利用全基因组连锁不平衡估计中国荷斯坦牛有效群体大小

有效群体大小是群体遗传学研究的一个重要内容,有助于我们更清楚地了解群体的遗传变异、进化和复杂性状的遗传机制等。随着高密度SNP标记的出现,越来越多的研究利用SNP标记间连锁不平衡估计有效群体大小。文章采集北京地区中国荷斯坦牛2 093个样本,并利用牛SNP芯片(Illumina BovineSNP50,含5 4001 SNPs)进行基因型测定,估计不同世代中国荷斯坦牛的有效群体大小。质量控制标准设定为SNP检出率0.95,最小等位基因频率>0.05,样本检出率0.95,哈代温伯格平衡检验显著性水平P<0.0001。经过质量控制,共1 968个样本和38 796个SNPs用于连锁不平衡分析。文章选取SNP间距0.1、0.2、0.5、1、2、5、10、15(Mb),估计中国荷斯坦牛在4世代之前有效群体大小。结果表明,中国荷斯坦牛的有效群体呈逐代下降趋势,至4世代前,中国荷斯坦牛平均有效群体为45头左右。     

利用PCR-RFLP检测中国荷斯坦牛遗传缺陷——瓜氨酸血症

瓜氨酸血症(Citrullinemia)是荷斯坦牛尿素循环发生代谢紊乱的一种常染色体隐性遗传缺陷。精氨酸琥珀酸合成酶基因外显子5发生突变(C-T)对这一紊乱负责。本研究应用PCR-RFLP方法和DNA测序技术检测济南市周边120头荷斯坦母牛和山东奥克斯生物技术有限公司种公牛站50头荷斯坦公牛的精氨酸琥珀酸合成酶基因外显子5。结果发现,所检测的公牛未发现瓜氨酸血症突变基因携带者,母牛中有2头为携带者,携带频率为1.18%。     

中国荷斯坦牛和鲁西黄牛mtDNAD-loop序列多态性分析

为了从母系遗传角度深入阐明中国荷斯坦牛和鲁西黄牛的群体遗传多样性以及起源进化,本研究采用PCR测序法测定了9头中国荷斯坦牛和11头鲁西黄牛的线粒体DNA D-loop区的部分序列,并经剪切后进行生物信息学软件分析.结果表明,20个个体D-loop区共711 bp,共检测到19种单倍型和50个多态位点.核苷酸多样性(Pi)为0.021 33±0.004 54,单倍型多样性(Hd)为0.994±0.019,平均核苷酸差异数(k)为15.146 20.构建的NJ网络进化树共分为两大支系,其中部分鲁西黄牛与瘤牛聚为一支,而中国荷斯坦牛和部分鲁西黄牛与普通黄牛聚为一支.说明中国荷斯坦牛和鲁西黄牛群体遗传多样性均较高;鲁西黄牛同时含有瘤牛和普通黄牛的血统,而中国荷斯坦牛只含有普通黄牛的血统.     

南方中国荷斯坦牛耐热性的数量遗传学研究

本研究采用公畜内半同胞相关法,分别从产奶量（350头）、耐热产奶量（116头）、直肠温度（200头）和红细胞钾（144头）四个方面估算奶牛群体遗传力,结果分别是0．334、0．196、0．20、0．318．利用SAS8．1版本对数据进行二元回归分析,得到回归方程：y=0．23853-0．0096xa＋0．000827x2（x1为直肠温度,x2为红细胞钾含量）．运用BLUP法估算公牛育种值,以色列公牛育种值最高,为85．12．     

