Sex-specific markers are powerful tools for identifying sex-determination system in various ani- mals. Bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix) are two of the most important edible fish in Asia, which have a long juvenility period that can lasts for 4–5years. In this study, we found one sex-specific marker by next-generation sequencing together with bioinformatics analysis in bighead carp. The male-specific markers were used to per- form molecular sexing in the progenies of artificial gynogenetic diploids and found all progenies (n¼ 160) were females. Meanwhile, around 1 : 1 sex ratio was observed in a total of 579 juvenile offspring from three other families. To further extend the male-specific region, we performed genome walking and got a male-specific sequence of 8,661 bp. Five pairs of primers were designed and could be used to efficiently distinguish males from females in bighead carp and sil- ver carp. The development of these male-specific markers and results of their molecular sexing in different populations provide strong evidence for a sex determination system of female homoga- metry or male heterogametry (XX/XY) in bighead carp and silver carp. To the best of our knowl- edge, this is the first report of effective sex-specific markers in these two large carp species. Key words: sex-specific markers, XX/XY sex determination system, bighead carp and silver carp 1. Introduction determination systems have showed to be highly diverse and com- 2–4 The mechanism of sex determination in ﬁsh has attracted a lot of plex. Different sex determination systems have been reported in biologist’s attention, as this mechanism has great implications both some closely related species or even within the same ﬁsh genus. in theory and practice. Fish are species of original vertebrate Studies of sex determination in ﬁsh provided insights into the genetic evolved from parthenogenesis to sexual reproduction, and their sex mechanisms of sex determination and origin and evolution of sex V C The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com 1 Downloaded from https://academic.oup.com/dnaresearch/advance-article-abstract/doi/10.1093/dnares/dsx054/4791395 by Ed 'DeepDyve' Gillespie user on 08 June 2018 2 Sex-specific markers in bighead carp 6,7 chromosomes. Many farmed ﬁsh exhibit signiﬁcant sexual In total, 160 young progenies from three diploid gynogenetic families dimorphism in body size, growth rate and ﬁrst time of sexual were sampled after 6-month culture in a pond for the detection of 2,8 maturation. Therefore, understanding the genetic basis of sex genetic sexes in the gynogenesis of bighead carp. In addition, 579 determination is critical for the implementation of breeding and pro- young progenies from three full-sib families were sampled after duction programs and sex control, and it can greatly add value by 6-month culture for the detection of sex ratio. The 16 silver carp 6,10 culturing monosex populations in some ﬁsh species. To date, sex samples used to verify the markers were sampled from Shishou Fish determination genes have been identiﬁed in few farmed ﬁsh species, Farm (Shishou, China), and they were caught from the Yangtze such as rainbow trout (Oncorhynchus mykiss), medaka (Oryzias River when they were young and then reared in the farm until 12 13 latipes), tiger pufferﬁsh (Takifugu rubripes), half-smooth tongue sexually mature. The sex of these ﬁsh was conﬁrmed by collecting sole (Cynoglossus semilaevis) and Nile tilapia (Oreochromis gametes released. Fin clips from each of the above mature or young niloticus). carp were collected and preserved in 95% alcohol at 4 C. Genomic Sex-speciﬁc markers are prerequisite for understanding the mech- DNA were extracted from alcohol-preserved ﬁn tissues following anisms of sex determination, identiﬁcation of sex-determining genes a standard phenol–chloroform protocol. The quality and quantity 2,16–18 and uncovering genetic architecture related to sex differences. of extracted DNA were checked by NanoDrop2000 spectrophotom- During the past few decades, different approaches have been used to eter (Thermo Scientiﬁc, USA) and 1% agarose gel electrophoresis. ﬁnd sex-speciﬁc DNA sequences in aquaculture ﬁshes. Traditional methods such as ampliﬁed fragment length polymorphism (AFLP) 2.2. 2b-RAD sequencing and male-specific tags filtering have been successfully used for the identiﬁcation of sex-speciﬁc To identify potential male-speciﬁc sequences in a bighead carp 10,19–24 markers in more than 20 aquaculture ﬁshes. With the devel- genome, we used a modiﬁed 2b-RAD genotyping method to ﬁnd opment of next-generation sequencing (NGS) technologies, many putative SNP loci that may be associated with each sex. In this study, new methods have been developed for screening genes or sex- we constructed 2b-RAD libraries for ﬁve sires and ﬁve dams of big- associated DNA fragments. For example, sex-speciﬁcally expressed head carp with known sex phenotypes (brood ﬁsh). A total of 250 ng 25–29 genes by RNA-seq, QTL mapping for sex-linked single nucleo- of genomic DNA from each ﬁsh was digested with BcgI restriction tide polymorphism (SNP) markers using high-density SNP linkage enzyme (New England Biolabs, USA) at 37 C for 4 h. The digestion maps through restriction site-associated DNA sequencing (RAD- products were heat-inactivated for 20 min at 65 C, and then linked 30–34 Seq), identiﬁcation of sex-related markers between several sexed to