Cloning, Expression Analysis and SNP Screening of the kiss1 Gene in Male Schizothorax biddulphi

Schizothorax biddulphi is an endemic fish distributed only in southern Xinjiang, China. Due to overfishing, water conservancy facilities, and other factors, as well as inherent biological limitations, resource recovery is quite difficult. For endangered fish with slow growth, late sexual maturity, and insufficient natural population supplementation, large-scale artificial reproduction and breeding are important for restoring resources. Therefore, it is urgent to optimize the reproductive regulation methods of the fish. The kiss1 gene is a key regulator of the reproductive regulation cascade, and identifying and analyzing the role of kiss1 are important for further elucidating the reproductive mechanism of S. biddulphi. To understand the characteristics of the kiss1 of S. biddulphi, the full-length cDNA sequence of kiss1 was obtained in this study, and its tissue expression specificity and association with phenotypic traits were analyzed in male fish. The full-length cDNA sequence of kiss1 in S. biddulphi was 658 bp, with an ORF of 327 bp, and encoded a 108-amino acid, unstable protein. Homology results indicated that kiss1 was highly conserved. qPCR showed kiss1 expression in different tissues in male S. biddulphi, with the highest expression in the gonads, followed by muscle, and significantly lower expression in the swim bladder, pituitary gland, heart, hypothalamus, gill, fin, liver, eye, and mid-kidney. qPCR revealed three SNP loci in the exonic region of kiss1. The c.3G>T locus was significantly correlated (p < 0.05) with gonad mass and the maturation coefficient in S. biddulphi. These results will help uncover the reproductive endocrinology network of S. biddulphi, improve artificial breeding technology for fish, and unveil new directions for breeding excellent strains of S. biddulphi and molecular marker-assisted breeding.


Introduction
The Tarim schizothoracin (Schizothorax biddulphi) in order Cypriniformes, family Cyprinidae, subfamily Schizothoracinae, and genus Schizothorax [1], is a special fish in Xinjiang. It is one of the most representative fish in the Tarim River basin and was once among the most important economic fish in Bosten Lake [2]. In the 1970s, S. biddulphi experienced a sharp decline in germplasm resources and numbers due to its biological features, such as slow growth, late sexual maturity, low fertility, and strict requirements for the spawning environment and living conditions, as well as constraints imposed by anthropogenic and natural conditions, resulting in severe deterioration of its habitat [3]. Its distribution is now extremely narrow, with individuals inhabiting only the Tarim River system and in very small numbers. In 1998, this species was included in the Red Book Single nucleotide polymorphisms (SNPs) are ideal genetic markers with promising applications in genetic diversity analysis, association analysis, variety identification, construction of high-density genetic linkage maps, and assisted breeding [33][34][35][36]. Therefore, in this study, we performed cloning, sequence alignment, and tissue differential expression analysis of the S. biddulphi kiss1 gene and conducted the first case of molecular marker screening for kiss1 gene polymorphism and reproductive traits in S. biddulphi, thereby providing technical support and a theoretical basis for molecular marker-assisted breeding in S. biddulphi.

Test Animals and Sample Collection
S. biddulphi were collected from August 2020 to August 2021 in the Kizil River with a geo-cage net (net length 10 m, 33 sections and 20 holes, frame 2 cm × 3 cm, mesh 4 mm). A total of 128 fish were caught, including 75 males and 53 females, with body lengths ranging from 95.05 to 328.52 mm and body masses from 8.02 to 415.02 g. Before the experiment, these fish were placed in a 2 m × 1 m tank for two weeks, and the experiment was carried out using an HZ-060 aerator for oxygenation. Floating compound feed (Sichuan Sterga Feed Ltd., Chengdu, China) was provided three times a day (9:30, 13:30, and 19:30). Fish were fed the diet at 3.0% to 5.0% of their total weight. Due to the small number of females, only a total of 62 males that developed to stage IV or V were selected (developmental stage I-III males were immature, and gonadosomatic indexes could not be calculated). The body lengths of males used in this study were 164.78 ± 32.52 mm, and the total weight was 63.85 ± 44.15 g. Such males were identified based on milky white spermatozoa and a small amount of semen flowing from the abdomen when lightly pressed. Morphological data were collected, and the fish were anesthetized using MS-222 and dissected to take the heart, liver, mid-kidney, muscle, gonads, eye, hypothalamus, pituitary gland, gill, fin, and swim bladder tissues, while the male gonads were weighed to calculate their maturation coefficients. The samples were immediately placed in liquid nitrogen and subsequently stored in a −80 • C freezer. A portion of the gonadal mixture was immersed in centrifuge tubes with anhydrous ethanol and stored in a −20 • C freezer. All animal experiments conformed to the standards of the Committee of Laboratory Animal Experimentation at the China Academy of Agriculture Sciences, China.

