Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a sui...Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a suitable model to analyze allele-specific expressi on (ASE) and allele-specific alter native splicing (ASS). Analysis of ASE and ASS can uncover the differences in cis-regulatory elements between closely related species, while eliminating interferenee of trans-regulatory elements. Here, we provide a detailed characterization of ASE and ASS from 19 and 10 transcriptome datasets across five tissues from reciprocal-cross hybrids of horsex don key (mule/hi nny) and cattlexyak (dzo), respectively. Results showed that 4.8%-8.7% and 10.8%-16.7% of genes exhibited ASE and ASS, respectively. Notably, IncRNAs and pseudogenes were more likely to show ASE than protein-coding genes. In addition, genes showing ASE and ASS in mule/hinny were found to be involved in the regulation of muscle strength, whereas those of dzo were involved in high-altitude adaptati on. In con clusi on, our study dem on strated that explorati on of genes showing ASE and ASS in hybrids of closely related species is feasible for species evolution research.展开更多
The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in ...The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.展开更多
A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly...A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru（bpy）3 ^2＋（TBR）-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.展开更多
We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treate...We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.展开更多
Hybrid crops often exhibit increased yield and greater resilience,yet the genomic mechanism(s)underlying hybrid vigor or heterosis remain unclear,hindering our ability to predict the expression of phenotypic traits in...Hybrid crops often exhibit increased yield and greater resilience,yet the genomic mechanism(s)underlying hybrid vigor or heterosis remain unclear,hindering our ability to predict the expression of phenotypic traits in hybrid breeding.Here,we generated haplotype-resolved T2T genome assemblies of two pear hybrid varieties,‘Yuluxiang’(YLX)and‘Hongxiangsu’(HXS),which share the same maternal parent but differ in their paternal parents.We then used these assemblies to explore the genome-scale landscape of allele-specific expression(ASE)and create a pangenome graph for pear.ASE was observed for close to 6000 genes in both hybrid cultivars.A subset of ASE genes related to aspects of fruit quality such as sugars,organic acids,and cuticular wax were identified,suggesting their important contributions to heterosis.Specifically,Ma1,a gene regulating fruit acidity,is absent in the paternal haplotypes of HXS and YLX.A pangenome graph was built based on our assemblies and seven published pear genomes.Resequencing data for 139 cultivated pear genotypes(including 97 genotypes sequenced here)were subsequently aligned to the pangenome graph,revealing numerous structural variant hotspots and selective sweeps during pear diversification.As predicted,the Ma1 allele was found to be absent in varieties with low organic acid content,and this association was functionally validated by Ma1 overexpression in pear fruit and calli.Overall,these results reveal the contributions of ASE to fruit-quality heterosis and provide a robust pangenome reference for high-resolution allele discovery and association mapping.展开更多
Despite the scientific and medicinal importance of diploid sika deer(Cervus nippon),its genome resources are limited and haplotype-resolved chromosome-scale assembly is urgently needed.To explore mechanisms underlying...Despite the scientific and medicinal importance of diploid sika deer(Cervus nippon),its genome resources are limited and haplotype-resolved chromosome-scale assembly is urgently needed.To explore mechanisms underlying the expression patterns of the allele-specific genes in antlers and the chromosome evolution in Cervidae,we report,for the first time,a high-quality haplotype-resolved chromosome-scale genome of sika deer by integrating multiple sequencing strategies,which was anchored to 32 homologous groups with a pair of sex chromosomes(XY).Several expanded genes(RET,PPP2R1A,PPP2R1B,YWHAB,YWHAZ,and RPS6)and positively selected genes(eIF4E,Wnt8A,Wnt9B,BMP4,and TP53)were identified,which could contribute to rapid antler growth without carcinogenesis.A comprehensive and systematic genome-wide analysis of allele expression patterns revealed that most alleles were functionally equivalent in regulating rapid antler growth and inhibiting oncogenesis.Comparative genomic analysis revealed that chromosome fission might occur during the divergence of sika deer and red deer(Cervus elaphus),and the olfactory sensation of sika deer might be more powerful than that of red deer.Obvious inversion regions containing olfactory receptor genes were also identified,which arose since the divergence.In conclusion,the high-quality allele-aware reference genome provides valuable resources for further illustration of the unique biological characteristics of antler,chromosome evolution,and multi-omics research of cervid animals.展开更多
