• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Chromosomal assembly of the Antarctic toothfish(Dissostichus mawsoni) genome using third-generation DNA sequencing and Hi-C technology

    2021-03-01 06:52:30SeungJaeLee,Jeong-HoonKim,EunaJo
    Zoological Research 2021年1期

    DEAR EDITOR,

    The Antarctic toothfish,Dissostichus mawsoni,belongs to the Nototheniidae family and is distributed in sub-zero temperatures below S60° latitude in the Southern Ocean.Therefore,it is an attractive model species to study the stenothermal cold-adapted character state.In this study,we successfully generated highly contiguous genome sequences ofD.mawsoni,which contained 1 062 scaffolds with a N50 length of 36.98 Mb and longest scaffold length of 46.82 Mb.Repetitive elements accounted for 40.87% of the genome.We also inferred 32 914 protein-coding genes usingin silicogene prediction and transcriptome sequencing and detected splicing variants using Isoform-Sequencing (Iso-Seq),which will be invaluable resource for further exploration of the adaptation mechanisms of Antarctic toothfish.This new high-quality reference genome ofD.mawsoniprovides a fundamental resource for a deeper understanding of cold adaptation and conservation of species.

    Many unique fish live in the Southern Ocean surrounding Antarctica within the coldest waters on Earth.It has been isolated by the Antarctic circumpolar current (Eastman,2005;Livermore et al.,2005) where sea temperatures range around the ice point (–1.9 °C) for most of the year.Antarctic fish,which include cold-adapted teleosts,are dominated by a single lineage belonging to the Perciformes suborder Notothenioidei.This suborder consists of eight families and>100 species and accounts for~90% of total fish biomass in the Antarctic Ocean (Eastman & De Vries,1981).From a common ancestor,a variety of closely related species with distinct ecological status as well as size,shape,and color have emerged in the Southern Ocean.Therefore,genomic research is essential to understand the environmental adaptation and evolution of these fish.

    The Antarctic toothfish,Dissostichus mawsoni,belongs to the family Nototheniidae of the order Perciformes and is native to the Southern Ocean.It is distributed below S60° latitude and is the largest of all Antarctic fish (2.0 m in length and 140 kg in mass) (Eastman & De Vries,1982).Its stenothermal cold-adapted state makes the species an attractive model for evolutionary and genomic studies among Antarctic fish.The Antarctic toothfish is an economically important fishery species,with a commercial catch in Subarea 88.1 of 2 680 tons in 2018 (Maschette et al.,2019)

    Recently,de novoassembly of the Antarctic toothfish genome and extensive transcriptomic characterization using short-read Illumina data have been reported (Chen et al.,2019),though the genome was fragmented into many scaffolds due to sequencing by synthesis technology.The development of third-generation single-molecule sequencing technology has enabled the production of long-read sequences and the discovery of the features of previously unavailable DNA regions.Here,we report on a re-assembled whole-genome ofD.mawsoniusing long-read sequencing and Hi-C technology,which should help provide comprehensive insight into its adaptive mechanisms.

    Antarctic toothfish (length~50 cm,sex not determined)were collected using a vertical setline in the eastern Ross Sea(Subarea 88.1),Antarctica (http://www.fao.org/fishery/area/Area88/en),during the austral summer season (December 2018).Specimens were killed for tissue sampling and then rapidly frozen for further analysis.All sample collection and experimental protocols were in compliance with the laws regarding activities and environmental protection in Antarctic and were approved by the Minister of Foreign Affairs and Trade of the Republic of Korea.

    To obtain sufficient high-quality DNA molecules for the PacBio Sequel platform (Pacific Biosciences,USA),oneD.mawsonifish was dissected and muscle tissue was used for DNA extraction using the phenol/chloroform extraction method.DNA quality was checked using a fragment analyzer system (Agilent Technologies,USA) and Qubit 2.0 fluorometer(Invitrogen,Life Technologies,USA).The single-molecule real-time (SMRT)bell library was sequenced using eight SMRT cells (Pacific Biosciences,SequelTMSMRT Cell 1M v2) with a Sequel Sequencing Kit 2.1 (Pacific Biosciences,USA) and 1×600 min movies were captured for each SMRT cell using the Sequel sequencing platform (Menlo Park,USA).The average coverage of the SMRT sequences was~81-fold(Supplementary Table S1).

    Muscle tissue from the same sample was used to construct a Hi-C chromatin contact map for chromosome-level assembly.Tissue fixation,chromatin isolation,and library construction were performed according to the manufacturer’s instructions (Dovetail Genomics,USA) (Belton et al.,2012).After checking the insert size,concentration,and effective concentration of the constructed libraries,the final libraries were sequenced using the Illumina NovaSeq 6 000 platform(San Diego,USA) with a 150-bp paired-end strategy.A total of 874 million raw reads were generated from the Hi-C libraries(Supplementary Table S1) and were mapped to the polishedD.mawsonicontigs using HiC-Pro (v2.8.0) with default parameters.

