Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole (Cynoglossus semilaevis)

Chinese tongue sole is a marine fish with ZW sex determination. Genome sequencing suggested that the Z-linked dmrt1 is a putative male determination gene, but direct genetic evidence is still lacking. Here we show that TALEN of dmrt1 efficiently induced mutations of this gene. The ZZ dmrt1 mutant fish developed ovary-like testis, and the spermatogenesis was disrupted. The female-related genes foxl2 and cyp19a1a were significantly increased in the gonad of the ZZ dmrt1 mutant. Conversely, the male-related genes Sox9a and Amh were significantly decreased. The dmrt1 deficient ZZ fish grew much faster than ZZ male control. Notably, we obtained an intersex ZW fish with a testis on one side and an ovary on the other side. This fish was chimeric for a dmrt1 mutation in the ovary, and wild-type dmrt1 in the testis. Our data provide the first functional evidence that dmrt1 is a male determining gene in tongue sole.


Suppl 1
Microinjection was done in three steps: First, because C. semilaevis embryos are floating, more than half of the medium was aspirated from the embryo holding device. Leaving too much fluid makes the embryos falling in the trough. Second, the embryos were rotated to a position where the cell can be injected by the microinjection needle perpendicularly. Third, the microinjection needle was placed close to a glass slide. The tip of the needle gently touched the rough surface of the glass slide, until a small amount of dmrt1-TALENs mRNA flew out because of pressure from the injector device.

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The PCR reaction contained 2µl gDNA (~50ng/µl) as template, 5µl 10x reaction buffer (with MgCl 2+ ), 4µl dNTPs, 0.5µl Takara Ex Taq DNA polymerase (Takara, Japan), 1.5µl of each primer (at 10µM) and 35.5µl ddH 2 O to a total volume of 50µl. PCR conditions were one cycle at 95°C for 5 min, followed by 35 cycles at 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, and a final elongation step at 72°C for 10 min. The PCR product was purified using Gel Extraction Kit (CWBIO, China). 5µl of purified PCR product added to 1.1µl 1x NEBuffer and 4.4µl ddH 2 O were heated to 95°C for 5min and then cooled to room temperature. Then T7E I (NEB, USA) was added and incubated at 37°C for 30 min. The reaction mixture was separated by agarose gel electrophoresis and detected by Gel Imaging System (Bio-Rad, USA). The purified PCR product was cloned into the PMD-18T vector (Takara, Japan) and transformed into Top10 competent cells. Individual bacterial colonies were randomly selected and screened by PCR in a total volume of 25µl as described above. Positive clones were sequenced and mutated sites compared to wild-type sequences. The mutation rate of each sample was calculated from the result of sequencing (mutated sequences/total sequences).

Figure S5
Expression of dmrt1, fox12 and cyp19a1a in dmrt1-deficient gonads of one year old fish determined by semiquantitavie RT-PCR. β-actin was used as control and for calibration of RNA amounts. A, mRNA expression of cyp19a1a in dmrt1-deficient testis is comparable to wt females, while only amplification barely above background is recorded in wild-type males. B, RT-PCR for dmrt1 with RNA from dmrt1-deficient testis revealed considerably less amplification compared with wild-type males. C, mRNA expression of foxl2 in dmrt1-deficient testis appears to be even higher in comparison with wild-type females. D, mRNA expression of β-actin in dmrt1-deficient tongue sole, wild-type females and wild-type males.

Figure S6
Dmrt1 sequences of ovary and testis from the hermaphroditic fish. A, sequences of wild-type and mutated target sites of dmrt1 retrieved from ovary, partial. B, sequences of only wild-type dmrt1 in testis, partial.

Figure S7
Expression of dmrt1, fox12 and cyp19a1a in gonads of the hermaphroditic fish. β-actin was used as control and for calibration of RNA amounts. A, mRNA expression of dmrt1 in intersexual testis is significantly higher than that in intersexual ovary. B, RT-PCR for cyp19a1a with RNA from intersexual testis revealed less amplification compared with intersexual ovary. C, mRNA expression of foxl2 in intersexual ovary appears to be higher in comparison with intersexual testis. D, mRNA expression of β-actin in gonads of intersexual tongue sole. Table S1. Primers used in this study.

Primer
Sequence