Applications of single-cell RNA sequencing in spermatogenesis and molecular evolution

Spermatogenic cell heterogeneity is determined by the complex process of spermatogenesis differentiation.However,effectively revealing the regulatory mechanisms underlying mammalian spermatogenic cell development and differentiation via traditional methods is difficult.Advances in technology have led to the emergence of many single-cell transcriptome sequencing protocols,which have partially addressed these challenges.In this review,we detail the principles of 10x Genomics technology and summarize the methods for downstream analysis of single-cell transcriptome sequencing data.Furthermore,we explore the role of single-cell transcriptome sequencing in revealing the heterogeneity of testicular ecological niche cells,delineating the establishment and disruption of testicular immune homeostasis during human spermatogenesis,investigating abnormal spermatogenesis in humans,and,ultimately,elucidating the molecular evolution of mammalian spermatogenesis.

Sequence and Molecular Evolution Analysis of Ubiquitin Proteins Encoded by Baculoviruses

[Objective] The aim of this study was to analyze the sequence characteristics and molecular evolution of ubiquitins encoded by baculoviruses.[Methods]Clustal W software was used for multiple sequence alignment analysis,and neighbor-joining method（NJ）and maximum parsimony method（MP）were used for the construction of phylogenetic tree.[Results]The baculoviral ubiquitins showed 73%-86% sequence identity to eukaryotic ubiquitin.Two heterogeneous regions of baculoviral ubiquitins were observed：one was the residues from 15-32,the other was located from residues 53 to 60.The else parts were conserved,where many functional amino acids were also observed.Phylogenetic analysis indicated that baculoviral ubiquitins could be divided into three sub-families,including sub-family GV,sub-family I and sub-family II.The molecular evolution of baculoviral ubiquitins might be under negative selection to maintain their functional and structural stability.[Conclusion]The analysis had provided reference for the researches on functional characterization of baculoviral ubiquitins.

Molecular evolution of hepatitis A virus in a human diploid cell line

AIM： To investigate the hoLspoLs, direction, and the time course of evolution of hepatitis A virus in the process of consecutive cell culture passage in human KMB17 diploid cells.METHODS： Wild type hepatitis A virus H2w was serially propagated in KMB17 cells until passage 30, and the fulllength genomes of H2w and its six chosen progenies were determined by directly sequencing RT-PCR products amplified from viral genomic RNA. Alignment comparison of sequences from H2w with its six progenies and phylogenetic analysis of the whole VP1 region from H2w, progenies of H2w, and other cell culture adapted hepatitis A virus were then carried out to obtain data on the molecular evolution of hepatitis A virus in the process of consecutive passage in KMB17 cells.RESULTS： Most of the mutations occurred by passage 5 and several hotspots related to adaptation of the virus during cell growth were observed. After that stage, few additional mutations occurred through the remaining duration of passage in KMB17 cells except for mutation in the virulence determinants, which occurred in the vicinity of passage 15. The phylogenetic analysis of the whole VP1 region suggested that the progenies of H2w evolved closely to other cell culture adapted hepatitis A virus, i.e. MBB, L-A-l, other than its progenitor H2w.CONCLUSION： Hepatitis A virus served as a useful model for studying molecular evolution of viruses in a given environment. The information obtained in this study may provide assistance in cultivating the next generation of a seed virus for live hepatitis A vaccine production.

Molecular Evolution of Visual Opsin Genes during the Behavioral Shifts between Different Photic Environments in Geckos

Reptiles are the most morphologically and physiologically diverse tetrapods,with the squamates having the most diverse habitats.Lizard is an important model system for understanding the role of visual ecology,phylogeny and behavior on the structure of visual systems.In this study,we compared three opsin genes(RH2,LWS and SWS1)among 49 reptile species to detect positively selected genes as well as amino acid sites.Our results indicated that visual opsin genes have undergone divergent selection pressures in all lizards and RH2 and LWS suffered stronger positive selection than SWS1.Twelve positively selected sites were picked out for RH2 and LWS.Moreover,many diagnostic sites were found between geckos and non-gecko lizards,most of which were located near the positively selected sites and some of them have already been reported to be responsible for significant shifts of the wavelength of maximum absorption(λ_(max)).The results indicated that the gecko lineage accelerated the evolution of these genes to adapt to the dim-light environment or nocturnality as well as the switch between nocturnality and diurnality.

