Identification of clade-wide putative cis-regulatory elements from conserved non-coding sequences in Cucurbitaceae genomes

Cis-regulatory elements regulate gene expression and play an essential role in the development and physiology of organisms.Many conserved non-coding sequences(CNSs)function as cis-regulatory elements.They control the development of various lineages.How-ever,predicting clade-wide cis-regulatory elements across several closely related species remains challenging.Based on the relationship between CNSs and cis-regulatory elements,we present a computational approach that predicts the clade-wide putative cis-regulatory elements in 12 Cucurbitaceae genomes.Using 12-way whole-genome alignment,we first obtained 632112 CNSs in Cucurbitaceae.Next,we identified 16552 Cucurbitaceae-wide cis-regulatory elements based on collinearity among all 12 Cucurbitaceae plants.Furthermore,we predicted 3271 potential regulatory pairs in the cucumber genome,of which 98 were verified using integrative RNA sequencing and ChIP sequencing datasets from samples collected during various fruit development stages.The CNSs,Cucurbitaceae-wide cis-regulatory elements,and their target genes are accessible at http://cmb.bnu.edu.cn/cisRCNEs_cucurbit/.These elements are valuable resources for functionally annotating CNSs and their regulatory roles in Cucurbitaceae genomes.

Infinite Sequence of Conservation Laws and Analytic Solutions for a Generalized Variable-Coefficient Fifth-Order Korteweg-de Vries Equation in Fluids

在这份报纸，为在液体的一个概括可变系数的第五顺序的 Korteweg-de Vries 方程的能量守恒定律的一个无限的序列基于 B 被构造 ? cklund 转变。Hirota 双线性的形式和符号的计算被使用获得三种答案。可变系数能影响典型的线的保存密度，联系流动，和外观。soliton 结构上的波浪数字的效果也被讨论并且 soliton 结构打字，例如，双 periodic soliton，平行 soliton 和 soliton 建筑群，被介绍。

Roles of flanking sequences in the binding between unimolecular parallel-stranded G-quadruplexes and ligands

G-quadruplexes attract more and more attention in recent years.Numerous small molecules which can induce or stabilize the formation of G-quadruplexes have been investigated on the purpose of anticancer drug development.As a motif existed in physiological condition,flanking sequences are an important part of G-quadruplexes but the study on the impact of flanking sequences on (G-quadruplex)-ligand binding is rarely reported.In this paper,the effects of flanking sequences on binding affinity between a series of unimolecular parallel-stranded G-quadruplex sequences derived from c-myc oncogene promoter (termed as c-myc G-quadruplexes) and their ligands are discussed in detail.The results showed that the flanking sequences on c-myc G-quadruplexes play key roles in (G-quadruplex)-ligand interaction.When a c-myc G-quadruplex is bound to its ligands,the flanking sequences might form a binding cavity above the terminal G-quartet,which could provide a suitable site for ligands to dock in.Moreover,the bases on flanking sequences could interact with ligand through π-π stacking,and finally form a sandwich-stacking mode (terminal G-quartet,ligand and bases on the flanking sequence).This mode could stabilize the (G-quadruplex)-ligand complex effectively and enhance the binding affinity dramatically.However,flanking sequences are also found to exhibit steric hindrance effect which could impede the (G-quadruplex)-ligand binding.

Comparative Analysis of Mitochondrial Control Region Sequence from Three Flatfish Species(Pleuronectidae)

The 5’-end of the mitochondrial control region sequences of three flatfishes (Pleuronectiformes: Pleuronectidae) were amplified and sequenced. These sequences were compared with those of other three Pleuronectids species retrieved from GenBank. A phylogenetic tree was constructed based on the partial control region sequences. The results of phyloge- netic analysis are consistent with those of conventional systematics. Compared to previous studies, the structure of the 5’-end of mitochondrial control region was analyzed. The terminal associated sequence motif and its complementary motif were i- dentified at the 5’-end of the sequences. A conserved sequence block, named as CM5’d, was identified in the 5’-end of con- trol region sequences in all Pleuronectids. Another central conserved sequence block, named as CSB-F, was detected in the central conserved blocks.