Cloning and Sequence Analysis of the kiss1 Gene
Total RNA was extracted from the gonadal tissue of S. biddulphi using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol, and contaminating genomic DNA was removed by treatment with DNaseI (TaKaRa, Shanghai, China). The quality of RNA was examined by spectrophotometry and agarose gel electrophoresis, and the first strand of cDNA was synthesized from the total RNA of mixed gonadal samples according to the manufacturer's 5 /3 RACE protocol and stored at −20 • C. Primers were designed using Primer Premier 6.0 based on a comparison of the coding regions of the kiss1 genes of C. auratus (NC_039278.1), D. rerio (NC_007122.7), and O. latipes (NC_019863.2) in the NCBI database. The primer names and sequences are shown in Table 1.
Specifically, the first round of PCR for 5 RACE was performed using the primers rkiss1-R1 and rkiss1-R2. The amplification procedure was as follows: 94 • C predenaturation for 2 min, followed by 38 cycles of 94 • C denaturation for 30 s, 70 • C for 30 s, and 66 • C for 30 s. The products of the first round of PCR were diluted 10-fold, and the second round of PCR was performed with conditions of 94 • C predenaturation for 2 min, 94 • C for 30 s, and 66 • C for 30 s. 3 RACE was performed using the primers rkiss1-F1 and rkiss1-F2 with the same reaction conditions and amplification procedure. PCR products were detected with 1.2% agarose gel electrophoresis and determined to have specific amplification. After specific amplification, cut-gel recovery and T vector splicing was performed, and the ligated products were cloned into the vector and sequenced separately. All sequencing was performed on an ABI377 DNA sequencer (Perkin-Elmer, Norwalk, CT, USA). The intermediate fragment and 5 RACE and 3 RACE ends of the obtained kiss1 gene were spliced using SeqMan to obtain the full-length cDNA of kiss1. The open reading frame (ORF) was queried for the kiss1 gene using DNAStar's EditSeq program, and the amino acid sequence was translated; the physicochemical properties of the protein and the secondary structure of the protein were analyzed using ExPASy (http://www.expasy.org, accessed on 28 February 2022), NetSurfP 2.0 (https://services.healthtech.dtu.dk/service. php?NetSurfP2.0, accessed on 28 February 2022) and SOPMA (http://metadatabase.org/ wiki/SOPMA, accessed on 28 February 2022), and the tertiary structure of the protein was predicted using Phyre2. SignalP5.0 Server (http://www.cbs.dtu.dk/services/SignalP/, accessed on 28 February 2022) was used for signal peptide analysis, NetPhos3.1 (http: //www.cbs.dtu.dk/s.ervices/NetPhos, accessed on 28 February 2022)for protein phosphorylation site prediction, TMHMM for transmembrane structural domain prediction, the Cell-PLoc 2.0 package for kiss1 subcellular localization, NCBI Blast for homology matching analysis, DNAMAN (version v 10.0.2.128; Lynnon Biosoft, Vaudreuil, QC, Canada) for multiple amino acid sequence comparison, and MEGA 7.0 (https://www.megasoftware.net/, accessed on 28 February 2022) for phylogenetic tree construction. Sequences were aligned using Muscle, and neighbor-joining trees were constructed using the p-distance model with 500 bootstrap replicates. The accession number and protein sequence of other fish in the phylogenetic tree analysis are provided in Table S1.