Taxonomy and molecular evolution of secondary metabolism pathway of isoflavones biosynthesis

We compared the cDNA sequences and amino acid sequences of Chalcone synthase （CHS）, Chalcone isomerase （CH1）, and lsoflavone synthase （IFS） in isoflavone biosynthesis pathway, found that their similarity were not always consistent; then we compared the amino acid sequences of CHS, CHI, and IFS, found that the clustering relationship of CHS and CHI were different in 13 kind of species; CHS and IFS were the same or similar in leguminous plants Medicago sativa, Medicago truncatula, Pisum sativum, Pueraria Montana, and Glycine max however disaccording with CHI. We prefer the amino acid sequence for cDNA sequence for evolution analysis, and think that the secondary metabolism mostly attributing to environmental stresses, and the functional genes on secondary metabolism pathway are unsuitable for taxonomy analysis.

Molecular evolution,virology and spatial distribution of HCV genotypes in Pakistan:A meta-analysis

Background:Hepatitis C,caused by the Hepatitis C Virus(HCV),is the second most common form of viral hepatitis.The geographical distribution of HCV genotypes can be quite complex,making it challenging to ascertain the most prevalent genotype in a specific area.Methods:To address this,a review was conducted to determine the prevalence of HCV genotypes across various provinces and as a whole in Pakistan.The scientific literature regarding the prevalence,distribution,genotyping,and epidemiology of HCV was gathered from published articles spanning the years 1996-2020.Results:Genotype 1 accounted for 5.1%of the patients,with its predominant subtype being 1a at 4.38%.The frequencies of its other subtypes,1b and 1c,were observed to be 1.0%and 0.31%respectively.Genotype 2 had a frequency of 2.66%,with the most widely distributed subtype being 2a at 2.11%of the patients.Its other subtypes,2b and 2c,had frequencies of 0.17%and 0.36%respectively.The most prevalent genotype among all isolates was 3(65.35%),with the most frequent subtype being 3a(55.15%),followed by 3b(7.18%).The prevalence of genotypes 4,5,and 6 were scarce in Pakistan,with frequencies of 0.97%,0.08%,and 0.32%respectively.The prevalence of untypeable and mixed genotypes was 21.34%and 3.53%respectively.Estimating genotypes proves to be a productive method in assisting with the duration and selection of antiviral treatment.Different HCV genotypes can exhibit variations in their response to specific antiviral treatments.Different genotypes may have distinct natural histories,including variations in disease progression and severity.Some genotypes may lead to more rapid liver damage,while others progress more slowly.Conclusions:This information can guide screening and testing strategies,helping to identify individuals at higher risk of developing severe complications.Studying the distribution of HCV genotypes in a population can provide valuable insights into the transmission dynamics of the virus.

Molecular Composition and Evolution of the Chalcone Synthase (CHS) Gene Family in Five Species of Camellia (Theaceae)

The molecular composition and evolution of the chalcone synthase (CHS) gene family from five species in Camellia (Theaceae) are explored in this study. Sixteen CHS exon 2 from four Camellia species were amplified from total DNA by PCR method. Three sequences of the fifth species in Camellia and two sequences of Glycine max as the designated outgroups were obtained from GenBank. Our results indicated that CHS gene family in Camellia was differentiated to three subfamilies (A, B, C) during the evolutionary history with six groups (A1, A2, A3, BI, B2, C). Among them, only group A2 was possessed by all five species in this study. However, the other five groups were detected only in some species of the plants studied. All members of CHS gene family in this study had high sequence similarity, more than 90% among the members in the same subfamily and more than 78% among different subfamilies at nucleotide level., According to the estimated components of amino acids, the function of CHS genes in Camellia had been diverged. The nucleotide substitutions of the different groups were not identical. Based on phylogenetic analyse inferred from sequences of CHS genes and their deduced amino acid sequences, we concluded that the CHS genes with new function in this genus were evolved either by mutations on several important sites or by accumulation of the mutations after the gene duplication. A further analysis showed that the diversification of CHS genes in Camellia still occurred recently, and the evolutionary models were different to some extant among different species. So we assumed that the different evolutionary models resulted from the impacts of variable environmental elements after the events of speciation.

