Duplicated chalcone synthase(CHS)genes modulate flavonoid production in tea plants in response to light stress

In tea plants,the abundant flavonoid compounds are responsible for the health benefits for the human body and define the astringent flavor profile.While the downstream mechanisms of flavonoid biosynthesis have been extensively studied,the role of chalcone synthase(CHS)in this secondary metabolic process in tea plants remains less clear.In this study,we compared the evolutionary profile of the flavonoid metabolism pathway and discovered that gene duplication of CHS occurred in tea plants.We identified three CsCHS genes,along with a CsCHS-like gene,as potential candidates for further functional investigation.Unlike the CsCHS-like gene,the CsCHS genes effectively restored flavonoid production in Arabidopsis chs-mutants.Additionally,CsCHS transgenic tobacco plants exhibited higher flavonoid compound accumulation compared to their wild-type counterparts.Most notably,our examination of promoter and gene expression levels for the selected CHS genes revealed distinct responses to UV-B stress in tea plants.Our findings suggest that environmental factors such as UV-B exposure could have been the key drivers behind the gene duplication events in CHS.

Targeted Mutagenesis of Duplicated Genes in Caenorhabditis elegans Using CRISPR-Cas9

CRISPR （clustered regularly interspaced short palindromic repeats）-Cas9-based genome editing has revolutionized func- tional genomics in many biological research fields. The specificity and potency of CR1SPR-Cas9 genome editing make it ideal for investigating the function of genes in vivo （Hsu et al., 2014）. Gene duplication is a key driver of evolu- tionary novelty （Taylor and Raes, 2004）. However, duplicated genes with near-identical sequences and functional redun- dancy have posed challenges for genetic analysis （Woollard, 2005）. The functions of duplicated genes can be assessed by simultaneous knockdown using homology-based methods such as RNA interference （RNAi） （Tischler et al., 2006）, Generation of double or triple mutants is an alternative way to assess functional redundancy of duplicated genes, However, generation of such compound mutants by forward or reverse genetic methods is time consuming.

Different expression patterns of duplicated PHANTASTICA-like genes in Lotus japonicus suggest their divergent functions during compound leaf development

Recent studies on leaf development demonstrate that the mechanism on the adaxial-abaxial polarity pattern formation could be well conserved among the far-related species, in which PHANTASTICA （PAHN）-Iike genes play important roles. In this study, we explored the conservation and diversity on functions of PHAN-Iike genes during the compound leaf development in Lotusjaponicus, a papilionoid legume. Two PHAN-Iike genes in L. japonicus, LjPHANa and LjPHANb, were found to originate from a gene duplication event and displayed different expression patterns during compound leaf development. Two mutants, reduced leafletsl （rell） and reduced leaflets3 （rel3）, which exhibited decreased adaxial identity of leaflets and reduced leaflet initiation, were identified and investigated. The expression patterns of both LjPHANs in rel mutants were altered and correlated with abnormalities of compound leaves. Our data suggest that LjPHANa and LjPHANb play important but divergent roles in regulating adaxial-abaxial polarity of compound leaves in L. japonicus.

The power of“controllers”:Transposon-mediated duplicated genes evolve towards neofunctionalization

Since the discovery of the first transposon by Dr.Barbara McClintock,the prevalence and diversity of transposable elements(TEs)have been gradually recognized.As fundamental genetic components,TEs drive organismal evolution not only by contributing functional sequences(e.g.,regulatory elements or“controllers”as phrased by Dr.McClintock)but also by shuffling genomic sequences.In the latter respect,TE-mediated gene duplications have contributed to the origination of new genes and attracted extensive interest.In response to the development of this field,we herein attempt to provide an overview of TEmediated duplication by focusing on common rules emerging across duplications generated by different TE types.Specifically,despite the huge divergence of transposition machinery across TEs,we identify three common features of various TE-mediated duplication mechanisms,including end bypass,template switching,and recurrent transposition.These three features lead to one common functional outcome,namely,TE-mediated duplicates tend to be subjected to exon shuffling and neofunctionalization.Therefore,the intrinsic properties of the mutational mechanism constrain the evolutionary trajectories of these duplicates.We finally discuss the future of this field including an in-depth characterization of both the duplication mechanisms and functions of TE-mediated duplicates.