Detection of kiss1 Expression in Different Tissues of S. biddulphi
Total RNA was extracted from the heart, liver, mid-kidney, muscle, gonad, eye, hypothalamus, pituitary gland, gill, fin, and swim bladder tissues of S. biddulphi (n = 3) using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the protocol of the US manufacturer. The amount of cDNA used in the qPCR system for different tissues was adjusted using β-actin as the internal reference gene. qPCR was performed according to the qPCR primers qkiss1-F and qkiss1-R, which were designed based on the kiss1 gene sequence (Table 1). qPCR was performed using a 15 µL system: 2 × SG Green qPCR Mix 7.5 µL, 0.25 µL of each upstream and downstream primer (10 µM), 1 µL of cDNA, and 6 µL of ddH2O. The PCR parameters included initial denaturation at 95 • C for 10 min, denaturation at 95 • C for 20 s, and annealing and extension at 60 • C for 30 s for 40 cycles. After PCR was completed, the melting curve was analyzed to determine the amplification of individual products. Three parallel PCRs were performed for each tissue sample, and the results obtained were analyzed using the comparative CT method (2 −∆∆Ct ) to obtain the relative expression level of each tissue. Origin 2021 software was used to create graphs.

SNP Locus Screening of the kiss1 Gene
Total RNA was extracted from each sample (n = 62) and reverse transcribed to cDNA as described above. Using this cDNA as a template, PCR amplification was performed using the kiss1-F and kiss1-R primers (Table 1). Primer design was performed on the NCBI website (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC= BlastHome, accessed on 28 February 2022) based on the conserved sequence on either side of the site. The main parameters are as follows: the length of primer was generally 18~24 bp; Tm 55 • C~65 • C; (G+C) content 40%~70%; production size was less than 800 bp. The products were purified by gel cutting and ligation transformation, followed by direct sequencing. The specific steps were performed as described above. Sequencing results were analyzed with sequence alignment using DNAMAN (version v 10.0.2.128; Lynnon BioSoft, Vaudreuil, QC, Canada) to find SNP sites.

Measurement of Reproductive Traits and Evaluation of its Association with SNP Loci
The gonadosomatic index (GSI) was calculated as follows: maturation coefficient = (gonadal weight/total weight) × 100%. The test data were expressed as the mean ± standard deviation (mean ± S.D.). The genotype frequency and allele frequency at each SNP locus of the kiss1 gene were calculated using Microsoft Excel, and the Hardy-Weinberg equilibrium (HWE) test was performed using the χ 2 test. ANOVA was performed in SPSS 26.0 to calculate the associations between different genotypes of the kiss1 gene and reproductive traits, and the results were expressed as the mean ± standard deviation. Duncan's new complex polarization method (Duncan's) was used to determine the significance of differences between the means of the groups, with significant differences indicated by p < 0.05.

Sequence Analysis of the kiss1 Gene
The kiss1 gene was 658 bp long, with a full-length ORF of 327 bp, and encoded 108 amino acids. The 5 UTR was 62 bp long, the 3 UTR was 269 bp long, the start codon was ATG, and the stop codon was TGA. Underlined was Kisspeptin-10 (YNLNSFGLRY) (Figure 1). There are 14 species that have greater than 80% amino acid sequence similarity with S. biddulphi (Table 2), which indicates that the protein encoded by the kiss1 gene cloned from S. biddulphi was highly conserved with that of other species. There are 14 species that have greater than 80% amino acid sequence similarity with S. biddulphi (Table 2), which indicates that the protein encoded by the kiss1 gene cloned from S. biddulphi was highly conserved with that of other species. The results of the evolutionary tree analysis of kiss1 proteins ( Figure 2) showed that the kiss1 protein of S. biddulphi was most closely related to that of S. richardsonii.

Characterization of kiss1-Encoded Proteins
The theoretical isoelectric point of the protein encoded by the kiss1 gene is 9.34, and the molecular weight of the kiss1 protein is 12.45 kDa. The 108 encoded amino acids comprise 19 unique amino acids, the most common of which is leucine (12.0%) and the least common of which is histidine (0.9%); there are 13 positively charged amino acids (Lys and Arg) (12.0%) and 10 negatively charged amino acids (Asp and Glu) (9.3%). The total number of protein atoms is 1733, and the instability index is 60.41. The protein is unstable. The secondary structure of the kiss1 protein mainly contained two secondary components, the extended strand and random coil ( Figure 3A), with the α-helix accounting for 23.15%, the extended strand accounting for 16.67%, and the random coil accounting for 60.19%. The similarity of the tertiary structure with this template was 45% ( Figure 3B). The shear site of the signal peptide was located between amino acids 15 and 16 ( Figure 3C). There were 10 serine phosphorylation sites located at sites 11, 20, 36, 51, 53, 57, 61, 74, 78, and 93; there were 8 threonine phosphorylation sites located at sites 5, 17, 31, 35, 43, 45, 48, and 80; and there were 7 tyrosine phosphorylation sites, located at sites 18, 25, 26, 79, 88, 89, and 98 ( Figure 3D). The kiss1 protein is not a transmembrane protein (Figure 3E), and its subcellular localization is mainly outside the cell; presumably, its function is performed mainly outside the cell.  Table 2.

