Supplementary Information for 2nd revision-lijt-20140819-2 · The BAC library was constructed using high molecular weight genomic DNA from a female C.carpio, which contained a total

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SUPPLEMENTARY INFORMATION FOR

Genome sequence and genetic diversity of the common carp, Cyprinus

carpio

Peng Xu1*, Xiaofeng Zhang2*, Xumin Wang3*, Jiongtang Li1*, Guiming Liu3*, Youyi Kuang2*,

Jian Xu1*, Xianhu Zheng2*, Lufeng Ren3, Guoliang Wang3, Yan Zhang1, Linhe Huo3, Zixia Zhao1,

Dingchen Cao2, Cuiyun Lu2, Chao Li2, Yi Zhou4, Zhanjiang Liu1,5, Zhonghua Fan3, Guangle Shan3,

Xingang Li3, Shuangxiu Wu3, Lipu Song3, Guangyuan Hou1, Yanliang Jiang1, Zsigmond Jeney6,

Dan Yu3, Li Wang3, Changjun Shao3, Lai Song3, Jing Sun3, Peifeng Ji1, Jian Wang1, Qiang Li1,

Liming Xu1, Fanyue Sun5, Jianxin Feng7, Chenghui Wang8, Shaolin Wang9, Baosen Wang1, Yan

Li1, Yaping Zhu1, Wei Xue1, Lan Zhao1, Jintu Wang1, Ying Gu2, Weihua Lv2, Kejing Wu3, Jingfa

Xiao3, Jiayan Wu3, Zhang Zhang3, Jun Yu3, Xiaowen Sun1,2

1Centre for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences, Beijing, 100141,

China 2Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin,

150001, China 3CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics,

Chinese Academy of Sciences, Beijing, 100029, China 4Stem Cell Program, Division of Hematology and Oncology, Boston Children’s Hospital and Dana

Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, 02115, USA 5The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and

Allied Aquacultures, Auburn University, Auburn, Alabama, 36849, USA 6Research Institute for Fisheries, Aquaculture and Irrigation, Szarvas, H‐5540, Hungary 7Henan Academy of Fishery Science, Zhengzhou, 450044, China 8College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, 201306, China 9Department of Psychiatry & Neurobiology Science, University of Virginia, Charlottesville,

Virginia, 22911, USA

* These authors contributed equally to this work. Correspondence and requests for

materials should be addressed to X.S. ([email protected]) and J.Y. ([email protected]).

Nature Genetics: doi:10.1038/ng.3098

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TABLE OF CONTENTS Supplementary Note ......................................................................................................................... 4

A. Genome sequencing, assembly and assessment ................................................................ 4

A1. Strain development, Sample preparation for genomic sequencing ........................... 4

A2. Whole genome shotgun sequencing .......................................................................... 4

A3. Estimation of the genome size .................................................................................... 5

A4. BAC library construction and BAC end sequencing ..................................................... 5

A5. WGS assembly ............................................................................................................. 5

A6. Quality assessment ..................................................................................................... 6

B. Genetic and physical map integration ................................................................................. 7

B1. High‐density linkage mapping ..................................................................................... 7

B2. Map integration .......................................................................................................... 8

C. Transcriptome sequencing ................................................................................................... 8

C1. ESTs from 454 platform ............................................................................................... 8

C2. RNA‐seq from Illumina platform ................................................................................. 9

D. Genome characterization .................................................................................................... 9

D1. GC content analysis ..................................................................................................... 9

D2. Repeat analysis ......................................................................................................... 10

D3. Gene prediction and functional annotation ............................................................. 11

E. Genome evolution ............................................................................................................. 12

F. Whole genome re‐sequencing analysis ............................................................................. 14

G. Comparative analysis of skin transcriptomes .................................................................... 16

Supplementary Figures ................................................................................................................... 17

Supplementary Figure 1: Genome size estimation by using different K‐mer length. ............. 17

Supplementary Figure 2. Distance distribution of mapped pair‐end/mate‐paired reads on the

genome assembly. ................................................................................................................... 18

Supplementary Figure 3. Comparison of the assembled genome with European C.carpio

scaffolds .................................................................................................................................. 19

Supplementary Figure 4. Genetic map of C.carpio. ................................................................ 21

Supplementary Figure 5. Genetic distance vs. physical distance. ........................................... 22

Supplementary Figure 6. Distribution of the genetic distance/physical distance on the

integrated genome of the C.carpio ......................................................................................... 23

Supplementary Figure 7. GC contents of C.carpio and other sequenced teleost genomes. ... 24

Supplementary Figure 8. Age distribution of interspersed repeats in C.carpio and D.rerio

genomes. ................................................................................................................................. 25

Supplementary Figure 9. Venn diagram of gene models. ....................................................... 26

Supplementary Figure 10. Comparison of gene structure of six teleost species .................... 27

Supplementary Figure 11: HOX gene clusters in the C.carpio genome ................................... 28

Supplementary Figure 12. Comparative analysis of HOX clusters between C.carpio and

Atlantic salmon genome. ........................................................................................................ 29

Supplementary Figure 13. A neighbor‐joining tree of 10 strains of common carp on the basis

of SNPs. ................................................................................................................................... 30

Supplementary Figure 14. Population structure of 10 strains of common carp from K=2 to


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K=8 on the basis of SNPs. ........................................................................................................ 31

Supplementary Figure 15. Deletion validation of three genes (ZPLD1, NLK and LRRC72) in

Hebao and Songpu. ................................................................................................................. 32

Supplementary Figure 16. Deletion investigation of FGFR1A1 gene in four strains. .............. 33

Supplementary Figure 17. Differentially expressed genes validated by qRT‐PCR. .................. 34

Supplementary Tables ..................................................................................................................... 35

Supplementary Table 1. Summaries of library construction and sequencing ......................... 35

Supplementary Table 2. Mapped reads to the genome assembly .......................................... 36

Supplementary Table 3. Gene region coverage assessed based on transcriptomes ............... 37

Supplementary Table 4. Gene region coverage assess by the CEGMA in teleosts .................. 38

Supplementary Table 5. Coverage of 19 duplicated gene pairs in the genome assembly ...... 39

Supplementary Table 6. All genotypes for the high density linkage map construction .......... 40

Supplementary Table 7. Repeat content in C.carpio genome ................................................. 41

Supplementary Table 8. Repeat contents and their classification .......................................... 42

Supplementary Table 9. Composition of repeat elements in 6 teleost genomes ................... 43

Supplementary Table 10. Transcriptome sequences used on genome annotation ................ 44

Supplementary Table 11. Statistics of gene models identified from C.carpio genome. .......... 45

Supplementary Table 12. Annotations against proteins with known function in each

database. ................................................................................................................................. 46

Supplementary Table 13. The statistics of gene structure of C.carpio and the comparison

with other teleosts. ................................................................................................................. 47

Supplementary Table 14. Non‐coding RNA genes in common carp genome .......................... 48

Supplementary Table 15. Systematic cross‐species comparative analysis and gene

classification. ........................................................................................................................... 48

Supplementary Table 16. List of C.carpio accessions for whole genome resequencing study 49

Supplementary Table 17. Statistics of genome resequencing data ......................................... 50

Supplementary Table 18. SNPs and small INDELs identified from re‐sequencing data of 33

accessions ............................................................................................................................... 51

Supplementary Table 19. List of genes in the selected regions (top 1% highest πHebao/πSongpu)

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Supplementary Table 20. List of genes in the selected regions (top 1% lowest πHebao/πSongpu)

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Supplementary Table 21. Enriched pathways in genes in the selected regions ...................... 52

Supplementary Table 22. Classification of SNPs identified from 326 genes. .......................... 52

Supplementary Table 23. List of genes containing unique non‐synonymous SNPs from

significantly diversified regions between Songpu and Hebao. ............................................... 52

Supplementary Table 24. Differentially expressed genes in the skin of Hebao and Songpu. . 52

Supplementary Table 25. List of primers for deletion validation and real‐time PCR analysis. 52

Supplementary References ..................................................................................................... 53


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Supplementary Note

A. Genomesequencing,assemblyandassessment

A1. Strain development, Sample preparation for genomic

sequencing

Common carp Songpu strain, or Songpu carp, was cultivated widely in northern China. Songpu strain was obtained by artificial selection with mirror carp that was introduced into China from Germany in 1984. Because the original stock had high mortality rate and low production, in order to improve cold resistance and production trait, original stock was selected to F4, and formed the Songpu strain. Eggs of a normal Songpu female C.carpio were activated with UV irradiated sperms and hatched at 22-25oC water temperature to single cell stage, then treated with heat shock at 39.5 oC for 105 seconds. All head-shocked fry were genotyped by using 17 microsatellite markers to assess their homozygosity. A gynogenetic Songpu C.carpio was selected as the genomic DNA source for whole genome DNA sequencing. Genomic DNA was extracted from blood using QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, Shanghai, China).

A2. Whole genome shotgun sequencing

The 454 sequencing was performed on Roche 454 genome sequencer at Seqwright, Huston, TX, USA by using the GS FLX Titanium chemistry. The total data set consisted of 11.6 gigabases (Gb) data with the average read length of 352 bp and 180 Mb mate-paired reads with jumping distances of 8 kb and the average read length of 302 bp (Supplementary Table 1).

The pair-end and mate-paired sequencing on Illumina and SOLiD platforms was performed at Beijing Institute of Genomics, Chinese Academy of Sciences with standard Manufacturing protocols. The sequenced libraries included short pair-end libraries with 250 bp, 300 bp and 500 bp insert length, and mate-paired libraries with 2 kb, 3 kb, 5 kb and 8 kb jumping distance. For the short insert libraries, a total of 728 M reads were generated, producing 75 Gb sequences and 44-fold coverage of the genome. From the large insert (2, 3, 5, 8 kb) libraries, 1,418 M reads or 79-fold coverage of the genome was obtained (Supplementary Table 1).


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A3. Estimation of the genome size

Based on analysis of sperm DNA content or C-values, the C.carpio genome size was estimated to be 1.85 Gb1. Nevertheless, C-value estimates can vary between 1.6 Gb2 and 2.0G3 for a genome with a lower genome size. We calculated genome size based on average sequencing depth, as determined by the peak in the frequency distribution of unique k-mers in the total raw sequencing read data set4. All paired-end reads from the short-insert libraries (250 bp, 300 bp and 500 bp) were used for this analysis. The low-quality bases were trimmed using the SOLEXAQA package5. The profiles of the k-mer distributions were obtained and the estimated sequencing depth can be calculated following Star et al.’s method6 (Supplementary Figure 1). The genome size is calculated by dividing the total amount of sequenced bases by sequencing depth. The length of the k-mers influences the final genome size as the peak depth decreases with an increase in k-mer length. The length of the k-mers increases from 15 bp to 31 bp. The peak depth did not increase to a saturation point until the k-mer size was longer than 26 bases in length. The maximal genome size is estimated to be 1.83 Gb (Supplementary Figure 1), slightly lower than the estimates based on sperm DNA content.

