Study of the Genetic Origin of the Mexican Creole Donkey (Equus asinus) by Means of the Analysis of the D-Loop Region of Mitochondrial DNA

The aim of this work was to analyse the genetic origin of the Mexican Creole donkey, as well as its genetic diversity, by comparison with Spanish and African donkey populations by means of the D-loop region of mitochondrial DNA. To this end, the genomic DNA of 68 Mexican Creole donkeys from eight geographical regions in six States of the Republic of México and from a Sicilian donkey was obtained. By the polymerase chain-reaction technique (PCR) a fragment of 541 bp was amplified, corresponding to the most informative region of the mitochondrial DNA, the D-loop. The fragments were subsequently sequenced. The analysed sequences revealed 10 new Mexican haplotypes that were different from those of the Spanish and African breeds with which they were compared, showing high levels of genetic diversity. Analysis of the phylogenetic relationships in the different Creole varieties showed a tendency of origin towards Spanish breeds, mainly the Andaluza, Zamorano-Leonesa and Majorera from the Canary Islands; these in turn showed an African origin, seven Mexican haplotypes and three haplotypes similar to those analysed by Aranguren and colleagues (2004) of Spanish and African breeds being obtained. This work allows us to reach the preliminary conclusion that the origin of Mexican Creole donkey populations in the different states of the Republic of México is clearly of Iberian origin, the Spanish donkey breed Andaluza being the main one contributing to the populations of the Mexican Creole donkeys, followed by the Spanish breeds Zamorano-Leonesa and Majorera from the Canary Islands, and that the populations possess high levels of genetic diversity.     

中国四个地方驴品种mtDNA D-Loop部分序列分析与系统进化研究

对中国4个家驴品种95个个体的mtDNAD—loop部分序列进行测序。用clustalX软件进行同源序列比对。Dnasp4-10软件用于遗传多样性分析，计算单倍型多样度、核苷酸多样度、平均核苷酸差异。MEGA3．1软件采用邻接法构建系统进化树并进行系统发生分析。以欧洲驴线粒体基因组为对照（Genbank登录号为X97337），中国家驴4个品种95个个体385bp序列共检测到核苷酸多态位点33个，其中31个位点为转换，1个位点为颠换，1个位点有插入现象。95个序列由31个单倍型组成，单倍型比例为32．6％。德州驴单倍型比例最高为100％，凉州驴单倍型比例最低为42．42％。31个单倍型与引用Genbank已登录的13条序列构建系统发育树，发现31个单倍型聚为两支，说明中国家驴可能有两个母系起源。以欧洲野驴线粒体D—loop为对照（登录号为AF403063、AF403063、AF403065），揭示本研究涉及的中国家驴品种与非洲野驴的进化亲缘关系较亚洲野驴近，并且从分子水平证明中国家驴可能起源于非洲野驴中的索马里野驴分支。     

Mitochondrial DNA variation and genetic relationships in Spanish donkey breeds (Equus asinus)

In this study, the mitochondrial DNA diversity of six Spanish donkey breeds and two African donkey populations (one from Morocco and the other from Zimbabwe) was analysed. A total of 79 animals were sequenced for 313 bp of the cytochrome b gene, and 91 individuals for 383 bp of the D‐loop region or control‐region. Sequence comparisons and phylogenetic analyses of both Spanish and African populations revealed low diversity. Only six and seven haplotypes respectively were found in cytochrome b and the D‐loop region. Relatively low nucleotide diversity (π) values were detected in these populations. The π values, from the D‐loop region, ranged from 0.0006 to 0.0169 for the Catalana and Andaluza breeds, respectively. The obtained results seem to confirm the existence of two divergent maternal lineages of African origin (Equus asinus africanus and E. a. somaliensis). In this paper the genetic relationships between these breeds are analysed and compared with those obtained in other European populations. Also, the data on the genetic relationships between the populations, obtained from nuclear DNA (microsatellites) and mitochondrial DNA (cytochrome b and D‐loop region) is argued and interpreted.     

