Genome-wide patterns of copy number variation in the diversified chicken genomes using next-generation sequencing

Background

Copy number variation (CNV) is important and widespread in the genome, and is a major cause of disease and phenotypic diversity. Herein, we performed a genome-wide CNV analysis in 12 diversified chicken genomes based on whole genome sequencing.

Results

A total of 8,840 CNV regions (CNVRs) covering 98.2 Mb and representing 9.4% of the chicken genome were identified, ranging in size from 1.1 to 268.8 kb with an average of 11.1 kb. Sequencing-based predictions were confirmed at a high validation rate by two independent approaches, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR). The Pearson’s correlation coefficients between sequencing and aCGH results ranged from 0.435 to 0.755, and qPCR experiments revealed a positive validation rate of 91.71% and a false negative rate of 22.43%. In total, 2,214 (25.0%) predicted CNVRs span 2,216 (36.4%) RefSeq genes associated with specific biological functions. Besides two previously reported copy number variable genes EDN3 and PRLR, we also found some promising genes with potential in phenotypic variation. Two genes, FZD6 and LIMS1, related to disease susceptibility/resistance are covered by CNVRs. The highly duplicated SOCS2 may lead to higher bone mineral density. Entire or partial duplication of some genes like POPDC3 may have great economic importance in poultry breeding.

Conclusions

Our results based on extensive genetic diversity provide a more refined chicken CNV map and genome-wide gene copy number estimates, and warrant future CNV association studies for important traits in chickens.

Electronic supplementary material

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Copy number variation in the speciation of pigs: a possible prominent role for olfactory receptors

Background

Unraveling the genetic mechanisms associated with reduced gene flow between genetically differentiated populations is key to understand speciation. Different types of structural variations (SVs) have been found as a source of genetic diversity in a wide range of species. Previous studies provided detailed knowledge on the potential evolutionary role of SVs, especially copy number variations (CNVs), between well diverged species of e.g. primates. However, our understanding of their significance during ongoing speciation processes is limited due to the lack of CNV data from closely related species. The genus Sus (pig and its close relatives) which started to diverge ~4 Mya presents an excellent model for studying the role of CNVs during ongoing speciation.

Results

In this study, we identified 1408 CNV regions (CNVRs) across the genus Sus. These CNVRs encompass 624 genes and were found to evolve ~2.5 times faster than single nucleotide polymorphisms (SNPs). The majority of these copy number variable genes are olfactory receptors (ORs) known to play a prominent role in food foraging and mate recognition in Sus. Phylogenetic analyses, including novel Bayesian analysis, based on CNVRs that overlap ORs retain the well-accepted topology of the genus Sus whereas CNVRs overlapping genes other than ORs show evidence for random drift and/or admixture.

Conclusion

We hypothesize that inter-specific variation in copy number of ORs provided the means for rapid adaptation to different environments during the diversification of the genus Sus in the Pliocene. Furthermore, these regions might have acted as barriers preventing massive gene flow between these species during the multiple hybridization events that took place later in the Pleistocene suggesting a possible prominent role of ORs in the ongoing Sus speciation.

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Detection of copy number variations and their effects in Chinese bulls

Background

Copy number variations (CNVs) are a main source of genomic structural variations underlying animal evolution and production traits. Here, with one pure-blooded Angus bull as reference, we describe a genome-wide analysis of CNVs based on comparative genomic hybridization arrays in 29 Chinese domesticated bulls and examined their effects on gene expression and cattle growth traits.

Results

We identified 486 copy number variable regions (CNVRs), covering 2.45% of the bovine genome, in 24 taurine (Bos taurus), together with 161 ones in 2 yaks (Bos grunniens) and 163 ones in 3 buffaloes (Bubalus bubalis). Totally, we discovered 605 integrated CNVRs, with more “loss” events than both “gain” and “both” ones, and clearly clustered them into three cattle groups. Interestingly, we confirmed their uneven distributions across chromosomes, and the differences of mitochondrion DNA copy number (gain: taurine, loss: yak & buffalo). Furthermore, we confirmed approximately 41.8% (253/605) and 70.6% (427/605) CNVRs span cattle genes and quantitative trait loci (QTLs), respectively. Finally, we confirmed 6 CNVRs in 9 chosen ones by using quantitative PCR, and further demonstrated that CNVR22 had significantly negative effects on expression of PLA2G2D gene, and both CNVR22 and CNVR310 were associated with body measurements in Chinese cattle, suggesting their key effects on gene expression and cattle traits.