A4. BAC library construction and BAC end sequencing

The BAC library was constructed using high molecular weight genomic DNA from a female C.carpio, which contained a total of 92,160 BAC clones with an average insert size of 141 kb and covered 7.7 × haploid genome equivalents. A total of 40,224 BAC clones were sequenced from both ends of individual BAC clones by the Sanger sequencing method, generating 65,720 clean BAC end sequences. Both BAC library and BAC end sequences had been published previously7,8. Those mate-paired BAC end sequences were used for contig scaffolding.

A5. WGS assembly

Firstly, the Celera assembler9 was used to assemble the next generation sequence reads on the 454 and the Sanger BAC end sequences. The 454 SFF files were converted to FRG file format required by Celera assembler using sffToCA. The Sanger BAC end sequences were converted to FRG using convert-fasta-to-v2.pl. The parameters were set as the following: utgErrorRate = 0.03; ovlErrorRate = 0.06; cnsErrorRate=0.10; cgwErrorRate = 0.10; overlapper = ovl; merSize = 22; unitigger = bog; utgBubblePopping = 1; ovlMemory = 8GB; ovlThreads = 32; ovlConcurrency = 24; ovlHashBlockSize = 10000000; ovlRefBlockSize = 64000000.

Secondly, reads from the Illumina libraries, SOLiD libraries and 454 8K mate-pair libraries were aligned to the 454 contigs using BWA10. Only uniquely aligned paired-end reads were used to construct scaffolds. Following Qiu et al.’s method11, we used a hierarchical assembly strategy in which we added data step by


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step from short paired-end reads to long paired-end reads. Thirdly, 65,720 BAC end sequences were mapped to the scaffolds using the

BLAT12 software with the default parameters (minimum overlap length 70% of the read length, minimum 90% identity in the overlap). Since the Celera assembly included the BAC end reads, the high mapping ratio is expected. We selected the best aligned region for each end sequences. If two end-reads from the same BAC were aligned to different scaffolds, these two scaffolds were merged into one. Finally, we used the paired-end information from the short-insert libraries with Gapcloser13 to fill gaps between scaffolds.

A6. Quality assessment

To examine the assembly integrity, the Illumina data, 454 data and SOLiD reads were aligned again onto the final assembly using BWA10 with default parameters. A total of 83% of raw reads can be mapped. This may be due to the incompleteness of the assembly.

The assembly was then aligned to five scaffolds picked from previously assembled European C.carpio genome by Henkel et al with an average length of 92kb14 and one previous assembled BAC. The alignment was carried out using BLAT. All of the five scaffolds could be mapped to five linkage groups with an average coverage of 90%. In additional, we also aligned assembled genome to the assembled BAC clones with coverage of them at 85.7% (Supplementary Figure 3).

The assembly was also evaluated using transcripts from NCBI database and 454 sequencing. The ESTs were aligned to the assembly sequences using BLAT with default parameters and an identity cutoff at 90%. The mapping ratio of ESTs was about 95% (Supplementary Table 3).

Core eukaryotic genes identified by CEGMA were also mapped to the assembled genome by BLAT to calculate the gene region coverage (Supplementary Table 4). The coverages of five CEG in teleost are between 82 and 95%.

Considering that C.carpio genome had a recent whole genome duplication event, It is important to assess whether our assembly could differentiate highly similar genes. 38 published paralogous mRNAs (19 pairs of paralogs) were used in the assessment. The 38 mRNAs were aligned with the assembly using BLAT and a region that aligned best with each of the mRNAs was selected. Only 6 genes in 3 homologous pairs, were merged into 3 single genes on the assembly while 32 genes in 16 homologous pairs were mapped to distinct genomic locations, indicating that 92% of homologous genes could be differentiated and that our assembly was of high quality (Supplementary Table 5).


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B. Geneticandphysicalmapintegration

B1. High‐density linkage mapping

B1.1.Genotyping

A total of 1,123 informative microsatellite markers were genotyped on the genetic mapping panel by using a tailed primer protocol 15,16. The PCR reaction mixture was composed of the following: 1× PCR buffer, 0.15 mM MgCl2, 0.2 mM of each dNTPs, 0.15 pmol of upper PCR primer, 6 pmol of lower PCR primer, 0.15 pmol of labeled tail primer, 0.5 U of DNA Taq polymerase (Fermentas, Vilnius, Lithuania), and 20 ng of genomic DNA, in a reaction volume of 15 μL. Amplifications were performed on an ABI 9700 thermocycler (Applied Biosystems, Foster City, CA, USA) as follows: an initial denaturation at 94 °C for 5 min, then 30 cycles consisting of 94 °C for 30 s, 56 °C for 45 s, and 72 °C for 45 s, and 10 cycles consisting of 94 °C for 30 s, 53 °C for 45 s, and 72 °C for 45 s, followed by a final extension at 72 °C for 10 min. The PCR products were analyzed on a 3130XL Genetic Analyzer (Applied Biosystems) with LIZ-500 size standards (Applied Biosystems) and genotyped with GeneMapper 4.0 software (Applied Biosystems).

We then genotyped a batch of SNP markers in the C.carpio F1 population using RAD (Restriction-site associated DNA) sequencing technology17. MseI and AluI were used for library construction. Fragment length was between 330 bp and 380 bp. The obtained RAD libraries were sequenced on an Illumina Genome Analyzer (GA) IIx. In total, 103.8M paired-end reads were generated. On average, 0.4 M reads were obtained for each descendant. Low quality markers and low-quality individuals with a lower number of usable data were excluded, and a total of 4,026 high quality SNP markers in 107 individuals were used for the linkage map construction.

B1.2.Linkagemapconstructionandnomenclature

JoinMap4.0 software was used to perform linkage analysis. Linkage between markers was examined by estimating LOD scores for recombination rate and map distances were calculated using Kosambi mapping function. Default significance levels from 4.0 to 10.0 LOD (logarithm (base 10) of odds) in steps of 1.0 LOD were used. A threshold of LOD 5.0 was set to detect suspect linkages resulting from possible allele-coding errors. Using these 1,123 microsatellite markers and 4,026 SNP markers, we first constructed a linkage map based on microsatellite markers, then added SNP markers gradually into the microsatellite map. We added SNP markers onto the maps as anchors if their additions didn’t change existing marker order. Otherwise, a SNP marker would be discarded. The final genetic linkage map comprises of 4,243


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markers, including 773 microsatellite markers and 3,474 SNP markers, covering an observed length of 3,946.7cM. The mean distance between adjacent markers was 0.9 cM (Supplementary Figure 4). The genotype information is included in Supplementary Table 6.

In order to facilitate further comparative analysis with zebrafish (Danio rerio) genome, we ordered the linkage groups based on the marker similarity to D.rerio. Due to one more round of whole genome duplication, C.carpio has twice the number of chromosomes (n = 50) than D.rerio (n=25). These markers were aligned to D.rerio genome by performing BLASTN searches with an e-value 1 × 10-5. The best aligned hit region in D.rerio genome was selected for each marker. If a majority of markers on a C. carpio group were linked to a D.rerio chromosome, this linkage group was named as either (2n-1) or (2n), where n is the D.rerio chromosome number.

B2. Map integration

The markers in the genetic map were aligned to the C.carpio scaffolds by performing BLAT search. The aligned length should cover at least 70% of the marker length. The best-aligned hit which covered the longest region of each marker was selected. If one marker had no unique best-aligned region, this marker was discarded. Finally a total of 3,511 markers were selected to anchor the scaffolds. If a scaffold contained over two markers, the orientation of this scaffold was consistent with the orders of these markers on the genetic map.

C. Transcriptomesequencing

C1. ESTs from 454 platform

Twelve tissues (brain, muscle, liver, intestine, blood, head kidney, trunk kidney, skin, gill, spleen, gonad and heart) were dissected and collected from a six-month-old gynogenic C.carpio. Total RNA was extracted from 12 tissues using TRIZOL (Invitrogen, Carlsbad, CA, USA). RNA samples were then treated by DNase I. Integrity and size distribution were checked on a Bioanalyzer 2100 chip (Agilent technologies, Santa Clara, CA, USA). Equal amounts of the high quality RNA samples from each tissue were pooled. RiboMinus™ Eukaryote Kit (Invitrogen) was used to deplete ribosomal RNA from pooled total RNA. Approximately 10 µg of processed total RNA were used for cDNA synthesis using M-MLV RTase cDNA Synthesis kit (TaKaRa, Dalian, China). A total of 10 µg cDNA were used for sequencing library construction and sequencing on a Roche Genome Sequencer FLX following the manufacture recommended procedures. A total of 1,418,591 clean reads with an average read length of 321 bp were collected (455 Mb) for transcriptome analysis. In addition, a small set of transcriptome sequences (242,261 reads) generated from brain, muscle and liver on the first generation of 454 platform were also used for


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gene annotation and transcriptome analysis. The entire set of reads had been submitted to the NCBI Sequence Read Archive under the accessions of SRA050545 and SRA009366. The data were summarized in Supplementary Table 10.

C2. RNA‐seq from Illumina platform

In addition, embryos were collected at 12 early development stages. Total RNA was extracted and processed as described above. The cDNA libraries were constructed and then sequenced on an Illumina HiSeq 2000 platforms following the manufacturer’s instructions. Briefly, mRNA was isolated from total RNA by Sera-mag Magnetic Oligo (dT) Beads. The mRNA in fragment buffer was sheared into short fragments about 200 bp in size. These mRNA fragments were used to synthesize first-strand cDNAs using random hexamers-primers and reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I, together with RNase H and dNTPs. The final products were purified using the QiaQuick PCR purification kit (Qiagen). The double-stranded cDNA was subjected to end-repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase. PE adapters were added to the repaired cDNA fragments by T4 DNA ligase. Fragment size selection was performed using an agarose gel, and fragments of 200~250 bp in size was collected and extracted from the agarose gel. The selected cDNA fragments were amplified by PCR. The constructed cDNA sequencing library was sequenced on an Illumina HiSeq 2000. A total of 435 M reads were collected from 12 samples. After quality filtration, approximate 344 M reads were used for transcriptome analysis and genome annotation (Supplementary Table 10).

