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Genetic variants in Epstein-Barr virus (EBV) have been strongly associated with nasopharyngeal carcinoma (NPC) in South China. However, different results regarding the most significant viral variants, with polymorphisms in EBER2 and BALF2 loci, have been reported in separate studies. In this study, we newly sequenced 100 EBV genomes derived from 61 NPC cases and 39 population controls. Comprehensive genomic analyses of EBV sequences from both NPC patients and healthy carriers in South China were conducted, totaling 279 cases and 227 controls. Meta-analysis of genome-wide association study revealed a 4-bp deletion downstream of EBER2 (coordinates, 7188–7191; EBER-del) as the most significant variant associated with NPC. Furthermore, multiple viral variants were found to be genetically linked to EBER-del forming a risk haplotype, suggesting that multiple viral variants might be associated with NPC pathogenesis. Population structure and phylogenetic analyses further characterized a high risk EBV lineage for NPC revealing a panel of 38 single nucleotide polymorphisms (SNPs), including those in the EBER2 and BALF2 loci. With linkage disequilibrium clumping and feature selection algorithm, the 38 SNPs could be narrowed down to 9 SNPs which can be used to accurately detect the high risk EBV lineage. In summary, our study provides novel insight into the role of EBV genetic variation in NPC pathogenesis by defining a risk haplotype of EBV for downstream functional studies and identifying a single high risk EBV lineage characterized by 9 SNPs for potential application in population screening of NPC. Author summary: Two genome-wide association studies (GWAS) of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) in South China have revealed different risk loci in EBER region and BALF2 gene of EBV, respectively. More EBV genomes isolated from healthy donors and NPC biopsies are included in this study. The meta-analysis of EBV genomes derived from 279 NPC cases and 227 healthy controls identifies a risk haplotype of EBV comprising of multiple genetic variants having strong linkage disequilibrium with each other. Furthermore, a high risk lineage of EBV characterized by 9 single nucleotide polymorphisms (SNPs) is found to be enriched in NPC of South China. The risk haplotype of EBV will be useful for downstream studies of disease mechanisms and the high risk EBV lineage may be included in screening protocol for early detection of NPC in the population. [ABSTRACT FROM AUTHOR]

Polygonatum cyrtonema Hua is a perennial herbaceous plant of the Polygonatum genus, belonging to the Liliaceae family, with significant medicinal and nutritional value. In China, this species is a traditional medicinal and edible herb with a long history of application and is widely appreciated by the people. However, as the demand for medicinal herbs continues to grow, excessive harvesting has led to the depletion of wild resources and the risk of genetic erosion. In addition, the chaotic cultivation of varieties and the lack of high quality germplasm resources have led to inconsistent quality of medical materials. Therefore, it is urgent to conduct genetic diversity evaluation of this species and establish a sound conservation plan. This study assessed the genetic diversity and population structure of 96 samples collected from seven regions in China using the simple sequence repeat (SSR) molecular marker technology. In this study, a total of 60 alleles (Na) were detected across the 10 polymorphic SSR markers used, with an average of 6.0 alleles generated per locus. The values of polymorphic information content (PIC) values ranged from 0.3396 to 0.8794, with an average value of 0.6430. The average value of the effective number of alleles (Ne) was 2.761, and the average value of the Shannon's information index (I) was 1.196. The population structure analysis indicates that the Polygonatum cyrtonema Hua germplasm can be classified into three subpopulations (JZ, QY, JD) at the molecular level, which corresponds to the previous subgroups identified based on individual plant phenotypic traits. Analysis of Molecular Variance (AMOVA) showed that 74% of the genetic variation was between individuals within populations in different regions. The phylogenetic analysis of the 96 germplasm samples divided them into three main populations. The QY and JD subpopulations are largely clustered together, which could be attributed to their mountainous distribution and the local climate environment. The genetic differentiation coefficient (Fst) value was low at 0.065, indicating relatively low population differentiation. The ratio of the genetic differentiation coefficient (Fst) between the JZ population and the other two populations (QY and JD) is much higher than the ratio between the QY and JD populations. Based on the clustering results and the ratio of the genetic differentiation coefficient (Fst), it can be inferred that the genetic relationship between the QY and JD subpopulations is closer, with a certain degree of genetic differentiation from the JZ subpopulation. This study supports the conservation of germplasm resources of Polygonatum cyrtonema Hua in China and provides new parental material for germplasm genetic improvement and breeding programs. [ABSTRACT FROM AUTHOR]