On the origin and evolution of new genes——a genomic and experimental perspective

The inherent interest on the origin of genetic novelties can be traced back to Darwin. But it was not until recently that we were allowed to investigate the fundamental process of origin of new genes by the studies on newly evolved young genes. Two indispensible steps are involved in this process： origin of new gene copies through various mutational mechanisms and evolution of novel functions, which fur- ther more leads to fixation of the new copies within populations. The theoretical framework for the former step formed in 1970s. Ohno proposed gene duplication as the most important mechanism producing new gene copies. He also believed that the most common fate for new gene copies is to become pseudogenes. This classical view was validated and was also challenged by the characterization of the first functional young gene jingwei in Drosophila. Recent genome-wide comparison on young genes of Drosophila has elucidated a compre- hensive picture addressing remarkable roles of various mechanisms besides gene duplication during origin of new genes. Case surveys revealed it is not rare that new genes would evolve novel structures and functions to contribute to the adaptive evolution of organisms. Here, we review recent advances in understanding how new genes originated and evolved on the basis of genome-wide results and ex- perimental efforts on cases. We would finally discuss the future directions of this fast-growing research field in the context of functional genomics era.

Genome-wide identification and co-expression network analysis of the Os NF-Y gene family in rice

Nuclear factor Y(NF-Y) is a ubiquitous transcription factor that regulates important physiological and developmental processes. In this study, we identified 34 Os NF-Y genes in rice, including 6 newly identified genes. Expression profile analysis covering the whole life cycle revealed that transcripts of Os NF-Y differentially accumulated in a tissue-specific,preferential or constitutive manner. In addition, gene duplication studies and expression analyses were performed to determine the evolutionary origins of the Os NF-Y gene family.Nine Os NF-Y genes were differentially expressed after treatment of seedlings with one or more abiotic stresses such as drought, salt and cold. Analysis of expression correlation and Gene Ontology annotation suggested that Os NF-Y genes were co-expressed with genes that participated in stress, accumulation of seed storage reserves, and plant development.Co-expression analysis also revealed that Os NF-Y genes might interact with each other,suggesting that NF-Y subunits formed complexes that take part in transcriptional regulation. These results provide useful information for further elucidating the function of the NF-Y family and their regulatory pathways.

Molecular evolution of the rice miR395 gene family

MicroRNAs (miRNAs) are 20-22 nucleotide non-coding RNAs that play important roles in plant and animal development. They are usually processed from larger precursors that can form stem-loop structures. Among 20 miRNA families that are conserved between Arabidopsis and rice, the rice miR395 gene family was unique because it was organized into compact clusters that could be transcribed as one single transcript. We show here that in fact this family had four clusters of total 24 genes. Three of these clusters were segmental duplications. They contained miR395 genes of both 120 bp and 66 bp long. However, only the latter was repeatedly duplicated. The fourth cluster contained miR395 genes of two different sizes that could be the consequences of intergenic recombination of genes from the first three clusters. On each cluster, both 1-duplication and 2-duplication histories were observed based on the sequence similarity between miR395 genes, some of which were nearly identical suggesting a recent origin. This was supported by a miR395 locus survey among several species of the genus Oryza, where two clusters were only found in species with an AA genome, the genome of the cultivated rice. A comparative study of the genomic organization of Medicago truncatula miR395 gene family showed significant expansion of intergenic spaces indicating that the originally clustered genes were drifting away from each other. The diverse genomic organizations of a conserved microRNA gene family in different plant genomes indicated that this important negative gene regulation system has undergone dramatic tune-ups in plant genomes.

Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice

To understand the expansion ofmulticopy microRNA （miRNA） families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miRl60 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.

