鸡繁殖性能近交衰退相关CpG岛差异甲基化基因的筛选

鸡繁殖性能近交衰退是地方鸡遗传资源活体保种过程中面临的重要问题之一,本研究旨在探讨全基因组CpG岛（CpG island,CGI）区DNA甲基化在鸡繁殖性能近交衰退中的作用。分别从狼山鸡高近交组和低近交组中各选取健康母鸡3只,即试验分2个组,每组3个重复,然后采用全基因组重亚硫酸盐测序（WGBS）技术,检测分析两组个体性腺轴组织（包括卵巢和下丘脑）全基因组DNA甲基化差异,筛选差异甲基化区域（DMRs）,并对CpG岛区差异甲基化基因进行功能注释和富集分析。结果表明,狼山鸡高近交组和低近交组比较,其卵巢和下丘脑基因组整体甲基化水平均不存在显著差异（P>0.05）;高、低近交组间差异甲基化区域检测发现,下丘脑和卵巢中分别检测到5 948和4 593个差异甲基化区域,其中1 798和995个差异甲基化区域位于基因组CpG岛区,分别注释到1 020和552个基因;下丘脑中,这些CpG岛区差异甲基化基因显著富集在信号转导、神经系统发育、生殖系统发育和卵母细胞成熟调控等繁殖相关的GO条目,以及转化生长因子β信号通路、乙型肝炎、脂肪酸代谢、胰岛素信号通路等19条KEGG信号通路（P<0.05）;卵巢中,CpG岛区差异甲基化基因显著富集于12条信号通路（P<0.05）,包括慢性骨髓白血病、流感A、精氨酸和脯氨酸代谢、粘着连接等,一些与卵子发育和性激素分泌相关的信号通路也被富集到,如黄体酮介导的卵母细胞成熟、卵母细胞减数分裂、GnRH信号通路、雌激素信号通路等,其中包含CDC27、ADCY8、AKT3等10个差异甲基化基因。因此,本研究在狼山鸡高、低近交组间检测到了大量差异甲基化区域,并发现大量差异甲基化基因与繁殖性状相关,推测这些基因CpG岛区DNA甲基化可能在狼山鸡繁殖性能近交衰退调控中发挥重要作用,研究结果为进一步深入探索鸡繁殖性能近交衰退调控机制奠定了基础,为物种资源保护和家禽育种工作提供了理论参考依据。     

近交对狼山鸡繁殖性状影响的研究

利用RAD-seq简化基因组测序鉴定狼山鸡保种群个体基因组SNP标记,计算个体(间)分子近交系数和分子亲缘系数,结合系谱信息组建高、低近交两个试验组。分析后代繁殖性状近交衰退系数,评价近交对繁殖性状的影响。结果显示:利用FROH、FGRM、FHOM和FUNI四种分子近交系数结合亲缘系数kin估算的后代分子近交系数较为一致。低近交组后代的平均分子近交系数小于0.04,高近交组(6个家系)后代的平均分子近交系数介于0.14~0.25。近交对各繁殖性状的效应表现并不一致。高近交组后代母鸡开产日龄、300日龄产蛋数发生显著衰退(P<0.05,P<0.01),且与分子近交系数呈显著相关(P<0.05,P<0.01);开产体重和开产蛋重未发生显著性衰退(P>0.05)。研究结果为进一步探讨狼山鸡繁殖性状近交衰退分子机制提供了基础。     

鸡繁殖性状近交衰退相关lncRNA的筛选

【目的】 筛选与鸡繁殖性状近交衰退相关的长链非编码RNA （long non-coding RNA，lncRNA），为深入探究lncRNA对鸡繁殖性能近交衰退调控机制提供参考。【方法】 以狼山鸡高、低近交群体为试验素材，通过转录组测序技术分析狼山鸡下丘脑和卵巢中lncRNA表达情况，筛选高、低近交组间差异表达lncRNA，并对其进行顺式调控靶基因预测及功能分析。【结果】 狼山鸡下丘脑和卵巢中共鉴定出4 222个lncRNAs，高、低近交组间比较发现，下丘脑中差异表达lncRNAs 35个，卵巢中差异表达lncRNAs 215个。下丘脑中差异lncRNAs中预测到顺式调控靶基因98个，这些靶基因显著富集于出生或孵化时胚胎发育终止、胚胎心导管发育、视黄酸应答等繁殖相关生物过程（P<0.05），涉及lncRNA MSTRG.9196.4、MSTRG.9195.2、MSTRG.6254.2以及相应靶基因DNAAF2和FKBP1B等胚胎发育相关基因；卵巢中差异lncRNAs预测到顺式调控靶基因414个，这些靶基因富集到卵母细胞减数分裂、MAPK、叶酸合成等信号通路，包括MSTRG.7683.1、MSTRG.13604.4、MSTRG.16570.1、MSTRG.8330.5、MSTRG.8330.4等神经内分泌调节及配子生成相关的lncRNAs。【结论】 本研究在下丘脑和卵巢中筛选到了一系列鸡胚胎发育及配子生成调控相关差异lncRNAs，这些lncRNAs可作为鸡繁殖性能近交衰退候选lncRNAs，为进一步揭示鸡繁殖性能近交衰退调控机制提供线索。     

