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While it seems a pronounced trend today, Eugene Tong’s innovative manner of dress in cut & sew menswear mixed with streetwear and high fashion was one of the first examples to polarize high against low. Admired by such photographers as Tommy Ton and Scott Schuman of The Sartorialist, Tong’s signature look is one that exudes a similar level of taste and consideration to his selection for this edition of HYPEBEAST Essentials. Style director for Condé Nast-owned Details Magazine, where the Taiwanese-born creative has held various positions over his career, Tong has also received acclaim for providing styling cues for New York label Public School. For a self-proclaimed shoe enthusiast, we did notice the absence of any form of footwear, yet Eugene did present a range of distinguished pieces from Dunhill, Louis Vuitton and Rolex, while locking down elements of sensibility with a basic Bread & Boxers T-shirt and a clean Public School cap.

Wen, R.-T., Granqvist, C. G. & Niklasson, G. A. Eliminating degradation and uncovering ion-trapping dynamics in electrochromic WO3 thin films. Nat. Mater. 14, 996–1001 (2015).

Kim, K.-W. et al. Electrostatic-force-assisted dispensing printing of electrochromic gels for low-voltage displays. ACS Appl. Mater. Interfaces 9, 18994–19000 (2017).

Wang, K., Wu, H., Meng, Y., Zhang, Y. & Wei, Z. Integrated energy storage and electrochromic function in one flexible device: an energy storage smart window. Energy Environ. Sci. 5, 8384–8389 (2012).

Xia, X. et al. Controllable growth of conducting polymers shell for constructing high-quality organic/inorganic core/shell nanostructures and their optical-electrochemical properties. Nano Lett. 13, 4562–4568 (2013).

Zhong, Y. et al. Electrochromic asymmetric supercapacitor windows enable direct determination of energy status by the naked eye. ACS Appl. Mater. Interfaces 9, 34085–34092 (2017).

Ginting, R. T., Ovhal, M. M. & Kang, J.-W. A novel design of hybrid transparent electrodes for high performance and ultra-flexible bifunctional electrochromic-supercapacitors. Nano Energy 53, 650–657 (2018).

Tian, Y. et al. Synergy of W18O49 and polyaniline for smart supercapacitor electrode integrated with energy level indicating functionality. Nano Lett. 14, 2150–2156 (2014).

Wei, H., Zhu, J., Wu, S., Wei, S. & Guo, Z. Electrochromic polyaniline/graphite oxide nanocomposites with endured electrochemical energy storage. Polymer 54, 1820–1831 (2013).

Mandal, D., Routh, P. & Nandi, A. K. A new facile synthesis of tungsten oxide from tungsten disulfide: structure dependent supercapacitor and negative differential resistance properties. Small 14, 1702881 (2018).

Cong, S., Tian, Y., Li, Q., Zhao, Z. & Geng, F. Single-crystalline tungsten oxide quantum dots for fast pseudocapacitor and electrochromic applications. Adv. Mater. 26, 4260–4267 (2014).

Xu, J. et al. High-energy lithium-ion hybrid supercapacitors composed of hierarchical urchin-like WO3/C anodes and MOF-derived polyhedral hollow carbon cathodes. Nanoscale 8, 16761–16768 (2016).

Ma, D., Shi, G., Wang, H., Zhang, Q. & Li, Y. Morphology-tailored synthesis of vertically aligned 1D WO3 nano-structure films for highly enhanced electrochromic performance. J. Mater. Chem. A 1, 684–691 (2013).

Yun, T. Y., Li, X., Kim, S. H. & Moon, H. C. Dual-function electrochromic supercapacitors displaying real-time capacity in color. ACS Appl. Mater. Interfaces 10, 43993–43999 (2018).

Shen, L. et al. Flexible electrochromic supercapacitor hybrid electrodes based on tungsten oxide films and silver nanowires. Chem. Commun. 52, 6296–6299 (2016).

Yang, P. et al. Large-scale fabrication of pseudocapacitive glass windows that combine electrochromism and energy storage. Angew. Chem. Int. Ed. 53, 11935–11939 (2014).

Cai, G. et al. Highly stable transparent conductive silver Grid/PEDOT:PSS electrodes for integrated bifunctional flexible electrochromic supercapacitors. Adv. Energy Mater. 6, 1501882 (2016).

Azam, A. et al. Two-dimensional WO3 nanosheets chemically converted from layered WS2 for high-performance electrochromic devices. Nano Lett. 18, 5646–5651 (2018).

Cai, G. et al. InkJet- printed all solid-state electrochromic devices based on NiO/WO3 nanoparticle complementary electrodes. Nanoscale 8, 348–357 (2016).

Scherer, M. R. J., Li, L., Cunha, R. M. S., Scherman, O. A. & Steiner, U. Enhanced electrochromism in gyroid-structured vanadium pentoxide. Adv. Mater. 24, 1217–1221 (2012).

Ryoo, R., Joo, S. H. & Jun, S. Synthesis of highly ordered carbon molecular sieves via template-mediated structural transformation. J. Phys. Chem. B 103, 7743–7746 (1999).

Zhang, J. et al. Ligand-assisted assembly approach to synthesize large-pore ordered mesoporous titania with thermally stable and crystalline framework. Adv. Energy Mater. 1, 241–248 (2011).

Mahoney, L. & Koodali, R. T. Versatility of evaporation-induced self-assembly (EISA) method for preparation of mesoporous TiO2 for energy and environmental applications. Materials 7, 2697–2746 (2014).

Xu, J., Xia, J. & Lin, Z. Evaporation-induced self-assembly of nanoparticles from a sphere-on-flat geometry. Angew. Chem. Int. Ed. 46, 1860–1863 (2007).

Byun, M., Bowden, N. B. & Lin, Z. Hierarchically organized structures engineered from controlled evaporative self-assembly. Nano Lett. 10, 3111–3117 (2010).

Yu, K., Hurd, A. J. & Eisenberg, A. Syntheses of silica/polystyrene-block-poly(ethylene oxide) films with regular and reverse mesostructures of large characteristic length scales by solvent evaporation-induced self-assembly. Langmuir 17, 7961–7965 (2001).

Yu, K., Smarsly, B. & Brinker, C. J. Self-assembly and characterization of mesostructured silica films with a 3D arrangement of isolated spherical mesopores. Adv. Funct. Mater. 13, 47–52 (2003).

Biccari, F. et al. Graphene-based electron transport layers in perovskite solar cells: a step-up for an efficient carrier collection. Adv. Energy Mater. 7, 1701349 (2017).

Taylor, D. J., Cromin, J. P., Allard, L. F. & Birnie, D. P. Microstructure of laser-fired, sol-gel-derived tungsten oxide films. Chem. Mater. 8, 1396–1401 (1996).

Deepa, M., Saxena, T. K., Singh, D. P., Sood, K. N. & Agnihotry, S. A. Spin coated versus dip coated electrochromic tungsten oxides films: structure, morphology, optical and electrochemical properties. Electrochim. Acta 51, 1974–1989 (2006).

Fang, Y., Sun, X. & Gao, H. Influence of PEG additive and annealing temperature on structural and electrochromic properties of sol-gel derived WO3 films. J. Sol.-Gel Sci. Technol. 59, 145–152 (2011).

Yoon, M., Mali, M. G., Kim, M.-W., Al-Deyab, S. S. & Yoon, S. S. Electrostatic spray deposition of transparent tungsten oxide thin-film photoanodes for solar water splitting. Catal. Today 260, 89–94 (2016).

Li, H. et al. Solution-processed porous tungsten molybdenum oxide electrodes for energy storage smart windows. Adv. Mater. Technol. 2, 1700047 (2017).

