【作者】 曹顶国；

【导师】 王宗礼；

【作者基本信息】 甘肃农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 我国地方家禽遗传资源非常丰富，分别具有特色的优良性状，但繁殖性能普遍较低，成为制约其产业化开发的瓶颈之一。运用传统育种手段，经过长期的持续选择，家禽繁殖性状的选育上已经获得重要的遗传进展，有些品种甚至已接近生理极限。随着分子生物学技术的快速发展，筛选与繁殖性状紧密相关的分子标记或功能基因，为这些性状的选择提供了新的、快捷的途径。本研究以山东地方鸡种为研究对象，采用直接测序、PCR-RFLP、RT-PCR和第二代Solexa测序技术，从DNA水平、mRNA水平以及全基因转录组和miRNA水平对鸡繁殖性状的遗传机制进行深入研究。一、VLDLR、BMPR-IB和BMP15基因的多态性与遗传效应选择山东3个地方鸡种(济宁百日鸡、汶上芦花鸡、莱芜黑鸡)和海兰褐蛋鸡为实验动物，共计1536只，分别检测VLDLR、BMPR-IB和BMP15基因的多态性，并分析与济宁百日鸡繁殖性状的相关性。VLDLR基因检测到4个SNPs位点：分别是内含子1内的C2500T (V2500)、外显子6内G8467A (V8467)、内含子15内G12321A (V12321)和内含子17内A13876G(V13876)。V8467位点D1D1基因型个体的43W蛋重极显著高于D2D2基因型个体(P<0.01)。V13876位点B1B1基因型个体的43W蛋重显著高于B2B2基因型个体(P<0.05)。BMPR-IB基因检测到3个SNPs位点：分别是内含子2内C217039T(BR217039)、内含子6内T230065C (BR230065)和内含子8内C233062T(BR233062)。BR217039位点E1E1基因型个体的43W产蛋量显著高于E2E2基因型个体(P<0.05)。BR230065位点F1F2基因型个体的开产日龄显著早于基因型F1F1个体(P<0.05)。BR233062位点G1G1基因型个体的开产体重显著高于G2G2基因型个体(P<0.05)，43W体重极显著高于G2G2基因型个体(P<0.01)。BMP15基因检测到3个SNPs位点：外显子1内A474G (BMP474)和C594T(BMP594)，外显子2内T2659C (BMP2659)。BMP474位点H2H2基因型个体的开产体重、开产日龄和43W体重显著高于H1H1基因型个体(P<0.05)，H1H2基因型个体具有最早的开产日龄和最高的43W产蛋量。BMP594位点，M1M1和M1M2基因型个体的43W体重显著高于M2M2基因型(P<0.05)。BMP2659位点N1N2基因型个体开产蛋重显著高于N2N2基因型个体(P<0.05)。二、VLDLR、BMPR-IB和BMP15基因的表达分析本研究分析了VLDLR、BMPR-IB和BMP15基因在济宁百日鸡不同时期卵巢、输卵管、下丘脑、肝脏以及各级卵泡中的表达规律。VLDLR基因在各级卵泡中的表达量变化趋势为4mm>6-8mm>15-19mm>33-34mm>23-29mm。同一组织在不同时期的表达量，肝脏和下丘脑中的表达量23W>19W>40W；卵巢中的表达量19W>40W>23W，其中在23W和40W的相对表达量相近。在同一时期在不同组织的表达量，3个时期的表达量均是卵巢>下丘脑>输卵管>肝脏，卵巢中的表达量极显著高于其他3种组织(P<0.01)。BMP15基因在各级卵泡中的表达量变化趋势为4mm>6-8mm>15-19mm>23-29mm>33-34mm。不同时期在卵巢中的表达量19W>40W>23W，肝脏中的表达量40W>23W>19W。在同一时期不同组织的表达规律与VLDLR基因相同，均是卵巢>下丘脑>输卵管>肝脏，卵巢中的表达量极显著高于其他3种组织(P<0.01)。BMPR-IB基因在各级卵泡中的表达量变化趋势与BMP15基因相同。在不同时期同一组织的表达量，肝脏和下丘脑中的表达量23W>19W>40W，卵巢中的表达量19W>23W>40W。在同一时期不同组织的表达量；19W时的表达量，下丘脑>卵巢>肝脏，23W和40W时的表达量，输卵管>下丘脑>卵巢>肝脏。三、高、低产汶上芦花鸡卵巢组织的转录组分析采用第二代测序技术对高、低产汶上芦花鸡的卵巢组织进行全基因组水平上的转录组测序，分别获得46,910,566个和62,855,432个reads，比对到参考基因组上的reads分别有38,632,004个和44,399,149个，占总读数的82.35%和70.64%；高、低产个体分别获得有功能注释的基因为4675个和4472个，分别占已有注释基因的82.39%和78.81%；在高、低产个体的卵巢组织中共获得158个差异表达基因，其中79个差异显著的(Q-value<0.05)，79个差异极显著(Q-value<0.01)。四、高、低产汶上芦花鸡卵巢差异表达miRNAs分析采用Illumina Solexa测序方法分析高产、低产汶上芦花鸡个体卵巢组织中差异表达的miRNAs，分别获得高达13.7和10.2百万条短序列读数，其中clean reads分别为13.0和9.8百万条，占源数据的95.06%和95.84%。在高、低产卵巢中均获得已知miRNAs142个，分别获得新miRNAs720个和591个。以低产鸡卵巢为对照，在高产鸡卵巢中共筛选到72个差异表达miRNAs，其中13个表达上调，59个表达下调。本研究采用候选基因法分析了VLDLR、BMPR-IB和BMP15基因多态性与繁殖性状的相关性，从时间和空间上对3个候选基因在不同组织中的mRNA表达水平进行了定量分析，并采用第二代深度测序技术筛选了高、低产汶上芦花鸡个体卵巢组织中的差异表达基因和miRNAs，为鸡繁殖性状的关键功能基因筛选及揭示鸡繁殖性状的内在分子遗传机制提供较为科学全面的理论依据。更多还原

【Abstract】 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.更多还原