Discovery of Tetrazolamide-benzimidazol-2-ones as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is one of the most valuable herbicide targets due to its unique biological functions. In search of HPPD inhibitors with promising biological performance, we designed and synthesized a series of novel tetrazolamide-benzimidazol-2-ones using a structure-based drug design strategy. Among the synthesized compounds, 1-(2-chlorobenzyl)-3-methyl-N-(1-methyl-1H-tetrazol-5-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-4-carboxamide, 25, IC50 = 10 nM, was identified to be the most outstanding HPPD inhibitor, which showed more than 36-fold increased Arabidopsis thaliana HPPD (AtHPPD) inhibition potency than mesotrione (IC50 = 363 nM). Our AtHPPD-25 complex indicated that one nitrogen atom on the tetrazole ring and the oxygen atom on the amide group formed a classical bidentate chelation interaction with the metal ion, the benzimidazol-2-one ring created a tight π-π stacking interaction with Phe381 and Phe424, and some hydrophobic interactions were also found between the ortho-Cl-benzyl group and surrounding residues. Compound 32 showed more than 80% inhibition against all four tested weeds at 150 g ai/ha by the postemergence application. Our results indicated that the tetrazolamide-benzimidazol-2-one scaffold may be a new lead structure for herbicide discovery.

A Chromosome-level Reference Genome of African Oil Palm Provides Insights into Its Divergence and Stress Adaptation.

The palm family (Arecaceae), consisting of ∼ 2600 species, is the third most economically important family of plants. The African oil palm (Elaeis guineensis) is one of the most important palms. However, the genome sequences of palms that are currently available are still limited and fragmented. Here, we report a high-quality chromosome-level reference genome of an oil palm, Dura, assembled by integrating long reads with ∼ 150נgenome coverage. The assembled genome was 1.7 Gb in size, covering 94.5% of the estimated genome, of which 91.6% were assigned into 16 pseudochromosomes and 73.7% were repetitive sequences. Relying on the conserved synteny with oil palm, the existing draft genome sequences of both date palm and coconut were further assembled into chromosomal level. Transposon burst, particularly long terminal repeat retrotransposons, following the last whole-genome duplication, likely explains the genome size variation across palms. Sequence analysis of the VIRESCENS gene in palms suggests that DNA variations in this gene are related to fruit colors. Recent duplications of highly tandemly repeated pathogenesis-related proteins from the same tandem arrays play an important role in defense responses to Ganoderma. Whole-genome resequencing of both ancestral African and introduced oil palms in Southeast Asia reveals that genes under putative selection are notably associated with stress responses, suggesting adaptation to stresses in the new habitat. The genomic resources and insights gained in this study could be exploited for accelerating genetic improvement and understanding the evolution of palms.

Long non-coding RNA ERVK13-1 aggravates osteosarcoma through the involvement of microRNA-873-5p/KLF5 axis.

OBJECTIVE: To explore the effect and mechanism of long noncoding RNA ERVK13-1 on osteosarcoma (OS) cell development by regulation of miR-873-5p/KLF5 axis. METHODS: The expression of ERVK13-1 in the collected tissue was detected by RT-qPCR, and then the relationship between ERVK13-1 expression and clinical characteristics of OS patients was analyzed. After OS cell lines were transfected with miR-873-5p inhibitor, si-ERVK13-1, si-KLF5 or their negative controls, the expression of ERVK13-1, miR-873-5p, and KLF5 in OS cell lines were measured, followed by determination of their effects on cell proliferation, migration, and invasion abilities. Moreover, the binding relationships of ERVK13-1 and miR-873-5p, as well as miR-873-5p and KLF5, were verified by the dual-luciferase reporter gene assay. RESULTS: Highly expressed ERVK13-1 was found in OS tissues, which was closely related to tumor size, tumor node metastasis, and distant metastasis. The overall survival of OS patients with high expression of ERVK13-1 was poorer than those with low expression of ERVK13-1. Elevated ERVK13-1 and KLF5 but suppressed miR-873-5p was observed in the OS cell lines U2OS and MG63. Transfection with miR-873-5p inhibitor enhanced the malignant potentials of OS cells, and transfection with si-ERVK13-1 or si-KLF5 reduced these abilities of OS cells. ERVK13-1 bound to miR-873-5p and KLF5 was a target gene of miR-873-5p. CONCLUSION: The ERVK13-1/miR-873-5p/KLF5 axis confers vital effect on the occurrence and progression of OS, thus providing possible guidance for the clinical treatment of OS.