D. Genomecharacterization

D1. GC content analysis

The GC content was calculated as the percentage of G and C in the non-N nucleotides within 10-kb non-overlapping windows in the assembled C.carpio genome. The C.carpio genome has a GC content of 37.0%. The GC contents of D. rerio (36.6%), Gasterosteus aculeatus (44.6%), Oryzias latipes (40.5%), Takifugu rubripes (45.5%) and Tetraodon nigroviridis (46.4%) genomes were also calculated for comparison purpose (Supplementary Figure 7). C.carpio has slightly higher GC content than D.rerio, but lower than the other sequenced teleosts.


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D2. Repeat analysis

We used two methods to identify the repeat contents in carp genome, homology-based and de novo prediction.

Homology-based analysis: we used Repbase (version 20120418) to perform the TE search by RepeatMasker(3.3.0) with WuBlast search engine. Because only ~3.7% transposons were characterized by this method, and all of transposal elements were included in de novo library, the result of homology-based analysis didn’t show separately.

De novo prediction: Transposable repeats were identified in the genome by RepeatMasker(version 3.3.0) using a de novo repeat library constructed by RepeatModeler( version 1.0.5). Because of a large amount of repeat elements that RepeatModeler failed to classify, several steps were taken to classify the remaining transposable elements manually: (i) blastn against RepBase library (version 20120418),(ii) blastx against transposable element proteins(version 20120418), (iii) blastx against SwissProt protein database, (iv) tblastx against Repbase, (v) post homologous search, unknown elements were classified using TEClass18 into four categories according to their transposal mechanism, DNA transposons, LTRs, LINEs and SINEs. The software used Support Vector Machine(SVM) engine with a Gaussian kernel to characterize the unknown transposal elements . The blast hit were filtered either at the DNA level: E-value <=1e-5, identity percent >=50%, alignment coverage>=50%, and the minimal matching length >=80bp or at the protein level: E-value <=1e-4, identity percent >=30%, alignment coverage>=30%, and the minimal matching length >=30amino acid. The elements matched with SwissProt database were first filtered out, and the remaining ones were processed by RepeatMasker. The tandem repeat(Simple repeats and satellites) were screened in the genome by TRF and RepeatMasker. To compare the repeat contents with other teleosts, the same method were done to identify the repeat content in the other five fish genome (D.rerio (ZV9), stickleback (BROADS1.64), medaka (MEDAKA1.64), fugu(FUGU4.64) and tetraodon (TETRAODON8.64)).

The de novo repeats library contained 2200 elements. After several classification steps as described above, 1899 elements were classified into 4 categories, 1139 DNA transposons, 406 LTRs, 234 LINEs. The carp genome contains 2.86 million repeats, which occupy about 37.3% of genome. Of these, 31.23% are interspersed repeats (Supplementary Table 7 and 8). Among them, the most abundant transposable elements are DNA transposons (17.53%). Of these DNA transposons, 4.35% are hAT and 3.12% TcMar. The substitute rate distribution shows a peak at 6% indicated there were a large amount of copies inserted after 17MYA (the rate of substitutions site per year was 3.51x10-9), and 0.4%~4.59% of genome were inserted after the fourth whole genome duplication(5.6~11.3MYA)19 (Supplementary Figure 8).

The repeats contents within six teleosts are following, C.carpio has a higher repeats than most teleosts, but is lower than D.rerio, mainly different in DNA transposons. On average, the substitution level of interspersed repeats from consensus


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sequence in carp genome(0.13) is slightly higher than that in D.rerio genome(0.11), but the copies and bases of young repeats(<5%) in C.carpio is lower than that in D.rerio genome(about 52Mb). In the other 5 teleosts, the repeats contents in Medaka genome had similar rate (30.68%) to C.carpio(31.23%). Medaka has a medium size genome(~860M), the genome size of C.carpio is about two times of Medaka genome, but the percent of repeats contents of the two genome are similar, this indicates that the copies of repeats contents in C.carpio doubled during the fourth whole genome duplication, but the average size of the elements didn’t change (Supplementary Table 9). There were 16 subclass elements of DNA transposons, LINEs and LTRs are shared by six teleosts, but none of SINEs are shared among these six species. This phenomenon indicated that teleosts have lineage-specific repeat elements, especially in SINE elements.

D3. Gene prediction and functional annotation

D3.1.Geneprediction

De novo gene prediction, sequence homology based predictions and RNA-seq data were used for gene prediction. All predicted gene structures are integrated into weighted consensus gene structures using EVidenceModeler (EVM).

Briefly, two de novo prediction software programs Augustus20 and Fgenesh (http://www.softberry.com) were used to predict genes on repeat-masked genome sequences, we did not to mask low complexity regions and simple repeats since they could be parts of coding sequences. Gene model parameters for the programs were trained from PASA-processed long genes and known genes from other fishes. Sequence homology based gene prediction included both raw and precise alignments. First, proteins sequences from NCBI non-redundant (NR) database and 68 species sequences in Ensembl (release version 68)21 were collected to build a database. Assembled genome sequences were aligned to protein sequences in the database by BlastX and the identified homologous proteins with an E-value threshold at 10-3 were selected and followed by a tBlastN alignment to the genome. Adjacent and overlap matches were merged together using Perl scripts, building the longest protein for each genomic sequence region. And then each target region in the genome was extended by 10 kb from both ends of the aligned region, to cover potential untranslated regions. The protein sequences were then aligned to these genome fragments by Genewise22. RNA-seq reads were mapped to genomic sequences by Tophat23 and then Cufflinks24 was used to get assemblies of transcripts. For a gene locus with several alternative splicing transcripts generated from Cufflinks, the transcript with the longest exon length was chosen. All the evidences were merged to form a comprehensive and consensus gene set by EVM. PASA25 was used to update the EVM consensus predictions by adding UTR annotations. The integrated annotation pipeline generated a total of 52,610 gene models (Supplementary Figure 9, Supplementary Table 11). About 91.4% of these genes were detected in assembled transcripts from RNA-seq


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reads. To determine how these structures of gene models differ from D. rerio, G.

aculeatus, Oryzias latipes, Takifugu rubripes and Tetraodon nigroviridis, we collected and analyzed the longest protein-coding transcript of each gene in all these species found in Ensembl. The analysis was restricted to only coding exons of the representative transcripts, to avoid bias resulted from different levels of data quality in UTR annotation among these species. The results are shown in Supplementary Table 13.

D3.2.Functionalassignment

To obtain gene function annotations, the BLAST search was conducted against NCBI nr, Swissprot and TrEMBL protein databases and homologs called with an E-value <10-5. Functional classification of Gene Ontology (GO) (http://www.geneontology.org/) of the unigenes was performed by the InterProScan26 program, which also generated family information from Interpro. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG)27 annotation service KAAS. We used the single-directional best hit (SBH) method to query the set of organisms representative for ‘genes’ as suggested on the KAAS website, with the default bitscore threshold of 60. The result was listed in Supplementary Table 12.

D3.3.Non‐codingRNAidentification

The tRNA genes were identified by tRNAscan-SE28. For rRNA identification, we first downloaded the human rRNA sequences from the Ensembl database. Then rRNAs in the database were aligned against the C.carpio genome using blastn with cutoff of E-value <1e-5, identity ≥85% and match length ≥50 bp. Based on sequence conservation, we identified 1,012 rRNA gene fragments, with an average length of 99 bp. MiRNAs were identified using MIREAP with our previously published small RNA sequencing29. The result was listed in Supplementary Table 14.

E. Genomeevolution

E1.Globalcomparisonofthegenesetstootherorganisms

Protein-coding genes and CDS (Coding DNA Sequence) from 11 species (D. rerio, G.aculeatus, O.latipes, T.rubripes, T.nigroviridis, Xenopus tropicalis, Anolis carolinensis, Homo sapiens, Mus musculus, Sus scrofa, Gallus gallus and Ciona intestinalis) were downloaded from Ensembl (release version 68). For the genes with


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alternative splicing variants, only the longest transcript was selected. The genes with less than 30 amino acids were discarded. The OrthoMCL pipeline30 was used to define gene family in the common ancestor of the species. The all-against-all similarities were performed using blastp with E-value cutoff at 10-5.

The 941 single-copy orthologs shared in all 12 species were identified using the OrthoMCL30. The well-aligned regions of each family alignments by MUSCLE31 were extracted with Gblocks32 with default parameters. Phylogenetic analysis of superalignments was performed using maximum-likelihood method implemented in PHYML33 with JTT model for amino acid substitutions, a gamma correction with four discrete classes, an estimated alpha parameter. C.intestinalis was selected as outgroup of the phylogenetic tree.

E2.ComparativeanalysiswithD.reriogenome

To identify syntenic blocks between C.carpio and D.rerio, we used MCScanX34, where genes can be set as anchors (minimum number of genes required to call synteny is 5). Firstly, we used BLASTP to align proteins of C.carpio and D.rerio with the parameters of "-e 10-5 –b 5 –v 5 –m 8". The alignments were then subjected to MCScanX to determine syntenic blocks, which were then displayed by using a Circos software35.

E3.Ksdistributionand4DTvanalysis

Analyses of homologous gene relationships provide strong evidence to support genome wide duplication events that may occur in the paleohistory of the C.carpio genome evolution. All-versus-all BLASTP comparisons (E-value < 10−5) were used to identify pairs of homologous genes. For a gene with alternative splicing mode, the longest protein was used. It’s possible that the BLAST output may contain noisy information when a gene has more than one conserved domains, or transposable element related genes were mis-annotated. So we removed gene families with more than 10 homologous genes in the following Ks analysis. For each duplicate gene pairs, we first aligned the sequence pairs using Clustalw. The corresponding codon alignments were produced using PAL2NAL. Finally, we estimated Ks of each paralogous pair using a maximum likelihood method in the CODEML program (run mode -2) of the PAML package. Ks distribution among D.rerio paralogous pairs was analyzed by following the same pipeline.

Orthologs between C.carpio and D.rerio were identified by Inparanoid as described in Fawcett et al. (2009). Then the Ks of each homolog pair was calculated with the above strategy.

Fourfold degeneration sites of paralogous pairs within a species and of orthologous pairs between species were then identified and the transversion rate (4DTv) of each gene pair was calculated using an in-house perl script.


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E4.HOXgenefamily

HOX gene cluster is a paradigm for studying the vertebrate genome evolution. D.rerio, which had undergone the third whole genome duplication, has 7 HOX gene clusters in the genome, missing HOXDb cluster. Atlantic salmon (Salmo salar), which had experienced the fourth WGD, has 13 clusters, missing three clusters (HOXAbβ, HOXDbа and HOXDbβ). We expect that C.carpio has much more HOX gene clusters than its close diploid relatives, e.g. D.rerio. To investigate this, we collected HOX genes and constructed gene clusters.

