Development of a target capture sequencing SNP genotyping platform for genetic analysis and genomic breeding in rapeseed

Rapeseed(Brassica napus)is an oil crop grown worldwide,making it a key plant species in molecular breeding research.However,the complexity of its polyploid genome increases sequencing costs and reduces sequencing accuracy.Target capture coupled with high-throughput sequencing is an efficient approach for detecting genetic variation at genomic regions or loci of interest.In this study,588 resequenced accessions of rapeseed were used to develop a target capture sequencing SNP genotyping platform named BnaPan50T.The platform comprised 54,765,with 54,058 resequenced markers from the pan-genome,and 855 variant trait-associated markers for 12 agronomic traits.The capture quality of BnaPan50T was demonstrated well in 12 typical accessions.Compared with a conventional genotyping array,BnaPan50T has a high SNP density and a high proportion of SNPs in unique physical positions and in annotated functional genes,promising wide application.Target capture sequencing and wholegenome resequencing in 90 doubled-haploid lines yielded 60%specificity,78%uniformity within tenfold coverage range,and 93%genotyping accuracy for the platform.BnaPan50T was used to construct a genetic map for quantitative trait loci(QTL)mapping,identify 21 unique QTL,and predict several candidate genes for yield-related traits in multiple environments.A set of 132 core SNP loci was selected from BnaPan50T to construct DNA fingerprints and germplasm identification resources.This study provides genomics resources to support target capture sequencing,genetic analysis and genomic breeding of rapeseed.

The impact of genotyping strategies and statistical models on accuracy of genomic prediction for survival in pigs

Background:Survival from birth to slaughter is an important economic trait in commercial pig productions.Increasing survival can improve both economic efficiency and animal welfare.The aim of this study is to explore the impact of genotyping strategies and statistical models on the accuracy of genomic prediction for survival in pigs during the total growing period from birth to slaughter.Results:We simulated pig populations with different direct and maternal heritabilities and used a linear mixed model,a logit model,and a probit model to predict genomic breeding values of pig survival based on data of individual survival records with binary outcomes(0,1).The results show that in the case of only alive animals having genotype data,unbiased genomic predictions can be achieved when using variances estimated from pedigreebased model.Models using genomic information achieved up to 59.2%higher accuracy of estimated breeding value compared to pedigree-based model,dependent on genotyping scenarios.The scenario of genotyping all individuals,both dead and alive individuals,obtained the highest accuracy.When an equal number of individuals(80%)were genotyped,random sample of individuals with genotypes achieved higher accuracy than only alive individuals with genotypes.The linear model,logit model and probit model achieved similar accuracy.Conclusions:Our conclusion is that genomic prediction of pig survival is feasible in the situation that only alive pigs have genotypes,but genomic information of dead individuals can increase accuracy of genomic prediction by 2.06%to 6.04%.

Molecular Genotyping of Human Papillomavirus (HPV) in Hypopharyngeal Cancer

Background: In recent years, head and neck cancers have become common worldwide, ranking sixth in incidence. In 2007, in France the incidence increased by 14,697 including 11,158 among men, which places them in fourth place. The same year, 32,268 patients were hospitalized for this pathology, but 95% are associated with alcohol and tobacco poisoning. Few data exist on these cancers in Africa and Senegal. In recent years, many studies have hypothesized that about 25% of head and neck cancers are associated with high-risk oncogenic human papillomaviruses (HPV) whose role in cervical cancer was already widely established. Objective: To know the prevalence and genotypes of HPV in head and neck cancers, particularly hypopharyngeal cancer. Material and method: This study was carried out on samples of biopsies of hypopharynx cancerous tissue (ulcerative-budding lesion) and healthy oropharyngeal tissue obtained from the ENT department of the Fann hospital, then sent to the Molecular Biology Unit of the Ouakam military hospital (HMO). The nucleic acids extraction was carried out using the standard method of the Zymo research kit “Quick-DNATM Miniprep Plus kit” https://www.zymoresearch.com/. Molecular HPV detection and genotyping were performed by multiplex RT-PCR with the Seegene AnyplexTM II HPV28 kit Detection on a Biorad CFX96 automaton according to the manufacturer’s protocol for the simultaneous genotyping of 28 types of HPV including 19 at High Risk (HR) and 9 low risk (LR). Results: 156 patients were sampled, 61 Hypopharynx cancer biopsies and 95 healthy tissues. The median age of the general population was 36.5 years [12, 73];the median age of the population with hypopharyngeal cancer of 40 years. Of the general study population 24.36% (38/156) was infected with HPV. In populations with hypopharyngeal cancer, HPV prevalence was 19.67% (12/61), 17.84% (5/28) in men and 21.21% (7/33) in women. HPV6 was the most frequently encountered genotype in the cancer population. Multiple infections have also been noted in cancer patients: HPV6+HPV18, HPV6+HPV56. For patients without hypopharyngeal cancer, the HPV prevalence was 27.36% (26/95), 9.59% (7/73) in women and 89.36% (19/22) in men. Several types of HPV-HR genotypes (HPV18, HPV26, HPV69), and HPV-LR genotypes (HPV42, HPV43, HPV70, HPV6) have been detected in healthy patients but also cases of co-infections (HPV6+HPV69;HPV56+HPV44;HPV58+HPV18). Conclusion: Our results showed a higher prevalence of HPV in non-cancer patients compared to hypopharyngeal cancer patients. The genotypes (HPV 6, 18 and 56) were observed in the study population. Molecular genotyping does not show a significant involvement of HPV in hypopharyngeal cancer.

