Identification,characterization, and utilization of single copy genes in 29 angiosperm genomes

Background

Single copy genes are common across angiosperm genomes. With the sufficiently high quality sequenced genomes, the identification of large-scale single copy genes among multiple species is possible. Although some characteristics have been reported, our study provides novel insights into single copy genes.

Results

We identified single copy genes across 29 angiosperm genomes. A significant negative correlation was found between the number of duplicate blocks and the number of single copy genes. We found that a considerable number of single copy genes are located in organelles, showing a preference for binding and catalytic activity. The analysis of effective number of codons (Nc) illustrates that single copy genes have a stronger codon bias than non-single copy genes in eudicots. The relative high expression level of single copy genes was partially confirmed by the RNA-seq data, rather than the Codon Adaptation Index (CAI). Unlike in most other species, a strongly negatively correlation occurs between Nc and GC3 among single copy genes in grass genomes. When compared to all non-single copy genes, single copy genes indicate more conservation (as indicated by Ka and Ks values). But our alternative splicing (AS) results reveal that selective constraints are weaker in single copy genes than in low copy family genes (1–10 in-paralogs) and stronger than high copy family genes (>10 in-paralogs). Using concatenated shared single copy genes, we obtained a well-resolved phylogenetic tree. With the addition of intron sequences, the branch support is improved, but striking incongruences are also evident. Therefore, it is noteworthy that inclusion of intron sequences seems more appropriate for the phylogenetic reconstruction at lower taxonomic levels.

Conclusions

Our analysis provides insight into the evolutionary characteristics of single copy genes across 29 angiosperm genomes. The results suggest that there are key differences in evolutionary constraints between single copy genes and non-single copy genes. And to some extent, these evolutionary constraints show some species-specific differences, especially between eudicots and monocots. Our preliminary evidence also suggests that the concatenated shared single copy genes are well suited for use in resolving phylogenetic relationships.

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A novel approach for multi-domain and multi-gene family identification provides insights into evolutionary dynamics of disease resistance genes in core eudicot plants

Background

Recent advances in DNA sequencing techniques resulted in more than forty sequenced plant genomes representing a diverse set of taxa of agricultural, energy, medicinal and ecological importance. However, gene family curation is often only inferred from DNA sequence homology and lacks insights into evolutionary processes contributing to gene family dynamics. In a comparative genomics framework, we integrated multiple lines of evidence provided by gene synteny, sequence homology and protein-based Hidden Markov Modelling to extract homologous super-clusters composed of multi-domain resistance (R)-proteins of the NB-LRR type (for NUCLEOTIDE BINDING/LEUCINE-RICH REPEATS), that are involved in plant innate immunity.

Results

To assess the diversity of R-proteins within and between species, we screened twelve eudicot plant genomes including six major crops and found a total of 2,363 NB-LRR genes. Our curated R-proteins set shows a 50% average for tandem duplicates and a 22% fraction of gene copies retained from ancient polyploidy events (ohnologs). We provide evidence for strong positive selection and show significant differences in molecular evolution rates (Ka/Ks-ratio) among tandem- (mean = 1.59), ohnolog (mean = 1.36) and singleton (mean = 1.22) R-gene duplicates. To foster the process of gene-edited plant breeding, we report species-specific presence/absence of all 140 NB-LRR genes present in the model plant Arabidopsis and describe four distinct clusters of NB-LRR “gatekeeper” loci sharing syntenic orthologs across all analyzed genomes.

Conclusion

By curating a near-complete set of multi-domain R-protein clusters in an eudicot-wide scale, our analysis offers significant insight into evolutionary dynamics underlying diversification of the plant innate immune system. Furthermore, our methods provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from any plant species.

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Ozone exposure,vitamin C intake,and genetic susceptibility of asthmatic children in Mexico City: a cohort study

Background

We previously reported that asthmatic children with GSTM1 null genotype may be more susceptible to the acute effect of ozone on the small airways and might benefit from antioxidant supplementation. This study aims to assess the acute effect of ozone on lung function (FEF_25-75) in asthmatic children according to dietary intake of vitamin C and the number of putative risk alleles in three antioxidant genes: GSTM1, GSTP1 (rs1695), and NQO1 (rs1800566).

