Differences in the intraspecies copy number variation of Arabidopsis thaliana conserved and nonconserved miRNA genes.

MicroRNAs (miRNAs) regulate gene expression by RNA interference mechanism. In plants, miRNA genes (MIRs) which are grouped into conserved families, i.e. they are present among the different plant taxa, are involved in the regulation of many developmental and physiological processes. The roles of the nonconserved MIRs-which are MIRs restricted to one plant family, genus, or even species-are less recognized; however, many of them participate in the responses to biotic and abiotic stresses. Both over- and underproduction of miRNAs may influence various biological processes. Consequently, maintaining intracellular miRNA homeostasis seems to be crucial for the organism. Deletions and duplications in the genomic sequence may alter gene dosage and/or activity. We evaluated the extent of copy number variations (CNVs) among Arabidopsis thaliana (Arabidopsis) MIRs in over 1000 natural accessions, using population-based analysis of the short-read sequencing data. We showed that the conserved MIRs were unlikely to display CNVs and their deletions were extremely rare, whereas nonconserved MIRs presented moderate variation. Transposon-derived MIRs displayed exceptionally high diversity. Conversely, MIRs involved in the epigenetic control of transposons reactivated during development were mostly invariable. MIR overlap with the protein-coding genes also limited their variability. At the expression level, a higher rate of nonvariable, nonconserved miRNAs was detectable in Col-0 leaves, inflorescence, and siliques compared to nonconserved variable miRNAs, although the expression of both groups was much lower than that of the conserved MIRs. Our data indicate that CNV rate of Arabidopsis MIRs is related with their age, function, and genomic localization.

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

Copy number variations (CNVs) greatly contribute to intraspecies genetic polymorphism and phenotypic diversity. Recent analyses of sequencing data for >1000 Arabidopsis (Arabidopsis thaliana) accessions focused on small variations and did not include CNVs. Here, we performed genome-wide analysis and identified large indels (50 to 499 bp) and CNVs (500 bp and larger) in these accessions. The CNVs fully overlap with 18.3% of protein-coding genes, with enrichment for evolutionarily young genes and genes involved in stress and defense. By combining analysis of both genes and transposable elements (TEs) affected by CNVs, we revealed that the variation statuses of genes and TEs are tightly linked and jointly contribute to the unequal distribution of these elements in the genome. We also determined the gene copy numbers in a set of 1060 accessions and experimentally validated the accuracy of our predictions by multiplex ligation-dependent probe amplification assays. We then successfully used the CNVs as markers to analyze population structure and migration patterns. Finally, we examined the impact of gene dosage variation triggered by a CNV spanning the SEC10 gene on SEC10 expression at both the transcript and protein levels. The catalog of CNVs, CNV-overlapping genes, and their genotypes in a top model dicot will stimulate the exploration of the genetic basis of phenotypic variation.

Polymorphisms in EGFR Gene Predict Clinical Outcome in Unresectable Non-Small Cell Lung Cancer Treated with Radiotherapy and Platinum-Based Chemoradiotherapy.

For non-small cell lung cancer (NSCLC), radiotherapy (RT) and platinum-based chemotherapy (CHT) are among the main treatment options. On the other hand, radioresistance and cytotoxic drug resistance are common causes of failure. The epidermal growth factor receptor (EGFR) plays an important role in radioresponse and therapy resistance. We hypothesized that single nucleotide polymorphisms (SNPs) in the EGFR gene might affect individual sensitivity to these treatments, and thus, therapy outcome and prognosis. The association between functional EGFR SNPs and overall (OS), locoregional recurrence-free (LFRS), and metastasis-free (MFS) survival was examined in 436 patients with unresectable NSCLC receiving RT and platinum-based CHTRT. In a multivariate analysis, the rs712830 CC homozygotes showed reduced OS in the whole group (p = 0.039) and in the curative treatment subset (p = 0.047). The rs712829 TT genotype was strongly associated with decreased LRFS (p = 0.006), and the T-C haplotype was a risk factor for locoregional recurrence in our patients (p = 0.003). The rs2227983 GG alone and in combination with rs712829 T was an indicator of unfavorable LRFS (p = 0.028 and 0.002, respectively). Moreover, significant independent effects of these SNPs on OS, LRFS, and MFS were observed. Our results demonstrate that inherited EGFR gene variants may predict clinical outcomes in NSCLC treated with DNA damage-inducing therapy.

Analysis of Arabidopsis non-reference accessions reveals high diversity of metabolic gene clusters and discovers new candidate cluster members.

Metabolic gene clusters (MGCs) are groups of genes involved in a common biosynthetic pathway. They are frequently formed in dynamic chromosomal regions, which may lead to intraspecies variation and cause phenotypic diversity. We examined copy number variations (CNVs) in four Arabidopsis thaliana MGCs in over one thousand accessions with experimental and bioinformatic approaches. Tirucalladienol and marneral gene clusters showed little variation, and the latter was fixed in the population. Thalianol and especially arabidiol/baruol gene clusters displayed substantial diversity. The compact version of the thalianol gene cluster was predominant and more conserved than the noncontiguous version. In the arabidiol/baruol cluster, we found a large genomic insertion containing divergent duplicates of the CYP705A2 and BARS1 genes. The BARS1 paralog, which we named BARS2, encoded a novel oxidosqualene synthase. The expression of the entire arabidiol/baruol gene cluster was altered in the accessions with the duplication. Moreover, they presented different root growth dynamics and were associated with warmer climates compared to the reference-like accessions. In the entire genome, paired genes encoding terpene synthases and cytochrome P450 oxidases were more variable than their nonpaired counterparts. Our study highlights the role of dynamically evolving MGCs in plant adaptation and phenotypic diversity.

MLPA-Based Analysis of Copy Number Variation in Plant Populations.

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.