Advances in Molecular Regulatory Mechanisms of Fruit Trees under Low Temperature Stress

The most recent research findings on the tolerance of fruit trees to cold stress are reviewed from a molecular perspective,including the perception and transduction of low temperature calcium signaling,CBF-dependent molecular regulatory mechanisms,non-CBF-dependent molecular regulatory mechanisms,and so forth.The objective is to provide a reference basis for further improving the cold resistance of fruit trees and cultivating new varieties of hardy plants.

Fast algorithm for constructing neighbor-joining phylogenetic trees

To improve the performance of Saitou and Nei＇s algorithm （SN） and Studier and Keppler＇s improved algorithm （SK） for constructing neighbor-joining phylogenetic trees and reduce the time complexity of the computation, a fast algorithm is proposed. The proposed algorithm includes three techniques. First, a linear array A[N] is introduced to store the sum of every row of the distance matrix （the same as SK）, which can eliminate many repeated computations. Secondly, the value of A [i] is computed only once at the beginning of the algorithm, and is updated by three elements in the iteration. Thirdly, a very compact formula for the sum of all the branch lengths of operational taxonomic units （OTUs） i and j is designed, and the correctness of the formula is proved. The experimental results show that the proposed algorithm is from tens to hundreds times faster than SN and roughly two times faster than SK when N increases, constructing a tree with 2 000 OTUs in 3 min on a current desktop computer. To earn the time with the cost of the space and reduce the computations in the innermost loop are the basic solutions for algorithms with many loops.

Molecular Characteristics and Phylogenetic Analysis of N Gene of Human Derived Rabies Virus

Objective To investigate the relationship between the molecular characteristics and phylogenetic evolution of rabies N gene.Methods Saliva samples were collected from rabies cases,and RT-PCR was used to amplify the N gene of rabies virus with the specific primers.The amplifying product of RT-PCR was cloned to pUCm-T vector and transformed into E.coli XL1-Blue and then the blue-white selection,PCR screening and gene sequencing were carried out to identify the positive clones.Finally,ExPASy and other bioinformatics softwares were used to analyze and predict the structure and biological characteristics of the N genome.Results The amplification product of RT-PCR was 1 353 bp,the recombinant plasmid pUCm-T/N was constructed,the whole length of the N gene open reading frame was composed of 1 353 nucleotide residues to code 450 amino acids （20 kinds）,the accession number submitted to the Genbank was HM756692,its sequence homology of nucleotides and amino acids compared with the vaccine strain CTN-1-V was 90% and 99% respectively.The evolutionary analysis showed that the isolated strain belonged to genotype I with certain geographic regionality.Conclusion The characteristics investigation and bioinformatics analysis of Hunan0806 N gene will provide fundament data to reveal the significance of the N gene characteristics for rabies epidemiology and its prevention control.

Characterization of Avian Influenza A(H7N9) Virus Prevalence in Humans and Poultry in Huai′an,China：Molecular Epidemiology,Phylogenetic,and Dynamics Analyses

Objective To trace the source of human H7N9 cases in Huai＇an and elucidate the genetic characterization of Huai＇an strains associated with both humans and birds in live poultry market.Methods An enhanced surveillance was implemented when the first human H7N9 case was confirmed in Huai＇an.Clinical specimens,cloacal swabs,and fecal samples were collected and screened by real-time reverse transcription-polymerase chain reaction（RT-PCR） for H7N9 virus.The positive samples were subjected to further RT-PCR and genome sequencing.The phylodynamic patterns of H7N9 virus within and separated from Huai＇an and evolutionary dynamics of the virus were analyzed.Results Six patients with H7N9 infection were previously exposed to live poultry market and presented symptoms such as fever（〉38.0 °C） and headaches.Results of this study support the hypothesis that live poultry markets were the source of human H7N9 exposure.Phylogenetic analysis revealed that all novel H7N9 viruses,including Huai＇an strains,could be classified into two distinct clades,A and B.Additionally,the diversified H7N9 virus circulated in live poultry markets in Huai＇an.Interestingly,the common ancestors of the Huai＇an H7N9 virus existed in January 2012.The mean nucleotide substitution rates for each gene segment of the H7N9 virus were（3.09-7.26）×10-3 substitutions/site per year（95% HPD：1.72×10-3 to 1.16×10-2）.Conclusion Overall,the source of exposure of human H7N9 cases in Huai＇an was live poultry market,and our study highlights the presence of divergent genetic lineage of H7N9 virus in both humans and poultry specimens in Huai＇an.

