Identification Of RNA Species In The Chicken Genome And Intestinal Microbiota Associated With The Duration Of Fertility Trait

Duration of fertility（DF）is a critical trait in chicken production,as short duration of fertility need more frequent artificial insemination which could hurt the reproductive organs and cause more labor cost.Thus,understanding the molecular mechanism of DF is important for developing new strategies for efficient production in chicken industry.Previous studies identified genes related to DF trait,but the roles of lncRNA s,which act as master regulators of gene expression and chromatin organization,have not been illustrated.Also,high levels of expression of lncRNA s in reproductive organs have been observed for the fulfilment of roles in sexual reproduction in poultry.To this end,we applied a genome-guided strategy to reconstruct the uterovaginal junction（UVJ）transcriptome of 14 egg-laying birds with long-and short-DF;and sought to uncover key lncRNAs related to duration of fertility traits by RNA-sequencing technology.Egg-laying hens’ lncRNAs were detected by qRT-PCR,and their functional roles in DF trait were clarified by KEGG and GO analysis.Next,the roles of lncRNA 8138.1 gene were validated by candidate gene association study.Additionally,we provided an in-depth study to evaluate the function of 16S rRNA gene on laying hens DF taking into account critical aspects,such as bacterial diversity and community composition of laying hens with（long-DF,or laying hens with short DF）without antibiotic/nutritional supplementation.The overall results are summarized.1.Phenotypic characterization and ultrastructural analysis of the UVJ1)Egg-laying hens were recorded daily by the DF trait:DN:the days postinsemination until the last fertile and FN:the numbers of fertile eggs laid after artificial insemination（AI）.In general,there is a wide range of variability in the DF trait.DN ranges between 8 and 19 days and FN ranges between 6 and 17 eggs.In this study,both DN and FN showed high individual variability between reproductively isolated hens coded as long-and short-DF hens.During the reproductive season,the estimated DF-trait for the two composite characteristics（DN and FN）was approximately（18.71±0.49;7.14±1.57）and（17.43±0.53;7.43±1.40）in the long-and short-DF hens respectively,even when we observed same and optimal multiple insemination conditions.2)Four long-DF and four short-DF individuals were selected for the ultrastructural analysis of UVJ tissue,and the results showed that more sperm storage tubules（SSTs）were embedded in the long-DF individuals when compared with the short-DF.There was a significant difference in the number of SSTs between the two groups（21.50±4.03 vs 4.00±0.91）（p<0.01）in long and short-DF hens respectively.These results were able to distinguish the long and short-DF UVJ tissues.2.LncRNA expression profiling from the egg-laying hens UVJ1)Examination of RNA-seq data revealed a total of 9977 lncRNAs and the majority of lncRNA s came from an intergenic region.We assessed what proportion of the lncRNAs were common to both long and short-DF and found that majority of the lncRNAs were shared between the two groups.Especially,about 9159 lncRNA transcripts were commonly expressed in all the libraries of two groups.Of this,3919 lncRNAs were constitutively expressed,and 5240 were expressed in at least one library within each group.Additionally,very few lncRNA s were uniquely expressed among the two groups,382 and 327 unique lncRNA s for long and short-DF,respectively.Further analysis revealed about 2576 novel lncRNA s in our dataset of which about 2472 and 2452 were expressed in long and short-DF respectively.These novel lncRNA transcripts were distributed among Chromosomes 1-28,Z,LGE64,and MT,of which Chromosomes 1-5 and Chromosome Z harbor the majority of the lncRNA transcripts.2)Differential expression（DE）analysis of lncRNA identified 223 lncRNAs differentially expressed between the two groups（q value<0.05）.Among these 223 lncRNAs,81 were up-regulated,and 142 were down-regulated in the long-DF group compared with the short-DF group.To confirm the results of the lncRNA sequencing analysis,DE-lncRNAs with a selection criterion of log2（fold-change）>2 were chosen for validation by qPCR.Among the selected lncRNA s genes,downregulated MSTRG.28487.2 and MSTRG.17455.2 expression fold change by qPCR were similar to the expression fold change obtained from the RNA-seq.Similarly,upregulated MSTRG.8138.1 and MSTRG.2982.4 also appeared to be expressed at the same level when compared with the RNA-seq data.3)DE-lncRNA target genes prediction uncovered over 200 lncRNA target genes and functional enrichment tests predict the potential functions of DE-lncRNAs.Gene ontology classification and pathway analysis revealed eight DE-lncRNAs,with the majority of their target genes enriched in biological functions such as reproductive structure development,developmental process involved in reproduction,response to cytokine,carbohydrate binding,in utero embryonic development,chromatin organization,positive regulation of MAPK cascade cellular response to