The loss of genetic diversity during captive breeding of the endangered sculpin, Trachidermus fasciatus, based on ISSR markers: implications for its conservation

Inter-simple sequence repeat (ISSR) markers were used to determine the genetic variation and genetic differentiation of cultured and wild populations of Trachidermus fasciatus, an endangered catadromous fish species in China. Six selected primers were used to amplify DNA samples from 85 individuals, and 353 loci were detected. Relatively low genetic diversity was detected in the cultured population (the percentage of polymorphic loci PPL = 73.80%, Nei's gene diversity h = 0.178 2, Shannon information index I = 0.276 9). However, the genetic diversity at the species level was relatively high (PPL = 91.78%; h = 0.258 3, I = 0.398 6). The UPGMA tree grouped together the genotypes almost according to their cultured and wild origin, showing distinct differences in genetic structure between wild and cultured populations. The pairwise Fst values confirmed significant genetic differentiation between wild and cultured samples. The cultivated population seems to be low in genetic diversity as a result of detrimental genetic effects in the captive population. The results suggest that ISSR markers are effective for rapid assessment of the degree of diversity of a population, thus giving important topical information relevant to preserving endangered species.

Novel Microsatellite Markers for Conservation of Australian Native <i>Samadera bidwillii</i>

Microsatellite markers were developed for Samadera bidwillii, a nationally listed vulnerable shrub or small tree, to enable investigation of its genetic structure and diversity within and among populations from its known distribution throughout coastal areas mainly in fragmented and disturbed lands from Mackay to Gympie, Queensland, Australia. The loci were tested for cross-amplification in related Samadera species. Ten polymorphic microsatellite markers were isolated and characterised from an enriched library of S. bidwillii, which exhibited di- and trinucleotide repeats. The mean number of alleles per locus ranged from 1.3 to 2.5 and mean expected and observed heterozygosities ranged from 0.06 to 0.33 and from 0.03 to 0.26, respectively in five populations. All loci successfully amplified in six other closely associated Samadera species also reported from Australia. Developed loci can be used in genetic diversity, population structure and gene flow studies with an emphasis on the conservation of S. bidwillii and related species.

Conservation and variability of hepatitis B core at different chronic hepatitis stages

BACKGROUND Since it is currently not possible to eradicate hepatitis B virus(HBV)infection with existing treatments,research continues to uncover new therapeutic strategies.HBV core protein,encoded by the HBV core gene(HBC),intervenes in both structural and functional processes,and is a key protein in the HBV life cycle.For this reason,both the protein and the gene could be valuable targets for new therapeutic and diagnostic strategies.Moreover,alterations in the protein sequence could serve as potential markers of disease progression.AIM To detect,by next-generation sequencing,HBC hyper-conserved regions that could potentially be prognostic factors and targets for new therapies.METHODS Thirty-eight of 45 patients with chronic HBV initially selected were included and grouped according to liver disease stage[chronic hepatitis B infection without liver damage(CHB,n=16),liver cirrhosis(LC,n=5),and hepatocellular carcinoma(HCC,n=17)].HBV DNA was extracted from patients’plasma.A region between nucleotide(nt)1863 and 2483,which includes HBC,was amplified and analyzed by next-generation sequencing(Illumina Mi Seq platform).Sequences were genotyped by distance-based discriminant analysis.General and intergroup nt and amino acid(aa)conservation was determined by sliding window analysis.The presence of nt insertion and deletions and/or aa substitutions in the different groups was determined by aligning the sequences with genotype-specific consensus sequences.RESULTS Three nt(nt 1900-1929,2249-2284,2364-2398)and 2 aa(aa 117-120,159-167)hyper-conserved regions were shared by all the clinical groups.All groups showed a similar pattern of conservation,except for five nt regions(nt 1946-1992,2060-2095,2145-2175,2230-2250,2270-2293)and one aa region(aa 140-160),where CHB and LC,respectively,were less conserved(P<0.05).Some group-specific conserved regions were also observed at both nt(2306-2334 in CHB and 1935-1976 and 2402-2435 in LC)and aa(between aa 98-103 in CHB and 28-30 and 51-54 in LC)levels.No differences in insertion and deletions frequencies were observed.An aa substitution(P79 Q)was observed in the HCC group with a median(interquartile range)frequency of 15.82(0-78.88)vs 0(0-0)in the other groups(P<0.05 vs CHB group).CONCLUSION The differentially conserved HBC and HBV core protein regions and the P79 Q substitution could be involved in disease progression.The hyper-conserved regions detected could be targets for future therapeutic and diagnostic strategies.

