Mutation of Genes Associated with Body Color, Growth, Intermuscular Bone, and Sex Differentiation in Onychostoma macrolepis Using CRISPR/Cas9

Abstract

Onychostoma macrolepis is not only a protected Cyprinid species in the wild but also an emerging commercial aquaculture fish in China. The objective of this research was to genetically modify the genes associated with commercial traits by CRISPR/Cas9 for the protection and utilization of the germplasm resources of O. macrolepis. To that end, one-cell stage embryos were obtained via hormone-induced ovulation and artificial insemination in O. macrolepis. Eight genes related to body color, growth, intermuscular bone, and sex differentiation were mutated in O. macrolepis using the CRISPR/Cas9 system by microinjection of gRNA/Cas9 mRNA. The optimal dose of gRNA/Cas9 mRNA was determined by injection of different concentrations of tyr (tyrosinase)-gRNA/Cas9 and examination of the mutation rate and hatching rate of embryos. Indels were detected by T7 endonuclease I digestion and Sanger sequencing. F0 mutants with high mutation rates were selected for phenotype analyses. Disruption of body color gene tyr, mpv17 (mitochondrial inner membrane protein MPV17), and csf1ra (colony-stimulating factor 1 receptor, a) resulted in obvious phenotype with decreased or even absence of melanophores, iridophores, and xanthophores, respectively. Mutation of mstnb (myostatin b) led to improved growth performance. Mutation of mc4r (melanocortin 4 receptor) led to no obvious phenotype. Mutation of runx2b (RUNX family transcription factor 2b) and bmp6 (bone morphogenetic protein 6) resulted in decreased or absence of intermuscular bones, as revealed by alizarin red S staining. Mutation of cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) resulted in ovarian degeneration as revealed by gonadal histological examination. Therefore, this study successfully obtained mutants with obvious phenotypes of genes associated with body color, growth, intermuscular bone, and sex differentiation by CRISPR/Cas9 in O. macrolepis.

1. Introduction

World fisheries and aquaculture production hit a new high in 2022, making an important contribution to global food security. China, with an annual production of 52,884 thousand tons, dominates global production. Aquaculture in different regions of China is/provides an important source of local food and economic income. Onychostoma macrolepis is a unique Chinese cyprinid fish species widely distributed in the Wei River, the Huai River, the upper and middle reaches of the Yangtze River, and the upper reaches of the Hai River. The adults of O. macrolepis are usually 50~150 g in weight and approximately 15~20 cm in length, with females larger than males and 5000~10,000 eggs per spawn in females [1]. It is known as a delicious and nutritious food fish due to its high percentages of protein and unsaturated fatty acids in muscle [2]. Unfortunately, its natural resources have declined sharply due to changes in the habitat environment and overfishing. The wild population of O. macrolepis was listed as a grade II protected animal on China’s list of national key protected wildlife in 2021. At present, in addition to being a commercial aquaculture species, artificially cultured O. macrolepis has to meet the requirements for stock enhancement under wild conditions. Even though O. macrolepis can spawn and breed on its own in the breeding waters, asynchronous spawning results in low breeding efficiency. Therefore, hormone-induced ovulation and artificial insemination are required to obtain large-scale fingerlings for aquaculture and stock enhancement in the river.

In recent years, genetics has been revolutionized by the emergence of gene editing technologies such as CRISPR/Cas9. This allows for the targeted alteration of organism traits by making precise changes to their genomes. Studies of gene editing in aquaculture species have confirmed that the CRISPR/Cas9 gene editing system is simple and efficient, with a low off-target propensity. So far, CRISPR/Cas9 gene editing technology has been developed and applied to more than 20 cultured fish species. A series of candidate genes associated with traits such as growth, intermuscular bone, sex, and body color have been mutated [3]. The mstnb (myostatin b), which has a significant impact on muscle growth, has been genetically edited in various farmed fish to obtain fast-growing individuals [4,5,6,7,8,9,10,11]. In addition, it has been found that mc4r (melanocortin 4 receptor) knockout also improves growth in channel catfish [12,13]. New strains without intermuscular bones have been created by mutating bmp6 (bone morphogenetic protein 6) and runx2b (RUNX family transcription factor 2b) in crucian carp (Carassius auratus) [14] and blunt snout bream (Megalobrama amblycephala) [15], respectively. Sex control was achieved through gene editing of genes associated with sex determination and differentiation in Nile tilapia (Oreochromis niloticus) [16,17], half-smooth tongue sole (Cynoglossus semilaevis) [18], and common carp (Cyprinus carpio L.) [19]. The acquisition of body color mutants is equally important in cultured fish. For example, mutation of tyr (tyrosinase) [20], mpv17 (mitochondrial inner membrane protein MPV17) [21], and csf1ra (colony-stimulating factor 1 receptor, a) [22] in the Nile tilapia resulted in the loss of melanophore, iridophore, and xanthophore, respectively, displaying albino, transparent, and gray body color.

