Molecular and genetic polymorphism in three cattle breeds

Determination of cattle breed and assignment of individual animals to that or another group based on distinctions of genotype is of great importance in genetics and selection. Using of PCR-analysis opened new possibilities for typing DNA. The procedure of PCR was optimized for animal DNA. Two methods of PCR, RAPD and SSRP, were applied for investigation of molecular-genetical polymorphism in three cattle breeds (Ukrainian red-motley lactescent, Golshtine and Simmental). A dendrogram reflecting genetical mutual relations between analyzed animals was drawn. The level of homo-heterozygosity and the distribution of microsatellite alleles were determined. System for investigation of molecular-genetical polymorphism in the cattle breeds and characterization of genotype of individual animals were developed and tested on a real population.     

Temporal changes in genetic variation of north European cattle breeds

Temporal changes in genetic variation within and between 13 North European cattle breeds were evaluated using erythrocyte antigen systems and transferrin protein as genetic markers. Current data on allele frequency distributions of markers in large commercial and smaller endangered native cattle breeds were compared to data published during 1956 to 1975. Intrabreed genetic variation was quantified by conventional parameters (e.g. heterozygosity, average number of alleles per locus) and migration by the effective migration rate. The neighbour-joining dendrogram of relationships between old and present cattle populations was constructed using Nei's standard genetic distance. Variance effective population size was estimated from changes in allele frequencies over time. Comparison of old and new data indicated some significant changes in allele frequencies. In six of the breeds, a few low-frequency alleles in the old data were absent in the present samples. Heterozygosity remained stable in most breeds. The harmonic means for variance effective population size ranged between 30 and 257. Current results indicate that despite marked declines in total population sizes, North European native cattle breeds have retained a reasonably high genetic diversity. However, their genes contribute less than previously to genetic variation of Nordic production breeds. Commercial breeds do not appear to have a larger effective population size than native breeds. The present effective population sizes imply that Nordic breeds could have lost from 1 to 11% of their heterozygosity over a 20-40-year period.     

Genetic variation of 5 SNPs of MC1R gene in Chinese indigenous sheep breeds

The purpose of this study was to assess genetic diversity, genetic differentiation relationship and population structure among 10 Chinese sheep populations using 5 single nucleotide polymorphisms (SNPs) in MC1R gene. The genetic diversity indices suggested that the intra-population variation levels of Chinese Merino and Large-tailed Han breeds were lowest than Kazakh Fat-Rumped. Chinese sheep breeds have maintained a high intra-population variation levels (95.23%). The genetic differentiation patterns and genetic relationships among Chinese sheep breeds displayed a high consistency with the traditional classification. The cluster trees were constructed by UPMGA method. The results showed that Chinese indigenous sheep populations have distinct genetic differentiation. The inter-population variation levels in Chinese sheep populations indicated three geographically independent domestication events have occurred. The Bayesian cluster analyses also showed a reliable clustering pattern, which revealed three major clusters in Chinese indigenous sheep populations (Mongolian group, Kazakh group and Tibetan group), except for Duolang and Minxian Black-fur. There were probably caused by different breeding history, geography isolation and different levels of inbreeding. The findings supported the related records in literature, ten sheep populations originated on different time stage from the primogenitor population and communicated genetically with each other in the process of natural and artificial selection, and in different ecological environment. It is concluded that Chinese indigenous sheep have higher genetic variation and diversity, genetic differentiation exist between Chinese sheep populations.     