Positional Information Storage in Sequence Patterns

We build a model of storage of well-defined positional information in probabilistic sequence patterns. Once a pattern is defined, it is possible to judge the effect of any mutation in it. We show that the frequency of beneficial mutations can be high in general and the same mutation can be either advantageous or deleterious depending on the pattern’s context. The model allows to treat positional information as a physical quantity, formulate its conservation law and to model its continuous evolution in a whole genome, with meaningful applications of basic physical principles such as optimal efficiency and channel capacity. A plausible example of optimal solution analytically describes phase transitions-like behavior. The model shows that, in principle, it is possible to store error-free information on sequences with arbitrary low conservation. The described theoretical framework allows one to approach from novel general perspectives such long-standing paradoxes as excessive junk DNA in large genomes or the corresponding G- and C-values paradoxes. We also expect it to have an effect on a number of fundamental concepts in population genetics including the neutral theory, cost-of-selection dilemma, error catastrophe and others.

耐盐转基因大豆事件AtARA6-A001外源插入片段侧翼序列及其应用

转基因大豆AtARA6-A001事件采用农杆菌介导大豆遗传转化法获得,其受体为沈农9号,具有耐盐特性,目前已经进入环境释放阶段。为明确该转基因大豆材料的分子特征及其转基因检测方法,推进该转基因事件生物安全评价工作。本研究以转基因大豆AtARA6-A001为研究对象,利用Southern杂交方法及基因组重测序技术鉴定了外源基因的拷贝数及插入位点的位置和方向。同时利用PCR扩增技术获得了外源T-DNA的左右侧翼序列,并基于左右旁侧序列,建立了转AtARA6基因耐盐大豆A001事件的特异性定性PCR检测方法。此方法能特异性检测转基因大豆植株AtARA6-A001根、茎、叶、花和种子样品,并且能够特异性识别转基因大豆事件的身份,这为后续转基因大豆及其产品的检测和监管提供技术支持。

Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single-Molecule Profiling

Recognition of CpG dinucleotide DNA in epigenetic information flow plays a pivotal role for cellular differentiation and development.The TET3 CXXC domain binds to CpG DNA,serving a basic epigenetic information reading mechanism.During the selective recognition of a CpG motif by a CXXC domain from crowded binding sites in a gene sequence,the protein-DNA interactions are beyond CpG dinu-cleotide.However,the selective binding dynamics of CpG within a long DNA context by epigenetic enzymes have been rarely exploit-ed,which is hard for ensemble methods to probe.Here,we used single-molecule magnetic tweezers to quantitatively examine the dynamics of TET3's CXXC domain on a Hoxa9 promoter DNA.Our single-molecule binding profile revealed that CXXC-DNA interactions involve both CpG motifs and their flanking sequences.The residence time of TET3 CXXC differs by about 1000 times in five distin-guished CpG clusters in the context of a CpG island.Moreover,we performed multi-state hidden Markov modeling analysis on the zip-ping/unzipping dynamics of a CpG hairpin,discovering TET3 CXXC's preference on CpG motifs regarding the-2 to+2 flanking bases.Our results shed light on the selective binding dynamics of a CXXC on a gene sequence,facilitating studies on epigenetic information reading mechanisms.