Briefly, HOX genes were searched by homologous method. First, we retrieved the HOX gene families data from TreeFam database including protein sequences, cDNA sequences and protein HMM profiles, then use these data to search the carp genome annotation data set by blast and HMMer. At a nucleotide level, the blast results were filtered by E value 10-10, identity percent >50% and coverage >=50%. At a protein level, amino acid sequence BLAST results were filtered by E value at 10-5, identity percent >50% and coverage >=30%. From BLAST and HMMer analyses, we got 114 candidate HOX genes. These genes were classified using sequence homology and manual curation examining conserved domains. We also aligned protein sequences of HOX gene in D.rerio, Human, Atlantic salmon and carp genome using Genewise. We then manually examined the alignment and made adjustment where necessary. After combining BLAST and Genewise results, we got 88 HOX genes that formed a total of 13 clusters in C.carpio genome where HOXCbβ, HOXDbа and HOXDbβ clusters have been lost (Supplementary Figure 11). These analyses revealed the difference of HOX clusters between carp and salmon in which the three lost clusters are HOXAbβ, HOXDbа and HOXDbβ. (Supplementary Figure 12). The results, however, do provide additional evidence for a fourth round of WGD in C.carpio.

F. Wholegenomere‐sequencinganalysis

F1.SamplingofrepresentativeC.carpioaccessions

A total of 33 individuals representing 10 strains of C. carpio were collected across Europe, America and China. Danube river carp and Szarvas 22 were collected from carp live gene bank at Research Institute for Fisheries, Aquaculture and Irrigation, Hungary (HAKI). American carp were collected from the Chattahoochee River, USA. All other strains or wild populations were collected in China, including Songpu carp from Heilongjiang Fishery Research Institute, Yellow River carp from Henan Academy of Fishery Sciences, Heilongjiang River carp from Fuyuan county of Heilongjiang province, Hebao carp from Wuyuan county of Jiangxi province, Xingguo red carp from Xingguo county of Jiangxi province, Oujiang color carp from Longquan county of Zhejiang province, koi from the breeding population at Beijing Fishery Research Institute. Fin chips or blood samples were collected from these


15

individual fish, and genomic DNA samples were extracted with DNeasy Blood & Tissue Kit (Qiagen). The sample accessions were listed in Supplementary Table 16.

F2.Wholegenomere‐sequencing,mappingandSNPcalling

DNA library preparation and sequencing were carried out at HudsonAlpha Genomic Services Laboratory (Huntsville, AL, USA) following the manufacturer’s instructions. After KAPA quantitation and dilution, libraries were sequenced on Illumina HiSeq 2000 with 101 bp pair-end reads. The pair-end reads from each accession were aligned to the reference genome using BWA10. After mapping, SNPs were identified on the basis of the mpileup files generated by SAMtools37. Filtering threshold was set as following, read depth no less than 10, quality score no less than 20. Genotypes supported by at least two reads and with minor allele frequency ≥ 0.1 were assigned to each genomic position and finally result in 18,949,596 SNPs. Totally 1,694,102 small INDELs were identified with a size range of 1-5 bp (Supplementary Table 17).

F3.Phylogeneticanalysisandpopulationstructureanalysis

In these analyses, we selected a subset of 2,873,733 SNPs that presented in all strains with MAF (minor allele frequency) over 0.1. In order to eliminate PSV (paralogous sequence variant) from duplicated genes (may come from multiple round of whole-genome and segmental duplications) in the carp genome, we performed all-against-all BLASTP between genes from the five teleosts (C. carpio, D. rerio, T. rubripes, O. latipes, and G. aculeatus), yielded 7,709 single-copy genes. We

constructed a maximum likelihood (ML) and a neighbor-joining (NJ) trees based on 8,375 SNPs

with PhyML and MEGA (Figure 3A and Supplementary Figure 13). The tree shows that C.carpio from Europe and Asia are well categorized separately. PCA was performed with EIGENSOFT which shows similar results to phylogenetic tree. We conducted population structure analysis using STRUCTURE with settings at 2,000 iterations and the number of clusters (K) of 2–8 (Figure 3C and Supplementary Figure 14). Finally we chose K=3 and 4 to illustrate the structure as it had the highest values of Ln. DISTRUCT software was used to plot the structure matrix.

F4.PidistributionanddifferencesbetweenHebaoandSongpu

With SNPs identified above, Pi distribution on each linkage group was calculated. Firstly SNP data were transformed to *.phy format by ClustalW software, and then Pi value of 50 linkage groups were generated using a sliding-window method by Variscan software. Window width was set to 50 kb and step-wise distance was 10kb. Pi value from Songpu and Hebao carp were compared and the ratios (πHebao/Songpu)

were sorted. According to the ratio values, highest 1% and lowest 1% of regions were assigned to annotated genes and GO enrichment was conducted on these genes.


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Totally 740 regions with highest ratio containing 288 genes were identified, covering 10.45Mb of genome. Meanwhile, 150 regions with lowest ratio containing 38 genes covered 2.22Mb of genome.

To identify potential diversified mutation loci between Hebao and Songpu, RNA-seq reads from skin tissue of Hebao and Songpu were mapped to those 326 candidate genes using BWA and SAMtools. We detected 160 non-synonymous SNPs in 82 genes from Songpu and 204 non non-synonymous SNPs in 106 genes from Hebao. We then identified 2 genes (SNED1 and TRIM9) and 5 genes (SNED1, RIN1, MLL3, CCNK, and SLC6A4) with pre-mature or skip-stop-codon SNPs in Hebao and Songpu, respectively (Supplementary Table 22 and 23). We also identified 3 genes in the Songpu (FGFR1A1, NLK and LRRC72) and 2 genes (ZPLD1 and VRK2) in the Hebao that have deletions in their coding region (Supplementary Figure 15 and 16). The primers were listed in Supplementary Table 25.

G. Comparativeanalysisofskintranscriptomes

Comparative transcriptome analysis between Songpu and Hebao was conducted using RNA-seq data from skin tissues of both strains. Raw reads was generated by HudsonAlpha Genomic Services Lab as previously described39. Reads with a low quality and a read length less than 10 bp were removed. All the cleaned reads were mapped to the assembled reference with Bowtie41. Then RSEM was then used to estimate and quantify the gene and isoform abundances. Finally, edgeR42 was used to normalize the expression levels in both strains and obtain the differentially expressed transcripts by pairwise comparisons. A total of 894 transcripts were identified that differentially expressed in the skin of Songpu and Hebao, and were listed in Supplementary Table 24. For quantitative RT-PCR (qRT-PCR) validation, total RNA was isolated and purified from all the samples using RNeasy Kit (Qiagen), and quantified using Nanodrop and Bioanalyzer 2100 (Agilent). qRT-PCR was performed on ABI PRISM 7500 Real-Time PCR System using QuantiTect SYBR Green PCR Kits (Qiagen), and the β-actin gene was used as the internal reference. A total of 15 genes with high level of significance were selected for qRT-PCR analysis (The primers in Supplementary Table 25). The expression patterns of 14 genes were in agreement across the RNA-Seq and qRT-PCR analyses with minor differences in the expression level (Supplementary Figure 17). GO annotation of genes was performed according to the orthologous relationship with the gene set of Danio rerio. The pathway analysis was performed using Ingenuity pathway analysis (IPA) tools.


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Supplementary Figures

SupplementaryFigure1:GenomesizeestimationbyusingdifferentK‐merlength.

(a)

(b)

(a) Distribution profiles of unique K-mer counts in the raw sequencing reads. The K-mer sizes are from 15 to 31. (b) The curve shows the genome size estimates based on the peak in the distribution profile for the K-mers at different sizes.


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SupplementaryFigure2.Distancedistributionofmappedpair‐end/mate‐pairedreadsonthegenomeassembly.

Distances were calculated by mapping the paired end or mate‐pair reads from Illumina, Roche

454 and SOLiD the scaffolds using BWA (settings: both reads in a pair should be uniquely

mapped).


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SupplementaryFigure3.ComparisonoftheassembledgenomewithEuropeanC.carpioscaffolds

(1)

(2)

(3)

(4)

(5)


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(6)

Sequence comparison analysis between scaffolds in European C.carpio previously assembled by

Henkel CV et al.(2012)14 (1-5) or assembled BAC (6) (blue lines) and our C.carpio genome (red

lines). The black lines represent perfectly matched sequences. Gaps between the lines represent

mismatches or gaps between the assemblies.


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SupplementaryFigure4.GeneticmapofC.carpio.

The genetic map was constructed by using 4,243 markers (microsatellite and SNP markers) on the

F1 mapping panel of Songpu with Joinmap. All genotypes of these markers on the mapping panel

were provided in Supplementary Table 6.


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60Cm HLJ13310.122Cm CC363807.848Cm CC1992410.028Cm HLJ301010.25Cm CC4547511.287Cm HLJ304011.65Cm CC637313.135Cm CC347114.371Cm CC946914.935Cm CC5326314.966Cm CC2346415.799Cm CAFS159816.406Cm HLJ236818.286Cm CC1576718.708Cm CC1014519.898Cm HLJ85319.937Cm CC844020.107Cm CC4925920.485Cm CC676721.783Cm CC1196622.124Cm CC1923522.318Cm CC6325522.373Cm CC214822.806Cm CC292023.545Cm CC2725923.828Cm HLJ363624.711Cm HLJ243425.111Cm CAFS238425.399Cm CC4023125.683Cm CAFS2152-226.324Cm CAFS65827.033Cm CC1332727.321Cm CC2980828.842Cm CC3224429.415Cm CC6632429.525Cm CC2619629.94Cm CC497130.387Cm CC2593530.672Cm CC5428831.003Cm CAFS8231.243Cm CC341831.256Cm CC5984031.47Cm CC1752031.78Cm CC1488032.156Cm CC2090632.406Cm CC120432.426Cm CC2934332.573Cm CC8035032.79Cm CC260133.212Cm CC1522133.437Cm CC2318833.586Cm CC314133.642Cm CC1278734.051Cm CC769034.497Cm CC997834.587Cm CC726134.68Cm CC2960234.866Cm CC1318935.257Cm CC480235.283Cm CC2476735.468Cm CC1288435.606Cm CC3327335.631Cm CC2864635.791Cm CC7139735.843Cm CC1813335.988Cm CC701736.091Cm CC6390736.198Cm CC6376236.271Cm CC2376536.445Cm CC430936.497Cm CC488536.542Cm CC304736.646Cm CC1470336.767Cm CC326336.78Cm CC420436.866Cm CC1397936.867Cm CC1204137.001Cm CC1700537.069Cm CC521137.074Cm CC3331037.074Cm CC6810837.115Cm CC383337.148Cm HLJ367537.193Cm CC596137.309Cm CC440137.443Cm CC156637.478Cm CC1872237.495Cm CC925637.633Cm CC7317337.758Cm CC7661437.891Cm CC977938.093Cm CC1214738.108Cm CC1933738.26Cm CC1763738.462Cm CC1182138.474Cm CC728438.682Cm CC5388638.818Cm CC364538.901Cm CC2251339.057Cm CC2525339.06Cm CC1443039.318Cm CC785139.558Cm CC6048239.635Cm HLJ255639.816Cm CC8037440.167Cm CC1622540.432Cm CAFS181440.728Cm CC872340.736Cm CC898141.17Cm CC4087641.58Cm CC2714141.748Cm CC2795942.08Cm CC8183442.659Cm CC2639242.826Cm CC7443243.159Cm CC807043.751Cm CAFS24644.54Cm HLJ253744.642Cm CC3808945.03Cm HLJ78245.821Cm CC6914446.303Cm CAFS152246.704Cm CC7376347.079Cm CC1605547.694Cm CC533647.968Cm MFW1548.822Cm HLJ147750.255Cm CAFS170851.432Cm HLJ276753.147Cm CC5923554.113Cm CC1799754.662Cm CAFS227855.745Cm HLJ265957.632Cm HLJ277158.48Cm CC6009059.858Cm CC4438360.674Cm CC7795062.354Cm CC108863.482Cm CC7664663.89Cm CC176364.794Cm CC2666365.032Cm CC1140365.135Cm HLJ57566.222Cm CC6131367.05Cm HLJ356268.529Cm CC3772970.689Cm CC1436872.834Cm CC2650579.982Cm CC2830