Phylogenetics and Molecular Divergence of Tilapia Fish (Oreochromis Species) Using Mitochondrial D-Loop and Cytochrome b Regions

Understanding the level of genetic diversity in any population is an important requisite towards strategizing measures for conservation and improvement of stocks. This study focused on the assessment of phylogenetics and molecular divergence of tilapia fish species obtained from two populations (Domita in South-South and Odeda in South-West, Nigeria) using the displacement loop (D-loop) and cytochrome b region of the mitochondrial deoxyribonucleic acid (mtDNA). A total of 28 samples (15 from South-South and 13 from South-West) were used for the genetic analysis. DNA was extracted from the tissue of all the samples using Quik-gDNATM miniPrep kit. The D-loop containing the hypervariable region was sequenced for all samples from the two populations, while cytochrome b (Cyt b) region of mtDNA was only sequenced for samples from South-South population. Chromatograms of the sequences were viewed and edited using Bioedit software. Multiple sequence alignment was carried out using molecular evolutionary genetic analysis (MEGA) software before subsequent genetic analyses. Phylogenetic analysis grouped the samples into two clusters based on population. Also, when the two mitochondrial regions were pooled together, they clustered into two major groups based on mitochondrial regions. Analysis of molecular variance (AMOVA) revealed 37.32% variation within population and 62.68% variation among population with a significant fixation index of 0.627 (p 0.05). The genetic distance inferred between D-loop regions of South-South and South-West populations was 0.243. Maternal lineage analysis revealed that the origin of tilapia fish from both populations could be traced to Oreochromis spirilus and Oreochromis leucostictus based on mitochondrial D-loop region. The findings of this study revealed molecular divergence among the tilapia populations and may serve as pivot information for the genetic improvement of this important species.

Molecular Cloning and Phylogenetic Analysis of a Chitin Deacetylase Isolated from the Epidermis of the Red Snow Crab <i>Chionoecetes japonicas</i>

Chitin deacetylase (CDA;EC 3. 5. 1. 41) catalyzes the deacetylation of chitin. In this study, we successfully cloned and sequenced a chitin deacetylase gene from the red snow crab Chionoecetes japonicas. By using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends, we obtained a 2141-bp amplicon containing a chitin deacetylase gene (CjCDA) from the epidermis of C. japonicas. The amplicon contains a 1575-bp open reading frame that is predicted to encode a 525-amino acid protein. The structure predicted from the deduced amino acid sequence included an N-terminal signal peptide, chitin-binding domain (CBD), low-density lipoprotein receptor class A domain (LDL-A), and catalytic domain. Comparative analysis of the deduced amino acid sequence of CjCDA revealed the highest homology (74%) to gastrolith protein 59 of Cherax quadricarinatus. We used RT-PCR to evaluate the expression of CjCDA in various tissues of C. japonicas, and we observed that CjCDA was expressed only in the epidermis. A phylogenetic analysis, using the amino acid sequences of CjCDA and other known chitin deacetylases, showed that CjCDA belonged to a group of crustacean chitin deacetylases. To our knowledge, this is the first study reporting the cDNA cloning of a chitin deacetylase from a crab.

