Marker-assisted conservation of European cattle breeds: An evaluation

Two methods have been developed for the assessment of conservation priorities on the basis of molecular markers. According to the Weitzman approach, contributions to genetic diversity are derived from genetic distances between populations. Alternatively, diversity within and across populations is optimized by minimizing marker-estimated kinships. We have applied, for the first time, both methods to a comprehensive data set of 69 European cattle breeds, including all cosmopolitan breeds and several local breeds, for which genotypes of 30 microsatellite markers in 25–50 animals per breed have been obtained. Both methods were used to calculate the gain in diversity if a breed was added to a set of nine non-endangered breeds. Weitzman-derived diversities were confounded by genetic drift in isolated populations, which dominates the genetic distances but does not necessarily increase the conservation value of a breed. Marker-estimated kinships across populations were less disturbed by genetic drift than the Weitzman diversities and assigned high conservation values to Mediterranean breeds, which indeed have genetic histories that differ from the non-endangered breeds. Prospects and limitations of marker-assisted decisions on conservation priorities are discussed.     

An assessment of European pig diversity using molecular markers: Partitioning of diversity among breeds

Genetic diversity within and between breeds (and lines) of pigs was investigated. The sample comprised 68 European domestic breeds (and lines), including 29 local breeds, 18 varieties of major international breeds, namely Duroc, Hampshire, Landrace, Large White and Piétrain, and 21 commercial lines either purebred or synthetic, to which the Chinese Meishan and a sample of European wild pig were added. On average 46 animals per breed were sampled (range 12–68). The genetic markers were microsatellites (50 loci) and AFLP (amplified fragment length polymorphism, 148 loci). The analysis of diversity showed that the local breeds accounted for 56% of the total European between-breed microsatellite diversity, and slightly less for AFLP, followed by commercial lines and international breeds. Conversely, the group of international breeds contributed most to within-breed diversity, followed by commercial lines and local breeds. Individual breed contributions to the overall European between- and within-breed diversity were estimated. The range in between-breed diversity contributions among the 68 breeds was 0.04–3.94% for microsatellites and 0.24–2.94% for AFLP. The within-breed diversity contributions varied very little for both types of markers, but microsatellite contributions were negatively correlated with the between-breed contributions, so care is needed in balancing the two types of contribution when making conservation decisions. By taking into account the risks of extinction of the 29 local breeds, a cryopreservation potential (priority) was estimated for each of them.     

Genetic distances within and across cattle breeds as indicated by biallelic AFLP markers

We tested the use of biallelic Amplified fragment length polymorphism (AFLP) polymorphisms for the estimation of relative genetic distances of cattle individuals within or across breeds. An allele permutation procedure was developed to estimate the stochastic variation of the genetic distance that is inherent to a given dataset. In a panel of 47 Holstein-Friesian cattle analysed with 248 polymorphic markers, the average genetic distance of bulls selected for breeding was slightly lower than the distance of the cows. The observed standard deviation (SD) of the distance indicated genetic subdivision, which for the bulls was explained by variation in the additive relationship derived from herdbook data. Animals from three different breeds, the highly selected Holstein-Friesian, the Italian Brown and the historic Maremmana, were compared on the basis of 106 polymorphic markers. No breed-specific fragments were observed. The mean pair-wise genetic distance within breeds was 85% of the value across breeds, but principal coordinates analysis clustered the animals according to their breed of origin. Calculation of distances between the breeds indicated a relatively divergent position of the Maremmana, relative to the two other breeds. However, biallelic markers indicate that the process of breed formation had only a limited effect on the diversity at marker loci.     

Differentiation of European cattle by AFLP fingerprinting

The Neolithic introduction of domestic cattle into Europe was followed by differential adaptation, selection, migration and genetic isolation, leading ultimately to the emergence of specialized breeds. We have studied the differentiation of European cattle by amplified fragment length polymorphism (AFLP) fingerprinting. Combining AFLP data sets from two laboratories yielded 81 biallelic polymorphic markers scored in 19-22 individual animals from 51 breeds. Model-based clustering differentiated Podolian cattle as well as French and Alpine breeds from other European cattle. AFLP genetic distances correlated well with microsatellite-based genetic distances calculated for the same breeds. However, the AFLP data emphasized the divergence of taurine and indicine cattle relative to the variation among European breeds and indicated an Eastern influence on Italian and Hungarian Podolian breeds. This probably reflects import from the East after the original introduction of domestic cattle into Europe. Our data suggest that Italian cattle breeds are relatively diverse at the DNA sequence level.     