Translational regulation is a critical step in the process of gene expression and governs the synthesis of proteins from mRNAs.Many studies have revealed translational regulation in plants in response to various envir...Translational regulation is a critical step in the process of gene expression and governs the synthesis of proteins from mRNAs.Many studies have revealed translational regulation in plants in response to various environmental stimuli.However,there have been no studies documenting the comprehensive landscape of translational regulation and allele-specific translational efficiency in multiple plant tissues,especially those of rice,a main staple crop that feeds nearly half of the world’s population.Here we used RNA sequencing and ribosome profiling data to analyze the transcriptome and translatome of an elite hybrid rice,Shanyou 63(SY63),and its parental varieties Zhenshan 97 and Minghui 63.The results revealed that gene expression patterns varied more among tissues than among varieties at the transcriptional and translational levels.We identified 3392 upstream open reading frames(uORFs),and the uORF-containing genes were enriched in transcription factors.Only 668 of 13492 long non-coding RNAs could be translated into peptides.Finally,we discovered numerous genes with allele-specific translational efficiency in SY63 and demonstrated that some cis-regulatory elements may contribute to allelic divergence in translational efficiency.Overall,these findings may improve our understanding of translational regulation in rice and provide information for molecular breeding research.展开更多
Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequenci...Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30%. The aim of this study was to evaluate allele-specific real-time PCR （ASPCR） to detect the resistance-related mutations located at positions 103, 184 and 215.Methods We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140clinical samples using this method.Results The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M1841, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Yemerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.展开更多
Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies hav...Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction （PCR） with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally. Methods In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results. Results Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment. Conclusions The drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.展开更多
Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions(DMRs),and aberrant genomic imprinted DNA methylation is associated with some human diseases,includ...Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions(DMRs),and aberrant genomic imprinted DNA methylation is associated with some human diseases,including Prader-Willi syndrome and cancer.Thus,the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases.To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system,which is an efficient genetargeting technique in various organisms,we examined the targeting efficiency of Staphylococcus aureus Cas9(SaCas9)and Streptococcus pyogenes Cas9(SpCas9)in response to DNA methylation interference.We found that the targeting efficiency of SaCas9,but not SpCas9,was enhanced by targeted DNA demethylation using the d Cas9-Tet1 catalytic domain(CD)but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-d Cas9.An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine(5mC)in a synthesized Cp G-containing context.Further analysis with Ch IP-Q-PCR demonstrated that the non-methylated sequence targeting of Sa Cas9 depends on the binding preference of SaCas9 to non-methylated sequences.Taking advantage of this feature of SaCas9,we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes,H19 and Snrpn,with relatively high efficiencies of 28.6%and 47.4%,respectively.These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation.By using SaCas9,we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.展开更多
RNA/DNA primer pairs and two polymerases were used to efficiently amplify DNA sequences using the conventional polymerase chain reaction(PCR).The reaction required the use of both DNA polymerase and reverse transcript...RNA/DNA primer pairs and two polymerases were used to efficiently amplify DNA sequences using the conventional polymerase chain reaction(PCR).The reaction required the use of both DNA polymerase and reverse transcriptase during each thermal cycle and formed a double-stranded DNA in which one terminus was an RNA/DNA hybrid.Because there is a higher sensitivity of the DNA polymerase to the mismatch at the 3?-end in the RNA/DNA hybrid duplex than in the DNA/DNA duplex,the RNA-primed PCR reveals much better specificity in the allele-specific PCR to detect single-nucleotide mutation.展开更多
Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania specie...Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.Methods:A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.Results:This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.Conclusions:Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.展开更多
Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress.Several tumor types,including non-small cell lung cancer(NSCLC),upregulate the system x-c cystine/glutamate antiporter(xCT)through overexp...Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress.Several tumor types,including non-small cell lung cancer(NSCLC),upregulate the system x-c cystine/glutamate antiporter(xCT)through overexpression of the cystine transporter SLC7A11,thus sustaining intracellular cysteine levels to support glutathione synthesis.Nuclear factor erythroid 2-related factor 2(NRF2)serves as a master regulator of oxidative stress resistance by regulating SLC7A11,whereas Kelch-like ECH-associated protein(KEAP1)acts as a cytoplasmic repressor of the oxidative responsive transcription factor NRF2.Mutations in KEAP1/NRF2 and p53 induce SLC7A11 activation in NSCLC.Extracellular cystine is crucial in supplying the intracellular cysteine levels necessary to combat oxidative stress.Disruptions in cystine availability lead to iron-dependent lipid peroxidation,thus resulting in a type of cell death called ferroptosis.Pharmacologic inhibitors of xCT(either SLC7A11 or GPX4)induce ferroptosis of NSCLC cells and other tumor types.When cystine uptake is impaired,the intracellular