The Institute of Experimental Animals, Hunan Province, China approved all sample protocols in this study. All fish were deeply anesthetized with 100 mg/L MS-222 (Sigma-Aldrich, St Louis, MO, United States) before tissue collection.

All RCC and 4nRR were selected from the State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Normal University, China. The generation of RCC and 4nRR was executed during the reproductive season of March 2021 within the Engineering Research Center of Polyploid Fish Breeding and Reproduction of the State Education Ministry, China, located at Hunan Normal University. All individuals were cultured in an open pool (0.067 ha) with suitable pH (7.0–8.5), water temperature (22°C–24°C), dissolved oxygen content (70%), and adequate forage. The cDNA and DNA of Gonad tissue was respectively used to clone CDS sequence and DNA composition. To determine Dmc1 expression characteristics at different age stages, gonads from RCC and 4nRR fish aged 3, 4, 5, 6, 7, and 11 months were collected, frozen in liquid nitrogen, and stored at −80°C for RNA extraction.

Chromosome preparations were prepared using kidney tissue of all samples in accordance with Qin et al. (2019a). The probes for fluorescence in situ hybridization (FISH) for the specific centromere repeats (263-bp, sequence number JQ086761) of RCC was performed for 4nRR and autodiploid gynogenetic offspring (2n = 100, RR) (G₁) and amplified via PCR using the primers 5′-TTC​GAA​AAG​AGA​GAA​TAA​TCT​A-3′ and 5′-AAC​TCG​TCT​AAA​CCC​GAA​CTA-3′. Detailed FISH steps were performed according to the method described by He et al. (2012).

Total RNA isolation was extracted using Trizol (Thermo Scientific, United States), while the quality and concentration were ascertained by agarose gel electrophoresis and a Nanodrop2000 spectrophotometer (Thermo Scientific, United States), respectively. The cDNA synthesis was achieved using SMARTerTM PCR cDNA Synthesis Kit, and the quality of cDNA was verified by the housekeeping gene (β-actin) and agarose gel electrophoresis. The degenerate primers of CDS and DNA full-length amplification of the Dmc1 gene in RCC were designed based on the nucleotide sequences of Cyprinidae fish and then the CDS and DNA full-length amplification primers of all Dmc1 in 4nRR were designed based on the genome sequence of 4nRR (unpublished) (Table 1). PCR amplification was respectively configured using cDNA and DNA of gonad as the template, and PCR products were cloned and sequenced to confirm the CDS and DNA specific sequence of Dmc1 in RCC and 4nRR. The identity and polypeptide sequence alignments of amino-acid sequences of Dmc1 were utilized with the DNAMAN version7.0. The qRT-PCR was carried out as directed by Wang et al. (2021). The qRT-PCR was conducted with triplicates. The qPCR-specific primers were designed and distinguished based on the specific SNP in the CDS of each Dmc1 gene and determined with cloning (Table 1). The results were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). SPSS 20.0 software was used to confirm the one-way Anova test for statistical analysis.

Due to the specific expression and alternative transcription of the Dmc1 in both RCC and 4nRR gonads, the DNA methylation level in both gonads was detected and analyzed. Comparison and analyses were made based on the expression level at different age stages, genomic DNA extraction of ovary from 5-month-old and testis from 6-month-old in RCC and 4nRR was obtained using TaKaRa MiniBEST universal Genomic DNA Extraction Kit Version. 5.0 (TaKaRa, Japan), while the quality and concentration checking were the same as the RNA. The extracted DNA was sodium bisulfite-modified using the EZ DNA Methylation-Gold™ Kit (Zymo Research, United States). Pseudogenization of Dmc1-2 sequences and failure of expression were observed in both RCC and 4nRR. Based on the cloned intron sequence of Dmc1-1 (located in intron 11 and intron 12) in RCC and Dmc1-1 (located in intron 11 and intron 12), Dmc1-3 (located in intron 5), Dmc1-4 (located in intron 3) in 4nRR, the design of methylation primers listed in Table 1, and the prediction of the CpG-rich islands were displayed on the MethPrimer website (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi). PCR amplification was configured using bisulfite-treated DNA as the template, and PCR products were cloned and sequenced to analyze the methylation status of different Dmc1.

We monitored gonadal development to understand Dmc1 expression at different age stages in response to the gonadal development in RCC and 4nRR. According to expression characteristics of Dmc1 in gonad structure at different age stages, ten females (from the age of 3, 5, 7, and 11 months) and ten males (from the age of 3, 4, 6, and 11 months) in RCC and 4nRR were randomly selected for histological observation of gonad structure. Then, detailed steps for observation of gonadal structure were performed following the method described by Qin et al. (2019b).

Autodiploid gynogenetic offspring were produced by artificial gynogenesis from the eggs of the 4nRR that were activated with UV-treated sterilized sperm of BSB without chromosome doubling treatment. The 263 probe was hybridized to the metaphase chromosomes of 4nRR and G₁, and the results of FISH were shown in Figure 1. Hybridization of the probe yielded one hundred specific centromere repeats loci in the chromosomal metaphases of 4nRR. And fifty specific centromere repeats loci were detected in the chromosomal metaphases of G₁. The G₁ possessed half sets of 4nRR-derived chromosomes, indicating that the chromosome from the eggs of the 4nRR was halved obviously, which is an important sign of meiosis of 4nRR.

