Tree Genome Research Articles

Genetic diversity and signatures of selection in various goat breeds revealed by genome-wide SNP markers

BackgroundThe detection of signatures of selection has the potential to elucidate the identities of genes and mutations associated with phenotypic traits important for livestock species. It is also very relevant to investigate the levels of genetic diversity of a population, as genetic diversity represents the raw material essential for breeding and has practical implications for implementation of genomic selection. A total of 1151 animals from nine goat populations selected for different breeding goals and genotyped with the Illumina Goat 50K single nucleotide polymorphisms (SNP) Beadchip were included in this investigation.ResultsThe proportion of polymorphic SNPs ranged from 0.902 (Nubian) to 0.995 (Rangeland). The overall mean HO and HE was 0.374 ± 0.021 and 0.369 ± 0.023, respectively. The average pairwise genetic distance (D) ranged from 0.263 (Toggenburg) to 0.323 (Rangeland). The overall average for the inbreeding measures FEH, FVR, FLEUT, FROH and FPED was 0.129, −0.012, −0.010, 0.038 and 0.030, respectively. Several regions located on 19 chromosomes were potentially under selection in at least one of the goat breeds. The genomic population tree constructed using all SNPs differentiated breeds based on selection purpose, while genomic population tree built using only SNPs in the most significant region showed a great differentiation between LaMancha and the other breeds. We hypothesized that this region is related to ear morphogenesis. Furthermore, we identified genes potentially related to reproduction traits, adult body mass, efficiency of food conversion, abdominal fat deposition, conformation traits, liver fat metabolism, milk fatty acids, somatic cells score, milk protein, thermo-tolerance and ear morphogenesis.ConclusionsIn general, moderate to high levels of genetic variability were observed for all the breeds and a characterization of runs of homozygosity gave insights into the breeds’ development history. The information reported here will be useful for the implementation of genomic selection and other genomic studies in goats. We also identified various genome regions under positive selection using smoothed FST and hapFLK statistics and suggested genes, which are potentially under selection. These results can now provide a foundation to formulate biological hypotheses related to selection processes in goats.

When COI barcodes deceive: complete genomes reveal introgression in hairstreaks.

Two species of hairstreak butterflies from the genus Calycopis are known in the United States: C. cecrops and C. isobeon Analysis of mitochondrial COI barcodes of Calycopis revealed cecrops-like specimens from the eastern US with atypical barcodes that were 2.6% different from either USA species, but similar to Central American Calycopis species. To address the possibility that the specimens with atypical barcodes represent an undescribed cryptic species, we sequenced complete genomes of 27 Calycopis specimens of four species: C. cecrops, C. isobeon, C. quintana and C. bactra Some of these specimens were collected up to 60 years ago and preserved dry in museum collections, but nonetheless produced genomes as complete as fresh samples. Phylogenetic trees reconstructed using the whole mitochondrial and nuclear genomes were incongruent. While USA Calycopis with atypical barcodes grouped with Central American species C. quintana by mitochondria, nuclear genome trees placed them within typical USA C. cecrops in agreement with morphology, suggesting mitochondrial introgression. Nuclear genomes also show introgression, especially between C. cecrops and C. isobeon About 2.3% of each C. cecrops genome has probably (p-value < 0.01, FDR < 0.1) introgressed from C. isobeon and about 3.4% of each C. isobeon genome may have come from C. cecrops. The introgressed regions are enriched in genes encoding transmembrane proteins, mitochondria-targeting proteins and components of the larval cuticle. This study provides the first example of mitochondrial introgression in Lepidoptera supported by complete genome sequencing. Our results caution about relying solely on COI barcodes and mitochondrial DNA for species identification or discovery.

Novel Insights into Tree Biology and Genome Evolution as Revealed Through Genomics.

Reference genome sequences are the key to the discovery of genes and gene families that determine traits of interest. Recent progress in sequencing technologies has enabled a rapid increase in genome sequencing of tree species, allowing the dissection of complex characters of economic importance, such as fruit and wood quality and resistance to biotic and abiotic stresses. Although the number of reference genome sequences for trees lags behind those for other plant species, it is not too early to gain insight into the unique features that distinguish trees from nontree plants. Our review of the published data suggests that, although many gene families are conserved among herbaceous and tree species, some gene families, such as those involved in resistance to biotic and abiotic stresses and in the synthesis and transport of sugars, are often expanded in tree genomes. As the genomes of more tree species are sequenced, comparative genomics will further elucidate the complexity of tree genomes and how this relates to traits unique to trees.