湖北省三品种猪27个微卫星座位的遗传变异

采用国际动物遗传学会（ISAG）和联合国粮农组织（FAO）共同推荐的27个微卫星标记,对湖北省3个主要地方猪种（通城猪、清平猪和阳新猪）的遗传变异进行了检测。计算出各个品种的基因杂合度、各个座位的多态信息含量及品种间的遗传距离。结果表明,3个地方猪种的平均基因杂合度分别为0.7489、0.6987和0.6273,遗传多样性比较丰富;通城猪和清平猪亲缘关系较近,而两者与阳新猪亲缘关系略远。     

Genetic variation and relationships of eighteen Chinese indigenous pig breeds

Chinese indigenous pig breeds are recognized as an invaluable component of the world''s pig genetic resources and are divided traditionally into six types. Twenty-six microsatellite markers recommended by the FAO (Food and Agriculture Organization) and ISAG (International Society of Animal Genetics) were employed to analyze the genetic diversity of 18 Chinese indigenous pig breeds with 1001 individuals representing five types, and three commercial breeds with 184 individuals. The observed heterozygosity, unbiased expected heterozygosity and the observed and effective number of alleles were used to estimate the genetic variation of each indigenous breed. The unbiased expected heterozygosity ranged between 0.700 (Mashen) and 0.876 (Guanling), which implies that there is an abundant genetic variation stored in Chinese indigenous pig breeds. Breed differentiation was shown by fixation indices (F_IT, F_IS, and F_ST). The F_ST per locus varied from 0.019 (S0090) to 0.170 (SW951), and the average F_ST of all loci was 0.077, which means that most of the genetic variation was kept within breeds and only a little of the genetic variation exists between populations. The Neighbor-Joining tree was constructed based on the Nei D_A (1978) distances and one large cluster with all local breeds but the Mashen breed, was obtained. Four smaller sub-clusters were also found, which included two to four breeds each. These results, however, did not completely agree with the traditional type of classification. A Neighbor-Joining dendrogram of individuals was established from the distance of – ln(proportions of shared alleles); 92.14% of the individuals were clustered with their own breeds, which implies that this method is useful for breed demarcation. This extensive research on pig genetic diversity in China indicates that these 18 Chinese indigenous breeds may have one common ancestor, helps us to better understand the relative distinctiveness of pig genetic resources, and will assist in developing a national plan for the conservation and utilization of Chinese indigenous pig breeds.     

Quantification and cell-to-cell variation of vascular endothelial growth factor receptors

The vascular endothelial growth factor receptors (VEGFR) play a significant role in angiogenesis, the formation of new blood vessels from existing vasculature. Systems biology offers promising approaches to better understand angiogenesis by computational modeling the key molecular interactions in this process. Such modeling requires quantitative knowledge of cell surface density of pro-angiogenic receptors versus anti-angiogenic receptors, their regulation, and their cell-to-cell variability. Using quantitative fluorescence, we systematically characterized the endothelial surface density of VEGFRs and neuropilin-1 (NRP1). We also determined the role of VEGF in regulating the surface density of these receptors. Applying cell-by-cell analysis revealed heterogeneity in receptor surface density and VEGF tuning of this heterogeneity. Altogether, we determine inherent differences in the surface expression levels of these receptors and the role of VEGF in regulating the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) receptors.     

Population genetic analyses of seven Chinese indigenous chicken breeds in a context of global breeds

Jiangxi province in China is rich in indigenous chicken breeds, which have diverse phenotypes and represent a valuable genetic resource for further genetic improvement of modern breeds. Here, we conducted a series of analyses to reveal genetic diversity, phylogenetic relationships and population structure of seven chicken breeds in Jiangxi province in the context of nine non‐local chicken breeds, using 600K SNP data. We show that Jiangxi indigenous breeds have more abundant nucleotide diversity than do European local and commercial breeds. Among Jiangxi breeds, Dongxiang Blue‐eggshell (DX) and Chongren Partride (CR) display remarkably reduced genetic diversity, as the two breeds exhibit increased inbreeding coefficients, runs of homozygosity, extent of linkage disequilibrium and reduced expected heterozygosity. DX, CR and Taihe Silkie (TH) represent three ancestral lineages of the Jiangxi chicken and display genetic differentiation from the other four Jiangxi breeds, which show a signature of admixture with European commercial breeds. These findings provide insight for the establishment of an efficient conservation program for Jiangxi chicken breeds. Considering the current status of genetic diversity and ancestral representativeness, particular attention should be paid to DX, CR and TH chickens.     

