拷贝数变异全基因组关联分析及数量性状基因座定位联合鉴定猪体高性状候选基因

旨在利用全基因组拷贝数变异区域（copy number variation regions,CNVRs）关联分析以及全基因组数量性状基因座（quantitative trait locus,QTLs）定位联合筛选出影响猪体高性状的候选基因。本研究利用快速检测基因组拷贝数变异软件CNVcaller对本实验室构建的大白×民猪F2代资源群体的重测序数据进行拷贝数变异检测。利用混合线性模型（mixed-linear model,MLM）将性别和胎次作为固定效应对体高性状进行拷贝数变异全基因组关联分析（CNVR-GWAS）。采用软件R/qtl进行QTL分析,并使用置换检验（permutation test,PT）进行检验。将CNVR-GWAS与QTL结果进行联合注释,结合GO富集和KEGG通路分析,对影响猪体高的位点和基因进行挖掘。利用实时荧光定量PCR（qPCR）方法验证候选基因。结果表明,本群体在全基因组范围内共有3 099个CNVRs,其中有两个CNVRs与体高性状在全基因组范围内显著相关,分别位于7号染色体的25 358 001~26 696 400 bp处（CNVR1）和54 087 201~54 090 000 bp处（CNVR2）。在混合线性模型分析的结果中发现,CNVR1拷贝数增加（P<0.01）和CNVR2拷贝数缺失（P<0.01）对猪的体高性状具有显著影响。基因组显著水平可找到2个显著影响猪体高的QTLs,分别为BH-1和BH-2,其中BH-2对体高性状的影响较大。CNVR1和BH-2重叠区存在1个嗅觉受体基因OR12D3和18个未被注释的基因。qPCR验证OR12D3的拷贝数变异与利用混合线性模型统计推断出的结果一致。初步推测,OR12D3基因的拷贝数变异可能与猪体高性状相关。     

A Male Genetic Map Designed for Quantitative Trait Loci Mapping in Norwegian Cattle

A male bovine linkage map was constructed using six half-sib families of the Norwegian Cattle population. The family material consisted of six sires and 285 sons, with the number of sons per sire ranging from 32 to 71. Of the 288 markers analysed, 261 were anonymous microsatellites and 27 were coding genes. Eleven of the coding genes were mapped by a two-step procedure involving intron/exon sequencing in the six sires and development of polymerase chain reaction-based methods for efficient genotyping in heterozygous families. Linkage analysis including both informative and non-informative sons was performed in order to obtain unbiased estimates of recombination fractions. The autosomal genome length of the Norwegian Cattle Map (NCM) was estimated as 2682 cM, with an average interval of 12.5 cM. The map provides sufficient marker density for scanning the whole bovine genome to locate quantitative trait loci. The large number of common markers between NCM and previously published maps will contribute to the comparison and integration of bovine maps.     

Evaluation of Prolactin Receptor (PRLR) as Candidate Gene for Male Fertility in Hanoverian Warmblood Horses

Stallion fertility has increasing importance as the artificial insemination is employed in horses more intensely. Molecular genetic markers may be useful tools to evaluate the stallion fertility before breeding. The prolactin receptor gene (PRLR) was chosen as a candidate for stallion fertility because of its influence on testicular and accessory sex gland function. Screening the equine PRLR gene for polymorphisms in Hanoverian stallions revealed two single nucleotide polymorphisms (SNPs). Association and haplotype analyses were performed in 162 Hanoverian warmblood stallions for these intragenic SNPs using the least square means (LSM) of the pregnancy rate per oestrus for stallions and the paternal component and embryonic component of the breeding values (BV) of the pregnancy rate per oestrus. The two SNPs (BIEC2‐589441, BIEC2‐560860) showed significant associations using single marker and haplotype analysis with the embryonic and paternal component of BV and one SNP (BIEC2‐560860) was also significantly associated with the LSM of the pregnancy rate per oestrus. This is the first report on an association of PRLR‐associated genetic markers with fertility traits in stallions.     