Conclusions

The results advanced our understanding of CNV as an important genomic structural variation in taurine, yak and buffalo. This study provides a highly valuable resource for Chinese cattle’s evolution and breeding researches.

Electronic supplementary material

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Genomic copy number variation in Mus musculus

Background

Copy number variation is an important dimension of genetic diversity and has implications in development and disease. As an important model organism, the mouse is a prime candidate for copy number variant (CNV) characterization, but this has yet to be completed for a large sample size. Here we report CNV analysis of publicly available, high-density microarray data files for 351 mouse tail samples, including 290 mice that had not been characterized for CNVs previously.

Results

We found 9634 putative autosomal CNVs across the samples affecting 6.87 % of the mouse reference genome. We find significant differences in the degree of CNV uniqueness (single sample occurrence) and the nature of CNV-gene overlap between wild-caught mice and classical laboratory strains. CNV-gene overlap was associated with lipid metabolism, pheromone response and olfaction compared to immunity, carbohydrate metabolism and amino-acid metabolism for wild-caught mice and classical laboratory strains, respectively. Using two subspecies of wild-caught Mus musculus, we identified putative CNVs unique to those subspecies and show this diversity is better captured by wild-derived laboratory strains than by the classical laboratory strains. A total of 9 genic copy number variable regions (CNVRs) were selected for experimental confirmation by droplet digital PCR (ddPCR).

Conclusion

The analysis we present is a comprehensive, genome-wide analysis of CNVs in Mus musculus, which increases the number of known variants in the species and will accelerate the identification of novel variants in future studies.

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Global copy number analyses by next generation sequencing provide insight into pig genome variation

Background

Copy number variations (CNVs) confer significant effects on genetic innovation and phenotypic variation. Previous CNV studies in swine seldom focused on in-depth characterization of global CNVs.

Results

Using whole-genome assembly comparison (WGAC) and whole-genome shotgun sequence detection (WSSD) approaches by next generation sequencing (NGS), we probed formation signatures of both segmental duplications (SDs) and individualized CNVs in an integrated fashion, building the finest resolution CNV and SD maps of pigs so far. We obtained copy number estimates of all protein-coding genes with copy number variation carried by individuals, and further confirmed two genes with high copy numbers in Meishan pigs through an enlarged population. We determined genome-wide CNV hotspots, which were significantly enriched in SD regions, suggesting evolution of CNV hotspots may be affected by ancestral SDs. Through systematically enrichment analyses based on simulations and bioinformatics analyses, we revealed CNV-related genes undergo a different selective constraint from those CNV-unrelated regions, and CNVs may be associated with or affect pig health and production performance under recent selection.

Conclusions

Our studies lay out one way for characterization of CNVs in the pig genome, provide insight into the pig genome variation and prompt CNV mechanisms studies when using pigs as biomedical models for human diseases.

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The genetic effect of copy number variations on the risk of type 2 diabetes in a Korean population

Background

Unlike Caucasian populations, genetic factors contributing to the risk of type 2 diabetes mellitus (T2DM) are not well studied in Asian populations. In light of this, and the fact that copy number variation (CNV) is emerging as a new way to understand human genomic variation, the objective of this study was to identify type 2 diabetes–associated CNV in a Korean cohort.

