Recent Progress in Elucidating the Structure, Function and Evolution of Disease Resistance Genes in Plants

Plants employ multifaceted mechanisms to fight with numerous pathogens in nature. Resistance （R） genes are the most effective weapons against pathogen invasion since they can specifically recognize the corresponding pathogen effectors or associated protein（s） to activate plant immune responses at the site of infection. Up to date, over 70 R genes have been isolated from various plant species. Most R proteins contain conserved motifs such as nucleotide-binding site （NBS）, leucine-rich repeat （LRR）, Toll-interleukin-1 receptor domain （TIR, homologous to cytoplasmic domains of the Drosophila Toll protein and the manamalian intefleukin-1 receptor）, coiled-coil （CC） or leucine zipper （LZ） structure and protein kinase domain （PK）. Recent results indicate that these domains play significant roles in R protein interactions with effector proteins from pathogens and in activating signal transduction pathways involved in innate immunity. This review highlights an overview of the recent progress in elucidating the structure, function and evolution of the isolated R genes in different plant-pathogen interaction systems.

Genome-wide identification and evolutionary analysis of MLO gene family in Rosaceae plants

Mutants lacking wild-type MLO(Mildew resistance Locus O)proteins show broad-spectrum resistance to the powdery mildew fungus,and dysregulated cell death control,with spontaneous cell death in response to developmental or abiotic stimuli.In order to understand the evolution and divergence patterns of the MLO gene family in Rosaceae plants,we analysed systematically genome-wide data from Fragaria vesca,Prunus persica,Prunus mume,Malus domestica,Pyrus bretschneideri and Rubus occidentalis based on bioinformatics methods.Using three phylogenetic methods(the neighbour-joining,maximum likelihood,and Bayesian methods),we identified 117 MLO genes from 6 Rosaceae species.The results of all three phylogenetic analysis methods supported that these genes were divided into six clades.Conserved motif analysis found that only motif 2 was present in all MLO proteins and had 3 nearly invariant amino acid residues.The findings indicated that motif 2 might be shared by the MLO gene family.The structural features of these genes showed large variations in sequence length among different species,although the lengths and the numbers of exons exhibited high degrees of similarity.Selective pressure analysis showed extremely significant differences in all 6 clades,with 2,1,and 1 site(s)under significant positive selection detected in clades III,IV,and VI,respectively.These positive selection sites were important driving forces for the promotion of the functional differentiation of the MLO genes.Functional divergence analysis showed that the significantly divergent sites were located within the domains of the MLO genes.Functional distance analysis showed that the clade V had more conservative functions and might have retained more original functions during the evolutionary process.However,clade I may have undergone extensive altered functional constraints as a specialised functional role.Moreover,the most original function of the MLO genes in Rosaceae could be related to the evolution of their resistance to powdery mildew,which then gradually evolved into functions such as the regulation of flower development,the control of root morphology,and seed evolution due to the different evolutionary rates after gene duplication.These results provide a theoretical basis for further studies of the molecular evolutionary patterns of the plant MLO gene family.

Three novel alleles of OsGS1 developed by base-editing-mediated artificial evolution confer glufosinate tolerance in rice

Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.

Markov Model Applied to Gene Evolution

The study of nucleotide substitution is very important both to our understanding of gene evolution and to reliable estimation of phylogenetic relationships. In this paper nucleotide substitution is assumed to be random and the Markov model is applied to the study of the evolution of genes. Then a non linear optimization approach is proposed for estimating substitution in real sequences. This substitution is called the 'Nucleotide State Transfer Matrix'. One of the most important conclusions from this work is that gene sequence evolution conforms to the Markov process. Also, some theoretical evidences for random evolution are given from energy analysis of DNA replication.

Evolution and phylogenetic application of the MC1R gene in the Cobitoidea(Teleostei: Cypriniformes)

Fish of the superfamily Cobitoidea sensu stricto （namely Ioaches） exhibit extremely high diversity of color patterns, but so far little is known about their evolutionary mechanism. Melanocortin 1 receptor gene （MCIR） plays an important role during the synthesis of melanin and formation of animal body color patterns. In this study, we amplified and sequenced the partial MCIR gene for 44 loach individuals representing 31 species of four families. Phylogenetic analyses yielded a topology congruent with previous studies using multiple nuclear loci, showing that each of the four families was monophyletic with sister relationships of Botiidae＋ （Cobitidae＋（Balitoridae＋Nemacheilidae））. Gene evolutionary analyses indicated that MCIR in Ioaches was under purifying selection pressure, with various sites having different dNIds values. Both Botiidae and Cobitidae had lower dN/ds values than those of background lineages, suggesting their evolution might be strongly affected by purifying selection pressure. For Balitoddae and Nemacheilidae, both had larger dNIds values than those of background lineages, suggesting they had a faster evolutionary rate under more relaxed selection pressure. Consequently, we inferred that the relatively stable color patterns in Botiidae and Cobitidae might result from the strong purifying selection pressure on the MC1R gene, whereas the complicated and diverse color patterns in Balitoridae and Nemacheilidae might be associated with the relaxed selection pressure. Given the easy experimental procedure for the partial MCTR gene and its excellent performance in reconstructing phylogeny, we suggest this gene could be used as a good molecular marker for the phylogenetic study of fish species.

