Development and application of three-tiered nuclear genetic markers for basal Hexapods using single-stranded conformation polymorphism coupled with targeted DNA sequencing

BMC Genetics
Ryan C Garrick, Paul Sunnucks

Abstract

Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates. Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and t...Continue Reading

References

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Citations

Oct 2, 2008·Bulletin of Entomological Research·W T TayP Batterham
Feb 24, 2010·BMC Genomics·Juan I Montoya-BurgosIlham Bahechar
May 19, 2010·International Journal of Molecular Sciences·Ryan C GarrickPaul Sunnucks
Feb 27, 2008·Evolution; International Journal of Organic Evolution·Ryan C GarrickPaul Sunnucks
Apr 5, 2007·Nature Protocols·Robin B GasserXingquan Zhu
Oct 25, 2007·Systematic Biology·Simon JolyBettine Jansen van Vuuren

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Methods Mentioned

BETA
PCR
genotyping
ESR
PCRs
electrophoresis

Software Mentioned

ALIGNIR
NEBCUTTER
PHYLIP
DNASP
STRUCTURE
TCS
BLAST

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