24个中外猪种(群)的AFLP多态性及其群体遗传关系

利用12对AFLP引物组合检测了19个中国地方猪种（群）,1个培育猪种和4个欧美引进猪种混合基因组DNA的遗传变异,根据AFLP分析结果计算了24个猪种（群）间的遗传相似系数,据此构建了UPGMA聚类关系图,结果表明,AFLP标记有着很高的多态检测效率（Ai),平均每个引物组合检测到17.3个多态标记,非常适合于猪种（群）遗传多样性分析和品种鉴定;中国地方猪种与欧美引进猪种间的遗传分化十分明显,两类猪群间的亲缘关系较远;南昌白猪与大白猪,铅山黑猪与玉山黑猪有着极近的亲缘关系,分别与其育成历史,地理分布和RAPD分析结果相一致。另外还对部分猪种（群）的聚类分析结果与其形态学,地理分布和现行分类情况不相一致的原因进行了探讨。     

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.     

Extensive spatial genetic structure revealed by AFLP but not SSR molecular markers in the wind-pollinated tree, Fagus sylvatica

Studies of fine-scale spatial genetic structure (SGS) in wind-pollinated trees have shown that SGS is generally weak and extends over relatively short distances (less than 30-40 m) from individual trees. However, recent simulations have shown that detection of SGS is heavily dependent on both the choice of molecular markers and the strategy used to sample the studied population. Published studies may not always have used sufficient markers and/or individuals for the accurate estimation of SGS. To assess the extent of SGS within a population of the wind-pollinated tree Fagus sylvatica, we genotyped 200 trees at six microsatellite or simple sequence repeat (SSR) loci and 250 amplified fragment length polymorphisms (AFLP) and conducted spatial analyses of pairwise kinship coefficients. We re-sampled our data set over individuals and over loci to determine the effect of reducing the sample size and number of loci used for SGS estimation. We found that SGS estimated from AFLP markers extended nearly four times further than has been estimated before using other molecular markers in this species, indicating a persistent effect of restricted gene flow at small spatial scales. However, our SSR-based estimate was in agreement with other published studies. Spatial genetic structure in F. sylvatica and similar wind-pollinated trees may therefore be substantially larger than has been estimated previously. Although 100-150 AFLP loci and 150-200 individuals appear sufficient for adequately estimating SGS in our analysis, 150-200 individuals and six SSR loci may still be too few to provide a good estimation of SGS in this species.     

Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.     

Contrasting patterns of genetic diversity at three different genetic markers in a marine mammal metapopulation

Many studies use genetic markers to explore population structure and variability within species. However, only a minority use more than one type of marker and, despite increasing evidence of a link between heterozygosity and individual fitness, few ask whether diversity correlates with population trajectory. To address these issues, we analysed data from the Steller's sea lion, Eumetiopias jubatus , where three stocks are distributed over a vast geographical range and where both genetic samples and detailed demographic data have been collected from many diverse breeding colonies. To previously published mitochondrial DNA (mtDNA) and microsatellite data sets, we have added new data for amplified fragment length polymorphism (AFLP) markers, comprising 238 loci scored in 285 sea lions sampled from 23 natal rookeries. Genotypic diversity was low relative to most vertebrates, with only 37 loci (15.5%) being polymorphic. Moreover, contrasting geographical patterns of genetic diversity were found at the three markers, with Nei's gene diversity tending to be higher for AFLPs and microsatellites in rookeries of the western and Asian stocks, while the highest mtDNA values were found in the eastern stock. Overall, and despite strongly contrasting demographic histories, after applying phylogenetic correction we found little correlation between genetic diversity and either colony size or demography. In contrast, we were able to show a highly significant positive relationship between AFLP diversity and current population size across a range of pinniped species, even though equivalent analyses did not reveal significant trends for either microsatellites or mtDNA.     

