Intron variation in marbled murrelets detected using analyses of single‐stranded conformational polymorphisms

Abstract

Combination of the targeted amplification of nuclear introns and the analysis of single-stranded conformational polymorphisms has the potential to provide an inexpensive, rapid, versatile and sensitive genetic assay for evolutionary studies and conservation. We are developing primers and protocols to analyse nuclear introns in vertebrates, and are testing them in a population genetic study of marbled murrelets Brachyramphus marmoratus. Here we present protocols and results for introns for aldolase B, alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase and lamin A. Results suggest that this approach presents a potentially powerful method for detecting genetic variation within and among local populations and species of animals: (i) a variety of genes can be surveyed, including genes of special interest such as those involved in disease resistance; (ii) assays are rapid and relatively inexpensive; (iii) large numbers of genes can be assayed, enabling accurate estimation of variation in the total genome; (iv) almost any mutation can be detected in the genes amplified; (v) the exact nature of variation can be investigated by sequence analysis if desired; (vi) statistical methods previously developed for proteins and/or sequence data can be used; (vii) protocols can be easily transferred to other species and other laboratories; and (viii) assays can be performed on old or degraded samples, blood or museum skins, so that animals need not be killed. Results of analyses for murrelets support earlier evidence that North American and Asiatic subspecies represent reproductively isolated species, and that genetic differences exist among murrelets from different sites within North America.

Phylogeny and comparative biogeography of Pionopsitta parrots and Pteroglossus toucans

“…Primers GapdL890 and GapdH950 were used for ampliWcation and sequencing of the Gapdh fragment, and EnoH and Eno1L for the Eno1 fragment (Friesen et al, 1997). PCRs were done using AmpliTaq or AmpliTaq Gold (Perkin-Elmer), beginning with 5 min at 94°C, followed by 30 cycles with an annealing temperature of 50°C.…”

Population genetic structure and conservation of marbled murrelets (Brachyramphus marmoratus)

“…Tissue samples were obtained from an additional 74 marbled murrelets, including 36 from northern California (Humboldt County), 30 from central California (San Mateo County) and eight from Alaska ( Figure 1). DNA was extracted using a standard protease-K phenol/ chloroform technique (Friesen et al 1997).…”

“…DNA was amplified using PCR primers BmaH600 (5¢-CAAAAGTGCCAA AAAGGTCGGATGTCG-3¢) and ND6 (5¢-CCT AGAAAAAGCACAAAATAAGTCAT-3¢; Kidd and Friesen 1998a) under standard conditions (Veit et al 2005) with annealing at 50°C. Variation was screened initially using single-stranded conformational polymorphisms (SSCPs; Hayashi 1991;Friesen et al 1997), and individuals were assigned tentative haplotypes. Two or more representatives of each haplotype (one for each unique haplotype) plus all ambiguous samples were re-amplified and gel-purified using Gel Extraction Spin Columns (Qiagen), and sequenced directly using either Thermosequenase TM cycle sequencing kits (Amersham Pharmacia) or an ABI Prism TM 373 Automated Sequencer (Mobix Labs, McMaster University).…”

“…Sequence variation in four nuclear introns (a-Enolase, Gpd, Ldh and RP40; Congdon et al 2000) was screened in the 74 new samples using a combination of SSCPs and direct sequencing (Friesen et al 1997(Friesen et al , 1999Congdon et al 2000), and combined with data from Congdon et al (2000). All 194 samples were assayed for length variation in three microsatellite loci (Uaa5-8, Ibarguchi et al 2000;Bma10-18 [forward: 5¢-GGTA GGAGCGGAGTAGGAGG-3¢; reverse: 5¢-GCAAA ATAAGGGTGAAGGCA-3¢] and Cco5-21 [forward: 5¢-TCAAGATGATGAAGACCCTAAT-3¢; reverse: 5¢-AGAGTTGCACAGGTTAAATACC-3¢], V.L.F.…”

A well-tested set of primers to amplify regions spread across the avian genome

“…In birds, there are several studies which have published sets of primers to amplify nuclear regions, particularly intron regions (e.g., Friesen et al, 1999Friesen et al, , 1997Primmer et al, 2002;Waltari and Edwards, 2002;Slade et al, 1993;Backstrom et al, 2008). However, many of these primers have been tested on a limited set of Table 1 Characteristics of tiie loci included in tliis study.…”