Recently, we have sequenced and assembled the genome of O. macrolepis at the chromosome level [23]. Meanwhile, the achievement of artificial insemination allows us to develop new varieties with excellent traits through gene editing technology, which is of great significance for the protection and utilization of the germplasm resources of O. macrolepis. The objective of this study is to mutate the genes associated with body color, growth, intermuscular bone, and sex differentiation using CRISPR/Cas9 in O. macrolepis to create new germplasm for aquaculture in the future.

2. Materials and Methods

2.1. Study Site

The artificial propagation and microinjection of O. macrolepis were conducted in the Lushui Ecological Agriculture Company (Langao County, Ankang, Shaanxi Province, China, 32°9′41.112″ N and 109°5′4.135″ E) from June to July 2022 and 2023. The experiments on gRNA design, synthesis, mutation detection, and phenotype detection were carried out in the Key Laboratory of Freshwater Fish Reproduction and Development (Southwest University, Chongqing, China).

2.2. Hormone-Induced Ovulation and Artificial Insemination

During the breeding season, males with a visible nuptial tubercle on the snout and anal fin, as well as females with a bulging abdomen, visible ovarian outlines on both sides of the genital pore, and protruding and reddish genital pore were screened and placed in pools. The water temperature was controlled at about 20 °C. After 2 days of adaptation, females were injected intramuscularly with a mixture of human chorionic gonadotropin (HCG) (Ningbo, China) (800–1000 units/kg) and domperidon (DOM) (Ningbo, China) (3 mg/kg) at the base of the dorsal fin and stimulated with slight water flow for ovulation. Males produce sperm without hormone induction. The female fish were checked for ovulation status every hour when males and females frequently chased each other, and females swam on the water surface for a long time without diving.

Fish eggs were collected by pressing the abdomen of the ovulated fish and artificially inseminated with sperm squeezed from mature males by the dry fertilization method. Fertilized eggs were evenly distributed in the floating nets and then placed in a flow-through pool for incubation with a water temperature controlled at 20–23 °C. The one-cell stage embryo was determined through microscopic observation. We began monitoring the embryos immediately after fertilization. Using the stereomicroscope SZ680 (Aote, Chongqing, China) with appropriate magnification, we were able to observe the animal pole at the upper end of the fertilized egg approximately 30 min after fertilization at a water temperature of 23 °C. The hatched fry were fed a commercial diet (Shengsuo, Yantai, Shandong, China) consisting mainly of 52% protein, 16.5% ash, and 8% lipids. The water temperature for fry cultivation was 20–23 °C, and the dissolved oxygen in the water was maintained above 7 mg/L.

2.3. gRNA Design, Synthesis, and Microinjection

We have sequenced and assembled the genome of O. macrolepis at the chromosome level (Sun et al., 2020). The gene sequences of tyr (Gene ID:131554175), mpv17 (Gene ID:131526710), csf1ra (Gene ID:131552990), mstnb (Gene ID:131547249), mc4r (Gene ID:131526202), runx2b (Gene ID:131526202), bmp6 (Gene ID:131533093), and cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) (Gene ID:131524768) were downloaded from the O. macrolepis genome database on the NCBI website. gRNA target sites were designed through the crisprscan online tool (https://www.crisprscan.org/sequence/, accessed on 10 May 2022). gRNA and Cas9 mRNA were synthesized as described previously for Nile tilapia [16]. To determine the optimal concentration of gRNA/Cas9 mixture for microinjection in O. macrolepis, 200 one-cell stage embryos were injected with a mixture of tyr-gRNA/Cas9 mRNA at three different concentrations (50/100 ng/μL, 100/250 ng/μL, and 250/500 ng/μL). The mutation efficiency and hatching rate of the injected embryos were then calculated. After the animal pole of the fertilized egg was observed under the stereomicroscope, the gRNA/Cas9 mRNA mixture of each gene of mpv17, csf1ra, mstnb, mc4r, runx2b, bmp6, and cyp19a1a was injected into one-cell stage embryos at a concentration of 100/250 ng/μL using an injection instrument (DMP-300) (Weike, Wuhan, China). Phenol red was added to the mixture as an indicator to determine whether the injection was successful.

