MLPA-Based Analysis of Copy Number Variation in Plant Populations

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.

Attention sports fans! The far-reaching contributions of bud sport mutants to horticulture and plant biology

“…More complex structural variations have been identified on three chromosomes of the grape sport Tempranillo blanco 32 , resembling a chromothripsis-like mechanism (clustered chromosomal rearrangements), which could have been generated by illegitimate re-joining of chromosome breaks in a unique event. Another example of structural variations that can alter gene expression and phenotype are copy number variants, which involve duplications or deletions of large segments of DNA 33 , 34 .…”

Copy number variation and disease resistance in plants

“…In the genome, there are regions that seem to be more prone to CNV than others, due to their specific structural features that will locally induce the mechanisms leading to CNV formation, e.g., non-allelic recombination (Zmienko et al 2016;Samelak-Czajka et al 2017). Differences in the DNA sequence of species' genomes contribute to their uniqueness.…”

AthCNV: A Map of DNA Copy Number Variations in the Arabidopsis Genome

“…The identified CNVs covered 1.8% of the reference genome and overlapped with nearly 500 genes. Subsequent copy number genotyping of several genes performed by our group using these 80 accessions indicated, however, that the number of genes affected by CNVs may in fact be much higher (Samelak-Czajka et al, 2017). Another study involved the detection of regions of deletions and duplications among 180 accessions, but these accessions represented a narrow local population from Sweden (Long et al, 2013).…”