cysteine pool can be sustained by the transsulfuration pathway,which is catalyzed by cystathionine-B-synthase(CBS)and cystathionine g-lyase(CSE).The involvement of exogenous cysteine/cystine and the transsulfuration pathway in the cysteine pool and downstream metabolites results in compromised CD8^(+)T cell function and evasion of immunotherapy,diminishing immune response and potentially reducing the effectiveness of immunotherapeutic interventions.Pyroptosis is a previously unrecognized form of regulated cell death.In NSCLCs driven by EGFR,ALK,or KRAS,selective inhibitors induce pyroptotic cell death as well as apoptosis.After targeted therapy,the mitochondrial intrinsic apoptotic pathway is activated,thus leading to the cleavage and activation of caspase-3.Consequently,gasdermin E is activated,thus leading to permeabilization of the cytoplasmic membrane and cell-lytic pyroptosis(indicated by characteristic cell membrane ballooning).Breakthroughs in KRAS G12C allele-specific inhibitors and potential mechanisms of resistance are also discussed herein.展开更多
High-density genetic markers are required for genotyping and linkage mapping in identifying genes from crops with complex genomes, such as barley. As the most common variation, single nucleotide polymorphisms(SNPs) ar...High-density genetic markers are required for genotyping and linkage mapping in identifying genes from crops with complex genomes, such as barley. As the most common variation, single nucleotide polymorphisms(SNPs) are suitable for accurate genotyping by using the next-generation sequencing(NGS) technology. Reduced representation libraries(RRLs) of five barley accessions and one mutant were sequenced using NGS technology for SNP discovery. Twenty million short reads were generated and the proportion of repetitive sequences was reduced by more than 56%. A total of 6061 SNPs were identified, and 451 were mapped to the draft sequence of the barley genome with pairing reads. Eleven SNPs were validated using length polymorphic allele-specific PCR markers.展开更多
Embryo abortion stage and rescue system of hybrids were studied in the distant hybridizationbetween plum and apricot. Identification of the hybrids was also made. The resultsshowed: (1) Embryo abortion started from th...Embryo abortion stage and rescue system of hybrids were studied in the distant hybridizationbetween plum and apricot. Identification of the hybrids was also made. The resultsshowed: (1) Embryo abortion started from three weeks after pollination. (2) The germinationand growth of embryos were different at different growth stages, which could germinateand grow with PF value>0.5, but failed with PF value<0.5. In embryo rescue system ofhybrids, the best germination and differentiation medium was MS+6-BA 2mgL-1+IAA 0.3mgL-1,the rate of germination and differentiation reached up to 80%, bud induction andmultiplication medium was MS+6-BA 1.5mgL-1+IAA 0.3mgL-1, rooting medium was 1/2 MS+IAA0.8mgL-1. Some hybrids were transplanted into the field successfully. (3) Leaf shapeinvestigation and identification by S allele-specific PCR and RAPDs showed that thehybrids were true ones.展开更多
Recent years have witnessed enormous progress in our understanding of the genetic predisposition to colorectal cancer (CRC). Estimates suggest that all or most genetic susceptibility mechanisms proposed so far, rangin...Recent years have witnessed enormous progress in our understanding of the genetic predisposition to colorectal cancer (CRC). Estimates suggest that all or most genetic susceptibility mechanisms proposed so far, ranging from high-penetrance genes to low-risk alleles, account for about 60% of the population-attributable fraction of CRC predisposition. In this context, there is increasing interest in the gene encoding the transforming growth factor β receptor 1 (TGFBR1 ); first when over a decade ago a common polymorphism in exon 1 (rs11466445, TGFBR1 *6A/9A) was suggested to be a risk allele for CRC, then when linkage studies identified the chromosomal region where the gene is located as susceptibility locus for familial CRC, and more recently when the allele-specific expression (ASE) of the gene was proposed as a risk factor for CRC. Published data on the association of TGFBR1 with CRC, regarding polymorphisms and ASE and including sporadic and familial forms of the disease, are often contradictory. This review gives a general overview of the most relevant studies in order to clarify the role of TGFBR1 in the field of CRC genetic susceptibility.展开更多
Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mt...Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mtDNA 10400 locus to design unrnodifled and 3 ＇ phosphorothioate-modified allele-specific primers for PCR, which was performed using polymerases with and without 3 ＇ exonuclease activities. The effects of these primers on primer-extension were evaluated by agarose gel electrophoresis. Results： The unmodified primers were extended by both exo and exo^＋ polymerase irrespective of whether the primers were matched or mismatched with the templates. However, the 3＇ phosphorothioate-modified primers with a terminal mismatch triggered an ＂off-switch＂ of exo~ polymerase when compared to exopolymerase. Conclusion： The＂ on/off＇switch constituted by the combination of 3 ＇ phosphorothioatemodified primers with exo^＋ polymerase is a cost-effective, high-throughput and reliable method for SNP typing, which will be of enormous application in association studies by single nucleotide polymorphism screening.展开更多