Comprehensive identification and analyses of the Hsf gene family in the whole-genome of three Apiaceae species

Apiaceae is a major family from Apiales and includes many important vegetable and medicinal crops.Heat shock transcription factors(Hsf)play important roles in heat tolerance during plant development.Here,we conducted systematic analyses of the Hsf gene family in three Apiaceae species,including 17 Apium graveolens(celery),32 Coriandrum sativum(coriander),and 14 Daucus carota(carrot).A total of 73 Hsf genes were identified in three representative species,including Arabidopsis thaliana,Vitis vinifera,and Lactuca sativa.Whole-genome duplication played important roles in the Hsf gene family’s expansion within Apiaceae.Interestingly,we found that coriander had more Hsf genes than celery and carrot due to greater expansion and fewer losses.Twenty-seven branches of the phylogenetic tree underwent considerable positive selection in these Apiaceae species.We also explored the expression patterns of Hsf genes in three plant organs.Collectively,this study will serve as a rich gene resource for exploring the molecular mechanisms of heat tolerance.Additionally,this is the first study to report on the Hsf gene family in Apiaceae;thus,our research will provide guidance for future comparative and functional genomic studies on the Hsf gene family and others in Apiaceae.

Genome-wide identification and expression analysis of Gossypium RING-H2 finger E3 ligase genes revealed their roles in fiber development,and phytohormone and abiotic stress responses

Background： RING H2 finger E3 ligase （RH2FE3） genes encode cysteine rich proteins that mediate E3 ubiquitin ligase activity and degrade target substrates. The roles of these genes in plant responses to phytohormones and abiotic stresses are well documented in various species, but their roles in cotton fiber development are poorly understood. To date, genome wide identification and expression analyses of Gossypium hirsutum RH2FE3 genes have not been reported. Methods： We performed computational identification, structural and phylogenetic analyses, chromosomal distribution analysis and estimated KJKs values of G hirsutum RH2FE3 genes. Orthologous and paralogous gene pairs were identified by all versus all BLASTP searches. We predicted cis regulatory elements and analyzed microarray data sets to generate heatmaps at different development stages. Tissue specific expression in cotton fiber, and hormonal and abiotic stress responses were determined by quantitative real time polymerase chain reaction （qRT PCR） analysis. Results： We investigated 140 G hirsutum, 80 G. orboreum, and evolutionary mechanisms and compared them with orthologs 89 G. roimondii putative RH2FB genes and their in Arobidopsis and rice. A domain based analysis of the G hirsutum RH2FE3 genes predicted conserved signature motifs and gene structures. Chromosomal localization showed the genes were distributed across all G hirsutum chromosomes, and 60 duplication events （4 tandem and 56 segmental duplications） and 98 orthologs were detected, cis elements were detected in the promoter regions of G hirsutum RH2FE3 genes. Microarray data and qRT PCR analyses showed that G hirsutum RH2FE3 genes were strongly correlated with cotton fiber development. Additionally, almost all the （brassinolide, gibberellic acid （GA）, indole 3-acetic acid drought, and salt）. dentified genes were up regulated in response to phytohormones （IAA）, and salicylic acid （SA）） and abiotic stresses （cold, heat, Conclusions： The genome wide identification, comprehensive analysis, and characterization of conserved domains and gene structures, as well as phylogenetic analysis, cis element prediction, and expression profile analysis of G hirsutum RH2FE3 genes and their roles in cotton fiber development and responses to plant hormones and abiotic stresses are reported here for the first time. Our findings will contribute to the genome wide analysis of putative RH2FE3 genes in other species and lay a foundation for future physiological and functional research on G hirsutum RH2FE3 genes.