Two Dmc1 copies, including Dmc1-1 and Dmc1-2, were obtained by validation at the DNA level, of which Dmc1-2 was pseudogenicized because of the deletion of first exon and second exon in both RCC and 4nRR. Moreover, two different duplicated genes (Dmc1-3 and Dmc1-4) possessing complete gene structure were observed in 4nRR (Figure 2). In RCC, Dmc1-1 and Dmc1-2 were located on Scaffold271 and Scaffold218 respectively, and Dmc1-1, Dmc1-2, Dmc1-3, Dmc1-4 were orderly located on chromosome 33A, 42A, 33B, and 42B in 4nRR respectively. However, in the analysis of Dmc1 transcript, we only noticed Dmc1-1 (GeneBank: MK140667.1) in RCC and Dmc1-3 (GeneBank: MH973697.1) in 4nRR with the same length of 1029bp CDS by cDNA cloning, whereas no Dmc1-1 and Dmc1-4 transcript were detected in 4nRR. Polypeptide sequence alignments revealed that the identity of Dmc1-1 in RCC and Dmc1-3 in 4nRR was 99.1%, while we analyzed and found only three missense-mutation sites (RCC ^Dmc1-1 /4nRR ^Dmc1-3 , V³⁶/M³⁶, I⁹⁵/V⁹⁵, P¹⁵⁴/T¹⁵⁴) among them. However, two specific nucleotide binding motifs (A and B) and two DNA binding domains (L1 and L2) were identified in both genes (Figure 3). Furthermore, the genetic and polypeptide sequence of Dmc1 in diploid Carassius auratus (2n = 100, abbreviated as 2nCC), autotriploid Carassius auratus (3n = 150, abbreviated as 3nCC), artificial autotriploid Carassius auratus (3n = 150, abbreviated as 3nRR) was identical to Dmc1-1 transcript (Figure 3). The DNA composition and transcript detection demonstrated that the gene variation and alternative transcription of Dmc1 did emerge following autotetraploidization.

For females in RCC and 4nRR, the ovarian development of individuals differentiated to stage II at 3 months, stage II at 5 months, stage III at 7 months, and stage Ⅳ at 11 months. Moreover, for males in RCC and 4nRR, the testis development of individuals differentiated to stage II at 3 months, stage III at 4 months, stage Ⅳ at 6 months, and stage Ⅴ at 11 months. The identical expression profiles were observed in the Dmc1-1 of RCC and Dmc1-3 of 4nRR by quantitative real-time PCR. Figure 4 shows the expression levels of Dmc1 in gonads of RCC and 4nRR at different age stages. For all female individuals, the Dmc1-1 in RCC and Dmc1-3 in 4nRR exhibited identical gene expression profiles in the ovary at different age stages, characterizing by that both genes were expressed only within developmental time point specific. First, the expression level was not detected at 3 months, then the expression level of both genes jumped from extremely low level at 4 months to the highest level at 5 months (p < 0.01) and finally dropped acutely to the zero (at 6 months) with no detectable expression level at 7 months and 11 months. However, in the male RCC and 4nRR individuals, the expression level of Dmc1 can be consistently detected that the expression level gradually increased to the maximum (at 6 months) and then continuously decreased to higher expression level (p < 0.01). In addition, in both RCC and 4nRR, different expression level of Dmc1 within the same development period in female and male can be observed that expression level in testis was significantly higher than the ovary.

The similar characteristics of gonadal development could be observed in both RCC and 4nRR. Figure 5 shows the gonad microstructure of different age stages of female and male in both RCC and 4nRR. The correlation analysis between expression level of Dmc1 and ovarian development depicted that large numbers of phase Ⅱ oocytes could be observed when the highest expression was detected in the ovary, however, the abrupt decrease in its expression level and non-expression did not affect the subsequent development of the ovary that the phase Ⅲ oocytes (at 7 months) and phase Ⅳ oocytes (at 11 months) can still be found in the ovary. For males in RCC and 4nRR, spermatogonium at 3 months and a small number of primary spermatocytes, secondary spermatocytes and spermatids at 4 months were formed in the testis as the expression level of Dmc1 increased. Then large groups of primary spermatocytes, secondary spermatocytes, and some spermatids were noticed at 6 months when the highest expression level was shown. After that, the spermatids and sperms in the testis increased at 11 months when the expression decreased. The above results demonstrated that the differential expression characteristic of Dmc1 between females and males in 4nRR remained the same as the RCC and Dmc1 is mainly expressed during the meiosis I.

Given that, in 4nRR, except for the original Dmc1-1 and duplicated Dmc1-4, the duplicated Dmc1-3 transcript was only detected, we hypothesized that this case was induced by epigenetic modification alteration, so that we assessed the DNA methylation status of the Dmc1-1, Dmc1-3, and Dmc1-4 in 4nRR and Dmc1-1 in RCC in gonads when the highest expression occurred. Table 2 and Figure 6 depict the methylation status of these genes in the ovary and testis. The results showed that, in RCC, the methylation levels of Dmc1-1 in 5-month-old ovaries were 0.500 and in 6-month-old testes were 0.267, but they turned into 1.000 in ovaries and testes of 4nRR. In terms of duplicated genes, methylation levels of Dmc1-3 in ovaries and testes were 0.375 and 0.300, respectively, which were much lower than that of Dmc1-4, reaching 0.912 in ovaries and 0.938 testes, respectively. This result suggested that the lowest methylation levels of duplicated Dmc1-3 was detected, supporting the hypothesis that the alteration of DNA methylation status may affect the transcription and expression of these genes.