    For transcriptome sequencing,we prepared 1 μg of pooled total RNA from the muscle and skin ofD.mawsoni.Using a SMARTer PCR cDNA Synthesis Kit (Clontech,USA),RNA was synthesized to cDNA. The SMRTbell library was constructed using the SMRTbellTMTemplate Prep Kit 1.0-SPv3.The SMRTbell library was sequenced using SMRT cells(Pacific Biosciences,SequelTMSMRT Cell 1M v2) and the Sequel Sequencing Kit 2.1.For each SMRT cell,1×600 min movies were captured with the use of the Sequel sequencing platform; the pre-extension time was 240 min (Supplementary Table S1).The Iso-Seq sequencing data were analyzed using SMRT Link (v6.0.0).

    Forde novogenome assembly, the FALCON-Unzip assembler (v0.4,Falcon,RRID:SCR 016089) was used(Chin et al.,2013) with parameters of length_cutoff=12 000 and length_cutoff_pr=10 000 and with filtered subreads from SMRT Link (v4.0.0) (minimum subread length=50). To improve the quality of the genome assembly,the FALCONUnzip assembler was polished using the Arrow algorithm with unaligned BAM files as raw data.

    A draftD.mawsonigenome was previously generated using Illumina short-read sequencing (Chen et al.,2019).However,since several gaps prevailed in the draft genome set and there was no information about the linkage group,it was difficult to compare the structure of the Antarctic toothfish genome at the chromosomal scale.To improve this genome resource,longread SMRT sequencing from Pacific Biosciences and Hi-C scaffolding were implemented.First,we performedde novoassembly of the long PacBio sequence reads using the FALCON-Unzip tool and obtained a genome assembly with a size of 924.75 Mb,an N50 contig size of 3.23 Mb,and longest contig size of 24.49 Mb (Supplementary Table S2).To construct the reference genome at the chromosome level,we constructed a Hi-C library and anchored the scaffolds into chromosomes after quality control using the HiC-Pro,Juicer(v1.5) (Durand et al.,2016) and 3D-DNA (v170123) pipeline(Dudchenko et al.,2017) based on the draft genome assembly(Figure 1A).The assembled genome was 926.3 Mb (GC content:41.57%) in length with a scaffold N50 of 36.98 Mb and longest scaffold of 46.82 Mb.In total,there were 1 062 scaffolds in theD.mawsonigenome assembly,with 24 chromosome-scale scaffolds occupying 91.3% of the assembly (Supplementary Tables S2,S3 and Figure S1).

    Benchmarking Universal Single-Copy Orthologs (BUSCO)v3.0 (Sim?o et al.,2015) (RRID:SCR 015008) was used along with the actinopterygii odb9 database to assess the completeness of the newD.mawsonigenome assembly.Among 4 584 BUSCO groups searched,4 197 and 194 BUSCO core genes were completely and partially identified,respectively,contributing to a total of 95.7% BUSCO genes in theD.mawsonigenome (Supplementary Table S4).

    The diploid chromosome number (2n) ofD.mawsoniis 48(Ghigliotti et al.,2007).Comparison of its chromosome-scale assemblies with those of theGasterosteus aculeatusgenome(2n=42) showed a highly similar synteny (Figure 1B).However,each of three chromosomes (from Groups 1,4,and 7) ofG.aculeatusmatched with two chromosomes inD.mawsoni(scaffolds 23 and 35,scaffolds 13 and 22,scaffolds 11 and 24,respectively) (Supplementary Figure S2).

    Ade novorepeat library was constructed using RepeatModeler (v1.0.3) (Bao & Eddy,2002),which included RECON (RRID:SCR 006345) and RepeatScout v1.0.5(RRID:SCR 014653) (Price et al.,2005) software with default parameters.The Tandem Repeats Finder (Benson,1999)program was used to predict the consensus sequences and classification information for each repeat.We analyzed the repetitive sequences in theD.mawsonigenome,including those in the tandem repeats and transposable elements (TEs).TEs play an important role in shaping genome architecture and are a source of regulatory mutations in evolution.A difficulty arises in representing TEs in genome assemblies based on short Illumina sequence reads.Therefore,our longread sequences greatly improved both the length and quantity of the TE repeats in theD.mawsonigenome assembly compared to the published short-read assembly.TheD.mawsonigenome contained 40.87% of repeat sequences,including 36.27% (336 Mb) of TEs such as long terminal repeats (LTRs,4.21%),short interspersed nuclear elements(SINES,0.50%),long interspersed nuclear elements (LINEs,(5.35%),and DNA transposons (15.51%) (Supplementary Table S5 and Figure S3).Divergence of TEs was examined using RepeatMasker software, where Kimura distances(Kimura, 1980) estimated for aligned TEs(calcDivergenceFromAlign.pl) were used to draw repeat landscapes (createRepeateLandscape.pl).TheD.mawsonigenome had a higher number of recent TE insertions (Kimura divergenceK-values≤5) that were strongly shaped by DNA transposons (Supplementary Figure S4).BecauseK-values calculated for TEs can reflect age and transposition history(Chalopin et al.,2015),we concluded that there have been recent transposable element bursts in the Antarctic toothfish.

