Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals

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Abstract

The first hurdle in developing microsatellite markers, cloning, has been overcome by next generation sequencing. The second hurdle is testing to differentiate polymorphic from non-polymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of alleles are sufficiently informative to be deployed in multiple studies. Both steps are laborious and still done manually. We have developed a strategy in which we first screen reads from multiple genotypes for repeats that show the most length variants, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Out of 48 tested two markers failed to amplify but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding duplicated loci will be difficult because the range of numbers of predicted alleles of highly polymorphic single- and multi-locus markers largely overlapped. Of the remainder, half were replicate markers (i.e., multiple primer pairs for one locus), indicating the difficulty of correctly filtering short reads containing repeat sequences. We subsequently refined the approach to eliminate multiple primer sets to the same loci. The remaining 18 markers were all highly polymorphic, amplifying on average 11.7 alleles per marker (range = 6 to 20) in 11 tetraploid roses, exceeding the 8.2 alleles per marker of the 24 most polymorphic markers genotyped previously. This strategy, therefore, represents a major step forward in the development of highly polymorphic microsatellite markers.
Original languageEnglish
Pages (from-to)17-27
JournalMolecular Ecology Resources
Volume15
Issue number1
DOIs
Publication statusPublished - 2015

Fingerprint

Transcriptome
transcriptome
Microsatellite Repeats
Alleles
microsatellite repeats
loci
Tetraploidy
alleles
Rosa
allele
Genes
tetraploidy
Organism Cloning
Genotype
marker
gardens
molecular cloning
gene
genes
garden

Keywords

  • est-ssr markers
  • genetic-linkage maps
  • in-silico
  • rose
  • l.
  • diversity
  • transferability
  • construction
  • variability
  • identification

Cite this

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title = "Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals",
abstract = "The first hurdle in developing microsatellite markers, cloning, has been overcome by next generation sequencing. The second hurdle is testing to differentiate polymorphic from non-polymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of alleles are sufficiently informative to be deployed in multiple studies. Both steps are laborious and still done manually. We have developed a strategy in which we first screen reads from multiple genotypes for repeats that show the most length variants, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Out of 48 tested two markers failed to amplify but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding duplicated loci will be difficult because the range of numbers of predicted alleles of highly polymorphic single- and multi-locus markers largely overlapped. Of the remainder, half were replicate markers (i.e., multiple primer pairs for one locus), indicating the difficulty of correctly filtering short reads containing repeat sequences. We subsequently refined the approach to eliminate multiple primer sets to the same loci. The remaining 18 markers were all highly polymorphic, amplifying on average 11.7 alleles per marker (range = 6 to 20) in 11 tetraploid roses, exceeding the 8.2 alleles per marker of the 24 most polymorphic markers genotyped previously. This strategy, therefore, represents a major step forward in the development of highly polymorphic microsatellite markers.",
keywords = "est-ssr markers, genetic-linkage maps, in-silico, rose, l., diversity, transferability, construction, variability, identification",
author = "M. Vukosavljev and G. Esselink and {van 't Westende}, W.P.C. and P. Cox and R.G.F. Visser and P. Arens and M.J.M. Smulders",
note = "This research was partly supported by the TTI Green Genetics project ŒHyperrose¹ and by the TKI Polyploids project (BO-26.03-002-001)",
year = "2015",
doi = "10.1111/1755-0998.12289",
language = "English",
volume = "15",
pages = "17--27",
journal = "Molecular Ecology Resources",
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T1 - Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals

AU - Vukosavljev, M.

AU - Esselink, G.

AU - van 't Westende, W.P.C.

AU - Cox, P.

AU - Visser, R.G.F.

AU - Arens, P.

AU - Smulders, M.J.M.

N1 - This research was partly supported by the TTI Green Genetics project ŒHyperrose¹ and by the TKI Polyploids project (BO-26.03-002-001)

PY - 2015

Y1 - 2015

N2 - The first hurdle in developing microsatellite markers, cloning, has been overcome by next generation sequencing. The second hurdle is testing to differentiate polymorphic from non-polymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of alleles are sufficiently informative to be deployed in multiple studies. Both steps are laborious and still done manually. We have developed a strategy in which we first screen reads from multiple genotypes for repeats that show the most length variants, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Out of 48 tested two markers failed to amplify but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding duplicated loci will be difficult because the range of numbers of predicted alleles of highly polymorphic single- and multi-locus markers largely overlapped. Of the remainder, half were replicate markers (i.e., multiple primer pairs for one locus), indicating the difficulty of correctly filtering short reads containing repeat sequences. We subsequently refined the approach to eliminate multiple primer sets to the same loci. The remaining 18 markers were all highly polymorphic, amplifying on average 11.7 alleles per marker (range = 6 to 20) in 11 tetraploid roses, exceeding the 8.2 alleles per marker of the 24 most polymorphic markers genotyped previously. This strategy, therefore, represents a major step forward in the development of highly polymorphic microsatellite markers.

AB - The first hurdle in developing microsatellite markers, cloning, has been overcome by next generation sequencing. The second hurdle is testing to differentiate polymorphic from non-polymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of alleles are sufficiently informative to be deployed in multiple studies. Both steps are laborious and still done manually. We have developed a strategy in which we first screen reads from multiple genotypes for repeats that show the most length variants, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Out of 48 tested two markers failed to amplify but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding duplicated loci will be difficult because the range of numbers of predicted alleles of highly polymorphic single- and multi-locus markers largely overlapped. Of the remainder, half were replicate markers (i.e., multiple primer pairs for one locus), indicating the difficulty of correctly filtering short reads containing repeat sequences. We subsequently refined the approach to eliminate multiple primer sets to the same loci. The remaining 18 markers were all highly polymorphic, amplifying on average 11.7 alleles per marker (range = 6 to 20) in 11 tetraploid roses, exceeding the 8.2 alleles per marker of the 24 most polymorphic markers genotyped previously. This strategy, therefore, represents a major step forward in the development of highly polymorphic microsatellite markers.

KW - est-ssr markers

KW - genetic-linkage maps

KW - in-silico

KW - rose

KW - l.

KW - diversity

KW - transferability

KW - construction

KW - variability

KW - identification

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JF - Molecular Ecology Resources

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