Variation and evolution of NP genes of human avian H_5N_1 virus strains

In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of strains were retrieved. The sequences were analyzed by DNAStar 5.0, and the evolutionary speed was studied with reference to the epidemiological data. It was found that NP genes of 45 strains during 1997-2006 were homologically classified into three groups： strains in 1997-1998, strains in 2004-2005 and strains from 2003 to 2006. There were 35 substitutions in NPs in all strains accounting for a ratio of 7.03% （35/498）. An additional glycoprotein domain （NGT430-432） was found in NP genes in the strains of 2003-2006, the mutation of N370S in GD-01-06 resulted in occurrence of one more glycoprotein domain （NES368-370）. In the synonymous variation, Ks values in NP were 2.03 × 10^-5-2.55 × 10^-5 Nt/d and K. values in NP were 1.58 × 10^-6-3.10 × 10^-6 Nt/d. There didn＇t exist obviously selective pressure. An additional glycoprotein domain in every strain of 2003-2006 and one more in strain GD-01-06 might change the antigenicity of human avian H5 N1 influenza virus. The variation on human avian H5 N1 influenza strains occurred frequently in the natural world, which would result in high probability of human-human transmission along with the natural evolution of the virus.

Plastid genome evolution of a monophyletic group in the subtribe Lauriineae(Laureae,Lauraceae)

Litsea,a non-monophyletic group of the tribe Laureae(Lauraceae),plays important roles in the tropical and subtropical forests of Asia,Australia,Central and North America,and the islands of the Pacific.However,intergeneric relationships between Litsea and Laurus,Lindera,Parasassafras and Sinosassafras of the tribe Laureae remain unresolved.In this study,we present phylogenetic analyses of seven newly sequenced Litsea plastomes,together with 47 Laureae plastomes obtained from public databases,representing six genera of the Laureae.Our results highlight two highly supported monophyletic groups of Litsea taxa.One is composed of 16 Litsea taxa and two Lindera taxa.The 18 plastomes of these taxa were further compared for their gene structure,codon usage,contraction and expansion of inverted repeats,sequence repeats,divergence hotspots,and gene evolution.The complete plastome size of newly sequenced taxa varied between 152,377 bp(Litsea auriculata)and 154,117 bp(Litsea pierrei).Seven of the 16 Litsea plastomes have a pair of insertions in the IRa(trnL-trnH)and IRb(ycf2)regions.The 18 plastomes of Litsea and Lindera taxa exhibit similar gene features,codon usage,oligonucleotide repeats,and inverted repeat dynamics.The codons with the highest frequency among these taxa favored A/T endings and each of these plastomes had nine divergence hotspots,which are located in the same regions.We also identified six protein coding genes(accD,ndhJ,rbcL,rpoC2,ycf1 and ycf2)under positive selection in Litsea;these genes may play important roles in adaptation of Litsea species to various environments.

Epididymis-specific lipocalin promoters

Our goal is to decipher which DNA sequences are required for tissue-specific expression of epididymal genes. At least 6 epididymis-specific lipocalin genes are known. These are differently regulated and regionalized in the epididymis. Lipocalin 5 （Lcn5 or mE-RABP） and Lipocalin 8 （Lcn8 or mEP17） are homologous genes belonging to the epididymis-specific lipocalin gene cluster. Both the 5 kb promoter fragment of the Lcn5 gene and the 5.3 kb promoter fragment of the Lcn8 gene can direct transgene expression in the epididymis （Lcn5 to the distal caput and Lcn8 to the initial segment）, indicating that these promoter fragments contain important cis-regulatory element（s） for epididymisspecific gene expression. To define further the fragments regulating gene expression, the Lcn5 promoter was examined in transgenic mice and immortalized epididymal cell lines. After serial deletion, the 1.8 kb promoter fragment of the Lcn5 gene was sufficient for tissue-specific and region-specific gene expression in transgenic mice. Transient transfection analysis revealed that a transcription factor forkhead box A2 （Foxa2） interacts with androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 kb and 1.3 kb and that Foxa2 expression inhibits androgen-dependent induction of the Lcn5 promoter activity. Immunohistochemistry indicated a restricted expression of Foxa2 in the epididymis where endogenous Lcn5 gene expression is suppressed and that the Foxa2 inhibition of the Lcn5 promoter is consistent with the lack of expression of Lcn5 in the corpus and cauda. Our approach provides a basic strategy for further analysis of the epididymal lipocalin gene regulation and flexible control of epididymal function. （Asian J Androl 2007 July; 9： 515-521）