Molecular evolution of Na^(+) channels in teleost fishes

Voltage-dependent sodium channels are critical for electrical excitability.Invertebrates possess a single sodium channel gene;two rounds of genome duplication early in vertebrates increased the number to four.Since the teleost-tetrapod split,independent gene duplications in each lineage have further increased the number of sodium channel genes to 10 in tetrapods and 8 in teleosts.Here we review how the occurrence of multiple sodium channel paralogs has influenced the evolutionary history of three groups of fishes:pufferfish,gymnotiform and mor-myriform electric fish.Pufferfish(tetraodontidae)produce a neurotoxin,tetrodotoxin,that binds to and blocks the pore of sodium channels.Pufferfish evolved resistance to their own toxins by amino acid substitutions in the pore of their sodium channels.These substitutions had to occur in parallel across multiple paralogs for organismal re-sistance to evolve.Gymnotiform and mormyriform fishes independently evolved electric organs to generate elec-tricity for communication and object localization.Two sodium channel genes are expressed in muscle in most fishes.In both groups of weakly electric fishes,one gene lost its expression in muscle and became compartmen-talized in the evolutionary novel electric organ,which is a muscle derivative.This gene then evolved at elevated rates,whereas the gene that is still expressed in muscle does not show elevated rates of evolution.In the electric organ-expressing gene,amino acid substitutions occur in parts of the channel involved in determining how long the channel will be open or closed.The enhanced rate of sequence evolution of this gene likely underlies the spe-cies-level variations in the electric signal.

Molecular evolution and species-specific expansion of the NAP members in plants

he NAP（NAC-Like, Activated by AP3/PI） subfamily is one of the important plant-specific transcription factors, and controls many vital biological processes in plants. In the current study, 197 NAP proteins were identified from 31 vascular plants,but no NAP members were found in eight non-vascular plants.All NAP proteins were phylogenetically classified into two groups（NAP I and NAP II）, and the origin time of the NAP I group might be relatively later than that of the NAP II group.Furthermore, species-specific gene duplications, caused by segmental duplication events, resulted in the expansion of the NAP subfamily after species-divergence. Different groups have different expansion rates, and the NAP group preference was found during the expansion in plants. Moreover, the expansion of NAP proteins may be related to the gain and loss of introns.Besides, functional divergence was limited after the gene duplication. Abscisic acid（ABA） might play an important role in leaf senescence, which is regulated by NAP subfamily. These results could lay an important foundation for expansion and evolutionary analysis of NAP subfamily in plants.

Habitat-dependent diversification and parallel molecular evolution： Water scavenger beetles as a case study

Habitat shift is a key innovation that has contributed to the extreme diversification of insects. Most groups are well-adapted to more or less specific environments and shifts usually only happen between similar habitats. To colonize a pro- foundly different habitat type does not only present ecological opportunities but also great challenges. We used Hydrophiloidea （water scavenger beetles） as a system to study transitions between terrestrial and aquatic environments. We estimated the diversi- fication rate of different clades using phylogenetic trees based on a representative taxon sampling and six genes. We also investi- gated possible evolutionary changes in candidate genes following habitat shifts. Our results suggest that the diversification rate is relatively slow （0.039-0.050 sp/My） in the aquatic lineage, whereas it is distinctly increased in the secondarily terrestrial clade （0.055-0.075 sp/My）. Our results also show that aquatic species have a G （Glycine） or S （Serine） amino acid at a given site of COI, while terrestrial species share an A （Alanine） amino acid with terrestrial outgroups. This indicates that habitat factors may create selection pressure on the evolution of functional genes and cause homoplasy in molecular evolution [Current Zoology 60 （5）： 561-570, 2014 ]

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants

Rubisco and fructose-1,6-bisphosphate aldolases （FBAs） are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sati- vum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former spe- cies. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro- Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.

Molecular evolution of methanogens based on their metabolic facets

The information provided by completely sequenced genomes of methanogens can yield insights into a deeper molecular understanding of evolutionary mechanisms.This review describes the advantages of using metabolic pathways to clarify evolutionary correlation of methanogens with archaea and prokaryotes.Metabolic trees can be used to highlight similarities in metabolic networks related to the biology of methanogens.Metabolic genes are among the most modular in the cell and their genes are expected to travel laterally,even in recent evolution.Phylogenetic analysis of protein superfamilies provides a perspective on the evolutionary history of some key metabolic modules of methanogens.Phage-related genes from distantly related organisms typically invade methanogens by horizontal gene transfer.Metabolic modules in methanogenesis are phylogenetically aligned in closely related methanogens.Reverse order reactions of methanogenesis are achieved in methylotrophic methanogens using metabolic and structural modules of key enzymes.A significant evolutionary process is thought to couple the utilization of heavy metal ions with energetic metabolism in methanogens.Over 30 of methanogens genomes have been sequenced to date,and a variety of databases are being developed that will provide for genome annotation and phylogenomic analysis of methanogens.Into the context of the evolutionary hypothesis,the integration of metabolomic and proteomic data into large-scale mathematical models holds promise for fostering rational strategies for strain improvement.