猪腹股沟/阴囊疝易感位点定位及其候选基因研究进展

腹股沟/阴囊疝是猪最常见的遗传缺陷之一,每年给世界养猪业造成巨大的经济损失,其发病率一般在1.7%~6.7%,但在不同的品种和群体间有较大差异。已有研究表明,猪腹股沟/阴囊疝的发生主要受遗传因素影响,本文综述了猪腹股沟/阴囊疝疾病易感位点和相关侯选基因的研究进展,旨在为系统解析猪猪腹股沟/阴囊疝的分子遗传及病理机制提供参考,并对发展抗猪腹股沟/阴囊疝分子育种技术提出展望。     

Implication of FKBP6 for Male Fertility in Horses

In stallions, impaired acrosome reaction (IAR) may often cause subfertility. Single nucleotide polymorphisms (SNPs) within FK506‐binding protein (FKBP6) seem to be associated with IAR in stallions. However, their effect on stallion fertility has not yet been quantified. Using whole‐genome sequence data of seven stallions, we searched FKBP6 for mutations to perform an association study in Hanoverian stallions with estimated breeding values for the paternal component of the pregnancy rate per oestrus cycle (EBV‐PAT) as target trait. Genotyping five exonic mutations within FKBP6 revealed a significant association of the SNP g.11040379C>A (p.167H>N) with EBV‐PAT in 216 Hanoverian stallions. The difference among the two homozygous genotypes was 7.62% in EBV‐PAT, corresponding to one standard deviation of EBV‐PAT. In conclusion, in Hanoverian stallions, the FKBP6‐associated SNP g.11040379C>A confers higher conception rates in A/A homozygous and lower conception rates in C/C homozygous Hanoverian stallions. Thus, an FKBP6‐associated missense mutation is significantly associated with stallion fertility.     

基于全基因组填充重测序关联分析鉴别影响苏山猪初生体尺与乳头数性状的遗传位点

旨在鉴别影响苏山猪初生体尺和乳头数性状的遗传位点,开发可用于辅助育种的分子标记,为苏山猪生长和繁殖性能的持续选育提供理论基础。本试验对269头苏山猪初生仔猪(出生后24 h内)的体尺和乳头数表型进行测定,并采集耳组织样品。基于全基因组重测序及基因型填充策略,利用全基因组关联分析(genome-wide association study, GWAS)和群体分化指数(fixation index,Fst)的方法挖掘与目的性状强关联位点,并对位置功能候选基因开展GO和KEGG分析,确定最有可能的主效基因。本研究共鉴别到4个候选位点,分布在13、14和X染色体上,其中与初生体尺性状显著关联的位点有2个,与乳头数性状显著关联的位点有2个。本研究筛选出1个与体长性状相关的候选基因ACTA2;1个与胸围性状相关的候选基因COL4A6;3个与乳头数性状相关的候选基因ACTA2、CRTAP和SEPTIN6,为苏山猪初生体尺性状和乳头数性状的遗传改良提供了重要的分子标记。     

Effect of Polymorphisms in Four Candidate Genes for Fertility on Litter Size in a German Pig Line

We carried out an SNP discovery project in pigs for candidate genes playing potentially important roles in embryonic development. Using eight pigs one each from eight breeds (Meishan, Mangalitza, Duroc, Pietrain, German Landrace, Hampshire, Husum Red Pied, German Large White), 36 SNPs were identified in intronic sequences of 21 porcine candidate genes based on sequencing of PCR products. The primer pairs were designed using porcine EST sequences allowing amplification of introns. These SNPs were tested for their association with the number of piglets born alive in German Large White sows using a discordant approach. Significant effects (p < 0.001 and p < 0.05, respectively) of intronic SNPs on litter size were found for four genes: mitogen‐activated protein kinase kinase kinase 3 (MAP3K3), vascular endothelial growth factor receptor (KDR), erbb2 interacting protein (ERBB2IP) and peroxisome proliferator‐activated receptor delta (PPARD). These SNPs can be further tested in upcoming association studies for their influence on litter size in different breeds using larger sample sizes.     