Objective: Familial focal epilepsy with variable foci (FFEVF) is a rare type of focal epilepsy syndrome; it is associated with NPRL3 variant. However, relevant reports are rare in China. We aimed to analyze the clinical features of Chinese patients with FFEVF to understand further the differences between various NPRL3 variants and explored the effect of NPRL3 variant on mRNA. Methods: We ran a full workup on a family with FFEVF (four patients, one healthy member): an inquiry of medical history, cranial magnetic resonance imaging (MRI), electroencephalogram (EEG), and whole exon sequencing. Their clinical features were compared with those of other FFEVF patients in published reports. The mRNA splicing changes were analyzed quantitatively and qualitatively using real-time quantitative—polymerase chain reaction (q-PCR) and reverse transcription (RT)-PCR and compared between our patients and healthy individuals. Results: Patients with NPRL3: c.1137dupT variant had a wide range of onset age (4 months to 31 years), diverse seizure types, variable foci (frontal lobe/temporal lobe), different seizure times (day/night) and frequencies (monthly/seldom/every day), different therapeutic effects (refractory epilepsy/almost seizure free), normal MRI, and abnormal EEG (epileptiform discharge, slow wave). The phenotypic spectrum with different NPRL3 variants was either similar or different. Significantly different relative quantities of mRNA were found between patients and healthy individuals in real-time qPCR. Abnormal splicing was observed in patients compared with healthy individual in RT-PCR. Despite having the same gene variant, different family members had different mRNA splicing, possibly causing different phenotypes. Conclusion: The clinical features of FFEVF varied, and auxiliary inspection was atypical. NPRL3: c.1137dupT could change the relative quantity of mRNA and cause abnormal splicing, which might produce different phenotypes in different family members. [ABSTRACT FROM AUTHOR]

Prunus sibirica is an economically important tree species that occurs in arid and semi-arid regions of northern China. For this species, creation of a core collection is critical for future ecological and evolutionary studies, efficient economic utilization, and development and management of the broader collection of its germplasm resources. In this study, we sampled 158 accessions of P. sibirica from Russia and China using 30 pair of simple sequence repeat molecular markers and 30 different schemes to identify candidate core collections. The 30 schemes were based on combinations of two different sampling strategies, three genetic distances, and five different sample sizes of the complete germplasm resource. We determined the optimal core collection from among the 30 results based on maximization of genetic diversity among groups according to Number of observed alleles (Na), Number of effective alleles (Ne), Shannon's information index (I), Polymorphic information content (PIC), Nei gene diversity (H) and compared to the initial collection of 158 accessions. We found that the optimal core collection resulted from preferred sampling at 25% with Nei & Li genetic distance these ratios of Na, Ne, I, PIC and H to the complete 158 germplasm resources were 73.0%, 113%, 102%, 100% and 103%, respectively, indicating that the core collection comprised a robust representation of genetic diversity in P. sibirica. The proposed core collection will be valuable for future molecular breeding of this species and management of its germplasm resources. [ABSTRACT FROM AUTHOR]

Leptocybe invasa Fisher et LaSalle is a global invasive pest that seriously damages Eucalyptus plants. Studying the genetic diversity, genetic structure and introgression hybridization of L. invasa in China is of great significance for clarifying the breeding strategy, future invasion and diffusion trends of L. invasa in China and developing scientific prevention and control measures. Genetic diversity and phylogenetic analyses of 320 L. invasa female adults from 14 geographic populations in China were conducted using 10 polymorphic microsatellite loci (SSRs) and mitochondrial DNA cytochrome oxidase I gene sequences (COIs). (1) The Bayesian phylogenetic tree and haplotype network diagram showed that only haplotype Hap3 existed in L. invasa lineage B in China, while haplotypes Hap1 and Hap2 existed in lineage A, among which haplotype Hap2 was found for the first time. The nucleotide and haplotype diversities of lineage A were higher than those of lineage B. (2) The SSR genetic diversity of the Wuzhou Guangxi, Ganzhou Jiangxi and Panzhihua Sichuan populations was higher than that of the other 11 populations, and the SSR genetic diversity of lineage A was higher than that of lineage B. (3) The AMOVA analysis of mitochondrial COI data showed that 75.55% of the variation was among populations, and 99.86% of the variation was between lineages, while the AMOVA analysis of nuclear SSR data showed that 35.26% of the variation was among populations, and 47.04% of the variation was between lineages. There were obvious differences in the sources of variation between the COI and SSR data. (4) The optimal K value of COI and SSR data in structure analysis was 2, and PCoA analysis also divided the dataset into two obvious categories. The UPMGA phylogenetic tree based on SSR data clustered 14 geographic species into two groups. The results of genetic structure analysis supported the existence of two lineages, A and B, in China. (5) Structural analysis showed that there was obvious introgressive hybridization in Wuzhou Guangxi, Ganzhou Jiangxi, Panzhihua Sichuan and other populations. These results suggest that lineage introgressive hybridization has occurred in the L. invasa population in China. The introgressive hybridization degree and genetic diversity of lineage A are obviously higher than those of lineage B. Lineage introgressive hybridization may be the driving force for further L. invasa invasion and diffusion in China in the future. [ABSTRACT FROM AUTHOR]