2.4. Mutation Detection

Embryos were collected 48 h after fertilization, including wild type and injected embryos (twenty embryos for each gene). Genomic DNA was isolated by phenol/chloroform extraction after digestion with protease (Sigma, St. Louis, MO, USA). DNA was precipitated with one volume of isopropanol, collected by centrifugation, and washed with 70% ethanol and absolute ethanol, then dried and dissolved in double distilled water. Agarose gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) were used for DNA quality assessment and DNA concentration measurement, respectively. Finally, DNA was diluted to 100 ng/μL and used as a template for PCR amplification. The DNA fragments containing the gene-editing target were amplified using specifically designed primer sets. The primers used to detect mutations are listed in Table S1 (Supplementary Materials). After amplification, PCR products were recovered by PCR Clean Up Kit D2000 (Omega, Norcross, GA, USA). Mutation of each gene was detected by T7 endonuclease I (NEB, Ipswich, MA, USA) digestion assay and Sanger sequencing. The sequencing results were analyzed using the Bioedit sequencing alignment editor. The mutation rate was calculated by subtracting the uncut band intensity from the total band intensity and divided by the total band intensity of a single digestion experiment. The band intensity was quantified using Quantity One software (Bio Rad, Hercules, CA, USA). The hatching rate was obtained by dividing the number of newly hatched fry at 3 daf (days after fertilization) by the number of fertilized eggs injected.

2.5. Imaging of Body Color Mutants

Fry at 3, 4, 5, 9, and 10 daf were placed in culture dishes and photographed by a Leica M165FC stereomicroscope (Leica, Wetzlar, Germany). Fish at 90 and 360 daf were anesthetized by Tricaine (Sigma-Aldrich, Shanghai, China) at a concentration of 20 ppm and 40 ppm, respectively. Photographs of 90 and 360 daf fish were taken with a Canon SLR camera 700D (Canon, Tokyo, Japan). The fin and scale of 90 dpf fish were imaged with an Olympus BX51 light microscope (Olympus, Shinjuku, Tokyo, Japan).

2.6. Growth Measurement and Muscle Fiber Analysis

The fish, both mutants and WT, were sampled at 90 daf. The body weight, length, height, and width of mstnb mutants (n = 10) and wild type (WT) fish (n = 10) were measured using vernier calipers and analytical balance. Photographs of 90 daf fish were taken with a Canon SLR camera 700D (Canon, Japan). After removing the internal organs, the entire fish was fixed in Bouin’s solution at room temperature for 24 h, dehydrated, and embedded in paraffin. All samples were cross-sectioned at 5 μm at the anterior edge of the dorsal fin of the body, and the sections were H&E-stained for muscle fiber analysis. Photographs were taken under an Olympus BX51 light microscope (Olympus, Japan).

2.7. Alizarin Red S Staining for Intermuscular Bone Detection

The staining was performed as described previously [24]. Briefly, WT fish and runx2b and bmp6 mutants at 90 daf were anesthetized with MS-222 (Sigma-Aldrich, Shanghai, China) and fixed in a freshly prepared fixative solution (5% formalin, 5% Triton X-100, and 1% KOH) and rocked for 12 h. After incubation with the fixative, the specimens were stained with 0.05% alizarin red S and washed with the clearing solution (20% Tween 20 and 1% KOH). Finally, the samples were immersed in glycerol. Photographs were taken by a Leica M165FC stereomicroscope (Leica, Germany).

2.8. Gonadal Histological Analysis

The gonads of WT fish and cyp19a1a mutants at 90 daf were sampled and fixed in Bouin’s solution for 24 h at room temperature, dehydrated, and embedded in paraffin. All tissue blocks were sectioned at 5 μm and the sections were stained with H&E. Photographs were taken under an Olympus BX51 light microscope (Olympus, Japan).