Characterization of the <Emphasis Type="Italic">GHR</Emphasis> gene genetic variation in Chinese indigenous goat breeds

The aim of the present work was to investigate single nucleotide polymorphism (SNP) of growth hormone receptor (GHR) gene exon 10, characterize the genetic variation in three Chinese indigenous goat breeds, and search for its potential association with cashmere traits. In this study, a polymerase chain reaction-single strand conformation polymorphism (PCR–SSCP) protocol has been developed for rapid genotyping of the GHR gene in goats. One hundred seventy-eight goats from Liaoning Cashmere (96), Inner Mongolia White Cashmere (40), and Chengdu Grey (42) breeds in China were genotyped at GHR locus using the protocol developed. In all goat breeds investigated, a SNP in exon 10 of GHR gene has been identified by analyzing genomic DNA. The polymorphism consists of a single nucleotide substitution A → G, resulting in two alleles named, respectively, A and G based on the nucleotide at the position. The allele A was found to be more common in the animals investigated, and seems to be more consistent with cattle and zebu at this polymorphic site found in goats. The Hardy–Weinberg equilibrium of genotype distributions of GHR locus was verified in Liaoning Cashmere, and Inner Mongolia White Cashmere breeds. According to the classification of polymorphism information content (PIC), Chengdu Grey was less polymorphic than Liaoning Cashmere and Inner Mongolia White Cashmere breeds at this locus. The phylogenetic tree of different species based on the nucleotide sequences of GHR gene exon 10 is generally in agreement with the known species relationship. No significant association was found between the polymorphism revealed and the cashmere traits analyzed in present work.     

Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers

China is regarded as one of the domestication cen-ters for chickens and archaeological studies provided evidence of chicken domestication in northern Chinaas early as 6000 BC[1]. At present, China has the larg-est chicken population in the world, represen…     

Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers

China is rich in chicken genetic resources, and many indigenous breeds can be found throughout the country. Due to poor productive ability, some of them are threatened by the commercial varieties from domestic and foreign breeding companies. In a large-scale investigation into the current status of Chinese poultry genetic resources, 78 indigenous chicken breeds were surveyed and their blood samples collected. The genomes of these chickens were screened using microsatellite analysis. A total of 2740 individuals were genotyped for 27 microsatellite markers on 13 chromosomes. The number of alleles of the 27 markers ranged from 6 to 51 per locus with a mean of 18.74. Heterozygosity (H) values of the 78 chicken breeds were all more than 0.5. The average H value (0.622) and polymorphism information content (PIC, 0.573) of these breeds suggested that the Chinese indigenous chickens possessed more genetic diversity than that reported in many other countries. The fixation coefficients of subpopulations within the total population (F _ST) for the 27 loci varied from 0.065 (LEI0166) to 0.209 (MCW0078), with a mean of 0.106. For all detected microsatellite loci, only one (LEI0194) deviated from Hardy-Weinberg equilibrium (HWE) across all the populations. As genetic drift or non-random mating can occur in small populations, breeds kept on conservation farms such as Langshan chicken generally had lower H values, while those kept on large populations within conservation regions possessed higher polymorphisms. The high genetic diversity in Chinese indigenous breeds is in agreement with great phenotypic variation of these breeds. Using Nei’s genetic distance and the Neighbor-Joining method, the indigenous Chinese chickens were classified into six categories that were generally consistent with their geographic distributions. The molecular information of genetic diversity will play an important role in conservation, supervision, and utilization of the chicken resources.     