The ChinaPower Podcast dissects critical issues underpinning China’s emergence as a global power. Hosted by Bonnie S. Glaser director of the CSIS China Power Project.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Additional file 2:Figure S1. Genomic synteny visualized using Oxford grids between linkage groups of C. c. haematopterus and LGs of C. c. carpio. (PDF 1100 kb)

Additional file 3:Figure S2. Genomic synteny visualized using Oxford grids between linkage groups of C. c. haematopterus and chromosomes Danio rerio. (PDF 32 kb)

Additional file 4:Figure S3. Genomic synteny visualized using Oxford grids between linkage groups of C. c. haematopterus and LGs of Ctenopharyngodon idellus. (PDF 31 kb)

Additional file 6:Figure S4. The QTL region for head length on LG5 of C. c. haematopterus and its homologous region in genomes of Danio rerio and Ctenopharyngodon idellus. (PDF 135 kb)

GUID: E4E061B4-903F-40BD-8DE2-B4A55B6EFA4BThe datasets supporting the findings of this article are included within the article and its supplementary information files. The raw sequence data from this study were deposited at the NCBI Sequence Read Archive (SRA) with the accession Number PRJNA377192.

A high-resolution genetic linkage map was constructed by using 7820 2b-RAD (2b-restriction site-associated DNA) and 295 microsatellite markers in a F2 family of the Yangtze River common carp (C. c. haematopterus). The length of the map was 4586.56 cM with an average marker interval of 0.57 cM. Comparative genome mapping revealed that a high proportion (70%) of markers with disagreed chromosome location was observed between C. c. haematopterus and another common carp strain (subspecies) C. c. carpio. A clear 2:1 relationship was observed between C. c. haematopterus linkage groups (LGs) and zebrafish (Danio rerio) chromosomes. Based on the genetic map, 21 QTLs for growth-related traits were detected on 12 LGs, and contributed values of phenotypic variance explained (PVE) ranging from 16.3 to 38.6%, with LOD scores ranging from 4.02 to 11.13. A genome-wide significant QTL (LOD = 10.83) and three chromosome-wide significant QTLs (mean LOD = 4.84) for sex were mapped on LG50 and LG24, respectively. A 1.4 cM confidence interval of QTL for all growth-related traits showed conserved synteny with a 2.06 M segment on chromosome 14 of D. rerio. Five potential candidate genes were identified by blast search in this genomic region, including a well-studied multi-functional growth related gene, Apelin.

We mapped a set of suggestive and significant QTLs for growth-related traits and sex based on a high-density genetic linkage map using SNP and microsatellite markers for Yangtze River common carp. Several candidate growth genes were also identified from the QTL regions by comparative mapping. This genetic map would provide a basis for genome assembly and comparative genomics studies, and those QTL-derived candidate genes and genetic markers are useful genomic resources for marker-assisted selection (MAS) of growth-related traits in the Yangtze River common carp.

A genetic linkage map is an essential tool in many genetics and genomics researches, such as quantitative trait loci (QTL) mapping for target traits of economic importance [1], positional or candidate gene cloning [2], construction of gene-centromere maps [3], comparative genome analysis [4] and genome assembly [5]. In most important aquaculture species, genetic maps have been constructed using amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers [6]. However, most of these maps have few molecular markers and low density owing to the high cost and laborious wet lab work of marker discovery and genotyping, which limits their abilities to perform fine-scale QTL mapping and other studies. With the development of the next-generation sequencing technologies (NGS), a variety of genotyping-by-sequencing (GBS) methods have been developed and applied for rapidly and cost-effectively developing and genotyping thousands of single nucleotide polymorphism (SNP) markers which are available for constructing high-resolution linkage maps [7]. Among those available GBS techniques, restriction site-associated DNA (RAD) sequencing [8] and its derivative methods, such as ddRAD [9], GGRS [10], SLAF [11] and 2b-RAD [12], are reduced representation approaches that construct sequencing libraries from a fraction of the genome produced by using restriction enzymes. RAD-related sequencing technologies have been successfully used for constructing high-resolution linkage maps in several aquaculture species, such as Atlantic salmon (Salmo salar) [13], Japanese flounder (Paralichthys olivaceus) [14], tilapia (Oreochromis niloticus L.) [15], Zhikong Scallop (Chlamys farreri) [5], pearl oyster (Pinctada fucata martensii) [16], Chinese mitten crab (Eriocheir sinensis) [17] and Asian seabass (Lates calcarifer) [18]. Among those RAD-related methods, 2b-RAD strategy may have the simplest protocol for library preparation without any extra procedures for fragment selection and purification, and has been used for map construction in aquaculture species [5, 19, 20]. Furthermore, 2b-RAD produces uniform fragments for sequencing, thus providing more effective utilization of sequencing data among all individuals investigated.

Growth, one of the most important economic traits for aquaculture fish, is a quantitative trait that controlled by multi-gene QTLs across the genome and environmental effects. Traditional strategies of genetic improvement for growth-related traits have mainly relied on family and individual selection based on phenotype and pedigree information [21]. Nowadays, marker-assisted selection (MAS) using markers linked to QTLs has become a valuable tool to improve the accuracy of selection and speed up the genetic improvement [22]. QTL mapping enables us not only to detect genetic markers associated with the genetic variation for important traits but also to identify the candidate genes involving physiological processes of the traits [23], showing its strong application potential in MAS program. QTL analyses of growth-related traits have been conducted in some aquaculture fishes, such as Atlantic salmon [24], rainbow trout (Oncorhynchus mykiss) [25], tilapia [26], Asian seabass [18], Japanese flounder [27] and half-smooth tongue sole (Cynoglossus semilaevis) [28]. In most cases, growth-related QTLs are mapped to several linkage groups (LGs), among them few QTLs are identified on a genome-wide scale and others on chromosome-wide significance levels. Significant QTLs for sex determination, disease resistance and anti-stress traits have also been investigated for fish species [29].

Common carp (Cyprinus carpio), one of the most important cyprinid species, is mainly cultured in Europe and Asia with a culture history of several thousand years, and contributes an annual production of 4.1 million metric tons in the world (FAO, 2013). Based on morphological characters, common carp was classified into three subspecies: C. c. carpio, C. c. haematopterus and C. c. rubrofuscus [30]. Owing to the important roles of common carp in aquaculture industry and biological studies, various genetic and genomic resources, such as genetic linkage maps [31], transcriptome [32], microRNA [33] and genome sequence [34] have been developed in the Songpu mirror carp strain which belongs to the subspecies C. c. carpio. The Yangtze River common carp, belonging to the subspecies C. c. haematopterus and as one of the most important aquacultured strains in China, has quite limited genetics and genomics resources for analysis of economic traits so far. The analysis of sequence variations obtained from genome resequencing has revealed that C. c. carpio and C. c. haematopterus are grouped into the European and Asian clades respectively [34]. Recently, a high-resolution genetic map has been constructed for another strain of C. c. haematopterus, Yellow River carp, revealing a puzzling finding that a high proportion (62.3%) of markers with disagreed chromosome location was observed between C. c. carpio and C. c. haematopterus [35]. Construction of the high-resolution map for the Yangtze River strain of common carp would provide more information to understand the genome structure and gene rearrangement between the two subspecies. QTLs for growth-related traits have been identified by several studies in the Songpu mirror carp strain mainly based on microsatellite markers [36–38]. Compared with SNPs, microsatellites have longer flanking DNA sequences which are effective for comparative mapping. However, the density of the map base on microsatellites is generally medium or low, which is not suitable for fine mapping of QTL.

The main objectives of this study include: (1) construction of a high-resolution genetic linkage map based on 2b-RAD markers and microsatellite markers in the Yangtze River common carp C. c. haematopterus, (2) comparative genome analysis between the genetic map and the assembled genomes of C. c. carpio, zebrafish (Danio rerio) and grass carp (Ctenopharyngodon idellus), (3) performing fine QTL mapping for growth-related traits and sex, and (4) identification of candidate genes associated with growth-related traits.

