Functional Analysis On Reproduction-associated Genes And Identification Of Ovarian Transcriptomes And MiRNAs In Chicken

There are many kinds of the chicken genetic resources and each has unique traitsin our country. Generally, most of them have low reproductive performance, and it isone of the bottlenecks in their industrial development. Using traditional breedingmethods in poultry breeding on reproductive traits has received important geneticprogress, and the reproductive performance of some varieties has even been close totheir physiological limits. With the rapid development of molecular biologytechniques, screening molecular markers or functional genes closely associated withreproductive traits provides a new and efficient way in selecting these traits.In this study, the reproductive traits were investigated in Shandong local chickensfrom DNA level, mRNA level, miRNA level and whole genome transcriptome, bydirect sequencing, PCR-RFLP, RT-PCR and second-generation Solexa sequencingtechnology.PartⅠ: The polymorphism and genetic effects of VLDLR, BMPR-IB and BMP15genes in Shandong local chickensIn this study, three Shandong local chicken breeds (Jining Bairi chicken, Laiwublack chicken and Wenshang barred chicken) and Hy-line brown chicken, totally1536individuals, were used to scan the polymorphisms of VLDLR, BMPR-IB and BMP15genes. The associations between polymorphisms and egg production traits wereanalyzed in Jining Bairi chickens.In VLDLR gene, four SNPs were found. They were C2500T in intron1, G8467Ain exon6, G12321A in intron15and A13876G in intron17. In V8467site, the eggweight at43W (EW43) of D1D1chickens was significantly higher than D2D2ones(P<0.01). In V13876site, the EW43of B1B1chickens was higher than B2B2ones(P<0.05).In BMPR-IB gene, three SNPs were detected. They were C217039T (BR217039)in intron2, T230065C (BR230065) in intron6and C233062T (BR233062) in intron8.In BR217039site, the Egg production at43W (E43) of E1E1chickens was higher than E2E2ones (P<0.05). In BR230065site, the age at first egg (AFE) of F1F2chickens was earlier than F1F1ones (P<0.05). In BR233062site, the significantdifferences of living weight at first egg (LWFE)(P<0.05), living weight at43W(LW43)(P<0.01) and EW43(P<0.01) appeared between G1G1chickens and G2G2ones.In BMP15gene, three SNPs were detected. They were A474G (BMP474) in exon1, C594T (BMP594) in exon1and T2659C (BMP2659) in exon2. In BMP474site,the difference of LWFE, AFE and LW43between H1H1chickens and H2H2oneswere significant (P<0.05). The H1H2chickens had earlier AFE and higher E43thanH1H1and H2H2ones. In BMP594site, the LW43of M2M2chickens wassignificantly lower than M1M1and M1M2ones (P<0.05). The egg weight at first egg(EWFE) of N1N2chickens was higher than N2N2ones in BMP2659site (P<0.05).Part Ⅱ: Analysis of expression of VLDLR, BMPR-IB and BMP15genes inShandong local chickensIn this study, we analyzed the expression in ovary, oviduct, hypothalamus, liverand ovarian follicle in different periods in Jining Bairi chickens. For VLDLR gene, theexpression level in ovarian follicle was4mm>6-8mm>15-19mm>33-34mm>23-29mm in diameter. The mRNA level in follicles of4mm and6-8mm indiameter were higher than that in the other follicles (P<0.01). In liver andhypothalamus, the expression level in23W was higher than that in other period, andit was lowest in40W. In ovary, the expression level was19W>40W>23W, themRNA level was similar in23W and40W. In different periods of19W,23W and40W, the trends of mRNA level of different issues was ovary> hypothalamus>oviduct>liver, at the same week, the highest mRNA level was found in ovary, and hadsignificant difference with that in the other issues (P<0.05) or (P<0.01).For BMP15gene, the expression level in ovarian follicle was4mm>6-8mm>15-19mm>23-29mm>33-34mm in diameter. The mRNA level in follicles of4mmand6-8mm in diameter were significantly higher than that in the other follicles(P<0.01). In ovary, the expression level was19W>40W>23W, the mRNA level in19W was higher than that in23W and40W (P<0.05). The expression level in liver was40W>23W>19W. In different periods of19W,23W and40W, the trends ofmRNA level of different issues was ovary> oviduct> liver. At the same week, thehighest mRNA level was found in ovary, and had significant difference with that inliver and oviduct (P<0.01).For BMPR-IB gene, the expression level in ovarian follicle was4mm>6-8mm>15-19mm>23-29mm>33-34mm in diameter. The mRNA level in follicles of4mmin diameter was significantly higher than that in follicles of15-19mm,23-29mm and33-34mm in diameter (P<0.01). In different developing periods, the expression levelwas23W>19W>40W in liver and hypothalamus, and the mRNA level in23W wassignificantly higher than that in40W (P<0.01). In ovary, the expression pattern was19W>23W>40W, and the mRNA level in19W was higher than that in40W(P<0.05).In19W, the trends of mRNA level of different issues was hypothalamus>ovary>liver, and the lower mRNA level was found in liver than that in the other issues(P<0.05). The expression trend was similar in23W and40W, it was oviduct>hypothalamus>ovary>liver, the highest mRNA level was found in oviduct, and hadsignificant difference with that in other issues (P<0.01).Part Ⅲ: Transcriptome analysis in high-yield and low-yield ovaries ofWenshang Barred chickensIn this study, transcriptome of high-yield (HY) and low-yield (LY) ovaries inWenshang Barred chickens were analyzed with the second-generation sequencingtechnology. Solexa sequencing provided a total of46,910,566and62,855,432readsfrom the HY and LY ovary tissue libraries, respectively. There are38,632,004and44,399,149reads mapped to the reference genome of chicken (Gallus gallus)respectively, which account for82.35%and70.64%of the total reads. In total,4675and4472genes, which account for82.39%and78.81%of genes in UCSC, weretermed as expressed genes in HY and LY ovaries. We identified158differentiallyexpressed genes in HY and LY ovaries, the difference of79genes between HY andLY ovaries was significant (Q-value<0.05), and the difference of the other79geneswas significant (Q-value<0.01). Part Ⅳ: Ovarian analysis of differentially expressed miRNAs in high-yield andlow-yield ovaries of Wenshang Barred chickensThe differentially expressed miRNAs in ovary of high-yield (HY) and low-yield(LY) chicken were screened by using Illumina Solexa sequencing method. A total of13.7million and10.2million reads from the HY and LY ovary tissue libraries wereobtained, respectively. After removing the low quality, adaptor, insufficiently taggedand sequences,13.0million and9.8million clean reads were ultimately obtained,account for95.06%and95.84%of the total reads.142known mature miRNAs wereobtained in the HY and LY ovary tissue, respectively.720and591novel miRNAswere identified in the HY and LY ovary tissue, respectively. Compared to the LYovary,72differentially expressed miRNAs were identified in HY ovary, in which13miRNAs were up-regulated and59miRNAs were down-regulated.In conclusion, the correlations between VLDLR BMPR-IB and BMP15genepolymorphism and reproductive traits were analyzed using the candidate geneapproach. The expression profile of different issues and in different developmentalstage was quantitatively characterized. Differently expression genes and miRNAswere identified in high-yield and low-yield ovary by the second-generation deepsequencing technology. These data may provide a comprehensive theoretical basis forthe identification of critical genes and revealing the molecular genetic mechanisms ofreproductive traits in chicken.