Survey of the rubber tree genome reveals a high number of cysteine protease-encoding genes homologous to Arabidopsis SAG12.

Arabidopsis thaliana SAG12, a senescence-specific gene encoding a cysteine protease, is widely used as a molecular marker for the study of leaf senescence. To date, its potential orthologues have been isolated from several plant species such as Brassica napus and Nicotiana tabacum. However, little information is available in rubber tree (Hevea brasiliensis), a rubber-producing plant of the Euphorbiaceae family. This study presents the identification of SAG12-like genes from the rubber tree genome. Results showed that an unexpected high number of 17 rubber orthologues with a single intron were found, contrasting the single copy with two introns in Arabidopsis. The gene expansion was also observed in another two Euphorbiaceae plants, castor bean (Ricinus communis) and physic nut (Jatropha curcas), both of which contain 8 orthologues. In accordance with no occurrence of recent whole-genome duplication (WGD) events, most duplicates in castor and physic nut were resulted from tandem duplications. In contrast, the duplicated HbSAG12H genes were derived from tandem duplications as well as the recent WGD. Expression analysis showed that most HbSAG12H genes were lowly expressed in examined tissues except for root and male flower. Furthermore, HbSAG12H1 exhibits a strictly senescence-associated expression pattern in rubber tree leaves, and thus can be used as a marker gene for the study of senescence mechanism in Hevea.

Ecological genomics of tropical trees: how local population size and allelic diversity of resistance genes relate to immune responses, cosusceptibility to pathogens, and negative density dependence.

In tropical forests, rarer species show increased sensitivity to species-specific soil pathogens and more negative effects of conspecific density on seedling survival (NDD). These patterns suggest a connection between ecology and immunity, perhaps because small population size disproportionately reduces genetic diversity of hyperdiverse loci such as immunity genes. In an experiment examining seedling roots from six species in one tropical tree community, we found that smaller populations have reduced amino acid diversity in pathogen resistance (R) genes but not the transcriptome in general. Normalized R gene amino acid diversity varied with local abundance and prior measures of differences in sensitivity to conspecific soil and NDD. After exposure to live soil, species with lower R gene diversity had reduced defence gene induction, more cosusceptibility of maternal cohorts to colonization by potentiallypathogenic fungi, reduced root growth arrest (an R gene-mediated response) and their root-associated fungi showed lower induction of self-defence (antioxidants). Local abundance was not related to the ability to induce immune responses whenpathogenrecognition was bypassed by application of salicylic acid, a phytohormone that activates defence responses downstream of R gene signalling. These initial results support the hypothesis that smaller local tree populations have reduced R gene diversity and recognition-dependent immune responses, along with greater cosusceptibility to species-specific pathogens that may facilitate disease transmission and NDD. Locallyrare species may be less able to increase their equilibrium abundance withoutgenetic boosts to defencevia immigration of novel R gene alleles from a larger and more diverse regional population.

De novo hybrid assembly of the rubber tree genome reveals evidence of paleotetraploidy in Hevea species

Para rubber tree (Hevea brasiliensis) is an important economic species as it is the sole commercial producer of high-quality natural rubber. Here, we report a de novo hybrid assembly of BPM24 accession, which exhibits resistance to major fungal pathogens in Southeast Asia. Deep-coverage 454/Illumina short-read and Pacific Biosciences (PacBio) long-read sequence data were acquired to generate a preliminary draft, which was subsequently scaffolded using a long-range “Chicago” technique to obtain a final assembly of 1.26 Gb (N50 = 96.8 kb). The assembled genome contains 69.2% repetitive sequences and has a GC content of 34.31%. Using a high-density SNP-based genetic map, we were able to anchor 28.9% of the genome assembly (363 Mb) associated with over two thirds of the predicted protein-coding genes into rubber tree’s 18 linkage groups. These genetically anchored sequences allowed comparative analyses of the intragenomic homeologous synteny, providing the first concrete evidence to demonstrate the presence of paleotetraploidy in Hevea species. Additionally, the degree of macrosynteny conservation observed between rubber tree and cassava strongly supports the hypothesis that the paleotetraploidization event took place prior to the divergence of the Hevea and Manihot species.

