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).