六个家驴品种mtDNA D—Loop部分序列的遗传多样性分析

对中国的6个家驴品种(关中驴、德州驴、庆阳驴、泌阳驴、广灵驴、晋南驴)利用PCR-SSCP技术分析了其mtDNA D-Loop部分序列的遗传多样性。87个个体共检测到19个单倍型,单倍型比例为21.8%。单倍型多样度以关中驴、晋南驴较高,分别为0.834和0.904;其次为泌阳驴和广灵驴,分别为0.629、0.529;德州驴和庆阳驴较低,分别为0.467、0.599。文章从分子水平上揭示了家驴的遗传多样性,为我国家驴品种资源的评价和利用提供了一些依据。     

中国家驴mtDNA D-loop 遗传多样性与起源研究

对我国12个家驴品种126个个体（包括引用26个个体）的mtDNA D-loop区399 bp进行分析,共检测到36种单倍型37个多态位点,其单倍型多样度为0.466 7-0.977 8,核苷酸多样度为0.001 2-0.028 5,表明我国家驴的遗传多态性丰富。与3条努比亚野驴、3条索马里野驴和6条亚洲野驴的序列构建NJ系统发育树,首次证明我国家驴的母系起源为非洲野驴中的索马里驴和努比亚驴,亚洲野驴不是中国家驴的祖先。本文还讨论了我国家驴可能的迁徙路线。     

Genetic analysis of Mexican hairless pig populations.

The main Creole pig population in Mexico, the hairless Mexican pig, remains as an unimproved and endangered genetic resource. In order to learn more about the genetic characteristics of this pig population, we assessed the allele frequency of 10 microsatellite loci in 177 unrelated hairless pigs from seven regions at Mexico and in 111 pigs of four commercial breeds (Landrace, Large White, Hampshire, and Duroc). Genetic diversity in each population was estimated by the unbiased average heterozygosity and the allele number. Nei's standard genetic distances and a neighbor-joining dendrogram were used to reveal the genetic relationships among these populations. In this report, we present data showing that the level of the genetic diversity in Mexican hairless pigs is high compared with previous reports, and that they belong to a genetic lineage divergent from commercial breeds. Furthermore, Mexican hairless pigs seem to have developed several genetically distinct lines associated with their geographic location. We conclude that the Mexican Creole pig populations may be a reservoir of genetic diversity that is important to preserve and evaluate as a source of new alleles for the future improvement of commercial pig lines.     

中国家驴生长激素基因内含子2的遗传多样性分析

为了从分子水平揭示家驴的遗传多样性,本研究以中国7个家驴品种(临县驴、关中驴、新疆驴、广灵驴、淮北驴、德州驴、晋南驴)174个个体为试验动物,利用PCR-SSCP技术研究生长激素(GH)基因第2内含子的遗传多样性并进行序列分析.结果显示:第2内含子表现多态性,174个个体检测到3个单倍型,单倍型比例为1.7%.单倍型多样度以临县驴、淮北驴较高,分别为0.678和0.542.广灵驴与关中驴的单倍型多样度(Haplotype diversity,H)比较接近,分别为0.409和0.462.其次为德州驴和晋南驴,分别为0.355和0.304,新疆驴最低为0.077.对该片段的纯合型分别测序发现,B单倍型在第735位碱基G→C.A单倍型在869位碱基G→T.上述结果首次证实驴GH基因内含子2存在多态性.     

