Fig 1.
Repetitive sequence expansion on microchromosomes.
(A) Pairwise comparison of all analyzed reads between hexaploid C. gibelio and tetraploid C. auratus. The X-axis and Y-axis show the numbers of similarity hits for each read in C. gibelio and C. auratus, respectively. Each spot corresponds to one read. The black ellipse indicates the repetitive sequences with expansion in C. gibelio compared to C. auratus. The red dots represent the reads of the most expanded satellite cluster (Cg-Ca-CL1). (B) The top 50 largest repeat clusters generated by C. gibelio-C. auratus (Cg-Ca) pairwise comparative analysis. The Y-axis shows the reads number in clusters, and the X-axis shows the cluster ID. (C, D) FISH analysis of satellite repeat cluster Cg-Ca-CL1 in female metaphase (C) and male metaphase (D) of C. gibelio. Scale bar = 5 μm. The white square indicates supernumerary microchromosomes with a male determination role in C. gibelio.
Fig 2.
Identification of three male-specific microchromosomes.
(A, B) FISH analysis on the male metaphase (A) and female metaphase (B) using PNAs as probe. The white rectangle indicates microchromosomes. Scale bar = 5 μm. (C) Electrophoresis of the amplified products from microdissected microchromosomes. (D) PCR detection of these amplified products using male-specific primers. M, marker; P, positive control using genomic DNA as template; N, negative control using water as template. (E-G) Co-localization of amplified DNAs and microchromosome-specific PNA probes. The amplified DNAs of MSM 1 (E), MSM 2 (F), and MSM 3 (G) were labeled with Digoxin, and the PNAs were labeled with Biotin, which appeared green and red fluorescence respectively. Scale bar = 5 μm. Mitotic cells of three males were used as replication for co-localization analysis.
Fig 3.
(A-C) Comparative analysis between MSM sequences and the reference genome of C. gibelio. MSM 1 (A), MSM 2 (B), and MSM 3 (C) blocks are shown at the right and the corresponding linkage groups of reference genome are displayed at the left. (D) Kimura divergence between the reference genome and homologous sequences of MSMs. The X axis represents the divergence and the Y axis displays the number of hits. (E) Pairwise comparative analysis among three MSMs. The X axis indicates the compared objects and the Y axis indicates the proportion of homologous sequences.
Fig 4.
Analysis of repetitive elements.
(A) The proportion of sequencing reads containing repetitive elements. The Y axis indicates the proportion of reads. (B) The proportion of repetitive elements including satellite, TE, and unknown. (C, D) Types and proportion of satellite repeats (C) and TEs (D). (E) Copy numbers of different types of TEs.
Fig 5.
Identification of genes and the corresponding transcripts.
(A) The hematoxylin-eosin staining of genotypic female and male gonads at 10 developmental stages. Scale bar = 50 μm. (B) The distribution of different types of gene families among nine species. The X axis shows nine species and the Y axis indicates the number of genes. (C) Numbers of identified genes on MSM 1, MSM 2 and MSM 3. (D) Results of gene integrity analysis. X axis indicates each integrity percentage bins and Y axis shows the number of genes. (E) KEGG analysis of the MSM-linked transcripts. (F) The distribution of MSM-linked transcripts in male and female gonads. (G) Sex-biased transcripts during the early stages of gonadal development. Dah, days after hatch.
Fig 6.
Potential male-specific gene fragments with transcriptional activity.
(A, B) Transcriptome subtraction (A) and subsequent genome subtraction (B) performed on MSM-linked transcripts. The grey dots represent transcripts with the coverage > 90% and identity > 98%. The pie charts indicate the number of discarded (grey) and remained transcripts (wathet and brown) (C) The pie chart of the second round of transcriptome/genome subtractions performed on gene fragments. (D) Heatmap of the gene fragments with higher expression in male gonads than in female gonads at least at one developmental stage from 18 to 35 dah. (E) The distribution of the gene fragments with higher expression in male gonads than in female gonads. The axis indicates the number of gene fragments. (F) qPCR detection of gene fragments with male-specific or male-biased expression at early gonadal developmental stages including 18, 22, 26, 30, and 35 dah. The X axis represents the stages of gonad development. The Y axis represents the relative expression, and the highest expression level of each gene fragment was used as control and defined as 1. F, female; M, male.
Fig 7.
Schematic diagram of MSM origin and male occurrence in C. gibelio.
Hexaploid C. gibelio was originated from ancestral tetraploid C. auratus via autopolyploidy, and the newly formed hexaploid C. gibelio reproduced via unisexual gynogenesis. Two possible evolutionary trajectories of MSM origin and male occurrence in C. gibelio were indicated by the dashed arrow and solid arrow respectively. One possibility is that MSMs and males might emerge at the beginning of C. gibelio formation via autopolyploidy, and the sex-determining gene/genes might be accumulated from the A chromosomes of sexual progenitor during autopolyploidy (dashed arrow). The other possibility is that MSMs and males did not emerge at the beginning of C. gibelio formation but formed during the evolutionary process after autopolyploidy. And the sex-determining gene/genes might be acquired from the duplicates of A chromosomes of C. gibelio or the DNA introgression of host sexual species (solid arrow).