    Genome annotation was conducted using MAKER v2.28(RRID:SCR_005309) (Holt & Yandell,2011),which is a portable and easily configurable genome annotation pipeline.Subsequently,repeat masked genomes were used forab initiogene prediction with SNAP v2006–07-28 (SNAP,RRID:SCR 002127) (Korf, 2004) and Augustus (Augustus: Gene Prediction,RRID:SCR_008417) software.MAKER was initially run in the est2genome mode based on the Iso-Seq data,including 57 406 full-length transcripts.Additionally,protein evidence was obtained from the genomes of 19 teleosts,including three Antarctic fish (Supplementary Table S6).Exonerate software,which provides integrated information for the SNAP program,was used to polish MAKER alignments.MAKER was then used to select and revise the final gene model considering all available information.Other non-coding RNAs in the Antarctic toothfish assembly were identified using Infernal (v1.1) (Nawrocki & Eddy,2013) and covariance models (CMs) from the Rfam database v12.1 (Rfam,RRID:SCR 007891) (Griffiths-Jones et al.,2005).Putative tRNA genes were identified using tRNAscan-SE v1.3.1(tRNAscan-SE,RRID:SCR 010835) (Lowe & Eddy,1997),which uses a CM that scores candidates based on their sequences and predicted secondary structures.

    The predicted genes were aligned to the NCBI nonredundant protein (nr) (Benson et al.,1999),SwissProt(RRID:SCR_002380) (Boeckmann et al.,2003),TrEMBL(RRID:SCR_002380) (Boeckmann et al., 2003), KOG(EuKaryotic Orthologous Groups) (Tatusov et al.,2001),and KEGG (Kyoto Encyclopedia of Genes and Genomes,RRID:SCR_001120) (Kanehisa & Goto,2000) databases using BLAST v2.2.31 (Altschul et al.,1990) with a maximum e-value of 1e-5.Gene Ontology (GO) (RRID:SCR_002811)terms (Dimmer et al.,2012) were assigned to the genes using the Blast2Go v4.0 pipeline (RRID:SCR_005828) (Conesa et al.,2005).

    A total of 32 914 protein-coding genes in theD.mawsonigenome were annotated using a combination ofab initiogene prediction,homology searching,and transcript mapping.The coding sequence comprised 51.2 Mb (exons 55.2 Mb) with an average of 9.7 exons per gene (Supplementary Table S7).Consequently,a total of 20 202 genes were annotated in >1 database (Supplementary Table S7).A total of 24 920,19 205,and 14 474 genes were annotated in the GO,KOG,and KEGG databases, respectively, and the functional classifications of these genes are presented in Supplementary Figures S5–S7.

    We identified orthologous gene clusters using the OrthoMCL (Li et al.,2003) pipeline,which applied the Markov Clustering Algorithm (MCL) with default options in all steps for the genome sequences of the 20 species (Supplementary Table S6).It was critical for analysis to include representative species of diverse phylogenetic clades and the 20 species were selected among those with well-annotated and wellassembled genomes.

    Phylogenetic tree construction was performed based on single-copy orthologous genes.The sequences of proteincoding genes were aligned using a Probabilistic Alignment Kit(PRANK) (L?ytynoja & Goldman,2005) with the codon alignment option. The maximum-likelihood method was applied to construct a phylogenetic tree using RAxML with 1 000 bootstraps,and divergence times were calibrated with TimeTree (median estimates of pairwise divergence time forD.rerioandG.morhua:230.4 million years ago) (Hedges et al.,2006).

    Ortholog gene families from each species were identified:7 731 orthologous gene families were commonly identified among the 20 teleosts, including 434 (1 431 genes)paralogous gene families that wereD.mawsoni-specific(Supplementary Table S8).The phylogenetic tree ofD.mawsoniand the 19 teleost species was constructed using 1 422 single-copy orthologs (Figure 1C).Among the 20 fish species,D.mawsoniand three other Antarctic fish were clustered together on the branch of a non-Antarctic fish,G.aculeatus,with a divergence time of around 105 million years ago.Furthermore,D.mawsonidiverged approximately 28 million years ago from the Antarctic fishChaenocephalus aceratus.Analysis of gene gain-and-loss among genomes enables the reconciliation of a species tree with the gene tree for each family.Here,D.mawsonihad 659 (including 2 114 genes) significantly expanded and 116 (including 136 genes)significantly contracted gene families (Figures 1C,D).The vast majority of the expanded biological pathways belonged to two functional categories:(i) involved in nervous system functions (neuron projection development, GO:0 031175;neuron development, GO:0 048666; cell morphogenesis involved in neuron differentiation,GO:0 048667; generation of neurons,GO:0 048699; neuron projection morphogenesis,GO:0 048812; axon development, GO:0061564) and (ii)cellular component morphogenesis (cell projection organization, GO:0 030030; cell part morphogenesis,GO:0 032990; cell projection morphogenesis,GO:0048858).In the molecular function category,peptidase regulator activity(endopeptidase regulator activity,GO:0 061135; peptidase inhibitor activity,GO:0 030414; endopeptidase inhibitor activity,GO:0004866),and signaling receptor binding (endopeptidase inhibitor activity,GO:0004866) were the major expanded pathways (Supplementary Tables S9,S10).In addition,14 055 orthologous gene families containing 16 162 genes inD.mawsoniwere commonly identified in the four Antarctic fish.Moreover,621 gene families wereD.mawsonispeciesspecific paralogs (Figure 1E) involved in DNA metabolic processes (DNA biosynthetic process,GO:0 071897; DNA integration,GO:0 015074; RNA-dependent DNA biosynthetic process,GO:0006278) (Supplementary Table S11).