Transposon-induced epigenetic silencing in the X chromosome as a novel form of dmrt1 expression regulation during sex determination in the fighting fish.

BACKGROUND: Fishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens. RESULTS: We conducted association mapping on four commercial and two wild populations of B. splendens. In three of the four commercial populations, the master sex determining (MSD) locus was found to be located in a region of ~ 80 kb on LG2 which harbours five protein coding genes, including dmrt1, a gene involved in male sex determination in different animal taxa. In these fish, dmrt1 shows a male-biased gonadal expression from undifferentiated stages to adult organs and the knockout of this gene resulted in ovarian development in XY genotypes. Genome sequencing of XX and YY genotypes identified a transposon, drbx1, inserted into the fourth intron of the X-linked dmrt1 allele. Methylation assays revealed that epigenetic changes induced by drbx1 spread out to the promoter region of dmrt1. In addition, drbx1 being inserted between two closely linked cis-regulatory elements reduced their enhancer activities. Thus, epigenetic changes, induced by drbx1, contribute to the reduced expression of the X-linked dmrt1 allele, leading to female development. This represents a previously undescribed solution in animals relying on dmrt1 function for sex determination. Differentiation between the X and Y chromosomes is limited to a small region of ~ 200 kb surrounding the MSD gene. Recombination suppression spread slightly out of the SD locus. However, this mechanism was not found in the fourth commercial stock we studied, or in the two wild populations analysed, suggesting that it originated recently during domestication. CONCLUSIONS: Taken together, our data provide novel insights into the role of epigenetic regulation of dmrt1 in sex determination and turnover of SD systems and suggest that fighting fish are a suitable model to study the initial stages of sex chromosome evolution.

The efficacy and safety of Xianling Gubao capsules in the treatment of knee osteoarthritis: A protocol for a randomized, double-blind, controlled trial.

BACKGROUND: Knee osteoarthritis (KOA) is a chronic degenerative joint disease, which is the most common type of osteoarthritis. The clinical manifestations are pain, swelling, and dysfunction of the knee joint, which seriously reduces the quality of life of patients and causes a huge social burden. At present, western medicine mainly focuses on symptomatic treatment, such as anti-inflammatory and pain relief, joint cavity injection, joint replacement, etc. The curative effect has certain limitations. Xianling Gubao capsule has some advantages in the treatment of KOA, but it lacks high-quality clinical research to verify it. Therefore, the purpose of this study is to evaluate the efficacy and safety of Xianling Gubao capsule in the treatment of KOA. METHODS: A randomized, double-blind, double-simulation, parallel controlled trial design was used to study the efficacy and safety of Xianling Gubao capsules in the treatment of KOA. The patients were randomly divided into a treatment group and the control group according to 1:1. The treatment group: Xianling Gubao capsule + glucosamine hydrochloride capsule simulation agent treatment; the control group: glucosamine hydrochloride capsule + Xianling Gubao capsule simulation agent treatment. Both groups received standard treatment for 8 weeks and followed up for 30 days. And at the same time, pay attention to its efficacy and safety indicators. Observation indicators include: the western Ontario and McMaster universities osteoarthritis index, hospital for special surgery knee score, liver and kidney function, adverse reactions, etc. Data analysis was performed using SPSS 25.0 software. DISCUSSION: This study will evaluate the efficacy and safety of Xianling Gubao capsule in the treatment of KOA. The results of this experiment will provide evidence support for Xianling Gubao capsule in the treatment of KOA. TRIAL REGISTRATION: OSF Registration number: DOI 10.17605/OSF.IO/ERM9C.

Comparative transcriptome analysis of the gills and hepatopancreas from Macrobrachium rosenbergii exposed to the heavy metal Cadmium (Cd2+).