中国荷斯坦牛ABCG2基因编码区多态性检测及生物信息学分析

目的：研究中国荷斯坦牛ABCG2基因编码区（CDS）多态性,并进行生物信息学分析。方法：以中国荷斯坦牛为材料,利用PCR-SSCP技术对ABCG2基因CDS多态性进行检测,然后预测蛋白质序列的改变,并用生物信息学软件对蛋白质序列突变前后的结构及性质进行分析。结果：在外显子9中存在一个A→G碱基突变,导致氨基酸由酪氨酸突变为半胱氨酸,将此突变命名为Y367C;在外显子14中存在一个G→A突变,导致氨基酸由精氨酸突变为谷氨酰胺,将此突变命名为R578Q。2个突变一个位于功能区与跨膜区之间,一个位于跨膜区。生物信息学分析发现,蛋白质二级结构增加了1个卷曲（C）和2个转角（T）,同时减少了3个β折叠（E）,且ABCG2蛋白的组成和一些性质也发生了改变。结论：检测到的2个单核苷酸多态性引起了ABCG2蛋白性质和二级结构的改变;为进一步研究ABCG2蛋白对产乳性状的影响奠定了基础。     

中国荷斯坦牛白细胞黏附缺陷症PCR-RFLP检测方法的研究

本试验根据已知牛染色体上CD18编码基因序列设计引物,提取牛血液和精液DNA,可扩增出338bp的DNA片段,将PCR产物克隆到pMD18-T载体中,对阳性重组质粒进行测序,确定为牛的CD18基因。由于CD18基因的383位碱基由A变为G,而引起牛白细胞黏附缺陷症(BLAD),通过对济南市11个奶牛场356头奶牛及53头荷斯坦种公牛进行了BLAD的PCR-RFLP检测,共检出3头杂合母牛(携带者),占检测母牛群的0.84%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者。     

中国荷斯坦牛线粒体DNA全序列多态性分析和分型

目的：阐明中国荷斯坦牛线粒体DNA全序列多态性及其起源关系。方法：从上海松江科研基地的10头中国荷斯坦牛外周血中抽提DNA,设计引物扩增全长线粒体DNA,通过对其进行序列测定、分析,绘制系统进化发育树。结果：得到了中国荷斯坦牛线粒体基因组的全序列和个体比对结果等相关统计数据,对多态性位点和突变位点等遗传信息进行了序列分析,并进一步分型。结论：研究结果对提高中国荷斯坦牛体细胞核移植和线粒体DNA多态性高通量分析效率有一定的指导意义,为线粒体DNA多态性检测芯片的研究奠定了基础。     

嘉黎牦牛和荷斯坦牛4项红细胞酶活性的测定

测定了西藏嘉黎牦牛和南京荷斯坦牛血液红细胞中乳酸脱氢酶 (LDH)、碱性磷酸酶 (AKP)、过氧化氢酶 (CAT)、超氧化物歧化酶 (SOD)等 4项酶的活性 ,结果分别为LDH 2 5 5 2 3 1 6± 71 4 0 3和 2 1 2 74 96± 6638 1 6(nmol s) ,AKP 4 4 4 4± 1 2 81和 36 5 3± 1 1 31 (nmol s) ,CAT 1 0 2 73± 32 2 2和 63 0 8± 1 2 4 7(U gHb) ,SOD 1 3979 1 6± 2 873 84和 92 85 37± 2 880 60 (U gHb)。西藏嘉黎牦牛的CAT和SOD极显著高于荷斯坦牛 (P <0 0 1 ) ;而LDH和AKP显著高于荷斯坦牛 (P <0 0 5 )。     

荷斯坦牛BoLA-DRA基因SNPs筛选及生物信息学分析

为了探讨BoLA-DRA基因多态性,本研究利用DNA混合池扩增产物直接测序的方法对荷斯坦牛BoLA-DRA基因编码区SNPs进行筛选,并利用生物信息学软件预测该基因m RNA二级结构。结果表明:荷斯坦牛BoLA-DRA基因编码区共有4个SNPs (exon2-A82T,exon2-G116A,exon2-C197T,exon2-C233A)。生物信息学预测结果显示,exon2-A82T、exon2-G116A和exon2-C233A增大了m RNA二级结构的稳定性,而exon2-C197T降低了mRNA二级结构的稳定性。本研究结果可为荷斯坦牛抗病和抗逆性能研究积累更多的分子遗传学数据,并为经济性状相关基因筛选提供理论依据。     