The mapping family in this study consists of 104 C. c. haematopterus progeny, and the phenotypic growth-related traits were all in concordance with normal distribution (P < 0.001 for all). The average values of body weight (BW), total length (TL), body length (BL), body height (BH) and head length (HL) were 369.9 ± 89.2 g, 27.4 ± 2.5 cm, 24.9 ± 2.4 cm, 7.9 ± 0.7 cm and 6.7 ± 0.6 cm, respectively. These growth-related traits showed a strong correlation with each other (r = 0.780–0.993, P < 0.001 for all) (Table 1). The highest correlation value (r = 0.993) was observed between TL and BL. The BW strongly correlated with TL (r = 0.934), BL (r = 0.934) and BH (r = 0.931). By phenotype sexing in the mapping family, 50 and 54 individuals were identified as males and females, respectively, with a sex ratio of 1:1.08.

Nearly 0.18 million potential BcgI restriction sites were estimated from the assembled genome sequence of common carp [34]. By single-end sequencing, a total of 263.98 million reads were generated from 2b-RAD libraries, including 7.61 million reads from the female parent, 10.22 million reads from the male parent and 246.15 million reads from the progenies (2.37 million reads per progeny). After quality filtering and sequence trimming, parental reads were clustered into 121,494 representative tags, including 91,591 parent-shared tags (codominant tags) and 29,903 parent-specific tags (dominant tags). After filtering low-quality tags with low (< 8) or high (> 3000) coverage, 83,924 codominant tags and 24,075 dominant tags were remained and used for constructing reference tags. The reads of offspring were mapped on the reference tags after quality filtering and sequence trimming. Finally, a total of 19,839 polymorphic markers including 12,084 codominant (parent-shared) markers and 7755 dominant (parent-specific) markers were heterozygous in at least one parent and genotyped in at least 80% of the offspring.

Among the 1500 microsatellites, 416 were reliably amplified polymorphic products in the mapping family. In total, 20,255 markers including above 2b-RAD and SSR markers were remained, and were tested for their segregation distortion. The results showed that 8921 markers including 3748 2b-RAD codominant markers, 4830 2b-RAD dominant markers and 343 SSR markers were in accordance with the Mendelian expectations (P ≥ 0.05) and were used for linkage analysis. These markers showed five segregation types:  (n = 14),  (n = 28),  (n = 373),  (n = 4971) and < nn × np > (n = 3535).

By using the JoinMap 4.1 software [39] with the logarithm of odds (LOD) threshold of 11.0, 8115 markers (7820 2b-RAD markers and 295 SSRs) (Additional file 1: Table S1) were successfully grouped into 50 linkage groups (LGs) which was consistent with the haploid chromosome number of common carp [38]. The sex-averaged genetic map spanned 4586.56 cM with an average marker interval of 0.57 cM. The genetic length of LGs ranged from 49.05 cM (LG38) to 139.52 cM (LG9) with an average of 91.73 cM, and the number of markers varied from 89 (LG24) to 262 (LG13) with an average of 162 (Fig. 1 and Table 2). Using two different methods [40, 41], the genome length was estimated to be 4587.70 cM (Ge1) and 4645.93 cM (Ge1), with an average of 4616.82 cM which was used as the expected genome length. Therefore, the genome coverage (Cof) of this genetic map was 99.3%. Since the genome size of common carp has been estimated to be 1.70 Gb based on cytogenetic method [42], the average recombination rate across all LGs was ~ 2.7 cM/Mb.

Linkage groupNumber of markersGenetic length (cM)Marker interval (cM)Linkage groupNumber of markersGenetic length (cM)Marker interval (cM)123084.810.3726117130.081.11

Through blast search, a total of 6002 (74.0%) markers were aligned to the assembled genome of C. c. carpio, among them, 3238 (53.9%) were uniquely mapped on the assembled LGs and were used for synteny analysis. Overall, a one-to-one correspondence was observed between LGs of C. c. haematopterus and C. c. carpio. However, for each LG of C. c. haematopterus, about an average proportion of 30% of markers were located on a single LG of C. c. carpio, others were dispersed on various LGs (Fig. 2a, Fig. ​Fig.2b2b and Additional file 2: Figure S1).

Circos diagram representing syntenic relationships between C. c. haematopterus (right) and (a and b) C. c. carpio (left) and (c) Danio rerio (left), and (d) Venn diagrams describing overlaps among uniquely aligned markers that mapped to genomes of C. c. carpio (Cc), D. rerio (Dr) and C. idellus (Ci). Only markers on each linkage group of C. c. haematopterus that were mapped to a single linkage group of C. c. carpio were shown in (b)

A total of 509 markers on linkage map of C. c. haematopterus were uniquely aligned to the chromosomes of D. rerio (Fig. ​(Fig.2c2c and Additional file 3: Figure S2), with 475 (93.3%) markers located into 50 syntenic boxes. Every two LGs of C. c. haematopterus were homologous with a single chromosome of D. rerio, showing a clear 2:1 relationship of C. c. haematopterus LGs and D. rerio chromosomes. A high level of genomic synteny was also detected between C. c. haematopterus and C. idellus (Additional file 4: Figure S3). Of the 667 markers uniquely anchored to the assembled LGs of C. idellus, 622 (93.3%) were located into syntenic boxes. Four LGs (LG19, LG20, LG43 and LG44) of C. c. haematopterus linkage map were mapped to LG24 of C. idellus, while a clear 2:1 syntenic relationship was observed between the remaining 46 LGs of C. c. haematopterus and 23 LGs of C. idellus. Among all aligned markers, 93 (55 2b-RAD markers and 38 SSRs) were uniquely aligned to all reference genomes (Fig. ​(Fig.2d),2d), revealing that these markers were highly conserved among the three cyprinid fishes.

The chromosome-wide and genome-wide LOD significance thresholds for growth-related traits varied from 3.3 to 6.2 and 6.1 to 10.2, respectively, based on permutation test. By using multiple QTL model (MQM), twenty one QTLs associated with growth-related traits were detected on 12 LGs, including two genome-wide significant QTLs (qBH27-a and qBW40-a) and 19 chromosome-wide significant QTLs, with LOD scores ranging from 4.02 to 11.13 (Fig. 3 and Table 3). Four QTLs (qTL22-a, qTL27-a, qTL39-a and qTL40-a) associated with TL were located at 68.57 cM, 70.23 cM, 41.29 cM and 29.36 cM along LG 22, LG 27, LG 39 and LG40, and contributed values of phenotypic variance explained (PVE) of 18.3, 20.3, 18.6 and 20.3%, respectively (Fig. ​(Fig.3a).3a). Owing to the high correlation value (r = 0.993) between TL and BL, QTLs for BL were located at the same confidence intervals along four LGs (Fig. ​(Fig.3b).3b). Five QTLs for BH were mapped to LG12, LG22, LG27, LG31 and LG46 with values of PVE of 18.0, 18.2, 24.2, 19.7 and 16.7%, respectively (Fig. ​(Fig.3c).3c). Four QTLs associated with HL were located on four LGs (LG5, LG27, LG28 and LG50) with values of PVE ranging from 16.3 to 18.4% (Fig. ​(Fig.3d).3d). For BW, four QTLs located on LG15, LG27, LG40 and LG45 contributed values of PVE of 21.2, 21.7, 38.6 and 32.5%, with LOD scores of 5.38, 5.51, 11.13 and 8.89, respectively (Fig. ​(Fig.3e).3e). Among all confidence intervals of QTLs, only the confidence interval on LG27 was associated with all five growth-related traits.

For sex, the genome-wide LOD significance threshold was 5.9. A genome-wide significant QTL was finely mapped on LG50 with the confidence interval ranging from 12.25 cM to 40.75 cM, and explained 38.1% of the phenotypic variance (LOD = 10.83) (Fig. ​(Fig.3f).3f). A total of 15 markers were identified in this QTL region, among them, nine continuous markers (from ref-20,177 to ref-119,060) with the average LOD score of 7.12 were all heterozygous in female parent and homozygous in male parent. The chromosome-wide LOD significance thresholds for sex varied from 3.2 (LG21) to 3.9 (LG32). Three chromosome-wide significant QTLs were detected on LG24, and contributed value of PVE of 18.7%, 16.9 and 22.2%, with LOD scores of 4.67, 4.19 and 5.66, respectively.