Evolutionary Mode of Metallothioneins Inferred from Cd-MT Genes of Tetrahymena

From Tetrahymena thermophila （strain BF5）, the coding region of Cd-MT gene was cloned and sequenced. and identified as MTT1 isoform. A serial duplicate structure is discovered in its amino acid sequence, which separates the coding region into three parts （Part 1：7-61; Part 2：64-118; Part 3：122-162）. The alignments among them and comparison with the corresponding parts of MT1 isoform suggest that MT1 and MTT1 isoforms both come from the same ancient gene that is homologous to Part 1, and Cd-MTs of Tetrahymena are aroused by such ancient gene duplication. The prediction of secondary structures and the analysis of the disulfide-bonding state of cysteine show that there are a lot of differences between MT1 and MTT1 isoforms, which maybe relate to their function mechanism.

Allelic Polymorphism,Gene Duplication and Balancing Selection of MHC Class ⅡB Genes in the Omei Treefrog(Rhacophorus omeimontis)

The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major histocompatibility complex（MHC） class Ⅱ molecules, the characterization and the evolution of amphibian MHC class Ⅱ genes have attracted increasing attention. The polymorphism of MHC class Ⅱ genes was found to be correlated with susceptibility to fungal pathogens in many amphibian species, suggesting the importance of studies on MHC class Ⅱ genes for amphibians. However, such studies on MHC class Ⅱ gene evolution have rarely been conducted on amphibians in China. In this study, we chose Omei treefrog（Rhacophorus omeimontis）, which lived moist environments easy for breeding bacteria, to study the polymorphism of its MHC class Ⅱ genes and the underlying evolutionary mechanisms. We amplified the entire MHC class ⅡB exon 2 sequence in the R. omeimontis using newly designed primers. We detected 102 putative alleles in 146 individuals. The number of alleles per individual ranged from one to seven, indicating that there are at least four loci containing MHC class ⅡB genes in R. omeimontis. The allelic polymorphism estimated from the 102 alleles in R. omeimontis was not high compared to that estimated in other anuran species. No significant gene recombination was detected in the 102 MHC class ⅡB exon 2 sequences. In contrast, both gene duplication and balancing selection greatly contributed to the variability in MHC class ⅡB exon 2 sequences of R. omeimontis. This study lays the groundwork for the future researches to comprehensively analyze the evolution of amphibian MHC genes and to assess the role of MHC gene polymorphisms in resistance against extracellular pathogens for amphibians in China.

Comparative transcriptome analysis between rhesus macaques(Macaca mulatta) and crab-eating macaques(M. fascicularis)

Understanding gene expression variations between species is pivotal for deciphering the evolutionary diversity in phenotypes. Rhesus macaques(Macaca mulatta, MMU)and crab-eating macaques(M. fascicularis, MFA) serve as crucial nonhuman primate biomedical models with different phenotypes. To date, however, large-scale comparative transcriptome research between these two species has not yet been fully explored. Here, we conducted systematic comparisons utilizing newly sequenced RNA-seq data from84 samples(41 MFA samples and 43 MMU samples)encompassing 14 common tissues. Our findings revealed a small fraction of genes(3.7%) with differential expression between the two species, as well as 36.5% of genes with tissue-specific expression in both macaques. Comparison of gene expression between macaques and humans indicated that 22.6% of orthologous genes displayed differential expression in at least two tissues. Moreover,19.41% of genes that overlapped with macaque-specific structural variants showed differential expression between humans and macaques. Of these, the FAM220A gene exhibited elevated expression in humans compared to macaques due to lineage-specific duplication. In summary,this study presents a large-scale transcriptomic comparison between MMU and MFA and between macaques and humans. The discovery of gene expression variations not only enhances the biomedical utility of macaque models but also contributes to the wider field of primate genomics.

