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Additional file 2: Fig S1. Genome-wide Hi-C interaction map. Fig S2. Divergence distributions of four TE sequences predicted by the de novo method (a) and comparison of the distribution of several features in the final gene set for seven fish species (b). TE, transport elements; DNA, DNA transposons; LTR, long terminal repeats; LINE, long interspersed nuclear elements; SINE, short interspersed nuclear element. The seven fish species: Misgurnus anguillicaudatus, Carassius auratus, Cyprinus carpio, Danio rerio, Sinocyclocheilus graham, S. rhinocerous, and Triplophysa tibetana. Fig S3. The construction of maximum likelihood tree of fos gene and generation of the fos (ID: Mis0086400.1) knockout loach (fos−/−) by CRISPR/Cas9 technology. (a) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of fos gene in fish species. Different branch colors represent different species. Red words indicate the knockout of fos gene in loach genome. (b) Schematic position of the CRISPR/Cas9 target site for fos gene knockout. (c) The transcription level of fos in livers of wild-type loach (WT) and fos−/− loach. (d) A statistics of survival rate of fertilized eggs of WT loach and heterozygous F1 generation (F1 generation self-crossed). *** extremely significant difference (p < 0.001). hpf, hours post fertilization; fos, Proto-oncogene c-Fos. The gene in red color is a positively selected gene in the loach genome. Fig S4. Identification and expression analysis of air-breathing- and digestion/absorption- related genes in loach Misgurnus anguillicaudatus. (a) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of hb gene family. Different color backgrounds represent different genes. Different branch colors and inner circle colors represent different species. hbb, hemoglobin subunit beta; hba, hemoglobin subunit alpha. (b) Expression levels of hbb and hba in posterior intestines of the loach under air exposure. (c) qPCR validation of RNA-seq data of loach posterior intestines under air exposure. (d) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of air-breathing-related genes. Different branch colors represent different species. (e) Maximum likelihood tree of digestion/absorption-related genes (TBLASTN (with e-value of 1×10−5)). Atp1a, sodium/potassium-transporting ATPase subunit; ryr, ryanodine receptor. Different color backgrounds represent different genes. Different branch colors represent different species. Npr2, atrial natriuretic peptide receptor 2; f9, coagulation factor IX; hspb1, heat shock protein beta-1; hyou1, hypoxia up-regulated protein 1; ptafr, platelet-activating factor receptor; scx, basic helix-loop-helix transcription factor scleraxis; galnt8, polypeptide N-acetylgalactosaminyltransferase 8; krt13, keratin, type I cytoskeletal 13 Cytokeratin-13; gp2, pancreatic secretory granule membrane major glycoprotein; smco3, single-pass membrane and coiled-coil domain-containing protein 3; ccl5, C-C motif chemokine 5; ifi44, interferon-induced protein 44; cldn5, claudin-5 Transmembrane protein deleted in VCFS; hspb1, heat shock protein beta-1; vegfr1 (flt1), vascular endothelial growth factor receptor 1; ctgf, connective tissue growth factor CCN family member 2. Fig S5. Identification of FMO and UGT gene families. (a) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of FMO gene family. Different colors represent different FMO gene families and different branch colors represent different species. (b) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of UGT gene family. Different colors represent different UGT gene families and different inner circle colors represent different species. Fig S6. qPCR validation of RNA-seq data from the drug stress trial. Fig S7. The expression levels of ugt genes in liver tissues of the loach under five drug stress. Mis115330.1 and Mis0135560.1 (ugt2a2), UDP-glucuronosyltransferase 2A2; Mis0072610.1 (ugt2a1), UDP-glucuronosyltransferase 2A1. Fig S8. Tissue expression analysis and the knockout of fmo5 (ID: Mis0185930.1) gene (fmo5−/−) in loach Misgurnus anguillicaudatus genome. (a) Tissue expression analysis of fmo5 (ID: Mis0185930.1) of the loach. (b) Schematic position of the CRISPR/Cas9 target site for fmo5 gene knockout. (c) The mRNA and protein expression levels of fmo5 in livers of wild-type (WT) and fmo5−/− loach. Different letters above error bars indicate significant difference among different tissues (p < 0.05); *** extremely significant difference (p < 0.001). Fig S9. Survival rates of wild-type (WT) and fmo5deletion (fmo5−/−) loach under five drug stress.