Methodology/Principal Findings

Using the Illumina HumanHap300 BeadChip (317,503 markers), genome-wide genotyping was performed to obtain signal and allelic intensities from 275 patients with type 2 diabetes mellitus (T2DM) and 496 nondiabetic subjects (Total n = 771). To increase the sensitivity of CNV identification, we incorporated multiple factors using PennCNV, a program that is based on the hidden Markov model (HMM). To assess the genetic effect of CNV on T2DM, a multivariate logistic regression model controlling for age and gender was used. We identified a total of 7,478 CNVs (average of 9.7 CNVs per individual) and 2,554 CNV regions (CNVRs; 164 common CNVRs for frequency>1%) in this study. Although we failed to demonstrate robust associations between CNVs and the risk of T2DM, our results revealed a putative association between several CNVRs including chr15:45994758–45999227 (P = 8.6E-04, P^corr = 0.01) and the risk of T2DM. The identified CNVs in this study were validated using overlapping analysis with the Database of Genomic Variants (DGV; 71.7% overlap), and quantitative PCR (qPCR). The identified variations, which encompassed functional genes, were significantly enriched in the cellular part, in the membrane-bound organelle, in the development process, in cell communication, in signal transduction, and in biological regulation.

Conclusion/Significance

We expect that the methods and findings in this study will contribute in particular to genome studies of Asian populations.     

Identification of copy number variants from exome sequence data

Background

With advances in next generation sequencing technologies and genomic capture techniques, exome sequencing has become a cost-effective approach for mutation detection in genetic diseases. However, computational prediction of copy number variants (CNVs) from exome sequence data is a challenging task. Whilst numerous programs are available, they have different sensitivities, and have low sensitivity to detect smaller CNVs (1–4 exons). Additionally, exonic CNV discovery using standard aCGH has limitations due to the low probe density over exonic regions. The goal of our study was to develop a protocol to detect exonic CNVs (including shorter CNVs that cover 1–4 exons), combining computational prediction algorithms and a high-resolution custom CGH array.

Results

We used six published CNV prediction programs (ExomeCNV, CONTRA, ExomeCopy, ExomeDepth, CoNIFER, XHMM) and an in-house modification to ExomeCopy and ExomeDepth (ExCopyDepth) for computational CNV prediction on 30 exomes from the 1000 genomes project and 9 exomes from primary immunodeficiency patients. CNV predictions were tested using a custom CGH array designed to capture all exons (exaCGH). After this validation, we next evaluated the computational prediction of shorter CNVs. ExomeCopy and the in-house modified algorithm, ExCopyDepth, showed the highest capability in detecting shorter CNVs. Finally, the performance of each computational program was assessed by calculating the sensitivity and false positive rate.

Conclusions

In this paper, we assessed the ability of 6 computational programs to predict CNVs, focussing on short (1–4 exon) CNVs. We also tested these predictions using a custom array targeting exons. Based on these results, we propose a protocol to identify and confirm shorter exonic CNVs combining computational prediction algorithms and custom aCGH experiments.

Electronic supplementary material

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Identification and functional characterization of copy number variations in diverse chicken breeds

Background

The detection and functional characterization of genomic structural variations are important for understanding the landscape of genetic variation in the chicken. A recently recognized aspect of genomic structural variation, called copy number variation (CNV), is gaining interest in chicken genomic studies. The aim of the present study was to investigate the pattern and functional characterization of CNVs in five characteristic chicken breeds, which will be important for future studies associating phenotype with chicken genome architecture.

Results

Using a commercial 385 K array-based comparative genomic hybridization (aCGH) genome array, we performed CNV discovery using 10 chicken samples from four local Chinese breeds and the French breed Houdan chicken. The female Anka broiler was used as a reference. A total of 281 copy number variation regions (CNVR) were identified, covering 12.8 Mb of polymorphic sequences or 1.07% of the entire chicken genome. The functional annotation of CNVRs indicated that these regions completely or partially overlapped with 231 genes and 1032 quantitative traits loci, suggesting these CNVs have important functions and might be promising resources for exploring differences among various breeds. In addition, we employed quantitative PCR (qPCR) to further validate several copy number variable genes, such as prolactin receptor, endothelin 3 (EDN3), suppressor of cytokine signaling 2, CD8a molecule, with important functions, and the results suggested that EDN3 might be a molecular marker for the selection of dark skin color in poultry production. Moreover, we also identified a new CNVR (chr24: 3484617–3512275), encoding the sortilin-related receptor gene, with copy number changes in only black-bone chicken.