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80Cm CAFS2444-28.409Cm HLJE59713.058Cm HLJ396517.48Cm CC1554218.667Cm CC7256920.035Cm CC4424423.009Cm CC7916826.17Cm CC5876226.687Cm CC1076427.027Cm CC7609727.454Cm CC1360828.587Cm CC263228.943Cm CC4946429.724Cm CC989430.226Cm CC2656530.435Cm CC2172930.686Cm CC5292030.741Cm CC1012431.051Cm CC6194131.128Cm CC2681531.182Cm CC2718531.5Cm CC926331.585Cm CC389031.59Cm CC583731.776Cm CC1683031.829Cm CC166732.061Cm CC166532.564Cm CC6194332.893Cm CC3028533.003Cm CC6850633.652Cm HLJ06433.755Cm CC2233934.107Cm CC4188634.359Cm CC2672534.755Cm CC7576235.188Cm CC3073935.618Cm CC107835.946Cm CC2375536.205Cm CC4768636.642Cm CC2836636.783Cm CC1241536.898Cm CC3065237.111Cm CC1323837.494Cm CC1108237.624Cm CC2209737.717Cm CC2523837.927Cm CC7948638.133Cm CC5107338.426Cm CC4581738.621Cm CC103239.244Cm HLJE41739.253Cm CAFS97539.544Cm HLJ214439.978Cm CAFS216640.38Cm CC1537240.939Cm CAFS230541.072Cm HLJ381541.723Cm HLJ366141.858Cm CAFS153342.227Cm CC2963042.506Cm CAFS73342.789Cm HLJ287543.498Cm HLJ405543.796Cm CC849444.178Cm CC3180644.331Cm CC1316544.674Cm HLJ320145.374Cm CC3193145.395Cm CC3210845.745Cm CC7058946.009Cm CC730046.52Cm CC7176046.912Cm CC1165747.419Cm CC5966747.683Cm CAFS228648.538Cm HLJE45049.286Cm CC1874349.918Cm CC1923350.335Cm CC190051.037Cm CC5565552.337Cm CC5040053.201Cm HLJ371653.379Cm CC2986955.085Cm CC1786055.317Cm CC2373856.671Cm CAFS225356.86Cm CC607457.216Cm CAFS2444-160.659Cm CC395460.825Cm CC2496362.359Cm CC3003762.626Cm CC3555763.86Cm CC849265.071Cm CC1466066.119Cm CC577366.858Cm CC3296567.636Cm CC3220868.072Cm CC1042668.686Cm CC1647469.215Cm HLJ395970.061Cm CC1714771.847Cm CC2500673.266Cm CC5122373.514Cm CC7119375.241Cm CC7720477.182Cm CC232677.457Cm HLJ283078.13Cm CC7732578.848Cm CC1750180.328Cm CC3140980.834Cm CC7714781.083Cm CC1648081.618Cm CC2787281.675Cm CC1776182.167Cm CC1012783.271Cm CC1978284.008Cm CC3387484.955Cm CC969385.639Cm CC7580586.532Cm CC3232887.584Cm CC247988.195Cm CC2921189.482Cm CC3529590.201Cm CC6561490.55Cm CC2858591.307Cm CC488892.667Cm HLJ232195.26Cm CC2661598.413Cm CC16010

90Cm HLJ24538.887Cm CC4061813.965Cm CC868816.839Cm CC1164718.977Cm CAFS92520.743Cm CC258222.818Cm CC329924.01Cm CC1652524.358Cm CC6164024.824Cm CC2839225.619Cm CC2967326.793Cm CC4707729.006Cm CC559329.536Cm CC820131.677Cm CC1241933.381Cm CC6835734.434Cm CC236735.684Cm CC2306137.498Cm CC866338.591Cm CC2861138.631Cm CC572039.201Cm CC1432540.583Cm CC3169241.802Cm CC548841.902Cm CC7469842.412Cm CC605742.929Cm CC7647643.523Cm CC3117643.942Cm CC7337744.407Cm CC6849044.876Cm CC6416545.171Cm HLJ190645.387Cm CC6619046.016Cm CC1855946.525Cm HLJ398547.138Cm HLJ399747.385Cm CAFS222547.752Cm CAFS214748.103Cm CC3311748.566Cm CC3913448.904Cm CC4620249.293Cm CC384949.505Cm CC1611350.265Cm CAFS211150.84Cm CC992651.289Cm CAFS64951.294Cm CAFS216051.997Cm CC4443552.237Cm CAFS52052.346Cm CC867152.507Cm CC340852.57Cm CC889552.73Cm CC1124852.764Cm CC567852.789Cm CC871452.855Cm CC4959852.994Cm CC1150153.235Cm CC6320953.27Cm CC5968153.669Cm CC1807253.821Cm CC2991454.172Cm CC693954.274Cm CC6274354.425Cm CC5198454.433Cm CC1263454.958Cm CAFS209955.601Cm CC5893256.154Cm CC763156.523Cm CC1186656.768Cm CC4891457.151Cm CC3369957.53Cm CC2928558.084Cm CC4418758.335Cm CC997158.779Cm CC8053359.161Cm CC1205960.136Cm CC7090160.745Cm CC3367061.184Cm CC7047662.762Cm CC682163.87Cm CC337365.114Cm CC2776766.518Cm CC1761369.408Cm CC5508070.9Cm CC774972.188Cm HLJ250472.408Cm CC7704473.434Cm CC1266775.241Cm HLJ232478.379Cm CC990380.28Cm CC803784.344Cm CC2909487.733Cm HLJ2516

100Cm HLJ578

18.919Cm CC84087

19.86Cm CC6707

21.241Cm CC7494

22.278Cm CC27938

23.095Cm CC17283

23.759Cm CC45049

24.892Cm CC46429

25.339Cm CC11846

26.163Cm CC12341

26.731Cm CC76386

27.025Cm CC7592

27.583Cm CC12297

29.264Cm CC58657

30.502Cm CC28460

31.422Cm CC77294

31.798Cm CC15217

31.991Cm CC18037

37.037Cm CC53526

38.973Cm CAFS867

39.93Cm CC30049

40.348Cm CC43487

42.702Cm CC4331

43.442Cm CC47313

44.14Cm CC17010

45.767Cm CAFS2332

47.984Cm CAFS1398

48.773Cm CAFS2191

49.334Cm CC36009

56.162Cm CC42236

59.885Cm HLJ693

60.151Cm CC20172

66.937Cm CC15593

69.229Cm CC58379

78.097Cm CC35178

81.576Cm CC29072

84.16Cm CC82593

85.594Cm CC29796

87.81Cm CC12496

88.296Cm CC32289

91.061Cm CAFS803

91.824Cm CC4144

92.194Cm CC25307

93.903Cm CC4386

95.798Cm CC60189

99.729Cm HLJ3486


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75.519Cm CC7445

76.212Cm CC4697

77.395Cm CC81574

78.428Cm HLJE373

79.196Cm CAFS1737

82.776Cm HLJ3407

126.423Cm HLJ3396

460Cm CC882715.701Cm CAFS244618.372Cm SNP093519.545Cm CC1403920.966Cm CC509123.029Cm CAFS222025.789Cm CC1802026.969Cm CC637228.133Cm CC898628.658Cm CC1562629.116Cm CC1581129.963Cm HLJ294530.987Cm CC2505432.261Cm CC4738832.782Cm CC2737133.229Cm CC2882233.403Cm CC3840333.627Cm CC6104334.027Cm CC2866435.029Cm CC352735.173Cm CC1688535.615Cm CC1738636.316Cm CC344336.591Cm CC619337.271Cm CC563637.764Cm CAFS88938.162Cm HLJ395138.412Cm CC325839.028Cm CC1418139.586Cm CC1161341.123Cm HLJ347841.502Cm CC162641.959Cm CC3048742.03Cm CC829142.221Cm CC4510742.585Cm CAFS24543.148Cm CC703943.43Cm CAFS178243.868Cm CC1352044.049Cm CC6854644.205Cm CC2396444.375Cm CC2692444.676Cm CC7044344.692Cm CC3358044.76Cm CC531145.095Cm CC477945.151Cm HLJ361445.181Cm CC594245.516Cm CC2061545.603Cm CC1616645.978Cm CC1684246.448Cm CAFS77347.781Cm CAFS153247.943Cm CC7989048.648Cm CC1795249.258Cm HLJ389850.266Cm CC4085450.916Cm CC7317751.591Cm CC4807752.257Cm HLJ317053.53Cm HLJ350653.849Cm CC653454.449Cm CAFS210555.065Cm CC1863455.49Cm CC435255.878Cm CC371157.048Cm CC8199057.563Cm CC1538758.124Cm CC2706058.947Cm CC194859.429Cm CC2664460.861Cm CC5816661.819Cm CC5213363.35Cm HLJ251064.471Cm CC3103666.972Cm CC755467.081Cm CC936968.479Cm CC1308768.695Cm CC8475169.382Cm CC1186070.682Cm CC890771.529Cm CAFS15273.513Cm CC3229273.913Cm CC4512489.095Cm HLJ2535