Additional file 1: Table S1. Statistics of assembly and annotation of loach Misgurnus anguillicaudatus genome. (1) Statistics of Illumina HiSeq sequencing data of the loach genome. (2) 17-kmer analysis for estimation of the loach genome size. (3) Hi-C library sequencing data of the loach. (4) Statistics of repeated sequence classification of the loach genome. (5) Statistics of gene annotation of the loach genome. (6) Statistics of gene functional annotation of the loach genome. Table S2. Detailed gene information related to mud-dwelling behavior and intestinal evolution (air-breathing and digestion/absorption) of loach Misgurnus anguillicaudatus. (1) GO enrichment analysis of the expanded myosin complex genes in the loach genome. (2) KEGG enrichment analysis of the positively selected gene involved in osteoclast differentiation in the loach genome. (3) GO enrichment analysis of the expanded gene families involved in oxygen transport in the loach genome. Red and black gene IDs present hbb and hba genes, respectively. (4) KEGG enrichment analysis of the positively selected genes involved in VEGF signaling pathway in the loach genome. (5) Expression analysis of some DEGs in posterior intestine transcriptomes between Leptobotia elongate (LE, without air-breathing) and the loach (MA, with intestinal air-breathing) (referred from our previous study). (6) Summary of detected microRNAs and target genes involved in vascular biology of loach posterior intestines (referred from our previous study). (7) The DEGs involved in intestinal air-breathing and nutrient uptake of the loach (referred from our previous studies) (8) Expression analysis of five key DEGs in posterior intestine transcriptomes of the loach between the control (C_chang) and air exposure (T_chang) group. Un means Mis0158000.1, which is a new gene and its KEGG annotation is interleukin 1 beta (Il1b). (9) KEGG enrichment analysis of the contracted gene families involved in digestion/absorption of the loach. (10) KEGG enrichment analysis of the expanded genes involved in digestion/absorption of the loach. hbb, hemoglobin subunit beta; hba, hemoglobin subunit alpha; VEGF, vascular endothelial growth factor; DEGs, differentially expressed genes; Il1b, Interleukin-1 beta; cldn5, Claudin-5 Transmembrane protein deleted in VCFS; hspb1, heat shock protein beta-1; vegfr1 (flt1), vascular endothelial growth factor receptor1; ctgf, Connective tissue growth factor CCN family member 2; hif1a, hypoxia-inducible factor 1-alpha. Table S3. Genes involved in detoxification function of loach Misgurnus anguillicaudatus. (1) The expanded genes involved in the detoxification function of the loach genome. (2) KEGG enrichment analysis of DEGs involved in the xenobiotics biodegradation and metabolism between the control and five drug stress groups. Red and black gene IDs present fmo and ugt genes, respectively. DEGs, differentially expressed genes. Table S4. Gene annotations for the zebrafish gene clusters in this study. Fos, proto-oncogene c-Fos; hba, hemoglobin subunit alpha; hbb, hemoglobin subunit beta; ryr, ryanodine receptor; atp1a, sodium/potassium-transporting ATPase subunit alpha; cldn5, claudin-5; vegfr1, vascular endothelial growth factor receptor 1; hspb1, heat shock protein beta-1; ctgf, connective tissue growth factor CCN family member 2; fmo, dimethylaniline monooxygenase [N-oxide-forming]; ugt, UDP-glucuronosyltransferase. Table S5. Primers used in this study. qPCR, quantitative PCR; # indicated T7 promoter sequences. Fos, proto-oncogene c-Fos; fmo5, dimethylaniline monooxygenase [N-oxide-forming] 5 (Mis0185930.1); hbb, hemoglobin subunit beta; hba, hemoglobin subunit alpha; atf6, cyclic AMP-dependent transcription factor; eif2ak3, eukaryotic translation initiation factor 2-alpha kinase 3; chop, DNA damage-inducible transcript 3 protein; muc2, mucin-2; Mis0185950.1, Mis0185940.1, Mis0185920.1, Mis0185970.1, Mis0186000.1, Mis0186010.1 (fmo5), dimethylaniline monooxygenase [N-oxide-forming] 5; Mis0115330.1, Mis0115330.1, Mis0135560.1 (ugt2a2), UDP-glucuronosyltransferase 2A2; Mis0072610.1 (ugt2a1), UDP-glucuronosyltransferase 2A1; npr2, atrial natriuretic peptide receptor 2; f9, coagulation factor IX; hspb1, heat shock protein beta-1; hyou1, hypoxia up-regulated protein 1; ptafr, platelet-activating factor receptor; scx, basic helix-loop-helix transcription factor scleraxis; galnt8, polypeptide N-acetylgalactosaminyltransferase 8; krt13, keratin, type I cytoskeletal 13 Cytokeratin-13; gp2, pancreatic secretory granule membrane major glycoprotein; smco3, single-pass membrane and coiled-coil domain-containing protein 3; ccl5, C-C motif chemokine 5; ifi44, interferon-induced protein 44; atf4, cyclic AMP-dependent transcription factor 4; ugt1a1, UDP-glucuronosyltransferase 1-1. Table S6. A summary of sequencing data used in genome assembly and annotation of Misgurnus anguillicaudatus.

学位の種別: 課程博士, 審査委員会委員 : （主査）東京大学教授 浅川 修一, 東京大学教授 塩見 美喜子, 東京大学准教授 伊藤 直樹, 東京大学准教授 木下 滋晴, 東京大学准教授 鈴木 道生

To predict the annual total yields of Chinese aquatic products in future five years (2011-2015), based on the theory and method of gray system, this paper firstly establishes a conventional GM (1,1) model and a gray metabolic GM (1,1) model respectively to predict the annual total yields of Chinese aquatic products in 2006-2009 and compare the prediction accuracy between these two models. Then, it selects the model with higher accuracy to predict the annual total yields of Chinese aquatic products in future five years. The comparison indicates that gray metabolic GM (1,1) model has higher prediction accuracy and smaller error, thus it is more suitable for prediction of annual total yields of aquatic products. Therefore, it adopts the gray metabolic GM (1,1) model to predict annual total yields of Chinese aquatic products in 2011-2015. The prediction results of annual total yields are 55.32, 57.46, 59.72, 62.02 and 64.43 million tons respectively in future five years with annual average increase rate of about 3.7%, much higher than the objective of 2.2% specified in the Twelfth Five-Year Plan of the National Fishery Development (2011 to 2015). The results of this research show that the gray metabolic GM (1,1) model is suitable for prediction of yields of aquatic products and the total yields of Chinese aquatic products in 2011-2015 will totally be able to realize the objective of the Twelfth Five-Year Plan.