Genetic Polymorphism of Eighteen Lycium barbarum Resources Based on nrDNA ITS Sequence

[Objective] The study aimed to investigate the genetic polymorphism of eighteen Lycium barbarum resources via nrDNA ITS sequencing. [Method] The genomic DNAs from Lycium barbarum leaves were isolated by modified CTAB method for PCR amplification on the nrDNA ITS region using specifically synthesized primers; the amplified fragments were cloned and sequenced, then the sequencing results were clustered. [Result] nrDNA ITS sequences of the tested eighteen Lycium barbarum were firstly obtained in the present study. For all eighteen tested materials, the variation range of whole ITS region was 559-634 bp, with an average of 612 bp; alignment analyses showed that the whole length of internal transcribed spacer (ITS1+ITS2) was 480 bp, within which there are 194 variation sites (accounting for 40.4%) and 286 conserved sites (accounting for 59.6%). The cluster results showed that the eighteen tested materials could be grouped into three classes. [Conclusion] Analysis of nrDNA ITS sequence may avail to identify the Lycium barbarum germplasm resources.

Isolation of Zizania latifolia Species-specific DNA Sequences and Their Utility in Identification of Z. latifolia DNA Introgressed into Rice

根据两个植物抗病基因N和RPS2中核酸结合位点 (NBS)和富亮氨酸重复区 (LRR)中的保守序列设计了一对特异引物 ,用PCR从具有水稻 (OryzasativaL .)改良所需要的许多优良性状的水稻近缘野生种菰 (Zizanialatifolia(Griseb .)Turcz.exStapf)的基因组DNA中扩增同源片段。PCR产物经克隆后 ,分别以菰和水稻的基因组DNA为探针 ,通过点杂交对所得克隆进行了分析。点杂交结果表明 ,在所分析的 6 0个克隆中有 2个克隆是菰专化的序列 ,即它们与水稻无杂交信号。基因组DNA的Southern杂交进一步证实了这 2个克隆的专化性。为了验证一些可能的“水稻_菰”渐渗杂交系是否确实含有源自供体菰的DNA ,以这 2个克隆为探针 ,与经EcoRⅠ酶切的 5个可能的渐渗杂交系进行了Southern杂交。结果表明 ,这 2个克隆均能检测出其中的一个系含有其同源序列。这一结果为曾经报道的经一种非常规有性杂交方法将菰DNA导入水稻提供了确凿的证据。

Characterization of Two Groups of Low_copy and Specific DNA Sequences Isolated from Chromosome 7B of Common Wheat

Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play a critical role in the stabilization and establishment of nascent plant polyploids as new species. To further study this issue, five wheat chromosome 7B_specific sequences, isolated from common wheat (Triticum aestivum L.) by chromosome microdissection, were characterized. The sequences were studied by genomic Southern hybridizations on a collection of polyploid wheats and their diploid progenitors. Four sequences hybridized to all polyploid species, but at the diploid level to only species closely related to the B_genome of polyploid wheat. This indicates that these sequences originated with the divergence of the diploid species, and was then vertically transmitted to polyploids. One sequence hybridized to all species at both the diploid and polyploid levels, suggesting its elimination after the polyploid wheat formation. The hybridization of this sequence to two synthetic polyploid wheats indicated that sequence elimination is a rapid event and probably related to methylation status of the sequence. Based on the above results, we suggest that selective changes of low_copy sequences occur rapidly after polyploid formation, which may contribute to the differentiation of chromosomes in newly formed allopolyploid wheats.

Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.

Genetic Diversity of Indocalamus Determined by Chloroplast DNA Sequence

[Objective]The aim was to research the relationship and genetic diversity of Indocalamus.[Method]Using 13 samples of Indocalamus and 3 samples of Sasa as materials,the intergenic regions of trnL-trnF gene in chloroplast were amplified by PCR,and sequence analysis and phylogenetic trees construction were carried out.[Result]Using the universal primer,the intergenic regions of trnL-trnF were amplified,the lengths of the segments varied from 1 008 bp to 1 103 bp,of which 940 bp was compared.The dendrogram of trnL-trnF sequences showed that Indocalamus and Sasa were clustered together and they were homologous by 99%.All the samples were divided into five groups,the first group included 12 samples such as Indosalamus pedalis,I.pumilus,I.victorialis,I.longiauritus,I.tessellatus,Sasa sinica,Sasa pygmaea,I.barbatus,I.guangdongensis,I.herklotsii,I.Hirtivaginatus and S.fortunei.I.decorus,I.lacunosus,I.Latifolius and I.Migoi were respectively divided into four groups.[Conclusion]The high homology of all samples showed the low evolution speed and little information sites which suggested that the phylogeny of Indocalamus could not be well resolved by the intergenic region of trnL-trnF.