By searching against the non-redundant (nr) protein database, the sequences of six QTL markers for growth-related traits showed high similarities to fish genes (Additional file 5: Table S2). On the other hand, the homologous regions of QTLs were identified in the assembled genomes of D. rerio and C. idellus. On LG27 of C. c. haematopterus, a confidence interval of QTL for all growth-related traits ranging from 69.2 cM to 70.6 cM was mapped to a 2.06 M region on chromosome 14 of D. rerio and a 1.20 M region on scaffold CI01000000 of C. idellus (Fig. 4). Based on the annotation information of D. rerio genome, 36 genes were located in this region. Among them, four genes, ZDHHC9 (zinc finger, DHHC-type containing 9), Apelin, PTTG1 (pituitary tumor-transforming 1) and Adrb2a (adrenoceptor beta 2, surface a) have been reported to play important roles in growth of skeletal muscle [43], obesity [44], tumorigenesis [45] and growth of muscle myotomal fibres [46], respectively, and a vimentin-like gene contains the sequence of marker ref-90,952. The confidence interval of the QTL (qHL5-a) on LG5 was mapped to a 0.87 M region on chromosome 3 of D. rerio and a 0.63 M region on Scaffold CI01000034 of C. idellus. However, this QTL region was observed inversion compared to genome sequence of D. rerio and C. idellus (Additional file 6: Figure S4), indicating that the rearrangement of chromosome might occur in this region between C. c. haematopterus and D. rerio and C. idellus. According to the annotation of D. rerio genome, two potential growth-related genes, Atf4b2 (activating transcription factor 4b2) and Pvalb6 (parvalbumin 6) were located in this region (Additional file 6: Figure S4). These candidate QTL genes may involve in the genetic control of growth-related traits, which are worthy of further studies.

In this study, 2b-RAD sequencing of a F2 family of common carp generated 8578 high-quality markers which were more than that obtained by RAD sequencing [34]. However, only 4% of these markers were polymorphic in both parents, which is similar to that reported in the sea cucumber [47]. Additional microsatellite markers were genotyped in this study, and 14% of them were parent-shared markers which help us to construct the first high-density sex-averaged genetic map of the Yangtze River common carp (C. c. haematopterus). The number of LGs (n = 50) (Fig. ​(Fig.1)1) is in agreement with the haploid chromosome number of common carp [48]. Compared with the genetic map of Songpu mirror carp (C. c. carpio) [34], this map had a slightly larger size (4586.56 vs 3946.7 cM) and a higher resolution (0.57 vs 0.93 cM). On LG21 and LG26, few markers were located at the terminal region, which may be caused by the presence of centromeres [3], and/or the effect of DNA methylation. Attention should be paid to the caution of mapped markers at the terminal region and the effect of DNA methylation as this might involve in the reality and accuracy of some SNP loci. However, it is unclear whether BcgI (type IIB endonuclease) used in the 2b-RAD sequencing of this study is sensitive to methylation or not [49–51], currently we have difficulties in dressing this issue clearly. In future, we should firstly clarify the sensitivity of BcgI to methylation and then investigate the relationship between the caution of mapped markers at the terminal region and the effect of DNA methylation.

With a long history of cultivation, common carp has been bred into numerous strains and local populations which are mainly grouped into European and Asian clades based on sequence variations [34]. In this study, the syntenic analysis was performed between the high-density genetic map of C. c. haematopterus (Asian) and the assembled genome of C. c. carpio (European) to investigate their evolutionary relationship. The results showed that a 1:1 relationship between LGs of C. c. haematopterus and C. c. carpio was observed based on syntenic boxes, however, only 30% of markers were located in these syntenic boxes (Additional file 2: Figure S1), which was similar to that observed from the strain of Yellow River carp C. c. haematopterus [35], indicating the extensive intra-chromosomal rearrangements between the two subspecies. Common carp is believed to have undergone a fourth round of genome duplication [34, 52], which was also validated in this study. A high level of conserved genomic synteny was observed between C. c. haematopterus and D. rerio (93.3%) with a clear 2:1 relationship between LGs and chromosomes (Fig. ​(Fig.2c2c and Additional file 3: Figure S2). Similarly, a clear 2:1 relationship was also observed between LGs of C. c. haematopterus and C. idellus except for LG24 of C. idellus, which showed synteny with LG19, LG20, LG43 and LG44 of C. c. haematopterus (Additional file 4: Figure S3). Therefore, two chromosomes (Chr10 and Chr22) of D. rerio were homologous with LG24 of C. idellus, which has also been reported in previous studies [53, 54]. According to the hypothesis that the ancestor of teleosts had 24 chromosomes [55, 56], the two chromosomes (Chr10 and Chr22) of D. rerio may be formed by the fission of an ancestral chromosome.

The high-density genetic map used for QTL analysis in this study had a higher resolution than previous used maps [36–38]. The 1.4 cM confidence interval of QTL for all growth traits showed synteny with a 2.06 M region on chromosome 14 of D. rerio which contains 36 genes (Fig. ​(Fig.4).4). Another 0.8 cM confidence interval of QTL for HL was mapped to a 0.87 M region on chromosome 3 of D. rerio containing 23 genes (Additional file 6: Figure S4). Potential candidate genes were identified based on previous study of gene function. Among them, two genes, Apelin and Pvalb6 are the most likely to be associated with growth. Apelin, an adipokine, is expressed and secreted by adipocytes, and has been reported to have significant effects on energy metabolism, insulin sensitivity and pituitary hormone release [45, 59, 60]. Parvalbumins are extremely abundant in fish muscle and play an important role in muscle relaxation [61]. A microsatellite polymorphism in Pvalb1 has been reported to be significantly associated with body weight and body length in Asian seabass [61]. Further studies are necessary to verify the associations between polymorphisms in these two genes with growth traits in common carp.

The sex determination of common carp is male-dominant (XX/XY) since sex ratios in conventional diploid offspring approximate 1:1 and gynogenetic offspring are all female [62]. However, like other fish species, it is difficult to distinguish between the sex chromosomes (X and Y), as well as between the sex and autosomal chromosomes based on current cytogenetic techniques [63, 64]. In this study, QTLs for sex determination of Yangtze River carp were identified on LG24 and LG50, which was not consistent with that observed in Yellow River carp with the QTLs on LG11and LG43 [35]. Chen et al. [65] identified a sex-specific DNA marker in Yellow River carp with the marker sequence mapped on an unplaced genomic scaffold. An earlier discovery has provided evidence for autosomal influences on sex determination in common carp [66]. These results may indicate that sex determination of common carp is polygenic and different genes may influence sex determination in different strains. Such polygenic sex determination mechanism has also been observed in other fish species, such as zebrafish [67, 68], tilapia [15, 69] and Atlantic salmon [70]. The sex-determining loci identified in this study would be useful for MAS breeding in the Yangtze River common carp.

Two wild populations of common carp were collected from two geographic areas (Jingzhou and Wuhan) along the Yangtze River, and were used to produce F1 populations. In late April 2011, a F2 family was generated by crossing of a dam and a sire from the the same F1 family, and was raised in a 0.3 ha muddy pond after disinfection and fertilizing at Zhangdu Lake Fish Farm (Wuhan, China). Fish were fed three times daily, with soy milk at the larval stage (about 15 days post-hatch) and pellet feed at subsequent stages. After 9 months of culture, 104 progeny used for QTL mapping were randomly selected for phenotypic measurements. Five parameters of growth-related traits including BW, TL, BL, BH and HL were recorded. The measured individuals were then PIT-tagged and continued being cultured in the pond. Phenotype sexing was performed for each progeny of the F2 family by gently press abdomen of the fish in late April 2012. In Wuhan, the time for fully maturation of common carp is one year for male and two years for females. In one year, testis is fully developed and full of sperm in spawning season (spring). If milt were observed from the genital opening, then the fish was recorded as a male, otherwise as a female (female fish also with a swollen belly, which has moderately developed or almost matured ovary inside in one year).