iBP-seq:An efficient and low-cost multiplex targeted genotyping and epigenotyping system

Inter-and intra-specific variations in phenotype are common and can be associated with genomic mutations as well as epigenomic variation.Profiling both genomic and epigenomic variants is at the core of dissecting phenotypic variation.However,an efficient targeted genotyping and epigenotyping system is lacking.We describe a new multiplex targeted genotyping and epigenotyping system called improved bulked-PCR sequencing(iBP-seq).We employed iBP-seq for the detection of genotypes and methylation levels of dozens of target regions in mixed DNA samples.iBP-seq can be adapted for the construction of linkage maps,fine mapping of quantitative-trait loci,and detection of genome editing mutations at a cost as low as$0.016 per site per sample.We developed an automated bioinformatics pipeline,including primer design,a series of bioinformatic analyses for genotyping and epigenotyping,and visualization of results.iBP-seq and its bioinformatics pipeline,available at http://zeasystemsbio.hzau.edu.cn/tools/ibp/,can be adapted to a wide variety of species.

Selenium Differentially Regulates Flavonoid Accumulation and Antioxidant Capacities in Sprouts of Twenty Diverse Mungbean(Vigna radiata(L.)Wilczek)Genotypes

Seed germination with selenium(Se)is promising for producing Se-biofortified foods.Mungbean(Vigna radiata(L.)Wilczek)sprout is freshly eaten as a salad dressed with sauce,making it superior for Se biofortification.Since the Se safety range for the human body is extremely narrow,it is imperative to evaluate the genotypic responses of mungbean sprouts to Se.This study evaluated the Se enrichment capacity and interaction withflavonoids and antioxidant systems in sprouts of 20 mungbean germplasms.Selenium treatment was done by immersing mung-bean seeds in 20μM sodium selenite solution for 8 h.Afterward,the biomass,Se amounts,flavonoid(particularly vitexin and isovitexin)contents,antioxidant capacity,and key biosynthetic gene expressions were measured.Sprout Se content was 2.0-7.0μg g^(-1) DW among the 20 mungbean germplasms.Selenium treatment differentially affected the biomass,totalflavonoid,vitexin,isovitexin,antioxidant enzyme activities,and antioxidant capacities of the mungbean germplasms.Eight germplasms showed increased biomass(p<0.05),the highest increasing by 127%,but 13 did not phenotypically respond to Se treatment.Seven and six germplasms showed varied levels of vitexin and isovitexin increment after Se treatment,the highest measuring 2.67-and 2.87-folds for vitexin and isovitexin,respectively.Two mungbeanflavonoid biosynthesis genes,chalcone synthase(VrCHS)and chalcone isomerase(VrCHI)were significantly up-regulated in the germplasms with increased vitexin and isovitexin levels(p<0.05).Moreover,Se enrichment capacity was significantly correlated with the vitexin,isovitexin,and antiox-idant capacities.In conclusion,mungbean sprouts could be a useful Se-biofortified food,but the Se enrichment capacity and nutritional response must be determined for each germplasm before commercialization.

