Analysis On Testis Transcriptome Sequencing And Related Genes Of Meiosis In Hybrid Of Red Crucian Carp × Common Carp

Crucian carp hybrid fish,was crossed by female red crucian carp（Carassius auratus red var.,（?）,2n=100,RCC）and male common carp（Cyprinus carpio,（?）,2n=100,CC）,which could break progeny F₁reproduction barrier to produce fertile hybrid strains.Crucian carp hybrid fish was hybrid progeny,which underwent a long meiosis arrest period in germ cells,composed by heterologous chromosomes.The functional expression of relevant meiosis genes during this process was directly related to whether germ cells could cross the reproduction barrier,which was a key point to analyze the molecular mechanism of breaking reproduction isolation and polyploidy in distant hybridization.In this study,diploid hybrid F₁（2n=100,JLF₁）in the non-breeding season and its hermaphroditic fertile female diploid RCC were used as experimental materials.Based on the testis transcriptome sequencing,the meiosis related genes were analyzed.Combined with the observation of testis histological microscopic structure and verified the expression of key genes by real-time fluorescence quantitative PCR（qRT-PCR）,we analyzed the correlation between the related gene differential expression and meiosis morphology.The main studies were as follows.1.Based on the Illumina platform,transcriptome sequencing was performed on the testis of RCC and JLF₁.Three testes of RCC produced69672536,66862654 and 53529812 clean reads,and three testes of JLF₁produced 58924616,67070840 and 61212184 clean reads,respectively.After splicing clean reads,47743 genes were obtained.According to FPKM quantitative results,the overall gene expression level of RCC was higher than that of JLF₁.DEseq analysis showed that there were 1843differentially expressed genes in RCC and JLF₁,of which 522 genes were up-regulated and 1321 genes were down-regulated.Through GO enrichment analysis,1245 differentially expressed genes were annotated to 2260 GO terms.In the classification of biological process,cellular component and molecular function,there were 772,474 and 1044differentially expressed genes,respectively.By means of KEGG analysis,820 differentially expressed genes were labeled into 122 KEGG signaling pathways,of which the most six significant pathways were progesterone-mediated oocyte maturation,cell cycle,oocyte meiosis,p53signaling pathway,cell adhesion molecules（CAMS）,ubiquitin mediated proteolysis.GO and KEGG databases were used to classify the annotated genes.According to the annotation results,ten significant candidate genes related to meiosis,cell cycle and gonadal differentiation were screened out.2.In order to analyze the abnormal characteristics of meiosis in hybrid fish,the testis microstructure of hybrid fish was observed by tissue section technique.The results showed that the fertile individuals of JLF₁needed two years to break through the reproduction barrier and achieve sexual maturity.A few male individuals could form mature sperm after delayed germ cell development.The major male individuals could not develop normally.Although spermatogonia（SPG）,primary spermatocyte（PS）,apoptotic cells（AP）were observed,germ cells were inhibited in the meiosis I metaphase and anaphase for a long time.3.To verify the expression of meiosis related genes,we selected eight key genes（Sun1,Rec8,Sycp3,Mlh1,Spo-11,Rad51,Dmc1,Smc5/6）involved in the regulation of meiosis and two genes（Cdc2,Cyclin B）involved in cell cycle from the differentially expressed genes for qRT-PCR analysis.The results showed that the expression trends of differentially expressed genes in the testis of RCC and JLF₁ were consistent with the RNA-seq quantification.Above ten genes were more highly expressed in the testis of JLF₁ than RCC.This was because RCC testis was not blocked during meiosis,and it was easy to develop into mature sperm,resulting in a relatively small number of spermatocytes in the same volume tissue.In the testis of non-breeding JLF₁,most spermatogenic cells were blocked and accumulated in meiosis I prophase,metaphase and anaphase,leading to richer expression of meiosis key genes and cell cycle genes.In this study,we analyzed transcriptome data associated with meiosis genes of crucian carp hybrid fish,discussed related gene expression characteristics of the JLF₁ testis in the meiosis arrest.It not only provided important molecular basis for breaking through the reproduction barrier of crucian carp hybrid fish,but also had important guide for fish distant hybridization research and genetic breeding.