adapter 1 and adapter 2 at 16 C overnight. The linked fragments 35–38 individuals using RAD-Seq without generating linkage maps, and were ampliﬁed with Phusion High-Fidelity DNA Polymerase 14,39–41 detecting sex-speciﬁc sequences by whole genome sequencing. (Thermo Scientiﬁc, USA) using a set of four primers by which Bighead carp and silver carp not only are important freshwater sample-speciﬁc barcode and sequencing primers were introduced. edible ﬁsh native to East Asia but also have been introduced into After 14 cycles of ampliﬁcation, the library was obtained by purify- many other countries for improving water quality and human con- 42,43 ing the products at 170 bp via retrieval from 8% polyacrylamide sumption. The global production of bighead carp and silver carp gels. Each library was pooled with an equal amount to make a ﬁnal reached 3.4 and 4.9 million tons in 2015, respectively (FAO). The library that was sequenced in a lane of the Illumina HiSeq 2500 age of ﬁrst maturation in bighead carp and silver carp normally takes SE50 platform (Illumina, USA). Rawdata were deposited in NCBI 4 to 5 years or more in central and south China and the incapability SRA PRJNA401338. of determining the gender before sex maturation has brought trou- Reads with low-quality bases were removed before the following bles for aquaculture. Thus, a sex-speciﬁc marker is desirable to dis- analyses were carried out. Consensus tags were constructed for each criminate male and female samples at an early life stage of the two sample with Stacks 1.31 with parameter ‘-m 4 -p 6 -M 1 -N 2 -d –r’. carps. The aim of this study is to identify sex-speciﬁc markers in the After that, with Perl scripts, we extracted male-speciﬁc tags present in two carps and then apply these sex markers to perform molecular all ﬁve sires but not in any of the ﬁve dams. The male-speciﬁc tags sexing and veriﬁcation in large number of samples including mature were then mapped to a female bighead carp genome (Shunping He, and young ﬁsh and progenies from gynogenetic and full-sib families. unpublished data), and those tags failing to align to the female refer- ence genome were considered as putative male-speciﬁc tags. 2. Materials and methods 2.3. Re-sequencing and screening of male-specific 2.1. Experimental fish genomic scaffolds All experimental procedures about dealing with the ﬁsh in this study Because the male-speciﬁc tags obtained from 2b-RAD sequencing were approved by the Committee for Animal Experiments of the method were only 32 bp in length, which are too short to design pri- Institute of Hydrobiology of the Chinese Academy of Sciences, mers for PCR experiment, we sequenced a whole genome of a male China. The methods used in this study were carried out in accord- bighead carp, Arp7, which was one of the ﬁve dams used in the ance with the Laboratory Animal Management Principles of China. screening of male-speciﬁc tags. The sequencing library was con- The rearing activities of bighead carp in Wuhan, Hubei were structed with small insert fragments (300–500 bp), and sequenced by approved by the owner of the pond. Illumina HiSeq 4000 sequencing platform using 150 bp paired-end A total of 136 sexually matured bighead carp (brood ﬁsh) were sampled from the Zhangdu Lake Fish Farm (Wuhan, China), all of reads. The raw reads were ﬁltered by removing adaptor sequences, which were harvested from the middle Yangtze River and sampled at contamination and low-quality reads. Then those ﬁltered reads least 6 years old. The sex phenotype of each sire and dam was indi- were mapped to a female bighead carp genome (Shunping He, unpublished data), and SOAPdenovo2 (version 2.04) was used to vidually conﬁrmed by collecting its gametes (sperm or eggs) released in artiﬁcial reproduction in May 2014. Artiﬁcial gynogenesis was assembly these unmapped reads to male-speciﬁc regions. The male- performed in bighead carp according to a previous method. speciﬁc 2b-RAD reads were aligned to the male-speciﬁc regions, and Downloaded from https://academic.oup.com/dnaresearch/advance-article-abstract/doi/10.1093/dnares/dsx054/4791395 by Ed 'DeepDyve' Gillespie user on 08 June 2018 H. Liu et al. 3 the targeted scaffolds were regarded as putative male-speciﬁc several rounds of genome walking experiments were carried out to scaffolds. gain ﬂanking sequences of the male-speciﬁc contigs. Genome walking was performed using Universal GenomeWalker 2.0 kit (Clontech) in accordance with the user’s manual. The primers used in genome 2.4. Development and validation of male-specific walking were summarized in Table 3. SCAR markers Three sequence characterized ampliﬁed region (SCAR) markers were designed based on each male-speciﬁc scaffolds using Primer Premier 5 3. Results (Premier Biosoft International, Palo Alto, CA, USA). Then PCR was 3.1. Male-specific 2b-RAD tags used to validate the sex speciﬁcity of male-speciﬁc SCAR markers. To ﬁnd sex-speciﬁc markers in bighead carp, we used 2b-RAD sequenc- PCR reactions were carried with a total volume of 25 ll, containing ing to genotype ﬁve male and ﬁve female samples, which were within 50 ng of template DNA, 2.5 llof 10 reaction buffer, 0.75 U of Taq the 136 brood ﬁsh. In total, 30,941,847 raw reads and 26,732,738 polymerase (TaKaRa, Japan), 0.8 ll of dNTP (2.5 mmol/l), 0.8 llof high-quality reads were obtained after ﬁltering low-quality reads forward and reverse primer mixture (2.5 lmol/l each) and water to (Table 1). A total of 16,137,193 high-quality reads were obtained from the ﬁnal volume. PCR programs were as follows: an initial denatura- ﬁve female samples and 10,595,545 high-quality reads from ﬁve male tion step of 94 C for 5 min; then 36 cycles of 94 C for 35 s, 50 C samples. Each individual gets 1,367,315 to 5,162,352 high-quality annealing for 35 s, and 72 C for 40 s; and ﬁnally an extension step of reads, and the average amount of data is 2,673,274 reads per sample. 