Phylogenetic relationships of Asian and European pig breeds determined by mitochondrial DNA D-loop sequence polymorphism

Phylogenetic relationships among Asian and European pig breeds were assessed using 1036 bp of mitochondrial DNA (mtDNA) D-loop sequences. An unweighted pair-group method with arithmetic mean (UPGMA) tree was constructed on the basis of maximum likelihood distances using sequences determined for three Cheju (Korea), 11 Chinese, one Westran (Australian feral origin) and two European pigs (Berkshire and Welsh), and also published sequences for four Japanese (including two Wild Boars), one Yucatan miniature, five European (including Large White, Landrace, Duroc, Swedish and Wild Boar) and two Meishan pigs. The Colombian collared peccary (Tayassu tajacu) sequence was also determined and used as an outgroup. The maximum parsimony with heuristic search method was used to determine bootstrap support values. Asian-type pigs clustered together (bootstrap support 33%), but were separate from European-type pigs that also clustered together (93%). The Westran pig, derived from the feral descendants of pigs inhabiting Kangaroo Island of South Australia, clustered with Asian pigs, demonstrating Asian origin of their mitochondria. Berkshire and Large White clustered with Asian pigs, indicating that Asian pigs were involved in the development of these breeds. Our findings clearly demonstrate that pigs indigenous to China, Korea and Japan are only recently diverged from each other and distinctly different from European-type pigs. European pig breeds consist of pigs with mitochondria of Asian and non-Asian type, some of which were formed from closely related maternal ancestors, if not from a single ancestor.     

The phylogeny of Chinese indigenous pig breeds inferred from microsatellite markers

A genetic study of 32 local Chinese, three foreign pig breeds [Duroc (DU), Landrace and Yorkshire], and two types of wild boar (Hainan and Dongbei wild boar) based on 34 microsatellite loci was carried out to clarify the phylogeny of Chinese indigenous pig breeds. The allele frequencies, effective numbers of alleles, and the average heterozygosity within populations were calculated. The results showed that the genetic variability of the Lingao pig was the largest, while the Jiaxing pig was the lowest. The greatest distance between domestic pigs was found between Shanggao and DU pig and the shortest was found between Wuzhishan and Lingao pig, respectively. A neighbour-joining tree constructed from Modified Cavalli-Sforza genetic distances divided Chinese pigs into two clusters; four subclusters were also identified. Our results only partly agree with the traditional types of classification and also provide a new relationship among Chinese local pig breeds. Our data also confirmed that Chinese pig breeds have a different origin from European/American breeds and can be utilized in programmes that aim to maintain Chinese indigenous pig breeds.     

Genetic diversity of eleven European pig breeds

A set of eleven pig breeds originating from six European countries, and including a small sample of wild pigs, was chosen for this study of genetic diversity. Diversity was evaluated on the basis of 18 microsatellite markers typed over a total of 483 DNA samples collected. Average breed heterozygosity varied from 0.35 to 0.60. Genotypic frequencies generally agreed with Hardy-Weinberg expectations, apart from the German Landrace and Schwäbisch-Hällisches breeds, which showed significantly reduced heterozygosity. Breed differentiation was significant as shown by the high among-breed fixation index (overall F_ST = 0.27), and confirmed by the clustering based on the genetic distances between individuals, which grouped essentially all individuals in 11 clusters corresponding to the 11 breeds. The genetic distances between breeds were first used to construct phylogenetic trees. The trees indicated that a genetic drift model might explain the divergence of the two German breeds, but no reliable phylogeny could be inferred among the remaining breeds. The same distances were also used to measure the global diversity of the set of breeds considered, and to evaluate the marginal loss of diversity attached to each breed. In that respect, the French Basque breed appeared to be the most "unique" in the set considered. This study, which remains to be extended to a larger set of European breeds, indicates that using genetic distances between breeds of farm animals in a classical taxonomic approach may not give clear resolution, but points to their usefulness in a prospective evaluation of diversity.     