Conclusions

Here, we report a genome-wide analysis of the CNVs in five chicken breeds using aCGH. The association between EDN3 and melanoblast proliferation was further confirmed using qPCR. These results provide additional information for understanding genomic variation and related phenotypic characteristics.

Electronic supplementary material

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Fine mapping QTL for female fertility on BTA04 and BTA13 in dairy cattle using HD SNP and sequence data

Background

Female fertility is important for the maintenance of the production in a dairy cattle herd. Two QTL regions on BTA04 and on BTA13 previously detected in Nordic Holstein (NH) and validated in the Danish Jersey (DJ) and Nordic Red (NR) were investigated further in the present study to further refine the QTL locations. Refined QTL regions were imputed to the full sequence data. The genes in the regions were then studied to ascertain their possible effect on fertility traits.

Results

BTA04 was screened for number of inseminations (AIS), 56-day non-return rate (NRR), days from first to last insemination (IFL), and the interval from calving to first insemination (ICF) in the range of 38,257,758 to 40,890,784 bp, whereas BTA13 was screened for ICF only in the range from 21,236,959 to 46,150,079 with the HD bovine SNP array for NH, DJ and NR. No markers in the DJ and NR breeds reached significance. By analyzing imputed sequence data the QTL position on BTA04 was narrowed down to two regions in the NH. In these two regions a total of 9 genes were identified. BTA13 was analyzed using sequence data for the NH breed. The highest –log₁₀(P-value) was 19.41 at 33,903,159 bp. Two regions were identified: Region 1: 33,900,143-33,908,994 bp and Region 2: 34,051,815-34,056,728 bp. SNPs within and between these two regions were annotated as intergenic.

Conclusion

Screening BTA04 and BTA13 for female fertility traits in NH, NR and DJ suggested that the QTL for female fertility were specific for NH. A missense mutation in CD36 showed the strongest association with fertility traits on BTA04. The annotated SNPs on BTA13 were all intergenic variants. It is possible that BTA13 at this stage is poorly annotated such that the associated polymorphisms are located in as-yet undiscovered genes. Fertility traits are complex traits as many different biological and physiological factors determine whether a cow is fertile. Therefore it is not expected that there is a simple explanation with an obvious candidate gene but it is more likely a network of genes and intragenic variants that explain the variation of these traits.

Electronic supplementary material

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Ancient DNA sheds light on the ancestry of pre-hispanic Canarian pigs

Background

Canarian Black (CB) pigs belong to an autochthonous and endangered breed, which is spread throughout the Canarian archipelago. It is commonly accepted that they represent a relic of the pig populations that were bred by the Berbers in North Africa over millennia. It is important to note that the geographic isolation of the Canary Islands has preserved this genetic legacy intact from foreign introgressions until the Spanish conquest of the archipelago in the 15^th century. Ten years ago, it was demonstrated that, in CB pigs, the frequency of the Asian A2 cytochrome-b haplogroup reached 73%. The current work aimed at investigating whether this observation is explained by either a recent or an ancient introgression of CB pigs with Far Eastern pigs.

Results

Genetic analyses of 23 ancient samples from pre-hispanic Canarian pigs (420 to 2500 years before present) showed that Near Eastern and Far Eastern genetic signatures were totally absent in the primitive Canarian pre-hispanic pigs. Indeed, the haplotypes detected in these pigs were closely related to those of North African and European wild boars.

Conclusions

Our results demonstrate that the high frequency of the Far Eastern mitochondrial cytochrome B A2 haplotype in modern Canarian Black pigs probably corresponds to a relatively recent introgression with British breeds.