72 C for 8 min. The ampliﬁed products were separated by 1% agar- ose or 8% polyacrylamide gel electrophoresis. After clustering, the tags per sample ranged from 59,506 to 70,463 with Eight male and eight female matured ﬁsh with known sexes were an average of 63,699. Average coverage depth per tag among samples ﬁrst used to verify the authenticity of SCAR markers by PCR/gel elec- varied from 22.28 to 86.75 (mean 42.69). The high-sequencing trophoresis assay. Then PCR products were sequenced via direct coverage depth guarantees the high reliability of sex-speciﬁc sequences generated in this study. With Perl scripts, we extracted those male- Sanger sequencing method to conﬁrm the identity of the nucleotide sequences of sex markers with the sequences of male-speciﬁc scaf- speciﬁc tags presented in all ﬁve males but not in any of ﬁve females. folds. To further validate that those SCAR markers are truly male- Finally, we got 13 tags presented in all ﬁve male ﬁsh but not in any of speciﬁc, and not simply speciﬁc to only several individuals, 120 ﬁve female ﬁsh. Reads supporting the 13 tags in all male samples were matured ﬁsh (broodstock population) with known phenotypic sexes summarized in Table 2. were used to verify the detection efﬁciency (accuracy) of those SCAR markers. Moreover, SCAR markers were also veriﬁed by 160 gyno- Table 1. Summary of 2b-RAD data for 10 bighead carp parents genetic diploid progenies of bighead carp from three dams. Parents Sex Raw reads High-quality reads Tags Sequencing depth 2.5. Analysis of sex ratio in full-sib families Arp1 $ 1,528,656 1,433,493 63,696 22.51 Arp2 $ 5,951,567 4,867,677 61,397 79.28 The male-speciﬁc markers were used to assess sex ratio (female to Arp3 $ 2,512,315 2,323,809 70,463 32.98 male) in three full-sib families of bighead carp (6 months old), which Arp4 $ 2,564,547 2,349,862 70,263 33.44 contained 90, 166, 324 young offspring, respectively. Chi-square Arp5 $ 6,344,744 5,162,352 59,506 86.75 tests were performed to determine the ﬁtness of sex ratio (female/ Arp6 # 1,578,254 1,479,263 64,451 22.95 male) to the expected 1 : 1. Arp7 # 5,911,337 4,852,376 60,226 80.57 Arp8 # 1,552,576 1,436,797 63,093 22.77 Arp9 # 1,450,791 1,367,315 61,362 22.28 2.6. Genome walking Arp10 # 1,547,060 1,459,794 62,534 23.34 The male-speciﬁc contigs did not have corresponding regions in Average # 3,094,185 2,673,274 63,699 42.69 female bighead carp genome (Shunping He, unpublished data), so Table 2. Information of 13 male-specific tags Name Tag Sequence Existence in the Scaffold of the female genome male genome 1 ref-22847 AGTAAACAGACGAGCAAACTGCAGCAAAAGAA No scaffold37502 2 ref-18178 AAATTGGCAACGAAAAAACTGCAGCGCGTATG No scaffold8218 3 ref-804 ACTCCTTTAACGATGAAGATGCAATTTTCGCC No — 4 ref-14673 CTGTTGAAGACGATGGAGGTGCGGATGACGTT Yes — 5 ref-18221 TGTCTTTTTACGAGACAGATGCTCCAGCATCA Yes — 6 ref-20508 ACCAAAGACACGATCACTGTGCTATAGCGTGC Yes — 7 ref-44705 CACATGAACCCGAGGGCAATGCATTCTGAATC Yes — 8 ref-45942 TCATCAGCATCGAGACTCCTGCCATTTCATTC Yes — 9 ref-46385 TGCATATGTCCGATAGGATTGCAACATTCAAA Yes — 10 ref-47639 TTATTTTCATCGACAAACTTGCCATTCGAGTC Yes — 11 ref-58079 TCTTGGATCCCGAAATAGCTGCCCGGAGGCGT Yes — 12 ref-63125 AGATGAGCTCCGAGTTCTGTGCCATCATTGCC Yes — 13 ref-68081 GCAACAATCTCGACCGCATTGCCGAAGAGGCC Yes — Downloaded from https://academic.oup.com/dnaresearch/advance-article-abstract/doi/10.1093/dnares/dsx054/4791395 by Ed 'DeepDyve' Gillespie user on 08 June 2018 4 Sex-specific markers in bighead carp We mapped these 13 potential male-speciﬁc tags to the female Table 3. Male-specific primers used for SCAR markers bighead carp genome sequence (Shunping He, unpublished data), 0 0 Name Reference sequences Primers (5 -3 ) and found 10 of them were mapped to a female genome. Among them, ﬁve had SNPs in the BcgI digestion recognition site ArS-9-1 scaffold37502 F: GCTCCTTACTCAGCAACT (N CGAN TGCN ) of the female genome, and other ﬁve tags R: TCAGTAAACAGACGAGCA 10 6 10 ArS-9-2 scaffold37502 F: GGTGCAGGATTTCCAGTT remained the same sequences as their corresponding female sequen- R: CCATTGATGTTGTCGCTCT ces. Therefore, only three tags without any mapping were male- ArS-9-3 scaffold37502 F: CAAAGACCGCAATAGGAG speciﬁc 2b-RAD tags (Table 2). R: GAGCATGTGAAATTAGTGAAG ArS-9-10 Genome walking F: GGCTATCTAAGTTTGGGC 3.2. Mapping male-specific tags to male-specific R: GGATGAGCATTGAAGGTG ArS-9-11 Genome walking F: GTAAGTTGAGTTTGTGGC genomic regions R: GATGAGCATTGAAGGTG Since male-speciﬁc 2b-RAD tags were too short to design primers for ArS-9-13 Genome walking F: CAAAGACCGCAATAGGAG PCR ampliﬁcation, one of the male individuals was subject to short- R: CCAGGACAAGGTGACATACT reads de novo sequencing. We got 48,157,939 pairs of 150 bp ArS-9-14 Genome walking F: TCGGCAAACAGAAAAGAC Illumina pair-end reads for this sample. After ﬁltering low-quality R: AATGGTGAATAGGGAGCG reads, 42,830,738 pair-end reads were left and mapped to a female ArS-9-15 Genome walking F: AGCAACTTTTGTCTGGTG bighead carp genome. As mapped reads were located in regions iden- R: AATGAATGGTGAATAGGG