CNV-seq, a new method to detect copy number variation using high-throughput sequencing

Background  

DNA copy number variation (CNV) has been recognized as an important source of genetic variation. Array comparative genomic hybridization (aCGH) is commonly used for CNV detection, but the microarray platform has a number of inherent limitations.     

Evolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens

Glycosyl hydrolase family 28 (GH28) is a set of structurally related enzymes that hydrolyze glycosidic bonds in pectin, and are important extracellular enzymes for both pathogenic and saprotrophic fungi. Yet, very little is understood about the evolutionary forces driving the diversification of GH28s in fungal genomes. We reconstructed the evolutionary history of family GH28 in fungi by examining the distribution of GH28 copy number across the phylogeny of fungi, and by reconstructing the phylogeny of GH28 genes. We also examined the relationship between lineage-specific GH28 expansions and fungal ecological strategy, testing the hypothesis that GH28 evolution in fungi is driven by ecological strategy (pathogenic vs. non-pathogenic) and pathogenic niche (necrotrophic vs. biotrophic). Our results showed that GH28 phylogeny of Ascomycota and Basidiomycota sequences was structured by specific biochemical function, with endo-polygalacturonases and endo-rhamnogalacturonases forming distinct, apparently ancient clades, while exo-polygalacturonases are more widely distributed. In contrast, Mucoromycotina and Stramenopile sequences formed taxonomically-distinct clades. Large, lineage-specific variation in GH28 copy number indicates that the evolution of this gene family is consistent with the birth-and-death model of gene family evolution, where diversity of GH28 loci within genomes was generated through multiple rounds of gene duplication followed by functional diversification and loss of some gene family members. Although GH28 copy number was correlated with genome size, our findings suggest that ecological strategy also plays an important role in determining the GH28 repertoire of fungi. Both necrotrophic and biotrophic fungi have larger genomes than non-pathogens, yet only necrotrophs possess more GH28 enzymes than non-pathogens. Hence, lineage-specific GH28 expansion is the result of both variation in genome size across fungal species and diversifying selection within the necrotrophic plant pathogen ecological niche. GH28 evolution among necrotrophs has likely been driven by a co-evolutionary arms race with plants, whereas the need to avoid plant immune responses has resulted in purifying selection within biotrophic fungi.     

Soil fungal communities of grasslands are environmentally structured at a regional scale in the Alps

Studying patterns of species distributions along elevation gradients is frequently used to identify the primary factors that determine the distribution, diversity and assembly of species. However, despite their crucial role in ecosystem functioning, our understanding of the distribution of below‐ground fungi is still limited, calling for more comprehensive studies of fungal biogeography along environmental gradients at various scales (from regional to global). Here, we investigated the richness of taxa of soil fungi and their phylogenetic diversity across a wide range of grassland types along a 2800 m elevation gradient at a large number of sites (213), stratified across a region of the Western Swiss Alps (700 km²). We used 454 pyrosequencing to obtain fungal sequences that were clustered into operational taxonomic units (OTUs). The OTU diversity–area relationship revealed uneven distribution of fungal taxa across the study area (i.e. not all taxa are everywhere) and fine‐scale spatial clustering. Fungal richness and phylogenetic diversity were found to be higher in lower temperatures and higher moisture conditions. Climatic and soil characteristics as well as plant community composition were related to OTU alpha, beta and phylogenetic diversity, with distinct fungal lineages suggesting distinct ecological tolerances. Soil fungi, thus, show lineage‐specific biogeographic patterns, even at a regional scale, and follow environmental determinism, mediated by interactions with plants.     

