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Spinyhead croaker (Collichthys lucidus) is a commercially important marine sciaenid fish, being found diverging into northern and southern lineages. A sex diagnostic marker has been developed in the croaker, but its application scope was limited in the populations belonging to southern lineage. In present study, novel diagnostic markers were developed to break the limitation. Resequencing was performed with samples from Zhousan (ZS) population and Ningde (ND), representing northern and southern lineages respectively. Based on the resulted data, the candidate sexually dimorphic SNPs and InDels were screened out, and their genomic distributions were visualized. For ZS population, 52,621 SNPs and 8594 InDels were screened out, mainly distributing on Chr.1 and Chr.8 along almost entire chromosome. For ND population, 2094 SNPs and 350 InDels were screened out, mainly clustering within a 0.55-Mb segment from 4.31–4.86 Mb on Chr.1 and a 1.58-Mb segment from 27.12–28.70 Mb on Chr.7. Based on a common male-specific delete on Chr.1, PCR primer pair CMS-1 was designed and demonstrated applicable to all four tested populations covering both lineages; while based on a male-specific delete of Chr.8 for ZS population, PCR primer pair ZS-8 was designed and demonstrated applicable only to the northern populations but not the southern populations. The novel sex-specific markers developed in the present study extend the toolkit for both sex control breeding and basic study on sex determination in the croaker. Besides, different number and location of sexually dimorphic SNPs/InDels between two lineages provide new insights in sex chromosome evolution in spinyhead croaker.

Sex-specific markers have important implications in genetic improvements of economical traits in aquacultured fish. Yellow drum (Nibea albiflora) is a commercially important fish species distributed in China, Southern Japan and Korea, exhibiting a sex-dependent dimorphic growth pattern where females grow much faster than males. Yet its sex determination mechanism was unclear and sex-specific markers were missing. In this study, a 45 bp deletion within the first intron of Dmrt1 was detected only in males through comparative analysis between males and females. Two pairs of primers (male-specific primers MS-F/R and male and female shared-primers MFS-F/R) were designed to efficiently distinguish the genetic sex of the yellow drum. Importantly, genotyping the deletion in large cohorts of both artificially bred and wild yellow drum confirmed its male-specific property. Our results showed that this male-specific genetic marker could be used to establish a simple and reproducible method for genetic sex identification. Furthermore, the fact in which males are heterozygotes and females are homozygotes of the marker indicates that yellow drum adopted the male heterogametic XX/XY sex determination system.

The gonadosomatic index (GSI) is an economically important trait in the large yellow croaker ( Larimichthys crocea ), a mariculture fish species with high commercial value in Southeast China. However, progress in the genetic research on this species is relatively lacking. To identify the relevant genes and chromosome regions associated with the GSI, we performed a genome-wide association study (GWAS) on a population of 500 individuals (237 males and 263 females), using single marker analysis and Bayesian methods. The genotyping-by-sequencing method was used to construct the libraries, and 29,748 SNPs were subsequently found in the genome of Larimichthys crocea . A total of six different SNPs, located on LG3, LG6, LG8, and LG16, were determined to be significantly associated with the GSI. Among them, only two SNPs were found by both GWAS methods. By integrating the results of two GWAS methods with the biological functions of the genes, six candidate genes (viz., FZD3 , DDX4 , EDC3 , CITED3 , SOX9-b, and LOC106602108 ) were revealed to be associated with the GSI in the large yellow croaker. These functional genes are involved in gonadal development or gonadogenesis, providing the basis for further study on genetic mechanisms and marker-assisted selection in this fish species. In this study, we found that the combination of single marker analysis and Bayesian methods could identify more SNP loci associated with the phenotypes. Thus, we suggest performing these two methods simultaneously in GWAS.

Understanding sex determination mechanisms is the first and most paramount step in the development of sex control breeding biotechnologies for fish. The large yellow croaker ( Larimichthys crocea ) is an economically vital fish in China and East Asia, which exhibits a sex-related growth dimorphism where females grow significantly faster than males. However, the lack of a rapid and accurate sex specific marker for the large yellow croaker has hampered the application of sex control in the breeding process, as well as sex-determination investigations of this species. In this study, a male-specific 15 bp deletion in the third intron of the Dmrt1 gene was identified by comparative analysis of genomic re-sequencing data from male and female samples. Two pairs of primers (MS and MFS) were carefully designed; thus, the male-specific marker could be genotyped by PCR amplification and agarose gel electrophoresis. More importantly, this male-specific marker could also be applied to precisely identify the genetic sex of multiple fish. This male specific DNA marker will have potential applications in the elucidation of the sex determination mechanism and will also accelerate the development of sex control breeding in the large yellow croaker. Moreover, our current data further validated an significant genetic evidence that an XY-male determination mechanism is utilized in the large yellow croaker.

Spinyhead croaker (Collichthys lucidus) is a commercially important marine sciaenid fish, exhibiting sexual dimorphism in growth, and being of a special multiple sex system X1X1X2X2/X1X2Y. In the present study, we developed the first PCR-based method for identifying the genetic sex in spinyhead croaker with a next generation sequencing (NGS)-based approach. After whole genome resequencing was performed on 10 individuals and 2 pools for each sex, approximately 404 G clean data and 2,857,406 high quality SNPs were obtained. With genome-wide association analysis (GWAS), 420 SNPs on 8 chromosomes were identified as sex significantly associated loci. Most of the sex associated loci aggregated on Chr. 1 and Chr. 7, suggesting that they might be the X1 and X2 chromosome. On the segment of Chr. 7 that aggregated sex associated SNPs, three candidate male specific deletions were detected by comparing the sequencing depth between sexes. Based on one of the putative male specific deletions (68 bp), a pair of primers for sex identification were designed and validated with PCR. The primers were applicable to ND and JR populations belonging to the southern group, but not to ZS population belonging the northern group. The methods for sex identification developed in the present study along with its limited versatility provided a necessary tool and important clues for deciphering the sex determination mechanism in spinyhead croaker.