tical between the male and female genome, we only kept 4,417,710 pairs of unmapped reads for de novo assembly of male-speciﬁc regions of bighead carp. Finally, we got 298,474 scaffolds with a total size of 79.0 Mb. To get the ﬂanking sequences of the three tags, we mapped them to assembled male-speciﬁc scaffolds. For the tag ref-22847, it was mapped to scaffold37502 (907 bp) with 100% identity. For the tag ref-18178, it was mapped to scaffold8218 (602 bp) of identical sequences. For the tag ref-804, we could not ﬁnd any mapping results in the scaffolds of the male genome. Therefore, scaffold37502 and scaffold8218 were candidate male-speciﬁc sequences in the big- head carp. 3.3. Development and validation of male-specific markers Three primers were designed for each male-speciﬁc scaffolds, and then their authenticity was veriﬁed by PCR between mature males and females (Table 3, Supplementary File S2). Three markers from scaffold37502 showed consistent ampliﬁcation of fragments in all eight mature males, while none of the eight mature females ampliﬁed Figure 1. PCR detection and genetic sexing in eight females and eight males those bands (Fig. 1). For scaffold8218, the PCR products from all of bighead carp by male-specific primer pairs designed based on male scaf- three primers showed no difference between male and female sam- fold37502. The DL 2000 DNA marker is shown on the left. ples (Supplementary Fig. S3). In order to detect universality of the male-speciﬁc markers, the three markers from scaffold37502 were The earlier results indicated that these sequences from male-speciﬁc tested in another 120 mature bighead carp ﬁsh, and the authenticity region are unique to the bighead carp male genome. of these sexing tests was 100% (Supplementary Fig. S1). These results strongly support high reliability of the male-speciﬁc markers 3.4. Genetic sex identification in full-sib families in other larger bighead carp samples, which conﬁrmed that these loci After verifying the authenticity of male-speciﬁc markers, they were are truly male-speciﬁc. These male-speciﬁc markers were also vali- used to analyse the ratio of females to males in three full-sib families of dated in 160 offspring from three diploid gynogenetic families, and young bighead carp containing 579 offspring, and the total segrega- all ﬁsh were identiﬁed as females (Supplementary Fig. S2). The male tion ratio of females to males was 295 : 284 (1.04 : 1) (Table 4)in all PCR products were Sanger sequenced, and the nucleotide sequences three families. The female-to-male segregation ratio of these three fam- were consistent with the male genomic sequences in scaffold37502. ilies were 46 : 44 (1.05 : 1), 82 : 84 (0.98 : 1) and 295 : 284 (1.04 : 1), However, three markers from scaffold8218 showed no difference respectively. Chi-square testing showed that the female-to-male ratio when ampliﬁed between female and male individuals. Therefore, of all three families did not have signiﬁcant differences with the 1 : 1 only scaffold37502 was conﬁrmed as a male-speciﬁc sequence in big- separation ratio (P¼ 1). head carp. We blasted the male-speciﬁc sequences of scaffold37502 against 3.5. Genome walking and more sex-specific markers the female genome of bighead carp, but no homologous sequences were found. Then we blasted the male-speciﬁc sequences of the scaf- After ﬁve rounds of genome walking (two for the upstream and three fold37502 against the NCBI NT database, and we could not ﬁnd for the downstream sequences), the corresponding male genomic sequence alignment of this region to any other vertebrate genomes. fragments were extended. The designed walking primers and the Downloaded from https://academic.oup.com/dnaresearch/advance-article-abstract/doi/10.1093/dnares/dsx054/4791395 by Ed 'DeepDyve' Gillespie user on 08 June 2018 H. Liu et al. 5 Table 4. The male and female ratio for three full-sib families of bighead carp Family Number of Number of Number of Ratio offspring females males 1 90 46 44 1.05 Figure 3. PCR detection and genetic sexing in eight females and eight males 2 166 82 84 0.98 of silver carp by male-specific primer pairs designed in bighead carp. The DL 3 323 167 156 1.07 2000 DNA marker is shown on the left. Total 579 295 284 1.04 4. Discussion Sex-associated markers’ identiﬁcation can facilitate the discovery of sex-determining region or even sex-determining genes. The results can also help us understand the sex-determination systems as well as origin and evolution of sex chromosomes. However, unlike the mammals or birds with signiﬁcant difference in sex chromosomes, large differentiated genomic regions are uncommon in ﬁsh. Thus, it is always difﬁcult to screen these sex-speciﬁc regions successfully. Traditional molecular marker technologies such as RAPD and AFLP have proved to be simple and effective ways to ﬁnd sex markers in 21–24,49 ﬁsh, but in some species they failed to detect any sex-speciﬁc 50–52 markers. In the last decade, the development of NGS technology allowed researchers to quickly and conveniently obtain millions of sequences at the genomic level, and it rendered new opportunities for sex differ- 37,53 ence analysis between males and females. With the combination of NGS and restriction digestion enzymes, RAD-Seq offers the possi- bility for generating thousands of SNPs in a short time, which could be used for identiﬁcation of sex-linked SNP markers or sex-speciﬁc DNA sequences and for the construction of