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  • Supplementary Information for

    Whole-genome sequence of a flatfish provides insights into ZW sex

    chromosome evolution and adaptation to a benthic lifestyle

    Songlin Chen1,10,11

    , Guojie Zhang2,10

    , Changwei Shao1,10

    , Quanfei Huang2,10

    , Geng Liu 2,10

    , Pei Zhang2,10

    , Wentao Song1, Na An

    2, Domitille Chalopin

    3, Jean-Nicolas Volff

    3,

    Yunhan Hong4, Qiye Li

    2, Zhenxia Sha

    1, Heling Zhou

    2, Mingshu Xie

    1, Qiulin Yu

    2, Yang

    Liu5, Hui Xiang

    6, Na Wang

    1, Kui Wu

    2, Changgeng Yang

    1, Qian Zhou

    2, Xiaolin Liao

    1,

    Linfeng Yang2, Qiaomu Hu

    1, Jilin Zhang

    2, Liang Meng

    1, Lijun Jin

    2, Yongsheng Tian

    1,

    Jinmin Lian2, Jingfeng Yang

    1, Guidong Miao

    1, Shanshan Liu

    1, Zhuo Liang

    1, Fang Yan

    1,

    Yangzhen Li1, Bin Sun

    1, Hong Zhang

    1, Jing Zhang

    1,Ying Zhu

    1, Min Du

    1, Yongwei

    Zhao1, Manfred Schartl

    7,11, Qisheng Tang

    1,11& Jun Wang

    2,8,9,11

    1Yellow Sea Fisheries Research Institute, CAFS, Key Laboratory for Sustainable

    Development of Marine Fisheries, Ministry of Agriculture, Qingdao 266071, China. 2BGI-Shenzhen, Shenzhen 518000, China.

    3Institut de Gnomique Fonctionnelle de Lyon,

    Universit de Lyon, CNRS, INRA, Ecole Normale Suprieure de Lyon, Lyon, France. 4Department of Biological Sciences, National University of Singapore, Science Drive 4,

    Singapore 117543, Singapore.5Dalian Ocean University, Heishijiao Street 52, Dalian

    116023, China.6State Key Laboratory of Genetic Resources and Evolution, Kunming

    Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, 650223, China. 7Physiologische Chemie I, University of Wrzburg, Biozentrum, Am Hubland, and

    Comprehensive Cancer Center, University Clinic Wrzburg, Josef Schneider Strae 6,

    D-97074 Wrzburg, Germany.8Department of Biology, University of Copenhagen,

    Universitetsparken 15, Kbenhavn, 2100, Denmark.9Princess Al Jawhara Center of

    Excellence in the Research of Hereditary Disorders, King Abdulaziz University, Jeddah,

    Saudi Arabia. 10

    Theseauthors contributed equally to this work.11

    These authors jointly

    directed this work.

    Correspondence should be addressed to J. W. (wangj@genomics.org.cn), S.C.

    (chensl@ysfri.ac.cn), M.S.(phch1@biozentrum.uni-wuerzburg.de) or Q.T.

    (ysfri@public.qd.sd.cn).

    Nature Genetics: doi:10.1038/ng.2890

  • 2

    Supplementary Information

    Supplementary Figures 1-38 .................................................................................................. 6

    Supplementary Figure 1. Distribution of sequencing depth of the assembled female

    genome by reads from the female and male samples. ........................................................................ 6

    Supplementary Figure 2. Distribution of 17-mers in the usable sequencing reads from the

    female sample. ......................................................................................................................................... 7

    Supplementary Figure 3. Distribution of 17-mers in the usable sequencing reads from the

    male sample. ............................................................................................................................................. 8

    Supplementary Figure 4. Phylogenetic tree of Cynoglossus semilaevis retroelements based

    on reverse transcriptase alignment. ....................................................................................................... 9

    Supplementary Figure 5. Phylogenetic tree of Cynoglossus semilaevis long terminal repeat

    (LTR) retroelements based on reverse transcriptase alignment. .................................................... 10

    Supplementary Figure 6. Phylogenetic tree of Cynoglossus semilaevis long interspersed

    nuclear elements (LINE) retroelements based on reverse transcriptase alignment. .................... 11

    Supplementary Figure 7. Distribution of divergence rate of each type of TEs in the tongue

    sole genome. .......................................................................................................................................... 12