Electronic supplementary material

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Detection of internal exon deletion with exon Del

Background

Exome sequencing allows researchers to study the human genome in unprecedented detail. Among the many types of variants detectable through exome sequencing, one of the most over looked types of mutation is internal deletion of exons. Internal exon deletions are the absence of consecutive exons in a gene. Such deletions have potentially significant biological meaning, and they are often too short to be considered copy number variation. Therefore, to the need for efficient detection of such deletions using exome sequencing data exists.

Results

We present ExonDel, a tool specially designed to detect homozygous exon deletions efficiently. We tested ExonDel on exome sequencing data generated from 16 breast cancer cell lines and identified both novel and known IEDs. Subsequently, we verified our findings using RNAseq and PCR technologies. Further comparisons with multiple sequencing-based CNV tools showed that ExonDel is capable of detecting unique IEDs not found by other CNV tools.

Conclusions

ExonDel is an efficient way to screen for novel and known IEDs using exome sequencing data. ExonDel and its source code can be downloaded freely at https://github.com/slzhao/ExonDel.

Electronic supplementary material

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Analysis of the genetics of boar taint reveals both single SNPs and regional effects

Background

Boar taint is an offensive urine or faecal-like odour, affecting the smell and taste of cooked pork from some mature non-castrated male pigs. Androstenone and skatole in fat are the molecules responsible. In most pig production systems, males, which are not required for breeding, are castrated shortly after birth to reduce the risk of boar taint. There is evidence for genetic variation in the predisposition to boar taint.A genome-wide association study (GWAS) was performed to identify loci with effects on boar taint. Five hundred Danish Landrace boars with high levels of skatole in fat (>0.3 μg/g), were each matched with a litter mate with low levels of skatole and measured for androstenone. DNA from these 1,000 non-castrated boars was genotyped using the Illumina PorcineSNP60 Beadchip. After quality control, tests for SNPs associated with boar taint were performed on 938 phenotyped individuals and 44,648 SNPs. Empirical significance thresholds were set by permutation (100,000). For androstenone, a ‘regional heritability approach’ combining information from multiple SNPs was used to estimate the genetic variation attributable to individual autosomes.

Results

A highly significant association was found between variation in skatole levels and SNPs within the CYP2E1 gene on chromosome 14 (SSC14), which encodes an enzyme involved in degradation of skatole. Nominal significance was found for effects on skatole associated with 4 other SNPs including a region of SSC6 reported previously. Genome-wide significance was found for an association between SNPs on SSC5 and androstenone levels and nominal significance for associations with SNPs on SSC13 and SSC17. The regional analyses confirmed large effects on SSC5 for androstenone and suggest that SSC5 explains 23% of the genetic variation in androstenone. The autosomal heritability analyses also suggest that there is a large effect associated with androstenone on SSC2, not detected using GWAS.

Conclusions

Significant SNP associations were found for skatole on SSC14 and for androstenone on SSC5 in Landrace pigs. The study agrees with evidence that the CYP2E1 gene has effects on skatole breakdown in the liver. Autosomal heritability estimates can uncover clusters of smaller genetic effects that individually do not exceed the threshold for GWAS significance.

Electronic supplementary material

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Rheumatoid factor and anti-citrullinated protein antibody positivity,but not level,are associated with increased mortality in patients with rheumatoid arthritis: results from two large independent cohorts

Introduction

This study aimed to investigate rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) status and levels as predictors of mortality in two large cohorts of patients with early inflammatory arthritis (EIA).

Methods

Data from the Norfolk Arthritis Register (NOAR) and Leiden Early Arthritis Clinic (EAC) cohorts were used. At baseline, patients had demographic data and smoking status recorded; RF, ACPA and inflammatory markers were measured in the local laboratories. Patients were flagged with national death registers until death or censor date. Antibody status was stratified as negative, low or high positive by RF and ACPA levels individually. In addition, patients were grouped as seronegative, RF positive, ACPA positive or double antibody (RF and ACPA) positive. Cox regression models explored associations between antibody status and mortality adjusting for age, sex, smoking status, inflammatory markers and year of enrolment.