Molecular Phylogeny of the “True Citrus Fruit Trees” Group (Aurantioideae, Rutaceae) as Inferred from Chloroplast DNA Sequence

The genus Citrus L. has a long controversial taxonomy history, and a well-resolved molecular phylogeny of the ＂true citrus fruit trees＂ group in the future will provide new information for advancing breeding techniques and developing better conservation strategies. In the present study, three cpDNA fragments （TrnL-TrnF, PsbH-PetB, and TrnS-TrnG） of 30 genotypes chosen from the six genera of the ＂true citrus fruit trees＂ group were analyzed. A molecular phylogenetic tree of the ＂true citrus fruit trees＂ group ＂~as reconstructed based on plastid DNA sequences. The results confirmed that the ＂true citrus fruit trees＂ group was monophyletic, and thereby the group was divided into genera as previously suggested based on morphological characters. The cpDNA data also suggested that Poncirus might be the first genus separated from the other five genera in the group. The genus Fortunella were of hybrid origin and Citrus might be as its putative paternal parent. The genera Microcitrus, Eremocitrus, and Clymenia were possibly monophyletic and their common ancestor might branch out from Citrus. Furthermore, the phylogenetic relationships within the Citrus genus were discussed.

Prevalence of cercarial infections in freshwater snails and morphological and molecular identification and phylogenetic trends of trematodes

Objective:To investigate the prevalence of cercarial infections in freshwater snails from several water sources in Nakhon Nayok,Nonthaburi,and Pathum Thani provinces of Central Thailand,and to reconstruct a phylogenetic tree for improved understanding of the relationships in the cercarial stage.Methods:The snail specimens were collected from 34 total sampling sites and investigated for cercarial infections using the crushing method.The cercarial specimens were classified and used for the phylogenetic tree analysis using the Internal Transcribed Spacer 2(ITS2).Results:A total of 1921 snail specimens were classified into five families and seven species.The results showed that four snail species were identified as intermediate hosts of the larval stages of trematodes,with an overall prevalence of infection of 2.45%(47/1921).The infected snail specimens included five groups of the cercarial type:cercariaeum cercariae,echinostome cercaria,megalurous cercaria,parapleurolophocercous cercaria,and xiphidiocercariae.This is particularly true of xiphidiocercariae,which was found to be the dominant type among cercarial infections in bithyniid snails by approximately 38.00%.With regard to molecular identification,the phylogenetic tree was reconstructed using the neighbor-joining method with 10000 bootstraps and separated the trematodes into three clades:Echinostomatoidea,Microphalloidea and Opisthorchioidea.Conclusions:The study reveals a high prevalence of cercarial infection for each cercarial type and maturation into a definite trematode genus and delineates morphological characteristics and evolutionary trends among each larval trematode in Nakhon Nayok,Nonthaburi and Pathum Thani provinces.In addition,the ITS2 sequence data of cercariae could be used to examine classification of these species at the family level.

A simple way to visualize detailed phylogenetic tree of huge genome-wide SNP data constructed by SNPhylo

Phylogenetic trees based on genome-wide single nucleotide polymorphisms （SNPs） among diverse inbreds could provide valuable and intuitive information for breeding and germplasm management in crops. As a result of sequencing technology developments, a huge amount of whole genome SNP data have become available and affordable for breeders. However, it is a challenge to perform quick and reliable plotting based on the huge amount of SNP data. To meet this goal, a visualization pipeline was developed and demonstrated based on publicly available SNP data from the current important maize inbred lines, including temperate, tropical, sweetcorn, and popcorn. The detailed phylogenetic tree plotted by our pipeline revealed the authentic genetic diversity of these inbreds, which was consistent with several previous reports and indicated that this straightforward pipeline is reliable and could potentially speed up advances in crop breeding.