Heavy metal Cadmium (Cd2+) pollution has become a severe environmental problem for aquatic organisms. In crustaceans, gills (Gi) and hepatopancreas (Hp) play a vital role in the toxicology. However, in Macrobrachium rosenbergill, there are few researches about gill and hepatopancreases responding to Cd2+ stress at a molecular level. In this study, transcriptomic analysis was applied to characterize gene expression profiles of gills and hepatopancreas of M. rosenbergill after Cd2+ exposure for 0 h, 3 h and 3 d. Six cDNA libraries (Gi 0 h, Gi 3 h, Gi 3 d, Hp 0 h, Hp 3 h, and Hp 3 d) were constructed and a total of 66,676 transcripts and 48,991 unigenes were annotated. Furthermore, differentially expressed genes (DEGs) were isolated by comparing the Cd2+ treated time-point libraries (3 h and 3 d group) with the control library (0 h group). The results showed that most of the DEGs were down-regulated after Cd2+ exposure and the number of DEGs among gill groups were significantly higher than those among hepatopancreas groups. GO functional and KEGG pathway analysis suggested many key DEGs in response to the Cd2+ stress, such as metallothionein and Hemocyanin. Additionally, a total of six DEGs were randomly selected to further identify their expressional profile by qPCR. The results indicated that these DEGs were involved in the response to Cd2+. This comparative transcriptome provides valuable molecular information on the mechanisms of responding to Cd2+ stress in M. rosenbergii, which lays the foundation for further understanding of heavy metal stress.

Transcriptional changes revealed water acidification leads to the immune response and ovary maturation delay in the Chinese mitten crab Eriocheir sinensis.

Nowadays, due to increasing carbon dioxide released, water acidification poses a series of serious impacts on aquatic organisms. To evaluate the effects of water acidification on crustaceans, we focused on the Chinese mitten crab Eriocheir sinensis, which is a spawning migration and farmed species in China. Based on histological and oocyte transparent liquid observation, we found that the acidified environment significantly delayed the ovarian maturation of E. sinensis. Moreover, RNA-seq was applied to obtain gene expression profile from the crab's gills and ovaries in response to acidified environment. Compared with control groups, a total of 5471 differentially expressed genes (DEGs) were identified in acidified gills and 485 DEGs were identified in acidified ovaries. Enrichment analysis indicated that some pathways also responded to the acidified environment, such as PI3K-Akt signaling pathway, Chemokine signaling pathway, apoptosis, and toll-like receptor signaling pathway. Subsequently, some DEGs involved in immune response (ALF, Cathepsin A, HSP70, HSP90, and catalase) and ovarian maturation (Cyclin B, Fem-1a, Fem-1b, and Fem-1c) were selected to further validate the influence of water acidification on gene expression by qRT-PCR. The results showed that the expression level of immune-related genes was significantly increased to response to the water acidification, while the ovarian maturation-related genes were significantly decreased. Overall, our data suggested that E. sinensis was sensitive to the reduced pH. This comparative transcriptome also provides valuable molecular information on the mechanisms of the crustaceans responding to acidified environment.

Genomic Basis of Striking Fin Shapes and Colors in the Fighting Fish.

Resolving the genomic basis underlying phenotypic variations is a question of great importance in evolutionary biology. However, understanding how genotypes determine the phenotypes is still challenging. Centuries of artificial selective breeding for beauty and aggression resulted in a plethora of colors, long-fin varieties, and hyper-aggressive behavior in the air-breathing Siamese fighting fish (Betta splendens), supplying an excellent system for studying the genomic basis of phenotypic variations. Combining whole-genome sequencing, quantitative trait loci mapping, genome-wide association studies, and genome editing, we investigated the genomic basis of huge morphological variation in fins and striking differences in coloration in the fighting fish. Results revealed that the double tail, elephant ear, albino, and fin spot mutants each were determined by single major-effect loci. The elephant ear phenotype was likely related to differential expression of a potassium ion channel gene, kcnh8. The albinotic phenotype was likely linked to a cis-regulatory element acting on the mitfa gene and the double-tail mutant was suggested to be caused by a deletion in a zic1/zic4 coenhancer. Our data highlight that major loci and cis-regulatory elements play important roles in bringing about phenotypic innovations and establish Bettas as new powerful model to study the genomic basis of evolved changes.

Genes, pathways and networks responding to drought stress in oil palm roots.