Characterization of kiss1-Encoded Proteins
The theoretical isoelectric point of the protein encoded by the kiss1 gene is 9.34, and the molecular weight of the kiss1 protein is 12.45 kDa. The 108 encoded amino acids comprise 19 unique amino acids, the most common of which is leucine (12.0%) and the least  Table 2.

Tissue Expression Profile of the kiss1 Gene
The expression of the kiss1 gene in different tissues of male S. biddulphi was detected by fluorescence quantitative PCR, and the results showed that the kiss1 gene was

Tissue Expression Profile of the kiss1 Gene
The expression of the kiss1 gene in different tissues of male S. biddulphi was detected by fluorescence quantitative PCR, and the results showed that the kiss1 gene was expressed in 11 tissues. kiss1 gene expression was highest in the gonads of male S. biddulphi, followed by the muscle, and then was ordered from high to low as follows: swim bladder, pituitary gland, heart, hypothalamus, gill, fin, liver, eye, and mid-kidney (Figure 4).

Polymorphic SNP Loci of the kiss1 Gene
Three SNP loci were detected in the exonic region of the kiss1 gene, and their base changes and amino acid changes, mutation types, and genetic structure information are shown in Table 2. SNP c.3G>T and SNP c.198T>G both have GG and TT genotypes, of which TT is the dominant genotype, with two alleles, G and T, of which T is the dominant allele. SNP c.12T>C has two genotypes, CC and TT, where C is the dominant genotype. The mutation types at these three loci were all missense mutations, and all deviated from Hardy-Weinberg equilibrium (p < 0.05, Table 3).
Genes 2023, 14, x FOR PEER REVIEW 10 of 16 expressed in 11 tissues. kiss1 gene expression was highest in the gonads of male S. biddulphi, followed by the muscle, and then was ordered from high to low as follows: swim bladder, pituitary gland, heart, hypothalamus, gill, fin, liver, eye, and mid-kidney ( Figure  4).

Polymorphic SNP Loci of the kiss1 Gene
Three SNP loci were detected in the exonic region of the kiss1 gene, and their base changes and amino acid changes, mutation types, and genetic structure information are shown in Table 2. SNP c.3G>T and SNP c.198T>G both have GG and TT genotypes, of which TT is the dominant genotype, with two alleles, G and T, of which T is the dominant allele. SNP c.12T>C has two genotypes, CC and TT, where C is the dominant genotype. The mutation types at these three loci were all missense mutations, and all deviated from Hardy-Weinberg equilibrium (p < 0.05, Table 3). The gonad weight and gonadosomatic index of individuals with the TT genotype were significantly higher than those of individuals with the GG genotype at SNP c.3G>T  The gonad weight and gonadosomatic index of individuals with the TT genotype were significantly higher than those of individuals with the GG genotype at SNP c.3G>T (p < 0.05). The gonad weight and gonadosomatic index of individuals with SNP c.12T>C and SNP c.198T>G were not significantly different (p > 0.05, Table 4). 0.72 ± 0.32 a 1.69 ± 0.03 a In the same SNPs with same small letter superscripts mean no significant differences (p > 0.05); different small letter superscripts mean significant differences (p < 0.05).