470Cm HLJ34529.428Cm CC4429133.023Cm HLJ114834.982Cm CAFS21336.045Cm CC4026438.386Cm CC4586938.992Cm CAFS171939.135Cm CC8055139.692Cm HLJ357540.067Cm CC618441.742Cm CC7679641.938Cm CC4723442.078Cm CAFS66043.022Cm CC1848244.282Cm HLJ375444.441Cm HLJE49944.736Cm CC1072746.043Cm CC8393847.986Cm HLJ194448.567Cm CC6204149.098Cm CC3159149.831Cm HLJ238150.524Cm CC1090451.126Cm CC870951.637Cm CC3838252.222Cm CC2302152.719Cm CC6578952.994Cm HLJ373353.508Cm CC5191654.128Cm CC2268654.327Cm CC2855354.536Cm HLJE51154.567Cm CC5813654.839Cm HLJ398854.934Cm CC2886555.27Cm CC3720555.803Cm CC728655.859Cm CC72955.881Cm CC646256.016Cm CC301956.078Cm CC5081056.258Cm CC8163556.475Cm CC1001756.555Cm CC8524456.708Cm CC1051056.821Cm CC4410057.029Cm CC553757.209Cm CC8047757.376Cm CC7453857.543Cm CC2292857.863Cm CC1400958.001Cm CC3392158.266Cm CC4008058.551Cm CC4159458.875Cm CC2890359.163Cm CC8056559.687Cm CC971260.407Cm CC308660.719Cm CC3233961.874Cm CAFS184262.269Cm HLJ31562.985Cm CC448263.429Cm CC1836763.765Cm CC2404165.22Cm CC2090165.589Cm CC839866.315Cm CC6748866.691Cm CC5161467.702Cm CC910268.223Cm CC3170168.838Cm CC1661369.576Cm CC6436170.093Cm CC2981670.562Cm CC3813472.635Cm CC1561573.842Cm CC1044074.172Cm CC3352875.419Cm HLJE38775.778Cm CC7713976.575Cm CC911980.022Cm CC352481.162Cm CC4533385.132Cm CAFS92690.126Cm CC2696194.937Cm CAFS1766

480Cm HLJ2270

6.83Cm CAFS2238

13.883Cm CC4721

16.315Cm CC28628

17.276Cm CC3725

17.746Cm HLJ3369

18.225Cm CC84737

18.728Cm CC1543

19.19Cm CC5632

19.818Cm CC27313

20.667Cm CC5083

21.918Cm CC14573

22.769Cm CC41296

25.098Cm HLJ2463

25.181Cm CC5434

29.197Cm CC83768

30.336Cm CC15857

30.767Cm CC9395

32.066Cm CC10849

32.558Cm CC18606

33.737Cm CC20551

35.985Cm CC16925

36.991Cm CC2622

37.086Cm HLJ3469

37.135Cm CC52103

38.337Cm CAFS2227

39.216Cm CC53340

40.292Cm CC54448

43.841Cm CC2428

45.613Cm CC73329

45.758Cm CC7184

46.616Cm CAFS1317

48.016Cm CC65211

50.142Cm CC9253

51.811Cm HLJ456

52.915Cm CC29989

52.966Cm CC65353

53.846Cm CC31800

54.279Cm CC32364

54.583Cm CC18667

55.956Cm CC13950

56.307Cm HLJ3633

57.225Cm CC14450

57.529Cm CC7919

57.807Cm CC4258

58.064Cm CC3888

58.381Cm CC52978

59.744Cm CC47360

59.991Cm HLJ2248

61.387Cm CC1143

61.899Cm CC80068

61.958Cm CC84318

63.028Cm CC56064

64.032Cm HLJ3554

65.988Cm CC38601

67.48Cm CC15529

68.853Cm CC19051

70.199Cm CC73945

71.703Cm CC10949

72.341Cm CAFS1932

73.539Cm HLJ3768

76.342Cm CC44390

77.832Cm HLJ1482

80.046Cm CC79226

80.726Cm HLJ3421

82.819Cm CC10798

86.248Cm CC17466

88.628Cm CC26780

89.83Cm CC16444

91.767Cm CC41127

94.045Cm HLJ2312

95.997Cm CC73779

98.278Cm CC33463

100.225Cm CC56167

102.798Cm CC24039

104.516Cm CC30292

110.233Cm CC74789

490Cm HLJ39085.675Cm CC341007.633Cm CC46539.555Cm CC1888113.958Cm CC4133321.407Cm CC7048123.872Cm CC3739524.642Cm CC1306826.432Cm CC523627.66Cm CC3592428.716Cm CC1531529.572Cm CAFS75130.679Cm CC3319631.756Cm CC987532.119Cm CC236632.401Cm CC352133.232Cm CC3821633.464Cm CC6532933.715Cm CC2406434.212Cm CC2220634.294Cm HLJ49534.431Cm CC5187335.069Cm CC3217035.608Cm CC2870335.78Cm CAFS218236.158Cm CC3728036.515Cm HLJ394836.541Cm CC6162437.041Cm CC7901937.177Cm CC3982837.416Cm CC1905837.562Cm CC785237.726Cm HLJ36037.845Cm CAFS642-237.986Cm CC325538.009Cm CC1172338.198Cm CC3191438.229Cm CC266338.245Cm CAFS110538.275Cm CAFS229838.299Cm CC6346438.389Cm CC7243238.541Cm CC3093438.692Cm CC1199738.876Cm CC343239.103Cm CC1801439.457Cm CC1897539.652Cm CC1288639.915Cm CAFS181340.19Cm CC1390540.469Cm CC671540.98Cm CC5058741.599Cm CC1118642.185Cm CC2395443.321Cm CC1785743.826Cm CC945844.326Cm HLJE36145.596Cm CC2318747.133Cm CC4574747.765Cm CC1518348.509Cm HLJ314650.082Cm HLJ357051.121Cm CC6322952.78Cm CC3369754.85Cm CC844255.818Cm CC1162160.405Cm CC4866663.274Cm CAFS55266.215Cm CC752367.906Cm CC3142969.83Cm CC6669769.855Cm CC4845073.13Cm CC4403477.418Cm CC826684.45Cm CAFS1422

500Cm CC182182.483Cm CC335694.593Cm CC663645.673Cm CC51046.376Cm CC848907.032Cm CC50477.877Cm CC196858.414Cm CC16689.044Cm CC42819.262Cm CC291189.515Cm CC465989.889Cm CC2830710.046Cm CC8423610.393Cm CC734210.874Cm CC2010011.162Cm CC4568811.233Cm CC3091511.45Cm CC940011.856Cm CC964811.884Cm CC933412.02Cm CC655912.289Cm CC2688512.41Cm CC576712.425Cm CC1869812.456Cm CC5970212.502Cm CC524612.518Cm CC127112.54Cm CC7890112.552Cm CC426312.564Cm CC1010412.574Cm CC7447612.843Cm CC1968013.035Cm CC3256213.222Cm CC6970013.445Cm CC7827813.779Cm CC493214.02Cm CC1051914.44Cm CC161114.55Cm CC1016515.086Cm CC535515.498Cm CC3362116.059Cm CC6899416.522Cm CC2791016.681Cm HLJ343216.922Cm HLJ43917.345Cm CC6280818.225Cm CC1900318.812Cm CC3213019.467Cm CC7437021.073Cm CAFS66423.043Cm CC414328.813Cm CC5094130.085Cm CC314834.808Cm HLJ343853.771Cm CC8516460.575Cm CAFS226762.852Cm CC2759764.772Cm CC31603


22

SupplementaryFigure5.Geneticdistancevs.physicaldistance.

Genetic position of the genetic markers was plotted against the corresponding physical position

on the 50 C.carpio pseudo chromosomes. In each plot, physical distance along the indicated

pseudo chromosomes is on the horizontal axis, and genetic distance is on the vertical axis (in cM).


23

SupplementaryFigure6.Distributionofthegeneticdistance/physicaldistanceontheintegratedgenomeoftheC.carpio


24

SupplementaryFigure7.GCcontentsofC.carpioandothersequencedteleostgenomes.


25

SupplementaryFigure8.AgedistributionofinterspersedrepeatsinC.carpioandD.reriogenomes.


26

SupplementaryFigure9.Venndiagramofgenemodels.

Gene models were supported by evidences from de novo prediction, protein‐based homology

searches and expression data.


27

SupplementaryFigure10.Comparisonofgenestructureofsixteleostspecies


28

SupplementaryFigure11:HOXgeneclustersintheC.carpiogenome

Each horizontal thick line represents a cluster, the paralogous cluster derived from the recent

duplication were named α andβ, and the intermediate letter a and b means the cluster was derived

from the third WGD. HOXA, HOXB, HOXC and HOXD indicate the four HOX gene cluster

derived from the second WGD.


29

SupplementaryFigure12.ComparativeanalysisofHOXclustersbetweenC.carpioandAtlanticsalmongenome.

ComparativeanalysisofHOXclustersbetweenC.carpio,human,D.rerioandAtlanticsalmon

genome.Eachblockrepresentacluster(A,B,CandD)fromthesecondWGD,theclusters

resultedfromthethirdWGDwerenameaandb(intermediatelow‐caseletter),andthe

clustersresultedfromthelastWGDwerenamedαandβ.AllHOXgenesaresymbolizedby

square,eachlinerepresentsaHOXcluster,whileeachcolumnindicatesparalogousgene.In

clusterA,Atlanticsalmonlostacluster,HOXAbβ,whileinclusterC,C.carpiolostoneof

clusterCb,therewasnotenoughevidencetodefinethisclusterasHOXCbαorHOXCbβ.


30

SupplementaryFigure13.Aneighbor‐joiningtreeof10strainsofcommoncarponthebasisofSNPs.

We use abbreviations to represent the 10 different strains: Sp for the Songpu carp, D for the Danube carp, Sz for the Szarvas carp, A for the American carp, Y for the Yellow River carp, H for the Heilongjiang carp, O for the Oujiang color carp, Hb for the Hebao carp, X for the Xingguo carp, and K for Koi.


31

SupplementaryFigure14.Populationstructureof10strainsofcommoncarpfromK=2toK=8onthebasisofSNPs.

We use abbreviations to represent the 10 different strains (the same as supplementary Figure 13). Higher Ln values indicate more reliable results (K=3 and K=4).