Genotyping ex situ trees of Abies nebrodensis translocated from the original Sicilian population to enrich the gene pool

As a dynamic ex situ conservation strategy,a clonal seed orchard was started in a nursery in Pomaio(POM)in central Italy in 1993 for an assisted migration experiment of Abies nebrodensis(Lojac.)Mattei.Two artifi cial ex situ populations were planted with this gene pool:a seedling arboretum in Pieve Santo Stefano(PSS)and a small dendrological collection in Papiano(PAP),both originating from the Sicilian relict population.Here,using AFLP markers,we estimated the relatedness among the relocated genotypes of the three collections to check whether the three collections had suffi cient genetic variability to be considered as additional sources of variability to the original gene pool for the assisted migration strategy.High individual genetic variability was found in the collections;each plant had a diff erent genotype and was confi rmed to belong to its population of origin.PAP and PSS trees were shown to be only from the original population of A.nebrodensis species and were derived from a limited set of maternal fertile genotypes.Based on the Sicilian fi r population inventory,nursery production in Sicily,and structure clustering analysis,close genetic relationships among POM,PAP and several PSS trees(1–35)were evident.Similarly,the PSS group(36–78)was genetically close to tree 1 of POM and in a lesser proportion to plants 7 and 9 of POM.The sampling of seedlings used to form batches in the nursery might have infl uenced the structure of the resultant plantations.All genotypes will be useful for enriching the original gene pool.

Identification of S-RNase genotype and analysis of its origin and evolutionary patterns in Malus plants

Identification of the S genotype of Malus plants will greatly promote the discovery of new genes,the cultivation and production of apple,the breeding of new varieties,and the origin and evolution of self-incompatibility in Malus plants.In this experiment,88 Malus germplasm resources,such as Aihuahong,Xishuhaitang,and Reguanzi,were used as materials.Seven gene-specific primer combinations were used in the genotype identification.PCR amplification using leaf DNA produced a single S-RNase gene fragment in all materials.The results revealed that 70 of the identified materials obtained a complete S-RNase genotype,while only one S-RNase gene was found in 18 of them.Through homology comparison and analysis,13 S-RNase genotypes were obtained:S_(1)S_(2)(Aihuahong,etc.),S_(1)S_(28)(Xixian Haitang,etc.),S_(1)S_(51)(Hebei Pingdinghaitang),S_(1)S_(3)(Xiangyangcun Daguo,etc.),S_(2)S_(3)(Zhaiyehaitang,etc.),S_(3)S_(51)(Xishan 1),S_(3)S_(28)(Huangselihaerde,etc.),S_(2)S_(28)(Honghaitang,etc.),S_(4)S_(28)(Bo 11),S_(7)S_(28)(Jiuquan Shaguo),S_(10)S_e(Dongchengguan 13),S_(10)S_(21)(Dongxiangjiao)and S_(3)S_(51)(Xiongyue Haitang).Simultaneously,the frequency of the S gene in the tested materials was analyzed.The findings revealed that different S genes had varying frequencies in Malus resources,as well as varying frequencies between intraspecific and interspecific.S_(3) had the highest frequency of 68.18%,followed by S_(1)(42.04%).In addition,the phylogenetic tree and origin evolution analysis revealed that the S gene differentiation was completed prior to the formation of various apple species,that cultivated species also evolved new S genes,and that the S_(50) gene is the oldest S allele in Malus plants.The S_(1),S_(29),and S_(33) genes in apple-cultivated species,on the other hand,may have originated in M.sieversii,M.hupehensis,and M.kansuensis,respectively.In addition to M.sieversii,M.kansuensis and M.sikkimensis may have also played a role in the origin and evolution of some Chinese apples.

Correlation between genotypes of Orientia tsutsugamushi and clinical characteristics of patients with scrub typhus in Guangzhou,China