high-density linkage 32,36,38,53 maps and sex QTL mapping. RAD-Seq has been success- fully used for sex-speciﬁc marker identiﬁcation in a variety of species using only multiple male and female individuals, which proved to be 35–38,53 an efﬁcient way for screening of sex-speciﬁc markers. But RAD-Seq always misses some of the digestion sites, which can cause Figure 2. PCR detection and genetic sexing in eight females and eight males the failure of identiﬁcation of sex-speciﬁc markers. of bighead carp by different male-specific primer pairs designed based on Compared to other RAD-Seq methods, the 2b-RAD method used genome walking sequences. The DL 2000 DNA marker is shown on the left. in this study has more advantages compared to traditional RAD-Seq. First, it can screen nearly every restriction site at the whole genome adapter primers were summarized in Table 3. Based on the ampliﬁed level whereas other RAD-Seq methods can only get a subset of total fragments from males, an 8,661 bp of male-speciﬁc sequences sites due to the multiple time size selection for efﬁcient PCR ampliﬁ- were assembled from the male-ampliﬁed fragments (Supplementary 54,55 cation and sequencing. The average sequence coverage for each File S1), and the whole sequence was submitted to NCBI at accession site in 2b-RAD is usually more than 40 coverage, which provided MG668998. According to the male-speciﬁc sequences, more sex- accuracy of 2b-RAD genotyping and high reliability of generated speciﬁc markers were designed, such as ArS-9-10, ArS-9-11, ArS-9- tags. Second, the sequences of 2b-RAD are easy for sex-speciﬁc 2b- 13, ArS-9-14 and ArS-9-15. The PCR results from different sex with RAD reads identiﬁcation, which is very convenient for PCR veriﬁca- these primers further identiﬁed that this region is male-speciﬁc region tion. With all of these positive attributes, the 2b-RAD method is very in the bighead carp genome (Fig. 2, Table 3). We blasted the genome suitable for sex-speciﬁc markers and sex-determining regions identiﬁ- walking sequence against the female genome, but no corresponding cation. Here, for the ﬁrst time, we utilize the 2b-RAD and genome homologous sequence was found. re-sequencing to efﬁciently and accurately identify the sex-speciﬁc markers in genomes of two closely related species, bighead carp and 3.6. Genetic sex identification in silver carp silver carp. Our study proved that combining 2b-RAD genotyping In order to verify the presence of the sex markers in other species of and genome re-sequencing is a very powerful and promising tool for the genus Hypophthalmichthys, the designed SCAR primers were sex-speciﬁc markers’ identiﬁcation. This strategy of SCAR-based also ampliﬁed in eight female and eight male silver carp and showed PCR detection in this study is the ﬁrst protocol successfully amplify- the same results with bighead carp (Fig. 3). We also found that the ing the Y-speciﬁc fragment in bighead carp and silver carp. PCR results matched with phenotype with a 100% overall accuracy. The studies of sex-determination in ﬁsh would contribute to the 1,3 The same ampliﬁcation results were found both on males and understanding of the origin and evolution of sex chromosomes. females of these two species, and sequences of PCR products of silver Although most ﬁsh species lack visually heteromorphic sex chromo- carp were also similar with that of bighead carp. somes, some species have evolved sex-determining regions (genes) Downloaded from https://academic.oup.com/dnaresearch/advance-article-abstract/doi/10.1093/dnares/dsx054/4791395 by Ed 'DeepDyve' Gillespie user on 08 June 2018 6 Sex-specific markers in bighead carp 12,23,30,32,34,38 and even nascent sex chromosome. Based on the 2b- carp showed a 1 : 1 best orthologues relationship based on compara- RAD sequencing and male ﬁsh re-sequencing, we got a scaffold span- tive genome mapping, meaning a low level of genetic diversity. In ning over 900 bp, which is totally associated with Y-speciﬁc frag- this study, bighead carp, silver carp and grass carp shared the same ments of bighead carp, suggesting that this scaffold is located on the sex-speciﬁc markers and sex-determining DNA sequence, suggesting Y chromosome or nascent Y-linked region. We extend this scaffold that they may have a homologous nascent Y chromosome and the to 8,661 bp by genome walking, but no homologous sequences cor- same pathways involved in sex determination systems. Therefore, the responding to this region was found in the female genome. This three carp should share the most recent common ancestor and the might be a signiﬁcant difference in male and female sex-determining sex determination systems probably arose before speciation. regions in bighead carp. The diversity between male and female ﬁsh We presented an approach that identiﬁes and validates sex- in this sex-related region may lead to recombination suppression as speciﬁc markers using 2b-RAD sequencing and genome re- an early stage of sex chromosome evolution. Our results and pre- sequencing from multiple male and female individuals in bighead vious karyotyping studies demonstrated that bighead carp has carp and silver carp. A male-speciﬁc scaffold was identiﬁed and three evolved a sex-determining region, but has not yet formed distinguish- sex-speciﬁc primers were designed ﬁrst according the male-speciﬁc able (heteromorphic) sex chromosomes. scaffold. The sex-speciﬁc markers could be used in genetic sexing To study whether