Identification of SNP markers for common CNV regions and association analysis of risk of subarachnoid aneurysmal hemorrhage in Japanese population

Copy number variation (CNV) is emerging as a new tool for understanding human genomic variation, but its relationship with human disease is not yet fully understood. The data for a total of 317,503 genotypes were collected for a genome-wide association study of subarachnoid aneurismal hemorrhage (SAH) in a Japanese population (cases and controls, n = 497) using Illumina HumanHap300 BeadChip^®. To identify multi-allelic CNV markers, we visually inspected all genotype clusters of 317,503 SNP markers covering the whole genome using Illumina’s BeadStudio 3.0^® software. As a result, we identified 597 multi-allelic CNV markers for common (copy loss frequency > 0.05) CNV regions in a Japanese population (n = 497). The identified CNV markers shared the following characteristics: enrichment of Hardy–Weinberg disequilibria, Mendelian inconsistency among families, and high missing genotype rate. All annotated information for those markers is summarized in our database (http://www.snp-genetics.com/user/srch.htm). In addition, we performed case-control association analyses of identified multi-allelic CNV markers with the risk of subarachnoid aneurysmal hemorrhage. One SNP marker (rs1242541) within a CNV region neighboring the Sel-1 suppressor of lin-12-like protein (SEL1L) was significantly associated with a risk of SAH (P = 0.0006). We also validated the CNV around rs1242541 using real-time quantitative polymerase chain reaction (PCR). Information and methods used in this study would be helpful for accurate genotyping of SNPs on CNV regions, which could be used for association analysis of SNP markers within CNV regions.     

Copy number variation associates with mortality in long‐lived individuals: a genome‐wide assessment

Copy number variants (CNVs) represent a significant source of genetic variation in the human genome and have been implicated in numerous diseases and complex traits. To date, only a few studies have investigated the role of CNVs in human lifespan. To investigate the impact of CNVs on prospective mortality at the extreme end of life, where the genetic component of lifespan appears most profound, we analyzed genomewide CNV data in 603 Danish nonagenarians and centenarians (mean age 96.9 years, range 90.0–102.5 years). Replication was performed in 500 long‐lived individuals from the Leiden Longevity Study (mean age 93.2 years, range 88.9–103.4 years). First, we assessed the association between the CNV burden of each individual (the number of CNVs, the average CNV length, and the total CNV length) and mortality and found a significant increase in mortality per 10 kb increase in the average CNV length, both for all CNVs (hazard ratio (HR) = 1.024, P = 0.002) and for duplications (HR = 1.011, P = 0.005), as well as per 100 kb increase in the total length of deletions (HR = 1.009, P = 0.0005). Next, we assessed the relation between specific deletions and duplications and mortality. Although no genome–wide significant associations were discovered, we identified six deletions and one duplication that showed consistent association with mortality in both or either of the sexes across both study populations. These results indicate that the genome–wide CNV burden, specifically the average CNV length and the total CNV length, associates with higher mortality in long‐lived individuals.     

The rpb2 gene represents a viable alternative molecular marker for the analysis of environmental fungal communities

Although the commonly used internal transcribed spacer region of rDNA (ITS) is well suited for taxonomic identification of fungi, the information on the relative abundance of taxa and diversity is negatively affected by the multicopy nature of rDNA and the existence of ITS paralogues. Moreover, due to high variability, ITS sequences cannot be used for phylogenetic analyses of unrelated taxa. The part of single‐copy gene encoding the second largest subunit of RNA polymerase II (rpb2) was thus compared with first spacer of ITS as an alternative marker for the analysis of fungal communities in spruce forest topsoil, and their applicability was tested on a comprehensive mock community. In soil, rpb2 exhibited broad taxonomic coverage of the entire fungal tree of life including basal fungal lineages. The gene exhibited sufficient variation for the use in phylogenetic analyses and taxonomic assignments, although it amplifies also paralogues. The fungal taxon spectra obtained with rbp2 region and ITS1 corresponded, but sequence abundance differed widely, especially in the basal lineages. The proportions of OTU counts and read counts of major fungal groups were close to the reality when rpb2 was used as a molecular marker while they were strongly biased towards the Basidiomycota when using the ITS primers ITS1/ITS4. Although the taxonomic placement of rbp2 sequences is currently more difficult than that of the ITS sequences, its discriminative power, quantitative representation of community composition and suitability for phylogenetic analyses represent significant advantages.     