Genomic inbreeding estimation in small populations: evaluation of runs of homozygosity in three local dairy cattle breeds

In the local breeds with small population size, one of the most important problems is the increase of inbreeding coefficient (F). High levels of inbreeding lead to reduced genetic diversity and inbreeding depression. The availability of high-density single nucleotide polymorphism (SNP) arrays has facilitated the quantification of F by genomic markers in farm animals. Runs of homozygosity (ROH) are contiguous lengths of homozygous genotypes and represent an estimate of the degree of autozygosity at genome-wide level. The current study aims to quantify the genomic F derived from ROH (F_ROH) in three local dairy cattle breeds. F_ROH values were compared with F estimated from the genomic relationship matrix (F_GRM), based on the difference between observed v. expected number of homozygous genotypes (F_HOM) and the genomic homozygosity of individual i (F_{MOL i}). The molecular coancestry coefficient (f_{MOL ij}) between individuals i and j was also estimated. Individuals of Cinisara (71), Modicana (72) and Reggiana (168) were genotyped with the 50K v2 Illumina BeadChip. Genotypes from 96 animals of Italian Holstein cattle breed were also included in the analysis. We used a definition of ROH as tracts of homozygous genotypes that were >4 Mb. Among breeds, 3661 ROH were identified. Modicana showed the highest mean number of ROH per individual and the highest value of F_ROH, whereas Reggiana showed the lowest ones. Differences among breeds existed for the ROH lengths. The individuals of Italian Holstein showed high number of short ROH segments, related to ancient consanguinity. Similar results showed the Reggiana with some extreme animals with segments covering 400 Mb and more of genome. Modicana and Cinisara showed similar results between them with the total length of ROH characterized by the presence of large segments. High correlation was found between F_HOM and F_ROH ranged from 0.83 in Reggiana to 0.95 in Cinisara and Modicana. The correlations among F_ROH and other estimated F coefficients were generally lower ranged from 0.45 (F_{MOL i}−F_ROH) in Cinisara to 0.17 (F_GRM−F_ROH) in Modicana. On the basis of our results, recent inbreeding was observed in local breeds, considering that 16 Mb segments are expected to present inbreeding up to three generations ago. Our results showed the necessity of implementing conservation programs to control the rise of inbreeding and coancestry in the three Italian local dairy cattle breeds.     

Effect of reduced genomic representation on using runs of homozygosity for inbreeding characterization

Genomic measures of inbreeding based on identical-by-descent (IBD) segments are increasingly used to measure inbreeding and mostly estimated on SNP arrays and whole-genome sequencing (WGS) data. However, some softwares recurrently used for their estimation assume that genomic positions which have not been genotyped are nonvariant. This might be true for WGS data, but not for reduced genomic representations and can lead to spurious IBD segments estimation. In this project, we simulated the outputs of WGS, two SNP arrays of different sizes and RAD-sequencing for three populations with different sizes and histories. We compare the results of IBD segments estimation with two softwares: runs of homozygosity (ROHs) estimated with PLINK and homozygous-by-descent (HBD) segments estimated with RZooRoH. We demonstrate that to obtain meaningful estimates of inbreeding, RZooRoH requires a SNPs density 11 times smaller compared to PLINK: ranks of inbreeding coefficients were conserved among individuals above 22 SNPs/Mb for PLINK and 2 SNPs/Mb for RZooRoH. We also show that in populations with simple demographic histories, distribution of ROHs and HBD segments are correctly estimated with both SNP arrays and WGS. PLINK correctly estimated distribution of ROHs with SNP densities above 22 SNPs/Mb, while RZooRoH correctly estimated distribution of HBD segments with SNPs densities above 11 SNPs/Mb. However, in a population with a more complex demographic history, RZooRoH resulted in better distribution of IBD segments estimation compared to PLINK even with WGS data. Consequently, we advise researchers to use either methods relying on excess homozygosity averaged across SNPs or model-based HBD segments calling methods for inbreeding estimations.     

