Capture of assay template by multiplex PCR of long amplicons for genotyping SNPs and InDels with MALDI-TOF mass spectrometry

Sexton, Timothy; Henry, Robert J.; McManus, Luke J.; Bowen, Stirling; Shepherd, Mervyn

doi:10.1007/s11032-009-9345-0

Cited by 14 publications

(11 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Published accounts of the SNP resource are still rare for eucalypts, but sequencing of the transcriptome and resequencing genome fractions or genes using conventional or next-generation sequencing has confirmed that large numbers of SNPs will be available (Külheim et al 2009;Novaes et al 2008;Poke et al 2003). Assay methods have been used that accommodate the complexity of SNP genotyping in the highly heterozygous eucalypt genome (Sexton et al 2010b), although at a relatively low throughput of less than 100 multiplexed SNPs.…”

Section: Molecular Marker Resourcesmentioning

confidence: 99%

“…Using the Sequenom mass spectrometry platform (Sexton et al 2010b) overcame this problem by generating fewer but much longer template amplicons instead of several short ones. When developing SNPs for the GGGT, stringent requirements of conservation in flanking sequences to the SNP both within and across species significantly enhanced SNP reliability and polymorphism of the assay, although such requirements reduced the number of assayable SNPs (Grattapaglia et al 2011a).…”

Section: Molecular Marker Resourcesmentioning

confidence: 99%

See 1 more Smart Citation

Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

Grattapaglia

Vaillancourt

Shepherd

et al. 2012

Tree Genetics & Genomes

228

188

View full text Add to dashboard Cite

Section: Molecular Marker Resourcesmentioning

confidence: 99%

Section: Molecular Marker Resourcesmentioning

confidence: 99%

Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

Grattapaglia

Vaillancourt

Shepherd

et al. 2012

Tree Genetics & Genomes

228

188

View full text Add to dashboard Cite

“…With the ever-decreasing costs of next-generation sequencing (NGS), large-scale sequencing projects in conservation programs are becoming increasingly feasible. Sequencing of PCR amplicons has benefitted greatly from high-throughput next-generation sequencing technology and it is now possible to simultaneously sequence large numbers of samples at multiple loci [ 30 ]. In species with a reference genome, it is possible to distribute markers across the genome and target protein coding regions to gain true insights of genome-wide and genic diversity.…”

Section: Introductionmentioning

confidence: 99%

Development of a SNP-based assay for measuring genetic diversity in the Tasmanian devil insurance population

et al. 2015

View full text Add to dashboard Cite

BackgroundThe Tasmanian devil (Sarcophilus harrisii) has undergone a recent, drastic population decline due to the highly contagious devil facial tumor disease. The tumor is one of only two naturally occurring transmissible cancers and is almost inevitably fatal. In 2006 a disease-free insurance population was established to ensure that the Tasmanian devil is protected from extinction. The insurance program is dependent upon preserving as much wild genetic diversity as possible to maximize the success of subsequent reintroductions to the wild. Accurate genotypic data is vital to the success of the program to ensure that loss of genetic diversity does not occur in captivity. Until recently, microsatellite markers have been used to study devil population genetics, however as genetic diversity is low in the devil and potentially decreasing in the captive population, a more sensitive genotyping assay is required.MethodsUtilising the devil reference genome and whole genome re-sequencing data, we have identified polymorphic regions for use in a custom genotyping assay. These regions were amplified using PCR and sequenced on the Illumina MiSeq platform to refine a set a markers to genotype the Tasmanian devil insurance population.ResultsWe have developed a set of single nucleotide polymorphic (SNP) markers, assayed by amplicon sequencing, that provide a high-throughput method for monitoring genetic diversity and assessing familial relationships among devils. To date we have used a total of 267 unique SNPs within both putatively neutral and functional loci to genotype 305 individuals in the Tasmanian devil insurance population. We have used these data to assess genetic diversity in the population as well as resolve the parentage of 21 offspring.ConclusionsOur molecular data has been incorporated with studbook management practices to provide more accurate pedigree information and to inform breeding recommendations. The assay will continue to be used to monitor the genetic diversity of the insurance population of Tasmanian devils with the aim of reducing inbreeding and maximizing success of reintroductions to the wild.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2020-4) contains supplementary material, which is available to authorized users.

show abstract

“…A total of 55 CCR sequences from Eucalyptus (n = 35), Corymbia (n = 18) and Angophora (n = 2) were aligned manually using the alignment explorer of Mega v4 (TAMURA et al, 2007). Fifty sequences were obtained from GenBank and a further five E. pilularis sequences were obtained by cloning and sequencing as described in Sexton et al (2010) (A list of the Genbank accession numbers and source organism is given in available from author upon request.…”

Section: Microsatellite Markers and Pcr Conditionsmentioning

confidence: 99%

Microsatellite markers for Eucalyptus pilularis (Subgenus Eucalyptus); sourcing genetic markers outside the subgenus

2013

View full text Add to dashboard Cite

Microsatellite markers remain the most broadly used molecular marker in eucalypt genetics. A major advantage of microsatellite markers is that they often transfer readily between related taxa circumventing the need to develop new markers de novo in each species. Markers have been developed for a number of species of major economic importance, mainly from the Subgenus Symphyomyrtus, but these may also be available for use in species of lesser economic importance from other subgenera. Here we report on the sourcing of microsatellite markers for E. pilularis (Subgenus Eucalyptus (Formerly Monocalyptus)) from species outside the subgenus. Ninety-seven precent (60 out of 62) of loci that amplified in the source taxon (E. grandis) also amplified in the target taxon E. pilularis. By characterising them on a diversity panel (n=24) and a pedigree, a subset of 41 loci were distilled out that could be scored reliably and were polymorphic (Mean unbiased heterozygosity= 0.81). Predictions of efficient microsatellite marker transfer among eucalypts based on low evolutionary divergence have largely been borne out and are congruent with accumulating evidence of low sequence divergence within Eucalyptus. Upon this favourable background for microsatellite marker transfer, this study indicates highly efficient transfer is possible by identifying loci with broad PCR optima and adoption of approaches that favour cross-species transfer.

show abstract

Capture of assay template by multiplex PCR of long amplicons for genotyping SNPs and InDels with MALDI-TOF mass spectrometry

Cited by 14 publications

References 26 publications

Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

Development of a SNP-based assay for measuring genetic diversity in the Tasmanian devil insurance population

Microsatellite markers for Eucalyptus pilularis (Subgenus Eucalyptus); sourcing genetic markers outside the subgenus

Contact Info

Product

Resources

About