Objective:To explore the correlation between genotypes of Orientia(O.)tsutsugamushi and clinical characteristics of scrub typhus patients.Methods:Clinical data of patients with scrub typhus admitted to different types of medical institutions in Guangzhou from September 2012 to December 2016 were collected using medical records.Demographic data,clinical manifestations,as well as hematological and biochemical indicators of patients infected with different genotypes were analyzed and compared.Results:A total of 192 patients were included in this study,including 121 patients with Karp genotype of O.tsutsugamushi infection(63.0%),36 patients with Gilliam genotype(19.0%),23 patients with Kato genotype(12.0%),and 12 patients with TA763 genotype(6.0%)infection.The median value of albumin in patients with Karp genotype infection was significantly lower than that of Gilliam-infected patients(P=0.032).Patients with Karp genotype infection had a significantly longer hospital stay(9 days)than those with Gilliam genotype(7 days)(P=0.009)and Kato genotype infection(6 days)(P=0.005).Karp-infected patients also represented for the largest number of patients with complicated organ involvement(88/133,66.2%).Furthermore,Karp-infected patients had higher risk of developing multiple organ dysfunction syndrome(18.2%)and requiring intensive care unit treatment(15.9%).Besides,patients with Gilliam genotype(8 days)and TA763 genotype infection(7.5 days)had shorter fever duration than those with Karp genotype(9 days)and Kato genotype(9 days)infection,respectively.Conclusions:Genotypes of Orientia tsutsugamushi were associated with varying clinical manifestations,organ involvement,and treatment outcomes,suggesting that genotypes ranged in virulence.

Occurrence of K1 and K2 serotypes and genotypic characteristics of extended spectrumβ-lactamases-producing Klebsiella pneumoniae isolated from selected hospitals in Malaysia

Objective:To determine the distribution,phenotypic and genetic background of extended spectrumβ-lactamases(ESBL)-producing Klebsiella(K.)pneumoniae clinical isolates associated with K1 and K2 serotypes in two selected hospitals in Malaysia.Methods:A total of 192 K.pneumoniae isolates were collected and subjected to antibiotic susceptibility,hypermucoviscosity test and multiplex PCR to detect the presence of K1-and K2-serotype associated genes.Multilocus sequence typing(MLST)was performed on ESBL-producing K.pneumoniae isolates presented with K1 and K2 serotypes,followed by phylogenetic analysis.Results:A total of 87 out of 192(45.3%)of the K.pneumoniae isolates collected were ESBL producers.However,only 8.3%(16/192)and 10.9%(21/192)of the total isolates were detected to carry K1-and K2-serotype associated genes,respectively.Statistical analysis showed that K1 and K2 capsular serotypes were not significantly associated with ESBL phenotype(P=0.196).However,they were significantly associated with hypervirulent,as demonstrated by the positive string test(P<0.001).MLST analysis revealed that ST23 as the predominant sequence type(ST)in the K1 serotype,while the ST in the K2 serotype is more diverse.Conclusions:Although the occurrence of ESBL-producing isolates among the hypervirulent strains was low,their coexistence warrants the need for continuous surveillance.MLST showed that these isolates were genetically heterogeneous.

Characterization of early maturing elite genotypes based on MTSI and MGIDI indexes:an illustration in upland cotton(Gossypium hirsutum L.)

Background Globally,the cultivation of cotton is constrained by its tendency for extended periods of growth.Early maturity plays a potential role in rainfed-based multiple cropping system especially in the current era of climate change.In the current study,a set of 20 diverse Gossypium hirsutum genotypes were evaluated in two crop seasons with three planting densities and assessed for 11 morphological traits related to early maturity.The study aimed to identify genotype(s)that mature rapidly and accomplish well under diverse environmental conditions based on the two robust multivariate techniques called multi-trait stability index(MTSI)and multi-trait genotype-ideotype distance index(MGIDI).Results MTSI analysis revealed that out of the 20 genotypes,three genotypes,viz.,NNDC-30,A-2,and S-32 accomplished well in terms of early maturity traits in two seasons.Furthermore,three genotypes were selected using MGIDI method for each planting densities with a selection intensity of 15%.The strengths and weaknesses of the genotypes selected based on MGIDI method highlighted that the breeders could focus on developing early-maturing genotypes with specific traits such as days to first flower and boll opening.The selected genotypes exhibited positive genetic gains for traits related to earliness and a successful harvest during the first and second pickings.However,there were negative gains for traits related to flowering and boll opening.Conclusion The study identified three genotypes exhibiting early maturity and accomplished well under different planting densities.The multivariate methods(MTSI and MGIDI)serve as novel approaches for selecting desired genotypes in plant breeding programs,especially across various growing environments.These methods offer exclusive benefits and can easily construe and minimize multicollinearity issues.