Molecular profiling and bioactive potential of an endophytic fungus Aspergillus sulphureus isolated from Sida acuta: a medicinal plant

Context: Sida acuta Burm.f. (Malvaceae) extracts are reported to have applications against malaria, diuretic, antipyretic, nervous and urinary diseases. No fungal endophytes of S. acuta are reported. Objective: Isolation, identification and evaluation of antibacterial, antioxidant, anticancer and haemolytic potential of fungal endophytes from the ethnomedcinal plant S. acuta. Materials and methods: Sida acuta stem segments were placed on PDA medium to isolate endophytic fungi. The fungus was identified by genomic DNA analysis and phylogenetic tree was constructed using ITS sequences (GenBank) to confirm species. The antibacterial efficacy of Aspergillus sulphureus MME12 ethyl acetate extract was tested against Gram-positive and Gram-negative pathogenic bacteria. DPPH free radical scavenging activity, anticancer and DNA fragmentation against EAC cells, and direct haemolytic activity (100–500 μg/mL) using human erythrocytes were determined. Results and discussion: The ethyl acetate extract of A. sulphureus (Fresen.) Wehmer (Trichocomaceae) demonstrated significant antibacterial potential against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhi compared to streptomycin. MIC against test pathogens was in the range of 15.6–62.5 μg/mL. The antioxidant results revealed significant RSA from 12.43% to 62.02% (IC50 = 350.4 μg/mL, p ≤ 0.05). MME12 offered considerable inhibition of EAC proliferation (23% to 84%, IC50 = 216.7 μg/mL, p ≤ 0.05) supported by DNA fragmentation studies. The extract also offered insignificant haemolysis (5.6%) compared to Triton X-100. Conclusions: A single endophytic fungus, A. sulphureus MME12 was isolated and identified using molecular profiling. The above-mentioned findings support the pharmacological application of A. sulphureus MME12 extract and demand for purification of the active principle(s).

Aquerium: A web application for comparative exploration of domain-based protein occurrences on the taxonomically clustered genome tree: Architecture Querying Podium

Aquerium: A web application for comparative exploration of domain-based protein occurrences on the taxonomically clustered genome tree: Architecture Querying Podium

Aquerium: A web application for comparative exploration of domain-based protein occurrences on the taxonomically clustered genome tree.

Gene duplication and loss are major driving forces in evolution. While many important genomic resources provide information on gene presence, there is a lack of tools giving equal importance to presence and absence information as well as web platforms enabling easy visual comparison of multiple domain-based protein occurrences at once. Here, we present Aquerium, a platform for visualizing genomic presence and absence of biomolecules with a focus on protein domain architectures. The web server offers advanced domain organization querying against the database of pre-computed domains for ∼26,000 organisms and it can be utilized for identification of evolutionary events, such as fusion, disassociation, duplication, and shuffling of protein domains. The tool also allows alternative inputs of custom entries or BLASTP results for visualization. Aquerium will be a useful tool for biologists who perform comparative genomic and evolutionary analyses. The web server is freely accessible at http://aquerium.utk.edu. Proteins 2016; 85:72-77. © 2016 Wiley Periodicals, Inc.

Phylogenetic analysis of full-length, early infection, hepatitis C virus genomes among people with intravenous drug use: the InC3 Study.

Cross-continental phylogenetic analysis is important to understand subtle molecular differences of currently circulating hepatitis C virus (HCV) subtypes. Existence of such differences can be crucial in pursuing a universal hepatitis C vaccine. We characterized molecular epidemiology of early HCV infections identified across nine cohorts [North America (n=4), Australia (n=4) and Europe (n=1)] in the International Collaborative of Incident HIV and Hepatitis C in Injecting Cohorts (InC3 ). One hundred and ninety-two full-length HCV genomes were amplified from plasma of incident infections and subjected to next generation sequencing to establish the largest cross-continental, full-length acute HCV genomic data set available to date. Genomes from the most common subtypes (1a: n=94, 2b: n=15 and 3a: n=68) were used in phylogenetic analysis. Using full genome trees, 78 sequences (44%) were found to lie within 29 phylogenetic clusters/pairs defined on the basis of molecular similarity of consensus sequences. Of these, 26 each had exclusively Australian or North American sequences indicating a strong geographical bias for molecular similarity. On further analysis of behavioural and demographic associations, binary logistic regression analysis showed that older age and non-Caucasian ethnicity were significantly associated with clustering. HCV probably evolves in micro-epidemics within geographically isolated communities.