Oil palm is the most productive oilseed crop and its oil yield is seriously affected by frequent drought stress. However, little is known about the molecular responses of oil palm to drought stress. We studied the root transcriptomic responses of oil palm seedlings under 14-day drought stress. We identified 1293 differentially expressed genes (DEGs), involved in several molecular processes, including cell wall biogenesis and functions, phenylpropanoid biosynthesis and metabolisms, ion transport and homeostasis and cellular ketone metabolic process, as well as small molecule biosynthetic process. DEGs were significantly enriched into two categories: hormone regulation and metabolism, as well as ABC transporters. In addition, three protein-protein interaction networks: ion transport, reactive nitrogen species metabolic process and nitrate assimilation, were identified to be involved in drought stress responses. Finally, 96 differentially expressed transcription factors were detected to be associated with drought stress responses, which were classified into 28 families. These results provide not only novel insights into drought stress responses, but also valuable genomic resources to improve drought tolerance of oil palm by both genetic modification and selective breeding.

miR-129-3p alleviates chondrocyte apoptosis in knee joint fracture-induced osteoarthritis through CPEB1.

BACKGROUND: Osteoarthritis (OA), a refractory disease, is one of the leading contributors for disability worldwide. Since chondrocyte is the only resident cell in cartilage, this study aims to explore the roles of miR-129-3p and CPEB1 in chondrocyte apoptosis in knee joint fracture-induced OA. METHODS: Cartilage was collected from 20 OA patients who underwent total knee replacement (OA group) and 20 patients with knee contusion (normal group). Then, miR-129-3p and CPEB1 levels in the cartilage were quantified by qRT-PCR. Primary rat chondrocytes in the knee were isolated and identified by toluidine blue staining and immunofluorescent staining of type II collagen. OA cellular models were induced by TNF-α treatment, in which miR-129-3p and CPEB1 expressions were assessed. Subsequently, cell viability, apoptosis, and the expression levels of apoptotic protein and caspase-3 were measured. Dual luciferase reporter assay identified the interaction between miR-129-3p and CPEB1. RESULTS: Patients in the OA group had decreased miR-129-3p expression and increased CPEB1 expression than those in the normal group. TNF-α treatment successfully induced the OA cellular model. Downregulated miR-129-3p and upregulated CPEB1 expressions were found in OA-treated chondrocytes. miR-129-3p overexpression or CPEB1 knockdown improved chondrocyte viability and attenuated apoptosis, and vice versa. miR-129-3p negatively regulated CPEB1, thus ameliorating apoptosis and enhancing cell viability. CONCLUSION: miR-129-3p negatively targeted CPEB1 to facilitate chondrocyte viability and hamper apoptosis.

Molecular characterization of ovary-specific gene Mrfem-1 and siRNA-mediated regulation on targeting Mrfem-1 in the giant freshwater prawn, Macrobrachium rosenbergii.

Characterized by ankyrin repeat motifs, the feminization-1 (fem-1) gene plays an essential role in sex determination/differentiation in Caenorhabditis elegans. However, there are only a few reports on fem-1 in crustaceans. In this study, a fem-1 gene (Mrfem-1) was first isolated from the giant freshwater prawn Macrobrachium rosenbergii. The full-length cDNA of Mrfem-1 was 2607 bp long, containing an open reading frame encoding 615 amino acids, and presenting eight ankyrin repeats. The full-length cDNA has been submitted to GenBank with the accession no. MT160093. According to the RT-PCR results, Mrfem-1 was exclusively expressed in the ovary. The expression level of Mrfem-1 had increased with ovarian maturation and reached the highest peak at vitellogenic stage. In situ hybridization results showed that positive signals were concentrated in the cytoplasm of previtellogenic stage, and scattered in the cytoplasm and follicular cells at vitellogenic stage, suggesting that Mrfem-1 might be associated with ovarian maturation. Moreover, two effective siRNAs targeting Mrfem-1 were found and their effectiveness verified in vitro. These results on Mrfem-1 will help us to better understand the fem family and provide a new resource for subsequent investigations of siRNA-mediated regulation on ovarian development in M. rosenbergii.

Mapping and Validating QTL for Fatty Acid Compositions and Growth Traits in Asian Seabass.