Hepatitis B virus genotypes in precision medicine of hepatitis Brelated hepatocellular carcinoma:Where we are now

Hepatitis B virus(HBV)infection is a major player in chronic hepatitis B that may lead to the development of hepatocellular carcinoma(HCC).HBV genetics are diverse where it is classified into at least 9 genotypes(A to I)and 1 putative genotype(J),each with specific geographical distribution and possible different clinical outcomes in the patient.This diversity may be associated with the precision medicine for HBV-related HCC and the success of therapeutical approaches against HCC,related to different pathogenicity of the virus and host response.This Editorial discusses recent updates on whether the classification of HBV genetic diversity is still valid in terms of viral oncogenicity to the HCC and its precision medicine,in addition to the recent advances in cellular and molecular biology technologies.

Genotypic effects on accelerated propagation of oil palm breeding materials selected(Elaeis guineensis jacq.)using somatic embryogenesis

Vegetable oil production from oil palm(Elaeis guineensis Jacq.)is an important industry due to the rising demand every year.The somatic embryogenesis culture can propagate oil palm duplicate as parent plant,which can be selected as breeding material to produce new planting germplasm with high production or disease resistance.This study aims to evaluate the genotypic effect of somatic embryogenesis,while immature leaflets were employed as explants.The culture used embryo induction medium based on Murashige and Skoog(MS)modifications that contained 5 mg/L Naphthalene Acetic acid(NAA)and 0.5 mg/L Benzyl Amino Purine(BAP).The genotypic effect was statistically significant in the percentage of callus induction,producing somatic embryos,and germination embryos.In this study,we successfully cloned thirteen oil palm genotypes(GE-02,GE-03,GE-06,GE-07,GE-09,GE-23,GE-24,GE-27,GE-28,GE-32,GE-33,GE-34,and GE-35),with the highest number of somatic embryos formed on GE-27 with a percentage of 70.1%.The cloning was successful in accelerating the propagation of oil palm for materials breeding programs to create new varieties with high production and disease resistance.It is necessary to observation the performance of these clones in the field in terms of mantle flower appearance.

Serological Investigation into the Infected Genotypes of Patients with Japanese Encephalitis in the Coastal Provinces of China

Objective Genotypes(G)1,3,and 5 of the Japanese encephalitis virus(JEV)have been isolated in China,but the dominant genotype circulating in Chinese coastal areas remains unknown.We searched for G5 JEV-infected cases and attempted to elucidate which JEV genotype was most closely related to human Japanese encephalitis(JE)in the coastal provinces of China.Methods In this study,we collected serum specimens from patients with JE in three coastal provinces of China(Guangdong,Zhejiang,and Shandong)from 2018 to 2020 and conducted JEV cross-neutralization tests against G1,G3,and G5.Results Acute serum specimens from clinically reported JE cases were obtained for laboratory confirmation from hospitals in Shandong(92 patients),Zhejiang(192 patients),and Guangdong(77 patients),China,from 2018 to 2020.Seventy of the 361 serum specimens were laboratory-confirmed to be infected with JEV.Two cases were confirmed to be infected with G1 JEV,32 with G3 JEV,and two with G5 JEV.Conclusion G3 was the primary infection genotype among JE cases with a definite infection genotype,and the infection caused by G5 JEV was confirmed serologically in China.

Multidimensional evaluation of salt tolerance in groundnut genotypes through biochemical responses

The manuscript explores the complex interplay between groundnut genotypes,salt tolerance and hormonal influence,shedding light on the dynamic responses of three specific groundnut genotypes,KDG-128,TG-37 A and GG-20,to salt treatments and gibberellic acid(GA3).The study encompasses germination,plant growth,total protein content and oil content as key parameters.Through comprehensive analysis,it identifies TG-37 A and KDG-128 as salt-tolerant genotypes,and GG-20 as salt-susceptible genotypes,which highlighting the potential for targeted breeding efforts to develop more resilient groundnut varieties.Moreover,the quantification of protein and oil content under different treatments provides vital data for optimizing nutritional profiles in groundnut cultivars.Principal Component Analysis(PCA) underscores the significance of the first principal component(PC1)in explaining the majority of variance,capturing primary trends and differences in plant length.Analysis of Variance(ANOVA) and hierarchical analysis confirm the presence of statistically significant differences in protein and oil content among the genotypes.Pearson's correlation coefficient matrix analysis reveals strong positive correlations between plant length and protein content,plant length and oil content,and a moderately positive correlation between protein content and oil content.These findings provide valuable insights into groundnut physiology,salt tolerance,and nutritional composition,with implications for future research in sustainable agriculture and crop improvement.