Genome-enable prediction for health traits using high-density SNP panel in US Holstein cattle

The objective of this study was to compare accuracies of different Bayesian regression models in predicting molecular breeding values for health traits in Holstein cattle. The dataset was composed of 2505 records reporting the occurrence of retained fetal membranes (RFM), metritis (MET), mastitis (MAST), displaced abomasum (DA), lameness (LS), clinical endometritis (CE), respiratory disease (RD), dystocia (DYST) and subclinical ketosis (SCK) in Holstein cows, collected between 2012 and 2014 in 16 dairies located across the US. Cows were genotyped with the Illumina BovineHD (HD, 777K). The quality controls for SNP genotypes were HWE P-value of at least 1 × 10⁻¹⁰; MAF greater than 0.01 and call rate greater than 0.95. The FImpute program was used for imputation of missing SNP markers. The effect of each SNP was estimated using the Bayesian Ridge Regression (BRR), Bayes A, Bayes B and Bayes Cπ methods. The prediction quality was assessed by the area under the curve, the prediction mean square error and the correlation between genomic breeding value and the observed phenotype, using a leave-one-out cross-validation technique that avoids iterative cross-validation. The highest accuracies of predictions achieved were: RFM [Bayes B (0.34)], MET [BRR (0.36)], MAST [Bayes B (0.55), DA [Bayes Cπ (0.26)], LS [Bayes A (0.12)], CE [Bayes A (0.32)], RD [Bayes Cπ (0.23)], DYST [Bayes A (0.35)] and SCK [Bayes Cπ (0.38)] models. Except for DA, LS and RD, the predictive abilities were similar between the methods. A strong relationship between the predictive ability and the heritability of the trait was observed, where traits with higher heritability achieved higher accuracy and lower bias when compared with those with low heritability. Overall, it has been shown that a high-density SNP panel can be used successfully to predict genomic breeding values of health traits in Holstein cattle and that the model of choice will depend mostly on the genetic architecture of the trait.     

基于高密度SNP标记的肉牛人工选择痕迹筛查

近年来随着遗传改良工作的实施,人工选择大大提高了肉牛的生产性能并使其遗传基础发生巨大改变。文章基于Illumina BovineSNP50(54K)和BovineHD(770K)两款芯片数据,采用FST检验方法分析牛群的遗传分化,并筛查人工选择在牛的基因组留下的印记。通过全基因组范围内的扫描,共发现47 104个"离群"位点和3064个群体特异的人工选择"候选基因",如CLIC5、TG、CACNA2D1、FSHR等。通过基因注释对基因的生物学过程和分子功能进行富集分析。文章构建了我国肉牛的全基因组的选择信号图谱,为深入研究人工选择和理解生物进化提供线索,且研究结果也显示人工选择对基因组的影响在牛品种遗传改良中发挥了重要作用。     

Evidence of inbreeding depression but not inbreeding avoidance in a natural house sparrow population

