Transcriptional Regulation Of Testes,Epididymis And Accessory Gonads Of Inbred Pig Based On Iso-seq And RNA-seq

The Banna mini-pig inbred line(BMI)is developed by utilizing the Diannan small-ear pigs(DSE)through full siblings and parent-child mating.The inbred line has a distinct genetic background and highly homozygous genes.It is an experimental material and organ donor in life sciences and xenotransplantation.However,the continuous high inbreeding seriously reduced the male fertility of BMI.Semen quality is the key factor determining boar fertility,and semen is produced by a combination of the testis,epididymis,prostate gland,vesicular gland,and bulbourethral gland.However,previous studies on the reproductivity of BMI boars mainly focused on the testes and epididymis,with little attention on the prostate gland,vesicular gland,and bulbourethral gland.In this study,we performed Iso-seq(Isoform-sequencing)on the samples of testis,epididymis,prostate gland,seminal vesicles,and bulbourethral gland from 12-month-old boars of BMI and DSE,and obtained full-length transcripts of the five tissues.Further,we conducted the quantitative analysis using RNA-seq to verify the Iso-seq result.The main research results were as follows:Using Iso-seq sequencing technology,a total of about 500 Gb of subreads were obtained from 10 samples of BMI and DSE,with an average of 50 Gb per tissue,and about 99.52% of the subreads were aligned to the pig reference genome,resulting in an average of 45,202 high-quality clusters after clustering.After removing redundant isoforms using c DNA＿Cupcake,a total of 110,996 isoforms were identified across the10 samples with length distribution ranging from 2-5 kb.Comparison of these non-redundant isoforms with the pig reference annotation revealed that they belonged to 22,298 genes,with 2,500 genes having more than 10 isoforms.Of the 110,996 isoforms,90,535 were identified as new isoforms,while 20,461 were known isoforms.The novel isoforms were annotated using five databases,and a total of 88,377 isoforms were annotated,with an annotation rate of 97.62%.To analyze the coding potential of the new isoforms,we obtained 5,986 lnc RNAs using four tools(CPAT,CPC2,GMST and Pfam database)with intergenic being the most common lnc RNA type.The number of predicted exons in the novel lnc RNAs had a similar trend to that of the reference lnc RNAs,with the length distribution ranging from 2-3 kb.SNP analysis using Iso Phase revealed that the overall number of SNPs in BMI was lower than that in DSE,and peak SNPs were detected in chromosome 11 of the testes in both pig breeds.Further analysis revealed that the gene detected with peak SNPs was MTRF1.A total of 3267 fusion genes were identified using SQANTI3 with the highest number of fusion genes in the testis and BMI having more fusion genes than DSE.Using RNA-seq sequencing technology,a total of approximately 232.12 Gb of raw data was obtained from 30 samples of BMI and DSE,with an average of 7 Gb per sample and about 96% of the reads met the quality control standard after filtering.By allocating the novel transcripts obtained from the third-generations sequencing and the known transcripts in Ensembl database,we mapped the clean reads obtained fromRNA-seq sequencing to pig genome(Sscrofa11.1)using STAR and obtained an average unique mapped rate of over 90%.Clustering analysis of the quantified TPM revealed that the correlation between samples of the same tissue was greater than 0.95,while the correlation between samples from different tissues was less than 0.5.We performed differential gene expression analysis between BMI and DSE using DESeq2,and a total of 665,66,362,565,and 214 differentially expressed genes were identified in the testis,epididymis,prostate gland,vesicular gland and bulbourethral gland,respectively.The testis had the highest number of differentially expressed genes,followed by the vesicular gland,and the epididymis had the least.Functional annotation revealed these differentially expressed genes were significantly enriched in important biological signaling pathways related to male reproductive organ development and regulation of sperm quality,including c AMP response,scavenger receptor activity,hyaluronic acid binding,regulation of circadian sleep/wake cycle,MAPK signaling pathway,Vitamin B6 metabolism,c GMP-PKG signaling pathway,Wnt signaling pathway,PI3K-Akt signaling pathway,Arachidonic acid metabolism.The analysis of alternative splicing(AS)revealed that the expression level of AS was highest in the testis and lowest in the vesicular gland,and skipped exon(SE)was the most AS event among the five tissues in the two breeds,while mutually exclusive exon(MX)was the least.Differential AS analysis between BMI and DSE showed that there were 756 differential AS events in the testis,which was the tissue with the most differential AS events,while the epididymis had the least differential AS events with only 236.In summary,we successfully obtained the full-length transcriptome sequences characteristics,transcriptional regulation differences and alternative splicing events of BMI and DSE,including testis,epididymis,prostate,vesicular gland,and bulbourethral gland by combining third-generation and second-generation sequencing technology,providing a data foundation for analyzing the expression regulation mechanism of reproductive differences in the five tissues between BMI and DSE,and providing data for the protection,utilization,genetic improvement,and further molecular breeding of BMI.