Cyprinidae ﬁsh shared the same sex- with 100% accuracy in brood ﬁsh and a large number of young ﬁsh determination system, we blasted the 8,661 bp sequences to the from full-sib families. In addition, all gynogenetic progenies were male-speciﬁc region in grass carp (Ctenopharyngodon idellus) genetically sexed as female by these sex-speciﬁc markers, conﬁrming genome, and found that most of the 4,148 bp of male-speciﬁc the female homogamety sex determination system in bighead carp. sequences in grass carp were presented in bighead carp male-speciﬁc Taken together, all these results conﬁrm that bighead carp and silver regions with 94% identify. Furthermore, one male-speciﬁc marker carp have a genetic mechanism of sex determination with an XX/XY has been found in common carp (Cyprinus carpio) from the Yellow sex determination system. The sex-speciﬁc markers developed in this River, but we found that sequence presented in gynogenetic study would be powerful and effective tools to uncover the sex deter- Songpu common carp genome. This demonstrates that this sequence mination system and identify potential genomic regions in bighead might be a strain-speciﬁc sex marker in common carp. And we found carp and silver carp. no similarity between it and male-speciﬁc marker in bighead carp. Unlike the big carp, the sex-determination of zebraﬁsh has been Acknowledgements found to be both genetic and environmental. Several markers have been found in the zebraﬁsh genome. Chromosome 4 are closely- This study was supported by grants from the NSFC (31472268 and related to its sex, but we found the male-speciﬁc region in bighead 31502153), FEBL (2016FBZ05) and DAC (2009045). We would like to thank carp cannot mapped to that region in zebraﬁsh genome. The reason Xinhua Wang, Baojiang Gan and Xueli Liu for sample collection and labora- tory technical assistance. We would like to thank Prof. Shunping He for offer- for above phenomena might be the ancestor of zebraﬁsh divided ing the bighead carp genome sequences. with the ancestor of Endemic Clad of East Asia Cyprinidae (ECEAC) ﬁsh at about 30 Mya, which is much older than speciﬁcation of ﬁsh within ECEAC (about 59 Mya). Based on this information, the Accession numbers bighead carp male-speciﬁc sequence was not only shared in silver PRJNA401338 and MG668998 carp but also in grass carp, and we predict that this male-speciﬁc sequence might emerge in the ancestor of ECEAC ﬁsh and was kept in all extent diploid ﬁsh in this group. As to the common carp, which Supplementary data is a tetraploid ﬁsh, it might have formed its own sex-determination region or genes in its speciation process. Supplementary data are available at DNARES online. Sex-speciﬁc markers have been successfully used to identify 18,24,35–38 the sex determination systems in many species, and it Conflict of interest is especially valuable in species that lack heteromorphic sex chromosomes. In this study, the presence of male-speciﬁc markers None declared. indicates an XX/XY sex determination system in bighead carp and silver carp. A larger sample size of bighead carp was used to further References verify the accuracy of the male-speciﬁc markers, and the veriﬁcation efﬁciency was 100% in all mature individuals. Besides, the female- 1. Devlin, R. H. and Nagahama, Y. 2002, Sex determination and sex differ- to-male segregation ratio in three full-sib families was 1 : 1. All those entiation in ﬁsh: an overview of genetic, physiological, and environmental results strongly indicated that bighead carp and silver carp have a inﬂuences, Aquaculture, 208, 191–364. genetic XX/XY sex determination system, which is similar to the 2. Kobayashi, Y., Nagahama, Y. and Nakamura, M. 2013, Diversity and plasticity of sex determination and differentiation in ﬁshes, Sex Dev., 7, grass carp’s XX/XY sex determination system. Based on these 115–25. results and the phylogenetic position of ﬁshes discussed earlier, we 3. Kikuchi, K. and Hamaguchi, S. 2013, Novel sex-determining genes in ﬁsh predicted that the ancestor of the whole Cyprinidae might have a and sex chromosome evolution, Dev. Dyn., 242, 339–53. XX/XY sex determination system. And this system might persist in 4. Mank, J. E. and Avise, J. C. 2009, Evolutionary diversity and turn-over of all extent species in Cyprinidae and facilitate the hybrid between dif- sex determination in teleost ﬁshes, Sex Dev., 3, 60–7. ferent species within this group. 5. Volff, J. N. 2005, Genome evolution and biodiversity in teleost ﬁsh, Bighead carp and silver carp are two of the most closely related Heredity (Edinb), 94, 280–94. species of the Cyprinidae family, cytogenetic analyses revealed that 6. Mei, J. and Gui, J. F. 2015, Genetic basis and biotechnological manipula- they have similar karyotypes and lack visually heteromorphic sex tion of sexual dimorphism and sex determination in ﬁsh, Sci. China Life 56,61 chromosomes. The linkage groups of bighead carp and silver Sci., 58, 124–36. Downloaded from https://academic.oup.com/dnaresearch/advance-article-abstract/doi/10.1093/dnares/dsx054/4791395 by Ed 'DeepDyve' Gillespie user on 08 June 2018 H. Liu et al. 7 7. Ellegren, H. 2011, Sex-chromosome evolution: recent progress and the 28. Chen, X., Mei, J., Wu, J., et al. 2015, A comprehensive transcriptome pro- vides candidate genes for sex determination/differentiation and SSR/SNP inﬂuence of male and female heterogamety, Nat. Rev. Genet., 12, markers in yellow catﬁsh, Mar. Biotechnol., 17, 190–8. 