Methods

257 asthmatic children from two cohort studies conducted in Mexico City were included. Stratified linear mixed models with random intercepts and random slopes on ozone were used. Potential confounding by ethnicity was assessed. Analyses were conducted under single gene and genotype score approaches.

Results

The change in FEF_25-75 per interquartile range (60 ppb) of ozone in persistent asthmatic children with low vitamin C intake and GSTM1 null was −91.2 ml/s (p = 0.06). Persistent asthmatic children with 4 to 6 risk alleles and low vitamin C intake showed an average decrement in FEF_25-75 of 97.2 ml/s per 60 ppb of ozone (p = 0.03). In contrast in children with 1 to 3 risk alleles, acute effects of ozone on FEF_25-75 did not differ by vitamin C intake.

Conclusions

Our results provide further evidence that asthmatic children predicted to have compromised antioxidant defense by virtue of genetic susceptibility combined with deficient antioxidant intake may be at increased risk of adverse effects of ozone on pulmonary function.     

Identification and distribution of the NBS-LRR gene family in the Cassava genome

Background

Plant resistance genes (R genes) exist in large families and usually contain both a nucleotide-binding site domain and a leucine-rich repeat domain, denoted NBS-LRR. The genome sequence of cassava (Manihot esculenta) is a valuable resource for analysing the genomic organization of resistance genes in this crop.

Results

With searches for Pfam domains and manual curation of the cassava gene annotations, we identified 228 NBS-LRR type genes and 99 partial NBS genes. These represent almost 1% of the total predicted genes and show high sequence similarity to proteins from other plant species. Furthermore, 34 contained an N-terminal toll/interleukin (TIR)-like domain, and 128 contained an N-terminal coiled-coil (CC) domain. 63% of the 327 R genes occurred in 39 clusters on the chromosomes. These clusters are mostly homogeneous, containing NBS-LRRs derived from a recent common ancestor.

Conclusions

This study provides insight into the evolution of NBS-LRR genes in the cassava genome; the phylogenetic and mapping information may aid efforts to further characterize the function of these predicted R genes.

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Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background

Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.

Results

Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.

Conclusions

The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.     

A high density GBS map of bread wheat and its application for dissecting complex disease resistance traits

Background

Genotyping-by-sequencing (GBS) is a high-throughput genotyping approach that is starting to be used in several crop species, including bread wheat. Anchoring GBS tags on chromosomes is an important step towards utilizing them for wheat genetic improvement. Here we use genetic linkage mapping to construct a consensus map containing 28644 GBS markers.

Results

Three RIL populations, PBW343 × Kingbird, PBW343 × Kenya Swara and PBW343 × Muu, which share a common parent, were used to minimize the impact of potential structural genomic variation on consensus-map quality. The consensus map comprised 3757 unique positions, and the average marker distance was 0.88 cM, obtained by calculating the average distance between two adjacent unique positions. Significant variation of segregation distortion was observed across the three populations. The consensus map was validated by comparing positions of known rust resistance genes, and comparing them to wheat reference genome sequences recently published by the International Wheat Genome Sequencing Consortium, Rye and Ae. tauschii genomes. Three well-characterized rust resistance genes (Sr58/Lr46/Yr29, Sr2/Yr30/Lr27, and Sr57/Lr34/Yr18) and 15 published QTLs for wheat rusts were validated with high resolution. Fifty-two per cent of GBS tags on the consensus map were successfully aligned through BLAST to the right chromosomes on the wheat reference genome sequence.

Conclusion

The consensus map should provide a useful basis for analyzing genome-wide variation of complex traits. The identified genes can then be explored as genetic markers to be used in genomic applications in wheat breeding.

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Identification of a common risk haplotype for canine idiopathic epilepsy in the ADAM23 gene

Background

Idiopathic epilepsy is a common neurological disease in human and domestic dogs but relatively few risk genes have been identified to date. The seizure characteristics, including focal and generalised seizures, are similar between the two species, with gene discovery facilitated by the reduced genetic heterogeneity of purebred dogs. We have recently identified a risk locus for idiopathic epilepsy in the Belgian Shepherd breed on a 4.4 megabase region on CFA37.