Genetic diversity in Spanish donkey breeds using microsatellite DNA markers

Genetic diversity at 13 equine microsatellite loci was compared in five endangered Spanish donkey breeds: Andaluza, Catalana, Mallorquina, Encartaciones and Zamorano-Leonesa. All of the equine microsatellites used in this study were amplified and were polymorphic in the domestic donkey breeds with the exception of HMS1, which was monomorphic, and ASB2, which failed to amplify. Allele number, frequency distributions and mean heterozygosities were very similar among the Spanish donkey breeds. The unbiased expected heterozygosity (H_E) over all the populations varied between 0.637 and 0.684 in this study. The low G_ST value showed that only 3.6% of the diversity was between breeds (P < 0.01). Significant deviations from Hardy-Weinberg equilibrium were shown for a number of locus-population combinations, except HMS5 that showed agreement in all analysed populations. The cumulative exclusion probability (PE) was 0.999 in each breed, suggesting that the loci would be suitable for donkey parentage testing. The constructed dendrogram from the D_A distance matrix showed little differentiation between Spanish breeds, but great differentiation between them and the Moroccan ass and also with the horse, used as an outgroup. These results confirm the potential use of equine microsatellite loci as a tool for genetic studies in domestic donkey populations, which could also be useful for conservation plans.     

Genetic analysis of Sicilian autochthonous horse breeds using nuclear and mitochondrial DNA markers

Genetic diversity and relationship among 3 Sicilian horse breeds were investigated using 16 microsatellite markers and a 397-bp length mitochondrial D-loop sequence. The analysis of autosomal DNA was performed on 191 horses (80 Siciliano [SIC], 61 Sanfratellano [SAN], and 50 Sicilian Oriental Purebred [SOP]). SIC and SAN breeds were notably higher in genetic variability than the SOP. Genetic distances and cluster analysis showed a close relationship between SIC and SAN breeds, as expected according to the breeds' history. Sequencing of hypervariable mitochondrial DNA region was performed on a subset of 60 mares (20 for each breed). Overall, 20 haplotypes with 31 polymorphic sites were identified: A higher haplotype diversity was detected in SIC and SAN breeds, with 13 and 11 haplotypes respectively, whereas only one haplotype was found in SOP. These were compared with 118 sequences from GenBank. BLAST showed that 17 of the 20 haplotypes had been reported previously in other breeds. One haplotype, found in SIC, traces back to a Bronze Age archaeological site (Inner Mongolia). The 3 Sicilian breeds are now rare, and 2 of them are officially endangered. Our results represent a valuable tool for management strategies as well as for conservation purposes.     

家兔遗传多样性及系统发生关系的微卫星分析

为了揭示我国家兔资源的遗传现状,本文从30个微卫星位点中筛选出10个位点,对22个家兔品种(系)的遗传变异进行了分析.结果表明:22个家兔品种(系)在10个微卫星座位上的平均有效等位基因数为3.O-4.6;平均多态信息含量在0.4997-0.7009之间,多为高度多态,说明家兔有丰富的遗传多样性.各品种之间的遗传距离(Ds)较近,在0.0087-1.2712之间,其中以美系獭兔和德系獭兔间距离最近,为0.0087.德国大白兔与长毛兔之间距离最远,为1.2712.根据遗传距离做聚类图,可以看出中国22个家兔品种被分为三大类:吉戎-Ⅰ系和吉戎-Ⅱ系成一类,塞北兔、哈白兔聚为一类,其他品种聚为一大类.该研究结果将为中国家兔品种的保护和利用提供重要的科学依据.     

Genetic diversity of Guangxi chicken breeds assessed with microsatellites and the mitochondrial DNA D-loop region