2.9. Data Analyses

The body weight, length, height, and width data are presented as mean ± standard deviation (mean ± SD). Significant differences in the data between the two groups were tested by a two-tailed independent Student’s t-test. Statistical analyses were performed using GraphPad Prism (v8.0.2) software package (GraphPad Software, Boston, MA, USA), and p < 0.05 was considered to be significantly different.

3. Results

3.1. Induced Ovulation and Artificial Insemination of O. macrolepis

During the breeding season (May to July each year), males naturally produce sperm without hormone induction. HCG and DOM were injected intramuscularly into the female parents to induce ovulation, and the effect time of the hormone was 14–16 h after injection at a water temperature of 20 °C. The average induced ovulation rate was 72%. Approximately 5000~10,000 eggs, depending on the size of the fish, were obtained from each female. In total, about 400,000 were obtained from 50 mature females in the two successive years. Fertilized eggs were quickly bonded into lumps when exposed to water and could be dispersed in 10 min. When incubated at a water temperature of 20–23 °C, an obvious animal pole was observed on the fertilized egg under the stereomicroscope 30 min after fertilization. About 1.5 h after fertilization, the fertilized eggs entered the two-cell stage. The embryos hatched from the egg membranes within 50–60 h. The average fertilization rate and hatching rate were 82% and 84.6%, respectively.

3.2. Generation of Tyr, mpv17, csf1ra, Mstnb, mc4r, runx2b, bmp6, and cyp19a1a Mutants by CRISPR/Cas9

The guide RNA target for each gene was designed on exons (Figure 1A–D,M–P). Injected embryos were collected 48 h after fertilization for genomic DNA extraction. Fragment amplification across the target site was performed for each target site. Mutation detection by T7 endonuclease I digestion revealed that indels occurred at all target sites. This was evidenced by the cleaved bands on the agarose gel electrophoresis (Figure 1E–H,Q–T). Indel types were identified by Sanger sequencing (Figure 1I–L,U–X).

3.3. Optimization of the gRNA/Cas9 mRNA Concentration for Injection

Three different concentrations of tyr-gRNA/Cas9 mRNA were microinjected to determine the optimal dose of gRNA/Cas9 mRNA mixture for gene editing in O. macrolepis. As demonstrated by the agarose gel electrophoresis assay, cleaved bands were observed in all three combinations, indicating indel formation in all three conditions (Figure 2A). Mutation efficiency was further calculated by the band intensity. The statistical data on the hatching rate of embryos injected with different concentrations of tyr-gRNA/Cas9 mRNA are shown in Table S2 (Supplementary material). The lowest mutation efficiency (34%) was detected with a gRNA/Cas9 mRNA concentration of 50/100 ng/μL. When the concentration of gRNA/Cas9 mRNA was increased to 100/250 ng/μL, the mutation efficiency increased to 44.5%, and the embryo hatching rate was maintained at 80%. Injection of gRNA/Cas9 mRNA at 250/500 ng/μL resulted in the highest mutation rate (55.3%) but also reduced the hatching rate (54%) (Figure 2B). Therefore, 100 ng/μL of gRNA and 250 ng/μL of Cas9 mRNA were selected as the optimal injection concentrations. Obvious phenotypes in tyr mutants injected with this optimal concentration were observed. At 10 daf, a large number of melanophores appeared in the eyes, head, and trunk of the WT O. macrolepis (Figure 2C–G), while in the tyr mutants with high mutation rates, the melanophores in these areas were decreased or even absent (Figure 2H–L). These results indicated that gene editing in O. macrolepis was successful and efficient.

