| Grass carp(Ctenopharyngodon idella)is one of the most important aquaculture species.It has a breeding history of more than 1,700 years in China.Artificial breeding of grass carp began in 1958,which is the most productive species in freshwater fish farming in the world and has great economic value.B lymphocytes are leukocytes with important immune functions.They play an important role in the process of resisting pathogenic infection by recognizing antigens to undergo immune activation,proliferation,differentiation,and finally secrete antibodies to mediate humoral immunity.In mammalian studies,B cells can be transdifferentiated into macrophages by gene editing and transgenic technology to acquire phagocytic capacity,while in teleost B cells phagocytosis can proceed spontaneously with pathogen clearance and antigen presentation.Therefore,some studies have proposed that B cells evolved from macrophages.However,function-based comparisons do not well illustrate the relationship between phagocytic B cells and innate immune cells,and more in-depth exploration at the molecular level is required.On the one hand,high-quality reference genome can provide guidance for genetic trait improvement,molecular-guided breeding and pathological research,and on the other hand,more complete genomic information can enable deeper studies on gene expression regulation.The main results of this paper are as follows:1.Whole-genome sequencing and comparative genomic analysis of grass carpThe grass carp genome was de novo sequenced and assembled using Pac Bio thirdgeneration sequencing and chromosome conformation capture technology,and a chromosome-level reference genome was obtained.The total genome length was 893.2 Mb,the Scaffold N50 was 35.7 Mb,and 891.9 Mb(99.85%)of the assembled sequences were anchored to 24 haploid chromosomes.A total of 30,342 protein-coding genes and 43.26%repeat sequences were annotated by the prediction method combining de novo,transcripts and homologous proteins.The DNA transposon content in repeat sequences is positively correlated with the genome size of cyprinids.Compared with the published grass carp genome based on second-generation sequencing,BUSCO analysis revealed that the genome assembly here is 95.7% complete,higher than 83.6% complete in the published genome based on second-generation sequencing technology.Species evolution analysis showed that grass carp and blunt snout bream are most closely related,with the estimated divergence time of approximately 23 million years.Comparative genome analysis revealed that grass carpspecific gene families,expansion and contraction gene families,positive selection genes,and genes with altered expression regulation were related to the adaptive evolution of grass carp.The functional enrichment analysis of these genes and gene families indicated that the coevolution of the immune system,nervous system and digestive system contributed to the herbivorous characteristics and unique body shape of grass carp.2.Comparative transcriptome analysis of phagocytic and non-phagocytic B cells derived from grass carp head kidneyTranscriptome analysis of phagocytic and non-phagocytic B cells found that the differentially expressed genes could be significantly divided into four trends,among which,P1 and P6 were phagocytic phenotype-dependent trends(P1 was down-regulated,P6 was up-regulated),and there was no difference between phagocytes.P0 and P7 are phagocytic ability-dependent trends(P0 was continuously down-regulated,P7 was continuously upregulated).Further functional enrichment analysis revealed that in the down-regulated trends,P1 was associated with transcription factor activity,P0 with BCR receptors and MHC protein complexes.In the up-regulated trends,P6 was associated with lysosomes,folds,and adherent spots,and P7 was associated with integrin complexes,cell adhesion,and presynaptic active zone cytoskeleton.GSEA analysis showed that activation of complement and coagulation cascade signaling pathway plays an important role in phagocytic B cells,whereas the B-cell receptor signaling pathway was inhibited in phagocytic B cells,and the inhibition became more significant with the enhancement of phagocytic ability.In addition,we identified multiple macrophage and monocyte phagocyte marker genes up-regulated in phagocytic B cells,such as cd11 b,f4/80,mrc1,m-csfr,g-csfr and marco.as well as upregulation of key transcription factors regulating macrophage development,such as pu.1,c/ebpα and c/ebpβ.Up-regulation of key genes and transcription factors regulating B cell development,such as cd79 a,cd79b,cd22,pax5,irf4,irf8,tcf4,etc.,were identified in non-phagocytic B cells.Notably,two key genes regulating epigenetic remodeling were extremely significantly downregulated in phagocytic B cells,such as kdm6(kdm6a,kdm6b)and egr(egr1,egr2,egr2 b,and egr3).In addition,based on cross-species comparative transcriptome analysis,we propose a vertebrate B cell evolution model: mammalian B1 ← teleost phagocytic B cells →teleost non-phagocytic B cells → mammalian B2(arrows indicate evolutionary direction).The above results illustrate the innate immunophenotype of phagocytic B cells.The acquisition of this phenotype is accompanied by the continuous negative regulation of the BCR signaling pathway,and the activation of genes and pathways related to cell adhesion and integrin complexes,which may be the molecular basis for the acquisition of phagocytic ability of teleost B cells.3.Chromosome accessibility analysis of phagocytic and non-phagocytic B cells derived from grass carp head kidneyFrom comparative transcriptome analysis we concluded that kdm6 b,a histone demethylase that specifically removes trimethylation of the 27 th lysine on histone H3(H3K27me3),may play an important role in regulating the phagocytic phenotype of B cells.H3K27 trimethylation is a repressive epigenetic mark that regulates chromatin state and gene silencing.We compared the chromatin accessibility of the three groups,and with the acquisition of the phagocytic phenotype and the enhancement of phagocytic capacity,the degree of chromatin openness in the promoter regions continued to decrease.Analysis of differential ACRs showed that most of the ACRs were closed as the phagocytic phenotype was acquired and phagocytosis was enhanced.Differential ACRs and differentially expressed gene association analysis showed that the degree of chromatin opening and gene expression were positively correlated in phagocytic and non-phagocytic B cells.Functional enrichment of down-regulated genes associated with the turned-off ACRs showed that the BCR signaling pathway was continuously negatively regulated with the acquisition of phagocytic phenotype and enhanced phagocytosis,and these genes included cd79 a,cd79b,lyn,blnk,etc.Chromatin accessibility was reduced in the promoter regions of IRF4 and IRF8,which are important transcription factors regulating B cell development.In mammalian studies,chromatin conformation is opened and genome-wide chromatin accessibility is increased along with activation of identity-determining genes and transcription factors during differentiation into downstream cell subsets after naive B cell activation.These are contrary to our results,indicating that phagocytic B cells are not downstream cells of normal differentiation after B cell activation and may undergo a process of transdifferentiation.4.Genome-wide DNA methylation analysis of phagocytic and non-phagocytic B cells derived from grass carp head kidneyDNA methylation is generally considered to be a relatively stable transcriptional repressive epigenetic mark.In our results,with the acquisition of the phagocytic phenotype and the enhancement of phagocytic capacity,the methylation level of genome-wide DNA gradually increased,and the differentially methylated regions were more hypermethylated than hypomethylated in phagocytic B cells.Functional enrichment analysis of DEGs associated with differentially methylated regions showed that cell adhesion,cell-matrix adhesion junctions,Rap1 signaling pathway,and BCR signaling pathway were significantly enriched in phagocytic B cells,suggesting that these pathways play important roles in the functional transformation of B cells.Studies in mammals have shown that activation-induced hypomethylation is a common and critical step in the differentiation of B cells into downstream B cell subsets(germinal center B cells,plasma cells,and memory B cells).This is the opposite of our results.Combined with chromatin accessibility and transcriptomic data,it again illustrates that phagocytic B cell is not a downstream branch of B cell differentiation. |
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