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.
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.
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.
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.
C3-N-5460 as above
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’.
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).