<p align="justify"> <span style="font-family:Verdana;"></span><span style="font-family:Verdana;"></span>Myeloproliferative neoplasms (MPNs) are a group of cl...<p align="justify"> <span style="font-family:Verdana;"></span><span style="font-family:Verdana;"></span>Myeloproliferative neoplasms (MPNs) are a group of clonal haematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. According to WHO classification, the Janus associated kinase 2 (<em>JAK</em>2) V617F mutation is one of the major diagnostic criteria in BCR-ABL1 negative myeloproliferative neoplasms. The aim of this study is to detect the <em>JAK</em>2 (V617F) mutation in patients with myeloproliferative neoplasms to get accurate diagnosis and proper management. A total of 90 clinically diagnosed MPN patients attending to Department of Clinical Haematology, Yangon General Hospital were enrolled in this study. The mean age was 53.4 ± 14 years which ranged from 16 to 81 years old and male and female ratio was 2.4:1. The identification of <em>JAK</em>2 (V617F) point mutation was found to be positive in 44/90 MPN patients (48.9%). According to MPN subtypes, the <em>JAK</em>2 mutation positivity was found in 19 out of 46 polycythemia vera patients (41.3%), 17 out of 25 essential thrombocythemia patients (68%), 8 out of 15 primary myelofibrosis patients (53.3%), 0 of 4 others myeloproliferative neoplasms (0%). Confirmation of each of nine <em>JAK</em>2 mutation positive and negative samples was done by Sanger sequencing. The arterial or venous thrombotic attack was found in 32/44 <em>JAK</em>2 mutation positive cases (72.7%) and 12/44 <em>JAK</em>2 mutation negative cases (27.3%). The association between thrombotic attack and presence of <em>JAK</em>2 mutation was statistically significance with p = 0.000. The diagnosis of myeloproliferative neoplasms mainly relies on the molecular genetics according to WHO classification. The Allele specific PCR reaction is sensitive, simple test and relatively cost-effective. Therefore, the identification of <em>JAK</em>2 (V617F) somatic point mutation by AS-PCR should be implemented as a routine diagnosis procedure for patients with chronic and suspected myeloproliferative neoplasms. </p>展开更多
Hybrid rice(Oryza sativa)generally outperforms its inbred parents in yield and stress tolerance,a phenomenon termed heterosis,but the underlying mechanism is not completely understood.Here,we combined transcriptome,pr...Hybrid rice(Oryza sativa)generally outperforms its inbred parents in yield and stress tolerance,a phenomenon termed heterosis,but the underlying mechanism is not completely understood.Here,we combined transcriptome,proteome,physiological,and heterosis analyses to examine the salt response of super hybrid rice Chaoyou1000(CY1000).In addition to surpassing the mean values for its two parents(mid-parent heterosis),CY1000 exhibited a higher reactive oxygen species scavenging ability than both its parents(over-parent heterosis or heterobeltiosis).Nonadditive expression and allele-specific gene expression assays showed that the glutathione S-transferase gene OsGSTU26 and the amino acid transporter gene OsAAT30 may have major roles in heterosis for salt tolerance,acting in an overdominant fashion in CY1000.Furthermore,we identified OsWRKY72 as a common transcription factor that binds and regulates OsGSTU26 and OsAAT30.The salt-sensitive phenotypes were associated with the OsWRKY72^(paternal)genotype or the OsAAT30^(maternal)genotype in core rice germplasm varieties.OsWRKY72^(paternal)specifically repressed the expression of OsGSTU26 under salt stress,leading to salinity sensitivity,while OsWRKY72^(maternal)specifically repressed OsAAT30,resulting in salinity tolerance.These results suggest that the OsWRKY72-OsAAT30/OsGSTU26 module may play an important role in heterosis for salt tolerance in an overdominant fashion in CY1000 hybrid rice,providing valuable clues to elucidate the mechanism of heterosis for salinity tolerance in hybrid rice.展开更多
Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexp...Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexplored.In this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active candidates.Candidatus hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting scope.Upon optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.Additionally,we identified nine mutations enhancing ChCas12b specificity.More importantly,we demonstrated that both ChCas12b and its high-fidelity variant,ChCas12b-D496A,enabled allelespecific disruption of genes harboring single nucleotide polymorphisms(SNPs).These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.展开更多
基金supported by grants from the National Natural Science Foundation of China(31572381)National Thousand Youth Talents Plan of the International Science and Technology Cooperation Project of China(2013DFA31420)Science and Technology Innovation Capability Promotion Program of the Science and Technology Department of Qinghai Province(2015-ZJ-712)
文摘Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a suitable model to analyze allele-specific expressi on (ASE) and allele-specific alter native splicing (ASS). Analysis of ASE and ASS can uncover the differences in cis-regulatory elements between closely related species, while eliminating interferenee of trans-regulatory elements. Here, we provide a detailed characterization of ASE and ASS from 19 and 10 transcriptome datasets across five tissues from reciprocal-cross hybrids of horsex don key (mule/hi nny) and cattlexyak (dzo), respectively. Results showed that 4.8%-8.7% and 10.8%-16.7% of genes exhibited ASE and ASS, respectively. Notably, IncRNAs and pseudogenes were more likely to show ASE than protein-coding genes. In addition, genes showing ASE and ASS in mule/hinny were found to be involved in the regulation of muscle strength, whereas those of dzo were involved in high-altitude adaptati on. In con clusi on, our study dem on strated that explorati on of genes showing ASE and ASS in hybrids of closely related species is feasible for species evolution research.