Segmental Translocation Contributed to the Origin of the Brassica S-locus

Self-incompatibility(SI),which has recurred during the evolution of plants,is one of the most important cross-pollination mating systems.Three S-loci have been reported in Brassicaceae,namely,Arabidopsis lyrata(Al),Brassica(Br),and Leavenworthia alabamica(La)S-loci.Here,through multi-genomic comparative analysis of 20 species,we revealed that the most ancient S-locus was formed prior to the divergence of Brassicaceae lineage I and II.Itwas retained and inherited by Arabidopsis,as the Al S-locus in Brassicaceae lineage I.Furthermore,we found that the Br S-locus,which has been widely used in the breeding of Brassica crops to generate hybrid seeds,was formed through segmental translocation(ST)in the hexaploid ancestor of Brassica in Brassicaceae lineage II.The Br S-locus was evolved through a ST from one of the triplicated ancestral S-locus paralogs in the Brassica hexaploidy ancestor,while the other two S-locus paralogs were lost.Together with the previous discovery that the La S-locus was formed through a secondary origin in Brassicaceae lineage I,we conclude the monophyletic origin of Al and Br S-loci and clarify the evolutionary route of S-loci in the Brassicaceae family.Our findings will contribute to evolutionary studies and breeding applications of the S-locus in Brassicaceae.

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.

当代中国人生存失范的峻切书写——新文化观与文学生存论观照下的《活着之上》

阎真《活着之上》因为对中国大学与社会生存失范状态的峻切书写,成为2015年当代文学界热门话题。从基因同异创化论与文学生存论视角,对《活着之上》所写的主题与人物作出分析,指出中国人近代以来面临新的历史境遇与资源竞争对象,竞争激化,造成了生存失范的文化历史境况。聂致远、蒙天舒等不仅是中国大学教师生存困境的体现者,更是整个中国当代社会的代表性人物,对于他们的叙述、虚构凸显了中国部分知识分子生存真实。

新的世界历史语境中的民族生存战略及其评价——新文化观观照下的新文化运动

以基因同异创化论新文化观观照新文化运动,认为这一运动是中华民族在不同的历史、文化因素的合力作用下的生存战略选择,并不只是新文化激进派的观念运动。这场运动既有得也有失,是整个民族作为基因群体在新的世界历史语境中的生存适应能力的表现。正视这样一种适应能力的根源与局限,进行深入的反思,冷静理性地探索一个世纪以后变化更剧烈的文化、历史语境中的民族生存新战略,并提出新战略的基本方向。

当代生物进化论研究新进展

分子进化学说着眼于分子层次的进化机制 ,自然选择学说着眼于群体水平的进化机制 ,两者结合把生物进化理论提高到一个新的水平。另外 ,基因调控下的细胞分化、分子驱动机制均从不同侧面揭示了生物进化原理。

Hox and ParaHox Genes in Evolution,Development and Genomics

The discovery of the homeobox motif and its presence in each gene of the Hox clusters revolutionized the fields of developmental biology and evolutionary developmental biology （1, 2）, providing a rapid entrance into investigating the mechanisms of development of almost any animal taxon as well as dramatically altering conceptions on the extent of genetic conservation across the animal kingdom.

进化的两种主要模式和主动进化

根据生物学现象的新发现和新生学科表观遗传学的内容,如基因表达的多个水平的调控、曾经的观点胎儿只受母体子宫羊水等环境的影响、现今的新发现推论胎儿可通过母体感受外界环境的变化致使其做出适应环境影响的变化。本人通过总结拉马克、达尔文以及达尔文之后的多个现代达尔文主义的学说观点,对亲代间性状变化的影响因素进行了分析和整理,得出了有关进化的两种主要模式的假说。即生物的进化,一受选择压力使优劣势性状的基因频率发生改变;二受环境的诱导使胎内子代基因的表达情况发生改变。主动进化假说即第二种模式,子代通过感知外界的环境做出适应性的改变,主要通过组蛋白及核苷酸的化学修饰,致使原本表达的基因沉默或原本沉默的基因表达。