基于全基因组Fst和nSL分析鉴别苏淮猪中性洗涤纤维表观消化率相关候选基因位点

旨在鉴定苏淮猪(含25％淮猪和75％大白猪血统的国家级新品种)在培育过程中受选择的与纤维表观消化率性状相关的基因片段,为解析苏淮猪耐粗饲遗传机制奠定基础.本研究首先检测分析了 331头160日龄苏淮猪个体的中性洗涤纤维(neutral detergent fiber,NDF)表观消化率,计算群体NDF表观消化率估计育种...     

孟德尔分离群体验证候选基因遗传效应的策略

由候选基因法得到的基因变异-目标性状的关联性,其准确性和可靠性受群体遗传背景、连锁不平衡和样本含量大小等因素的影响,所以变异-性状关联性在某群体建立后,应进一步在其它群体中加以验证。在存在低等位基因频率的情况下,如果想得到一定数量低频率等位基因纯合子基因型个体的表型记录,就需要维持很大的试验群体,且试验费用和工作量非常大。本研究提出了一个解决群体内纯合子频率低的策略,即利用杂合子交配产生孟德尔分离群,不但可以获得符合要求的群体规模,而且群体的遗传背景更为相近,同时可以充分考虑家系效应,可以提高验证候选基因遗传效应的准确性。本研究以验证鸡垂体特异转录因子基因POU1F1-6位点对鸡生长性状的遗传效应为例,表明此策略在低等位基因频率情况下验证候选基因遗传效应是可取的,且大大降低了工作量和试验成本。     

Multivariate Cluster Analysis Regression Procedures as Tools to Identify Motile Sperm Subpopulations in Rabbit Semen and to Predict Semen Fertility and Litter Size

Computerized motility analysis (CASA) shows that four separate subpopulations of spermatozoa with different motility characteristics co-exist in rabbit ejaculates. There were significant (p < 0.01) differences in the distribution of these subpopulations among separate genetic lines, total sperm abnormalities and the percentage of altered acrosomes. Furthermore, logistic and linear multivariate regressions among several parameters of rabbit semen quality analysis were tested for use as predictive tools for the fertilizing ability of a specific artificial insemination semen sample. Logistic regression analysis rendered two mathematical, significant (p < 0.01) models: one between sperm viability and conception rate and the other between total sperm abnormalities and conception rate. Multiple linear regression analyses also yielded some significant relationships between both fertility (p < 0.001) and litter size (p < 0.05), with respect to some semen characteristics. Our results support the hypothesis that the predictive in vivo fertility use of the standard rabbit semen quality analysis coupled with a CASA determination could be reasonably achieved by applying linear and logistic regression analyses among several parameters of rabbit semen quality analysis.     

绵羊不同妊娠时期卵巢转录组差异表达比较分析

[目的]探究随着绵羊体内胎儿的发育,母体卵巢转录组表达水平的变化。[方法]试验绵羊为苏格兰黑面羊和特克赛尔羊F₁代杂交品种,3个妊娠时间分别为妊娠23 d、妊娠35 d和妊娠100 d。从NCBI的高通量测序数据SRA存储库下载3个妊娠时期绵羊卵巢转录组的18个样本数据。使用Z-Score方法对转录组数据进行标准化处理。将妊娠23 d、妊娠35 d的绵羊卵巢组织进行比对,妊娠35 d、妊娠100 d的绵羊卵巢组织进行比对,使用R语言的limma包进行基因差异表达分析,筛选DEGs,以P-Value<0.05和|logFC|≥2作为筛选DEGs条件。利用DAVID在线软件对DEGs进行注释及功能富集分析,并以P-Value<0.05作为信号通路显著富集标志。[结果]在妊娠23 d与妊娠35 d组绵羊卵巢中筛选出54个DEGs,妊娠35 d与妊娠100 d组绵羊卵巢中筛选出68个DEGs,两组均包含HSP90AA1、HSP90AB1、MMP2和HSP90B1基因。妊娠23 d与35 d组绵羊卵巢的54个DEGs显著富集到包括内质网蛋白质加工信号通路、核糖体信号通路、雌激素信号通路、癌症蛋白聚糖信号通路、扩张型心肌病信号通路、肥厚型心肌病信号通路6条通路;妊娠35 d、妊娠100 d组绵羊卵巢的68个DEGs显著富集到包括癌症相关信号通路、黏着斑信号通路、抗原加工与提呈信号通路、细胞外基质受体相互作用信号通路、细菌侵袭上皮细胞信号通路、内质网蛋白质加工信号通路、核糖体信号通路、雌激素信号通路、PI3K-Akt信号通路、癌症蛋白聚糖信号通路和前列腺癌信号通路11条通路。[结论]在妊娠期间雌激素信号通路上的HSP90AA1、HSP90AB1、MMP2和HSP90B1基因表达有很大变化,这些基因可能对绵羊妊娠维持有着重要的作用。     