A Proposal for a Genome Similarity-Based Taxonomy for Plant-Pathogenic Bacteria that Is Sufficiently Precise to Reflect Phylogeny, Host Range, and Outbreak Affiliation Applied to Pseudomonas syringae sensu lato as a Proof of Concept.

Taxonomy of plant pathogenic bacteria is challenging because pathogens of different crops often belong to the same named species but current taxonomy does not provide names for bacteria below the subspecies level. The introduction of the host range-based pathovar system in the 1980s provided a temporary solution to this problem but has many limitations. The affordability of genome sequencing now provides the opportunity for developing a new genome-based taxonomic framework. We already proposed to name individual bacterial isolates based on pairwise genome similarity. Here, we expand on this idea and propose to use genome similarity-based codes, which we now call life identification numbers (LINs), to describe and name bacterial taxa. Using 93 genomes of Pseudomonas syringae sensu lato, LINs were compared with a P. syringae genome tree whereby the assigned LINs were found to be informative of a majority of phylogenetic relationships. LINs also reflected host range and outbreak association for strains of P. syringae pathovar actinidiae, a pathovar for which many genome sequences are available. We conclude that LINs could provide the basis for a new taxonomic framework to address the shortcomings of the current pathovar system and to complement the current taxonomic system of bacteria in general.

Identification and characterisation of Short Interspersed Nuclear Elements in the olive tree (Olea europaea L.) genome.

Short Interspersed Nuclear Elements (SINEs) are nonautonomous retrotransposons in the genome of most eukaryotic species. While SINEs have been intensively investigated in humans and other animal systems, SINE identification has been carried out only in a limited number of plant species. This lack of information is apparent especially in non-model plants whose genome has not been sequenced yet. The aim of this work was to produce a specific bioinformatics pipeline for analysing second generation sequence reads of a non-model species and identifying SINEs. We have identified, for the first time, 227 putative SINEs of the olive tree (Olea europaea), that constitute one of the few sets of such sequences in dicotyledonous species. The identified SINEs ranged from 140 to 362bp in length and were characterised with regard to the occurrence of the tRNA domain in their sequence. The majority of identified elements resulted in single copy or very lowly repeated, often in association with genic sequences. Analysis of sequence similarity allowed us to identify two major groups of SINEs showing different abundances in the olive tree genome, the former with sequence similarity to SINEs of Scrophulariaceae and Solanaceae and the latter to SINEs of Salicaceae. A comparison of sequence conservation between olive SINEs and LTR retrotransposon families suggested that SINE expansion in the genome occurred especially in very ancient times, before LTR retrotransposon expansion, and presumably before the separation of the rosids (to which Oleaceae belong) from the Asterids. Besides providing data on olive SINEs, our results demonstrate the suitability of the pipeline employed for SINE identification. Applying this pipeline will favour further structural and functional analyses on these relatively unknown elements to be performed also in other plant species, even in the absence of a reference genome, and will allow establishing general evolutionary patterns for this kind of repeats in plants.

Population genomics of divergence within an obligate pollination mutualism: Selection maintains differences between Joshua tree species.

Speciation is a complex process that can be shaped by many factors, from geographic isolation to interspecific interactions. In Joshua trees, selection from pollinators on style length has been hypothesized to contribute to the maintenance of differentiation between two hybridizing sister species. We used population genomics approaches to measure the extent of genetic differentiation between these species, test whether selection maintains differences between them, and determine whether genetic variants associated with style length show signatures of selection. Using restriction-site-associated DNA (RAD)-sequencing, we identified 9516 single nucleotide polymorphisms (SNPs) across the Joshua tree genome. We characterized the genomic composition of trees in a narrow hybrid zone and used genomic scans to search for signatures of selection acting on these SNPs. We used a genome-wide association study to identify SNPs associated with variation in phenotypic traits, including style length, and asked whether those SNPs were overrepresented among the group under selection. The two species were highly genetically differentiated (FST = 0.25), and hybrids were relatively rare in the hybrid zone. Approximately 20% of SNPs showed evidence of selection maintaining divergence. While SNPs associated with style length were overrepresented among those under selection (P << 0.0001), the same was true for SNPs associated with highly differentiated vegetative traits. The two species of Joshua tree are clearly genetically distinct, and selection is maintaining differences between them. We found that loci associated with differentiated traits were likely to be under selection. However, many traits other than style length appeared to be under selection. Together with the dearth of intermediate hybrids, these findings reveal that these taxa are more strongly diverged than previously suspected and that selection, likely on many targets, is maintaining separation where the two species meet and hybridize.