The domestic chicken (Gallus gallus domesticus) is an excellent model for genetic studies of phenotypic diversity. The Guangxi Region of China possesses several native chicken breeds displaying a broad range of phenotypes well adapted to the extreme hot-and-wet environments in the region. We thus evaluated the genetic diversity and relationships among six native chicken populations of the Guangxi region and also evaluated two commercial breeds (Arbor Acres and Roman chickens). We analyzed the sequences of the D-loop region of the mitochondrial DNA (mtDNA) and 18 microsatellite loci of 280 blood samples from six Guangxi native chicken breeds and from Arbor Acres and Roman chickens, and used the neighbor-joining method to construct the phylogenetic tree of these eight breeds. Our results showed that the genetic diversity of Guangxi native breeds was relatively rich. The phylogenetic tree using the unweighed pair-group method with arithmetic means (UPGAM) on microsatellite marks revealed two main clusters. Arbor Acres chicken and Roman chicken were in one cluster, while the Guangxi breeds were in the other cluster. Moreover, the UPGAM tree of Guangxi native breeds based on microsatellite loci was more consistent with the genesis, breeding history, differentiation and location than the mtDNA D-loop region. STRUCTURE analysis further confirmed the genetic structure of Guangxi native breeds in the Neighbor-Net dendrogram. The nomenclature of mtDNA sequence polymorphisms suggests that the Guangxi native chickens are distributed across four clades, but most of them are clustered in two main clades (B and E), with the other haplotypes within the clades A and C. The Guangxi native breeds revealed abundant genetic diversity not only on microsatellite loci but also on mtDNA D-loop region, and contained multiple maternal lineages, including one from China and another from Europe or the Middle East.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Genetic variation in two conserved local Romanian pig breeds using type 1 DNA markers

Analysis of the genetic variation of an endangered population is an important component for the success of conservation. Animals from two local Romanian pig breeds, the Mangalitsa and Bazna, were analysed for variation at a number of genetic loci using PCR-based DNA tests. Polymorphism was assessed at loci which 1) are known to cause phenotypic variation, 2) are potentially involved in trait differences or 3) are putative candidate genes. The traits considered are disease resistance, growth, coat colour, meat quality and prolificacy. Even though the populations are small and the markers are limited to specific genes, we found significant differences in five of the ten characterised loci. In some cases the observed allele frequencies were interesting in relation to gene function and the phenotype of the breed. These breeds are part of a conservation programme in Romania and marker information may be useful in preserving a representative gene pool in the populations. The use of polymorphisms in type 1 (gene) markers may be a useful complement to analysis based on anonymous markers.     

中国5个地方牦牛品种遗传多样性的微卫星分析

本文以大额牛（Bos frontalis）为外群,应用16对微卫星DNA标记结合荧光-多重PCR技术,评估了5个中国地方牦牛（Bos grunniens）（帕里牦牛、斯布牦牛、西藏高山牦牛、麦洼牦牛和九龙牦牛）品种内遗传变异和品种间遗传关系。6个群体的16个微卫星座位上共检测到159个等位基因,其中有33个等位基因为5个牦牛品种所特有。6个群体的有效等位基因数（Ne）在2.2043-3.2754之间,平均杂合度（H）在0.4858-0.6153之间,平均多态信息含量（PIC）在0.4230-0.5711之间。5个牦牛品种的微卫星座位有丰富的遗传多样性;而大额牛的遗传多样性相对较贫乏。5个牦牛群体间的遗传分化系数（Gst）为0.0527,表明牦牛亚群体间遗传分化水平很低。采用邻近结合法构建聚类图和模糊聚类分析表明,5个牦牛品种分为两大类,其中斯布牦牛、西藏高山牦牛、帕里牦牛和麦洼牦牛为一大类,九龙牦牛为一类。研究结果将为中国地方牦牛品种的保护和利用提供重要的理论依据。     

中国部分家养山羊mtDNA D环区遗传多样性与进化

对我国10个家养山羊品种140只个体的mtDNA D-loop区进行测序分析, 测得结果: 整个D-loop区为 1 211~1 213 bp, 共检测到84种单倍型, 171个多态位点。其中核苷酸多样性(Nucleotide diversity, Pi)为: 0.02063 ± 0.00225, 单倍型多样性(Haplotype gene diversity, Hd)为: 0.988 ± 0.003, 平均核苷酸差异数(Average number of nucleotide differences) k为: 24.896。表明我国家养山羊品种遗传多样性丰富。通过构建NJ网络进化树, 得出中国家养山羊主要分为两大支系, 并且其中一支与角骨羊(Capra aegagrus)聚在一起, 而旋角野山羊(Capra fal-coneri)单独聚为一支, 说明角骨羊对中国家养山羊贡献较大。     