3.4. Phenotype Observation in Body Color Gene Mutants

Four main types of pigment cells—melanophores, xanthophores, iridophores, and erythrophores—were identified in adult O. macrolepis. The morphology and the first appearance time of pigment cells are shown in Figure S1 (Supplementary materials). The melanophores, xanthophores, iridophores, and erythrophores first appeared at 4, 5, 5, and 360 daf, respectively. At 90 daf, the tyr, mpv17, and csf1ra mutants exhibited different body colors compared to the WT fish. A decrease or even absence (albino) of melanophores was observed in the tyr mutant with a high mutation rate. Different from the WT fish, the albino displayed red eyes and a golden body color (Figure 3A,B). In the mpv17 mutants with a high mutation rate, some areas on the body were transparent, with most of the gills and black peritoneum visible because of iridophores loss, while the melanophores and xanthophores developed normally (Figure 3C). The csf1ra mutant with a high mutation rate displayed a grey body color due to the absence of xanthophores (Figure 3D). No melanophores and xanthophores were observed in the caudal fin of the tyr and csf1ra mutants, respectively (Figure 3F,H). Many iridophores were observed on the scales of the WT fish and the tyr and csf1ra mutants (Figure 3I,J,L), while no iridophores were observed on the scales of the mpv17 mutants (Figure 3K).

3.5. Improved Growth Performance by Mstnb Mutation

Growth performance was evaluated with randomly selected WT fish (n = 10) and mstnb mutants (n = 10) from the same parents, at the same age (90 daf), and raised under the same conditions. Body weight, height, and width were significantly higher in the mstnb mutants compared to the WT fish, but no significant differences in body length were observed between them (Figure 4A–D). Compared with the WT fish, individuals with high mutation rates had obviously increased body height and width (Figure 4E–H). On the cross-section of the fish body, the muscle area was larger in the mutant than WT fish (Figure 4I). Histological observation revealed that the increase in muscle area was caused by an increase in muscle fibers rather than an increase in muscle fiber size (Figure 4J,K). In contrast, no obvious phenotype was observed in the mc4r mutants.

3.6. Reduced Intermuscular Bones in the runx2b and bmp6 Mutants

Alizarin red S staining was used to detect intermuscular bones of the WT fish and runx2b, bmp6 mutants at 90 daf. Compared to the WT fish (Figure 5A,B), intermuscular bones were reduced or even absent in the runx2b (Figure 5C,D) and bmp6 (Figure 5E,F) mutants.

3.7. Ovary Degeneration in cyp19a1a Mutants

Gonadal histology analysis was performed for both WT fish and mutants at 90 daf. A large number of spermatogonia and oocytes existed in the testis and ovary of the WT O. macrolepis, respectively (Figure 6A,C). In the cyp19a1a mutants, the testis developed normally (Figure 6B), while the ovaries underwent oocyte degeneration (Figure 6D).

4. Discussion

After years of trial and effort, the technology for hormone-induced ovulation and artificial insemination in O. macrolepis has become increasingly mature. Stable ovulation rate, fertilization rate, and hatching rate were achieved, which allowed us to apply gene editing technology to O. macrolepis with reference to the existing gene editing protocol for Nile tilapia in our group [16].

4.1. Tyr Mutants with Golden Body Color

The tyr gene encodes a key rate-limiting enzyme in melanin biosynthesis. Loss of tyrosinase results in a lifelong lack of melanogenesis, giving rise to the typical albino phenotype in vertebrates [25,26,27,28,29]. In fish, the tyrb mutants display red eyes and white skin without melanin deposition, but other types of pigment cells are unaffected in medaka (Oryzias latipes) [30] and Nile tilapia [20,22]. The tyr F0 mutants also display varying degrees of chimeric albino, complete albino, or a golden-colored phenotype due to defective melanin synthesis in rainbow trout (Oncorhynchus mykiss) [31], large-scale loach (Paramisgurnus dabryanus) [32], yellow catfish (Tachysurus fulvidraco) [33], white crucian carp (Carassius auratus cuvieri) [34], and goldfish (Carassius auratus) [35]. In O. macrolepis, the tyr mutants with a high mutation rate also exhibited an albino phenotype with no melanophore pigmentation. The mutants displayed a golden-yellow body color as the xanthophores on the skin were not affected. These results suggest that the function of the tyr gene is highly conserved in vertebrates.

4.2. mpv17 Mutants with Transparent Body Color

The mpv17 encodes a mitochondrial inner membrane protein. In mammals, Mpv17 deletion results in mitochondrial metabolic syndrome [36]. In zebrafish (Danio rerio), mpv17 mutants induced by CRISPR/Cas9 display abnormal growth and survive failure during developmental stages [37], while natural mutants of this gene are viable and display a significant deletion of the iridophores and a small decrease in the melanophores [38,39]. Similarly, an almost complete loss of iridophores and a decrease in melanophores were observed in the mpv17 mutants in tilapia in our lab [21]. In the present study, the O. macrolepis mpv17 mutants also showed a loss of iridophores. However, as only F0 chimeras have been obtained so far, it is unclear whether the mpv17 homozygous mutation is crucial for the survival of O. macrolepis.