Identification and evaluation of reference genes for normalization in quantitative real-time PCR analysis in the premodel tree Betula luminifera

Betula luminifera is a commercial tree species that is emerging as a new model system for tree genomics research. A draft genomic sequence is expected to be publicly available in the near future, which means that an explosion of gene expression studies awaits. Thus, the work of selecting appropriate reference genes for qPCR normalization in different tissues or under various experimental conditions is extremely valuable. In this study, ten candidate genes were analyzed in B. luminifera subjected to different abiotic stresses and at various flowering stages. The expression stability of these genes was evaluated using three distinct algorithms implemented using geNorm, NormFinder and BestKeeper. The best-ranked reference genes varied across different sample sets, though RPL39, MDH and EF1a were determined as the most stable by the three programs among all tested samples. RPL39 and EF1a should be appropriate for normalization in N-starved roots, while the combination of RPL39 and MDH should be appropriate for N-starved stems and EF1a and MDH should be appropriate in N-starved leaves. In PEG-treated (osmotic) roots, MDH was the most suitable, whereas EF1a was suitable for PEG-treated stems and leaves. TUA was also stably expressed levels in PEG-treated plants. The combination of RPL39 and TUB should be appropriate for heat-stressed leaves and flowering stage. For reference gene validation, the expression levels of SOD and NFYA-3 were investigated. This work will be beneficial to future studies on gene expression under different abiotic stress conditions and flowering status in B. luminifera.

Development of single nucleotide polymorphism markers in the large and complex rubber tree genome using next-generation sequence data

The development of single nucleotide polymorphism (SNP) markers provides the opportunity to improve many areas of plant breeding and population genetics. Unfortunately, for species such as the rubber tree (Hevea brasiliensis), the use of next-generation sequencing for genomic SNP discovery is very difficult because of the large genome size and the abundance of repeated sequences. Access to a set of validated SNP markers is a significant advantage for rubber researchers who wish to apply SNPs in scientific research. Here, we performed genomic sequencing of H. brasiliensis and generated 10,993,648 short reads, which were assembled into 10,071 contigs (N50 = 3078) by a de novo assembly strategy. A total of 2446 contigs presented no hits in the current H. brasiliensis genome assembly and may therefore be considered novel genomic sequences of rubber tree. A total of 143 putative polymorphic positions were selected, gene annotations were available for 58.7 % of the markers, and all of the sequences could be anchored to the released H. brasiliensis genome. These SNPs were validated in eight genotypes of H. brasiliensis and 15 F1 plants from a mapping population, resulting in 30 (20.9 %) positions correctly classified. The analysis revealed key candidate genes responsible for defence mechanisms and provided markers for further genetic improvement of Hevea in breeding programmes.

The rubber tree genome shows expansion of gene family associated with rubber biosynthesis.

Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis’s capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree.

Genome-wide analysis of rubber tree (Hevea brasiliensis Muell. Arg.) aquaporin genes reveals a crucial role of the PIP subfamily in the water balance of laticifers

The movement of water is a matter of great importance to all plant species. This is particularly true for the rubber tree ( Hevea brasiliensis Muell. Arg.) which is tapped for the laticifer cytoplasm in the form of aqueous latex. Due to the lack of plasmodesmata, the laticifer water balance is mediated largely by aquaporins (AQPs), a class of channel-forming integral membrane proteins that transport water and other small solutes. Our studies reported the characterization of 51 full-length AQP genes from the rubber tree genome, and further assigned them to five subfamilies, i.e. plasma membrane intrinsic proteins (PIPs: 15), tonoplast intrinsic proteins (TIPs: 17), NOD26-like intrinsic proteins (NIPs: 9), small basic intrinsic proteins (SIPs: 4) and X intrinsic proteins (XIPs: 6). Although the classification of subfamily/subgroup is the same as in Ricinus communis , genome-wide comparative analysis revealed the PIP and TIP subfamilies in rubber tree are highly expanded, corresponding to their high water permeability and the particular importance of water balance in a big tree and a highly differentiated laticifer tissue. Functional prediction based on the analysis of the aromatic/arginine (ar/R) selectivity filter, Froger’s positions and specificity-determining positions (SDPs) further suggested the potentially key role of HbPIPs and HbTIPs in the laticifer water balance. Moreover, deep sequencing of the latex transcriptome and qRT-PCR analysis supported a crucial role of several PIP subfamily members in the laticifer water balance under both normal and ethephon-stimulated conditions.