Rapid Evolution of the Mammalian HILS1 Gene and the Nuclear Condensation Process during Mammalian Spermiogenesis

Many genes associated with reproduction show rapid evolution across diverse animal groups, a result commonly due to adaptive evolution driven by positive selection （Swanson and Vacquier, 2002）. Different theories have been proposed to explain the elevated rates of evolution （Swanson and Vacquier, 2002）, including sperm competition, where sperm compete to fertilize eggs leading to the proteins in the sper- matozoa adaptively evolving to increase their ability to fertilize eggs; sexual conflict, where the egg experiences a loss of fitness when sperm are too abundant; sexual selection, where eggs bind sperm carrying adaptive alleles （Palumbi, 1999）; and cryptic female choice （reviewed in Swanson and Vacquier, 2002）.

Base-Editing-Mediated Artificial Evolution of OsALS1 In Planta to Develop Novel Herbicide-Tolerant Rice Germplasms

Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.

A well-supported nuclear phylogeny of Poaceae and implications for the evolution of C4 photosynthesis

Poaceae(the grasses)includes rice,maize,wheat,and other crops,and is the most economically important angiosperm family.Poaceae is also one of the largest plant families,consisting of over 11000 species with a global distribution that contributes to diverse ecosystems.Poaceae species are classified into 12 subfamilies,with generally strong phylogenetic support for their monophyly.However,many relationships within subfamilies,among tribes and/or subtribes,remain uncertain.To better resolve the Poaceae phylogeny,we generated 342 transcriptomic and seven genomic datasets;these were combined with other genomic and transcriptomic datasets to provide sequences for 357 Poaceae species in 231 genera,representing 45 tribes and all 12 subfamilies.Over 1200 low-copy nuclear genes were retrieved from these datasets,with several subsets obtained using additional criteria,and used for coalescent analyses to reconstruct a Poaceae phylogeny.Our results strongly support the monophyly of 11 subfamilies;however,the subfamily Puelioideae was separated into two non-sister clades,one for each of the two previously defined tribes,supporting a hypothesis that places each tribe in a separate subfamily.Molecular clock analyses estimated the crown age of Poaceae to be101 million years old.Ancestral character reconstruction of C3/C4 photosynthesis supports the hypothesis of multiple independent origins of C4 photosynthesis.These origins are further supported by phylogenetic analysis of the ppc gene family that encodes the phosphoenolpyruvate carboxylase,which suggests that members of three paralogous subclades(ppc-aL1a,ppc-aL1b,and ppcB2)were recruited as functional C4 ppc genes.This study provides valuable resources and a robust phylogenetic framework for evolutionary analyses of the grass family.

Origin of new genes after zygotic genome activation in vertebrate

New genes are drivers of evolutionary innovation and phenotypic evolution. Expression of new genes in early development raises the possibility that new genes could originate and be recruited for functions in embryonic development, but this remains undocu- mented. Here, based on temporal gene expression at different developmental stages in Xenopus tropicolis, we found that young protein-coding genes were significantly enriched for expression in developmental stages occurring after the midblastula trans- ition （MBT）, and displayed a decreasing trend in abundance in the subsequent stages after MBT. To complement the finding, we demonstrate essential functional attributes of a young orphan gene, named as Fog2, in morphological development. Our data indicate that new genes could originate after MBT and be recruited for functions in embryonic development, and thus provide insights for better understanding of the origin, evolution, and function of new genes.

Phylogenetic Analysis of the Plant-specific Zinc Finger-Homeobox and Mini Zinc Finger Gene Families

Zinc finger-homeodomain proteins （ZHD） are present in many plants; however, the evolutionary history of the ZHD gene family remains largely unknown. We show here that ZHD genes are plant-specific, nearly all intronless, and related to MINI ZINC FINGER （MIF） genes that possess only the zinc finger. Phylogenetic analyses of ZHD genes from representative land plants suggest that non.seed plant ZHD genes occupy basal positions and angiosperm homologs form seven distinct clades. Several clades contain genes from two or more major angiosperm groups, including eudicots, monocots, magnoliids, and other basal angiosperms, indicating that several duplications occurred before the diversification of flowering plants. In addition, specific lineages have experienced more recent duplications. Unlike the ZHD genes, MIFs are found only from seed plants, possibly derived from ZHDs by loss of the homeodomain before the divergence of seed plants. Moreover, the MIF genes have also undergone relatively recent gene duplications. Finally, genome duplication might have contributed substantially to the expansion of family size in angiosperms and caused a high level of functional redundancy/overlap in these genes.