Genetic diversity and relationships of Portuguese and other horse breeds based on protein and microsatellite loci variation

There are three native Portuguese horse breeds: Lusitano, Sorraia and Garrano. This study compares diversity patterns of 17 protein and 12 microsatellite markers in these three as well as 30 other breeds to infer relationships among the breeds and to compare levels of polymorphism of these breeds for use in conservation efforts. The Garrano and the Lusitano showed a high level of genetic diversity, similar to that observed for most of the other analysed breeds, while the Sorraia and Friesian breeds showed low levels of variation for both genetic marker types. The combined protein and microsatellite data produced a tree that fit historical records well and with greater confidence levels than those for either data set alone. The combined genetic diversity and relationship information provides important baseline data for future breed conservation efforts, especially for a critically endangered breed such as the Sorraia.     

Comparison of microsatellite and blood protein diversity in sheep: inconsistencies in fragmented breeds

Finnsheep, Romanov, Oxford Down and three local breeds from Finland or northwestern Russia were assessed at 15 microsatellite and 7 protein loci. A novel albumin allele was identified. Diversity patterns were mostly concordant between marker types, but discrepancies appeared for the local Viena and Vepsia sheep, both demonstrating frequent linkage disequilibria for both marker types and excess of homozygotes for microsatellites, and in the case of Vepsia also for proteins as signs of breed fragmentation. On the basis of microsatellite data, the neighbour-joining tree and two-dimensional map constructed from DA distances suggested that difference in longitude of breed origin would relate to breed relationship, whereas on the basis of protein data latitude would have this quality. These different impressions resulted because genetic distances involving Vepsia sheep were relatively low for protein variation compared with microsatellites. Microsatellite variation correlated positively with protein variation, but for the local Viena sheep protein variation was comparatively low. Populations had significant differences in allelic richness, but not in genetic diversity. Analysis implied that at least 30 polymorphic loci were needed to detect a difference in diversity between populations using a paired t-test, if the true mean diversity difference was 0.2. In the total sample, proteins demonstrated larger theta-values, but this was reversed for Finnsheep, for which model-based clustering of microsatellite genotypes revealed a structure associated with coat colour. Imported and rare sheep exhibited lowered allelic variability and increased frequency of pairwise disequilibria between unlinked markers. Our results emphasize that more loci are required for studying fragmented breeds.     

An empirical assessment of individual-based population genetic statistical techniques: application to British pig breeds

Recently developed Bayesian genotypic clustering methods for analysing genetic data offer a powerful tool to evaluate the genetic structure of domestic farm animal breeds. The unit of study with these approaches is the individual instead of the population. We aimed to empirically evaluate various individual-based population genetic statistical methods for characterization of genetic diversity and structure of livestock breeds. Eighteen British pig populations, comprising 819 individuals, were genotyped at 46 microsatellite markers. Three Bayesian genotypic clustering approaches, principle component analysis (PCA) and phylogenetic reconstruction were applied to individual multilocus genotypes to infer the genetic structure and diversity of the British pig breeds. Comparisons of the three Bayesian genotypic clustering methods (, and ) revealed some broad similarities but also some notable differences. Overall, the methods agreed that majority of the British pig breeds are independent genetic units with little evidence of admixture. The three Bayesian genotypic clustering methods provided complementary, biologically credible clustering solutions but at different levels of resolution. detected finer genetic differentiation and in some cases, populations within breeds. Consequently, it estimated a greater number of underlying genetic populations (K, in the notation of Bayesian clustering methods). Two of the Bayesian methods ( and ) and phylogenetic reconstruction provided similar success in assignment of individuals, supporting the use of these methods for breed assignment.     