Comparative analysis of SIMILAR to RCD ONE(SRO)family from tetraploid cotton species and their diploid progenitors depict their significance in cotton growth and development

Background SRO(Similar to RCD1)genes family is largely recognized for their importance in the growth,develop-ment,and in responding to environmental stresses.However,genome-wide identification and functional characteri-zation of SRO genes from cotton species have not been reported so far.Results A total of 36 SRO genes were identified from four cotton species.Phylogenetic analysis divided these genes into three groups with distinct structure.Syntenic and chromosomal distribution analysis indicated uneven distribu-tion of GaSRO,GrSRO,GhSRO,and GbSRO genes on A2,D5 genomes,Gh-At,Gh-Dt,Gb-At,and Gb-Dt subgenomes,respectively.Gene duplication analysis revealed the presence of six duplicated gene pairs among GhSRO genes.In promoter analysis,several elements responsive to the growth,development and hormones were found in GhSRO genes,implying gene induction during cotton growth and development.Several miRNAs responsive to plant growth and abiotic stress were predicted to target 12 GhSRO genes.Organ-specific expression profiling demonstrated the roles of GhSRO genes in one or more tissues.In addition,specific expression pattern of some GhSRO genes dur-ing ovule development depicted their involvement in these developmental processes.Conclusion The data presented in this report laid a foundation for understanding the classification and functions of SRO genes in cotton.

Electrophoretic Analysis of Isozymes and Discussion about Species Differentiation in Three Species of Genus Gymnocypris

By using the method of electrophoresis,three isozymes (lactate dehydrogenase,malate dehydrogenase and esterase) of three species of genus Gymnocypris were described and analyzed from North Tibet in this paper. The results showed that all three isozymes presented interspecific difference and distinct differentiation among individuals in the same population,and there was no electrophorectic difference between males and females. Analysis of relationships among three naked carps indicated a high degree of similarity between G. selincuoensis and G. cuoensis ,whereas low degree between G. selincuoensis and G. namensis . Furthermore,three isozymes presented expression of null alleles,and the duplicate genes of LDH A 2,LDH B 2,s MDH A 2 and m MDH B 2 also expressed in some individuals. Compared to other tetraploid fishes,three naked carps retained more functional duplicate genes and null alleles. This suggests fishes of genus Gymnocypris are at the early stage of evolution after polyploidization than that of fishes of Catostomidae,it directly related to the later time of schizothoracine fishes originate as well as severe environment.

Inheritance of Ear Tip-Barrenness Trait in Maize

The aim of this paper is to study the inheritance pattern of ear tip-barrenness trait in maize （Zea mays L.）. Ear tipbarrenness trait in maize can be classified into two types, tip-barren and tip-barrenless. Two inbred lines, lx01-3 （tipbarrenless type）, wx04-1 （tip-barren type）, and their F1, F2, BC1, BC2 generations were analyzed on their ear tip-barrenness types. Results showed that F1 was tip-barren type; the ratio of tip-barren type versus tip-barrenless type followed a 12.78： 1 ratio in F2 segregation population and a 2.75：1 ratio in BC1. Z2 test indicated that the trait of ear tip-barrenness type followed an inheritance pattern of 2 duplicate dominant genes. SPSS analysis indicated that the trait of ear tip-barrenness length is of abnormal distribution. Above results mean that： （1） The trait of maize ear tip-barrenness type is controlled by 2 duplicate dominant genes; tip-barren type is dominant over tip-barrenless type; （2） the trait of tip-barrenness length is a quantitative character controlled by polygene with major genes expected.

Science Letters:Two ancient rounds of polyploidy in rice genome

An ancient genome duplication (PPP1) that predates divergence of the cereals has recently been recognized. We report here another potentially older large-scale duplication (PPP2) event that predates monocot-dicot divergence in the genome of rice (Oryza sativa L.), as inferred from the age distribution of pairs of duplicate genes based on recent genome data for rice. Our results suggest that paleopolyploidy was widespread and played an important role in the evolution of rice.