Discussion
The developmental process in animals is extremely complex and precise, and the central nervous system and the endocrine system work together to jointly regulate the developmental process, with the kisspeptin system playing a key role in reproductive development and energy metabolism in fish [28,37]. In this study, the 658-bp cDNA sequence of kiss1 was cloned from S. biddulphi for the first time, which encoded a 108-amino acid protein. The C-terminal core decapeptide of this sequence is YNLNSFGLRY (Y-Y pattern), the shortest mature sequence that stimulates phosphatidylinositol conversion at the amino acid level [38], which is highly consistent with the C-terminal decapeptide sequences of D. rerio [39], O. latipes [40] and other carps, showing that the core sequence of the kiss1 gene, kisspeptin-10, is highly conserved in carps. In contrast, in mammals and amphibians, the corresponding amino acid at position 3 of this core sequence is W [41,42]. In fish, it corresponds to L, immediately following the core decapeptide with GLR as the splitting site and amidation site of this sequence. This short peptide is equally highly conserved in mammals and fish [38,39]. kiss1 and kiss2 encode different functional proteins, and in the kiss2 protein, the C-terminal core decapeptide YNLNSFGLRY of the kiss1 protein corresponds to FNYNPFGLRF, where the same motif of -N-N-FGLR-is present [32,43]. The kiss1 and kiss2 genes are RY-and RF-amide-type compounds, respectively. Generally, only kiss1 is present in mammals, and some scholars speculate that it may be due to the loss of the kiss2 gene during evolution. Fish generally contain two kiss genes, kiss1 and kiss2. Anguilla anguilla is the only teleost species having two kiss genes (kiss1 and kiss2) and three kissr types (kissr1, kissr2, and kissr3) reported to date, indicating that the evolutionary process varies with species [13]. The secondary structure of the kiss1 gene of S. biddulphi mainly includes an extended strand and irregular curl, while the irregular curl structure often dominates changes in enzyme activity and functional site composition [44], which is presumed to have an important role in the formation of specific functional sites, and its inclusion of two plus-tail signals (AATAAA) suggests selective shearing during transcription [45]. There were 14 fish species with an amino acid sequence similarity more significant than 80%, and the homology results showed that the kiss1 amino acid sequence of S. biddulphi had the highest similarity of 98% with that of the congener S. richardsonii. In the phylogenetic tree, they were clustered onto one branch, indicating the closest genetic relationship.
Exploring the expression of a gene in different tissues helps analyze the mechanism of gene regulation, and the gene expression pattern indicates, to some extent, the functional diversity among tissues [23]. It has been confirmed that the kisspeptin system mediates the reproductive endocrine process in animals. In this study, the kiss1 gene had the highest expression in the gonads, followed by the muscle, swim bladder, pituitary gland, heart, hypothalamus, and gill, and low expression in the fin, liver, eye, and mid-kidney, similar to the kiss1 gene tissue expression results of Acipenser schrenckii [30]. In other teleosts, there were differences in the pattern of kiss1 gene expression, with D. rerio's kiss1 being most expressed in the brain and pituitary gland and the intestine, pancreas, kidney, and adipose tissue [39]. kiss1 of S. japonicus was expressed in the brain, gonads, gills, heart, and spleen, but kiss1 expression was not present in the pituitary gland, and the pattern of expression varied by reproductive cycle [28]. In T. putitora, the expression of kiss1 was inconsistent between ovarian development and spermatogenesis [46], and the expression of kiss1 increased as the organism developed. These results indicate that the expression of kiss1 varies among species, gender, and reproductive cycles. The high expression in the gonads of S. biddulphi is consistent with findings for O. latipes [40], C. auratus [47], and S. japonicus [28], confirming the involvement of kiss1 in the reproductive function of fish. In some species, Oryzias latipes, zebrafish, Dicentrarchus labrax, and Scomber japonicus, it has been demonstrated that kiss1 is regularly expressed at different developmental stages in the male brain [23,40,48,49]. In the brain of Scomber japonicus males, kiss1 mRNA expression level gradually decreased from the immature stage to the spermiation stage, followed by a significant decline during the post-spawning stage [23]. The kiss1 gene in the brain of S. biddulphi may follow a similar expression pattern during the gonadal development stage. In the present study, the kiss1 gene expression levels in the pituitary and hypothalamus were not very high, probably because gonads were developed to stage IV or V. They may not be at the peak of expression in the temporal expression pattern.