牦牛发情期卵巢比较转录组学研究

旨在进一步了解牦牛发情期卵巢的分子机制,解析牦牛繁殖的特殊性。本研究应用RNA-seq技术对牦牛和平原黄牛发情期卵巢进行转录组高通量测序及全基因组差异表达模式比对分析。通过比较分析牦牛和黄牛卵巢转录组数据,共筛选出1 307个差异表达基因,其中661个基因表达量上调和646个基因表达量下调。进一步功能分析表明,这些差异基因涉及多种GO分类及KEGG通路。其中GO分类注释显示,差异基因与细胞粘附、激素调控等生物学过程存在密切关联,同时钙离子结合、阳离子跨膜转运等分子事件表现活跃。KEGG通路分析显示,补体和凝血级联通路的富集水平最高,其次为细胞色素P450相关通路。昼夜节律等一些新型通路,尽管与生殖功能没有明显关联,但也表现出显著富集。本研究首次对比牦牛和黄牛发情期卵巢转录组数据,筛选并分析相关差异基因。该研究结果为进一步阐述牦牛卵巢的基本分子机理提供基础,同时也为全面理解牦牛繁殖特异性提供新的思路。     

多浪羊与小尾寒羊皮下脂肪组织转录组分析

旨在探索多浪羊（D组）与小尾寒羊（X组）皮下脂肪组织中的特异性表达基因,并研究其潜在的作用,为理解绵羊脂肪组织发育规律以及对脂代谢相关疾病的预防和治疗研究提供依据。本研究选取脂肪沉积能力存在差异、健康无病、体况良好、种内个体体重相近（约50 kg）的雌性成年多浪羊和小尾寒羊为试验材料,分为D组（试验组）和X组（对照组）,每组3个重复,采集位于背最长肌的皮下脂肪组织,应用RNA-Seq技术和生物信息学方法进行转录组测序并对结果进行分析。以|Fold change|≥2,P adjust≤0.05为标准筛选差异表达基因,通过对差异表达基因进行GO功能注释和KEGG通路富集分析,得到与脂肪沉积和脂代谢有关的差异基因。为了验证测序数据的可靠性,本研究随机选取了6个差异表达基因进行qRT-PCR验证。结果显示,在6个样本中共检测到38 672个已知的mRNAs,新的mRNAs为1 606个,在两组中共有839个差异表达基因,其中有320个差异基因在多浪羊组中上调表达,有519个差异基因在多浪羊组中下调表达。通过GO功能注释分析发现,差异表达基因主要参与脂质分解代谢过程、脂质生物合成过程、脂质分解代谢负调控过程、MAPK级联反应调控、对甘油三酯的反应等生物学过程。KEGG通路富集结果显示,差异表达基因显著富集到了PI3K-Akt、MAPK、胰岛素以及PPAR等信号通路中。qRT-PCR结果与测序结果一致,表明测序结果可靠。通过对多浪羊和小尾寒羊皮下脂肪组织进行转录组测序以及生物信息学分析,筛选到与脂肪沉积和脂代谢相关的差异表达基因,这些基因主要参与脂质生物合成、脂质代谢等过程,其中COL1A1、AKT2、SCD、LPL、PCK1与PPP2R5A可能在多浪羊与小尾寒羊的皮下脂肪组织的沉积与代谢中发挥重要作用。     