生态脆弱煤矿区水体中微生物群落特征及矿井充水指示

我国生态脆弱矿区水资源整体匮乏,但局部地区矿井涌水却异常巨大。对生态脆弱矿区异常矿井充水水源识别,对矿区生态环境保护意义重大。研究以涌水量超过1 000 m^(3)/h的榆树湾煤矿为背景,在分析矿井涌水量特征的基础上,采集了研究区主要含水层和矿井涌水点的30个水样,开展了水样高通量微生物测序研究。对测序结果采用Alpha多样性分析、Beta多样性分析及微生物构成差异分析,系统研究了矿井水体中微生物群落特征,分析了矿井充水来源。研究结果表明:高通量测试结果通过了Coverage指数检验,验证了其用于矿井充水识别的可行性。但人类活动密切接触的水样显示出微生物更加丰富,需要在取样中有效规避。榆树湾煤矿浅表松散含水层中微生物丰富度和多样性都较高,以新鞘脂菌属(Novosphingobium)、梭杆菌属(Fusobacterium)和硫属(Sulfuricum)等微生物丰度最高;直罗组为代表的基岩含水层微生物丰富度较低,但微生物多样性较高,以丛毛单胞菌属(Comamonas)、蛲虫属(Vermiphilaceae)和巴氏杆菌(Paeniglutamicibacter)等微生物丰度最高。所有水样中最为优势的门类为变形菌门,占比达到35.5%~89.7%。Alpha多样性、PCoA和NSDM分析揭示了榆树湾矿井充水水源主要来自松散含水层和直罗组含水层。但Beta多样性分析发现,随着直罗组静储量的释放,松散层中离石组黄土充水比例进一步增大。综合分析显示,榆树湾煤矿目前为基岩含水层快速释水+离石组含水层持续释水模式的充水模式,矿井涌水统计分析结果验证了这一结论。研究为相似化学成分含水层充水识别,提供了新的方法。

Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x＋35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x＋35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

不同pH处理下宣纸微生物群落的变化及影响

宣纸是我国传统手工纸的重要门类,更是古籍书画文物的重要载体,对中华文化的流传起着重要的作用。然而现存的宣纸文物面临着各种病害的侵袭,微生物侵害和纸张酸化就是其中的主要威胁之一。本研究用一批清末民初的手工纸样品制备混合菌液,通过回植培养的方式研究了pH4.01、pH7.00、pH10.00条件下微生物群落结构动态和对宣纸的侵蚀。结果显示,不同pH处理会显著影响宣纸中细菌的群落组成,随着pH的升高,样本中细菌的群落多样性更加丰富。不同pH下真菌的群落结构则没有明显的聚类关系。同时,环境因子关联分析的结果显示细菌群落结构变化与纸张表面pH相关性高,而真菌群落变化与纸张重量变化相关性高。纤维素降解与细菌和真菌群落的相关性相差不大。

基于全基因组重测序分析中国地方鸡的群体结构和保护优先权

[目的]旨在对中国本土鸡品种群体结构和保护优先权进行分析,促进畜牧业的可持续健康发展。[方法]收集了8个品种、共96个个体的重测序数据。通过主成分PCA分析、构建系统进化树及近交和遗传距离分析来了解群体结构,计算去除每个品种后总体基因和等位基因多样性的增减、模拟计算品种对具有最大基因多样性和最大等位基因多样性合成群体的贡献占比,进而系统评估品种保护优先权。[结果]所研究的品种可分为4个集群,其中藏鸡与其他品种分化最高,其自身分为2个亚群,其他品种则分为南北2个集群。藏鸡对遗传多样性的贡献最大;其次是文昌鸡和瑶鸡,此三者应考虑作为优先保护的品种。[结论]该研究结果将可为地方鸡品种保护与开发利用提供参考。

Population genomic data reveal low genetic diversity,divergence and local adaptation among threatened Reeves's Pheasant(Syrmaticus reevesii)

Population genomic data could provide valuable information for conservation efforts;however,limited studies have been conducted to investigate the genetic status of threatened pheasants.Reeves’s Pheasant(Syrmaticus reevesii)is facing population decline,attributed to increases in habitat loss.There is a knowledge gap in understanding the genomic status and genetic basis underlying the local adaptation of this threatened bird.Here,we used population genomic data to assess population structure,genetic diversity,inbreeding patterns,and genetic divergence.Furthermore,we identified candidate genes linked with adaptation across the current distribution of Reeves’s Pheasant.The present study assembled the first de novo genome sequence of Reeves’s Pheasant and annotated 19,458 genes.We also sequenced 30 individuals from three populations(Dabie Mountain,Shennongjia,Qinling Mountain)and found that there was clear population structure among those populations.By comparing with other threatened species,we found that Reeves’s Pheasants have low genetic diversity.Runs of homozygosity suggest that the Shennongjia population has experienced serious inbreeding.The demographic history results indicated that three populations experienced several declines during the glacial period.Local adaptative analysis among the populations identified 241 candidate genes under directional selection.They are involved in a large variety of processes,including the immune response and pigmentation.Our results suggest that the three populations should be considered as three different conservation units.The current study provides genetic evidence for conserving the threatened Reeves’s Pheasant and provides genomic resources for global biodiversity management.