    Figure 1 Genome analysis of D.mawsoni

    Splicing variants were analyzed using SQANTI2 (Tardaguila et al.,2018) with the Iso-Seq data as full-length transcript sequences.The Iso-Seq data were aligned to the assembled genome using Minimap2 (Li,2018) and the collapsed highquality isoforms were aligned to unique isoforms using the Cupcake ToFU pipeline (Tseng,2017).SQANTI2 extracted various types of splicing variants using the collapsed isoforms and the gene prediction information of the assembled genome.After excluding novel,mono exonic,and antisense transcripts,genes were selected based on the ascending order of the number of isoforms.Enrichment analysis of genes of splicing variants was performed using the Blast2GO v4.0 pipeline (RRID:SCR_005828) (Conesa et al.,2005) with FDR<0.5.

    Iso-Seq data analysis identified 31 480 unique isoforms in 14 565 unique genes.Most novel genes were located in the intergenic region (Supplementary Tables S12, S13).Functional annotation using enrichment analysis by Fisher’s Exact Test for genes with more than 10 splicing variants(Supplementary Table S14) identified genes related to development,anatomical structure development,functional annotations (system development,GO:0 048731; animal organ development, GO:0 048513;tissue development,GO:0 009888; cell development, GO:0 048468; embryo development,GO:0 009790; muscle structure development,GO:0 061061; epithelium development,GO:0 060429; and circulatory system development, GO:0072359), and organization related function (cytoskeleton organization,GO:0 007010;protein-containing complex subunit organization,GO:0 043933; actin cytoskeleton organization,GO:0 030036; supramolecular fiber organization,GO:0 097435;and organelle organization,GO:0006996) (Supplementary Table S15).

    In the current study, we presented a high-quality chromosome-level genome assembly of the Antarctic toothfish,D.mawsoni,using PacBio Sequel sequencing and Hi-C chromatin contact maps.TheD.mawsonigenome assembly (926 Mb) included 24 chromosomes that accounted for 91% (840 Mb) of all genome sequences.TheD.mawsonigenome contained 32 914 protein-coding genes and 434 paralogousD.mawsoni-specific gene families among 20 teleost fish and 621 paralogousD.mawsoni-specific gene families among the four Antarctic teleost fish. This chromosome-length genome assembly will not only provide insights into the molecular and ecological adaptations of Antarctic fish to extreme environments but will also facilitate exploration of genomic adaptations to a wide range of evolutionary, ecological, metabolic, developmental, and biochemical features of Antarctic fish.

    DATA AVAILABILITY

    TheDissostichus mawsonigenome project was deposited in NCBI under BioProject No.PRJNA574770 and the Whole-Genome Shotgun project was deposited at DDBJ/ENA/GenBank under accession No.JAAKFY000000000.The version described in this paper is JAAKFY010000000.The genome browser, assembly, and annotation data are accessible on http://genome.kusglab.org/.

    SUPPLEMENTARY DATA

    Supplementary data to this article can be found online.

    COMPETING INTERESTS

    The authors declare that they have no competing interests.

    AUTHORS’ CONTRIBUTIONS

    H.P.and H.-W.K.conceived the study.S.J.L.,E.J.,S.-G.C.,S.C.E.C.,J.K.,and H.P.performed genome sequencing,assembly, and annotation. E.J. and S.J.L. performed experiments.S.J.L.,J.-H.K,H.-W.K.,and H.P.mainly wrote the manuscript.All authors contributed to writing and editing the manuscript as well as collating the supplementary information and creating the figures.All authors read and approved the final version of the manuscript.

    ACKNOWLEDGEMENTS

    We would like to thank Sunwoo Corporation for providing the Antarctic toothfish samples.