Discriminant Classification Model of DNA Sequence

[Objective] The research aimed to construct the discriminant classification model of DNA sequence by combining with the biology knowledge and the mathematical method.[Method] According to the polarity nature of side chain radical in the amino acid,the classification information of amino acid which represented the sequence characteristic from the content and array situation of base was extracted from the different sequences that the amino acid content was different.The four-dimension vector was used to represent.Mahalanobis distance and Fisher discriminant methods were used to classify the given sequence.[Result] In the model,the back substitution rates of sample obtained by two kinds of classification methods were both 100%,and the consistent rate of classification was 90%.[Conclusion] In the model,the calculation method was simple,and the accuracy of classification result was higher.It was superior to the discriminant classification model which was only based on the base content.

Sequence Analysis of Mitochondrial DNA D-loop Region in Xinjiang Goose

[Objective] The sequences of mitochondrial DNA D-loop region of Xinjiang Goose with three different colors of plumage were analyzed in order to study the genetic diversity of Xinjiang Goose, as well as the phylogeny and evolution. [Method] Ten geese were selected randomly from the core populations of grey-, mosaic- and white-plumaged Xinjiang Goose respectively with a total number of thirty as experi- mental materials, of which the blood samples were collected from the largest vein under the wing （brachial vein） for DNA extraction. Sequences of mitochondrial DNA D-loop regions were determined using DNA sequencing technology to analyze the polymorphism. In addition, the genetic distances among different populations were estimated through the comparison with the reference sequences. [Resull] The con- tents of A, G, C and T nucleotides in the D-loop region of Xinjiang Goose were 28.85%, 17.05%, 25.38% and 28.72%, respectively. The average haplotype diversity and nucleotide diversity of Xinjiang Goose were 0.583 and 0.056. Xinjiang Goose and Greylag Goose were clustered into the same group. [Conclusion] The results showed that Xinjiang Geese with three different colors of plumage all descend from Greylag Goose （Anser anser）.

p21^(WAF1/CIP1) Gene DNA Sequence Change and Their Relationship with the Phenotype of Human Osteosarcoma

Objective: To investigate the p21WAF1 /CIP1gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. Methods: p21WAF1 /CIP1gene DNA of 36 osteosarcoma spec- imens was examined by using polymerase chain reaction-single strand conformation polymorphism (PCR- SSCP) method. The PCR products were sequenced directly. Results: In p21WAF1 /CIP1 gene exon3 of 36 cases of human osteosarcoma, the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 17 cases with the incidence being 44.4%. In 10 normal blood samples, DNA sequence analysis showed the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 8 cases with the incidence being 80%. Conclusion: The novel location of p21WAF1 /CIP1gene polymorphism of osteosarcoma, but not mutation was de?ned, and this location might provide the meaningful reference for the further research of p21WAF1/CIP1 gene.p2lWAF1/CIP1基因DNA序列分析及其与骨肉瘤表型的关系

新型超声快速处理活检标本保存不同年限对DNA质量的影响

背景:新型超声组织处理技术越来越多地被用来进行分子生物学分析,研究新型超声处理不同存储年限组织DNA的质量,对进一步分子检测的标本质控具有重要意义。目的:探讨新型超声处理活检标本存储不同年限对DNA质量的影响,以期为分子检测探索最佳的标本存储时间。方法:收集40例乳腺穿刺小活检组织,采用超声技术制作石蜡标本,按照存储年限分为4组:<1年组、1-3年组、>3-5年组及>5年组,每组10例,对石蜡标本进行切片,每张切片厚3μm,切片10-15张,提取DNA后通过Nanophotometer N60超微量分光光度计和Qubit 4.0荧光计检测DNA的质量浓度,记录A_(260)/A_(280)比值判定DNA的纯度,利用全自动毛细管电泳核酸分析仪(Qsep 100)检测DNA片段完整性,以评估DNA片段的质量。结果与结论:4组样本A_(260)/A_(280)均值在1.8-2.0之间,达到纯度要求,无明显差异。4组样本的DNA质量浓度(Qubit浓度)均值分别为30.39,14.33,2.52,1.95 ng/μL;DNA的平均N/Q比值分别为6.48,14.18,24.56,29.86;DNA质量数均值分别为5.64,1.76,1.24,0.80;大片段占比均值分别为56.08%,17.72%,12.68%,7.90%。PCR检测内控基因Ct均值分别为15.32,17.09,18.39,21.24。与<1年组相比,其余3组DNA浓度显著降低,N/Q比值显著增加,DNA质量数和大片段占比均值显著降低,Ct值升高,差异有显著性意义(P<0.05)。实验结果表明,对于新型超声处理活检标本,应优先选择存储<1年的样本进行日常分子检测,储存3年内的样本可满足二代测序等检测要求,5年内样本仅可尝试进行PCR等检测,存储超过5年的样本不建议进行后续分子检测。