157–66. 29. Tao, W. J., Yuan, J., Zhou, L., et al. 2013, Characterization of gonadal 8. Cnaani, A. and Levavi-Sivan, B. 2009, Sexual development in ﬁsh, practi- transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differen- cal applications for aquaculture, Sex Dev., 3, 164–75. tially expressed genes, PLoS One, 8. DOI: 10.1371/journal.pone. 9. Martı´nez, P., Vinas, ~ A. M., Sa ´ nchez, L., et al. 2014, Genetic architecture of sex determination in ﬁsh: applications to sex ratio control in aquacul- 30. Palaiokostas, C., Bekaert, M., Khan, M. G. Q., et al. 2015, A novel ture, Front. Genet., 5, 340. sex-determining QTL in Nile tilapia (Oreochromis niloticus), BMC 10. Wang, D., Mao, H. L., Chen, H. X., Liu, H. Q. and Gui, J. F. 2009, Genomics, 16. DOI: 10.1186/S12864-015-1383-X. Isolation of Y- and X-linked SCAR markers in yellow catﬁsh and applica- 31. Palaiokostas, C., Bekaert, M., Taggart, J. B., et al. 2015, A new tion in the production of all-male populations, Anim. Genet., 40, 978–81. SNP-based vision of the genetics of sex determination in European sea 11. Yano, A., Guyomard, R., Nicol, B., et al. 2012, An immune-related gene bass (Dicentrarchus labrax), Genet. Sel. Evol., 47. DOI: 10.1186/s12711- evolved into the master sex-determining gene in rainbow trout, 015-0148-y. Oncorhynchus mykiss, Curr. Biol., 22, 1423–8. 32. Palaiokostas, C., Bekaert, M., Davie, A., et al. 2013, Mapping the sex 12. Matsuda, M., Nagahama, Y., Shinomiya, A., et al. 2002, DMY is a determination locus in the Atlantic halibut (Hippoglossus hippoglossus) Y-speciﬁc DM-domain gene required for male development in the medaka using RAD sequencing, BMC Genomics, 14. DOI: 10.1186/1471-2164- ﬁsh, Nature, 417, 559–63. 14-566. 13. Kamiya, T., Wataru, K., Satoshi, T., et al. 2012, A trans-species missense 33. Anderson, J. L., Marı´, A. R., Braash, I., et al. 2012, Multiple SNP in Amhr2 is associated with sex determination in the tiger pufferﬁsh, sex-associated regions and a putative sex chromosome in zebraﬁsh Takifugu rubripes (Fugu), PLoS Genet., 8. DOI: 10.1371/journal.pgen. revealed by RAD mapping and population genomics, PLoS One, 7. DOI: 10.1371/journal.pone.0040701. 14. Chen, S. L., Zhang, G., Shao, C., et al. 2014, Whole-genome sequence of 34. Palaiokostas, C., Bekaert, M., Khan, M. G. Q., et al. 2013, Mapping and a ﬂatﬁsh provides insights into ZW sex chromosome evolution and adap- validation of the major sex-determining region in Nile tilapia tation to a benthic lifestyle, Nat. Genet., 46, 253–þ. (Oreochromis niloticus L.) using RAD sequencing, PLoS One, 8. DOI: 15. Li, M. H., Yunlv, S., Jiue, Z., et al. 2015, A tandem duplicate of 10.1371/journal.pone.0068389. anti-mullerian hormone with a missense SNP on the Y chromosome is 35. Carmichael, S. N., Bekaert, M., Taggart, J. B., et al. 2013, Identiﬁcation essential for male sex determination in Nile tilapia, Oreochromis niloti- of a sex-linked SNP marker in the salmon louse (Lepeophtheirus salmo- cus, PLoS Genet., 11. DOI: 10.1371/journal.pgen.1005678. nis) using RAD sequencing, PLoS One, 8. DOI: 10.1371/journal.pone. 16. Gui, J. F. and Zhu, Z. Y. 2012, Molecular basis and genetic improvement of economically important traits in aquaculture animals, Chinese Sci. 36. Kafkas, S., Khodaeiaminjan, M., Guney, M. and Kafkas, E. 2015, Bull, 57, 1751–60. Identiﬁcation of sex-linked SNP markers using RAD sequencing suggests 17. Gui, J., Zhou, L. and Wu, Q. 2007, Genetic Basis and Artiﬁcial Control ZW/ZZ sex determination in Pistacia vera L., BMC Genomics, 16. DOI: of Sexuality and Reproduction in Fish. Science Press: Beijing. 10.1186/s12864-015-1326-6. 18. Charlesworth, D. and Mank, J. E. 2010, The birds and the bees and the 37. Gamble, T. and Zarkower, D. 2014, Identiﬁcation of sex-speciﬁc molecu- lar markers using restriction site-associated DNA sequencing, Mol. Ecol. ﬂowers and the trees: lessons from genetic mapping of sex determination Resour., 14, 902–13. in plants and animals, Genetics, 186, 9–31. 38. Fowler, B. L. and Buonaccorsi, V. P. 2016, Genomic characterization of 19. Kovacs, B., Egedi, S., Bartfai, R. and Orban, L. 2000, Male-speciﬁc DNA sex-identiﬁcation markers in Sebastes carnatus and S. chrysomelas rock- markers from African catﬁsh (Clarias gariepinus), Genetica, 110, 267–76. ﬁshes, Mol. Ecol., 25, 2165–75. 20. Vale, L., Dieguez, R., Sa ´ nchez, L., Martı´nez, P. and Vinas, ~ A. 2014, A 39. Wang, Y. P., et al. 2015, The draft genome of the grass carp sex-associated sequence identiﬁed by RAPD screening in gynogenetic indi- (Ctenopharyngodon idellus) provides insights into its evolution and vege- viduals of turbot (Scophthalmus maximus), Mol. Biol. Rep., 41, 1501–9. tarian adaptation, Nat. Genet., 47, 625–31. 21. Felip, A., Martinez-Rodriguez, G., Piferrer, F., Carrillo, M. and Zanuy, S. 40. Green, J. E., Dalikova, M., Sahara, K., Marec, F. and Akam, M. 2016, 2000, AFLP analysis conﬁrms exclusive maternal genomic contribution of XX/XY system of sex determination in the geophilomorph centipede meiogynogenetic sea bass (Dicentrarchus labrax L.), Mar. Biotechnol., 2, Strigamia maritima, PLoS One, 11. DOI: 10.1371/journal.pone.0150292. 301–6. 41. Koerich, L. B., Dupim, E. G., Faria, L. L., et al. 2016, First report of 22. Felip, A., Young, W. P., Wheeler, P. A. and Thorgaard, G. H. 2005, An Y-linked genes in the kissing bug Rhodnius prolixus, BMC Genomics, AFLP-based approach for the identiﬁcation of sex-linked markers in rain- 17,1. bow trout (Oncorhynchus mykiss), Aquaculture, 247, 35–43. 