Genetic diversity of the Mexican Lidia bovine breed and its divergence from the Spanish population

Lidia bovine breed exists since the XIV century in the Iberian Peninsula. These animals were initially produced for meat but some, showing an aggressive behaviour which difficulted their management, were used to participate in popular traditional and social events. A specialization of the breed giving rise to the original Lidia population is documented in Spain since mid-XVIII century. Following the same tradition than in the Spanish population, Mexico used aggressive animals at the beginning of the XX century until two families of breeders started importing Lidia breed bovines from Spain with the aim of specializing their production. Each family (Llaguno and González) followed different breeding managements, and currently, most of the Lidia Mexican population derives from the Llaguno line. Although genetic structure and diversity of the Spanish population have been studied (using autosomal microsatellite markers, Y chromosome DNA markers and mitochondrial DNA sequences), the Mexican population is not analysed. The aim of the study was to assess both the genetic structure and diversity of the Mexican Lidia breed and its relationship with the original Spanish population using the same molecular tools. A total of 306 animals belonging to 20 breeders issued from both existing Mexican families were genotyped, and the genetic information was compared to the previously existing Spanish information. Slightly higher levels of genetic diversity in Mexican population were found when comparing to the Spanish population, and the variability among populations accounted for differences within them showing mean values of 0.18 and 0.12, respectively. Animals from the Mexican breeders, belonging to each of the two families, clustered together, and there was little evidence of admixture with the Spanish population. The analysis of Y chromosome diversity showed a high frequency of the H6 haplotype in the Mexican population, whereas this haplotype is rare in the Spanish, which is only found in the Miura (100%) and Casta Navarra (38%) lineages. Mitochondrial DNA revealed similar haplotypic pattern in both Spanish and Mexican populations, which is in accordance with most of the Mediterranean bovine breeds. In conclusion, as the Mexican Lidia population had initially a small number of founders and its current population has been reared isolated from their Spanish ancestors since a long time, these bottleneck effects and a combination of mixed cattle origin are the factors that might erase any trace of the Spanish origin of this population.     

新疆驴和青海驴MSTN基因多态性分析

 为揭示中国家驴 MSTN 基因的遗传多样性,以新疆驴和青海驴为研究对象,根据GenBank上公布的马的 MSTN 序列,设计9对特异性引物,利用驴混合DNA池为模板对该 MSTN 基因部分序列进行PCR扩增和测序分析。结果显示,扩增出的驴 MSTN 基因部分序列长度为3242 bp,包括5'-UTR区(671 bp)、第一外显子(373 bp)、第一内含子(1825 bp)和第二外显子(373 bp)。在该基因中共发现4个SNPs,分别为65 bp处T→Ｃ（5'-UTR）,872 bp处A→G（第一外显子）,2017 bp处G→A (第一内含子),2395处C→G突变（第一内含子）。利用PCR-RFLP技术对新疆驴和青海驴共计80个样本进行基因分型,4个突变位点共检测到6种单倍型（H1-H6）,其中H1是最主要的单倍型。两个家驴品种 MSTN 基因的遗传多样性很丰富（H=0.6044）,这对中国驴的遗传资源保护具有重要作用。     

中国9个家驴品种mtDNA D-loop部分序列分析与系统进化研究

本研究利用Dnasp4.10软件对中国9个家驴品种162个个体的mtDNA D-loop区385bp进行遗传多样性分析,共检测到32种单倍型35个核苷酸多态位点,其单倍型多样度为0.8137～0.9722,核苷酸多样度为0.0182～0.0270,表明我国家驴的遗传多态性丰富;利用MEGA3.1软件采用邻接法与3个努比亚野驴、3个索马里野驴和6个亚洲野驴的序列构建NJ系统发育树,并进行系统进化分析。结果表明:我国家驴的母系起源是非洲野驴中的努比亚野驴和索马里野驴,亚洲野驴不是中国家驴的母系祖先。     

关中驴线粒体DNA D-loop多态性分析

本文对 6头关中驴线粒体DNAD loop区 399bp的核苷酸序列进行了分析。结果发现 ,关中驴的D loop区核苷酸变异只有转换 1种形式。 6头关中驴D loop区的核苷酸序列组成 3种单倍型 ,单倍型比例为 5 0 .0 0 % ,说明关中驴mtDNA遗传多样性正逐步丧失 ,需要加强驴种质资源保护。以欧洲驴D loop序列为对照 ,关中驴 3种单倍型的核苷酸变异率分别为 4 .2 1%、4 .5 1%和 0 .2 5 %。在获得的 399bpD loop碱基序列中 ,共检测出核苷酸多态位点18个 ,多态位点比例为 4 .5 1%。从D loop区核苷酸序列的 3种单倍型分析 ,发现关中驴可能有 2种不同的母系起源     