Results

We have expanded a previous study replicating the association with a combined analysis of 157 cases and 179 controls in three additional breeds: Schipperke, Finnish Spitz and Beagle (p_c = 2.9e–07, p_GWAS = 1.74E-02). A targeted resequencing of the 4.4 megabase region in twelve Belgian Shepherd cases and twelve controls with opposite haplotypes identified 37 case-specific variants within the ADAM23 gene. Twenty-seven variants were validated in 285 cases and 355 controls from four breeds, resulting in a strong replication of the ADAM23 locus (p_raw = 2.76e–15) and the identification of a common 28 kb-risk haplotype in all four breeds. Risk haplotype was present in frequencies of 0.49–0.7 in the breeds, suggesting that ADAM23 is a low penetrance risk gene for canine epilepsy.

Conclusions

These results implicate ADAM23 in common canine idiopathic epilepsy, although the causative variant remains yet to be identified. ADAM23 plays a role in synaptic transmission and interacts with known epilepsy genes, LGI1 and LGI2, and should be considered as a candidate gene for human epilepsies.

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Association between risk of oral precancer and genetic variations in microRNA and related processing genes

Background

MicroRNAs have been implicated in cancer but studies on their role in precancer, such as leukoplakia, are limited. Sequence variations at eight miRNA and four miRNA processing genes were studied in 452 healthy controls and 299 leukoplakia patients to estimate risk of disease.

Results

Genotyping by TaqMan assay followed by statistical analyses showed that variant genotypes at Gemin3 and mir-34b reduced risk of disease [OR = 0.5(0.3–0.9) and OR = 0.7(0.5–0.9) respectively] in overall patients as well as in smokers [OR = 0.58(0.3–1) and OR = 0.68(0.5–0.9) respectively]. Among chewers, only mir29a significantly increased risk of disease [OR = 1.8(1–3)]. Gene-environment interactions using MDR-pt program revealed that mir29a, mir34b, mir423 and Xpo5 modulated risk of disease (p < 0.002) which may be related to change in expression of these genes as observed by Real-Time PCR assays. But association between polymorphisms and gene expressions was not found in our sample set as well as in larger datasets from open access platforms like Genevar and 1000 Genome database.

Conclusion

Variations in microRNAs and their processing genes modulated risk of precancer but further in-depth study is needed to understand mechanism of disease process.     

Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness

Background

Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate intracellular pathogen that infects many ruminant species. The acquisition of foreign genes via horizontal gene transfer has been postulated to contribute to its pathogenesis, as these genetic elements are absent from its putative ancestor, M. avium subsp. hominissuis (MAH), an environmental organism with lesser pathogenicity. In this study, high-throughput sequencing of MAP transposon libraries were analyzed to qualitatively and quantitatively determine the contribution of individual genes to bacterial survival during infection.

Results

Out of 52384 TA dinucleotides present in the MAP K-10 genome, 12607 had a MycoMarT7 transposon in the input pool, interrupting 2443 of the 4350 genes in the MAP genome (56%). Of 96 genes situated in MAP-specific genomic islands, 82 were disrupted in the input pool, indicating that MAP-specific genomic regions are dispensable for in vitro growth (odds ratio = 0.21). Following 5 independent in vivo infections with this pool of mutants, the correlation between output pools was high for 4 of 5 (R = 0.49 to 0.61) enabling us to define genes whose disruption reproducibly reduced bacterial fitness in vivo. At three different thresholds for reduced fitness in vivo, MAP-specific genes were over-represented in the list of predicted essential genes. We also identified additional genes that were severely depleted after infection, and several of them have orthologues that are essential genes in M. tuberculosis.

Conclusions

This work indicates that the genetic elements required for the in vivo survival of MAP represent a combination of conserved mycobacterial virulence genes and MAP-specific genes acquired via horizontal gene transfer. In addition, the in vitro and in vivo essential genes identified in this study may be further characterized to offer a better understanding of MAP pathogenesis, and potentially contribute to the discovery of novel therapeutic and vaccine targets.

