Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 17-27 |
| Journal | Molecular Ecology Resources |
| Volume | 15 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2015 |
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Keywords
- est-ssr markers
- genetic-linkage maps
- in-silico
- rose
- l.
- diversity
- transferability
- construction
- variability
- identification
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Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals. / Vukosavljev, M.; Esselink, G.; van 't Westende, W.P.C.; Cox, P.; Visser, R.G.F.; Arens, P.; Smulders, M.J.M.
In: Molecular Ecology Resources, Vol. 15, No. 1, 2015, p. 17-27.Research output: Contribution to journal › Article › Academic › peer-review
TY - JOUR
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
U2 - 10.1111/1755-0998.12289
DO - 10.1111/1755-0998.12289
M3 - Article
VL - 15
SP - 17
EP - 27
JO - Molecular Ecology Resources
JF - Molecular Ecology Resources
SN - 1755-098X
IS - 1
ER -