中国地方猪种线粒体基因组遗传多样性及其系统发育分析

为从分子水平上探究中国地方猪种遗传多样性和分类地位,本试验采用生物信息学方法比较了6个类型共22个中国地方猪种的线粒体基因组全序列,分析了其多态性,并构建了6个类型猪种线粒体D-loop区单倍型的网络中介图以及基于线粒体D-loop序列、Cytb基因、完整编码区序列的系统进化树。结果表明,6个类型22个猪种中共检测到了144个多态位点,22种单倍型,说明地方猪种具有丰富的遗传多样性;地方猪线粒体基因组序列中核苷酸变异以转换为主,且Ti/Tv大于转换/颠换比临界值（2.0）,变异位点均符合中性突变。6个类型猪种间遗传距离均较小,且有共享单倍型。系统进化树结果表明,6种类型地方猪种主要聚为两个支系。表明线粒体D-loop序列及Cytb基因均可作为研究种内系统发育、起源进化的分子标记。     

江淮流域9个家鸭品种的分子生物地理演化过程分析

通过对江淮流域9个家鸭品种106个样本的线粒体D-loop区的序列分析，探讨了各品种体现在该特征的遗传多样性的现代分布格局及其地理演化过程。对检测出的31个单倍型的分析表明：长江中游地区的家鸭品种遗传多样性高于东部地区的家鸭品种。品种之间基因流同地理距离之间呈弱负相关。歧点分布分析和Tajima＇s D值中性检验符合中性突变，提示江淮流域鸭在过去没有出现群体扩张，群体大小稳定。各单倍型在最大简约树中的分布和实际地理分布无显著关系，各地理单元的单倍型都相互散布在不同的分布群中，呈现一种混杂的分布格局。     

Genetic Diversity and Phylogenetic Relationships of the Complete Mitochondrial Genome in Chinese Indigenous Pig Breeds

In order to explore the genetic diversity and the classification status of the Chinese domestic pigs at the molecular level, the complete mitochondrial DNA (mtDNA) genomic sequences of 22 Chinese indigenous pig breeds from 6 types were downloaded from GenBank for the analysis using the bioinformatics method. The polymorphism of nucleic acid sequences was analyzed,the molecular phylogenetic tree based on D-loop sequences, Cytb gene, complete coding region mtDNA sequences and the Median-Joining network of the mtDNA D-loop haplotypes were constructed for 22 pig breeds from 6 types. The results showed that a total of 144 mutation sites among 22 pig breeds were detected, 22 haplotypes were discovered, which indicated that Chinese indigenous pig breeds had abundant genetic diversity. According to the complete mtDNA genomic sequences analysis, which suggested that the main mutations was transition, the transition/transversion ratio was greater than 2.0 and all mutations were in line with the neutral mutation. Our findings clearly demonstrated that there was a near genetic distance among 6 types, meanwhile, the shared haplotypes were found. The phylogenetic tree showed that Chinese indigenous pigs from 6 types were diverged from two ancestors.The results indicated that mtDNA D-loop and Cytb gene might serve as molecular markers for phylogenesis, origin and evolution.     

Genetic characterization of the autochthonous sheep populations from Chiapas, Mexico

The Tzotziles, a Mayan native group located in Chiapas, Southern Mexico, have sheep breeding as one of their principal means of subsistence. Sheep were introduced in the Americas by the Spaniards in the first half of the XVI Century. Three populations of sheep from Chiapas—Café, Chamula and Chiapas—were typed with 27 microsatellites. Genetic distances were calculated for three Iberian breeds, Spanish Merino, Churra, two Canary Islands breeds, Canaria and Palmera, and the French Mutton Merino (Precoce). In the case of Chiapas sheep most of the markers showed Hardy–Weinberg equilibrium and the fixation index of Fst (0.095) showed a moderate level of genetic differentiation. The three distinct sheep subpopulations (Café, Chamula and Chiapas) are genetically differentiated inside the Chiapas sheep population. These breeds could be historically related to Spanish sheep populations, but have diverged significantly as a result of genetic drift and selection.     