应用微卫星标记分析中国地方鸡种的遗传变异

利用 8个微卫星位点对中国 9个地方鸡种和 1个引进品种进行了遗传检测。计算出了各品种的平均杂合度、平均多态信息含量 (PIC)及品种间的遗传距离 ,并进行了系统聚类。结果表明 :8个微卫星位点上共检测到了5 4个等位基因 ,每个位点上平均为 6 .75个。各位点平均多态信息含量为 0 .5 0 71～ 0 .74 34,均表现出了高度多态性。各群体平均杂合度较高 ,为 0 .5 5 6 4～ 0 .7135 ,说明我国地方鸡种有着较丰富的遗传多样性。地方鸡种间的遗传距离相对较远 ,10个鸡种共分为三大类。研究结果对我国鸡种资源的评估、保存和预测杂种优势具有一定的指导意义     

Multiple trait genetic evaluation of clinical mastitis in three dairy cattle breeds

In 2010, a routine genetic evaluation on occurrence of clinical mastitis in three main dairy cattle breeds – Montbéliarde (MO), Normande (NO) and Holstein (HO) – was implemented in France. Records were clinical mastitis events reported by farmers to milk recording technicians and the analyzed trait was the binary variable describing the occurrence of a mastitis case within the first 150 days of the first three lactations. Genetic parameters of clinical mastitis were estimated for the three breeds. Low heritability estimates were found: between 2% and 4% depending on the breed. Despite its low heritability, the trait exhibits genetic variation so efficient genetic improvement is possible. Genetic correlations with other traits were estimated, showing large correlations (often>0.50, in absolute value) between clinical mastitis and somatic cell score (SCS), longevity and some udder traits. Correlation with milk yield was moderate and unfavorable (ρ=0.26 to 0.30). High milking speed was genetically associated with less mastitis in MO (ρ=−0.14) but with more mastitis in HO (ρ=0.18). A two-step approach was implemented for routine evaluation: first, a univariate evaluation based on a linear animal model with permanent environment effect led to pre-adjusted records (defined as records corrected for all non-genetic effects) and associated weights. These data were then combined with similar pre-adjusted records for others traits in a multiple trait BLUP animal model. The combined breeding values for clinical mastitis obtained are the official (published) ones. Mastitis estimated breeding values (EBV) were then combined with SCSs EBV into an udder health index, which receives a weight of 14.5% to 18.5% in the French total merit index (ISU) of the three breeds. Interbull genetic correlations for mastitis occurrence were very high (ρ=0.94) with Nordic countries, where much stricter recording systems exist reflecting a satisfactory quality of phenotypes as reported by the farmers. They were lower (around 0.80) with countries supplying SCS as a proxy for the international evaluation on clinical mastitis.     

采用微卫星标记分析13个中外牛品种的遗传变异和品种间的遗传关系

本研究应用联合国粮农组织(FAO)和国际动物遗传学会(ISAG)推荐的10对微卫星引物,结合荧光–多重PCR技术,检测了10个中国地方黄牛品种和3个外来牛品种的基因型。通过计算基因频率、多态信息含量和遗传杂合度,以Nei’s遗传距离和Nei’s标准遗传距离为基础,采用非加权组对算术平均聚类法构建了聚类图,分析了13个牛品种的群体内遗传变异和群体间遗传关系。并以聚类分析和群体结构分析为基础,将13个中外黄牛品种分为三类:Ⅰ类属于普通黄牛品种,包括延边牛、沿江牛、长白地方牛、蒙古牛、阿勒泰白头牛、哈萨克牛、复州牛和西藏牛;Ⅱ类属于含有瘤牛血统的黄牛品种,包括日喀则驼峰牛和阿沛甲咂牛;Ⅲ类属于外来牛品种,包括德国黄牛、西门塔尔牛和夏洛来牛。研究结果为加强我国地方黄牛品种种质特性研究以及地方牛品种资源的保护与利用提供了科学的依据。     