Before the library preparation, potential restriction sites were calculated based on the assembled genome sequence of common carp (http://www.carpbase.org). 2b-RAD libraries were prepared for two parents and 104 progeny by following the standard protocol [12] with some modifications. 200 ng of genomic DNA from each individual was digested with BcgI restriction enzyme (New England Biolabs, USA) at 37 °C for 4 h. The digestion product was heat-inactivated for 20 min at 65 °C, and then ligated to adapter 1 and adapter 2 at 16 °C over night. The ligated fragments were amplified with Phusion High-Fidelity DNA Polymerase (Thermo Scientific, USA) using a set of four primers that introduce sample-specific barcode and sequencing primers. After 15 cycles of PCR, the library was obtained by purifying the amplification products at ~ 170 bp via retrieval from 8% polyacrylamide gels. Libraries were pooled with equal amount to make the final library which was sequenced in a lane of the Illumina HiSeq2500 SE50 platform (Illumina, USA).

A total of 1500 genomic and transcript-associated SSR markers previously published for common carp [73, 74] were also used for initial segregation screening in the mapping family. Polymorphic loci segregated in either female or male parents were genotyped in the progeny through PCR amplification. PCR was performed on a veritiTM 96 well thermal cycler (Applied Biosystems, USA) with a total volume of 12.5 μl, containing 30 ng template DNA, 1.25 μl 10× PCR buffer (TaKaRa, Japan), 0.25 U Taq DNA polymerase (TaKaRa, Japan), 50 μM each dNTP, 0.2 μM each primer and water to the final volume. The thermal cycling was programmed as follows: 5 min at 94 °C, followed by 37 cycles of 94 °C for 30 s, 35 s at appropriate annealing temperature, and 72 °C for 40 s, and the last extension at 72 °C for 10 min. PCR products were size-fractionated on 8% polyacrylamide gels and visualized by ethidium bromide staining.

Microsatellite loci were separated into three segregation patterns: 1:1 (type lm × ll or nn × np), 1:2:1 (type hk × hk) and 1:1:1:1 (type ab × cd or ef × eg), and 2b-RAD markers just showed the first two segregation patterns. The genotyping data of 2b-RAD and microsatellite markers were integrated for further analysis. Segregating markers that could not be genotyped in at least 20% of the offspring were removed. The sex-averaged genetic linkage map was constructed using JoinMap 4.1 [39] under the CP algorithm. The “Locus genot. Freq.” function was used for a chi-square test to assess the goodness of fit to the expected segregation ratios for each locus at the confidence level of 0.05. Markers showing significant departure from the expected segregation ratios were excluded. Linkage between markers was examined by estimating logarithm of the odds (LOD) scores for recombination fraction. Markers were grouped at a LOD threshold score of 11.0 and a maximum recombination fraction of 0.35. The regression mapping algorithm was selected for mapping. The Kosambi mapping function was used to convert the recombination frequencies into map distances in centiMorgans (cM). MapChart 2.2 software was used for graphical visualization of the linkage groups (LGs) [75].

Figure S3. Genomic synteny visualized using Oxford grids between linkage groups of C. c. haematopterus and LGs of Ctenopharyngodon idellus. (PDF 31 kb)

Figure S4. The QTL region for head length on LG5 of C. c. haematopterus and its homologous region in genomes of Danio rerio and Ctenopharyngodon idellus. (PDF 135 kb)

This research was supported by the Chinese Academy of Sciences (XDA0810405), FEBL Program (2016FBZ05) and MOST 973 Project (2010CB126305) of China. We would like to thank Profs. X. Sun and P. Xu from the Chinese Academy of Fishery Sciences for their assistance in data analysis, and Drs. W. Guo, C. Zhu and Y. Sun for their technical assistance in both laboratory and field work.

The datasets supporting the findings of this article are included within the article and its supplementary information files. The raw sequence data from this study were deposited at the NCBI Sequence Read Archive (SRA) with the accession Number PRJNA377192.

1. Sun XW, Liang LQ. A genetic linkage map of common carp (Cyprinus carpio L.) and mapping of a locus associated with cold tolerance. Aquaculture.2004;238:165–172. doi: 10.1016/S0044-8486(03)00445-9. [CrossRef]

2. Tamura Y, Hattori M, Yoshioka H, Yoshioka M, Takahashi A, Wu J, et al. Map-based cloning and characterization of a brown planthopper resistance gene BPH26 from Oryza sativa L. ssp indica cultivar ADR52. Sci Rep.2014;4:5872. doi: 10.1038/srep05872. PubMed] [CrossRef]

3. Feng X, Wang X, Yu X, Zhang X, Lu C, Sun X, et al. Microsatellite-centromere mapping in common carp through half-tetrad analysis in diploid meiogynogenetic families. Chromosoma.2015;124:67–79. doi: 10.1007/s00412-014-0485-6. [PubMed] [CrossRef]

4. Zhu C, Tong J, Yu X, Guo W. Comparative mapping for bighead carp (Aristichthys nobilis) against model and non-model fishes provides insights into the genomic evolution of cyprinids. Mol Gen Genomics.2015;290:1313–1326. doi: 10.1007/s00438-015-0992-z. [PubMed] [CrossRef]

5. Jiao W, Fu X, Dou J, Li H, Su H, Mao J, et al. High-resolution linkage and quantitative trait locus mapping aided by genome survey sequencing: building up an integrative genomic framework for a bivalve mollusc. DNA Res.2014;21:85–101. doi: 10.1093/dnares/dst043. PubMed] [CrossRef]

6. Yue GH. Recent advances of genome mapping and marker-assisted selection in aquaculture. Fish Fish.2014;15:376–396. doi: 10.1111/faf.12020. [CrossRef]

7. Davey JW, Hohenlohe PA, Etter PD, Boone JQ, Catchen JM, Blaxter ML, et al. Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nat Rev Genet.2011;12:499–510. doi: 10.1038/nrg3012. [PubMed] [CrossRef]

13. Gonen S, Lowe NR, Cezard T, Gharbi K, Bishop SC, Houston RD. Linkage maps of the Atlantic salmon (Salmo salar) genome derived from RAD sequencing. BMC Genomics.2014;15:166. doi: 10.1186/1471-2164-15-166. PubMed] [CrossRef]

14. Shao C, Niu Y, Rastas P, Liu Y, Xie Z, Li H, et al. Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis. DNA Res.2015;22:161–170. doi: 10.1093/dnares/dsv001. PubMed] [CrossRef]

16. Shi Y, Wang S, Gu Z, Lv J, Zhan X, Yu C, et al. High-density single nucleotide polymorphisms linkage and quantitative trait locus mapping of the pearl oyster, Pinctada fucata martensii dunker. Aquaculture.2014;434:376–384. doi: 10.1016/j.aquaculture.2014.08.044. [CrossRef]

19. Liu H, Fu B, Pang M, Feng X, Yu X, Tong J. A high-density genetic linkage map and QTL fine mapping for body weight in crucian carp (Carassius auratus) using 2b-RAD sequencing. G3. 2017;7:2473–87.

