Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals
The first hurdle in developing microsatellite markers, cloning, has been overcome by next‐generation sequencing. The second hurdle is testing to differentiate polymorphic from nonpolymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of al...
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Published in | Molecular ecology resources Vol. 15; no. 1; pp. 17 - 27 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Blackwell Publishing Ltd
01.01.2015
Wiley Subscription Services, Inc |
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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 nonpolymorphic 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 performed 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. 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 multilocus 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 = 6–20) 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. |
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AbstractList | The first hurdle in developing microsatellite markers, cloning, has been overcome by next-generation sequencing. The second hurdle is testing to differentiate polymorphic from nonpolymorphic 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 performed 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. 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 multilocus 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 = 6-20) 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. 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. 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 nonpolymorphic 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 performed 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. 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 multilocus 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 = 6–20) 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. |
Author | Vukosavljev, M. Arens, P. Cox, P. Smulders, M. J. M. van 't Westende, W. P. C. Esselink, G. D. Visser, R. G. F. |
Author_xml | – sequence: 1 givenname: M. surname: Vukosavljev fullname: Vukosavljev, M. organization: Wageningen UR Plant Breeding, Wageningen University & Research Centre, P.O. Box 386, NL-6700AJ, Wageningen, the Netherlands – sequence: 2 givenname: G. D. surname: Esselink fullname: Esselink, G. D. organization: Wageningen UR Plant Breeding, Wageningen University & Research Centre, P.O. Box 386, NL-6700AJ, Wageningen, the Netherlands – sequence: 3 givenname: W. P. C. surname: van 't Westende fullname: van 't Westende, W. P. C. organization: Wageningen UR Plant Breeding, Wageningen University & Research Centre, P.O. Box 386, NL-6700AJ, Wageningen, the Netherlands – sequence: 4 givenname: P. surname: Cox fullname: Cox, P. organization: Roath BV, Eindhoven, the Netherlands – sequence: 5 givenname: R. G. F. surname: Visser fullname: Visser, R. G. F. organization: Wageningen UR Plant Breeding, Wageningen University & Research Centre, P.O. Box 386, NL-6700AJ, Wageningen, the Netherlands – sequence: 6 givenname: P. surname: Arens fullname: Arens, P. organization: Wageningen UR Plant Breeding, Wageningen University & Research Centre, P.O. Box 386, NL-6700AJ, Wageningen, the Netherlands – sequence: 7 givenname: M. J. M. surname: Smulders fullname: Smulders, M. J. M. email: Correspondence: Marinus Smulders, Fax: +31 317 418094;, rene.smulders@wur.nl organization: Wageningen UR Plant Breeding, Wageningen University & Research Centre, P.O. Box 386, NL-6700AJ, Wageningen, the Netherlands |
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Copyright | 2014 John Wiley & Sons Ltd 2014 John Wiley & Sons Ltd. Copyright © 2015 John Wiley & Sons Ltd Wageningen University & Research |
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Notes | TTI Green Genetics project 'Hyperrose' Table S1 Reads produced and microsatellite motifs foundTable S2 Overview of studies reporting microsatellite development in polyploids ark:/67375/WNG-SV3LP8RD-F ArticleID:MEN12289 TKI Polyploids project - No. BO-26.03-002-001 istex:DA8D41E6B8D41A2C8379B35655CA2E5F70E6803A ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
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Snippet | The first hurdle in developing microsatellite markers, cloning, has been overcome by next‐generation sequencing. The second hurdle is testing to differentiate... The first hurdle in developing microsatellite markers, cloning, has been overcome by next-generation sequencing. The second hurdle is testing to differentiate... Abstract The first hurdle in developing microsatellite markers, cloning, has been overcome by next‐generation sequencing. The second hurdle is testing to... The first hurdle in developing microsatellite markers, cloning, has been overcome by next generation sequencing. The second hurdle is testing to differentiate... |
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Title | Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals |
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