鸡快慢羽分子鉴定方法的建立及其应用

利用找到的鸡快羽和慢羽基因,采用双重PCR方法建立了一种高效的鸡快、慢羽分子鉴定方法,在快慢羽自别雌雄鸡群体当中验证表明准确率达100%。采用该方法和经典表型鉴定方法对同一群鸡进行快慢羽判定,结果发现,除表型鉴定为微长型的个体与分子鉴定结果符合率为94.1%外,其它羽型符合率均达100%。对该鸡群快、慢羽个体早期生长速度比较研究发现,除出壳重差异不显著、第1周差异显著(P0.05),在其后各周,快、慢羽个体间体重差异均极显著(P0.01)。     

A亚型禽白血病的遗传抗性位点与肉鸡重要经济性状的关联分析

为了研究A亚型禽白血病病毒遗传抗性与重要经济性状的关联性,本试验组建了肉鸡F_2代资源群体,在788只健康鸡中筛选了tva基因的抗性位点,分别测量记录每只鸡6、8、10、12周龄体重、屠宰体重、胸肌重、腿肌重等重要经济性状指标,并与tva基因位点进行关联分析。研究发现,在该试验群体中存在3个突变型tva^r3、tva^r5和tva^r6,命名为tva^mut502-516。通过关联分析发现,tva^mut502-516位点在屠体性状方面,只与腹脂重(AFW)和腹脂率(AFR)呈显著相关(P<0.05),与生长、饲料转化率及其他屠宰性状的相关性均未达到显著水平(P>0.05)。研究结果表明,A亚型禽白血病病毒的抗性遗传突变可以显著降低肉鸡的腹脂重(AFW)和腹脂率(AFR),并且对肉鸡的生长、饲料转化率等其他重要经济性状没有产生不良的影响,因此,该抗性位点可以兼顾抗性和生长发育,可应用于A亚型禽白血病抗病育种。     

8个微卫星位点在甘肃高山细毛羊肉毛兼用品系中的遗传多样性研究

利用8个微卫星位点对甘肃高山细毛羊肉毛兼用品系进行遗传检测,计算了等位基因频率、有效等位基因数、群体杂合度和多态信息含量。结果表明,位点BM3501的有效等位基因数、群体杂合度和多态信息含量都最高（Ne=7.30,H=0.866,PIC=0.848）;位点BM3413的有效等位基因数、群体杂合度和多态信息含量都最低（Ne=2.33,H=0.573,PIC=0.478）;8个微卫星位点在甘肃高山细毛羊肉毛兼用品系中多态性丰富,除位点BM3413表现为中度多态外,其余位点均表现为高度多态。因此,甘肃高山细毛羊肉毛兼用品系遗传杂合度较高,遗传多样性丰富,选择余地较大;8个微卫星位点可用于甘肃高山细毛羊肉毛兼用品系遗传多样性评估,并可用于对其羊毛性状和肉用性状的进一步研究。     