Genetic relationships of five Indian horse breeds using microsatellite markers

The genetic relationships of five Indian horse breeds, namely Marwari, Spiti, Bhutia, Manipuri and Zanskari were studied using microsatellite markers. The DNA samples of 189 horses of these breeds were amplified by polymerase chain reaction using 25 microsatellite loci. The total number of alleles varied from five to 10 with a mean heterozygosity of 0.58 ± 0.05. Spiti and Zansakari were the most closely related breeds, whereas, Marwari and Manipuri were most distant apart with Nei's D_A genetic distance of 0.071 and 0.186, respectively. In a Nei's D_A genetic distances based neighbour joining dendrogram of these breeds and a Thoroughbred horse outgroup, the four pony breeds of Spiti, Bhutia, Manipuri and Zanskari clustered together and then with the Marwari breed. All the Indian breeds clustered independently from Thoroughbreds. The genetic relationships of Indian horse breeds to each other correspond to their geographical/environmental distribution.     

Phylogenetic relationships of Cheju horses to other horse breeds as determined by mtDNA D-loop sequence polymorphism

Historical records suggest that horses inhabiting the island of Cheju in Korea are descendants of Mongolian horses introduced in 1276. Other studies, however, suggest that horses may have been present on the island prior to the Mongolian introduction. To determine the origin of the Cheju horses we used a phylogenetic analysis of sequences of the mitochondrial DNA (mtDNA) D-loop region, including tRNA Pro and parts of tRNA thr and tRNA Phe sequences (1102-bp excluding the tandem repeat region). Maximum parsimony and neighbor-joining trees were constructed using sequences determined for seven Cheju, four Mongolian, one Przewalskii and two Chinese Yunnan horses, and published sequences for one Swedish and three Thoroughbred horses. Donkey mtDNA was used as an outgroup. We found that the mtDNA D-loop sequence varies considerably within Mongolian, Cheju and Thoroughbred horse breeds, and that Cheju horses clustered with Mongolian horses as well as with horses from other distantly related breeds. On the basis of these findings we propose that horses on Cheju Island are of mixed origin in their maternal lineage, and that horses may have existed and been traded on the island before the Mongolian introduction.     

应用微卫星标记分析23个中国地方马种的遗传多样性

为了调查中国马种的群体遗传分化与遗传结构状况, 本研究应用FAO和ISAG推荐的25对微卫星引物, 结合荧光标记PCR分析技术, 对中国23个马群体和1个英纯血马群体进行了分子遗传学研究。结果表明: 中国地方马群体的遗传多样性比较丰富, 23个中国马群体的等位基因数、多态信息含量和遗传杂合度等都高于英国纯血马。根据群体遗传距离构建的系统进化树, 能够清晰地将英纯血马从中国马中独立出来, 同时可将中国马群体分成不同的支, 基本上与其地理分布格局相符。我们用MVSP软件进行群体遗传分化分析可见, 前三个主成分的三维散点图可以明显地把英纯血马从所有群体中独立出来, 并把中国的马群体分成几个相对独立的支。进一步分析第一、二主成分的二维散点图, 可将中国的马群体分化成南方支系、藏马支系、新疆和青海支系、内蒙古支系及东北支系等5个部分。根据Structure软件分析, 推测中国马群体含有5个潜在的支系, 基本上代表了我国现在主要家马来源的基础遗传支系。这些信息可以为我国现有马种类型的划分与马种资源遗传多样性的保护提供科学依据。     

利用InDel标记分析中国香菇菌株的遗传多样性与群体结构

通过对5个香菇菌株重测序,以香菇L808-1菌株的全基因组序列为参考基因组,分析了这些菌株中插入/缺失（InDel）标记位点在香菇基因组10条染色体上的分布,并筛选了插入/缺失碱基数≥15的位点,合成了449对InDel标记引物。经过PCR和电泳检测,其中237对引物条带清晰。最终筛选出107个PIC≥0.3的标记作为核心InDel标记对来自国内的44份香菇菌株进行遗传多样性分析。聚类分析显示栽培菌株和野生菌株各自聚为一支,所选香菇菌株间存在明显的群体分层。群体结构分析显示香菇种质资源可分为4个亚群,主成分分析显示香菇菌株之间的位置及距离与聚类分析和群体结构分析结果相符。香菇InDel分子标记的开发与应用,为香菇核心种质的构建和种质资源育种引用提供了基础。     