Results

A total of 4962 patients were included, 64% were female. Median age at onset was 56 (NOAR) and 54 (EAC) years. In NOAR and EAC respectively, 35% and 42% of patients were ACPA/RF positive. When antibody status was stratified as negative, low or high positive, there were no consistent findings between the two cohorts. Double antibody positivity was associated with excess mortality in both cohorts compared to seronegative patients: NOAR and EAC respective adjusted HR (95% confidence interval) 1.35 (1.09 to 1.68) and 1.58 (1.16 to 2.15).

Conclusions

Patients with EIA who are seropositive for both RF and ACPA have increased mortality compared to those who are single positive or seronegative. Antibody level in seropositive patients was not consistently associated with excess mortality.

Electronic supplementary material

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Genome wide CNV analysis reveals additional variants associated with milk production traits in Holsteins

Background

Milk production is an economically important sector of global agriculture. Much attention has been paid to the identification of quantitative trait loci (QTL) associated with milk, fat, and protein yield and the genetic and molecular mechanisms underlying them. Copy number variation (CNV) is an emerging class of variants which may be associated with complex traits.

Results

In this study, we performed a genome-wide association between CNVs and milk production traits in 26,362 Holstein bulls and cows. A total of 99 candidate CNVs were identified using Illumina BovineSNP50 array data, and association tests for each production trait were performed using a linear regression analysis with PCA correlation. A total of 34 CNVs on 22 chromosomes were significantly associated with at least one milk production trait after false discovery rate (FDR) correction. Some of those CNVs were located within or near known QTL for milk production traits. We further investigated the relationship between associated CNVs with neighboring SNPs. For all 82 combinations of traits and CNVs (less than 400 kb in length), we found 17 cases where CNVs directly overlapped with tag SNPs and 40 cases where CNVs were adjacent to tag SNPs. In 5 cases, CNVs located were in strong linkage disequilibrium with tag SNPs, either within or adjacent to the same haplotype block. There were an additional 20 cases where CNVs did not have a significant association with SNPs, suggesting that the effects of those CNVs were probably not captured by tag SNPs.

Conclusion

We conclude that combining CNV with SNP analyses reveals more genetic variations underlying milk production traits than those revealed by SNPs alone.

Electronic supplementary material

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Non-synonymous mutations mapped to chromosome X associated with andrological and growth traits in beef cattle

Background

Previous genome-wide association analyses identified QTL regions in the X chromosome for percentage of normal sperm and scrotal circumference in Brahman and Tropical Composite cattle. These traits are important to be studied because they are indicators of male fertility and are correlated with female sexual precocity and reproductive longevity. The aim was to investigate candidate genes in these regions and to identify putative causative mutations that influence these traits. In addition, we tested the identified mutations for female fertility and growth traits.

Results

Using a combination of bioinformatics and molecular assay technology, twelve non-synonymous SNPs in eleven genes were genotyped in a cattle population. Three and nine SNPs explained more than 1% of the additive genetic variance for percentage of normal sperm and scrotal circumference, respectively. The SNPs that had a major influence in percentage of normal sperm were mapped to LOC100138021 and TAF7L genes; and in TEX11 and AR genes for scrotal circumference. One SNP in TEX11 was explained ~13% of the additive genetic variance for scrotal circumference at 12 months. The tested SNP were also associated with weight measurements, but not with female fertility traits.

Conclusions

The strong association of SNPs located in X chromosome genes with male fertility traits validates the QTL. The implicated genes became good candidates to be used for genetic evaluation, without detrimentally influencing female fertility traits.

Electronic supplementary material

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A genome-wide scan for signatures of directional selection in domesticated pigs

Background

Animal domestication involved drastic phenotypic changes driven by strong artificial selection and also resulted in new populations of breeds, established by humans. This study aims to identify genes that show evidence of recent artificial selection during pig domestication.