Molecular phylogenetic analysis of sulfate-reducing bacteria from deep sediment layers of the tropical West Pacific warm pool

The diversity of sulfate-reducing bacteria （SRB） from deep layers of deep-sea sediments [ more than 2 m bsf （below seafloor） ] of two sites （WO1 -3 and WPO1 -4） in a tropical West Pacific warm pool region was characterized by using molecular phylogenetic analysis. The results of culture-independent samples demonstrated that the dominant clones from both sites were related to Grampositive spore forming genus, Desulfotomaculum, which accounted for 36.8% of all the sequencing clones from Site WP01 - 3 and 62.8% from Site WP01 -4. However, the other SRB group which was generally reported to be predominant in the deep-sea sediments of other regions, δ- subclass of the proteobacteria was found to be in very low percentages. Therefore, it could be speculated that there existed a unique chemical environment in the deep-sea sediment of this warm pool region. When comparing the Desulfotomaculum sp. related sequences from both sites, it was revealed that though the Desulfotomaculum-like sequences from Site WP01 -3 were more diverse than those from Site WP01 -4, all these sequences from both sites showed high similarity and formed a new phylogenetically homogeneous cluster in the Desulfotomaculum genus which had never been reported before. Successful enrichment of SRB was only achieved from samples of Site WP01 -4 and the sequence analysis of culture-dependent samples further confirmed the dominance of Desulfotomaculum genus. But Desulfotomaculum-related sequences from culture-dependent and culture-independent samples belonged to two different clusters respectively. This difference showed the choice of cultivation to the microorganisms.

Predicting the Underlying Structure for Phylogenetic Trees Using Neural Networks and Logistic Regression

Understanding an underlying structure for phylogenetic trees is very important as it informs on the methods that should be employed during phylogenetic inference. The methods used under a structured population differ from those needed when a population is not structured. In this paper, we compared two supervised machine learning techniques, that is artificial neural network (ANN) and logistic regression models for prediction of an underlying structure for phylogenetic trees. We carried out parameter tuning for the models to identify optimal models. We then performed 10-fold cross-validation on the optimal models for both logistic regression?and ANN. We also performed a non-supervised technique called clustering to identify the number of clusters that could be identified from simulated phylogenetic trees. The trees were from?both structured?and non-structured populations. Clustering and prediction using classification techniques were?done using tree statistics such as Colless, Sackin and cophenetic indices, among others. Results from 10-fold cross-validation revealed that both logistic regression and ANN models had comparable results, with both models having average accuracy rates of over 0.75. Most of the clustering indices used resulted in 2 or 3 as the optimal number of clusters.

Phylogenetic Relationship and Molecular Divergence Dating Using SRY Gene Polymorphism about Four Ladoum Sheep Lineages in Senegal

Animal genetic resources are playing a vital role in livestock production and are essential to food security. The present study aims to contribute to a better understanding genetic local sheep breeds and to elucidate the phylogenetic relationships through the evolution of the SRY gene in four different lineages of Ladoum sheep raised in Senegal. After a brief analysis of genetic diversity, the phylogenetic relationships and molecular dating were inferred through haplotype networks and four phylogenetic reconstruction methods. The different haplotype networks are constructed with NETWORK ver. 5.0.0.0 using the Median-Joining method. Phylogenetic trees were reconstructed using neighbor-joining, maximum parsimony, maximum likelihood and Bayesian inference. The robustness of the nodes in phylogenetic trees of the three first methods was assessed by 1000 bootstraps. For Bayesian inference, the posterior probability distribution of the trees was estimated by 4 MCMC chains. 5,000,000 generations were performed for each of the chains by sampling the different parameters every 1000 generations. Results show a low polymorphism. Haplotypic diversity is much higher than the average nucleotide divergence between all pairs of haplotypes. The majority and central haplotype indicates a close relationship between “Batling” and “Tyson” individuals. “Birahim” lineage is very distinct from the rest. Phylogenetic trees confirm two genetically separate clades between “Birahim” and the other lineages. The period of divergence between “Birahim” lineage versus the common ancestor of the other three lineages was 2504 years ago. The polyphyly revealed in “Birahim” lindicates that this lineage does not contain the common ancestor of all individuals who compose it. It could therefore be derived from two or more sheep breeds with a common ancestor, Ovis aries. The monophyletic clade appears to be a group including a common ancestor and all of its genetic descendants. This group, bringing together the other three lineages, is in the process of being structured into sub-lineages. This study is the first to show that there are only two genetic lines within ladoum sheep in Senegal.