32

SupplementaryFigure15.Deletionvalidationofthreegenes(ZPLD1,NLKandLRRC72)inHebaoandSongpu.


33

SupplementaryFigure16.DeletioninvestigationofFGFR1A1geneinfourstrains.

The figure illustrates a 306-bp deletion in FGFR1A1 gene in Songpu carp, which is causative

mutation of reduced scale. A) Hebao carp; B)Songpu carp; C) Xingguo red carp; D) Yellow

river carp.


34

SupplementaryFigure17.DifferentiallyexpressedgenesvalidatedbyqRT‐PCR.

Comparison between RNA-Seq results and qRT-PCR validation results. X-axis shows genes validated in this study; Y-axis shows Log2Ratio of expression of Hebao versus Songpu.


35

Supplementary Tables

SupplementaryTable1.Summariesoflibraryconstructionandsequencing

Libraries (insert size) Platform Total Data (Gb) Read length (bp) Sequence Coverage (X)

Single-end (N/A) 454 11.6 352 7

Paired-end (250 bp) Illumina 20.5 76 12

Paired-end (300 bp) Illumina 18.4 100 10.8

Paired-end (500 bp) Illumina 35.7 110 21

Mate-paired (2 kb) Illumina 5.8 80 3.4




Mate-paired (8 kb) 454 0.18 302 0.11

Mate-paired (8 kb) SOLiD 74 50 43.5

BAC library (141 kb) Sanger 0.043 647 0.03

Total 221.42 130.24


36

SupplementaryTable2.Mappedreadstothegenomeassembly

Libraries Clean reads Mapped reads Mapping ratio

Illumina 250 bp 270,766,288 254,152,676 93.86%

Illumina 300 bp 133,609,098 125,305,365 93.79%

Illumina 500 bp 324,605,540 304,867,293 93.92%

Illumina 2 kb 73,213,150 65,691,354 89.73%

Illumina 3 kb 256,747,542 153,340,679 59.72%

Illumina 5 kb 88,946,434 73,079,990 82.16%

Illumina 8 kb 251,423,074 153,767,798 61.16%

454 8 kb 957,508 938,813 98.04%

SOLiD 8 kb 748,046,808 55,835,484 74.60%

BAC 72,973 72,100 98.80%


37

SupplementaryTable3.Generegioncoverageassessedbasedontranscriptomes

Dataset Number Total length

(bp)

Covered by

assembly Percent

≥90% of sequence

covered by one scaffold

≥50% of sequence

covered by one scaffold

Number Percent Number Percent

≥100 bp 636,084 94,241,302 566,572 89.1% 506,601 79.6% 559,484 88.0%

≥200 bp 1,175,029 374,243,029 1,043,138 88.8% 918,762 78.2% 1,002,667 85.3%

≥500 bp 40,309 25,324,026 33,831 83.9% 26,943 66.8% 32,426 80.4%

≥2000 bp 105 250,785 103 98.1% 86 81.9% 97 92.4%

≥3000 bp 58 251,593 58 100% 50 86.2% 55 94.8%

≥100 bp 636,084 94,241,302 566,572 89.1% 506,601 79.6% 559,484 88.0%


38

SupplementaryTable4.GeneregioncoverageassessbytheCEGMAinteleosts

Dataset CEG in

teleost

Mapped

proteins

Completeness

Complete 191 172 90%

Group 1 35 29 83%

Group 2 39 32 82%

Group 3 56 53 95%

Group 4 61 58 95%


39

SupplementaryTable5.Coverageof19duplicatedgenepairsinthegenomeassembly

Pairs Gene Name Accession Identity Length Aligned length location

1 gonadotropin type I M37379 84% 820 790 000000319:47753-48912:-

gonadotropin type II M37380 784 616 000002077:163010-163983:-

2 Gh1 AJ640135 95% 633 633 000029063:481686-484013:+

Gh2 AJ640136 633 627 000012601:7508- 9331:+

3 cERK1 AB006038 77% 1539 1539 000000441:1588610-1625743:-

cERK2 AB006039 1658 1658 000028992: 1955587-1977378:-

4* C1rs-A AB042609 88% 2550 2343 000001243: 980208-988780:-

C1rs-B AB042610 2249 2223 000001243: 806022-811380:-

5 CfI-A AB072912 83% 1837 1482 000000198:79163-83752:-

CfI-B AB072913 2316 2295 000028834:808927-918173:-

6* Mbl1 AB110825 97% 913 889 000028941:537322-539760:+

Mbl2 AB110826 1023 1009 000028941:537322-539867:+

7* interleukin 1 beta 2-1 AJ401030 97% 875 875 000028918:628805-630834:+

interleukin 1 beta 2-2 AJ401031 1007 989 000028918:614607-640025:+

8 NILT1 AJ811994 84% 1185 1170 000006917:110387-112551:-

NILT2 AJ811995 1287 1253 000028978:2074672-2079749:-

9 leptin-II AJ830744 89% 706 699 000028869: 551482-552283:-

leptin-I AJ830745 960 924 000001162: 739679-741076:+

10 erythropoietin-I AJ831393 94% 990 986 000001141:428204-436851:-

erythropoietin-II AJ831394 469 452 000001124: 650412-660809:+

11* ifng2a AM168523 90% 780 763 000000341:1688587-1690701:-

ifng2b AM168524 749 749 000000341:1688591-1690701:-

12 Pitx2 I EF051103 96% 1036 1036 000028943:482003-483824:+

Pitx2 II EF051104 393 393 000028943:412283-412676:+

13 CD8a1 EU025118 83% 964 949 000000492:549999-554385:+

CD8a2 EU025119 972 628 000028977:459227-461280:+

14 cyp19a EU375455 68% 1807 1798 000000032:634927-641239:+

cyp19b EU375456 2846 2815 000001476:38402-46251:-

15 FGFR1a1 EU919569 95% 2454 2454 000000184:194505-224759:+

FGFR1a2 EU919570 2457 2457 000002146:712005-740223:-

16 gpx4a FJ656211 79% 560 560 000029057: 246640-249567:+

gpx4b FJ656212 519 480 000001057: 808493-978227:+

17 MyD88a GU809230 92% 2389 2366 000002126 :158214-162203:+

MyD88b GU809231 1566 1539 000000519:1095435-1099167:-

18 pro-opiomelanocortin-II Y14617 93% 959 931 000000378:1412003-1414404:-

pro-opiomelanocortin-I Y14618 955 928 000000009:19065-23542:-

19 CD8b1 EU025120 92% 1160 1140 000028977:449703-453659:+

CD8b2 EU025121 1157 1115 000000492:540682-544942:+

* Two duplicated genes were collapsed into one gene.


40

SupplementaryTable6.Allgenotypesforthehighdensitylinkagemapconstruction

(Included in a separated excel file)


41

SupplementaryTable7.RepeatcontentinC.carpiogenome

Repeat

Elements

Carp

Copies Bases Percent (%)

SINE 60436 9314658 0.55

LINE 169647 60640105 3.58

LTR 113023 38668676 2.28

DNA 1237555 232389411 13.71

Unclassified 1281659 188341161 11.11

total 2862320 529354011 31.23

All of the data were based on the RepeatMasker(version 3.3.0), with de novo library on C.carpio

genome assembly constructed by RepeatModeler (version 1.04).


42

SupplementaryTable8.Repeatcontentsandtheirclassification

Classes copies bases percent

DNA transposons

DNA/CMC 170276 22468462 1.33

DNA/Harbinger 2409 452406 0.03

DNA/hAT 462891 73785723 4.35

DNA/IS4EU 559 202290 0.01

DNA/Kolobok 75645 16783388 0.99

DNA/MULE 415 93767 0.01

DNA/PIF 60048 10259836 0.61

DNA/PiggyBac 36691 9121587 0.54

DNA/Sola 16204 1431846 0.08

DNA/TcMar 165723 52844723 3.12

DNA/other 204507 35651258 2.10

DNA/Helitron 42187 9294125 0.55

Retrotransposons

LINE/I 6633 1694810 0.10 LINE/L1 7185 3040045 0.18

LINE/L2 112883 40333840 2.38

LINE/Penelope 329 69020 0.00

LINE/Rex 39433 14524811 0.86

LINE/RTE 3184 977579 0.06

LTR/Copia 745 371121 0.02

LTR/DIRS 55769 17990259 1.06

LTR/ERV1 2112 416540 0.02

LTR/Gypsy 32312 13153896 0.78

LTR/Ngaro 3118 403639 0.02

LTR/Pao 1581 796967 0.05

LTR/Other 17386 5536254 0.33

SINE/Deu 3314 659784 0.04

SINE/ID 18791 1868768 0.11

SINE/L2 1518 170957 0.01

SINE/tRNA 3589 506116 0.03

SINE/Other 33224 6109033 0.36

Unclassified 1281659 188341161 11.11

Total 2862320 529354011 31.23


43

SupplementaryTable9.Compositionofrepeatelementsin6teleostgenomes

D.rerio G.aculeatus O.latipes T.rubripes T.nigroviridiscopies bases percent percent percent percent percent percent