Background: Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup. Methodology/Principal Findings: In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as L. interrogans, L. borgpetersenii, and L. kirschneri, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic Leptospira circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs. Conclusions/Significance: Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the complicated relationship between STs and serovars indicates an urgent need to develop an improved scheme for Leptospira serotyping. Author Summary: Leptospirosis, caused by pathogenic Leptospira spp, is a globally widespread zoonosis. In this study, our focusing on serogroup Icterohaemorrhagiae strains of Leptospira using MLST as a tool for phylogenetic analysis that has led to a better understanding of evolution of Leptospira. This totally consisted of 120 epidemic strains from five geographically diverse regions were isolated over the past 50 years in China and 8 strains from seven different countries. 17 STs were identified in these 128 strains by MLST analysis. Adding 116 serogroup Icterohaemorrhagiae in the Leptospira MLST database and studies previously described, 22 STs were identified in the 244 isolates. The genetic diversity of Leptospira belonging to serogroup Icterohaemorrhagiae from China was generally different from that of isolates elsewhere. Results of the 16S rRNA sequencing typing and MLST genotyping method were nearly consistent. Here, MLST revealed the high diversity of STs among the serogroup Icterohaemorrhagiae strains in China. Our present study provides a blueprint for further phylogenetic research. More convenient molecular techniques have to be developed to identify and characterize Leptospira species and STs. [ABSTRACT FROM AUTHOR]

Background: Schistosoma japonicum still causes severe parasitic disease in mainland China, but mainly in areas along the Yangtze River. However, the genetic diversity in populations of S. japonicum has not been well understood across its geographical distribution, and such data may provide insights into the epidemiology and possible control strategies for schistosomiasis. Methodology/Principal Findings: In this study infected Oncomelania snails were collected from areas in the middle and lower (ML) reaches of the Yangtze River, including Hubei, Hunan, Anhui, Jiangxi and Jiangsu provinces, and in the upper reaches of the river, including Sichuan and Yunnan provinces in southwest (SW) China. The adult parasites obtained from experimentally infected mice using isolated cercariae were sequenced individually for several fragments of mitochondrial regions, including Cytb-ND4L-ND4, 16S-12S and ND1. Populations in the ML reaches exhibited a relatively high level of diversity in nucleotides and haplotypes, whereas a low level was observed for populations in the SW, using either each single fragment or the combined sequence of the three fragments. Pairwise analyses of F-statistics (Fst) revealed a significant genetic difference between populations in the ML reaches and those in the SW, with limited gene flow and no shared haplotypes in between. It is rather obvious that genetic diversity in the populations of S. japonicum was significantly correlated with the geographical distance, and the geographical separation/isolation was considered to be the major factor accounting for the observed difference between populations in the ML reaches and those in the SW in China. Conclusions: S. japonicum in mainland China exhibits a high degree of genetic diversity, with a similar pattern of genetic diversity as observed in the intermediate host snails in the same region in China. Author Summary: Despite the existing threat of schistosomiasis in some rural areas along the Yangtze River, the genetic diversity of Schistosoma japonicum has not been investigated across its wide geographical distribution in China, and such information may provide insight into the disease epidemiology and the development of its control measures. In this study, the adult parasites, obtained through infecting mice with cercariae from snails of the genus Oncomelania collected from a wide range of localities in currently endemic areas of schistosomiasis in the middle and lower (ML) reaches of the Yangtze River, and in Sichuan and Yunnan provinces in the upper reaches of the river in southwest (SW) China, were sequenced individually for mitochondrial genes. In general, a relatively high degree of genetic variation was observed in populations in the ML reaches in terms of nucleotide and haplotype diversity, but a low level was observed in populations in the SW. The significant difference in genetic diversity as revealed by F-statistics, and the existence of no shared haplotypes, were observed between populations in the ML reaches and those in the SW, indicating the effect of geographical separation/isolation upon the schistosomes and probably the parasite-snail system in China. [ABSTRACT FROM AUTHOR]