The rubber tree genome reveals new insights into rubber production and species adaptation.

The Para rubber tree (Hevea brasiliensis) is an economically important tropical tree species that produces natural rubber, an essential industrial raw material. Here we present a high-quality genome assembly of this species (1.37 Gb, scaffold N50 = 1.28 Mb) that covers 93.8% of the genome (1.47 Gb) and harbours 43,792 predicted protein-coding genes. A striking expansion of the REF/SRPP (rubber elongation factor/small rubber particle protein) gene family and its divergence into several laticifer-specific isoforms seem crucial for rubber biosynthesis. The REF/SRPP family has isoforms with sizes similar to or larger than SRPP1 (204 amino acids) in 17 other plants examined, but no isoforms with similar sizes to REF1 (138 amino acids), the predominant molecular variant. A pivotal point in Hevea evolution was the emergence of REF1, which is located on the surface of large rubber particles that account for 93% of rubber in the latex (despite constituting only 6% of total rubber particles, large and small). The stringent control of ethylene synthesis under active ethylene signalling and response in laticifers resolves a longstanding mystery of ethylene stimulation in rubber production. Our study, which includes the re-sequencing of five other Hevea cultivars and extensive RNA-seq data, provides a valuable resource for functional genomics and tools for breeding elite Hevea cultivars.

First Case of Human Brucellosis Caused by Brucella melitensis in Korea.