Estimation of fungal biomass in forest litter and soil

The contents of fungal biomass markers were analysed in the fruit bodies of dominant basidiomycetes from an ectomycorrhiza-dominated coniferous forest, and used to estimate the fungal biomass content in the litter and soil. The content of ergosterol (3.8 ± 2.0 mg g^?1 dry fungal biomass) and the phospholipid fatty acid 18:2ω6,9 (11.6 ± 4.3 mg g^?1) showed less variation than the internal transcribed spacer (ITS) copy numbers (375 ± 294 × 10⁹ copies g^?1). A high level of variation in the ITS copy numbers (per ng DNA) was also found among fungal taxa. The content of fungal biomass in the litter and soil, calculated using the mean contents, varied between 0.66 and 6.24 mg g^?1 fungal biomass in the litter, and 0.22 and 0.68 mg g^?1 in the soil. The ratio of fungal biomass in the litter to that in the soil varied greatly among the markers. The estimates of fungal biomass obtained with different biomarkers are not exactly comparable, and caution should be used when analysing taxon abundance using PCR amplification of fungal rDNA.     

Finding single copy genes out of sequenced genomes for multilocus phylogenetics in non-model fungi

Historically, fungal multigene phylogenies have been reconstructed based on a small number of commonly used genes. The availability of complete fungal genomes has given rise to a new wave of model organisms that provide large number of genes potentially useful for building robust gene genealogies. Unfortunately, cross-utilization of these resources to study phylogenetic relationships in the vast majority of non-model fungi (i.e. "orphan" species) remains an unexamined question. To address this problem, we developed a method coupled with a program named "PHYLORPH" (PHYLogenetic markers for ORPHans). The method screens fungal genomic databases (107 fungal genomes fully sequenced) for single copy genes that might be easily transferable and well suited for studies at low taxonomic levels (for example, in species complexes) in non-model fungal species. To maximize the chance to target genes with informative regions, PHYLORPH displays a graphical evaluation system based on the estimation of nucleotide divergence relative to substitution type. The usefulness of this approach was tested by developing markers in four non-model groups of fungal pathogens. For each pathogen considered, 7 to 40% of the 10-15 best candidate genes proposed by PHYLORPH yielded sequencing success. Levels of polymorphism of these genes were compared with those obtained for some genes traditionally used to build fungal phylogenies (e.g. nuclear rDNA, β-tubulin, γ-actin, Elongation factor EF-1α). These genes were ranked among the best-performing ones and resolved accurately taxa relationships in each of the four non-model groups of fungi considered. We envision that PHYLORPH will constitute a useful tool for obtaining new and accurate phylogenetic markers to resolve relationships between closely related non-model fungal species.     

Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses

Background  

Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32) into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi.     

Extreme copy number variation at a tRNA ligase gene affecting phenology and fitness in yellow monkeyflowers

Copy number variation (CNV) is a major part of the genetic diversity segregating within populations, but remains poorly understood relative to single nucleotide variation. Here, we report on a tRNA ligase gene (Migut.N02091; RLG1a) exhibiting unprecedented, and fitness‐relevant, CNV within an annual population of the yellow monkeyflower Mimulus guttatus. RLG1a variation was associated with multiple traits in pooled population sequencing (PoolSeq) scans of phenotypic and phenological cohorts. Resequencing of inbred lines revealed intermediate‐frequency three‐copy variants of RLG1a (trip+; 5/35 = 14%), and trip+ lines exhibited elevated RLG1a expression under multiple conditions. trip+ carriers, in addition to being over‐represented in late‐flowering and large‐flowered PoolSeq populations, flowered later under stressful conditions in a greenhouse experiment (p < 0.05). In wild population samples, we discovered an additional rare RLG1a variant (high+) that carries 250–300 copies of RLG1a totalling ~5.7 Mb (20–40% of a chromosome). In the progeny of a high+ carrier, Mendelian segregation of diagnostic alleles and qPCR‐based copy counts indicate that high+ is a single tandem array unlinked to the single‐copy RLG1a locus. In the wild, high+ carriers had highest fitness in two particularly dry and/or hot years (2015 and 2017; both p < 0.01), while single‐copy individuals were twice as fecund as either CNV type in a lush year (2016: p < 0.005). Our results demonstrate fluctuating selection on CNVs affecting phenological traits in a wild population, suggest that plant tRNA ligases mediate stress‐responsive life‐history traits, and introduce a novel system for investigating the molecular mechanisms of gene amplification.     