利用Bayesian-MCMC方法进行畜禽远交群多家系离散性状QTL连锁检测

利用Bayesian—MCMC方法对不同家系结构的畜禽远交群体的二级离散性状进行QTL连锁检测，在分析中，基于IBD方差组分的随机模型的定位策略，同时利用MCMC的3种不同抽样技术（Gibbs抽样、Metropolis抽样和Reversible jump MCMC抽样）产生相应QTL参数的后验样本，在此基础上进行目标参数的Bayesian统计推断。结果表明：Bayesian—MCMC方法能够对QTL数目进行准确估计，并且在不同家系结构下得到较为理想的参数估计结果。     

Efficacy for a New Live Attenuated Salmonella Enteritidis Vaccine Candidate to Reduce Internal Egg Contamination

To evaluate the efficacy of a novel attenuated Salmonella Enteritidis (△lon△cpxR) vaccine candidate (JOL919), chickens were immunized through oral and intramuscular routes to reduce egg contamination against S. Enteritidis challenge. Birds were orally immunized with JOL919 on the first day of life and were subsequently boosted in the 6th and 16th weeks through oral (group B) or intramuscular (group C) route, while control birds were unimmunized (group A). The chickens of all groups were challenged intravenously with the virulent S. Enteritidis strain in the 24th week. The immunized groups B and C showed significantly higher plasma IgG and intestinal secretory IgA levels as compared to those of the control group. The lymphocyte proliferation response and CD45⁺CD3⁺ T‐cell number in the peripheral blood of the groups B and C were significantly increased. In addition, the egg contamination rates were significantly lower in the group B (0%, 10.7% and 0%) and the group C (3.6%, 14.3% and 3.6%) as compared to the group A (28.6%, 42.8% and 28.6%) in the 1st, 2nd and 3rd weeks post‐challenge. All animals in the groups B and C showed lower organ lesion scores in the liver and spleen and lower bacterial counts in the liver, spleen and ovary at the 3rd week post‐challenge. These results indicate that this vaccine candidate can be an efficient tool for prevention of Salmonella infections by inducing protective humoral and cellular immune responses. In addition, this vaccine did not prevent egg contamination, but did appear to reduce incidence. Booster immunizations, especially via oral administration route, showed an efficient protection against internal egg contamination with S. Enteritidis.     

Genetic and Environmental Correlations Among Female Fertility Traits,and Between the Ability to Show Oestrus and Milk Production in Dairy Cattle

Genetic and environmental correlations were estimated both between the ability to show oestrus and milk production, and among different fertility traits (heat-intensity score, number of days between consecutive inseminations, number of inseminations per service period, interval between calving and first or last insemination, and interval between first and last insemination). Milk production was measured as the average of the energy-corrected milk yield on second and third monthly test days. The number of records were approximately 450000, 350000, 180000 and 75000 in the heifer period, first, second and third lactations, respectively. A linear, trivariate model that included the effects of herd-year, year, month, age and cow's sire was applied. The results indicated that further selection for increased milk production is not expected to deteriorate heat intensity. The number of days between calving and first insemination, the number of inseminations and the heat intensity were complementary, and can be recommended for a selection index for fertility.     

Genetic Parameters of Disease Incidence,Fertility and Milk Yield of First Parity Cows and the Relation to Feed Intake of Growing Bulls

Abstract

Not much is known about the effect of light sources on behavioural development of laying hens. The effect of natural and artificial light, respectively, on feeding and perching of layers was studied. 126-day-old chicks were divided into 1) A8: 8h artificial light+16h darkness, 2) A16: 16h artificial light+8h darkness, 3) N8: 8h daylight+16h darkness. A8 and N8 birds did not differ in their feeding behaviour, whereas the mean proportion of A16 birds feeding was 0.056 lower than in A8 birds in daytime (P<0.001) and 0.019 lower in nighttime (P<0.0001). N8 birds tended to start nighttime perching earlier than A8 birds (hazard ratio=8.5; P=0.056). These results suggest that chicks can change their feeding behaviour depending on day length. Natural daylight was found to precipitate the onset of nighttime perching in the life of the pullet.