Preliminary Study On Molecular Mechanism Of Sex Determination And Differentiation In Loach

The dojo loach Misgurnus anguillicaudatus is an endemic freshwater species in Asia that belongs to the Cobitidae family.Due to its nutritional value and broad environmental adaptability,it has become an important economic species in Asia.Females grow significantly faster than males,but sex determination and differentiation were affected by temperature,which will affect economic benefits.However,the molecular mechanism of sex determination and differentiation is still unclear.In the present study,M.anguillicaudatus was selected as the research object.Firstly,we conducted a genome survey of M.anguillicaudatus using next-generation sequencing technology,and we tried to obtain sex-related molecular markers by comparative genome technology.Meanwhile,we developed microsatellite and SNP markers based on the information of genome survey sequencing.Secondly,the transcriptome profiles were comparatively analyzed between male and female gonads to obtain differentially expressed genes.Thirdly,effects of temperature-genetic interactions on the sex determination and differentiation in loach were investigated,and the expression of sexrelated genes at the early developmental stage in different temperatures were analyzed;Fourthly,we cloned sex-related gene Rspo1 and analyzed its expression pattern in different tissues,the effect of temperature on the methylation levels and expression of Rspo1 in gonads were studied.Main results are shown as follows:Its genome size was estimated to be approximately 1105.97 Mb by using K-mer analysis,and its heterozygosity ratio,repeat sequence content,and GC content were1.45%,58.98%,and 38.03%,respectively.A total of 376,357 microsatellite motifs were identified.Mononucleotides,with a frequency of 42.57%,were the most frequently repeated motifs,followed by 40.83% dinucleotide,7.49% trinucleotide,8.09%tetranucleotide,and 0.91% pentanucleotide motifs.The AC/GT,AAT/ATT,and ACAG/CTGT repeats were the most abundant motifs among dinucleotide,trinucleotide,and tetranucleotide motifs,respectively.Twelve male-specific tags were obtained by comparing the male and female genomes,and primers were designed to verify the sex of individuals.The results did not achieve the purpose of identifying the sex of individuals.We identified 20 polymorphic microsatellite markers in the 30 wild population.The number of alleles,observed and expected heterozygosity per locus ranged from 7to 19,0.400 to 0.933,and 0.752 to 0.938,respectively.All twenty loci were highly informative(PIC > 0.700).Eight loci deviated from Hardy–Weinberg equilibrium after Bonferroni correction.Meanwhilie,112 novel single nucleotide polymorphisms(SNPs)were screened based on 2b-RAD sequencing database,and 57 SNP markers were developed and characterized by genotyping 40 individuals using SNa Pshot method.The observed heterozygosity(Ho)ranged from 0.025 to 0.675,while the expected heterozygosity(He)varied from 0.025 to 0.500.The minor allele frequency(MAF)ranged from 0.013 to 0.500.Among these SNPs,18 loci were found to deviate significantly from the Hardy–Weinberg equilibrium after Bonferroni correction.A total of 55143 differential expressed genes(DEGs)were identified between male and female gonads,including 44051 DEGs upregulated in testis and 11092 DEGs upregulated in ovary.52 differential expressed genes were obtained and annotated for follow-up research.Compared with low temperature,high temperature(31℃)treatment produced more males.Sex ratio response to elevated temperature is family-specific.Histological sections displayed that high temperature accelerated the development of gonad.In the31℃ treatment,the gonad started to differentiate into ovary at 20 dph,and the gonad started to differentiate into testis at 25 dph,while ovary appeared at 30 dph in the 25℃treatment.In the temperature range of 25 ℃ ～31 ℃,the critical period of gonad differentiation in loach was 20～30dph.The expression of the sex-related genes Foxl2,Cyp19 ala,Sox9,and Dmrt1 were affected by temperature at the early developmental stage.The sex-related gene expression were detected at 10 days,earlier than the morphological differentiation.With the development process(10 ～ 60 dph),the gene expression first increased and then decreased.The expression of Foxl2,Cyp19 ala and Sox9 reached the peak at 20 d,the highest peak of Dmrt1 expression appeared at 15 d.Rspo1 was successfully cloned from M.anguillicaudatus,and its expression profile was analyzed.The full-length cDNA of M.anguillicaudatus Rspo1(Ma Rspo1)comprised 1322 bp and included an open reading frame(ORF)of 795 bp,which encoded a predicted polypeptide measuring 264 amino acids in length.Phylogenetic and gene structure analyses showed a highly conserved sequence of Ma Rspo1(identical to the Rspo1 genes of other species),consisting of an N-terminal signal peptide(SP),two furin-like cysteine-rich domains(FU1 and FU2),a thrombospondin type 1 repeat(TSP1)and a C-terminal region.Real-time PCR revealed the female-biased expression profile of Ma Rspo1,with the highest expression level among tested tissues detected in ovary.Investigation of Ma Rspo1 expression levels throughout the early development stage(10～60 days post hatching)under three temperature treatments(25℃,28℃,and31℃)revealed significantly differential expression of Ma Rspo1 among the three temperature groups,with decreased Ma Rspo1 expression in the high-temperature(31℃)group.The results of DNA methylation analysis indicated that exposure to high temperature during early development can increase the average promoter methylation level of Ma Rspo1 in both females and males.