Genotype-based precision nutrition strategies for the prediction and clinical management of type 2 diabetes mellitus

Globally,type 2 diabetes mellitus(T2DM)is one of the most common metabolic disorders.T2DM physiopathology is influenced by complex interrelationships between genetic,metabolic and lifestyle factors(including diet),which differ between populations and geographic regions.In fact,excessive consumptions of high fat/high sugar foods generally increase the risk of developing T2DM,whereas habitual intakes of plant-based healthy diets usually exert a protective effect.Moreover,genomic studies have allowed the characterization of sequence DNA variants across the human genome,some of which may affect gene expression and protein functions relevant for glucose homeostasis.This comprehensive literature review covers the impact of gene-diet interactions on T2DM susceptibility and disease progression,some of which have demonstrated a value as biomarkers of personal responses to certain nutritional interventions.Also,novel genotype-based dietary strategies have been developed for improving T2DM control in comparison to general lifestyle recommendations.Furthermore,progresses in other omics areas(epigenomics,metagenomics,proteomics,and metabolomics)are improving current understanding of genetic insights in T2DM clinical outcomes.Although more investigation is still needed,the analysis of the genetic make-up may help to decipher new paradigms in the pathophysiology of T2DM as well as offer further opportunities to personalize the screening,prevention,diagnosis,management,and prognosis of T2DM through precision nutrition.

Risk factors for hepatocellular carcinoma associated with hepatitis C genotype 3 infection:A systematic review

BACKGROUND Hepatitis C virus(HCV)is a blood-borne virus which globally affects around 79 million people and is associated with high morbidity and mortality.Chronic infection leads to cirrhosis in a large proportion of patients and often causes hepatocellular carcinoma(HCC)in people with cirrhosis.Of the 6 HCV genotypes(G1-G6),genotype-3 accounts for 17.9%of infections.HCV genotype-3 responds least well to directly-acting antivirals and patients with genotype-3 infection are at increased risk of HCC even if they do not have cirrhosis.AIM To systematically review and critically appraise all risk factors for HCC secondary to HCV-G3 in all settings.Consequently,we studied possible risk factors for HCC due to HCV-G3 in the literature from 1946 to 2023.METHODS This systematic review aimed to synthesise existing and published studies of risk factors for HCC secondary to HCV genotype-3 and evaluate their strengths and limitations.We searched Web of Science,Medline,EMBASE,and CENTRAL for publications reporting risk factors for HCC due to HCV genotype-3 in all settings,1946-2023.RESULTS Four thousand one hundred and forty-four records were identified from the four databases with 260 records removed as duplicates.Three thousand eight hundred and eighty-four records were screened with 3514 excluded.Three hundred and seventy-one full-texts were assessed for eligibility with seven studies included for analysis.Of the seven studies,three studies were retrospective case-control trials,two retrospective cohort studies,one a prospective cohort study and one a cross-sectional study design.All were based in hospital settings with four in Pakistan,two in South Korea and one in the United States.The total number of participants were 9621 of which 167 developed HCC(1.7%).All seven studies found cirrhosis to be a risk factor for HCC secondary to HCV genotype-3 followed by higher age(five-studies),with two studies each showing male sex,high alpha feto-protein,directly-acting antivirals treatment and achievement of sustained virologic response as risk factors for developing HCC.CONCLUSION Although,studies have shown that HCV genotype-3 infection is an independent risk factor for end-stage liver disease,HCC,and liver-related death,there is a lack of evidence for specific risk factors for HCC secondary to HCV genotype-3.Only cirrhosis and age have demonstrated an association;however,the number of studies is very small,and more research is required to investigate risk factors for HCC secondary to HCV genotype-3.

Subclinical hepatitis E virus genotype 1 infection:The concept of“dynamic human reservoir”