Gene-based copy number variation study reveals a microdeletion at 12q24 that influences height in the Korean population

Height is a classic polygenic trait with high heritability (h² = 0.8). Recent genome-wide association studies have revealed many independent loci associated with human height. In addition, although many studies have reported an association between copy number variation (CNV) and complex diseases, few have explored the relationship between CNV and height. Recent studies reported that single nucleotide polymorphisms (SNPs) are highly correlated with common CNVs, suggesting that it is warranted to survey CNVs to identify additional genetic factors affecting heritable traits such as height.This study tested the hypothesis that there would be CNV regions (CNVRs) associated with height nearby genes from the GWASs known to affect height. We identified regions containing > 1% copy number deletion frequency from 3667 population-based cohort samples using the Illumina HumanOmni1-Quad BeadChip. Among the identified CNVRs, we selected 15 candidate regions that were located within 1 Mb of 283 previously reported genes. To assess the effect of these CNVRs on height, statistical analyses were conducted with samples from a case group of 370 taller (upper 10%) individuals and a control group of 1828 individuals (lower 50%).We found that a newly identified 17.7 kb deletion at chromosomal position 12q24.33, approximately 171.6 kb downstream of GPR133, significantly correlated with height; this finding was validated using quantitative PCR. These results suggest that CNVs are potentially important in determining height and may contribute to height variation in human populations.     

Landscape‐scale genetic differentiation of a mycangial fungus associated with the ambrosia beetle,Xylosandrus germanus (Blandford) (Curculionidae:Scolytinae) in Japan

In this study, we examined the genetic structures of the ambrosia fungus isolated from mycangia of the scolytine beetle, Xylosandrus germanus to understand their co‐evolutionary relationships. We analyzed datasets of three ambrosia fungus loci (18S rDNA, 28S rDNA, and the β‐tubulin gene) and a X. germanus locus dataset (cytochrome c oxidase subunit 1 (COI) mitochondrial DNA). The ambrosia fungi were separated into three cultural morphptypes, and their haplotypes were distinguished by phylogenetic analysis on the basis of the three loci. The COI phylogenetic analysis revealed three distinct genetic lineages (clades A, B, and C) within X. germanus, each of which corresponded to specific ambrosia fungus cultural morphptypes. The fungal symbiont phylogeny was not concordant with that of the beetle. Our results suggest that X. germanus may be unable to exchange its mycangial fungi, but extraordinary horizontal transmission of symbiotic fungi between the beetle's lineages occurred at least once during the evolutionary history of this symbiosis.     

Whole Genome Distribution and Ethnic Differentiation of Copy Number Variation in Caucasian and Asian Populations

Although copy number variation (CNV) has recently received much attention as a form of structure variation within the human genome, knowledge is still inadequate on fundamental CNV characteristics such as occurrence rate, genomic distribution and ethnic differentiation. In the present study, we used the Affymetrix GeneChip® Mapping 500K Array to discover and characterize CNVs in the human genome and to study ethnic differences of CNVs between Caucasians and Asians. Three thousand and nineteen CNVs, including 2381 CNVs in autosomes and 638 CNVs in X chromosome, from 985 Caucasian and 692 Asian individuals were identified, with a mean length of 296 kb. Among these CNVs, 190 had frequencies greater than 1% in at least one ethnic group, and 109 showed significant ethnic differences in frequencies (p<0.01). After merging overlapping CNVs, 1135 copy number variation regions (CNVRs), covering approximately 439 Mb (14.3%) of the human genome, were obtained. Our findings of ethnic differentiation of CNVs, along with the newly constructed CNV genomic map, extend our knowledge on the structural variation in the human genome and may furnish a basis for understanding the genomic differentiation of complex traits across ethnic groups.     

