Cargando…
Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat
KEY MESSAGE: Conversion of SNP chip assays into locus-specific KASP markers requires adapted strategies in polyploid species with high genome homeology. Procedures are exemplified by QTL-associated SNPs in hexaploid wheat. ABSTRACT: Kompetitive allele-specific PCR (KASP) markers are commonly used in...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360542/ https://www.ncbi.nlm.nih.gov/pubmed/32500260 http://dx.doi.org/10.1007/s00122-020-03608-x |
_version_ | 1783559230803935232 |
---|---|
author | Makhoul, M. Rambla, C. Voss-Fels, K. P. Hickey, L. T. Snowdon, R. J. Obermeier, C. |
author_facet | Makhoul, M. Rambla, C. Voss-Fels, K. P. Hickey, L. T. Snowdon, R. J. Obermeier, C. |
author_sort | Makhoul, M. |
collection | PubMed |
description | KEY MESSAGE: Conversion of SNP chip assays into locus-specific KASP markers requires adapted strategies in polyploid species with high genome homeology. Procedures are exemplified by QTL-associated SNPs in hexaploid wheat. ABSTRACT: Kompetitive allele-specific PCR (KASP) markers are commonly used in marker-assisted commercial plant breeding due to their cost-effectiveness and throughput for high sample volumes. However, conversion of trait-linked SNP markers from array-based SNP detection technologies into KASP markers is particularly challenging in polyploid crop species, due to the presence of highly similar homeologous and paralogous genome sequences. We evaluated strategies and identified key requirements for successful conversion of Illumina Infinium assays from the wheat 90 K SNP array into robust locus-specific KASP markers. Numerous examples showed that commonly used software for semiautomated KASP primer design frequently fails to achieve locus-specificity of KASP assays in wheat. Instead, alignment of SNP probes with multiple reference genomes and Sanger sequencing of relevant genotypes, followed by visual KASP primer placement, was critical for locus-specificity. To identify KASP assays resulting in false calling of heterozygous individuals, validation of KASP assays using extended reference genotype sets including heterozygous genotypes is strongly advised for polyploid crop species. Applying this strategy, we developed highly reproducible, stable KASP assays that are predictive for root biomass QTL haplotypes from highly homoeologous wheat chromosome regions. Due to their locus-specificity, these assays predicted root biomass considerably better than the original trait-associated markers from the Illumina array. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00122-020-03608-x) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-7360542 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-73605422020-07-16 Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat Makhoul, M. Rambla, C. Voss-Fels, K. P. Hickey, L. T. Snowdon, R. J. Obermeier, C. Theor Appl Genet Original Article KEY MESSAGE: Conversion of SNP chip assays into locus-specific KASP markers requires adapted strategies in polyploid species with high genome homeology. Procedures are exemplified by QTL-associated SNPs in hexaploid wheat. ABSTRACT: Kompetitive allele-specific PCR (KASP) markers are commonly used in marker-assisted commercial plant breeding due to their cost-effectiveness and throughput for high sample volumes. However, conversion of trait-linked SNP markers from array-based SNP detection technologies into KASP markers is particularly challenging in polyploid crop species, due to the presence of highly similar homeologous and paralogous genome sequences. We evaluated strategies and identified key requirements for successful conversion of Illumina Infinium assays from the wheat 90 K SNP array into robust locus-specific KASP markers. Numerous examples showed that commonly used software for semiautomated KASP primer design frequently fails to achieve locus-specificity of KASP assays in wheat. Instead, alignment of SNP probes with multiple reference genomes and Sanger sequencing of relevant genotypes, followed by visual KASP primer placement, was critical for locus-specificity. To identify KASP assays resulting in false calling of heterozygous individuals, validation of KASP assays using extended reference genotype sets including heterozygous genotypes is strongly advised for polyploid crop species. Applying this strategy, we developed highly reproducible, stable KASP assays that are predictive for root biomass QTL haplotypes from highly homoeologous wheat chromosome regions. Due to their locus-specificity, these assays predicted root biomass considerably better than the original trait-associated markers from the Illumina array. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00122-020-03608-x) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2020-06-04 2020 /pmc/articles/PMC7360542/ /pubmed/32500260 http://dx.doi.org/10.1007/s00122-020-03608-x Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Original Article Makhoul, M. Rambla, C. Voss-Fels, K. P. Hickey, L. T. Snowdon, R. J. Obermeier, C. Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat |
title | Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat |
title_full | Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat |
title_fullStr | Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat |
title_full_unstemmed | Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat |
title_short | Overcoming polyploidy pitfalls: a user guide for effective SNP conversion into KASP markers in wheat |
title_sort | overcoming polyploidy pitfalls: a user guide for effective snp conversion into kasp markers in wheat |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360542/ https://www.ncbi.nlm.nih.gov/pubmed/32500260 http://dx.doi.org/10.1007/s00122-020-03608-x |
work_keys_str_mv | AT makhoulm overcomingpolyploidypitfallsauserguideforeffectivesnpconversionintokaspmarkersinwheat AT ramblac overcomingpolyploidypitfallsauserguideforeffectivesnpconversionintokaspmarkersinwheat AT vossfelskp overcomingpolyploidypitfallsauserguideforeffectivesnpconversionintokaspmarkersinwheat AT hickeylt overcomingpolyploidypitfallsauserguideforeffectivesnpconversionintokaspmarkersinwheat AT snowdonrj overcomingpolyploidypitfallsauserguideforeffectivesnpconversionintokaspmarkersinwheat AT obermeierc overcomingpolyploidypitfallsauserguideforeffectivesnpconversionintokaspmarkersinwheat |