Abstract
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.
Highlights
The rise of high-throughput genomics techniques – DNA arrays and, more recently, wholegenome sequencing (WGS) – has revealed the structural complexity and dynamics of eukaryotic genomes
We described the protocol for the simple MLPAbased Copy number variants (CNVs) genotyping in plants, with particular emphasis on the model plant Arabidopsis
We provided a description of the probe design process, experimental setup, and data analysis
Summary
The rise of high-throughput genomics techniques – DNA arrays and, more recently, wholegenome sequencing (WGS) – has revealed the structural complexity and dynamics of eukaryotic genomes. The number of species for which CNV regions have been inferred on the genome-wide scale is growing rapidly This list includes maize, rice, sorghum, Arabidopsis (Arabidopsis thaliana), soybean, wheat, and barley (Springer et al, 2009; Beló et al, 2010; Swanson-Wagner et al, 2010; Cao et al, 2011; Saintenac et al, 2011; Zheng et al, 2011; McHale et al, 2012; Muñoz-Amatriaín et al, 2013; Duitama et al, 2015; Bai et al, 2016). It is anticipated that the number of genetic studies focused on individual CNVs of interest will grow and that new CNV-associated traits will be revealed
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