Inbreeding is common in small and threatened populations and often has a negative effect on individual fitness and genetic diversity. Thus, inbreeding can be an important factor affecting the persistence of small populations. In this study, we investigated the effects of inbreeding on fitness in a small, wild population of house sparrows (Passer domesticus) on the island of Aldra, Norway. The population was founded in 1998 by four individuals (one female and three males). After the founder event, the adult population rapidly increased to about 30 individuals in 2001. At the same time, the mean inbreeding coefficient among adults increased from 0 to 0.04 by 2001 and thereafter fluctuated between 0.06 and 0.10, indicating a highly inbred population. We found a negative effect of inbreeding on lifetime reproductive success, which seemed to be mainly due to an effect of inbreeding on annual reproductive success. This resulted in selection against inbred females. However, the negative effect of inbreeding was less strong in males, suggesting that selection against inbred individuals is at least partly sex specific. To examine whether individuals avoided breeding with close relatives, we compared observed inbreeding and kinship coefficients in the population with those obtained from simulations of random mating. We found no significant differences between the two, indicating weak or absent inbreeding avoidance. We conclude that there was inbreeding depression in our population. Despite this, birds did not seem to actively avoid mating with close relatives, perhaps as a consequence of constraints on mating possibilities in such a small population.     

Genome-wide identification of SSR and SNP markers from the non-heading Chinese cabbage for comparative genomic analyses

Background

Non-heading Chinese cabbage (NHCC), belonging to Brassica, is an important leaf vegetable in Asia. Although genetic analyses have been performed through conventional selection and breeding efforts, the domestication history of NHCC and the genetics underlying its morphological diversity remain unclear. Thus, the reliable molecular markers representative of the whole genome are required for molecular-assisted selection in NHCC.

Results

A total of 20,836 simple sequence repeats (SSRs) were detected in NHCC, containing repeat types from mononucleotide to nonanucleotide. The average density was 62.93 SSRs/Mb. In gene regions, 5,435 SSRs were identified in 4,569 genes. A total of 5,008 primer pairs were designed, and 74 were randomly selected for validation. Among these, 60 (81.08%) were polymorphic in 18 Cruciferae. The number of polymorphic bands ranged from two to five, with an average of 2.70 for each primer. The average values of the polymorphism information content, observed heterozygosity, Hardy-Weinberg equilibrium, and Shannon’s information index were 0.2970, 0.4136, 0.5706, and 0.5885, respectively. Four clusters were classified according to the unweighted pair-group method with arithmetic average cluster analysis of 18 genotypes. In addition, a total of 1,228,979 single nucleotide polymorphisms (SNPs) were identified in the NHCC through a comparison with the genome of Chinese cabbage, and the average SNP density in the whole genome was 4.33/Kb. The number of SNPs ranged from 341,939 to 591,586 in the 10 accessions, and the average heterozygous SNPs ratio was ~42.53%. All analyses showed these markers were high quality and reliable. Therefore, they could be used in the construction of a linkage map and for genetic diversity studies for NHCC in future.

Conclusions

This is the first systematic and comprehensive analysis and identification of SSRs in NHCC and 17 species. The development of a large number of SNP and SSR markers was successfully achieved for NHCC. These novel markers are valuable for constructing genetic linkage maps, comparative genome analysis, quantitative trait locus (QTL) mapping, genome-wide association studies, and marker-assisted selection in NHCC breeding system research.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1534-0) contains supplementary material, which is available to authorized users.     

Establishment of paternity testing system using microsatellite markers in Chinese Holstein

To estimate the efficiency of microsatellite markers in paternity testing among Chinese Holstein, 30 microsatellite loci were used to differentiate 330 Chinese Holstein genotypes, according to the calculation of the allele frequency, number of alleles, effective number of alleles, genetic heterozygosity, polymorphic information content (PIC), and the exclusion probability in this cattle population. The results demonstrated that the exclusion probability ranged from 0.620 in locus BM1818 to 0.265 in locus INRA005 with the average of 0.472 and 11 microsatellite markers exceeding 0.5. The combined exclusion probability of nine microsatellite markers was over 0.99. The result showed that paternity testing of Chinese Holstein was basically resolved using the nine microsatellite markers selected.     