Electronic supplementary material

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Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica

Background

Powdery mildew (PM) is a major fungal disease of thousands of plant species, including many cultivated Rosaceae. PM pathogenesis is associated with up-regulation of MLO genes during early stages of infection, causing down-regulation of plant defense pathways. Specific members of the MLO gene family act as PM-susceptibility genes, as their loss-of-function mutations grant durable and broad-spectrum resistance.

Results

We carried out a genome-wide characterization of the MLO gene family in apple, peach and strawberry, and we isolated apricot MLO homologs through a PCR-approach. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Homologs that are candidates for being PM susceptibility genes were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen Podosphaera leucotricha.

Conclusions

Genomic tools available for Rosaceae were exploited in order to characterize the MLO gene family. Candidate MLO susceptibility genes were identified. In follow-up studies it can be investigated whether silencing or a loss-of-function mutations in one or more of these candidate genes leads to PM resistance.

Electronic supplementary material

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Global epigenomic analysis indicates that Epialleles contribute to Allele-specific expression via Allele-specific histone modifications in hybrid rice

Background

For heterozygous genes, alleles on the chromatin from two different parents exhibit histone modification variations known as allele-specific histone modifications (ASHMs). The regulation of allele-specific gene expression (ASE) by ASHMs has been reported in animals. However, to date, the regulation of ASE by ASHM genes remains poorly understood in higher plants.

Results

We used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to investigate the global ASHM profiles of trimethylation on histone H3 lysine 27 (H3K27me3) and histone H3 lysine 36 (H3K36me3) in two rice F1 hybrids. A total of 522 to 550 allele-specific H3K27me3 genes and 428 to 494 allele-specific H3K36me3 genes were detected in GL × 93-11 and GL × TQ, accounting for 11.09% and 26.13% of the total analyzed genes, respectively. The epialleles between parents were highly related to ASHMs. Further analysis indicated that 52.48% to 70.40% of the epialleles were faithfully inherited by the F1 hybrid and contributed to 33.18% to 46.55% of the ASHM genes. Importantly, 66.67% to 82.69% of monoallelic expression genes contained the H3K36me3 modification. Further studies demonstrated a significant positive correlation of ASE with allele-specific H3K36me3 but not with H3K27me3, indicating that ASHM-H3K36me3 primarily regulates ASE in this study.

Conclusions

Our results demonstrate that epialleles from parents can be inherited by the F1 to produce ASHMs in the F1 hybrid. Our findings indicate that ASHM-H3K36me3, rather than H3K27me3, mainly regulates ASE in hybrid rice.

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High correlation between genotypes and phenotypes of environmental bacteria Comamonas testosteroni strains

Background

Members of Comamonas testosteroni are environmental microorganisms that are usually found in polluted environment samples. They utilize steroids and aromatic compounds but rarely sugars, and show resistance to multiple heavy metals and multiple drugs. However, comprehensive genomic analysis among the C. testosteroni strains is lacked.

Results

To understand the genome bases of the features of C. testosteroni, we sequenced 10 strains of this species and analyzed them together with other related published genome sequences. The results revealed that: 1) the strains of C. testosteroni have genome sizes ranging from 5.1 to 6.0 Mb and G + C contents ranging from 61.1% to 61.8%. The pan-genome contained 10,165 gene families and the core genome contained 3,599 gene families. Heap’s law analysis indicated that the pan-genome of C. testosteroni may be open (α = 0.639); 2) by analyzing 31 phenotypes of 11 available C. testosteroni strains, 99.4% of the genotypes (putative genes) were found to be correlated to the phenotypes, indicating a high correlation between phenotypes and genotypes; 3) gene clusters for nitrate reduction, steroids degradation and metal and multi-drug resistance were found and were highly conserved among all the genomes of this species; 4) the genome similarity of C. testosteroni may be related to the geographical distances.

Conclusions

This work provided an overview on the genomes of C. testosteroni and new genome resources that would accelerate the further investigations of this species. Importantly, this work focused on the analysis of potential genetic determinants for the typical characters and found high correlation between the phenotypes and their corresponding genotypes.

Electronic supplementary material

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