    欧美日韩亚洲综合一区二区三区_| 人人妻人人澡人人爽人人夜夜| 国内毛片毛片毛片毛片毛片| 少妇人妻久久综合中文| 午夜91福利影院| 激情视频va一区二区三区| 午夜福利一区二区在线看| 黄色视频,在线免费观看| 亚洲天堂av无毛| 国产激情久久老熟女| 亚洲精品国产av成人精品| 国产成人欧美| 久久久久精品人妻al黑| 如日韩欧美国产精品一区二区三区| 精品一区在线观看国产| 一级黄色大片毛片| 亚洲国产看品久久| 精品免费久久久久久久清纯 | 免费观看av网站的网址| 精品欧美一区二区三区在线| av电影中文网址| 成年av动漫网址| 十八禁网站免费在线| 91国产中文字幕| 伦理电影免费视频| 欧美成人午夜精品| 另类亚洲欧美激情| 久久精品亚洲av国产电影网| 亚洲精品久久午夜乱码| 91字幕亚洲| 国产精品二区激情视频| 国产黄频视频在线观看| 久久青草综合色| 欧美成人午夜精品| 深夜精品福利| 中文欧美无线码| 欧美人与性动交α欧美精品济南到| 大片电影免费在线观看免费| xxxhd国产人妻xxx| 国产成人欧美| 成在线人永久免费视频| 精品欧美一区二区三区在线| 九色亚洲精品在线播放| 亚洲av美国av| 久久精品久久久久久噜噜老黄| 精品国内亚洲2022精品成人 | 一级毛片女人18水好多| 91字幕亚洲| 女性生殖器流出的白浆| 国产区一区二久久| 大片免费播放器 马上看| 在线亚洲精品国产二区图片欧美| 国产精品.久久久| 亚洲色图 男人天堂 中文字幕| 91老司机精品| 午夜精品国产一区二区电影| 国产成人精品在线电影| 国产一区二区在线观看av| 午夜福利视频精品| 丝袜喷水一区| 亚洲七黄色美女视频| 色综合欧美亚洲国产小说| 久久精品亚洲熟妇少妇任你| 一区二区日韩欧美中文字幕| 久久性视频一级片| 日本av手机在线免费观看| 国产亚洲av高清不卡| 日韩电影二区| 超色免费av| 国产人伦9x9x在线观看| 黄色 视频免费看| 91麻豆精品激情在线观看国产 | 999久久久国产精品视频| 欧美激情极品国产一区二区三区| 久久精品久久久久久噜噜老黄| 少妇猛男粗大的猛烈进出视频| 纯流量卡能插随身wifi吗| 亚洲精品粉嫩美女一区| 亚洲伊人久久精品综合| 午夜福利影视在线免费观看| 国产精品久久久av美女十八| 亚洲精华国产精华精| 久久ye,这里只有精品| 97精品久久久久久久久久精品| 欧美在线黄色| 亚洲第一青青草原| 黄色怎么调成土黄色| 如日韩欧美国产精品一区二区三区| 香蕉国产在线看| 久9热在线精品视频| 亚洲一区二区三区欧美精品| 嫁个100分男人电影在线观看| 麻豆国产av国片精品| 中文字幕色久视频| 桃花免费在线播放| 欧美少妇被猛烈插入视频| 国产成人a∨麻豆精品| av免费在线观看网站| 自线自在国产av| 黄片小视频在线播放| 纵有疾风起免费观看全集完整版| 日本一区二区免费在线视频| 久久久久精品国产欧美久久久 | 高清在线国产一区| 在线观看免费高清a一片| 精品高清国产在线一区| 国产成人免费无遮挡视频| 免费在线观看日本一区| 免费久久久久久久精品成人欧美视频| 免费在线观看日本一区| 国产精品国产三级国产专区5o| 搡老熟女国产l中国老女人| 久久热在线av| 国产99久久九九免费精品| 午夜福利影视在线免费观看| 啦啦啦视频在线资源免费观看| 亚洲中文字幕日韩| 新久久久久国产一级毛片| 一区福利在线观看| videosex国产| 亚洲精华国产精华精| 欧美日韩亚洲综合一区二区三区_| 欧美人与性动交α欧美精品济南到| 亚洲伊人久久精品综合| 色播在线永久视频| 久久久久久免费高清国产稀缺| 日韩视频在线欧美| 中文字幕人妻丝袜制服| 两性夫妻黄色片| 制服人妻中文乱码| 美女国产高潮福利片在线看| 999久久久精品免费观看国产| 欧美日韩亚洲高清精品| 国产又爽黄色视频| 国产主播在线观看一区二区| 国产淫语在线视频| 91麻豆精品激情在线观看国产 | 久久久欧美国产精品| 欧美日本中文国产一区发布| 捣出白浆h1v1| 国产一区二区激情短视频 | 亚洲精品成人av观看孕妇| av在线app专区| 十八禁网站网址无遮挡| 他把我摸到了高潮在线观看 | 欧美黑人精品巨大| 午夜福利视频在线观看免费| 岛国在线观看网站| 国精品久久久久久国模美| 两性午夜刺激爽爽歪歪视频在线观看 | 18禁黄网站禁片午夜丰满| 大香蕉久久成人网| 免费人妻精品一区二区三区视频| 久久精品成人免费网站| 