基金Financial support received from Department of Biotechnology,Government of India vide grant No.BT/PR-8953/BCE/08/533/2007project sanctioned against grant No.BT/04/NE/2009financial support from Department of Science&Technology,Government of India in the form of a research fellowship under the INSPIRE program
文摘The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.
基金the National Natural Science Foundation of China （Nos. 30600128, 30670507,30470494） the Natural Science Foundation of Guangdong Province （No. 015012）.
文摘A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru（bpy）3 ^2＋（TBR）-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.
文摘We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.
基金funded by the National Key Research and Development Program of China(2022YFF1003100-02)the National Natural Science Foundation of China(32172511)+5 种基金the Jiangsu Agricultural Science and Technology Innovation Fund(CX(22)2025)the Natural Science Foundation of Jiangsu Province(BK20210397)the Seed Industry Promotion Project of Jiangsu(JBGS(2021)022)the Guidance Foundation of the Hainan Institute of Nanjing Agricultural University(NAUSY-MS08)the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,the Jiangsu Provincial Key Research and Development Program(BE2023365)the Earmarked Fund for China Agriculture Research System(CARS-28).This study was supported by the High-Performance Computing Platform of the Bioinformatics Center,Nanjing Agricultural University.
文摘Hybrid crops often exhibit increased yield and greater resilience,yet the genomic mechanism(s)underlying hybrid vigor or heterosis remain unclear,hindering our ability to predict the expression of phenotypic traits in hybrid breeding.Here,we generated haplotype-resolved T2T genome assemblies of two pear hybrid varieties,‘Yuluxiang’(YLX)and‘Hongxiangsu’(HXS),which share the same maternal parent but differ in their paternal parents.We then used these assemblies to explore the genome-scale landscape of allele-specific expression(ASE)and create a pangenome graph for pear.ASE was observed for close to 6000 genes in both hybrid cultivars.A subset of ASE genes related to aspects of fruit quality such as sugars,organic acids,and cuticular wax were identified,suggesting their important contributions to heterosis.Specifically,Ma1,a gene regulating fruit acidity,is absent in the paternal haplotypes of HXS and YLX.A pangenome graph was built based on our assemblies and seven published pear genomes.Resequencing data for 139 cultivated pear genotypes(including 97 genotypes sequenced here)were subsequently aligned to the pangenome graph,revealing numerous structural variant hotspots and selective sweeps during pear diversification.As predicted,the Ma1 allele was found to be absent in varieties with low organic acid content,and this association was functionally validated by Ma1 overexpression in pear fruit and calli.Overall,these results reveal the contributions of ASE to fruit-quality heterosis and provide a robust pangenome reference for high-resolution allele discovery and association mapping.
基金the National Key R&D Program of China(Grant No.2018YFC1706601)the Natural Science Foundation of Heilongjiang Province of China(Grant No.C2017012)。
文摘Despite the scientific and medicinal importance of diploid sika deer(Cervus nippon),its genome resources are limited and haplotype-resolved chromosome-scale assembly is urgently needed.To explore mechanisms underlying the expression patterns of the allele-specific genes in antlers and the chromosome evolution in Cervidae,we report,for the first time,a high-quality haplotype-resolved chromosome-scale genome of sika deer by integrating multiple sequencing strategies,which was anchored to 32 homologous groups with a pair of sex chromosomes(XY).Several expanded genes(RET,PPP2R1A,PPP2R1B,YWHAB,YWHAZ,and RPS6)and positively selected genes(eIF4E,Wnt8A,Wnt9B,BMP4,and TP53)were identified,which could contribute to rapid antler growth without carcinogenesis.A comprehensive and systematic genome-wide analysis of allele expression patterns revealed that most alleles were functionally equivalent in regulating rapid antler growth and inhibiting oncogenesis.Comparative genomic analysis revealed that chromosome fission might occur during the divergence of sika deer and red deer(Cervus elaphus),and the olfactory sensation of sika deer might be more powerful than that of red deer.Obvious inversion regions containing olfactory receptor genes were also identified,which arose since the divergence.In conclusion,the high-quality allele-aware reference genome provides valuable resources for further illustration of the unique biological characteristics of antler,chromosome evolution,and multi-omics research of cervid animals.
基金supported by the National Natural Science Foundation of China(31871269 and 32270712)the Hubei Provincial Natural Science Foundation of China(2019CFA014)a starting research grant for High-level Talents from Guangxi University.