The evolution and functional divergence of 10 Apolipoprotein D-like genes in Nilaparvata lugens

Apolipoprotein D(ApoD),a member of the lipocalin superfamily of proteins,is involved in lipid transport and stress resistance.Whereas only a single copy of the ApoD gene is found in humans and some other vertebrates,there are typically several ApoD-like genes in insects.To date,there have been relatively few studies that have examined the evolution and functional differentiation of ApoD-like genes in insects,particularly hemi-metabolous insects.In this study,we identified 10 ApoD-like genes(NlApoD1−10)with distinct spatiotemporal expression patterns in Nilaparvata lugens(BPH),which is an important pest of rice.NlApoD1−10 were found to be distributed on 3 chromosomes in a tandem array of NlApoD1/2,NlApoD3−5,and NlApoD7/8,and show sequence and gene structural divergence in the coding regions,indicating that multiple gene duplication events occurred during evolution.Phylogenetic analysis revealed that NlApoD1−10 can be clustered into 5 clades,with NlApoD3−5 and NlApoD7/8 potentially evolving exclusively in the Delphacidae family.Functional screening using an RNA interference approach revealed that only NlApoD2 was essential for BPH development and survival,whereas NlApoD4/5 are highly expressed in testes,and might play roles in reproduction.Moreover,stress response analysis revealed that NlApoD3−5/9,NlApoD3−5,and NlApoD9 were up-regulated after treatment with lipopolysaccharide,H2O2,and ultraviolet-C,respectively,indicating their potential roles in stress resistance.

Plastome characteristics and species identification of Chinese medicinal wintergreens(Gaultheria,Ericaceae)

Wintergreen oil is a folk medicine widely used in foods,pesticides,cosmetics and drugs.In China,nine out of 47 species within Gaultheria(Ericaceae)are traditionally used as Chinese medicinal wintergreens;however,phylogenetic approaches currently used to discriminating these species remain unsatisfactory.In this study,we sequenced and characterized plastomes from nine Chinese wintergreen species and identified candidate DNA barcoding regions for Gaultheria.Each Gaultheria plastome contained 110 unique genes(76 protein-coding,30 tRNA,and four rRNA genes).Duplication of trnfM,rps14,and rpl23 genes were detected,while all plastomes lacked ycf1 and ycf2 genes.Gaultheria plastomes shared substantially contracted SSC regions that contained only the ndhF gene.Moreover,plastomes of Gaultheria leucocarpa var.yunnanensis contained an inversion in the LSC region and an IR expansion to cover the ndhF gene.Multiple rearrangement events apparently occurred between the Gaultheria plastomes and those from several previously reported families in Ericales.Our phylogenetic reconstruction using 42 plastomes revealed well-supported relationships within all nine Gaultheria species.Additionally,seven mutational hotspot regions were identified as potential DNA barcodes for Chinese medicinal wintergreens.Our study is the first to generate complete plastomes and describe the structural variations of the complicated genus Gaultheria.In addition,our findings provide important resources for identification of Chinese medicinal wintergreens.

Fifteen Million Years of Evolution in the Oryza Genus Shows Extensive Gene Family Expansion

In analyzing gene families in the whole-genome sequences available for O. sativa （AA）, O. glaberrima （AA）, and O. brachyantha （FF）, we observed large size expansions in the AA genomes compared to FF genomes for the superfamilies F-box and NB-ARC, and five additional families： the Aspartic proteases, BTB/POZ proteins （BTB）, Glutaredoxins, Trypsin a-amylase inhibitor proteins, and Zf-Dof proteins. Their evolutionary dynamic was investigated to understand how and why such important size variations are observed between these closely related species. We show that expansions resulted from both amplification, largely by tandem duplications, and contraction by gene losses. For the F-box and NB-ARC gene families, the genes conserved in all species were under strong purifying selection while expanded orthologous genes were under more relaxed purifying selection. In F-box, NB-ARC, and BTB, the expanded groups were enriched in genes with little evidence of expression, in comparison with conserved groups. We also detected 87 loci under positive selection in the expanded groups. These results show that most of the duplicated copies in the expanded groups evolve neutrally after duplication because of functional redundancy but a fraction of these genes were preserved following neofunctionalization. Hence, the lineage-specific expansions observed between Oryza species were partly driven by directional selection.