国产精品 国内视频| 欧美精品一区二区大全| 免费观看av网站的网址| 嫁个100分男人电影在线观看| 91精品三级在线观看| 女性生殖器流出的白浆| 黄频高清免费视频| 老司机影院毛片| 国产一区二区三区在线臀色熟女 | 99精国产麻豆久久婷婷| 老司机影院毛片| 一区二区三区精品91| 婷婷丁香在线五月| 日日摸夜夜添夜夜添小说| 精品一区二区三区av网在线观看 | 亚洲第一av免费看| 国产淫语在线视频| 亚洲精品美女久久av网站| 国产成人精品无人区| 永久免费av网站大全| 国产精品久久久久久人妻精品电影 | 一边摸一边做爽爽视频免费| 午夜激情av网站| 狠狠精品人妻久久久久久综合| 久久精品熟女亚洲av麻豆精品| 99精品欧美一区二区三区四区| 99精国产麻豆久久婷婷| 两个人免费观看高清视频| 天天影视国产精品| 久久毛片免费看一区二区三区| 成人亚洲精品一区在线观看| 亚洲人成77777在线视频| 午夜福利一区二区在线看| 亚洲久久久国产精品| 精品人妻熟女毛片av久久网站| 国产精品av久久久久免费| 欧美日韩成人在线一区二区| 免费观看a级毛片全部| 欧美亚洲 丝袜 人妻 在线| 亚洲精品日韩在线中文字幕| 99香蕉大伊视频| 大码成人一级视频| 男男h啪啪无遮挡| 岛国在线观看网站| 精品福利观看| av在线app专区| h视频一区二区三区| 欧美激情 高清一区二区三区| 亚洲精品美女久久av网站| 国产精品九九99| 18在线观看网站| 精品国产一区二区三区久久久樱花| 美女脱内裤让男人舔精品视频| 国产av精品麻豆| 精品福利永久在线观看| 在线观看免费午夜福利视频| 中文字幕最新亚洲高清| 美国免费a级毛片| 国产日韩欧美视频二区| 国产精品影院久久| 他把我摸到了高潮在线观看 | av在线老鸭窝| 中文字幕人妻丝袜一区二区| 日韩欧美免费精品| 亚洲精华国产精华精| 精品一区二区三区四区五区乱码| 精品一区二区三区av网在线观看 | 18禁黄网站禁片午夜丰满| 欧美久久黑人一区二区| 咕卡用的链子| 国产亚洲一区二区精品| 狠狠精品人妻久久久久久综合| 日韩欧美一区二区三区在线观看 | 久久久久精品国产欧美久久久 | 午夜精品国产一区二区电影| 欧美老熟妇乱子伦牲交| 最近最新中文字幕大全免费视频| 桃花免费在线播放| 脱女人内裤的视频| 午夜福利影视在线免费观看| av线在线观看网站| 成年人免费黄色播放视频| 亚洲av欧美aⅴ国产| 中文字幕色久视频| 中文字幕av电影在线播放| 国产亚洲av高清不卡| 在线永久观看黄色视频| 性色av乱码一区二区三区2| 国产欧美日韩一区二区精品| 亚洲免费av在线视频| 黑人欧美特级aaaaaa片| 男男h啪啪无遮挡| 不卡一级毛片| 久久久欧美国产精品| 午夜福利在线观看吧| 大码成人一级视频| 亚洲av成人一区二区三| 97精品久久久久久久久久精品| h视频一区二区三区| 亚洲av日韩在线播放| 久久久久久亚洲精品国产蜜桃av| 少妇猛男粗大的猛烈进出视频| 精品国产超薄肉色丝袜足j| 另类亚洲欧美激情| 久久人妻熟女aⅴ| 国产麻豆69| 这个男人来自地球电影免费观看| 99久久精品国产亚洲精品| 亚洲一卡2卡3卡4卡5卡精品中文| 男人操女人黄网站| 久久热在线av| 悠悠久久av| 日本黄色日本黄色录像| 电影成人av| 国产色视频综合| 欧美日韩福利视频一区二区| 国产高清国产精品国产三级| 亚洲一区中文字幕在线| 丁香六月天网| 女人久久www免费人成看片| 嫁个100分男人电影在线观看| 老司机福利观看| 美国免费a级毛片| 国产一级毛片在线| 久久亚洲精品不卡| 狠狠狠狠99中文字幕| 日韩制服骚丝袜av| 久久热在线av| 亚洲国产精品成人久久小说| 乱人伦中国视频| 精品人妻一区二区三区麻豆| 在线永久观看黄色视频| 大陆偷拍与自拍| 中文字幕人妻熟女乱码| 国产精品亚洲av一区麻豆| 成年女人毛片免费观看观看9 | 日日夜夜操网爽| 国产精品熟女久久久久浪| videos熟女内射| 91精品三级在线观看| 女人精品久久久久毛片| 黄色怎么调成土黄色| 久久人人爽人人片av| 在线精品无人区一区二区三| 这个男人来自地球电影免费观看| 大片电影免费在线观看免费| 五月天丁香电影| 精品人妻1区二区| 亚洲精品粉嫩美女一区| 最黄视频免费看| 国产成人免费无遮挡视频| 午夜视频精品福利| 欧美日韩中文字幕国产精品一区二区三区 | 国产伦人伦偷精品视频| 