Random Penetrance of Mutations Among Individuals:A New Type of Genetic Drift in Molecular Evolution

The determinative view of mutation penetrance is a fundamental assumption for the building of molecular evolutionary theory:individuals in the population with the same genotype have the same fitness effect.Since this view has been constantly challenged by experimental evidence,it is desirable to examine to what extent violation of this view could affect our under-standing of molecular evolution.To this end,the author formulated a new theory of molecular evolution under a random model of penetrance:for any individual with the same mutational genotype,the coefficient of selection is a random variable.It follows that,in addition to the conventional Ne-genetic drift(Ne is the effective population size),the variance of penetrance among individuals(ε^(2))represents a new type of genetic drift,coined by theε^(2)-genetic drift.It has been demonstrated that these two genetic drifts together provided new insights on the nearly neutral evolution:the evolutionary rate is inversely related to the log-of-Ne when theε^(2)-genetic drift is nontrivial.This log-of-Ne feature ofε^(2)-genetic drift did explain well why the dN/dS ratio(the nonsynonymous rate to the synonymous rate)in humans is only as twofold as that in mice,while the effective population size(Ne)of mice is about two-magnitude larger than that of humans.It was estimated that,for the first time,the variance of random penetrance in mammalian genes was approximatelyε^(2)≈5.89×10^(-3).

Massive Molecular Parallel Evolution of the HSP90AA1 Gene between High-elevation Anurans

HSP90 AA1 is part of the heat shock protein 90 gene family and has important functions against heat stress. We report a case of molecular level parallel evolution of the HSP90 AA1 gene in high elevation amphibians. HSP90 AA1 gene sequences of four high-elevation anurans, Bufo gargarizans, Nanorana parkeri, Rana kukunoris, and Scutiger boulengeri, were compared along with five of their low-elevation relatives. A total of 16 amino-acid sites were identified as parallel evolution between N. parkeri and R. kukunoris. We generated both model based（Zhang and Kumar＇s test） and empirical data based（parallel/divergence plotting） null distributions for non-parallel evolution, and both methods clearly determined that the observed number of parallel substitutions were significantly more than the null expectation. Furthermore, on the HSP90 AA1 gene tree, N. parkeri and R. kukunoris formed a strongly supported clade that was away from their respective relatives. This study provides a clear case of molecular parallel evolution, which may have significant implications in understanding the genetic mechanisms of high-elevation adaptation.

Research Progress of Molecular Clock

In the paper, related research and progress of molecular clock hypothesis were summarized, including definition of molecular clock, supporting proofs, contro- versy, significance and perfection, application and accompanying challenges.

Evolution of COP9 Signalosome and Proteasome Lid Complex

The COP9 signalosome and the regulatory lid of the 26S proteasome are both eight-subunit protein complexes which are present in most eukaryotes. There is a one-to-one relationship between the corresponding subunits of the two protein complexes in terms of their size and amino acid sequences. Eight groups of subunits from the COP9 signalosome and the proteasome lid complex of different organisms are collected from all the databases at the NCBI website. The corresponding subunits of COP9 signalosome and proteasome lid complex share at least 12% amino acid identity and some conserved regions, and the conserved sites spread evenly over the entire length of the subunits, suggesting that the two complexes have a common evolutionary ancestor. Phylogenetic analyses based on the amino acid sequences of the corresponding subunits of two protein complexes indicate that every tree consists of two clades. The subunits from one of the two protein complexes of different organisms are grouped into one of the two clades respectively. The sequences of single-cell organisms are always the basal groups to that of multi-cell animal and plant species. These results imply that the duplication/divergence events of COP9 signalosome and regulatory lid of the proteasome genes have occurred before the divergence of single-cell and multi-cell eukaryotes, and the genes of the two complexes are independently evolved. The analyses of dN/dS correlation show significant Pearson's correlations between 21 and 15 pairs of subunit-encoding sequences within the COP9 signalosome and the proteasome lid complex respectively, suggesting that those subunits pairs might have related functions and interacted with one another, and resulted in co-evolution.