转录因子MhGL3对薄荷表皮毛发育的影响

【目的】探明调控薄荷表皮毛发育的候选基因,为深入研究薄荷表皮毛发育、地方种质资源的保护与开发利用和新品种选育提供参考。【方法】以本地具少量表皮毛薄荷及突变的具大量表皮毛薄荷为研究材料,采用转录组测序、组装和注释等方法筛选影响薄荷表皮毛发育的候选基因并进行克隆、生物信息及表达分析研究。【结果】从多毛和少毛薄荷中分别获得39853和30249条Unigene基因,N50长度分别为1380和1434 bp,序列平均长度分别为789.44和779.03 bp。克隆测序发现,多毛薄荷转录因子MhGL3序列在920 bp位置缺少碱基A,导致其转录因子MhGL3序列长度延伸至1848 bp,获得HLH保守功能区域,从而调控薄荷表皮毛发育。【结论】转录因子MhGL3突变是导致薄荷表皮毛发育的重要因素。

人WASH468和WASH465蛋白质特性辨析

目的探讨两种较为公认但序列不同的Wiskott-Aldrich综合征蛋白和富含脯氨酸同源物(Wiskott-Aldrich syndrome protein and SCAR homolog,WASH)的差异。方法使用免疫荧光、免疫共沉淀和激光微辐射等实验分析两种WASH在定位模式、与FAM21或Ku蛋白的相互作用、向DNA损伤位点募集速率和蛋白质稳定性方面的差异;比较多种生物中的WASH蛋白质序列和检测多种在生物和医学研究中常用的细胞中的WASH编码序列,分析两种人WASH序列的普遍性和保守性。结果两种WASH展现出类似的内体(endosome)定位模式。WASH468表现出与FAM21更强的相互作用,并且WASH468展现出更强的稳定性。WASH465表现出与Ku蛋白更强的相互作用,并且WASH465展现出向DNA损伤位点更强的募集。多种生物中的WASH序列与人WASH468序列的一致性明显高于WASH465,并且多种在生物和医学研究中常用的细胞中的WASH氨基酸序列均与WASH468一致。结论WASH468和WASH465的生物学特性存在差异,WASH468序列的普遍性和保守性明显高于WASH465,因此WASH468是更保守的人WASH序列。

Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy

AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also analyzed.RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.

Generation and Analysis of Pathogenicity-related Gene Mutants of Colletotrichum gloeosporioides Using a Novel Promoter Trapping System

Agrobactedum tumefac/ens-mediated transformation （ATMT） is an efficient tool for insertional mutagenesis and is used in a wide variety of plants. This paper reports a promoter trapping method to generate mutants in the filamentous fungus, Colletotrichum gloeosporioides, by ATMT insertion of a trapping vector （pCAHPH） that carries a promoterless hygromycin phosphotransferase （hph） gone. Transformants were selected on the media containing 200 ~mL hy^omycin B, and screened for pathogenicity-related gene mdtants. Their pathogenicity-related mutants T-DNA flanking sequences were then cloned and analyzed. Hph genes were amplified from mutant genomic DNA but not from wild-type DNA, indicating that the phenotypic alternations of these mutants were the results of T-DNA inser- tion. T-DNA flanking sequences were obtained using modified themud asymmetric interlaced PCR. Two right-sided flanking sequences were highly homologous to proteins from other species.

Comparative population genetic analyses suggest hybrid origin of Rhododendron pubicostatum,an endangered plant species with extremely small populations endemic to Yunnan,China

Gene flow between sympatric congeneric plants is thought to be very common and may pose serious threats to endangered species.In the present study,we evaluate the genetic diversity and divergence of three sympatric Rhododendron species in Jiaozi Mountain using newly developed microsatellites through the Illumina MiSeq sequencing approach.Genetic diversity of all three Rhododendron species studied was moderate in comparison to genetic parameters previously reported from species of this genus.Interestingly,genetic structure analysis of the three species identified a possible hybrid origin of the threatened Rh.pubicostatum.This sympatry should be considered a unimodal hybrid zone,since Rh.pubicostatum is predominant here.Unimodal hybrid zones are uncommon in Rhododendron,despite the fact that hybridization frequently occurs in the genus.Issues pertaining to the conservation of Rh.pubicostatum resulting from admixture of genetic material from its parental species are discussed.