20. Fu B, Liu H, Yu X, Tong J. A high-density genetic map and growth related QTL mapping in bighead carp (Hypophthalmichthys nobilis). Sci Rep. 2016;6(28679) PubMed]

24. Gutierrez AP, Lubieniecki KP, Davidson EA, Lien S, Kent MP, Fukui S, et al. Genetic mapping of quantitative trait loci (QTL) for body-weight in Atlantic salmon (Salmo salar) using a 6.5 K SNP array. Aquaculture.2012;358:61–70. doi: 10.1016/j.aquaculture.2012.06.017. [CrossRef]

25. Wringe BF, Devlin RH, Ferguson MM, Moghadam HK, Sakhrani D, Danzmann RG. Growth-related quantitative trait loci in domestic and wild rainbow trout (Oncorhynchus mykiss) BMC Genet.2010;11:63. doi: 10.1186/1471-2156-11-63. PubMed] [CrossRef]

26. Liu F, Sun F, Xia JH, Li J, Fu GH, Lin G, et al. A genome scan revealed significant associations of growth traits with a major QTL and GHR2 in tilapia. Sci Rep.2014;4:7256. doi: 10.1038/srep07256. PubMed] [CrossRef]

27. Song W, Pang R, Niu Y, Gao F, Zhao Y, Zhang J, et al. Construction of high-density genetic linkage maps and mapping of growth-related quantitative trail loci in the Japanese flounder (Paralichthys olivaceus) PLoS One.2012;7:e50404. doi: 10.1371/journal.pone.0050404. PubMed] [CrossRef]

28. Song W, Li Y, Zhao Y, Liu Y, Niu Y, Pang R, et al. Construction of a high-density microsatellite genetic linkage map and mapping of sexual and growth-related traits in half-smooth tongue sole (Cynoglossus semilaevis) PLoS One.2012;7:e52097. doi: 10.1371/journal.pone.0052097. PubMed] [CrossRef]

29. Tong JG, Sun XW. Genetic and genomic analyses for economically important traits and their applications in molecular breeding of cultured fish. Sci China Life Sci.2015;58:178–186. doi: 10.1007/s11427-015-4804-9. [PubMed] [CrossRef]

30. Zhou J, Wu Q, Wang Z, Ye Y. Molecular phylogeny of three subspecies of common carp Cyprinus carpio, based on sequence analysis of cytochrome b and control region of mtDNA. J Zool Syst Evol Res.2004;42:266–269. doi: 10.1111/j.1439-0469.2004.00266.x. [CrossRef]

32. Ji P, Liu G, Xu J, Wang X, Li J, Zhao Z, et al. Characterization of common carp transcriptome: sequencing, de novo assembly, annotation and comparative genomics. PLoS One.2012;7:e35152. doi: 10.1371/journal.pone.0035152. PubMed] [CrossRef]

34. Xu P, Zhang X, Wang X, Li J, Liu G, Kuang Y, et al. Genome sequence and genetic diversity of the common carp, Cyprinus carpio. Nat Genet.2014;46:1212–1219. doi: 10.1038/ng.3098. [PubMed] [CrossRef]

36. Laghari MY, Zhang Y, Lashari P, Zhang X, Xu P, Xin B, et al. Quantitative trait loci (QTL) associated with growth rate trait in common carp (Cyprinus carpio) Aquacult Int.2013;21:1373–1379. doi: 10.1007/s10499-013-9639-4. [CrossRef]

37. Zhang Y, Wang S, Li J, Zhang X, Jiang L, Xu P, et al. Primary genome scan for complex body shape-related traits in the common carp Cyprinus carpio. J Fish Biol.2013;82:125–140. doi: 10.1111/j.1095-8649.2012.03469.x. [PubMed] [CrossRef]

38. Laghari MY, Lashari P, Zhang X, Xu P, Narejo NT, Liu Y, et al. Mapping QTLs for swimming ability related traits in Cyprinus carpio L. Mar Biotechnol.2014;16:629–637. doi: 10.1007/s10126-014-9578-8. [PubMed] [CrossRef]

39. Van Ooijen JW. JoinMap 4, software for the calculation of genetic linkage maps in experimental populations. Netherlands: Kyazma BV, Wageningen; 2006.

40. Chakravarti A, Lasher LK, Reefer JE. A maximum likelihood method for estimating genome length using genetic linkage data. Genetics.1991;128:175–182. PubMed]

41. Fishman L, Kelly AJ, Morgan E, Willis JH. A genetic map in the Mimulus guttatus species complex reveals transmission ratio distortion due to heterospecific interactions. Genetics.2001;159:1701–1716. PubMed]

43. Maak S, Boettcher D, Tetens J, Swalve HH, Wimmers K, Thaller G, et al. Expression of microRNAs is not related to increased expression of ZDHHC9 in hind leg muscles of splay leg piglets. Mol Cell Probes.2010;24:32–37. doi: 10.1016/j.mcp.2009.09.001. [PubMed] [CrossRef]

44. Boucher J, Masri B, Daviaud D, Gesta S, Guigné C, Mazzucotelli A, et al. Apelin, a newly identified adipokine up-regulated by insulin and obesity. Endocrinology.2005;146:1764–1771. doi: 10.1210/en.2004-1427. [PubMed] [CrossRef]

45. Genkai N, Homma J, Sano M, Tanaka R, Yamanaka R. Increased expression of pituitary tumor-transforming gene (PTTG)-1 is correlated with poor prognosis in glioma patients. Oncol Rep.2006;15:1569–1574. [PubMed]

46. Mareco EA, de la Serrana DG, Johnston IA, Dal-Pai-Silva M. Characterization of the transcriptome of fast and slow muscle myotomal fibres in the pacu (Piaractus mesopotamicus) BMC Genomics.2015;16:182. doi: 10.1186/s12864-015-1423-6. PubMed] [CrossRef]

47. Tian M, Li Y, Jing J, Mu C, Du H, Dou J, et al. Construction of a high-density genetic map and quantitative trait locus mapping in the sea cucumber Apostichopus japonicus. Sci Rep.2015;5:14852. doi: 10.1038/srep14852. PubMed] [CrossRef]

48. Yu X, Zhou T, Li K, Li Y, Zhou M. On the karyosystematics of cyprinid fishes and a summary of fish chromosome studies in China. Genetica.1987;72:225–235. doi: 10.1007/BF00116227. [CrossRef]

49. Saupe S, Bernard P, Laurent-Brun E, Derancourt J, Roizès G. Construction of a human BcgI DNA fragment library. Gene.1998;213:17–22. doi: 10.1016/S0378-1119(98)00216-9. [PubMed] [CrossRef]

50. Smith RM, Jacklin AJ, Marshall JJT, Sobott F, Halford SE. Organization of the BcgI restriction–modification protein for the transfer of one methyl group to DNA. Nucleic Acids Res.2013;41:405–417. doi: 10.1093/nar/gks1000. PubMed] [CrossRef]

52. Wang JT, Li JT, Zhang XF, Sun XW. Transcriptome analysis reveals the time of the fourth round of genome duplication in common carp (Cyprinus carpio) BMC Genomics.2012;13:96. doi: 10.1186/1471-2164-13-96. PubMed] [CrossRef]

53. Wang Y, Lu Y, Zhang Y, Ning Z, Li Y, Zhao Q, et al. The draft genome of the grass carp (Ctenopharyngodon idellus) provides insights into its evolution and vegetarian adaptation. Nat Genet.2015;47:625–631. doi: 10.1038/ng.3280. [PubMed] [CrossRef]

54. Xia JH, Liu F, Zhu ZY, Fu J, Feng J, Li J, et al. A consensus linkage map of the grass carp (Ctenopharyngodon idella) based on microsatellites and SNPs. BMC Genomics.2010;11:135. doi: 10.1186/1471-2164-11-135. PubMed] [CrossRef]

55. Nakatani Y, Takeda H, Kohara Y, Morishita S. Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in early vertebrates. Genome Res.2007;17:1254–1265. doi: 10.1101/gr.6316407. PubMed] [CrossRef]

56. Kasahara M, Naruse K, Sasaki S, Nakatani Y, Qu W, Ahsan B, et al. The medaka draft genome and insights into vertebrate genome evolution. Nature.2007;447:714–719. doi: 10.1038/nature05846. [PubMed] [CrossRef]

57. Gjerde B, Gjedrem T. Estimates of phenotypic and genetic parameters for carcass traits in Atlantic salmon and rainbow trout. Aquaculture.1984;36:97–110. doi: 10.1016/0044-8486(84)90057-7. [CrossRef]

58. Luo W, Zeng C, Deng W, Robinson N, Wang W, Gao Z. Genetic parameter estimates for growth-related traits of blunt snout bream (Megalobrama amblycephala) using microsatellite-based pedigree. Aquac Res.2014;45:1881–1888. doi: 10.1111/are.12014. [CrossRef]

