Transcriptome Analysis Of Layers Ovaries Between Egg-Laying And Broody Periods And Identifition For Candidate Genes Associated With Ovarian Development

Broodiness,which is a maternal behavior and instinct in most domestic fowls,reduces laying performance that is a major economic concern in the poultry industry.The maintenance of ovarian atrophy in broody chickens is directly related to the broody period length and recovery to egg-laying.The growth and development of laying hen ovary is regulated by various regulatory factors.However,most previous studies focused on exploring candidate genes of bird broodiness,information concerning systematical explanation for the molecular mechanisms of ovarian atrophy in broody chickenremains scarce.Thus,we collected atrophic ovaries(AO)from broody green-shell chickens(BC)which kept the status of typical broodiness for about 30 days and normal ovaries(NO)from even-aged egg-laying hens(EH)for transcriptome sequencing,identified m RNA,mi RNA and lnc RNA of each sequencing library,and analyzed their genomic features.The main results as follows:1.At 30-54 weeks of age,the average ovipository cycle for green-shell chicken is 3.37 days,and it is 32.34% hens for ovipository cycle to range from 3 to 4 days.The intervals between adjacent ovipository cycles were in a large range,with the shortest less than 2 days and the longest was more than 5 days.At 54 th week of age,46.7 % green-shell chicken laid eggs from 8:00 am to 12:00 am,with a laying peak of 12.17 % between 9am and 10 am.The average egg-laying interval for green-shell chicken is 26.54 hours,and the largest proportion with 32.49% is period from 24 to 26 hours.2.Plasma concentrations of follicle stimulating hormone(FSH)and luteinizing hormone(LH)in broody chickens were significantly lower than that in egg-laying hens(P <0.05),and prolactin(PRL)was significantly higher in broody chickens than egg-laying hens(P < 0.05).Egg-laying hens had plump ovaries with many visible follicles and a gradually increasing volume,whereas ovaries of broody chickens showed obvious atrophy with visible characteristics.Ovary weights and ratio(Ovary weight / Body weight *100%),ovary volume,and stroma weights of broody chickens were significantly lower than those of egg-laying hens(P < 0.05),and small yellow follicles(SYF,5-10 mm in diameter and have not entered the hierarchy),and large yellow follicles(LYF,preovulatory follicles,> 10 mm in diameter and have entered the hierarchy)were not observed in broody hens.Observation under the light microscope revealed an unconsolidated ovary with many primaryfollicles(one layer of cuboidal granulosa cells,PFs)and secondary follicles(two to six layers of granulosa cells,SFs)in egg-laying hens,while the broody chickens had numerous PFs but few SFs within the more compact ovary.3.We performed largescale cDNA sequencing experiments in six libraries using the Illumina paired-end RNA-seq approach,resulting in a total of 508.93 M(million)paired-end reads of the 125 nucleotide length,which yielded 63.3 Gb(giga bases)of raw data,and an average of 84.8 M of raw reads was obtained per library.And about 98.7%(502.42 M)of raw reads passed initial quality thresholds and were deemed as clean reads for the subsequent analyses,where an average clean reads was 83.74 M per library.In the all sequencing libraries,we identified 49,461 reliable m RNA transcripts,including 15,952 known and 33,509 novel transcripts.A total of 48,992 m RNA transcripts were co-expressed in normal ovary(NO)and atrophic ovary(AO),while 171 and 298 m RNA transcripts were specifically expressed in NO and AO,respectively.We found 2,198,928 mutation sites in six libraries,including 2,083,946 SNPs and 114,982 In Dels.We also detected an average alternative splicing events was 54,010 per library.In the expression analysis,3,480 significantly differentially expressed(DE)m RNAs including 1,719 down-regulated(49.4%)and 1,761 up-regulated(50.6%)were discovered in AO.The most of DE m RNAs were found to be associated with anatomical structure development,lipid and polysaccharide metabolism,cellular signaling process,cell proliferation and reproduction.We also detected some genes associated with reproductive endocrine system and growth and development of ovarian cells.4.We obtained a total of 78.32 M raw reads from all six small-RNA sequencing libraries,and an average of 13.05 M of raw reads was detected per library.And about 95.48%(74.78 M)of raw reads passed initial quality thresholdsand were deemed as clean reads,while an average clean reads was 12.46 M per library.In the all sequencing libraries,we identified2,827 reliable mi RNAs,including 634 known mi RNAs(22.43%),1,423 conserved mi RNAs(50.34%)and 770 predicted novel mi RNAs(27.24%).A total of 1,777 mi RNA were co-expressed in normal ovary(NO)and atrophic ovary(AO),while 592 and 458 mi RNA were specifically expressed in NO and AO,respectively.In the expression analysis,116 significantly DE mi RNAs including 37 up-regulated(31.9%)and 79 down-regulated(68.1%)were discovered in AO.A total of 9,081 target protein-coding genes for DE mi RNAs were identified,including 508 intersection genes which were detected to be mainly related to developmental process,cell adhesion,ECM-receptor interaction and organ morphogenesis.We also identified some mi RNAs closely related with ovarian development.5.In the all sequencing libraries,we identified 8,714 reliable lncRNA transcripts,including 7,241 intergenic lnc RNA and846 antisense lnc RNA.A total of 8,531 lnc RNA transcripts were co-expressed in normal ovary(NO)and atrophic ovary(AO),while 30 and153 lnc RNA transcripts were specifically expressed in NO and AO,respectively.Compared with protein-coding genes,these lnc RNAs had some obvious genomic features involved lower expression level,shorter transcripts length and lower exon number.In the expression analysis,959 significantly DE lnc RNAs including 903 up-regulated(94.16%)and 56down-regulated(5.84%)were discovered in AO.Cis and trans target genes of lnc RNAs were detected to be mainly related to response to stimulus,signal transducer activity,transmembrane transport and cellular basic metabolism.Though ce RNA(competing endogenous RNAs)network analysis,we identified some lnc RNAs closely related with ovarian development.In summary,we characterized mRNA,miRNA,and lncRNA transcripts profiles of atrophic ovaries from broody chickens and normal ovaries from egg-laying hens using transcriptome sequencing.We identified and characterized RNA transcripts(especially mi RNA and lnc RNA)that were involved in growth and development of laying hen ovary.This study further improved the molecular regulatory network of ovarian growth and development in chicken,and also provided an available reference for the study of non-coding RNAs related to chicken ovarian atrophy.