国产精品亚洲av一区麻豆| 精品高清国产在线一区| 自线自在国产av| 搡老熟女国产l中国老女人| 91精品三级在线观看| 国产有黄有色有爽视频| 日韩制服骚丝袜av| 久久 成人 亚洲| 老熟妇仑乱视频hdxx| 国产一卡二卡三卡精品| 免费女性裸体啪啪无遮挡网站| 99久久精品国产亚洲精品| 王馨瑶露胸无遮挡在线观看| 欧美日韩av久久| 亚洲第一av免费看| 高清视频免费观看一区二区| 中文字幕人妻丝袜制服| 国产麻豆69| 50天的宝宝边吃奶边哭怎么回事| 手机成人av网站| 久久国产精品男人的天堂亚洲| 人人妻人人爽人人添夜夜欢视频| 亚洲精品自拍成人| 国产激情久久老熟女| 九色亚洲精品在线播放| 欧美日韩黄片免| 亚洲av美国av| 亚洲国产av新网站| 黑人操中国人逼视频| 丝瓜视频免费看黄片| 老熟妇仑乱视频hdxx| 每晚都被弄得嗷嗷叫到高潮| av欧美777| 嫁个100分男人电影在线观看| 国产成人免费无遮挡视频| 乱人伦中国视频| 黄频高清免费视频| 大片电影免费在线观看免费| 又紧又爽又黄一区二区| 国产在线观看jvid| 国产伦人伦偷精品视频| 欧美久久黑人一区二区| 亚洲全国av大片| 热re99久久精品国产66热6| 涩涩av久久男人的天堂| 中文字幕另类日韩欧美亚洲嫩草| 99热网站在线观看| 免费日韩欧美在线观看| 一区福利在线观看| 久久精品亚洲av国产电影网| 亚洲精品国产一区二区精华液| 不卡av一区二区三区| 少妇粗大呻吟视频| 久久精品国产亚洲av香蕉五月 | 男女高潮啪啪啪动态图| 精品国内亚洲2022精品成人 | 欧美老熟妇乱子伦牲交| 香蕉国产在线看| 黄色a级毛片大全视频| 久久精品国产a三级三级三级| 黄色视频,在线免费观看| 国产国语露脸激情在线看| 精品高清国产在线一区| 午夜免费成人在线视频| 国产精品一区二区在线观看99| 日本vs欧美在线观看视频| 午夜福利影视在线免费观看| 国产成人系列免费观看| 国产伦人伦偷精品视频| 久久久国产一区二区| 777久久人妻少妇嫩草av网站| 一区福利在线观看| 精品福利永久在线观看| 伦理电影免费视频| 狠狠精品人妻久久久久久综合| 大陆偷拍与自拍| 高清在线国产一区| 国产在线一区二区三区精| 日韩熟女老妇一区二区性免费视频| 精品一区二区三区四区五区乱码| 免费日韩欧美在线观看| 精品人妻在线不人妻| 伊人亚洲综合成人网| 久久青草综合色| 亚洲成人免费av在线播放| 色精品久久人妻99蜜桃| 啦啦啦中文免费视频观看日本| 精品亚洲成a人片在线观看| 成年美女黄网站色视频大全免费| 精品欧美一区二区三区在线| 亚洲欧美日韩另类电影网站| 国产在线免费精品| 久久久精品国产亚洲av高清涩受| 不卡av一区二区三区| 久久人人97超碰香蕉20202| 国产精品九九99| 亚洲综合色网址| 97人妻天天添夜夜摸| av欧美777| 国产黄频视频在线观看| 久久久久久久久免费视频了| 成人av一区二区三区在线看 | 2018国产大陆天天弄谢| 99热全是精品| videosex国产| 少妇人妻久久综合中文| 搡老熟女国产l中国老女人| 亚洲伊人色综图| 国产成人精品无人区| 国产精品一区二区免费欧美 | 男女边摸边吃奶| 看免费av毛片| 新久久久久国产一级毛片| 99国产精品一区二区蜜桃av | 男人添女人高潮全过程视频| 免费看十八禁软件| 久久青草综合色| 91成人精品电影| 色精品久久人妻99蜜桃| 日日爽夜夜爽网站| 午夜免费观看性视频| 一级,二级,三级黄色视频| 国产免费视频播放在线视频| 天天影视国产精品| 视频在线观看一区二区三区| 脱女人内裤的视频| 91成人精品电影| 亚洲精品自拍成人| 热re99久久国产66热| avwww免费| 亚洲色图 男人天堂 中文字幕| 在线观看免费日韩欧美大片| 精品一区二区三区四区五区乱码| 女人爽到高潮嗷嗷叫在线视频| 热99久久久久精品小说推荐| 亚洲天堂av无毛| 免费日韩欧美在线观看| 久久天堂一区二区三区四区| 菩萨蛮人人尽说江南好唐韦庄| 国产亚洲av高清不卡| 亚洲黑人精品在线| 国产欧美日韩一区二区精品| 国产免费一区二区三区四区乱码| 国产免费av片在线观看野外av| 久久精品久久久久久噜噜老黄| 色婷婷久久久亚洲欧美| 亚洲第一青青草原| 两人在一起打扑克的视频| 老司机在亚洲福利影院| 亚洲国产日韩一区二区| 成年女人毛片免费观看观看9 | 国产欧美日韩一区二区三区在线| 狠狠婷婷综合久久久久久88av| 