MLPA-Based Analysis of Copy Number Variation in Plant Populations
| Autor: | Marek Figlerowicz, Piotr Kozlowski, Malgorzata Marszalek-Zenczak, Agnieszka Zmienko, Malgorzata Marcinkowska-Swojak, Anna Samelak-Czajka |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0106 biological sciences
0301 basic medicine Whole genome sequencing Genetics education.field_of_study multiplexing Population structural variation Plant Science CNV genotyping Biology 1001 Arabidopsis Genomes project 01 natural sciences MLPA Structural variation 03 medical and health sciences 030104 developmental biology Genotype Multiplex Multiplex ligation-dependent probe amplification Copy-number variation education Genotyping Protocols 010606 plant biology & botany |
| Zdroj: | Frontiers in Plant Science |
| ISSN: | 1664-462X |
| Popis: | Copy number variants (CNVs) are intraspecies duplications/deletions of large DNA segments (>1 kb). A growing number of reports highlight the functional and evolutionary impact of CNV in plants, increasing the need for appropriate tools that enable locus-specific CNV genotyping on a population scale. Multiplex ligation-dependent probe amplification (MLPA) is considered a gold standard in genotyping CNV in humans. Consequently, numerous commercial MLPA assays for CNV-related human diseases have been created. We routinely genotype complex multiallelic CNVs in human and plant genomes using the modified MLPA procedure based on fully synthesized oligonucleotide probes (90–200 nt), which greatly simplifies the design process and allows for the development of custom assays. Here, we present a step-by-step protocol for gene-specific MLPA probe design, multiplexed assay setup and data analysis in a copy number genotyping experiment in plants. As a case study, we present the results of a custom assay designed to genotype the copy number status of 12 protein coding genes in a population of 80 Arabidopsis accessions. The genes were pre-selected based on whole genome sequencing data and are localized in the genomic regions that display different levels of population-scale variation (non-variable, biallelic, or multiallelic, as well as CNVs overlapping whole genes or their fragments). The presented approach is suitable for population-scale validation of the CNV regions inferred from whole genome sequencing data analysis and for focused analysis of selected genes of interest. It can also be very easily adopted for any plant species, following optimization of the template amount and design of the appropriate control probes, according to the general guidelines presented in this paper. |
| Databáze: | OpenAIRE |
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