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.

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. 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. 2c 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.

Strong correlations among the five growth-related traits (BW, TL, BL, BH and HL) have been reported in some aquaculture species, such as Atlantic salmon [57], blunt snout bream (Megalobrama amblycephala) [58] and Zhikong Scallop [5]. Similarly in this study, five growth-related traits were highly correlated with each other, with the highest correlation value (r = 0.99) between TL and BL and relatively low correlation value (mean r = 0.76) between HL and other four traits. As expected, QTLs for TL and BL had the same distribution patterns on four LGs, while three of four QTLs for HL were located on three LGs which contain no QTL for other growth-related traits (Table 3). On LG27, a QTL-containing interval was identified for all growth-related traits, suggesting the influence of one QTL on all growth-related traits in the Yangtze River carp. QTLs for growth on LG27 have also been identified in Yellow River carp [36], further comparative analysis should be performed to verify the common loci that have genetic effects on growth in both strains of C. c. haematopterus.

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. 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.

A high-density genetic linkage map of the Yangtze River carp (C. c. haematopterus) was constructed based on 7820 2b-RAD markers and 295 microsatellite markers in this study. Comparative genome mapping revealed that common carp had undergone extensive intra-chromosomal rearrangements during the domestication process. A high level of syntenic relationship was observed between Yangtze River carp and zebrafish and grass carp. A total of 21 QTLs for growth-related traits were identified on 12 LGs. A set of candidate genes were identified based on the sequences of QTL-containing intervals. These genetic markers or candidate genes associated with growth traits provide a basis for marker-assisted selection (MAS) in the Yangtze River carp and/or other common carp strains.