We describe here a procedure for the purification of high molecular weight genomic DNA that combines the economies of ‘do‐it‐yourself’, single‐tube protocols with the sample throughput and DNA quality of microplate‐based DNA extraction and purification kits from commercial suppliers. The procedure allows the preparation of genomic DNA of a quality suitable for polymerase chain reaction‐based studies of large populations at around one‐tenth of the cost of commercially available kits. Furthermore, 96 samples can be purified from crude tissue digests in around 30 min and are produced in microtitre plate format to allow efficient downstream processing of samples.
We present a predicted passerine genome map consisting of 196 microsatellite markers distributed across 25 chromosomes. The map was constructed by assigning chromosomal locations based on the sequence similarity between 550 publicly available passerine microsatellites and the draft chicken genome sequence published by the International Chicken Genome Sequencing Consortium. We compared this passerine microsatellite map with a recently published great reed warbler (Acrocephalus arundinaceus) linkage map derived from the segregation of marker alleles in a pedigree of a natural population. Twenty-four microsatellite markers were shared between the two maps, distributed across ten chromosomes. Synteny was maintained between the predicted passerine microsatellite map and the great reed warbler linkage map, confirming the validity and accuracy of our approach. Possible applications of the predicted passerine microsatellite map include genome mapping; quantitative trait locus (QTL) discovery; understanding heterozygosity-fitness correlations; investigating avian karyotype evolution; understanding microsatellite mutation processes; and for identifying loci conserved in multiple species, unlinked loci for use in genotyping sets and sex-linked markers.
We describe a convenient, cost-effective and flexible medium-throughput single nucleotide polymorphism (SNP) genotyping method, Multiplex SNP-SCALE, which enables the simultaneous amplification by polymerase chain reaction (PCR) of up to 25 (or potentially more) loci followed by electrophoresis in an automated DNA sequencer. We extended the original SNP-SCALE method to include (i) use of a commercial multiplex PCR kit, (ii) a four-dye system, (iii) much-reduced (2-µL) reaction volumes, (iv) drying down of template DNA before PCR, (v) use of pig-tailed primers, (vi) a PCR product weighting system, (vii) a standard optimized touchdown PCR thermocycling programme, and (viii) software (SNP-SCALE Primer Designer) that automatically designs suitable SNP-SCALE primers for a batch of loci. This new protocol was validated for different types of SNPs. The method is cost- and time-effective for medium-scale evolutionary and ecological projects involving 10s to 100s of loci.
We identified microsatellite sequences of potential utility in the house sparrow (Passer domesticus) and assigned their predicted genome locations. These sequences included newly isolated house sparrow loci, which we fully characterized. Many of the newly isolated loci were polymorphic in two other species of Passeridae: Berthelot's pipit Anthus berthelotii and zebra finch Taeniopygia guttata. In total, we identified 179 microsatellite markers that were either isolated directly from, or are of known utility in, the house sparrow. Sixty-seven of these markers were designed from unique sequences that we isolated from a house sparrow genomic library. These new markers were combined with 36 house sparrow markers isolated by other studies and 76 markers isolated from other passerine species but known to be polymorphic in the house sparrow. We utilized sequence homology to assign chromosomal locations for these loci in the assembled zebra finch genome. One hundred and thirty-four loci were assigned to 25 different autosomes and eight loci to the Z chromosome. Examination of the genotypes of known-sex house sparrows for 37 of the new loci revealed a W-linked locus and an additional Z-linked locus. Locus Pdoμ2, previously reported as autosomal, was found to be Z-linked. These loci enable the creation of powerful and cost-effective house sparrow multiplex primer sets for population and parentage studies. They can be used to create a house sparrow linkage map and will aid the identification of quantitative trait loci in passerine species.
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