Genetic variations of the porcine PRKAG3 gene in Chinese indigenous pig breeds

Four missense substitutions (T30N, G52S, V199I and R200Q) in the porcine PRKAG3 gene were considered as the likely candidate loci affecting meat quality. In this study, the R200Q substitution was investigated in a sample of 62 individuals from Hampshire, Chinese Min and Erhualian pigs, and the genetic variations of T30N, G52S and V199I substitutions were detected in 1505 individuals from 21 Chinese indigenous breeds, 5 Western commercial pig breeds, and the wild pig. Allele 200R was fixed in Chinese Min and Erhualian pigs. Haplotypes II-QQ and IV-QQ were not observed in the Hampshire population, supporting the hypothesis that allele 200Q is tightly linked with allele 199V. Significant differences in allele frequencies of the three substitutions (T30N, G52S and V199I) between Chinese indigenous pigs and Western commercial pigs were observed. Obvious high frequencies of the "favorable" alleles 30T and 52G in terms of meat quality were detected in Chinese indigenous pigs, which are well known for high meat quality. However, the frequency of the "favorable" allele 199I, which was reported to have a greater effect on meat quality in comparison with 30T and 52G, was very low in all of the Chinese indigenous pigs except for the Min pig. The reasons accounting for this discrepancy remain to be addressed. The presence of the three substitutions in purebred Chinese Tibetan pigs indicates that the three substitutions were ancestral mutations. A novel A/G substitution at position 51 in exon 1 was identified. The results suggest that further studies are required to investigate the associations of these substitutions in the PRKAG3 gene with meat quality of Chinese indigenous pigs, and to uncover other polymorphisms in the PRKAG3 gene with potential effects on meat quality in Chinese indigenous pigs.     

Inhibition of vascular endothelial growth factor expression by TRUE gene silencing

Pathogenic angiogenesis in various diseases including cancer, autoimmune diseases, and age-related macular degeneration is thought to be regressed with anti-angiogenic drugs. TRUE gene silencing is a new technology to eliminate a specific mRNA using synthetic sgRNA and cellular tRNase Z^L. To discover anti-angiogenic sgRNAs, we applied TRUE silencing to the VEGF gene. We examined eight sgRNAs for efficacy in targeting exogenous human VEGF mRNA. Many of them worked efficiently in 293 and HeLa cells. Two of them downregulated the endogenous VEGF gene expression in HeLa cells very efficiently, and the efficacy of these two sgRNAs surpassed that of siRNA extremely.     

微卫星标记对12个中外牛品种群体遗传结构的研究

选用联合国粮农组织(FAO)和国际动物遗传学会(ISAG)推荐的12对微卫星引物, 采用荧光标记–多重PCR技术, 检测了9个中国地方黄牛品种和3个外来牛品种的遗传多样性。利用等位基因频率计算出各群体的平均遗传杂合度(H)、多态信息含量(PIC)和群体间的DA及DS遗传距离。基于DA遗传距离, 用UPGMA法进行聚类分析, 结果12个中外牛品种被聚为4类: Ⅰ类属于南方黄牛品种, 包括恩施牛、黎平牛、昭通牛和川南山地牛; Ⅱ类属于中原黄牛品种, 包括郏县红牛、早胜牛和平陆山地牛; Ⅲ类属于北方黄牛, 包括延边牛和长白地方牛; Ⅳ类属于外来牛品种, 包括西门塔尔牛、夏洛来牛和德国黄牛。研究结果为中国地方牛品种的保护和利用提供了理论依据。     

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.     

Expression of the chick vascular endothelial growth factor D gene during limb development

Vascular endothelial growth factor D (VEGF-D) is a member of the VEGF/PDGF superfamily that has been implicated in angiogenesis and lymphangiogenesis. We have isolated a chick cDNA that shows homology with VEGF-D (also known as FIGF, c-fos-induced growth factor) of other species. Here, we describe the expression pattern of cVegf-D in chick embryos. In the limb buds, cVegf-D shows a dynamic expression pattern that is restricted to the mesenchyme of the posterior region. cVegf-D expression is also detected in the ectoderm and mesenchyme of the head region, somites, notochord and pharyngeal arches. We also report on the capability of Sonic hedgehog and retinoic acid to regulate cVegf-D expression.