Dear Editor, Human brucellosis is the most common zoonosis worldwide and is caused by gram-negative bacteria, Brucella spp. [1]. Among the Brucella spp., B. abortus, B. canis, B. melitensis, and B. suis can cause human brucellosis. Human brucellosis involves various organs including bone marrow (BM), shows non-specific clinical manifestations including fever, fatigue, back pain, and arthralgia, and can progress to septicemia or multi-organ failure [1,2]. In Korea, since the first case of human brucellosis in 2002 [3], all reported 747 cases have been caused by B. abortus, and the majority were related to the out-break of bovine brucellosis in mid-2000 [4]. We report the first Korean case of human brucellosis caused by B. melitensis. A 34-yr-old man presented with high fever (39.2℃), significant weight loss (10 kg/month), and back pain that persisted for three weeks. He was a Korean resident living in northwestern China (Yanji, Jilin) who had been working on a stock farm in Pyeongchang, Gangwon Province, Korea for two months before visiting our hospital. He had hepatosplenomegaly, elevated aminotransferases, and pancytopenia. His complete blood count showed the following: hemoglobin, 8.8 g/dL; white blood cells, 2.1×109/L; and platelets, 43×109/L. His BM was normocellular with intact trilineage hematopoiesis, and neither granuloma nor necrosis was present. Plasma cells (CD19+/CD56-) were elevated (11.7%), and polyclonal gammopathy was detected via serum protein electrophoresis. A magnetic resonance imaging to evaluate back pain revealed pyogenic spondylitis at L3-4. The first two blood cultures yielded gram-negative bacilli, which were identified as B. melitensis by using Vitek 2 (bioMerieux, Marcy-l'Etoile, France). In a microagglutination test (MAT), the antibody titer was 1:80. The patient received doxycycline and rifampicin for three weeks and was discharged with no symptoms except mild leukopenia. The MAT titer was not rechecked during the recovery period, because he declined further treatment. The Vitek 2 system database has a limitation in identifying Brucella spp.: it misidentifies B. abortus as B. melitensis. Therefore, the identification result of B. melitensis was further confirmed at the Division of Zoonoses at the Korea Centers for Disease Control and Prevention after the approval of the Institutional Review Board of Konkuk University Medical Center, Seoul, Korea. After a blood culture, the cultivated product was streaked on both a blood agar plate and a Brucella agar plate and incubated at 37℃ and 5% CO2 atmosphere. After several passages through the Brucella agar plate, the pure culture product (the isolate) tested negative for H2S production and positive for oxidase and urease. DNA was extracted from the isolate to amplify the Brucella-specific genes (16S rRNA, BCSP31, and Omp2) [5]. PCR targeting Omp31, IS711, and BCSS confirmed this isolate as B. melitensis [6,7,8] (Table 1). Draft whole genome sequence analysis assembled by CLC bio CLC Genomics Workbench 7.5.1 (Waltham, MA, USA) revealed that the isolate was B. melitensis (Fig. 1). Fig. 1 Average nucleotide identity tree of Brucella melitensis. The genome tree was constructed by using BM2015 (the isolate) and related Brucella spp. Table 1 Primers used in this study and the results of PCR and sequencing To identify Brucella spp. when brucellosis is suspected, a careful and complete history should be taken of the patient's occupation, travel, and unpasteurized diet history [9]. The present patient had worked on a sheep farm for two months before admission and experienced nonspecific symptoms (fever, weight loss, and back pain) for three weeks, indicating B. melitensis infection. Before he was in Korea, he had lived in north-western China, where B. melitensis has been dominant [10]. It is not clear whether he had raw food from infected animals in China or in Korea, or whether he acquired brucellosis occupationally by direct skin contact or inhalation of aerosolized infected particles [9]. Although the clinical history appears insufficient, considering that he had been healthy before developing these clinical symptoms, it is unlikely that he already had brucellosis in China. In the present case, H2S was negative, and oxidase and urease were positive in the biochemical analysis, suggesting the possibility of B. melitensis infection together with the patient's working history on the sheep farm. However, H2S production can be also negative in the other Brucella spp., and molecular identification is necessary to confirm the diagnosis. We used multiple gene targets to detect the Brucella genus using a common PCR assay [5] and also used Omp31, IS711, and BCSS gene targets to distinguish B. melitensis from the other Brucella spp. [6,7,8] (Table 1). Conclusively, this is the first official case of human brucellosis caused by B. melitensis in Korea that was confirmed by using molecular methods. Careful recording of patient history and molecular identification are necessary in patients with suspected brucellosis to confirm the causative Brucella spp.

Structural and functional characterization of tree proteins involved in redox regulation: a new frontier in forest science

This paper describes how the combination of genomics, genetic engineering, and 3D structural characterization has helped clarify the redox regulatory networks in poplar with consequences not only in system biology in plants but also in bacteria and mammalian systems. Tree genomes are increasingly available with a large number of orphan genes coding for proteins, the function of which is still unknown. Modern techniques of genome analysis coupled with recombinant protein technology and massive 3D structural determination of tree proteins should help elucidate the function of many of the proteins encoded by orphan genes. X-ray crystallography and NMR will be the methods of choice for protein structure determination. In this review, we provide examples illustrating how the above-mentioned techniques improved our understanding of redox regulatory circuits in poplar, the first forest tree species sequenced. We showed that poplar peroxiredoxins use either thioredoxin or glutaredoxin as electron donors to reduce hydrogen peroxide. That glutaredoxin could be a reductant was unknown at the time of this discovery even in other biological organisms and was later confirmed notably by the observation that the two genes are fused in some bacteria and by the resolution of the structure of the bacterial hybrid protein. Similarly, genome analysis coupled to in vitro analysis of enzymatic properties led to the discovery that some plant methionine sulfoxide reductases can also use both thioredoxins and glutaredoxins as electron donors. Besides their disulfide reductase activity, it has been demonstrated that some poplar glutaredoxins are also involved in iron-sulfur center biogenesis and assembly. The original 3D structure determination has been made with poplar glutaredoxin C1 and then confirmed in a variety of other biological organisms including human. Our work also showed that in plants, so-called glutathione peroxidases use thioredoxins and not glutathione as electron donors. This is true for all non-selenocysteine-containing glutathione peroxidases. Finally, connections between the thioredoxin and glutaredoxin systems have been elucidated through the study of atypical poplar thioredoxins. Altogether, these data illustrate how the combination of genetic engineering and structural biology improves our understanding of biological processes and helps fuel systems biology for trees and other biological species.