Genetic diversity of Cheju horses (Equus caballus) determined by using mitochondrial DNA D-loop polymorphism

We used sequence polymorphism of the mitochondrial DNA D-loop (968 bp excluding the tandem repeat region) to determine genetic diversity of horses inhabiting Cheju (a southern island of Korea). Seventeen haplotypes with frequencies from 1.5 to 21.5% were found among 65 Cheju horse samples. Genetic diversity (h) of the 17 haplotypes was calculated to be 0.91, indicating that the extant Cheju horse population consists of diverse genetic groups in their maternal lineage. Phylogenetic analysis showed that 17 types of Cheju (D-loop sequences determined), 5 Mongolian, 6 Arabian, 3 Belgian, 2 Tsushima, 2 Yunnan, 1 Przewalskii, and 3 Thoroughbred horses (published sequences for the latter seven breeds) showed that Cheju horses were distributed into many different clusters in the tree. Four Mongolian horses clustered with separate Cheju horse groups, showing that some Cheju horses are clearly of Mongolian origin. The analysis of partial sequences (284 bp) of the D-loop of 109 horses showed that Thoroughbred, Mongolian, Lipizzan, and Arabian breeds are as diverse as Cheju horses. Our data together with others' suggest that most horse breeds tested with reasonably sufficient numbers of samples are diverse in their maternal lineages and also are not uniquely different from each other.     

Genetic diversity in Chinese modern wheat varieties revealed by microsatellite markers

Genetic diversity of 1680 modern varieties in Chinese candidate core collections was analyzed at 78 SSR loci by fluorescence detection system. A total of 1336 alleles were detected, of which 1253 alleles could be annotated into 71 loci. For these 71 loci, the alleles ranged from 4 to 44 with an average of 17.6, and the PIC values changed from 0.19 to 0.89 with an average of 0.69. (1) In the three genomes of wheat, the average genetic richness was B>A>D, and the genetic diversity indexes were B>D>A. (2) Among the seven homoeologous groups, the average genetic richness was 2=7>3>4>6>5>1, and the genetic diversity indexes were 7>3>2>4>6>5>1. As a whole, group 7 possessed the highest genetic diversity, while groups 1 and 5 were the lowest. (3) In the 21 wheat chromosomes, 7A, 3B and 2D possessed much higher genetic diversity, while 2A, 1B, 4D, 5D and 1D were the lowest. (4) The highest average genetic diversity index existed in varieties bred in the 1950s, and then it declined continually. However, the change tendency of genetic diversity among decades was not greatly sharp. This was further illustrated by changes of the average genetic distance between varieties. In the 1950s it was the largest (0.731). Since the 1960s, it has decreased gradually (0.711, 0.706, 0.696, 0.695). The genetic base of modern varieties is becoming narrower and narrower. This should be given enough attention by breeders and policy makers.     

Phylogenetic relationships of Northeast Asian cattle to other cattle populations determined using mitochondrial DNA D-loop sequence polymorphism

Phylogenetic relationships of Northeast Asian cattle to various other cattle breeds including Bos taurus, Bos indicus, and Bison bison were assessed using mtDNA D-loop sequences. A neighbor-joining tree was constructed using sequences determined for 4 Cheju Black, 4 Cheju Yellow, 4 Korean Yellow cattle (Bos taurus), and 2 American Brahman cattle (Bos indicus), and also published sequences for 31 Japanese Black cattle, 45 European breed cattle, 6 African zebus, 2 African taurines, and 6 Indian zebus. Five American bisons (Bison bison) were used as an outgroup. The neighbor-joining tree showed that American bisons and Indian zebus are clearly separate from other cattle breeds, respectively, and African cattle clustered together, although with a low bootstrap probability (<50%). Results indicate that cattle in Northeast Asia, Europe, and Africa are closely related to each other–suggesting their recent divergence, but are separate from Indian zebus.