The Y chromosome sequence of the channel catfish suggests novel sex determination mechanisms in teleost fish

Abstract

Background: Sex determination mechanisms in teleost fish broadly differ from mammals and birds, with sex chromosomes that are far less differentiated and recombination often occurring along the length of the X and Y chromosomes, posing major challenges for the identification of specific sex determination genes. Here, we take an innovative approach of comparative genome analysis of the genomic sequences of the X chromosome and newly sequenced Y chromosome in the channel catfish.

Results: Using a YY channel catfish as the sequencing template, we generated, assembled, and annotated the Y genome sequence of channel catfish. The genome sequence assembly had a contig N50 size of 2.7 Mb and a scaffold N50 size of 26.7 Mb. Genetic linkage and GWAS analyses placed the sex determination locus within a genetic distance less than 0.5 cM and physical distance of 8.9 Mb. However, comparison of the channel catfish X and Y chromosome sequences showed no sex-specific genes. Instead, comparative RNA-Seq analysis between females and males revealed exclusive sex-specific expression of an isoform of the breast cancer anti-resistance 1 (BCAR1) gene in the male during early sex differentiation. Experimental knockout of BCAR1 gene converted genetic males (XY) to phenotypic females, suggesting BCAR1 as a putative sex determination gene.

Conclusions: We present the first Y chromosome sequence among teleost fish, and one of the few whole Y chromosome sequences among vertebrate species. Comparative analyses suggest that sex-specific isoform expression through alternative splicing may underlie sex determination processes in the channel catfish, and we identify BCAR1 as a potential sex determination gene.

Keywords: Catfish; PacBio; RNA-Seq; Sex determination; Y chromosome.

Fig. 1

QTL mapping of sex chromosome of channel catfish. a The distribution of LOD values of QTL of sex of channel catfish on each linkage group are shown in chromosomal order. b The distribution of LOD values of QTL of sex on linkage group (LG) 4 are shown in red line, and 95% Bayes confidence interval is shown within the blue dotted lines

Fig. 2

Genome-wide association study (GWAS) of sex determination region of channel catfish. a The distribution of significant GWAS SNPs across the whole genome of channel catfish. SNPs with false discovery rate (FDR) P value ≤ 1E−10 are shown as red dots. SNPs with FDR 1E−5 > P value > 1E−10 are shown as purple triangles. SNPs with FDR P value > 1E−5 are shown as green squares. b The distribution of significant GWAS SNPs on chromosome 4. The sex determination region is indicated within blue dotted lines

Fig. 3

Comparison of sequence similarity of channel catfish X and Y chromosomes. Genomic reads were mapped to chromosome assemblies. X axis is the position of the sex chromosome. Y axis is the percent similarity. Purple dots indicate the forward read match, and blue dots indicate the reverse read match

Fig. 4

Differentially expressed genes on the channel catfish sex chromosome. Teal bars indicate the genes highly expressed in females, and maroon bars indicate the genes highly expressed in males. The X axis shows the position along the sex chromosome, and Y axis indicates the fold change in the gene expression. Blue dotted lines indicate the border of the sex determination region. a Differentially expressed genes from 10 to 14 days post fertilization (dpf). b Differentially expressed genes from 15 to 19 dpf

Fig. 5

Structure of BCAR1 gene in channel catfish. The BCAR1 genes contain 8 exons, and four transcript isoforms of BCAR1 gene exist. Isoform 2 and isoform 4 start in exon 1 and contain exons 3–8, but have slightly different splicing sites from each other. Isoforms 1 and 3 begin in exon 2 and contain exons 3–8 but slightly different splicing sites. The red lines indicate the position and sequences of the male-specific transcript and that sequence from the YY genome is shown at the bottom

Fig. 6

Gonadal tissues from targeted knockout of the BCAR1 gene in channel catfish. a Histological examination of gonads 90 days post fertilization from 15 microinjected fish. Arrows indicate the position of the gonad. The agarose gel analysis indicates the genotypic sex of each fish. Males are shown as two bands, and females are shown as one band. Two fish (#9, #15) with male genotypes but female phenotypes are labeled in red. b Sequencing analysis of two KO clones. Target sequence is shown in red, mutations and indels are highlighted in green, and short black lines denote deletions WT, wild type

Fig. 7

One hypothetical mechanism of BCAR1 involvement in sex determination. The Y-linked BCAR1 isoform inhibits estrogen/estrogen receptor alpha-mediated signal transduction in the undifferentiated gonad and drives development toward the testis

Fig. 8

Generation of YY catfish. Channel catfish were fed testosterone to produce generation 1 sex-reversed XY females which were then mated with normal XY males. Generation 2 YY males were identified using molecular markers, and YY genotype was validated through production of all male offspring in generation 3