Hepatitis E virus(HEV)is hyperendemic in South Asia and Africa accounting for half of total Global HEV burden.There are eight genotypes of HEV.Among them,the four common ones known to infect humans,genotypes 1 and 2 are prevalent in the developing world and genotypes 3 and 4 are causing challenge in the industrialized world.Asymptomatic HEV viremia in the general population,especially among blood donors,has been reported in the literature worldwide.The clinical implications related to this asymptomatic viremia are unclear and need further exploration.Detection of viremia due to HEV genotype 1 infection,apparently among healthy blood donors is also reported without much knowledge about its infection rate.Similarly,while HEV genotype 3 is known to be transmitted via blood transfusion in humans and has been subjected to screening in many European nations,instances of transmission have also been documented albeit without significant clinical consequences.Epidemiology of HEV genotype 1 in endemic areas often show waxing and waning pattern.Occasional sporadic occurrence of HEV infection interrupted by outbreaks have been frequently seen.In absence of known animal reservoir,where HEV exists in between outbreak is a mystery that needs further exploration.However,occurrence of asymptomatic HEV viremia due to HEV genotype 1 during epidemiologically quiescent period may explain that this phenomenon may act as a dynamic reservoir.Since HEV genotype 1 infection cannot cause chronicity,subclinical transient infection and transmission of virus might be the reason it sustains in interepidemic period.This might be the similar phenomenon with SARS COVID-19 corona virus infection which is circulating worldwide in distinct phases with peaks and plateaus despite vaccination against it.In view of existing evidence,we propose the concept of“Dynamic Human Reservoir.”Quiescent subclinical infection of HEV without any clinical consequences and subsequent transmission may contribute to the existence of the virus in a community.The potential for transmitting HEV infection by asymptomatic HEV infected individuals by fecal shedding of virus has not been reported in literature.This missing link may be a key to Pandora's box in understanding epidemiology of HEV infection in genotype 1 predominant region.

Microsatellite Genotyping for Four Expected Inbred Mouse Strains from KM Mice

Chinese Kun Ming （KM） mouse, an outbreed strain of laboratory animal, has been widely utilized in related pharmaceutical and genetic studies throughout China. However, the value of KM mice to the research community has been severely limited, partially due to the fact that well-characterized inbred strain of KM mice is not available. Several expected inbred strains from KM mice have been bred, but their genetic purity remains uncertain. In this study, four expected inbred strains of KM mice （A1, T2, N2, and N4） were chosen and their inbred degree were compared with two classical inbred mouse lines （BALB/c and C57BL/6） by analyzing the genotypes of about 30 microsatellite markers. In the four strains, A1 and N4 were homozygous at all genotyped loci, but N2 and T2 were only heterozygous at locus D15Mit16. These results indicate that the level of genetic purity/homozygousity of A1, N4, N2, and T2 inbred line is comparable to those of BALB/c and C57BL/6. This study provided the first and solid evidence for genetic purity of four expected inbred strains of KM mice. These 4 inbred mice strains should be well maintained for further characterization and utilization in genetic studies.

Pedigree tracing of Fenneropenaeus chinensis by microsatellite DNA markers genotyping

Two microsateUite DNA loci were used to trace the pedigree structure of six families in the shrimp Fenneropenaeus chinensis. Four of the families were natural mating, and the others were mated by artificial insemination. Eleven alleles were acquired at two microsatellite DNA loci （locus RS0622 and locus EN0033 ） by investigating 145 offsprings and 11 parents. Five alleles were acquired from locus RS0622 and six from locus EN0033. As analyzed, the gene frequencies were between 0. 024 1 and 0. 493 1, the heterozygosity was 0.652 2 and 0.688 8, and the polymorphism information content （PIC） was 0.585 7 and 0.652 9 for the locus RS0622 and the locus EN0033, respectively. Twenty-three genotypes were detected and the genotypes of the losing parents were also inferred. The pedigrees of three F1 and three F2 generations were determined by matching the genotype at each locus.

Improved Allele-specific Polymerase Chain Reaction for Single Nucleotide Polymorphism Genotyping

An improved allele-specific PCR（AS-PCR） approach was applied to investigating -55C/T polymorphism in promoter region of the uncoupling protein 3（UCP3）gene. AS-PCR is a competitive PCR method which is based on positioning the 3＇ base of a PCR primer to match one single nucleotide polymorphism（SNP） allele and accurately extend only the correctly matched primer. But it is limited in use because of its poor specificity. In this study, we improved the specificity of AS-PCR by introducing additional mismatch at the penultimate base of 3＇ end of AS-PCR primer in combination with decreasing the level of dNTP in the reaction mixture. Sensitivity, specificity and reliability of this method were assessed for both simple plasmid model and complex human genomic SNP targets. The -55C/T（rs1800849） polymorphisrn of the UCP3 gene was analyzed via this AS-PCR and restriction fragment length polymorphism（RFLP）, the latter was used as a gold standard. The results suggest that the increase in AS-PCR discrimination with this method should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping application.