNTTYGT
snfR GAACAGCATGGACAGCATCATYTCRTT
T3/snfL ATTAACCCTCACTAAAGGAAGATGCGGGGCCARGCNTTYGT
T7/snfR AATACGACTCACTATAGGAACAGCATGGACAGCATCATYTCRTT
ntidF1 GGGCCGCATCTTCGARCAYAARTGG
ntidR1 TGGAGGGGTAGGTGTTCCARCARTA
T3/ntidF1 ATTAACCCTCACTAAAGGGGCCGCATCTTCGARCAYAARTGG
T7/ntidR1 AATACGACTCACTATAGTGGAGGGGTAGGTGTTCCARCARTA
glass1 TTTCGATTGCGGCGGNTGYTTYGA
glass2 GCCGTGGTGCATGGTCATRTTCAT
T3/glass1 ATTAACCCTCACTAAAGTTTCGATTGCGGCGGNTGYTTYGA
T7/glass2 AATACGACTCACTATAGGCCGTGGTGCATGGTCATRTTCAT
sia1 TCGAGTGCCCCGTGTGYTTYGAYTA
sia2 GAAGTGGAAGCCGAAGCAGSWYTGCATCAT
T3/sia1 ATTAACCCTCACTAAAGTCGAGTGCCCCGTGTGYTTYGAYTA
T7/sia2 AATACGACTCACTATAGGAAGTGGAAGCCGAAGCAGSWYTGCATCAT
T3/fkhL ATTAACCCTCACTAAAGTCCCTACTCCTACATCTCCCTGATHACNATG
T7/fkhR AATACGACTCACTATAGCGCAGGTAGCAGCCGTTYTCRAACATRT
weeL GCCTGGGCCGAGGAYGAYCAYATG
weeR TCACGTGGCCCAGGTCNCCDATYTT
escL GGCCATCAACGAGCTGAARTTYCAYCC
escR TTCCAGCACACGATGGCRTTYTCRCA
T3/escL ATTAACCCTCACTAAAGGGCCATCAACGACGTGAARTTYCAYCC
T7/escR AATACGACTCACTATAGCGAACCACTGCACGCAGTCNACRTARTT
wgL GCAGTTCCGGAACCGGMGNTGGAAYTG
wgR GGACATGCCGTGGCACTTRCAYTCYTG
T3/amyF1 ATTAACCCTCACTAAAGCGCCCCTGGTGGGARMGNTA
T7/amyR1 AATACGACTCACTATAGCGCGCAGGCCCACNARYTCRCA
T3/magoL ATTAACCCTCACTAAAGCCACAAGGGCAAGTTCGGNCAYGARTT
T7/magoR AATACGACTCACTATAGCACTTCAGGTCCTGCACCARRTARTARAA
References: Adams, M.D., et al., 2000, The genome sequence of D. melanogaster. Science 287: 2185-2215; Brower, A., and R. DeSalle 1994, Practical and theoretical considerations for choice of DNA
sequence region in insect molecular systematics, with a short review of published
studies using nuclear gene regions. Ann. Rev. Entomol. 87: 702-716; Simon, C., F. Frati, A. Beckenbach, B. Crespi, H. Liu, and
P. Flook 1994, Evolution, weighting,
and phylogenetic utility of mitochondrial gene sequences and a compilation
of conserved polymerase chain reaction primers. Ann. Rev. Entomol. 87: 651-701; Zilversmit et al., 2002, High
Throughput Sequencing Protocols for a Survey of Genomic Characters in the
Family Drosophilidae. Dros. Inf. Serv. 84: (this issue).