Asian seabass is an important food fish species. While improving growth, increasing the nutritional value is important, omega-3 fatty acids are indispensable to human health. Identifying and validating DNA markers associated with traits is the first step towards marker-assisted selection (MAS). We quantified 13 different fatty acids and three growth traits in 213 F2 Asian seabass from a family at the age 270 days post hatch, and screened QTL for these traits. The content of total fatty acids in 100 g flesh was 2.57 ± 0.80 g, while the proportions of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were 16.96 ± 2.20% and 5.42 ± 0.90%, respectively. A linkage map with 2424 SNPs was constructed and used for QTL mapping. For fatty acid compositions, 14 significant QTL were identified on three linkage groups (LG5, LG11 and LG14), with phenotypic variance explained (PVE) from 12.8 to 24.6%. Thirty-nine suggestive QTL were detected on 16 LGs. Two significant QTL for EPA were identified on LG5 and LG14, with PVE of 15.2% and 15.1%, respectively. No significant QTL was identified for DHA. For growth traits, six significant and 13 suggestive QTL were identified on two and seven LGs, respectively. Only a few significant QTL for fatty acids overlapped with previously mapped QTL for these traits, suggesting that most QTL detected in a family are family-specific and could only be used in MAS in the family per se. To facilitate population-wide molecular breeding, more powerful methods (e.g. GWAS) should be used to identify SNPs for genomic selection.

Constructing High-Density Genetic Maps and Developing Sexing Markers in Northern Snakehead (Channa argus).

High-density genetic maps are essential for mapping QTL, improving genome assembly, comparative genomics, and studying sex chromosome evolution. The northern snakehead (Channa argus) is an economically important foodfish species with significant sexual dimorphism, where the males grow much faster and bigger than the females. However, to date, the sex determination pattern is still not clear, limiting identification of sex chromosomes, even sex determination genes and development of monosex populations that are valuable for both sex evolution of vertebrates and aquaculture practices. Here, a sex-averaged map and two sex-specific genetic maps were constructed with 2974, 2323, and 2338 SNPs, respectively. Little difference was observed in the pattern of sex-specific recombination between female- and male-specific genetic maps. Genome scan identified a major locus for sex determination at LG16. Females and males are, respectively, homogametic and heterogametic, suggesting an XY sex determination system for this species. By resequencing genomes, InDels in the sex-associated QTL region were discovered and used for developing sex-specific PCR assays for fast sexing of snakehead. These high-density genetic maps provide useful resources for future genomic studies in snakehead and its related species. The PCR assays for sexing are of importance in developing all male populations for aquaculture.

Transcriptome analysis of the brain of the Chinese mitten crab, Eriocheir sinensis, for neuropeptide abundance profiles during ovarian development.

Neuropeptides, important messenger molecules, regulate various physiological processes, such as growth, development, and reproduction. In the present study, cDNA libraries from brains of E. sinensis were constructed and sequenced using the Illumina technique for transcript analysis and neuropeptides discovery. There were 233,887 transcripts assembled for 194,286 unigenes. According to the annotations of NCBI non-redundant protein (NR) database, 2487 (11.31%) unigenes were annotated successfully. In total, 1273 transcripts were assigned to the "signal transduction mechanisms" category using KOG analysis. The results of KEGG indicate signal transduction and translation pathways were the dominant and enriched signal pathways. Additionally, results indicated C2H2 was the main transcription factor (TF) family. Analysis of the assembled transcripts indicated there were 22 neuropeptide transcripts, such as allatostatin, crustacean female sex hormone, crustacean hyperglycemic hormone, diuretic hormone 31, and eclosion hormone. The detection of these neuropeptides provide for a basic understanding for future study of functions in development, reproduction, and sexual maturation in crustaceans.

QTL Mapping for Resistance to Iridovirus in Asian Seabass Using Genotyping-by-Sequencing.

Identifying quantitative trait loci (QTL) for viral disease resistance is of particular importance in selective breeding programs of fish species. Genetic markers linked to QTL can be useful in marker-assisted selection (MAS) for elites resistant to specific pathogens. Here, we conducted a genome scan for QTL associated with Singapore grouper iridovirus (SGIV) resistance in an Asian seabass (Lates calcarifer) family, using a high-density linkage map generated with genotyping-by-sequencing. One genome-wide significant and three suggestive QTL were detected at LG21, LG6, LG13, and LG15, respectively. The phenotypic variation explained (PVE) by the four QTL ranged from 7.5 to 15.6%. The position of the most significant QTL at LG21 was located between 31.88 and 36.81 cM. The SNP marker (SNP130416) nearest to the peak of this QTL was significantly associated with SGIV resistance in an unrelated multifamily population. One candidate gene, MECOM, close to the peak of this QTL region, was predicted. Evidence of alternative splicing was observed for MECOM and one specific category of splicing variants was differentially expressed at 5 days post-SGIV infection. The QTL detected in this study are valuable resources and can be used in the selective breeding programs of Asian seabass with regard to resistance to SGIV.