文摘Translational regulation is a critical step in the process of gene expression and governs the synthesis of proteins from mRNAs.Many studies have revealed translational regulation in plants in response to various environmental stimuli.However,there have been no studies documenting the comprehensive landscape of translational regulation and allele-specific translational efficiency in multiple plant tissues,especially those of rice,a main staple crop that feeds nearly half of the world’s population.Here we used RNA sequencing and ribosome profiling data to analyze the transcriptome and translatome of an elite hybrid rice,Shanyou 63(SY63),and its parental varieties Zhenshan 97 and Minghui 63.The results revealed that gene expression patterns varied more among tissues than among varieties at the transcriptional and translational levels.We identified 3392 upstream open reading frames(uORFs),and the uORF-containing genes were enriched in transcription factors.Only 668 of 13492 long non-coding RNAs could be translated into peptides.Finally,we discovered numerous genes with allele-specific translational efficiency in SY63 and demonstrated that some cis-regulatory elements may contribute to allelic divergence in translational efficiency.Overall,these findings may improve our understanding of translational regulation in rice and provide information for molecular breeding research.
基金This work was supported-by a grant from the National Natural Science Foundation of China （No. 30830088 and No. 30800938）.
文摘Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30%. The aim of this study was to evaluate allele-specific real-time PCR （ASPCR） to detect the resistance-related mutations located at positions 103, 184 and 215.Methods We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140clinical samples using this method.Results The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M1841, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Yemerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.
基金Beijing Natural Science Foundation，Beijing City Talent Training Project Fund
文摘Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction （PCR） with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally. Methods In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results. Results Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment. Conclusions The drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.
基金supported by the National Key R&D Program of China(2016YFC0905901,2018YFC1004700)the National Natural Science Foundation of China(31471400)
文摘Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions(DMRs),and aberrant genomic imprinted DNA methylation is associated with some human diseases,including Prader-Willi syndrome and cancer.Thus,the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases.To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system,which is an efficient genetargeting technique in various organisms,we examined the targeting efficiency of Staphylococcus aureus Cas9(SaCas9)and Streptococcus pyogenes Cas9(SpCas9)in response to DNA methylation interference.We found that the targeting efficiency of SaCas9,but not SpCas9,was enhanced by targeted DNA demethylation using the d Cas9-Tet1 catalytic domain(CD)but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-d Cas9.An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine(5mC)in a synthesized Cp G-containing context.Further analysis with Ch IP-Q-PCR demonstrated that the non-methylated sequence targeting of Sa Cas9 depends on the binding preference of SaCas9 to non-methylated sequences.Taking advantage of this feature of SaCas9,we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes,H19 and Snrpn,with relatively high efficiencies of 28.6%and 47.4%,respectively.These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation.By using SaCas9,we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.
基金supported by the Chinese Academy of Sciences(Hundreds of Talents Program)the National Natural Science Foundation of China(21172215 and 21102139)the Innovation Program of the Chinese Academy of Sciences(KSCX2-EW-J-22)
文摘RNA/DNA primer pairs and two polymerases were used to efficiently amplify DNA sequences using the conventional polymerase chain reaction(PCR).The reaction required the use of both DNA polymerase and reverse transcriptase during each thermal cycle and formed a double-stranded DNA in which one terminus was an RNA/DNA hybrid.Because there is a higher sensitivity of the DNA polymerase to the mismatch at the 3?-end in the RNA/DNA hybrid duplex than in the DNA/DNA duplex,the RNA-primed PCR reveals much better specificity in the allele-specific PCR to detect single-nucleotide mutation.
文摘Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.Methods:A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.Results:This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.Conclusions:Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.
基金supported by a Spanish Association Against Cancer(AECC)grant,(grant No.PROYE18012ROSE)support from Julián Santamaría Vali?o to the IOR Foundation。
文摘Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress.Several tumor types,including non-small cell lung cancer(NSCLC),upregulate the system x-c cystine/glutamate antiporter(xCT)through overexpression of the cystine transporter SLC7A11,thus sustaining intracellular cysteine levels to support glutathione synthesis.Nuclear factor erythroid 2-related factor 2(NRF2)serves as a master regulator of oxidative stress resistance by regulating SLC7A11,whereas Kelch-like ECH-associated protein(KEAP1)acts as a cytoplasmic repressor of the oxidative responsive transcription factor NRF2.Mutations in KEAP1/NRF2 and p53 induce SLC7A11 activation in NSCLC.Extracellular cystine is crucial in supplying the intracellular cysteine levels necessary to combat oxidative stress.Disruptions in cystine availability lead to iron-dependent lipid peroxidation,thus resulting in a type of cell death called ferroptosis.Pharmacologic inhibitors of xCT(either SLC7A11 or GPX4)induce ferroptosis of NSCLC cells and other tumor types.When cystine uptake is impaired,the intracellular cysteine pool can be sustained by the transsulfuration pathway,which is catalyzed by cystathionine-B-synthase(CBS)and cystathionine g-lyase(CSE).The involvement of exogenous cysteine/cystine and the transsulfuration pathway in the cysteine pool and downstream metabolites results in compromised CD8^(+)T cell function and evasion of immunotherapy,diminishing immune response and potentially reducing the effectiveness of immunotherapeutic interventions.Pyroptosis is a previously unrecognized form of regulated cell death.In NSCLCs driven by EGFR,ALK,or KRAS,selective inhibitors induce pyroptotic cell death as well as apoptosis.After targeted therapy,the mitochondrial intrinsic apoptotic pathway is activated,thus leading to the cleavage and activation of caspase-3.Consequently,gasdermin E is activated,thus leading to permeabilization of the cytoplasmic membrane and cell-lytic pyroptosis(indicated by characteristic cell membrane ballooning).Breakthroughs in KRAS G12C allele-specific inhibitors and potential mechanisms of resistance are also discussed herein.
