Primer Announcement - PCR Primers for Nuclear Gene

Q. Quentin Fang qfang at GSAIX2.CC.GASOU.EDU
Mon Oct 7 14:35:38 EST 1996


Dear Colleagues,

	Over the last years, we at Maryland have methodically tested
several nuclear genes for their phylogenetic informativeness within
Lepidoptera.  Our goal is to use the most promising candidates for
resolving deep-level relationships within Noctuoidea, Bombycoidea and
Macrolepidoptera generally.

	In addition to developing PCR primers, we have aimed to determine
taxonomic levels at which particular gene sequences recover
previously-well-established groups with acceptably low levels of
homoplasy.  We hope that the extra effort taken at the beginning of a
project to do such "concordance" studies will be rewarded by greater
efficiency later on, both for us and for others who may wish to use the
primers.  We have previously published primer sequences and concordance
studies for elongation factor 1a (EF-1a)  and phosphoenolpyruvate
carboxykinase (PEPCK). In a forthcoming paper in Systematic Biology, we
report a test of the phylogenetic utility of the nuclear gene coding for
dopa decarboxylase (DDC) within the noctuid moth subfamily Heliothinae
(mid- to late-Tertiary in origin?).  This gene had not previously been
used for phylogeny reconstruction. Relationships inferred from DDC were in
close agreement with those implied by morphology, allozymes and EF-1a.
Both synonymous and nonsynonymous changes are positively informative of
relationships, although there are many more synonymous changes. Synonymous
changes in DDC appear to evolve several times faster than in EF-1a.  In a
further study now in preparation we examined DDC at a deeper taxonomic
level, across the superfamily Noctuoidea. These are probably early- to
mid-Tertiary divergences.  At this level, homoplasy for synonymous changes
becomes more problematical.  There is, however, a clear signal for amino
acid changes, and pairwise divergence values indicate that the sequence as
a whole is well removed from saturation. However, the short internodes
suggest either that the groups radiated quickly or that amino acid
character evolution is slow.  Although our evidence is not yet conclusive,
we are hopeful that DDC amino acid sequences will provide good information
for Mesozoic and early Tertiary splits, a time span for which relatively
few useful genes have been identified.  The DDC primer sequences listed
below should amplify within Noctuoidea and Bombycoidea, where our own
research is presently focused.  Some primers will also work across
Lepidoptera and Trichoptera, although not necessarily for all taxa. Given
that the initial primers were designed based on a Drosophila sequence,
they should also work within Diptera.  For other groups, we recommend that
you go to GenBank, align the available sequences, map the primer sequences
below onto the published sequences, and decide where to add greater
degeneracy in order to enhance your chances of success.  (Accession
numbers up to 1994 can be easily retrieved from Friedlander et al.,
Systematic Biology 43:511-525 1994.  Regardless of how well your primer
sequences match, you will want to compare RT-PCR and DNA-PCR.  Multiple,
large introns are present in Lepidoptera in the region amplified by the
primers 1.7dF and 4ddRC, although this may not be the case in Diptera.
Good luck!

--Q.Quentin Fang (qfang at gsaix2.cc.gasou.edu), Jerry Regier, Charles Mitter

			DDC PRIMER SEQUENCES

1.4F	TTT CAT GCT TAT TTT CCT ACT GC  [424]
1.7dF	GC(C/T) TG(CT) AT(CT) GG(AT) TT(CT) AC(CT) TGG AT   [493]
1.9dF	ATG (CT)T(GA) GA(CT) TGG (CT)T(AG) GG(CT) CA(GA) ATG   [551]
3.2dF	TGG (TC)T(AG) CA(TC) GT(CG) GA(TC) GC(ATG) GC(CT) TA(TC) GC [1000]
3.2dRC	TA (GA)GC (TAC)GC (AG)TC (GC)AC (AG)TG (TC)A(AG) CCA   [975]
4ddRC	GG (GT)AT (TC)TG CCA (GA)TG (AC)CG (AG)TA (AG)TC   [1203]

COMMENTS ON PRIMER SEQUENCES:  Primers except 1.4F included an M13
sequence at the 5' end to facilitate automated sequencing.  Primer names
ending in F identify forward primers, which bind to the anti-sense strand
of DNA.  Primer names ending in RC identify reverse complement primers.
Numbers in brackets at the 3' end of each primer sequence refer to its
nucleotide position relative to the DDC sequence (sense strand) in the
moth Manduca sexta (GenBank accession no. U03909).  1.4F was defined by
comparison among human, rat, Drosophila melanogaster and Manduca sexta
sequences already in GenBank. It was used to sequence across and to better
define primer region 1.7 in a few moth taxa.  1.4F was not routinely used
for PCR or sequencing.  All other primer sequences incorporated
additional degeneracy based on new moth sequences.



More information about the Mol-evol mailing list