cytokine stimulus and immune pathways.Multiple lines of evidence point to lncRNA s as master regulators,suggesting some lncRNA s might perform more specific roles in reproductive organs and/or tissuespecific manner such as MSTRG.20950.12,MSTRG.17455.2,MSTRG.23167.4.MSTRG.28487.2,MSTRG.14719.6,MSTRG.2982.4,MSTRG.24725.1 and MSTRG.8138.1.4)According to KEGG analysis,we consider pathway which might be correlated with sexual reproduction（"mTOR signaling pathway","Metabolic pathways",and "RIGI-like receptor signaling pathway"）.Most of the DF lncRNAs might play the role of master regulators of biological pathways and chromatin organization suited for coordination and control of molecular processes involved in fertility.3.Identification of lncRNA 8138.1 as a candidate gene for DF traitTo comprehensively study the functionality of lncRNA 8138.1 gene,we identified the regulatory pathway and biological processes involved in the putative targets of lncRNA 8138.1 gene.Due to knowing function of lncRNA 8138.1 gene in response to growth factor,reproductive system development,positive regulation of cell differentiation,developmental process involved in reproduction,regulation of osteoblast differentiation,and carbohydrate binding.We performed candidate gene association studies in a big population of egg-laying hens（n=1042）to identify the regulatory region that showed a significant relationship with DF trait.1)The sequence analysis of lncRNA8138.1 gene containing about 1.6 k nucleotide was observed with four single nucleotide polymorphisms（SNPs）including SNPs r.4937159A>G;r.4937219T>C;r.4937258G>C;and r.4937318C>G.Next,the genomic DNAs from the hen population（n=1,042）were subjected to polymerase chain reaction（PCR）and restriction fragment length polymorphism（RFLP）to detect a region of 457 bp carrying lncRNA8138.1 SNP A/G at Chr:18-4937159.Further inspection of the region containing r.4937159A>G revealed three genotypes viz.,AA,GA,and GG were observed with respective frequencies of 0.106,0.607,and 0.287 in laying hens population.2)Furthermore,we experimentally predict the secondary structure of lncRNA8138.1 r.4937159A>G mutation for chicken,which may be used to upgrade sequence/structure alignments for other species.The results showed lncRNA8138.1 A>G substitution had significant predictive consequences on the common secondary structure of individuals with AA genotype when compared to GG genotype.3)Statistical analysis indicated that lncRNA8138.1 genotypes obtained from the region of r.4937159A>G substitution was associated with DF-traits（EN,FN,and DN）respectively in egg-laying hens（n;mean±standard deviation of the mean（SD））according to genotype AA（110;12.99±0.29,8.56±0.24,10.63±0.18）;AG（633;13.56±0.12,8.90±0.10,10.74±0.07）;AA（299;14.50±0.17,9.57±0.14,11.04±0.11）（GG>AA,GG>AG;p<0.0001）.These findings provide a rich genomic resource for the function and possible involvement of the lncRNA8138.1 gene in reproductive function and DF regulation.4.Identification of bacterial diversity and community structure in egg-laying hens with long and short-DF by 16S rRNA gene sequencing.1)Faecal samples from laying hens with long and short-DF were collected for microbial DNA extraction,high-throughput sequencing by amplification of the 16S rRNA V3-V4 hypervariable regions,and bioinformatics analysis.Clean sequences（403,243）clustered into 392 operational taxonomic units（OTUs）（97%identity）,representing 98 species belonging to 119 genera,45 families,27 orders,21 classes,and 13 phyla.Long and short-DF groups shared 378 OTUs,indicating the presence of a core microbiome.2)Alpha diversity analysis showed that the microbial diversity in long-DF group exceeded that of the short-DF group.The dominant phyla of laying hens gut microbiota included Firmicutes,Bacteroidetes,Fusobacteria,and Proteobacteria,together accounting（97.5%and 98.1%of the total sequences）in the long and short-DF groups respectively.The dominant genera included Lactobacillus,Fusobacterium,Bacteroides,Intestinibacter,Rikenellaceae_RC9_gut_group,Parabacteroides and Desulfovibrio.Beta diversity analysis showed that the gut microbial community structure was similar in the two groups.Phylum Saccharibacteria,class Epsilonproteobacteria,order Campylobacterales,family Helicobacteraceae,including the genera Helicobacter domain were identified as potential biomarkers by Metastats.3)The most abundant genera（50）,potentially contributed to the differential distributions of faecal microbiota between the two groups were identified by SparCC including Clostridium_sensu_stricto₁,Enterococcus,Intestinibacter,Lactobacillus,Prevotellaceae_NK3B31_group,Rikenellaceae_RC9_gut_group,Sphaerochaeta and Terrisporobacter.4)Microbial functional predictions analysis revealed a large proportion of metabolic processes are involved,including carbohydrate metabolism,amino acid metabolism,membrane transport,metabolism of cofactors and vitamins,energy metabolism,nucleotide metabolism,and lipid metabolism.Together,these results significantly expand the knowledge of microbial interactions between long and short-DF hens and functional output of microbiome in regulating DF.