基金supported by the National Natural Science Foundation of China (31000711, 31370032)China Agriculture Research System (CARS-05)the Agricultural Science and Technology Innovation Program
文摘High-density genetic markers are required for genotyping and linkage mapping in identifying genes from crops with complex genomes, such as barley. As the most common variation, single nucleotide polymorphisms(SNPs) are suitable for accurate genotyping by using the next-generation sequencing(NGS) technology. Reduced representation libraries(RRLs) of five barley accessions and one mutant were sequenced using NGS technology for SNP discovery. Twenty million short reads were generated and the proportion of repetitive sequences was reduced by more than 56%. A total of 6061 SNPs were identified, and 451 were mapped to the draft sequence of the barley genome with pairing reads. Eleven SNPs were validated using length polymorphic allele-specific PCR markers.
基金supported by the Technological Production Transformation Foundation by the Ministry of Science and Technology of China(2002370010495)the foundation of Shandong Fruit Tree Three-Zero Project
文摘Embryo abortion stage and rescue system of hybrids were studied in the distant hybridizationbetween plum and apricot. Identification of the hybrids was also made. The resultsshowed: (1) Embryo abortion started from three weeks after pollination. (2) The germinationand growth of embryos were different at different growth stages, which could germinateand grow with PF value>0.5, but failed with PF value<0.5. In embryo rescue system ofhybrids, the best germination and differentiation medium was MS+6-BA 2mgL-1+IAA 0.3mgL-1,the rate of germination and differentiation reached up to 80%, bud induction andmultiplication medium was MS+6-BA 1.5mgL-1+IAA 0.3mgL-1, rooting medium was 1/2 MS+IAA0.8mgL-1. Some hybrids were transplanted into the field successfully. (3) Leaf shapeinvestigation and identification by S allele-specific PCR and RAPDs showed that thehybrids were true ones.
基金Supported by The Spanish Ministry of Science and Innovation(Grant BFU2009-10281 and Ramón y Cajal contract)the Scientific Foundation of Asociación Espa ola Contra el Cáncer
文摘Recent years have witnessed enormous progress in our understanding of the genetic predisposition to colorectal cancer (CRC). Estimates suggest that all or most genetic susceptibility mechanisms proposed so far, ranging from high-penetrance genes to low-risk alleles, account for about 60% of the population-attributable fraction of CRC predisposition. In this context, there is increasing interest in the gene encoding the transforming growth factor β receptor 1 (TGFBR1 ); first when over a decade ago a common polymorphism in exon 1 (rs11466445, TGFBR1 *6A/9A) was suggested to be a risk allele for CRC, then when linkage studies identified the chromosomal region where the gene is located as susceptibility locus for familial CRC, and more recently when the allele-specific expression (ASE) of the gene was proposed as a risk factor for CRC. Published data on the association of TGFBR1 with CRC, regarding polymorphisms and ASE and including sporadic and familial forms of the disease, are often contradictory. This review gives a general overview of the most relevant studies in order to clarify the role of TGFBR1 in the field of CRC genetic susceptibility.
基金supported by a grant from the "135" Foundation of Jiangsu Province(RC2002052).
文摘Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mtDNA 10400 locus to design unrnodifled and 3 ＇ phosphorothioate-modified allele-specific primers for PCR, which was performed using polymerases with and without 3 ＇ exonuclease activities. The effects of these primers on primer-extension were evaluated by agarose gel electrophoresis. Results： The unmodified primers were extended by both exo and exo^＋ polymerase irrespective of whether the primers were matched or mismatched with the templates. However, the 3＇ phosphorothioate-modified primers with a terminal mismatch triggered an ＂off-switch＂ of exo~ polymerase when compared to exopolymerase. Conclusion： The＂ on/off＇switch constituted by the combination of 3 ＇ phosphorothioatemodified primers with exo^＋ polymerase is a cost-effective, high-throughput and reliable method for SNP typing, which will be of enormous application in association studies by single nucleotide polymorphism screening.