Results

Whole-genome resequencing of 30 individual pigs from domesticated breeds, Landrace and Yorkshire, and 10 Asian wild boars at ~16-fold coverage was performed resulting in over 4.3 million SNPs for 19,990 genes. We constructed a comprehensive genome map of directional selection by detecting selective sweeps using an F_ST-based approach that detects directional selection in lineages leading to the domesticated breeds and using a haplotype-based test that detects ongoing selective sweeps within the breeds. We show that candidate genes under selection are significantly enriched for loci implicated in quantitative traits important to pig reproduction and production. The candidate gene with the strongest signals of directional selection belongs to group III of the metabolomics glutamate receptors, known to affect brain functions associated with eating behavior, suggesting that loci under strong selection include loci involved in behaviorial traits in domesticated pigs including tameness.

Conclusions

We show that a significant proportion of selection signatures coincide with loci that were previously inferred to affect phenotypic variation in pigs. We further identify functional enrichment related to behavior, such as signal transduction and neuronal activities, for those targets of selection during domestication in pigs.

Electronic supplementary material

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Litter quality and decomposability of species from a Mediterranean succession depend on leaf traits but not on nitrogen supply

Background and Aims

The rate of plant decomposition depends on both the decomposition environment and the functional traits of the individual species (e.g. leaf and litter quality), but their relative importance in determining interspecific differences in litter decomposition remains unclear. The aims of this study were to: (a) determine if species from different successional stages grown on soils with low and high nitrogen levels produce leaf and litter traits that decompose differently under identical conditions; and (b) assess which trait of living leaves best relates to litter quality and litter decomposability

Methods

The study was conducted on 17 herbaceous species representative of three stages of a Mediterranean successional sere of Southern France. Plants were grown in monocultures in a common garden under two nitrogen levels. To elucidate how different leaf traits affected litter decomposition a microcosm experiment was conducted to determine decomposability under standard conditions. Tests were also carried out to determine how successional stage and nitrogen supply affected functional traits of living leaves and how these traits then modified litter quality and subsequent litter decomposability.

Key Results

The results demonstrated that leaf traits and litter decomposability varied according to species and successional stage. It was also demonstrated that while nitrogen addition affected leaf and litter traits, it had no effect on decomposition rates. Finally, leaf dry matter content stood out as the leaf trait best related to litter quality and litter decomposability

Conclusions

In this study, species litter decomposability was affected by some leaf and litter traits but not by soil nitrogen supply. The results demonstrated the strength of a trait-based approach to predict changes in ecosystem processes as a result of species shifts in ecosystems.Key words: Leaf traits, litter quality, litter decomposability, nitrogen addition, secondary succession     

Gene transfer using micellar nanovectors inhibits choroidal neovascularization in vivo

Purpose

Age-related macular degeneration caused by choroidal neovascularization (CNV) remains difficult to be treated despite the recent advent of several treatment options. In this study, we investigated the in vivo angiogenic control by intravenous injection of polyion complex (PIC) micelle encapsulating plasmid DNA (pDNA) using a mice CNV model.

Methods

The transfection efficiency of the PIC micelle was investigated using the laser-induced CNV in eight-week-old male C57 BJ/6 mice. Firstly, each mouse received intravenous injection of micelle encapsulating pDNA of Yellow Fluorescent Protein (pYFP) on days 1,3 and 5. The expression of YFP was analyzed using fluorescein microscopy and western blotting analysis. In the next experiments, each mouse received intravenous injection of micelle encapsulating pDNA of soluble Fms-like tyrosine kinase-1 (psFlt-1) 1,3 and 5 days after the induction of CNV and the CNV lesion was analyzed by choroidal flatmounts on day 7.

Results

Fluorescein microscopy and western blotting analysis revealed that the expression of YFP was confirmed in the CNV area after injection of the PIC micelle, but the expression was not detected neither in mice that received naked pDNA nor those without CNV. Furthermore, the CNV area in the mice that received intravenous injection of the psFlt-1-encapsulated PIC micelle was significantly reduced by 65% compared to that in control mice (p<0.01).

Conclusions

Transfection of sFlt-1 with the PIC micelle by intravenous injection to mice CNV models showed significant inhibition of CNV. The current results revealed the significant potential of nonviral gene therapy for regulation of CNV using the PIC micelle encapsulating pDNA.