In addition to the gonads, kiss1 is also highly expressed in muscle tissues. It has been confirmed that muscle tissue is one of the main regions for energy accumulation and metabolism, and the kiss1 gene has an important role in maintaining energy balance [24]. Kisspeptin can act as an anorexigenic factor, but its effect on energy balance is not mediated by food intake but by regulating energy digestion, which is related to the number of kisspeptin ARC neurons and can have an effect on body weight changes in mice [21,50]. The results of a large number of metabolic stress models show that reproductive regulation is inhibited by the metabolic stress response, and leptin and NPY factors, which are closely related to energy metabolism, can inhibit the expression of the kiss gene, which in turn affects the reproduction of the organism [51]. However, the specific controlling mechanisms still need to be investigated. The pituitary gland, as an indispensable component of the HPG axis, mutually regulates its function with the hypothalamus and gonads. The hypothalamus releases gonadotropin-releasing hormone (GnRH) that binds to receptors that promote the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland, forming a neuroendocrine network that ultimately affects the reproductive function of fish and this series of cascade reactions cannot be separated from either organ. The expression of kiss1 in the pituitary gland and hypothalamus is also consistent with their respective functions [52]. Similar results were obtained for O. latipes [40], C. auratus [47], L. rohita [53], D. labrax [21], etc. Interestingly, kiss1 gene expression was also detected in the fins of fish, which play an important role in maintaining balance and assisting forward movement during swimming, suggesting that kiss1 has some function other than regulating reproduction.
Reproductive traits are important indicators for assessing the reproductive potential of individuals. The important role of kiss1 in animal reproduction can make it a candidate gene for fertility, and by identifying its SNP loci and analyzing the association between genotype and phenotype, effective molecular markers for the genetic improvement of animal reproductive traits can be obtained [54]. The SNP g.1311578G>T locus found in Ovis aries plays a key role in lambing and can be screened for the TT dominant genotype to improve reproductive and production potential [55]. Maitra et al. (2014) found three SNP loci (G296C, G2510A, and C2540T) related to the litter size of Indian C. hircus in intron 1 of the kiss1 gene [56]. An et al. (2013) detected two SNP loci (G2124T>A and G2270C>T) significantly associated with the lambing number of C. hircus in intron 1 of the kiss1 gene in three different sheep populations [57]. Similar mutations at the G227C and T486A loci of the kiss1 gene were reported to be associated with lambing numbers in Northern Guizhou C. hircus [58].
In this study, for the first time, SNP loci in exons of the kiss1 gene were correlated with reproductive traits in male S. biddulphi, and the results showed that the gonad weight and maturation coefficients of individual S. biddulphi males with the TT genotype were significantly higher than those of individuals with the GG genotype at SNP c.3G>T. Mutation of single nucleotides at the SNP c.3G>T locus resulted in amino acid types that significantly affected gonad mass and maturation coefficients of S. biddulphi males. The exonic region belongs to the coding region of the gene, and this single base substitution leads to an amino acid change; this nonsynonymous mutation may affect the stability of the protein and function of the gene, which may be responsible for the differences in reproductive traits among individuals; therefore, it is hypothesized that the dominant allele T improves the reproductive ability of S. biddulphi to some extent. The results also indicated that the pure TT genotype at the SNP c.3G>T locus in S. biddulphi yielded better reproductive traits and could be used to select parents for breeding progeny with obvious reproductive advantages. Since reproductive traits are influenced not only by genes but also by the environment, it is not clear whether the kiss1 gene is the main-effect gene affecting S. biddulphi or whether there is perhaps a linkage with other main-effect genes controlling reproductive traits, and the mechanism needs to be further explored.
Although the size of the studied population was relatively small, the present results have important and practical implications for the recovery and continuation of resources of S. biddulphi and the breeding of superior breeds, which may lead to significant improvement in reproductive characteristics. However, differences in gonad weight and maturation coefficients among genotypes need to be confirmed based on a larger amount of additional data to confirm their significant effects. The next step could be the identification and screening of associated SNP loci for highly reproductive females, which could be applied in actual reproduction practices in combination with the present results.

Conclusions
In conclusion, the kiss1 gene of S. biddulphi is homologous to that in other fishes, and the widespread expression of kiss1 in different tissues implies that it may be involved in functions other than reproduction. However, in-depth research is still needed regarding its expression pattern and involvement in other functions during gonad development. In this study, we searched for mutated loci of kiss1 in S. biddulphi and found that the SNP c.3G>T locus was significantly associated with both gonad mass and the maturation coefficient in S. biddulphi, and individuals with the genotype TT had significantly larger reproductive traits than those with other genotypes. The identified SNPs could be used as markers for higher fertility and, thus, to improve the reproductive performance of this population.