Background: Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum, which causes schistosomiasis endemic in the Far East, and especially in mainland China. O. hupensis largely determines the parasite's geographical range. How O. hupensis's genetic diversity is distributed geographically in mainland China has never been well examined with DNA sequence data. Methodology/Principal Findings: In this study we investigate the genetic variation among O. hupensis from different geographical origins using the combined complete internal transcribed spacer 1 (ITS1) and ITS2 regions of nuclear ribosomal DNA. 165 O. hupensis isolates were obtained in 29 localities from 7 provinces across mainland China: lake/marshland and hill regions in Anhui, Hubei, Hunan, Jiangxi and Jiangsu provinces, located along the middle and lower reaches of Yangtze River, and mountainous regions in Sichuan and Yunnan provinces. Phylogenetic and haplotype network analyses showed distinct genetic diversity and no shared haplotypes between populations from lake/marshland regions of the middle and lower reaches of the Yangtze River and populations from mountainous regions of Sichuan and Yunnan provinces. The genetic distance between these two groups is up to 0.81 based on Fst, and branch time was estimated as 2–6 Ma. As revealed in the phylogenetic tree, snails from Sichuan and Yunnan provinces were also clustered separately. Geographical separation appears to be an important factor accounting for the diversification of the two groups of O. hupensis in mainland China, and probably for the separate clades between snails from Sichuan and Yunnan provinces. In lake/marshland and hill regions along the middle and lower reaches of the Yangtze River, three clades were identified in the phylogenetic tree, but without any obvious clustering of snails from different provinces. Conclusions: O. hupensis in mainland China may have considerable genetic diversity, and a more complex population structure than expected. It will be of significant importance to consider the genetic diversity of O. hupensis when assessing co-evolutionary interactions with S. japonicum. Author Summary: The intermediate host of Schistosoma japonicum in Asia is the snail Oncomelania hupensis, which can be separated phenotypically into ribbed- and smooth-shelled morphotypes. In China, the typical morphotype is ribbed-shelled, with its distribution restricted to mainland China. Smooth-shelled snails with varix are also distributed in China, which are considered to belong to the same subspecies as the ribbed-shelled snails. In this study we investigate the genetic variation among O. hupensis from different geographical origins using combined complete ITS1 and ITS2 regions of nuclear ribosomal DNA. Snails including ribbed-shelled and smooth-shelled (but with varix on the shell) from the lake/marshland region of the middle and lower reaches of the Yangtze River, and smooth-shelled snails from mountainous regions of Sichuan and Yunnan provinces, were genetically distinct with no shared haplotypes detected. Furtheremore, the snails from Sichuan and Yunnan provinces were clustered in separate clades in the phylogenetic tree, and three clades were observed for snails from the middle and lower reaches of the Yangtze River. The population diversity of O. hupensis in China is thus considered large, and evolutionary relationships in the host-parasite system of O. hupensis-S. japonicum may be of interest for further research. [ABSTRACT FROM AUTHOR]

Eight traditional subspecies of tiger (Panthera tigris), of which three recently became extinct, are commonly recognized on the basis of geographic isolation and morphological characteristics. To investigate the species' evolutionary history and to establish objective methods for subspecies recognition, voucher specimens of blood, skin, hair, and/or skin biopsies from 134 tigers with verified geographic origins or heritage across the whole distribution range were examined for three molecular markers: (1) 4.0 kb of mitochondrial DNA (mtDNA) sequence; (2) allele variation in the nuclear major histocompatibility complex class II DRB gene; and (3) composite nuclear microsatellite genotypes based on 30 loci. Relatively low genetic variation with mtDNA, DRB, and microsatellite loci was found, but significant population subdivision was nonetheless apparent among five living subspecies. In addition, a distinct partition of the Indochinese subspecies P. t. corbetti into northern Indochinese and Malayan Peninsula populations was discovered. Population genetic structure would suggest recognition of six taxonomic units or subspecies: (1) Amur tiger P. t. altaica; (2) northern Indochinese tiger P. t. corbetti; (3) South China tiger P. t. amoyensis; (4) Malayan tiger P. t. jacksoni, named for the tiger conservationist Peter Jackson; (5) Sumatran tiger P. t. sumatrae; and (6) Bengal tiger P. t. tigris. The proposed South China tiger lineage is tentative due to limited sampling. The age of the most recent common ancestor for tiger mtDNA was estimated to be 72,000–108,000 y, relatively younger than some other Panthera species. A combination of population expansions, reduced gene flow, and genetic drift following the last genetic diminution, and the recent anthropogenic range contraction, have led to the distinct genetic partitions. These results provide an explicit basis for subspecies recognition and will lead to the improved management and conservation of these recently isolated but distinct geographic populations of tigers. Genetic analysis provides the basis for subspecies recognition among tigers, and will lead to improved conservation strategies for these endangered animals. [ABSTRACT FROM AUTHOR]