Invasion potential and host shifts of Australian and African ectomycorrhizal fungi in mixed eucalypt plantations

? Transportation of forestry materials results in unintended co-introduction of nonnative species that may cause enormous ecological or economic damage. While the invasion ecology of plants and animals is relatively well-known, that of microorganisms, except aboveground pathogens, remains poorly understood. ? This work addresses host shifts and invasion potential of root symbiotic ectomycorrhizal fungi that were co-introduced with Australian eucalypts and planted in clear-cut miombo woodlands in Zambia, south-central Africa. ? By use of rDNA and plastid intron sequence analysis for identification and phylogenetic techniques for inferring fungal origin, we demonstrated that host shifts were uncommon in the Australian fungi, but frequent in the African fungi, especially in mixed plantations where roots of different trees intermingle. ? There was evidence for naturalization, but not for invasion by Australian ectomycorrhizal fungi. Nevertheless, the fungi introduced may pose an invasion risk along with further adaptation to local soil environment and host trees. Inoculation of eucalypts with native edible fungi may ameliorate the potential invasion risks of introduced fungi and provide an alternative source of nutrition.     

The Association between DNA Copy Number Aberrations at Chromosome 5q22 and Gastric Cancer

Background

Gastric cancer is common cancer. Discovering novel genetic biomarkers might help to identify high-risk individuals. Copy number variation (CNV) has recently been shown to influence risk for several cancers. The aim of the present study was sought to test the association between copy number at a variant region and GC.

Methods

A total of 110 gastric cancer patients and 325 healthy volunteers were enrolled in this study. We searched for a CNV and found a CNV (Variation 7468) containing part of the APC gene, the SRP19 gene and the REEP5 gene. We chose four probes targeting at APC-intron8, APC-exon9, SRP19 and REEP5 to interrogate this CNV. Specific Taqman probes labeled by different reporter fluorophores were used in a real-time PCR platform to obtain copy number. Both the original non-integer data and transformed integer data on copy number were used for analyses.

Results

Gastric caner patients had a lower non-integer copy number than controls for the APC-exon9 probe (Adjusted p = 0.026) and SRP19 probe (Adjusted p = 0.002). The analysis of integer copy number yielded a similar pattern although less significant (Adjusted p = 0.07 for APC-exon9 probe and Adjusted p = 0.02 for SRP19 probe).

Conclusions

Losses of a CNV at 5q22, especially in the DNA region surrounding APC-exon 9, may be associated with a higher risk of gastric cancer.     

Genetic monogamy despite variable ecological conditions and social environment in the cooperatively breeding apostlebird

Mating strategies may be context‐dependent and may vary across ecological and social contexts, demonstrating the role of these factors in driving the variation in genetic polyandry within and among species. Here, we took a longitudinal approach across 5 years (2006–2010), to study the apostlebird (Struthidea cinerea), an Australian cooperatively breeding bird, whose reproduction is affected by ecological “boom and bust” cycles. Climatic variation drives variation in the social (i.e., group sizes, proportion of males and females) and ecological (i.e., plant and insect abundance) context in which mating occurs. By quantifying variation in both social and ecological factors and characterizing the genetic mating system across multiple years using a molecular parentage analysis, we found that the genetic mating strategy did not vary among years despite significant variation in rainfall, driving primary production, and insect abundance, and corresponding variation in social parameters such as breeding group size. Group sizes in 2010, an ecologically good year, were significantly smaller (mean = 5.8 ± 0.9, n = 16) than in the drought affected years, between 2006 and 2008, (mean = 9.1 ± 0.5, n = 63). Overall, apostlebirds were consistently monogamous with few cases of multiple maternity or paternity (8 of 78 nests) across all years.     

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.     

Mycorrhizas: Gene to Function

Substantial progress has been made toward development of molecular tools for identification and quantification of mycorrhizal fungi in roots and evaluation of the diversity of ectomycorrhizal (ECM) fungi and the phylogeny and genetic structure of arbuscular mycorrhizal (AM) fungi. rDNA analysis confirms high diversity of ECM fungi on their hosts, and for AM fungi has revealed considerable genetic variation within and among morphologically similar AM fungal species. The fungal and plant genes, regulation of their expression, and biochemical pathways for nutrient exchange between symbiotic partners are now coming under intense study and will eventually be used to define the ecological nutritional role of the fungi. While molecular biological approaches have increased understanding of the mycorrhizal symbiosis, such knowledge about these lower-scale processes has yet to influence our understanding of larger-scale responses to any great extent.