4.3. csf1ra Mutants with Gray Body Color

The csf1r gene encodes a macrophage surface receptor that receives signals from its ligand colony stimulating factor-1 (Csf1). Studies in mammals show that the CSF1/CSF1R signaling pathway regulates macrophage function and differentiation and promotes monocyte survival, migration, and proliferation. However, it is not associated with a neural crest or pigment cell development [40]. Two homologous genes, csf1ra and csf1rb, have been identified in teleosts due to the third round of genome duplication. Mutation of both genes completely suppresses microglia development in zebrafish [41,42]. Furthermore, csf1ra has been shown to be critical in the differentiation of xanthophores in zebrafish [42,43], guppy (Poecilia reticulata) [44], and Nile tilapia [22]. Mutation of this gene resulted in the absence of xanthophores and a decrease in melanophores in these fishes. In the present study, the absence of xanthophores was also observed in the csf1ra mutants with a high mutation rate in O. macrolepis.

4.4. Mstnb Mutants with Enhanced Muscle Mass

In addition to the body color genes, we also mutated genes associated with growth, intermuscular bone, and sex differentiation. Mutation of mstnb leading to activation of muscle growth has been reported in zebrafish [45,46], common carp [47], olive flounder (Paralichthys olivaceus) [4], blunt snout bream [5], loach (Misgurnus anguillicaudatus) [6], channel catfish [7], Nile tilapia [10], and Culter alburnus [11]. The mstnb mutants of most fish species show an increase in the number of muscle fibers leading to weight gain. Larger muscle fiber size has been observed in F1 heterozygous blunt snout bream [5]. In this study, the mstnb mutants of O. macrolepis showed a significant increase in body weight, height, and width compared to the WT fish due to the increase in the muscle fiber number, instead of muscle fiber size in the mutants. In contrast, no obvious phenotype was observed in the mc4r mutants in O. macrolepis, even though there are reports showing that mc4r knockout improves growth in channel catfish [12,48].

4.5. bmp6 and runx2b Mutants with Less or No Intermuscular Bones

In zebrafish, mutation of runx2b [49] and bmp6 [50] eliminated intermuscular bone without affecting growth and reproduction. Later, it was validated in aquaculture species such as blunt snout bream [15], crucian carp [14], and gibel carp (Carassius gibelio) [51]. In the present study, mutation of both runx2b and bmp6 resulted in a remarkable decrease or even absence of intermuscular bones in the mutants as revealed by alizarin red S staining.

4.6. cyp19a1a Mutants with Degenerated Ovaries

cyp19a1a, the gene encoding 17β-estradiol synthetase, is critical for the development of the ovary. Mutation of cyp19a1a leads to female-to-male sex reversal in zebrafish [52,53], Nile tilapia [54], and medaka [55]. In this study, we mutated the cyp19a1a to achieve sex control in O. macrolepis. Mutation of cyp19a1a resulted in degenerated ovaries, suggesting that cyp19a1a is also important for ovarian development in O. macrolepis. Whether these degenerated ovaries will be reversed to testes remains to be tested.

5. Conclusions

We have established artificial propagation in O. macrolepis for stock enhancement in the wild and aquaculture. The successful acquisition of one-cell stage embryos allowed us to establish CRISPR/Cas9 gene editing successfully in this species. Eight genes related to body color, growth, intermuscular bone, and sex differentiation were mutated in O. macrolepis. Phenotypes with golden, transparent, and gray body colors were observed in the tyr, mpv17, and csf1ra mutants, respectively. Improved growth performance was observed in the mstnb but not in the mc4r mutants. A decrease or even absence of intermuscular bones was observed in the runx2b and bmp6 mutants. Degenerated ovaries were observed in the cyp19a1a mutants. Only F0 founder mutants were obtained in this study. However, this type of gene editing will be commercially viable once these mutations are passed to the F2 homozygous mutants and used for larger-scale production of new germplasm with body color, fast growth, lacking intermuscular bones, and sex control.