蒙古马高负荷运动训练前后转录组差异表达分析

旨在对蒙古马高负荷运动训练前后转录组差异表达进行分析。本试验对6匹进行了4个月高负荷运动训练的蒙古马进行研究,分别在训练前后两个时期采集肌肉样品,利用二代测序技术对肌肉样品建立转录组文库,对发生显著变化的差异基因进行GO功能富集和KEGG Pathway分析,寻找训练前后发生显著变化的代谢通路及相关候选基因。结果表明,在转录组筛选到的1 102个显著差异表达基因中,有299个在训练后发生下调,803个发生上调。这些差异基因被注释到3 398条GO term上。其中与运动学相关的生物学过程有:肌肉结构发育、心血管系统发育、循环系统发育、肌肉组织发育、肌肉器官发育、肌肉收缩和肌肉细胞分化等。对差异表达基因进行KEGG pathway分析,结果表明,差异基因被富集到如扩张型心肌病、肥厚型心肌病、心肌收缩、钙信号通路、肌动蛋白骨架调节等与运动相关的193条通路中。本研究结果为探究蒙古马强耐力的基本分子机理提供理论基础,同时也为理解蒙古马运动学特性提供新的思路。     

sRNA伴侣蛋白Hfq敲除条件下沙门菌的转录组分析

为探明沙门菌(Salmonella)在sRNA伴侣蛋白Hfq敲除条件下转录组变化情况。本试验采用HiSeq测序平台对沙门菌LT2菌株及其Hfq敲除株进行高通量测序,通过DESeq2差异分析方法筛选敲除sRNA伴侣蛋白Hfq条件下的差异表达基因,对其进行生物信息学GO功能显著性富集分析和Pathway显著性富集分析。结果显示,对照组和试验组分别得到12 753 534条、8 254 308条Clean Reads。通过DESeq2差异分析获得差异基因1 055个,其中表达上调基因516个,表达下调基因539个。GO功能显著性富集分析表明显著差异表达基因主要富集在钴胺素代谢过程、钴胺素生物合成过程、碳水化合物代谢过程等生物过程中。Pathway显著性富集分析表明显著差异表达基因富集到细菌趋化性、丙酸代谢和碳代谢等11个信号通路中。结果表明,本试验应用RNA-seq技术丰富了sRNA伴侣蛋白Hfq调控基因数量,筛选出20个受伴侣蛋白Hfq调控的显著差异表达基因,其中4个基因功能及编码蛋白未知,注释了差异表达基因的生物学功能及信号通路,推断Hfq在细菌对数期主要通过影响营养提供和趋化性等途径,继而影响细菌的正常生理活动,为Hfq协同sRNA的调控机理研究及sRNA靶基因的筛选奠定了基础。     

利用配对RNA-seq数据筛选显著差异背膘厚度松辽黑猪背最长肌差异表达基因

本研究旨在利用转录组测序技术鉴定松辽黑猪背最长肌组织中的差异表达基因（DEGs）,找出与猪脂肪沉积相关的潜在候选基因。通过对松辽黑猪进行活体背膘厚测定,选择具有极端背膘厚的3对个体（高、低各3头）为研究对象。取背最长肌组织提取RNA,并对其进行双末端转录组测序,然后,基于edgeR包的配对方法筛选差异基因,并进行生物学功能富集分析。结果鉴定出590个差异表达基因,其中,188个基因在高背膘厚组猪背最长肌组织中高表达,402个基因在低背膘厚组猪背最长肌组织中高表达。通过生物学功能分析发现,有42条显著富集通路,鉴定出的与脂肪沉积相关的通路有脂肪酸代谢、不饱和脂肪酸的生物合成以及PPAR信号通路等,筛选出FABP3、FADS1、FADS2、OLR1、ACSL1、LIPA和PLIN2为脂肪沉积性状的候选基因。该研究结果为进一步探究松辽黑猪肌内脂肪分子调控机制提供了参考。     

基于转录组测序探究地塞米松在前体脂肪细胞成脂分化过程中的作用机制

试验旨在研究地塞米松（DEX）在前体脂肪细胞成脂分化过程中的作用机制,为后续体外研究猪肌内脂肪（IMF）沉积的调控机制奠定前期基础。试验分别使用DEX及常用成脂诱导剂MDI(3-异丁基-1-甲基黄嘌呤0.5 mmol/L、地塞米松1μmol/L、胰岛素10μg/mL)处理3T3-L1细胞,利用GO Term和KEGG PATHWAY对差异基因进行功能及信号通路分析。结果发现：相比阴性对照组,MDI组和DEX组分别筛选到2280个、1188个差异表达基因,差异基因富集到血管相关平滑肌收缩、脂质生物合成、脂肪细胞分化等生物学过程;对2处理组间的差异基因集进行Venn分析,获得779个共有差异表达基因,其中上下调模式一致的差异表达基因751个,且显著富集到脂肪细胞分化、脂肪酸代谢过程和PPAR信号通路等。本研究鉴定出通过DEX诱导前体脂肪细胞成脂分化过程的关键基因Selenbp1、Arid5b、Rgs2、Metrnl、Acsl6、Hmgcs1等。     