丰满迷人的少妇在线观看| 国产在线观看jvid| 如日韩欧美国产精品一区二区三区| 美国免费a级毛片| 国产91精品成人一区二区三区 | 久久这里只有精品19| 久热爱精品视频在线9| 亚洲国产日韩一区二区| 午夜成年电影在线免费观看| 一个人免费看片子| 欧美人与性动交α欧美精品济南到| 国产在视频线精品| www.999成人在线观看| 黑人猛操日本美女一级片| 亚洲专区中文字幕在线| 天天操日日干夜夜撸| 王馨瑶露胸无遮挡在线观看| 日韩欧美一区二区三区在线观看 | 国产成人av激情在线播放| 国产日韩欧美亚洲二区| 亚洲人成电影观看| 欧美乱码精品一区二区三区| 99re6热这里在线精品视频| 日韩中文字幕视频在线看片| 黄网站色视频无遮挡免费观看| 大陆偷拍与自拍| 青春草亚洲视频在线观看| 搡老岳熟女国产| 日本一区二区免费在线视频| 精品福利观看| 国产一区二区三区在线臀色熟女 | 午夜久久久在线观看| 99久久人妻综合| 亚洲色图 男人天堂 中文字幕| 国产精品一区二区精品视频观看| 黄色视频不卡| 久久ye,这里只有精品| kizo精华| 久久人人爽av亚洲精品天堂| 亚洲,欧美精品.| 十分钟在线观看高清视频www| 又黄又粗又硬又大视频| 久久精品人人爽人人爽视色| av一本久久久久| 久久精品熟女亚洲av麻豆精品| 日本vs欧美在线观看视频| 老汉色∧v一级毛片| 一级,二级,三级黄色视频| 欧美人与性动交α欧美软件| 国产一区二区三区综合在线观看| 亚洲avbb在线观看| 丝袜喷水一区| 操出白浆在线播放| 另类精品久久| 国产色视频综合| 欧美日韩精品网址| 国产成人精品久久二区二区91| 搡老熟女国产l中国老女人| 欧美黄色片欧美黄色片| 高清av免费在线| 久久久国产欧美日韩av| 人人妻人人澡人人看| 久久久久久免费高清国产稀缺| 亚洲国产欧美一区二区综合| 美国免费a级毛片| 国产三级黄色录像| 黄色a级毛片大全视频| 蜜桃在线观看..| 少妇的丰满在线观看| 亚洲全国av大片| 免费观看a级毛片全部| 一边摸一边做爽爽视频免费| 国产福利在线免费观看视频| 王馨瑶露胸无遮挡在线观看| 99国产精品免费福利视频| 国产成人啪精品午夜网站| av在线老鸭窝| 性少妇av在线| 婷婷成人精品国产| 十八禁人妻一区二区| 9热在线视频观看99| 国产精品久久久久久人妻精品电影 | 99精国产麻豆久久婷婷| 啦啦啦中文免费视频观看日本| 亚洲视频免费观看视频| 国产精品免费大片| 亚洲成人免费av在线播放| 少妇人妻久久综合中文| 老熟妇仑乱视频hdxx| 后天国语完整版免费观看| 美女国产高潮福利片在线看| 国产成人精品在线电影| 99九九在线精品视频| videosex国产| 国产成人免费无遮挡视频| 女性生殖器流出的白浆| 久久国产亚洲av麻豆专区| 成年人免费黄色播放视频| 久久热在线av| 久久久欧美国产精品| 一级毛片电影观看| 男女无遮挡免费网站观看| 91麻豆av在线| 欧美97在线视频| 久久久久久亚洲精品国产蜜桃av| 欧美+亚洲+日韩+国产| 精品熟女少妇八av免费久了| 美女视频免费永久观看网站| 18在线观看网站| 一二三四在线观看免费中文在| 欧美精品一区二区大全| 色老头精品视频在线观看| 51午夜福利影视在线观看| 高清欧美精品videossex| 国产av精品麻豆| 亚洲av成人一区二区三| 人人妻人人澡人人爽人人夜夜| 少妇猛男粗大的猛烈进出视频| videosex国产| 国产99久久九九免费精品| 人人妻人人澡人人看| 99热全是精品| 在线看a的网站| 亚洲中文日韩欧美视频| 亚洲中文av在线| 99久久综合免费| 亚洲性夜色夜夜综合| 午夜福利影视在线免费观看| 丝袜美腿诱惑在线| 超碰成人久久| 久久人妻熟女aⅴ| 国产亚洲精品一区二区www | 高清在线国产一区| 亚洲精品久久成人aⅴ小说| 人妻久久中文字幕网| 日韩视频在线欧美| 国产人伦9x9x在线观看| 亚洲av美国av| 在线观看免费高清a一片| 国产伦人伦偷精品视频| 亚洲欧美精品综合一区二区三区| 1024视频免费在线观看| 性色av一级| bbb黄色大片| 极品少妇高潮喷水抽搐| 777米奇影视久久| 亚洲精品久久成人aⅴ小说| 正在播放国产对白刺激| 高清视频免费观看一区二区| 精品国内亚洲2022精品成人 | 精品国产一区二区久久| 人人妻,人人澡人人爽秒播| av又黄又爽大尺度在线免费看| 男人添女人高潮全过程视频|