Assessment of Sperm DNA Fragmentation in Stallion (Equus caballus) and Donkey (Equus asinus) Using the Sperm Chromatin Dispersion Test

Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.     

驴辅助繁育技术研究进展

目前,驴产业正在由短期育肥向繁育兼顾的新型可持续发展模式转变,同其他畜禽品种类似,驴产业的转型也是以扩大优良种群和提高品种质量为前提基础,而数量的增加和质量的提高都离不开良种繁育这一关键环节。然而,母驴繁殖力低是制约驴产业成功转型的主要瓶颈之一。驴繁殖力低不仅导致现有驴群的扩繁速度慢,存栏量与日益增长的皮、肉、奶的消费需求之间差距越来越大,而且使新品种的培育周期延长。已有研究表明,辅助繁育技术在提高母驴繁殖性能和改良驴的遗传性状等方面发挥重要作用,如同期发情、排卵控制、人工授精和胚胎移植等辅助繁殖技术可大幅提高母驴的繁殖能力;同时,微卫星标记技术、线粒体DNA测序技术、单核苷酸多态性研究和全基因组选择技术等分子育种手段也能够进一步加快驴的遗传改良进程。文章简要介绍了驴的生殖生理,系统阐述了同期发情、排卵控制、人工授精、胚胎移植、分子标记辅助育种和全基因组选择等辅助繁育技术在驴上的研究进展,并对未来研究方向进行了展望,以期对提高母驴繁殖性能提供借鉴,为开展驴分子育种研究提供有效线索。     

浙江省本地及引进鹅种遗传多样性研究

本研究利用PCR和DNA克隆测序技术扩增并分析了浙江省5个地方鹅种和1个引进家鹅品种96个个体的线粒体DNA D环区(D-loop)全序列。结果表明:浙江省境内的家鹅线粒体DNA D-loop全序长为1 166～1 179 bp,共发现89个变异位点,定义了36种单倍型,群体总核苷酸多样度达到了0.007 25,单倍型多样度为0.862,说明鹅群的遗传资源较为丰富,其中磐石灰鹅的遗传多样性最丰富,朗德鹅的遗传多样性最匮乏,而且没有发现朗德鹅与其他本地鹅种之间的显著差异。同时,个体聚类图中同品种内的个体没有聚在一起,说明浙江省鹅品种的母系来源比较混杂,品种选育偏重于父系,应同时结合基因组水平对其遗传结构进行分析。     

Genetic analysis of Mexican Criollo cattle populations

The objective of this study was to evaluate the genetic structure of Mexican Criollo cattle populations using microsatellite genetic markers. DNA samples were collected from 168 animals from four Mexican Criollo cattle populations, geographically isolated in remote areas of Sierra Madre Occidental (West Highlands). Also were included samples from two breeds with Iberian origin: the fighting bull (n = 24) and the milking central American Criollo (n = 24) and one Asiatic breed: Guzerat (n = 32). Genetic analysis consisted of the estimation of the genetic diversity in each population by the allele number and the average expected heterozygosity found in nine microsatellite loci. Furthermore, genetic relationships among the populations were defined by their genetic distances. Our data shows that Mexican cattle populations have a relatively high level of genetic diversity based either on the mean number of alleles (10.2-13.6) and on the expected heterozygosity (0.71-0.85). The degree of observed homozygosity within the Criollo populations was remarkable and probably caused by inbreeding (reduced effective population size) possibly due to reproductive structure within populations. Our data shows that considerable genetic differentiation has been occurred among the Criollo cattle populations in different regions of Mexico.