60. Castan-Laurell I, Dray C, Knauf C, Kunduzova O, Valet P. Apelin, a promising target for type 2 diabetes treatment? Trends Endocrin Met.2012;23:234–241. doi: 10.1016/j.tem.2012.02.005. [PubMed] [CrossRef]

61. Xu YX, Zhu ZY, Lo LC, Wang CM, Lin G, Feng F, et al. Characterization of two parvalbumin genes and their association with growth traits in Asian seabass (Lates calcarifer) Anim Genet.2006;37:266–268. doi: 10.1111/j.1365-2052.2006.01423.x. [PubMed] [CrossRef]

62. Nagy A, Csanyi V. A new breeding system using gynogenesis and sex-reversal for fast inbreeding in carp. Theor Appl Genet.1984;67:485–490. doi: 10.1007/BF00264890. [PubMed] [CrossRef]

63. Devlin RH, Nagahama Y. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture.2002;208:191–364. doi: 10.1016/S0044-8486(02)00057-1. [CrossRef]

64. Takehana Y, Naruse K, Hamaguchi S, Sakaizumi M. Evolution of ZZ/ZW and XX/XY sex-determination systems in the closely related medaka species, Oryzias hubbsi and O. dancena. Chromosoma.2007;116:463–470. doi: 10.1007/s00412-007-0110-z. [PubMed] [CrossRef]

65. Chen J, Wang Y, Yue Y, Xia X, Du Q, Chang Z. A novel male-specific DNA sequence in the common carp, Cyprinus carpio. Mol Cell Probes.2009;23:235–239. doi: 10.1016/j.mcp.2009.04.004. [PubMed] [CrossRef]

66. Komen J, Yamashita M, Nagahama Y. Testicular development induced by a recessive mutation during gonadal differentiation of female common carp (Cyprinus carpio, L.) Develop Growth Differ.1992;34:535–544. doi: 10.1111/j.1440-169X.1992.00535.x. [CrossRef]

68. Anderson JL, Marí AR, Braasch I, Amores A, Hohenlohe P, Batzel P, et al. Multiple sex-associated regions and a putative sex chromosome in zebrafish revealed by RAD mapping and population genomics. PLoS One.2012;7:e40701. doi: 10.1371/journal.pone.0040701. PubMed] [CrossRef]

69. Eshel O, Shirak A, Weller JI, Slossman T, Hulata G, Cnaani A, et al. Fine-mapping of a locus on linkage group 23 for sex determination in Nile tilapia (Oreochromis niloticus) Anim Genet.2011;42:222–224. doi: 10.1111/j.1365-2052.2010.02128.x. [PubMed] [CrossRef]

70. Eisbrenner WD, Botwright N, Cook M, Davidson EA, Dominik S, Elliott NG, et al. Evidence for multiple sex-determining loci in Tasmanian Atlantic salmon (Salmo salar) Heredity.2014;113:86–92. doi: 10.1038/hdy.2013.55. PubMed] [CrossRef]

71. Taggart JB, Hynes RA, Prodöuhl PA, Ferguson A. A simplified protocol for routine total DNA isolation from salmonid fishes. J Fish Biol.1992;40:963–965. doi: 10.1111/j.1095-8649.1992.tb02641.x. [CrossRef]

73. Wang D, Liao X, Cheng L, Yu X, Tong J. Development of novel EST-SSR markers in common carp by data mining from public EST sequences. Aquaculture.2007;271:558–574. doi: 10.1016/j.aquaculture.2007.06.001. [CrossRef]

74. Ji P, Zhang Y, Li C, Zhao Z, Wang J, Li J, et al. High throughput mining and characterization of microsatellites from common carp genome. Int J Mol Sci.2012;13:9798–9807. doi: 10.3390/ijms13089798. PubMed] [CrossRef]

77. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Res.2009;19:1639–1645. doi: 10.1101/gr.092759.109. PubMed] [CrossRef]

Supporting Information Scheme S1 shows the synthetic routes of the target compounds (see supporting file and paper35 for complete details of synthesis). The peptides LV-OBzl and Orn(Boc)-NBzl (2E) were prepared according to the standard condensation reaction procedure. ZD-E-1M was obtained by converting the amino acid side chain to chloroacetamidine group. The structures and purities of ZD-BM and ZD-E-1M were confirmed by Fourier-transform mass spectra, Fourier-transform infrared spectroscopy, 1H NMR, 13C NMR, HPLC, UV and fluorescence spectrum (Supporting Information Figs. S1‒S25).

We described the PAD4 inhibitor YW3-5635, which consisted of a naphthalene ring and an ornithine skeleton with chloramidine and benzylamine. We used YW3-56 as a lead compound to design and synthesize PAD4 inhibitors. ZD-BM and ZD-E-1M were screened by docking, scoring, and analyzing their interaction with PAD436,37. CDOCKER Energy in Supporting Information Table S1 shows that the interactions of ZD-BM and ZD-E-1M with PAD4 were both strong (48.755 and 47.628, respectively) and greater than that of YW3-56 (43.91). Fig. 1A and Supporting Information Fig. S26 showed the key amino acids and interaction forces between PAD4 and ZD-E-1M. Asp350 and Asp473 could stabilize chloroacetamidine group by hydrogen bonding and halogen, respectively, and properly position the imine carbon for nucleophilic attack. The departing amine was then protonated by His640. The carbonyl group could form a hydrogen bond with Arg374, which was specific to the peptidyl-arginine substrates compared to free arginine. The high potency of ZD-E-1M might be attributed to a hydrogen bond between benzoxadiazole and Trp347, and a π‒alkyl with Arg639, which is unique to PAD438. To further illustrate the mechanism of compounds inhibiting PAD4, we determined the PAD4 inhibition activities as shown in Supporting Information Fig. S27A by a colorimetric method (Fig. S27B). Briefly, ZD-E-1M, ZD-BM and YW3-56 compete with the model substrate (BAEE) for the active site of PAD4. The IC50 of ZD-E-1M inhibiting PAD4 was 2.390 μmol/L, which was similar to that of YW3-56. This suggested that ZD-E-1M inhibited PAD4 catalyzing histone citrullination by simulating the PAD4 substrate bound to the PAD4 active site (Fig. S27C).

Importantly, computer-aided drug design and PAD4 inhibition activities cannot guarantee that adequate concentration of drugs will be delivered to target cells; the delivered concentration is also related to the bioavailability of drugs, which is dependent on the shape and size of nanocarriers8,33,34,39. Hence, we introduced the pH-responsive moiety nitrobenzoxadiazole (NBD) at the N-terminus of ornithine to confer pH-responsiveness (Fig. 1B). In addition, NBD served as a label because of its strong fluorescence in hydrophobic environments30,31.

The pH-responsive self-assembly properties of ZD-B and ZD-E-1 were assessed from three aspects. We observed the shape and size by TEM (Hitachi) and SEM (Hitachi) (Fig. 1C‒F). The average hydrodynamic diameters were measured by dynamic light-scattering (DLS, Brookhaven, Nano-ZS90, Holtsville, NY, USA), and the zeta potential was determined for six days (Fig. 1G). At pH 7.4, TEM and SEM images showed that ZD-B had a dendritic crystal structure with length over 1000 nm, width 100–300 nm, and nanospheres with size 60–240 nm (Fig. 1C). ZD-E-1 was spherical with diameters from 30 to 100 nm (Fig. 1E). At pH 6.5, TEM and SEM images showed that ZD-B and ZD-E-1 self-assembled into a flower-like and loose nanostructure, 400 and 200 nm, respectively (Fig. 1D and F). The sizes of the nanodrugs increased in the acidic condition. This increase may have resulted from a looser structure induced by the acidic microenvironment13,14,40,41.