Pse-in-One 2.0: An Improved Package of Web Servers for Generating Various Modes of Pseudo Components of DNA, RNA, and Protein Sequences

Pse-in-One 2.0 is a package of web-servers evolved from Pse-in-One (Liu, B., Liu, F., Wang, X., Chen, J. Fang, L. & Chou, K.C. Nucleic Acids Research, 2015, 43:W65-W71). In order to make it more flexible and comprehensive as suggested by many users, the updated package has incorporated 23 new pseudo component modes as well as a series of new feature analysis approaches. It is available at http://bioinformatics.hitsz.edu.cn/Pse-in-One2.0/. Moreover, to maximize the convenience of users, provided is also the stand-alone version called “Pse-in-One-Analysis”, by which users can significantly speed up the analysis of massive sequences.

Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone(Haliotis discus hannai)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the mi-crosatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber (1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats (13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (< 20 repeats) were most abundant, accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatellite isolation in other abalone species.

Genome-wide identification of RNA editing in seven porcine tissues by matched DNA and RNA high-throughput sequencing

Background: RNA editing is a co/posttranscriptional modification mechanism that increases the diversity of transcripts, with potential functional consequences. The advent of next-generation sequencing technologies has enabled the identification of RNA edits at unprecedented throughput and resolution. However, our knowledge of RNA editing in swine is still limited.Results: Here, we utilized RES-Scanner to identify RNA editing sites in the brain, subcutaneous fat, heart, liver,muscle, lung and ovary in three 180-day-old Large White gilts based on matched strand-specific RNA sequencing and whole-genome resequencing datasets. In total, we identified 74863 editing sites, and 92.1% of these sites caused adenosine-to-guanosine(A-to-G) conversion. Most A-to-G sites were located in noncoding regions and generally had low editing levels. In total, 151 A-to-G sites were detected in coding regions(CDS), including 94 sites that could lead to nonsynonymous amino acid changes. We provide further evidence supporting a previous observation that pig transcriptomes are highly editable at PRE-1 elements. The number of A-to-G editing sites ranged from 4155(muscle) to 25001(brain) across the seven tissues. The expression levels of the ADAR enzymes could explain some but not all of this variation across tissues. The functional analysis of the genes with tissuespecific editing sites in each tissue revealed that RNA editing might play important roles in tissue function.Specifically, more pathways showed significant enrichment in the fat and liver than in other tissues, while no pathway was enriched in the muscle.Conclusions: This study identified a total of 74863 nonredundant RNA editing sites in seven tissues and revealed the potential importance of RNA editing in tissue function. Our findings largely extend the porcine editome and enhance our understanding of RNA editing in swine.

Molecular Taxonomy of Conogethes punctiferalis and Conogethes pinicolalis(Lepidoptera: Crambidae) Based on Mitochondrial DNA Sequences

Conogethes punctiferalis（Guenée）（Lepidoptera： Crambidae） was originally considered as one species with fruit-feeding type（FFT） and pinaceae-feeding type（PFT）, but it has subsequently been divided into two different species of Conogethes punctiferalis and Conogethes pinicolalis. The relationship between the two species was investigated by phylogenetic reconstruction using maximum-likelihood（ML） parameter estimations. The phylogenetic tree and network were constructed based upon sequence data from concatenation of three genes of mitochondrial cytochrome c oxidase subunits I, II and cytochrome b which were derived from 118 samples of C. punctiferalis and 24 samples of C. pinicolalis. The phylogenetic tree and network showed that conspecific sequences were clustering together despite intraspecific variability. Here we report the results of a combined analysis of mitochondrial DNA sequences from three genes and morphological data representing powerful evidence that C. pinicolalisand C. punctiferalis are significantly different.

Chaos game representation(CGR)-walk model for DNA sequences

Chaos game representation （CGR） is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to determine the coordinates of their positions in a continuous space. This distribution of positions has two features： one is unique, and the other is source sequence that can be recovered from the coordinates so that the distance between positions may serve as a measure of similarity between the corresponding sequences. A CGR-walk model is proposed based on CGR coordinates for the DNA sequences. The CGR coordinates are converted into a time series, and a long-memory ARFIMA （p, d, q） model, where ARFIMA stands for autoregressive fractionally integrated moving average, is introduced into the DNA sequence analysis. This model is applied to simulating real CGR-walk sequence data of ten genomic sequences. Remarkably long-range correlations are uncovered in the data, and the results from these models are reasonably fitted with those from the ARFIMA （p, d, q） model.