Isolation, Molecular and Phylogenetic Analysis of Porcine Encephalomyocarditis Virus Strain HLJ in China

Encephalomyocarditis virus(EMCV)is a positive single-stranded small RNA virus without envelope,which can infect a variety of mammals.Swines are the most susceptible animals,which can cause acute myocarditis and respiratory failure in piglets and reproductive failure in pregnant sows.Diseases caused by EMCV have a wide range of effects on the global swine industry.In this study,a strain of EMCV was isolated from a swine aborted fetus in northeast China.It was identified by reverse transcriptase polymerase chain reaction(RT-PCR),electron microscopic observation and indirect immunofluorescence assay.The subsequent results showed that the virus titer of HLJ strain grew to 8.3 lgTCID50 on baby hamster kidney 21(BHK-21)cells.And HLJ strain caused the specific cytopathic effect(CPE)on BHK-21 cells and severe pathological changes in mice.Complete genome sequencing and multiple sequence alignment showed that the homology between HLJ strain and other isolates worldwide was 71.5%-99.7%.Phylogenetic analysis showed that EMCV isolates fell into five clusters:lineageⅠ,Ⅱ,Ⅲ,ⅣandⅤ,based on the nucleotide sequences of the entire open reading frame(ORF)and VP1 gene.HLJ isolate was grouped into lineage I.The analyses of amino acid mutation sites of VP1 protein showed that the amino acids at positions 20 and 54 in VP1 junction were unique to HLJ strain.The isolation of HLJ strain enriched the epidemiological database of EMCV.

Using DNA Sequences and Phylogenetic Trees as Tools for Teaching Entomology to Undergraduate Students: A Simple Approach

This technical note aims to show how any instructor teaching entomology can use the Basic Local Alignment Search Tool (BLAST) and the “one click” mode of Phylogeny.fr to teach undergraduate students about insect DNA similarity in a simple way. Teaching an entomology course requires the use of numerous tools to help students grasp different concepts. Knowing that there are more than one million described species of insects means that teaching students about insect identification and taxonomy can be challenging. However, here we present two easy exercises that could be used as classroom or take-home assignments to demonstrate various levels of DNA similarity among different insect taxa. Such exercises unlock students’ creativity and break the barrier of fear of bioinformatics. Moreover, they open up new ways for them to understand insect taxonomy through molecular biology and allow them to develop new skills that contribute to strengthening their scientific performance in the future, especially when they do research as graduate students. Finally, this note is an example of how to integrate simple bioinformatics tools into the teaching of entomology.

Molecular cloning,expression pattern and phylogenetic analysis of Guanine nucleotide exchange factor Vav2 in lamprey,Lampetra japonica

The Guanine nucleotide exchange factor Vav2（Vav2） is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog（Lj-Vav2） of Vav2 was identified in the lamprey（Lampetra japonica）. To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology（CH） domain, Dbl-homologous（DH） domain, Pleckstrin homology（PH） domain, Cysteine-rich（C1）domains, Src homology 3（SH3） domains and Src homology 2（SH2） domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the LjVav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The LjVav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.

Application of Molecular Marker Technology in the Study of Forest Tree Species

Due to its unique advantages, molecular marker technology is widely applied in the research of forest tree species. This paper reviewed the application of molecular marker technology in tree species resource diversity, germplasm identification, genetic map construction, gene mapping and marker-assisted selection (MAS) breeding. In addition, it elaborated the great significance of molecular marker technology to promote the sustainable development of forestry production in China.