The size distribution (Supporting Information Fig. S28A‒D) of ZD-B and ZD-E-1 at pH 7.4 and 6.5 in TEM photos were similar to the diameter measured by light scattering in solution (Fig. 1G). The size of ZD-B remained ∼300 nm stably in water and did not change with decreasing pH for six days. As the pH decreased, the size of ZD-E-1 increased notably, from 80 nm (pH 7.4) to 208 nm (pH 6.5). With increasing time, the sizes of ZD-B and ZD-E-1 were stable in solution at pH 6.5 and 7.4. In addition, the zeta potentials of ZD-B and ZD-E-1 were stable in solution at pH 6.5 and 7.4, from −5 to −15 mV (Fig. 1H). The negative zeta potential was likely the result of the strongly electron-withdrawing nitro group. As the pH decreased, the zeta potential of ZD-B changed slightly, whereas the zeta potential of ZD-E-1 decreased, from −15 to −10 mV. This decreased zeta potential indicated that the nitrobenzoxadiazole group was protonated to form a hydrophilic chain from a hydrophobic chain7,42. Besides, we also found that the particle size and zeta potential of ZD-E-1 remain stable when the concentration is greater than 0.001 mg/mL concentration (Fig. S28E and S28F).

To characterize the self-assembly properties of nanoparticles, the critical aggregation concentration (CAC)43 and drug release curve44 at different pH values (Fig. 1J‒K and Supporting Information Fig. S29) were determined. At pH 7.4, the CAC of ZD-B was similar to that of ZD-E-1, about 15 μmol/L. With the decrease of pH value, CAC values of ZD-B and ZD-E-1 were increased to 30 and 50 μmol/L, respectively. This suggested that the nanoflowers structure made it easier for nanodrugs to release. As shown in Fig. S29C, there was almost no release of ZD-B at pH 7.4 and 6.5, whereas ZD-E-1 could be released over time. The accumulative drug release rate of ZD-E-1 at pH 6.5 reached 99.0% at 8 h, while it reached about 90% at pH 7.4. This illustrated that the nanoflower structure was contributed to drug release rate at tumor site.

We used Fourier-transform mass spectra, NOSEY 2D NMR spectroscopy, and material studio (BIOVIA, San Diego, CA, USA) to identify molecules in solution45,46. Fig. 2A and B show the FTMS spectra of ZD-B and ZD-E-1 in ultrapure water. There was an existence of dimeric (967.4319 and 919.3070) and trimeric (1450.6859 and 1378.4527) supermolecules in ZD-B and ZD-E-1 spectra, respectively, and even the tetramer (1933.9432) and pentamer (2418.1988) were detected in ZD-B spectrum47. These larger forms indicated that several small molecules first aggregated into self-assembled units in solution. The FTMS spectra of ZD-B and ZD-E-1 at pH 6.5 (Supporting Information Fig. S30A and S30B) show the nanodrug self-assembly units are similar to that in neutral environment.

Self-assembly characteristics of ZD-B and ZD-E-1. (A and B) Fourier-transform mass spectrometry of ZD-B and ZD-E-1 in pH 7.4 solution. (C and D) NOSEY 2D NMR spectrum of ZD-B and ZD-E-1. (E and F) Molecular dynamics simulation of ZD-B and ZD-E-1 and interaction of molecules in self-assembled nano-units.

To investigate the interaction pattern, we recorded NOESY spectra of ZD-B and ZD-E-1 at pH 6.5 and 7.4 in deuterated solvent. Fig. 2C shows one interesting cross-peak of ZD-B resulted from the interaction of H near the nitro of the NBD residue of one molecule with the chiral carbon H of the valine residue of another molecule. This interaction indicated that the distance between the NBD residue and benzylamine residue was less than 4 Å. Fig. 2D shows four cross-peaks of ZD-E-1. Cross-peaks 1 and 4 resulted from the interaction of H near the nitro of the NBD residue of one molecule with the benzene ring H and methylene H of benzylamine of another molecule, respectively. Cross-peaks 2 and 3 resulted from the interaction of H away from nitro of the NBD residue of one molecule with the chloromethyl H of amino acid side chain and benzene ring H of benzylamine of another molecule, respectively. This interaction suggested that the distances between the NBD residue of one molecule and the benzylamine residue or side chain of another molecule were both less than 4 Å. The NOSEY 2D NMR spectrum in pH 6.5 deuterated solvent was similar to the spectrum at pH 7.4 (Fig. S30C and S30D). The main difference was that cross-peak 1 became deeper and cross-peak 3 became lighter in acidic environments, which indicated that the intermolecular interaction angle of protonation was adjusted slightly.

By combining the results of FTMS spectra and NOSEY 2D NMR, we clarified the interaction of the self-assembled unit. First, FTMS spectra confirmed that there were five molecules of ZD-BM and three molecules of ZD-E-1M in the self-assembled units, respectively. Second, cross-peaks in NOSEY spectrum identified the spatial position of groups. Finally, five molecules of ZD-BM or three molecules of ZD-E-1M were packed together in Discovery Studio (Neotrident, Beijing, China) to give the conformation of self-assembled units (Fig. 2E and F).

We also used a Material Studio molecular dynamics (MD) simulation technique to examine self-assembly of the nanodrugs in normal and acidic pH. The MD simulation of ZD-BM showed that 1200 molecules could form a spherical structure of diameter ∼118 nm at pH 7.4 (Fig. 2E) and a multi-space spherical structure of diameter ∼128 nm at pH 6.5 (Fig. S30E). The MD simulation of ZD-E-1M (Fig. 2F) showed that 900 molecules formed a nanosphere of diameter ∼96 nm in normal pH, while that of ZD-E-1M with H+ (Fig. S30F) showed a looser structure of diameter ∼118 nm. The simulated particle size was related to the number of molecules in the box. Therefore, the simulated particle size is not exactly equal to the actual particle size. The simulated particle size is equal to or a fraction of the actual particle size.

To study the pH-responsive transformation of ZD-B and ZD-E-1 on the cell surface and inside the cell, we incubated normal HL7702 cells and 4T1 tumor cells with the nanoparticles for 48 h. Intracellular morphology and localization of the nanoparticles were observed by fluorescence and CLSM (Leica). Fluorescence microscope images (Fig. 3A) showed dendritic fluorescent structures self-assembled on the surface of HL7702 cells treated with 50 μmol/L ZD-B. This dendritic appearance was similar to the shape in solution at pH 7.4 (Fig. 1C). To observe the nanodrug inside the cells, we acquired CLSM images of HL7702 cells treated with 20 μmol/L ZD-B (Fig. 3A). Only a few fluorescent spots were observed and dendritic fluorescence signal was not determined, which indicated that the dendritic structures did not enter the normal cells. Similarly, HL7702 cells treated with 20 μmol/L ZD-E-1 (Fig. 3B) showed only a few fluorescent spots in the cytoplasm and no fluorescence in the nucleus. This result suggested that only a small amount of ZD-E-1 entered the normal cell cytoplasm.

Intracellular morphology, localization, and cytotoxicity of self-assembled molecules. (A) Intracellular morphology of 50 μmol/L ZD-B after 48 h observed by fluorescence microscopy and localization of 20 μmol/L ZD-B observed by confocal laser scanning microscopy (CLSM, Leica) in HL7702 cells. Scale bar = 25 μm. (B) Localization of 20 μmol/L ZD-E-1 after 48 h observed by CLSM (Leica) in HL7702 cells. Scale bar = 25 μm. (C‒D) Cellular localization of 20 μmol/L ZD-B and ZD-E-1 after 48 h observed by CLSM (Leica) in 4T1 tumor cells. Scale bar = 25 μm. (E) The mean fluorescence intensity of drugs 48 h after administration determined by ImageJ software. (F and G) Cell viability of HL7702 and 4T1 tumor cells treated with 3.25, 6.25, 12.5, 25, 50, and 100 μmol/L ZD-B and ZD-E-1 for 48 h. (H) IC50 of ZD-B and ZD-E-1 of HL7702 and 4T1 cells. (I) Representative CLSM images from different time points. Scale bar = 25 μm. (J) Curves of mean fluorescence intensity determined by ImageJ at the different time points. Data are presented as mean ± SD (n = 3). ∗∗P < 0.01.

Fig. 3C shows that, in 4T1 tumor cells, Z