Molecular Characterization of Four ADF Genes Differentially Expressed in Cotton

Actin depolymerizing factor （ADF）, highly conserved in all eukaryotic cells, is a low molecular mass of actin-binding protein, which plays a key role in modulating the polymerizing and depolymerizing of the actin filaments. Four cDNAs （designated GhADF2, GhADF3, GhADF4, and GhADF5, respectively） encoding ADF proteins were isolated from cotton （Gossypium hirsutum） fiber cDNA library. GhADF2 cDNA is 705 bp in length and deduces a protein with 139 amino acids. GhADF3 cDNA is 819 bp in length and encodes a protein of 139 amino acids. GhADF4 cDNA is 804 bp in length and deduces a protein with 143 amino acids. GhADF5 cDNA is 644 bp in length and encodes a protein of 141 amino acids. The molecular evolutionary relationship of these genes was analyzed by means of bioinformatics. GhADF2 is closely related to GhADF3 （99% identity） and PetADF2 （89% identity）. GhADF4 is closely related to AtADF6 （78% identity）, and GhADF5 is closely related to AtADF5 （83% identity）. These results demonstrated that the plant ADF genes are highly conserved in structure. RT-PCR analysis showed that GhADF2 is predominantly expressed in fiber, whereas, GhADF5 is mainly expressed in cotyledons. On the other hand, it seems that GhADF3 and GhADF4 have no tissue specificity. Expression levels of different ADF genes may vary considerably in the same cell type, suggesting that they might be involved in regulating tissue development of cotton and the each ADF isoform may diverge to form the functional difference from the other ADFs during evolution.

ANALYSIS OF EVOLUTIONARY RELATIONSHIP BETWEEN ASTASIA LONGA AND EUGLENA GRACILIS BY USING RAPD TECHNIQUE AND CLADISTIC ANALYSIS

Although both Astasia longa and Euglena gracilis belong to different genera, they share many morphological characters except that A. longa has no chloroplast. In the 1940’s, on the basis of the finding that in darkness or upon addition of some chemicals, E. gracilis would fade reversibly or irreversibly, some scholars hypothesised that A. longa evolved from E. gracilis by losing chloroplast. The authors’ use of RAPD and cladistic analyses in a study on the evolutionary relationship between A .longa and E. gracilis showed that the A. longa ’s relationship with E. gracilis was closer than that with other green euglenoids. This proves the hypothesis that A. longa evolved from E. gracilis is reasonable. The results of this study suggest that saprophytic colorless euglenoids were transformed from green euglenoids by losing their choroplasts.

Fluctuation theorem for the mutation process in in vitro evolution

A proposition based on the fluctuation theorem in thermodynamics is formulated to quantitatively describe molecular evolution processes in biology. Although we cannot give full proof of its generality, we demonstrate via computer simulation its applicability in an example of DNA in vitro evolution. According to this theorem, the evolution process is a series of exponentially rare fluctuations fixed by the force of natural selection

Spatio-temporal evolution of Allium L. in the Qinghaie-Tibet-Plateau region: Immigration and in situ radiation

A plethora of studies investigating the origin and evolution of diverse mountain taxa has assumed a causal link between geological processes （orogenesis） and a biological response （diversification）. Yet, a substantial delay （up to 30 Myr） between the start of orogenesis and diversification is often observed. Evolutionary biologists should therefore identify alternative drivers of diversification and maintenance of biodiversity in mountain systems. Using phylogenetic, biogeographic, and diversification rate analyses, we could identify two independent processes that most likely explain the diversity of the widespread genus Allium in the Qinghai-Tibet Plateau （QTP） region： （1） While the QTP-related taxa of the subgenus Melanocrommyum diversified in situ, （2） QTP-related taxa of other subgenera migrated into the QTP from multiple source areas. Furthermore, shifts in diversification rates within Allium could not be attributed spatially and temporally to the uplift history of the QTP region. Instead, global cooling and climate oscillations in the Quaternary were major contributors to increased speciation rates in three clades of AUium. Our study therefore adds to the growing evidence supporting the ＂mountain-geo-biodiversity hypothesis＂, which highlights the role of climate oscillations for the diversification of mountain organisms.