A novel color image encryption scheme using fractional-order hyperchaotic system and DNA sequence operations

In this paper, Adomian decomposition method （ADM） with high accuracy and fast convergence is introduced to solve the fractional-order piecewise-linear （PWL） hyperchaotic system. Based on the obtained hyperchaotic sequences, a novel color image encryption algorithm is proposed by employing a hybrid model of bidirectional circular permutation and DNA masking. In this scheme, the pixel positions of image are scrambled by circular permutation, and the pixel values are substituted by DNA sequence operations. In the DNA sequence operations, addition and substraction operations are performed according to traditional addition and subtraction in the binary, and two rounds of addition rules are used to encrypt the pixel values. The simulation results and security analysis show that the hyperchaotic map is suitable for image encryption, and the proposed encryption algorithm has good encryption effect and strong key sensitivity. It can resist brute-force attack, statistical attack, differential attack, known-plaintext, and chosen-plaintext attacks.

Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing

Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.

Phylogeny of Ptychostomum (Bryaceae,Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.

IDENTIFICATION AND SEQUENCE OF A cDNA CLONE CORRESPONDING TO A GENE INVOLVED IN DEVELOPMENT OF UNDARIA PINNATIFIDA

During the induction of gamete-producing gametangia, induced gametophytes werecollected at 4 days intervals (0,4,8, 12 d) and total RNAs were isolated by CsCl gradient ultracentrifu-gation. Some stage-specific expressed mRNAs were identified by differential display of mRNAs from dif-ferent developing stages of the gametophytes. The cDNA of one specific mRNA was verified, cloned andsequenced. This gene was specifically expressed during 4 days of induction, and had partial homologoussequence with tobacco IAA-binding protein gene. It suggests that this cDNA may represent a gene whichis related to the LAA regulating function during the development of the gametophytes.

Variation of microbiological and small molecule metabolite profiles of Nuodeng ham during ripening by high-throughput sequencing and GC-TOF-MS

The internal microbial diversity and small molecular metabolites of Nuodeng ham in different processing years(the first,second and third year sample)were analyzed by high-throughput sequencing technology and gas chromatography-time of flight mass spectrography(GC-TOF-MS)to study the effects of microorganisms and small molecular metabolites on the quality of ham in different processing years.The results showed that the dominant bacteria phyla of Nuodeng ham in different processing years were Proteobacteria and Firmicutes,the dominant fungi phyla were Ascomycota and Basidiomycota,while Staphylococcus and Aspergillus were the dominant bacteria and fungi of Nuodeng ham,respectively.Totally,252 kinds of small molecular metabolites were identified from Nuodeng ham in different processing years,and 12 different metabolites were screened through multivariate statistical analysis.Further metabolic pathway analysis showed that 23 metabolic pathways were related to ham fermentation,of which 8 metabolic pathways had significant effects on ham fermentation(Impact>0.01,P<0.05).The content of L-proline,phenyllactic acid,L-lysine,carnosine,taurine,D-proline,betaine and creatine were significantly positively correlated with the relative abundance of Staphylococcus and Serratia,but negatively correlated with the relative abundance of Halomonas,Aspergillus and Yamadazyma.

Mechanism of Guangdong Shenqu in regulating intestinal flora in mice with food stagnation and internal heat based on 16S rDNA sequencing

Objective:To investigate the effect of Guangdong Shenqu(GSQ)on intestinal flora structure in mice with food stagnation through 16S rDNA sequencing.Methods: Mice were randomly assigned to control,model,GSQ low-dose(GSQL),GSQ medium-dose(GSQM),GSQ high-dose(GSQH),and lacidophilin tablets(LAB)groups,with each group containing 10 mice.A food stagnation and internal heat mouse model was established through intragastric administration of a mixture of beeswax and olive oil(1:15).The control group was administered normal saline,and the model group was administered beeswax and olive oil to maintain a state.The GSQL(2 g/kg),GSQM(4 g/kg),GSQH(8 g/kg),and LAB groups(0.625 g/kg)were administered corresponding drugs for 5 d.After administration,16S rDNA sequencing was performed to assess gut microbiota in mouse fecal samples.Results: The model group exhibited significant intestinal flora changes.Following GSQ administration,the abundance and diversity index of the intestinal flora increased significantly,the number of bacterial species was regulated,andαandβdiversity were improved.GSQ administration increased the abundance of probiotics,including Clostridia,Lachnospirales,and Lactobacillus,whereas the abundance of conditional pathogenic bacteria,such as Allobaculum,Erysipelotrichaceae,and Bacteroides decreased.Functional prediction analysis indicated that the pathogenesis of food stagnation and GSQ intervention were primarily associated with carbohydrate,lipid,and amino acid metabolism,among other metabolic pathways.Conclusion: The digestive mechanism of GSQ may be attributed to its role in restoring diversity and abundance within the intestinal flora,thereby improving the composition and structure of the intestinal flora in mice and subsequently influencing the regulation of metabolic pathways.