文摘<p align="justify"> <span style="font-family:Verdana;"></span><span style="font-family:Verdana;"></span>Myeloproliferative neoplasms (MPNs) are a group of clonal haematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. According to WHO classification, the Janus associated kinase 2 (<em>JAK</em>2) V617F mutation is one of the major diagnostic criteria in BCR-ABL1 negative myeloproliferative neoplasms. The aim of this study is to detect the <em>JAK</em>2 (V617F) mutation in patients with myeloproliferative neoplasms to get accurate diagnosis and proper management. A total of 90 clinically diagnosed MPN patients attending to Department of Clinical Haematology, Yangon General Hospital were enrolled in this study. The mean age was 53.4 ± 14 years which ranged from 16 to 81 years old and male and female ratio was 2.4:1. The identification of <em>JAK</em>2 (V617F) point mutation was found to be positive in 44/90 MPN patients (48.9%). According to MPN subtypes, the <em>JAK</em>2 mutation positivity was found in 19 out of 46 polycythemia vera patients (41.3%), 17 out of 25 essential thrombocythemia patients (68%), 8 out of 15 primary myelofibrosis patients (53.3%), 0 of 4 others myeloproliferative neoplasms (0%). Confirmation of each of nine <em>JAK</em>2 mutation positive and negative samples was done by Sanger sequencing. The arterial or venous thrombotic attack was found in 32/44 <em>JAK</em>2 mutation positive cases (72.7%) and 12/44 <em>JAK</em>2 mutation negative cases (27.3%). The association between thrombotic attack and presence of <em>JAK</em>2 mutation was statistically significance with p = 0.000. The diagnosis of myeloproliferative neoplasms mainly relies on the molecular genetics according to WHO classification. The Allele specific PCR reaction is sensitive, simple test and relatively cost-effective. Therefore, the identification of <em>JAK</em>2 (V617F) somatic point mutation by AS-PCR should be implemented as a routine diagnosis procedure for patients with chronic and suspected myeloproliferative neoplasms. </p>
基金supported by grants from the National Natural Science Foundation of China(Grant No.32272050 and U21A20208)the National Center of Technology Innovation for Saline-Alkali Tolerant Rice(2022PT1005)+2 种基金the Hunan Natural Science Foundation(Grant No.2022JJ30021)the Science and Technology Innovation Program of Hunan Province(2023NK1010)the Changsha Science and Technology Project(Grant No.kq2202221)。
文摘Hybrid rice(Oryza sativa)generally outperforms its inbred parents in yield and stress tolerance,a phenomenon termed heterosis,but the underlying mechanism is not completely understood.Here,we combined transcriptome,proteome,physiological,and heterosis analyses to examine the salt response of super hybrid rice Chaoyou1000(CY1000).In addition to surpassing the mean values for its two parents(mid-parent heterosis),CY1000 exhibited a higher reactive oxygen species scavenging ability than both its parents(over-parent heterosis or heterobeltiosis).Nonadditive expression and allele-specific gene expression assays showed that the glutathione S-transferase gene OsGSTU26 and the amino acid transporter gene OsAAT30 may have major roles in heterosis for salt tolerance,acting in an overdominant fashion in CY1000.Furthermore,we identified OsWRKY72 as a common transcription factor that binds and regulates OsGSTU26 and OsAAT30.The salt-sensitive phenotypes were associated with the OsWRKY72^(paternal)genotype or the OsAAT30^(maternal)genotype in core rice germplasm varieties.OsWRKY72^(paternal)specifically repressed the expression of OsGSTU26 under salt stress,leading to salinity sensitivity,while OsWRKY72^(maternal)specifically repressed OsAAT30,resulting in salinity tolerance.These results suggest that the OsWRKY72-OsAAT30/OsGSTU26 module may play an important role in heterosis for salt tolerance in an overdominant fashion in CY1000 hybrid rice,providing valuable clues to elucidate the mechanism of heterosis for salinity tolerance in hybrid rice.
基金supported by the National Key Research and Development Program of China(2021YFC2701103,2021YFA0910602,and 2019YFA0802804)the National Natural Science Foundation of China(82070258 and 31925011)+1 种基金Open Research Fund of State Key Laboratory of Genetic Engineering,Fudan University(SKLGE-2104)Science and Technology Research Program of Shanghai(19DZ2282100)。
文摘Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexplored.In this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active candidates.Candidatus hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting scope.Upon optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.Additionally,we identified nine mutations enhancing ChCas12b specificity.More importantly,we demonstrated that both ChCas12b and its high-fidelity variant,ChCas12b-D496A,enabled allelespecific disruption of genes harboring single nucleotide polymorphisms(SNPs).These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.