DNA total 1195368 223095286 13.16 41.84 2.99 8.52 1.41 0.96

Academ 0.01 0.01

CMC 170276 22468462 1.33 6.74 0.31 0.03 0.04 0.01

En‐Spm 0.02

Ginger 0.04 0.01 0.01

Harbinger 2409 452406 0.03 0.01 0.02

hAT 462891 73785723 4.35 10.7 1.24 3.04 0.39 0.52

IS4EU 559 202290 0.01

Kolobok 75645 16783388 0.99 1.92

Maverick 0.05 0.09 0

MULE 415 93767 0.01 0.24 0.05 0.01 0.03

Novosib 0.01 0

P 0.15

PIF 60048 10259836 0.61 2.32 0.3 1.04 0.2 0

PiggyBac 36691 9121587 0.54 2.06 0.01 0.8

Sola 16204 1431846 0.08 0.13 0.01 0.01

TcMar 165723 52844723 3.12 5.43 0.65 2.62 0.72 0.39

other 204507 35651258 2.1 12.04 0.3 0.96 0.04 0

LINE total 169647 60640105 3.58 3.2 3.29 4.4 2.99 1.63

CR1 0.07

Dong 0.35 0.16

I 6633 1694810 0.1 0.17 0.02 0.01 0.02 0.03

L1 7185 3040045 0.18 0.46 0.11 0.46 0.2 0.1

L2 112883 40333840 2.38 1.96 1.67 1.75 1.37 0.17

Penelope 329 69020 0 0.33 0.12 0.2 0.23

Proto2 0.03

R2 0.02 0.02 0.18

Rex 39433 14524811 0.86 0.35 0.86 0.41 0.46 0.62

RTE 3184 977579 0.06 0.26 0.3 0.95 0.55 0.31

LTR total 113023 38668676 2.28 4.71 1.9 1.39 1.03 0.49

Copia 745 371121 0.02 0.03 0.03 0.05

DIRS 55769 17990259 1.06 1.22 0.02 0.01 0.06

ERV1 2112 416540 0.02 0.26 0.42 0.08 0.29 0.05

ERV 0.01

ERVK 0.02 0.07 0.01 0.02

Gypsy 32312 13153896 0.78 1.27 1.22 0.64 0.61 0.23

Ngaro 3118 403639 0.02 1.1 0.01 0.62 0.07 0.08

Pao 1581 796967 0.05 0.03 0.13 0.01 0.01

Other 17386 5536254 0.33 0.81 0.03 0.01

SINE total 60436 9314658 0.55 2.71 0.51 0.89 0.2 0.1

5S 0.29 0.01 0 0.02 0.01

Alu 0

BovA 0

Deu 3314 659784 0.04 0.2 0

ID 18791 1868768 0.11 0.04

MIR 0.2 0.29 0

L2 1518 170957 0.01 0.02

RTE 0.02

tRNA 3589 506116 0.03 0.19 0.02 0.09

V 1.73 0.16 0.26 0.04

Other 33224 6109033 0.36 0.26 0.07 0.24 0.18 0.05

RC Helitron 42187 9294125 0.55 2.47 0.02 0.01 0.02 0.02

Unclassified 1281659 188341161 11.11 4.84 4.77 15.47 1.45 2.49

total 2862320 529354011 31.23 59.78 13.48 30.68 7.1 5.7

Classes SubclassesC.carpio


44

SupplementaryTable10.Transcriptomesequencesusedongenomeannotation

Samples Number of total reads Number of Cleaned reads Total Bases of

Cleaned reads

Sequencing platform

Mixed tissues 2,116,226 1,418,591 455,367,711 Roche 454

Mixed tissues 242,261 241,170 52,812,898 Roche 454

Embyro‐0hpf 41,427,198 32,162,356 2,344,233,594 Illumina 100 PE










Fry‐1dph 27,492,778 23,497,838 1,792,303,590 Illumina 100 PE

Fry‐6dph 36,546,630 28,824,010 2,192,612,594 Illumina 100 PE

Note: RNA samples from mixed tissues including brain, blood, gill, heart, liver, spleen, intestine,

head kidney, kidney, muscle, gonad and ovary are pooled. “hpf” indicates hour‐post‐fertilization

and “dph” indicates day‐post‐hatchery.


45

SupplementaryTable11.StatisticsofgenemodelsidentifiedfromC.carpiogenome.

Source Number

De novo (Fgenesh) 105739

De novo (Augustus) 51721

Homolog 38092

RNAseq 73930

Total (EVM) 52610


46

SupplementaryTable12.Annotationsagainstproteinswithknownfunctionineachdatabase.

Number Percent (%)

Total 52610 --

Annotated Swissprot 43641 82.95%

TrEMBL 47708 90.68%

InterPro 38331 72.86%

KEGG 13015 24.74%

GO 31792 60.43%

Total 47795 90.85%

Un-annotated 4815 9.15%


47

SupplementaryTable13.ThestatisticsofgenestructureofC.carpioandthecomparisonwithotherteleosts.

Gene Set

Genome

assembly

size (Mb)

No.

genes

Mean

CDS

length

Mean

introgenic

region (bp)

No. exons

per gene

Mean

exon size

(bp)

Mean intron

size (bp)

C.carpio 1700 52610 1487.25 12145.20 7.48 198.78 1811.62

D.rerio 1412 26163 1853.73 22746.50 7.97 232.47 2923.24

G.aculeatus 461 20787 1592.57 8510.57 9.88 161.15 771.14

O.latipes 868 19686 1553.13 12619.6 10.04 154.66 1215.21

T.rubripes 393 18523 1617.17 7465.52 10.69 151.33 600.60

T.nigroviridis 358 19602 1517.67 6033.84 10.14 149.62 491.01


48

SupplementaryTable14.Non‐codingRNAgenesincommoncarpgenome

Non‐coding RNAs Copy number Average length (bp) Total Length (kb)

miRNAs 914 22 20.4

rRNAs 5S 927 85 78.5

5.8S 9 140 1.26

18S 34 329 11.2

28S 42 228 9.6

Summary 1012 99 100.2

tRNAs 3,622 74 268.1

SupplementaryTable15.Systematiccross‐speciescomparativeanalysisandgeneclassification.

1:1:1 X:X:X vertebrate Teleost Cyprindae Patchy Homology Undetectable

specific specific specific similarity

C. intestinalis 941 3,373 2,847 4,654 4,843

O. latipes 941 5,167 2,970 607 5,786 3,578 637

G. aculeatus 941 5,403 3,126 618 6,540 3,410 748

T. rubripes 941 5,386 3,104 588 6,094 2,363 47

D. rerio 941 5,834 3,500 1032 1,777 7,915 4,935 271

C. carpio 941 9,372 4,665 955 2,037 8,647 22,784 3,209

X. tropicalis 941 4,505 2,851 6,698 3,369 65

M. musculus 941 4,573 2,724 6,779 7,076 945

S. scrofa 941 4,704 2,822 5,945 7,069 159

H. sapiens 941 4,768 2,823 7,291 5,703 958

G. gallus 941 4,082 2,597 4,741 2,488 1,887

A. carolinensis 941 4,411 3,029 5,939 3,184 301

'1:1:1' indicates universal single-copy genes. 'X:X:X' indicates any other orthologous group

(missing in one species allowed), with 'X' meaning one or more orthologs per species. 'Patchy'

indicates other orthologs that are present in at least one teleost and one tetrapod genome.

'Homology' indicates partial homology detected with E < 10-6 but no orthology classified.


49

SupplementaryTable16.ListofC.carpioaccessionsforwholegenomeresequencingstudy

Accession Population or strain Group Geographic location Notes

D_1 Danube River Wild Europe (Danube River)



Sz_1 Szarvas 22 Domesticated Europe (Hungary)



A_1 American Wild North America (Chattahoochee River)



Sp_1 Songpu Domesticated Asia (Heilongjiang Province, China) Mirror scale




K_1 Koi Domesticated Asia (Japan) Kōhaku variety



Y_1 Yellow River Domesticated Asia (Yellow River, China)




H_1 Heilongjiang River Wild Asia (Heilongjiang River, China)



O_1 Oujiang color carp Domesticated Asia (Oujiang River, China) Various body color



P_1 Hebao carp Domesticated Asia (Jiangxi Province, China) Special body shape; red skin




X_1 Xingguo red carp Domesticated Asia (Jiangxi Province, China) Red skin




50

SupplementaryTable17.Statisticsofgenomeresequencingdata

Accession Raw bases (Gb) Mapped bases (Gb) Mapping rate (%) Coverage rate (%) Depth

D_1 13.05 10.49 80.35 89.83 6.19

D_2 14.94 13.30 89.02 91.13 7.84

D_3 15.28 13.58 88.87 91.14 8.01

Sz_1 14.63 11.17 76.39 84.73 6.59

Sz_2 17.57 15.53 88.37 91.85 9.16

Sz_3 12.08 9.12 75.49 80.51 5.38

Sp_1 11.71 9.90 84.54 89.32 5.84

Sp_2 12.39 10.40 83.93 89.09 6.14

Sp_3 12.66 10.64 84.10 89.35 6.28

Sp_4 11.45 9.62 84.02 88.58 5.68

A_1 13.50 11.82 87.60 86.98 6.98

A_2 12.41 10.91 87.94 83.45 6.44

A_3 9.38 8.23 87.76 87.22 4.86

K_1 11.81 10.00 84.65 88.06 5.90

K_2 14.09 11.90 84.47 89.33 7.02

K_3 6.75 5.78 85.61 77.59 3.41

Y_1 10.40 8.30 79.87 86.62 4.90

Y_2 10.43 8.33 79.84 87.04 4.91

Y_3 11.92 9.08 76.16 87.19 5.36

Y_4 14.01 11.11 79.31 89.18 6.56

H_1 13.40 9.21 68.75 86.73 5.44

H_2 16.13 12.25 75.93 89.72 7.23

H_3 15.22 9.46 62.20 87.49 5.58

O_1 11.98 9.65 80.52 87.00 5.69

O_2 11.39 8.23 72.27 85.65 4.86

O_3 10.66 8.26 77.48 85.63 4.87

P_1 9.86 7.92 80.32 85.60 4.67

P_2 13.63 9.53 69.90 85.63 5.62

P_3 13.07 10.36 79.23 88.28 6.11

P_4 12.54 10.21 81.44 88.11 6.03

X_1 13.01 11.42 87.76 89.46 6.74

X_2 13.70 12.04 87.93 89.24 7.11

X_3 12.55 11.01 87.74 88.96 6.50


51

SupplementaryTable18.SNPsandsmallINDELsidentifiedfromre‐sequencingdataof33accessions

Category No. SNPs No. small INDELs

Danube River 3,819,739 448,313

Szarvas 22 3,692,292 493,202

Songpu 3,324,223 292,432

American 3,818,139 334,345

Koi 4,984,190 409,550

Yellow River 3,841,719 261,928

Heilongjiang River 3,819,859 267,109

Oujiang color carp 2,480,140 168,430

Hebao carp 4,395,749 322,164

Xingguo red carp 5,413,133 479,845

Non-redundant 18,949,596 1,694,102


52

SupplementaryTable19.Listofgenesintheselectedregions(top1%highestπHebao/πSongpu)


SupplementaryTable20.Listofgenesintheselectedregions(top1%lowestπHebao/πSongpu)


SupplementaryTable21.Enrichedpathwaysingenesintheselectedregions


SupplementaryTable22.ClassificationofSNPsidentifiedfrom326genes.

SNP classification Number of unique SNPs

Songpu Hebao

Synonymous 320 495

Non-synonymous 160 204

mis-sense 154 200

pre-mature 3 3

skip-stop-codon 3 1

Total 480 699

SupplementaryTable23.Listofgenescontaininguniquenon‐synonymousSNPsfromsignificantlydiversifiedregionsbetweenSongpuandHebao.


SupplementaryTable24.DifferentiallyexpressedgenesintheskinofHebaoandSongpu.


SupplementaryTable25.Listofprimersfordeletionvalidationandreal‐timePCRanalysis.



53

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Documents

Supplementary Information for 2nd revision-lijt-20140819-2 · The BAC library was constructed using high molecular weight genomic DNA from a female C.carpio, which contained a total