肾上腺素对民猪脂肪细胞基因表达模式的影响

为了探究冷刺激对民猪脂肪细胞代谢的影响,试验以体外培养的前脂肪细胞为试验材料,通过添加终浓度为1 000 nmol/L的肾上腺素模拟冷刺激环境,同时设置未添加肾上腺素的对照组,诱导分化7 d后收集细胞进行转录组测序(RNA-seq)分析,筛选出表达水平发生变化的差异基因,对差异表达基因进行GO功能注释和KEGG信号通路富集分析,最后通过qRT-PCR验证试验检测RNA-seq结果的准确性。结果表明：肾上腺素会改变脂肪细胞中基因的表达模式,引起128个基因显著上调,93个基因显著下调,其中游离脂肪酸受体4(FFAR4)基因上调倍数最大,为4.63倍。对差异表达基因进行GO功能注释,在分子功能注释分析中有8个条目显著富集,在生物过程注释分析中有128个条目显著富集,在细胞组分注释分析中无显著富集的条目。对差异基因进行KEGG信号通路富集分析,仅神经活性配体-受体相互作用通路被富集。说明肾上腺素可以改变脂肪细胞原有的代谢模式,与脂肪酸代谢和糖原合成等相关的基因发生了显著变化,同时细胞内外的信号转导也发生了改变。     

基于RAD-seq测序的狼山鸡基因组选择信号分析

试验旨在分析狼山鸡基因组选择信号,发掘狼山鸡重要种质特性基因。采用简化基因组RAD-seq测序鉴定狼山鸡及其他18个地方鸡种基因组SNP标记,构建系统进化树阐明狼山鸡的遗传结构,采用ZHp选择信号检测方法鉴定狼山鸡基因组受选择区域（基因）。结果显示,在狼山鸡中鉴定出320 874个SNPs。19个地方鸡种总体上聚为五大类,与品种形成历史和区域分布基本一致。狼山鸡16个常染色体上的46个区域受到选择作用（ZHp<-3.5）,包含122个受选择基因,其中部分区域在遗传聚类同分支的安义瓦灰鸡（15个区域）、边鸡（14个区域）、大骨鸡（11个区域）和北京油鸡（13个区域）中也受到选择。GO分析结果显示,狼山鸡122个受选择基因显著富集在血细胞生成、转录调控、嗜酸性粒细胞趋化、骨化等生物学过程（P<0.05）。KEGG分析结果显示,狼山鸡122个受选择基因显著富集在心肌细胞肾上腺素能信号传导、Toll样受体信号通路、Ca²⁺信号通路、细胞质DNA传感通路、NOD样受体信号通路等（P<0.05）。选择作用主要体现在对狼山鸡刺激响应、先天免疫、代谢、神经系发育等方面的塑造。研究结果可为狼山鸡品种评价、保护及利用提供重要遗传信息。     

Yearling trait comparisons among inbred lines and selected noninbred and randomly bred control groups of Rambouillet, Targhee and Columbia ewes

Inbreeding with concurrent selection was used to develop 26 Rambouillet, 20 Targhee and 10 Columbia inbred lines of sheep. Inbreeding coefficients averaged 30, 29 and 30% for the three breeds, respectively, at the conclusion of the study. A selected noninbred control group and a randomly bred unselected control group were maintained for each breed. Yearling traits were evaluated for 545 Rambouillet, 572 Targhee and 411 Columbia yearling ewes, each belonging to one of the inbred lines or control groups. In each breed, the selected controls were generally of greatest overall merit, the unselected controls intermediate and the inbred lines of least merit. Only a few yearling traits of only a few inbred lines were superior (P less than .05) to those of their appropriate selected control groups. Selection within inbred lines was generally ineffective in offsetting inbreeding depression. However, single trait selection for traits of high heritability, notably yearling weight, clean fleece weight and staple length, appeared to compensate for inbreeding effects on those traits. Deleterious consequences of inbreeding were particularly apparent in yearling weight, average daily gain, type and condition scores, grease and clean fleece weights and index of overall merit. Inbreeding also resulted in fewer neck folds among inbreds of all three breeds. Correlations between the rankings of inbred lines at weaning and yearling ages were high for traits of higher heritability. Superiority of the selected controls in most traits was of about the same magnitude at weaning and yearling ages. In no case did the final overall merit (index value) of an inbred line of any of the three breeds significantly exceed the overall merit of its respective selected control group.     