    Supplementary Figure 8. Venn diagram showing supporting evidence for the reference

    gene set. ................................................................................................................................................. 13

    Supplementary Figure 9. Comparisons of gene parameters among tongue sole, medaka,

    Takifugu, Tetraodon, stickleback and zebrafish genomes. .............................................................. 14

    Supplementary Figure 10. Statistics of orthologous families for zebrafish, tongue sole,

    Tetraodon, Takifugu, stickleback, and medaka (representing Osteichthyes), human

    (representing mammals), and chicken (representing birds). ........................................................... 15

    Supplementary Figure 11. Venn diagram showing shared orthologous groups for

    Pleuronectiformes (tongue sole), Tetraodontidae (Takifugu and Tetraodon), Smegmamorpha

    (medaka and stickleback), and Cypriniformes (zebrafish). ............................................................ 16

    Supplementary Figure 12. Distribution of protein identities of orthologs between human

    and fish species and chicken in all single-copy families. ............................................................... 17

    Supplementary Figure 13. Dynamic evolution of gene families. ............................................... 17

    Supplementary Figure 14. qRT-PCR analysis of positively selected genes and

    differentially expressed genes between pre- and post-metamorphosis fish. ................................ 18

    Supplementary Figure 15. Phylogenetic tree using all single-copy orthologs. ......................... 19

    Supplementary Figure 16. Estimation of divergence time. ......................................................... 19

    Supplementary Figure 17. Reconstructed vertebrate ancestral chromosomes. ......................... 20

    Supplementary Figure 18. Model of teleost genome evolution. ................................................. 21

    Supplementary Figure 19. Rectangular dot plots show chromosomal locations of

    Z-orthologous genes. ............................................................................................................................ 22

    Supplementary Figure 20. Structure of sex chromosomes. ......................................................... 23

    Supplementary Figure 21. Distribution of Ks for Z-W gene pairs in the non-PAR region. .... 24

    Supplementary Figure 22. Dosage compensation of the Z chromosome in tongue sole. ....... 25

    Supplementary Figure 23. Up-regulation of Z gene expression in females. ............................. 26

    Nature Genetics: doi:10.1038/ng.2890

  • 3

    Supplementary Figure 24. Methylation status across the differentially methylated region

    (DMR) of dmrt1, sf-1, patched1, follistatin, and neurl3 genes. ..................................................... 27

    Supplementary Figure 25. Gonad histological structure at different developmental stages

    in Cynoglossus semilaevis. .................................................................................................................. 29

    Supplementary Figure 26. Expression of Z chromosome sex-related genes. ........................... 30

    Supplementary Figure 27. RT-PCR analysis of sf-1_chr.Z, dmrt1, patched1_chr.Z, and

    follistatin expression from various tissues from female and male Cynoglossus semilaevis. ..... 30

    Supplementary Figure 28. Expression pattern of sex-related genes (dmrt1, sf-1_chr.Z,

    patched1_chr.Z, and follistatin) during the sex reversal period treatment with high

    temperature. ........................................................................................................................................... 31

    Supplementary Figure 29. Comparison of Z and W-linked sex-related genes. ........................ 32

    Supplementary Figure 30. qPCR analysis of the dmrt1 gene in the whole fish. ...................... 33

    Supplementary Figure 31. Location of dmrt1 gene in the tongue sole genome: metaphases

    from male and female showing the hybridization signal of BAC probe containing dmrt1. ...... 34

    Supplementary Figure 32. Gonad in situ hybridization using a sense RNA probe to dmrt1

    and no RNA probe as a control. .......................................................................................................... 35

    Supplementary Figure 33. Expression of the Z-linked E3 ubiquitin ligase gene, neurl3. ...... 36

    Supplementary Figure 34. Gonad in situ hybridization using a neurl3 sense RNA probe. .... 37

    Supplementary Figure 35. Apparent absence of W sperm from pseudo-males using

    W-linked SSR marker. ......................................................................................................................... 37

    Supplementary Figure 36. Gene expression profiling in sexual reversals. ............................... 38

    Supplementary Figure 37. RT-PCR analysis of aqp1, gas8, ropn1l, nme5, tekt1, plcz1,

    tbpl1, spag6, gal3st1, dnajb13, cldn11, gpr64 expression from three individuals of female

    and pseudomale C. semilaevis. ........................................................................................................... 39

    Supp