Assessment of relationships between pigs based on pedigree and genomic information

Relationships play a very important role in studies on quantitative genetics. In traditional breeding, pedigree records are used to establish relationships between animals; while this kind of relationship actually represents one kind of relatedness, it cannot distinguish individual specificity, capture the variation between individuals or determine the actual genetic superiority of an animal. However, with the popularization of high-throughput genotypes, assessments of relationships among animals based on genomic information could be a better option. In this study, we compared the relationships between animals based on pedigree and genomic information from two pig breeding herds with different genetic backgrounds and a simulated dataset. Two different methods were implemented to calculate genomic relationship coefficients and genomic kinship coefficients, respectively. Our results show that, for the same kind of relative, the average genomic relationship coefficients (G matrix) were very close to the pedigree relationship coefficients (A matrix), and on average, the corresponding values were halved in genomic kinship coefficients (K matrix). However, the genomic relationship yielded a larger variation than the pedigree relationship, and the latter was similar to that expected for one relative with no or little variation. Two genomic relationship coefficients were highly correlated, for farm1, farm2 and simulated data, and the correlations for the parent-offspring, full-sib and half-sib were 0.95, 0.90 and 0.85; 0.93, 0.96 and 0.89; and 0.52, 0.85 and 0.77, respectively. When the inbreeding coefficient was measured, the genomic information also yielded a higher inbreeding coefficient and a larger variation than that yielded by the pedigree information. For the two genetically divergent Large White populations, the pedigree relationship coefficients between the individuals were 0, and 62 310 and 175 271 animal pairs in the G matrix and K matrix were greater than 0. Our results demonstrated that genomic information outperformed the pedigree information; it can more accurately reflect the relationships and capture the variation that is not detected by pedigree. This information is very helpful in the estimation of genomic breeding values or gene mapping. In addition, genomic information is useful for pedigree correction. Further, our findings also indicate that genomic information can establish the genetic connection between different groups with different genetic background. In addition, it can be used to provide a more accurate measurement of the inbreeding of an animal, which is very important for the assessment of a population structure and breeding plan. However, the approaches for measuring genomic relationships need further investigation.     

Genetic variation in eight Chinese cattle breeds based on the analysis of microsatellite markers

Genetic variability and genetic relationships were investigated among eight Chinese cattle breeds using 12 microsatellite markers. Three hundred and fifty-two alleles were detected and the average number of alleles per locus ranged from 8.33 ± 1.67 in the Jiaxian breed to 21.33 ± 5.60 in the Qinchuan breed with a mean value of 13.91. The total number of alleles per microsatellite ranged from 21 (INRA005, HEL1) to 40 (HEL13), with a mean of 29.33 per locus. The fixation indices at the 12 loci in the eight breeds were very low with a mean of 0.006. A principal components analysis and the construction of a neighborjoining tree showed that these eight Chinese cattle breeds cluster into three groups i.e. the Yanbian andChineseHolstein, theNanyang and Jiaxian, and the four remaining breeds.This clustering agrees with the origin and geographical distributions of these Chinese breeds.     

中国荷斯坦牛POU1F1基因与PRL基因的多态性及其聚合效应对产奶性状的影响

文章采用DNA测序、PCR-RFLP和CRS-PCR技术对979头中国荷斯坦牛POU1F1基因与PRL基因进行研究,发现了3个新SNPs,分别是POU1F1基因第二外显子G1178C、PRL基因5侧翼区A906G和A1134G。采用SAS统计软件GLM程序,利用最小二乘法拟合线性模型,分析基因多态性与产奶性状的关系。结果表明:POU1F1基因1178位点GC基因型在产奶量、乳蛋白量、乳脂量方面均为优良基因型。PRL基因5侧翼区906位点AG基因型在产奶量方面为优良基因型,1134位点不同基因型产奶性状差异不显著。对PRL基因5侧翼区的906位点和POU1F1基因的1178位点进行基因互作分析,结果在乳脂率、乳蛋白率、产奶量、乳蛋白量和乳脂量方面各基因型组合之间均未观察到显著差异,说明基因聚合效应并不是单基因效应的简单相加,基因聚合效应在分子育种中具有更重要的意义。