42. Kolar, C. S., et al. Asian carps of the genus Hypophthalmichthys (Pisces, 23. Dan, C., Mei, J., Wang, D. and Gui, J. F. 2013, Genetic differentiation Cyprinidae)-a biological synopsis and environmental risk assessment. and efﬁcient sex-speciﬁc marker development of a pair of Y- and X-linked markers in yellow catﬁsh, Int. J. Biol. Sci., 9, 1043–9. 43. Chen, J., Xie, P., Zhang, D. W. and Lei, H. H. 2007, In situ studies on the 24. Pan, Z. J., Li, X. Y., Zhou, F. J., Qiang, X. G. and Gui, J. F. 2015, distribution patterns and dynamics of microcystins in a biomanipulation Identiﬁcation of sex-speciﬁc markers reveals male heterogametic sex deter- ﬁsh-bighead carp (Aristichthys nobilis), Environ. Pollut., 147, 150–7. mination in Pseudobagrus ussuriensis, Mar. Biotechnol., 17, 441–51. 44. Zhu, C. K., Sun, Y. H., Yu, X. M. and Tong, J. G. 2013, Centromere 25. Lamatsch, D. K., Adolfsson, S., Senior, A. M., et al. 2015, A transcrip- localization for bighead carp (Aristichthys nobilis) through half-tetrad tome derived female-speciﬁc marker from the invasive western mosquito- analysis in diploid gynogenetic families, PLoS One, 8. DOI: 10.1371/jour ﬁsh (Gambusia afﬁnis), PLoS One, 10. DOI: 10.1371/journal.pone. nal.pone.0082950. 45. Chong, L. 2001, Molecular cloning: a laboratory manual, 3rd edition, 26. Lu, J. G., Zheng, M., Zheng, J., et al. 2015, Transcriptomic analyses Science, 292, 446. reveal novel genes with sexually dimorphic expression in yellow catﬁsh 46. Catchen, J., Hohenlohe, P. A., Bassham, S., Amores, A. and Cresko, W. (Pelteobagrus fulvidraco) brain, Mar Biotechnol, 17, 613–23. A. 2013, Stacks: an analysis tool set for population genomics, Mol. Ecol., 27. Diaz, N. and Piferrer, F. 2015, Lasting effects of early exposure to temper- 22, 3124–40. ature on the gonadal transcriptome at the time of sex differentiation in the 47. Luo, R. B., et al. 2012, SOAPdenovo2: an empirically improved European sea bass, a ﬁsh with mixed genetic and environmental sex deter- memory-efﬁcient short-read de novo assembler, Gigascience, 1. DOI: 10. mination, BMC Genomics, 16. DOI: 10.1186/S12864-015-1862-0. 1186/2047-217x-1-18. Downloaded from https://academic.oup.com/dnaresearch/advance-article-abstract/doi/10.1093/dnares/dsx054/4791395 by Ed 'DeepDyve' Gillespie user on 08 June 2018 8 Sex-specific markers in bighead carp 48. Piferrer, F., Ribas, L. and Diaz, N. 2012, Genomic approaches to study 55. Wang, S., Meyer, E., McKay, J. K. and Matz, M. V. 2012, 2b-RAD: a genetic and environmental inﬂuences on ﬁsh sex determination and differ- simple and ﬂexible method for genome-wide genotyping, Nat. Methods, entiation, Mar. Biotechnol., 14, 591–604. 9, 808–10. 49. Wang, D. W., Li, Y. and Li, Z. Q. 2011, Identiﬁcation of a male-speciﬁc 56. Kong, Q. L., LI, Z. Y., Fu, M. L., Wang, Q. and WANG, H. Y. 2006, ampliﬁed fragment length polymorphism (AFLP) and a sequence charac- Analysis of DAPI karyotype of bighead carp (Aristichthys nobilis) chro- terized ampliﬁed region (SCAR) marker in Eucommia ulmoides Oliv, Int. mosomes, Sichuan J. Zool., 25, 64–7. J. Mol. Sci., 12, 857–64. 57. Chen, J., Wang, Y., Yue, Y., et al. 2009, A novel male-speciﬁc DNA 50. Gao, Z. X., Wang, H. P., Yao, H., Tiu, L. and Wang, W. M. 2010, No sequence in the common carp, Cyprinus carpio, Mol. Cell. Probes, 23, sex-speciﬁc markers detected in bluegill sunﬁsh Lepomis macrochirus by 235–9. AFLP, J. Fish Biol., 76, 408–14. 58. Howe, K., Clark, M. D., Torroja, C. F., et al. 2013, The zebraﬁsh refer- 51. Yarmohammadi, M., Pourkazemi, M., Ghasemi, A., Hassanzadeh, M. ence genome sequence and its relationship to the human genome, Nature. and Chakmehdouz, F. 2011, AFLP reveals no sex-speciﬁc markers in 496, 498–503. Persian sturgeon (Acipenser persicus) or beluga sturgeon (Huso huso) 59. Tao, W., Zou, M., Wang, X., et al. 2010, Phylogenomic analysis resolves from the southern Caspian Sea, Iran, Progr. Biol. Sci., 1, 55–114. the formerly intractable adaptive diversiﬁcation of the endemic clade of 52. Sriphairoj, K., Na-Nakorn, U., Brunelli, J. P. and Thorgaard, G. H. 2007, east Asian Cyprinidae (Cypriniformes), PLoS One, 5, e13508. No AFLP sex-speciﬁc markers detected in Pangasianodon gigas and P. 60. Xu, P., Zhang, X., Wang, X., et al. 2014, Genome sequence and hypophthalmus, Aquaculture, 273, 739–743. genetic diversity of the common carp, Cyprinus carpio, Nat. Genet. 46, 53. Gamble, T., Coryell, J., Ezaz, T., et al. 2015, Restriction site-associated 1212–19. DNA sequencing (RAD-seq) reveals an extraordinary number of transi- 61. Su, Z. G., Xu, K. S., Chen, S. P. and Bai, G. D. 1984, Studies on triploid tions among gecko sex-determining systems, Mol. Biol. Evol., 32, silver carp and its karyotype, Zool. Res. 1984(S2), 15–20. 1296–1309. 62. Zhu, C. K., Tong, J. O., Yu, X. M. and Guo, W. J. 2015, Comparative 54. Baird, N. A., et al. 2008, Rapid SNP discovery and genetic mapping using mapping for bighead carp (Aristichthys nobilis) against model and sequenced RAD markers, PLoS One, 3. DOI: 10.1371/journal.pone. non-model ﬁshes provides insights into the genomic evolution of cypri- 0003376 nids, Mol. Genet. Genomics, 290, 1313–26. Downloaded from https://academic.oup.com/dnaresearch/advance-article-abstract/doi/10.1093/dnares/dsx054/4791395 by Ed 'DeepDyve' Gillespie user on 08 June 2018
DNA Research – Oxford University Press
Published: Jan 5, 2018
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