Genome-Wide Association Study Identifies Loci Associated with Resistance to Viral Nervous Necrosis Disease in Asian Seabass.

Viral nervous necrosis disease (VNN), caused by nervous necrosis virus (NNV), is one major threat to mariculture. Identifying loci and understanding the mechanisms associated with resistance to VNN are important in selective breeding programs. We performed a genome-wide association study (GWAS) using genotyping-by-sequencing (GBS) to study the genomic architecture of resistance to NNV infection in Asian seabass. We genotyped 986 individuals from 43 families produced by 15 founders with 44498 bi-allelic genetic variants using GBS. The GWAS identified three genome-wide significant loci on chromosomes 16, 19, and 20, respectively, and six suggestive loci on chromosomes 1, 8, 14, 15, 21, and 24, respectively, associated with resistance to NNV infection measured as binary and quantitative traits. Using the 500 most significant markers in combination with a training population of 800 samples could reach a genomic prediction accuracy of 0.7. Candidate genes significantly associated with resistance to NNV, including lysine-specific demethylase 2A, beta-defensin 1, and cystatin-B, which play important roles in immune responses against virus infection, were identified. Almost all the candidate genes were differentially expressed in different tissues against NNV infection. The significant genetic variants can be used in genomic selection and help understand the mechanism of resistance to VNN. Future studies should use populations of large effective size and whole genome resequencing to identify more useful genetic variants.

Construction of high-resolution recombination maps in Asian seabass.

BACKGROUND: A high-density genetic map is essential for de novo genome assembly, fine mapping QTL for important complex traits, comparative genomic studies and understanding the mechanisms of genome evolution. Although a number of genomic resources are available in Asian seabass (Lates calcarifer), a high-density linkage map is still lacking. To facilitate QTL mapping for marker-assisted selection and genome assembly, and to understand the genome-wide recombination rates, we constructed high density linkage maps using three families and genotyping by sequencing. RESULTS: A high-density consensus linkage map consisting of 8, 274 markers was constructed based on sex-averaged genetic maps. The genetic maps were then aligned and integrated with the current genome assembly of Asian seabass. More than 90% of the genome contig sequences were anchored onto the consensus genetic map. Evidence of assembly errors in the current genome assembly was identified. A fragment of up to 2.5 Mb belonging to LG14 was assembled into Chr15. The length of family-specific sex-averaged maps ranged from 1348.96 to 1624.65 cM. Female maps were slightly longer than male maps using common markers. Female-to-male ratios were highly variable both across chromosomes within each family and throughout three families for each chromosome. However, the distribution patterns of recombination along chromosomes were similar between sexes across the whole genome. The overall recombination rates were significantly correlated with genome-wide GC content and the correlations were revealed to be stronger in females than in males. CONCLUSIONS: These high-density genetic maps provide not only essential tools for facilitating de novo genome assembly and comparative genomic studies in teleosts, but also critical resources for fine mapping QTL and genome-wide association mapping for economically important traits in Asian seabass.

Charactering the ZFAND3 gene mapped in the sex-determining locus in hybrid tilapia (Oreochromis spp.).

Zinc finger AN1-type domain 3 (ZFAND3) is essential for spermatogenesis in mice. However, its function in teleosts remains unclear. In this study, we characterized the ZFAND3 gene (termed as OsZFAND3) in an important food fish, tilapia. The OsZFAND3 cDNA sequence is 1,050 bp in length, containing an ORF of 615 bp, which encodes a putative peptide of 204 amino acid residues. Quantitative real-time PCR revealed that the OsZFAND3 transcripts were exclusively expressed in the testis and ovary. In situ hybridization showed that the high expression of OsZFAND3 transcripts was predominantly localized in the spermatocyte and spermatid. These results suggest that OsZFAND3 is involved in male germ cell maturation. Three single nucleotide polymorphisms (SNPs) were detected in the introns of OsZFAND3. The OsZFAND3 gene was mapped in the sex-determining locus on linkage group 1 (LG1). The three SNPs in the OsZFAND3 gene were strictly associated with sex phenotype, suggesting that the OsZFAND3 gene is tightly linked to the sex-determining locus. Our study provides new insights into the functions of the OsZFAND3 gene in tilapia and a foundation for further detailed analysis of the OsZFAND3 gene in sex determination and differentiation.