The effect of inbreeding on the manifestations of some physiological traits in the Kladrub Black Horse strain

Inbreeding was used in the regeneration process in the breeding of the Kladrub Black Horse. Inbreeding rate is realized in practice, as related to the manifestation of the selected characteristics of this unique horse population. The evaluation of the effect of inbreeding on conception rate and fertility did not demonstrate any differences between the group of inbred mares and non-inbred mares. The study was conducted in mares used for breeding for 10 years. The results provide evidence that inbreeding causes no depression in this characteristics in the Kladrub Black Horse which would otherwise be the first physiological function in the reproduction process to be altered as a result of inbreeding. The evaluation of the effect of inbreeding on growth rate by comparing it with the standard growth curves which were characterized by the exponential function y = A (1 -- e-k(t-to)) did not indicate any statistically significant differences in growth dynamics between foals with a zero Fx value and inbred foals. This study was based on the evaluation of growth rate in the basic body traits and weight. It is inferred that inbreeding does not reduce growth dynamics of different body measurements and weight. Similarly, the evaluation of the body dimensions and weight of inbred and non-inbred adults showed no significant differences. Similar results were obtained from the evaluation of the relation between inbreeding and body constitution, character, and temperament. All the correlation coefficients are very low, almost equal to zero.     

Inbreeding depression in Zebu cattle traits

The productivity of herds may be negatively affected by inbreeding depression, and it is important to know how intense is this effect on the livestock performance. We performed a comprehensive analysis involving five Zebu breeds reared in Brazil to estimate inbreeding depression in productive and reproductive traits. Inbreeding depression was estimated for 13 traits by including the individual inbreeding rate as a linear covariate in the standard genetic evaluation models. For all breeds and for almost all traits (no effect was observed on gestation length), the performance of the animals was compromised by an increase in inbreeding. The average inbreeding depression was ?0.222% and ?0.859% per 1% of inbreeding for linear regression coefficients scaled on the percentage of mean (β_m) and standard deviation (β_σ), respectively. The means for β_m (and β_σ) were ?0.269% (?1.202%) for weight/growth traits and ?0.174% (?0.546%) for reproductive traits. Hence, inbreeding depression is more pronounced in weight/growth traits than in reproductive traits. These findings highlight the need for the management of inbreeding in the respective breeding programmes of the breeds studied here.     

产蛋和停产武定鸡卵巢转录组SNP分析

为提高武定鸡生产性能,探索繁殖相关差异基因,对270日龄产蛋和停产一周以上的武定鸡卵巢组织进行转录组测序,将获得的数据进行SNP检测,利用GO富集和KEGG富集分析SNP所在基因。结果显示:产蛋武定鸡特有的SNP有116235个,停产武定鸡特有的SNP有156020个,两者共有的SNP有276467个;GO富集分析发现,SNP所在基因大量富集在生物进程中;KEGG富集分析发现,SNP所在基因显著富集在代谢途径、剪接、细胞周期、细胞凋亡、基础转录因子等通路;对GnRH信号通路中的5个基因SNP分析发现,有9个SNP位于外显子区,其中有4个SNP位于编码区并引起了氨基酸的改变。研究结果为武定鸡繁殖性能的相关研究提供了理论依据。     

Effects of inbreeding and heterosis in Hereford females on fertility, calf survival and preweaning growth

Effects of inbreeding and heterosis and the difference between them were estimated by comparing linecross (L), topcross (T), inbred (I) and control line (C) Hereford females for reproductive and preweaning growth traits of their progeny. Inbred females (average inbreeding coefficient = 26.5%) originated from four single-sire inbred lines. Control females (average inbreeding coefficient = 6.9%) were produced by a four-sire, 60-cow line. Linecross females were produced from all possible reciprocal crosses of the I lines. Topcross females were produced by mating I bulls to C cows. Differences in pregnancy rate among these lines were not detected. Effects of maternal heterosis were positive for both prenatal and postnatal survival. The weaning rate by L females exceeded the weaning rate by I females as a result. Prenatal survival was reduced in calves from I females relative to those from C females, resulting in corresponding differences in birth and weaning rates. Differences in the magnitudes of maternal heterosis and inbreeding effects were not detected, except for birth weight. For birth weight the effect of maternal inbreeding was of greater magnitude than the effect of maternal heterosis. Within the I and C lines regressions of pregnancy rate, prenatal survival, birth rate, postnatal survival, weaning rate, weaning weight/cow exposed, birth weight and weaning weight on the inbreeding coefficients of the cows indicated significant inbreeding depression on all traits except pregnancy rate and postnatal survival.