Molecular phylogeny and taxonomy of Phlomoides(Lamiaceae subfamily Lamioideae)in China:Insights from molecular and morphological data

Phlomoides,with 150-170 species,is the second largest and perhaps most taxonomically challenging genus within the subfamily Lamioideae(Lamiaceae).With about 60 species,China is one of three major biodiversity centers of Phlomoides.Although some Phlomoides species from China have been included in previous molecular phylogenetic studies,a robust and broad phylogeny of this lineage has yet to be completed.Moreover,given the myriad new additions to the genus,the existing infrageneric classification needs to be evaluated and revised.Here,we combine molecular and morphological data to investigate relationships within Phlomoides,with a focus on Chinese species.We observed that plastid DNA sequences can resolve relationships within Phlomoides better than nuclear ribosomal internal and external transcribed spacer regions(nrITS and nrETS).Molecular phylogenetic analyses confirm the monophyly of Phlomoides,but most previously defined infrageneric groups are not monophyletic.In addition,morphological analysis demonstrates the significant taxonomic value of eight characters to the genus.Based on our molecular phylogenetic analyses and morphological data,we establish a novel section Notochaete within Phlomoides,and propose three new combinations as well as three new synonyms.This study presents the first molecular phylogenetic analyses of Phlomoides in which taxa representative of the entire genus are included,and highlights the phylogenetic and taxonomic value of several morphological characters from species of Phlomoides from China.Our study suggests that a taxonomic revision and reclassification for the entire genus is necessary in the future.

Resolving tumor evolution:a phylogenetic approach

The evolutionary dynamics of cancer,characterized by its profound heterogeneity,demand sophisticated tools for a holistic understanding.This review delves into tumor phylogenetics,an essential approach bridging evolu-tionary biology with oncology,offering unparalleled insights into cancer’s evolutionary trajectory.We provide an overview of the workflow,encompassing study design,data acquisition,and phylogeny reconstruction.No-tably,the integration of diverse data sets emerges as a transformative step,enhancing the depth and breadth of evolutionary insights.With this integrated perspective,tumor phylogenetics stands poised to redefine our understanding of cancer evolution and influence therapeutic strategies.

基于SRAP分子标记的五十份木槿种质资源亲缘关系的分析

以木槿(Hibiscus syriacus L.)为试材,采用UPGMA法,通过16对SRAP引物组合研究50份木槿种质亲缘关系,以期为加速木槿新品种的选育进程提供参考依据。结果表明:16对SRAP引物组合共扩增出390条条带,其中有388条具有多态性,多态性比率平均为99.49%。在遗传距离为0.3620处,50份木槿样本被分为5组,分类结果显示木槿品种间的亲缘关系与木槿花花瓣基部色斑和花瓣型这2个形态特征呈现密切相关,而与花色以及植株、叶片以及果实等形态特征未呈现相关性。综上所述,SRAP标记技术对木槿基因组检测多态性水平高,可用于木槿种质资源鉴定以及分子标记辅助育种等工作。

Reanalysis of the phylogenetic relationships of the Pentatomomorpha(Hemiptera: Heteroptera) based on ribosomal, Hox and mitochondrial genes

Pentatomomorpha is one of the most biodiverse infraorders among the true bugs（Hemiptera： Heteroptera）. Phylogenetic relationships among the superfamilies within this infraorder have been uncertain, especially for the Eutrichophora. The previous studies were based on morphological characters, or just mitochondrial or nuclear genes, or only partial 18 S rDNA and COI. In this study, we used maximum likelihood（ML） and Bayesian inference（BI） based on massive molecular datasets（18S rDNA, 28 S rDNA, Hox and mitochondrial genes totaling 21 loci and 12,538 characters） to infer a robust phylogeny for this terrestrial group. Results strongly support the monophyly of all superfamilies; the superfamily status of Aradoidea and the following relationships：（Aradoidea ＋（Pentatomoidea ＋（Coreoidea ＋（Lygaeoidea ＋ Pyrrhocoroidea）））） in Pentatomomorpha, and（Coreoidea ＋（Lygaeoidea ＋ Pyrrhocoroidea）） in Eutrichophora. Our results suggest that sampling greater numbers of genes is an effective tool for resolving phylogenetic problems.