Assessing genetic diversity in indigenous Veneto chicken breeds using AFLP markers

Genetic variation in four indigenous chicken breeds from the Veneto region of Italy was assessed using amplified fragment length polymorphism (AFLP) markers. A total of 99 individuals were analysed using three AFLP primer combinations that produced 70 polymorphisms. Four indigenous Veneto chicken breeds (Ermellinata, Padovana, Pépoi and Robusta) and a reference broiler line were included in the analysis. Breed-specific markers were identified in each breed. The expected heterozygosity did not differ significantly among the indigenous Veneto chicken breeds and the broiler line. The coefficient of gene variation (Gst) value across loci indicated that almost half of the total variability was observed among breeds. Nei's standard genetic distance between pairs of breeds showed that the distance between the broiler line and the Pépoi breed was greater than the distances between the broiler line and the other three chicken breeds. Cluster analysis based on standard genetic distances between breeds indicated that the Padovana and Pépoi breeds were closely related. Factorial analysis based on a binary matrix of the AFLP data showed a clear distinction of all breeds.     

Morphological and molecular diversity among populations of Quercus brantii Lindl. in western forest of Iran

Abstract

This study represents a preliminary step toward understanding the genetic structure of Persian oak in Iran. The genetic variability of Quercus brantii in Western forest of Iran was evaluated by amplified fragment length polymorphism (AFLP), chloroplast microsatellite and leaf morphology. Fifty-five trees from eight regions were sampled from across the range of Chaharmahal va Bakhtiari province of Iran. Twenty morphological traits were analyzed through clustering and ordination method. At morphological level, the applied statistics suggest that macromorphological traits significantly differentiate between populations. The overall sample shows a proportion of AFLP polymorphic markers of 92.1%, denoting a high level of variability. Based on AFLP data, differences among populations within geographic regions account for 11.6% of the total variation and only 0.57% is attributed to variation among regions. Based on chloroplast microsatellite (cpSSR), 34% of total variation was found among populations, suggesting a high within-population haplotype diversity. The dendrogram obtained from cpSSR showed a general pattern quite different from the pattern obtained from morphological analysis and AFLP markers.     

Relationships between the endangered Pustertaler-Sprinzen and three related European cattle breeds as analysed with 20 microsatellite loci

We estimated the genetic relationships between the endangered German Pustertaler-Sprinzen cattle breed and the Pinzgauer, Vosges and Simmental breeds--decided upon after consultation of the available historical literature. Within-breed diversity of the four breeds was also assessed. Twenty microsatellite markers were amplified in 27-50 unrelated individuals from populations of each breed. Within-breed variation was estimated from average heterozygosity values and mean number of alleles. Breed relationships were evaluated by genetic distance and a neighbour-joining tree was calculated from these estimates. Bootstrap resampling of loci tested the robustness of the tree topology obtained. A tree was also constructed from distance matrices using individual animals as operational taxonomic units. From both the average heterozygosity values and mean number of alleles calculated, the Pustertaler breed appears to be no more genetically impoverished than the other breeds analysed. The breed tree showed an 85% support for the Pustertaler-Pinzgauer grouping, and this result is echoed in the genetic distance values and allele-sharing individual tree.     

Assessing genetic diversity in Italian goat populations using AFLP® markers

Amplified fragment length polymorphism (AFLP) markers were used to investigate the genetic variation in a sample of seven goat (Capra hircus) populations. A total of 210 individuals (30 per population) were analysed using seven selected AFLP primer combinations that produced 219 clear polymorphisms. Four autochthonous goat breeds (Bionda dell'Adamello, Frisa, Orobica and Verzaschese), two primary populations, one from the Lombardy Alps (Val di Livo) and the other from Sardinia island (Sarda) and a reference cosmopolitan breed (Saanen) were included in the analysis. The expected heterozygosity (Het) did not differ significantly among breeds (range 0.21-0.24). No breed specific markers were identified. The variability at AFLP loci was largely maintained within breeds, as indicated by the coefficient of genetic differentiation (Gst) value (0.11). Dice similarities calculated between pairs of individuals belonging to the same or to different breeds largely overlapped. Bootstrapping on markers indicated that the coefficient of variation (CV) of the genetic indexes tested decreases only marginally by adding markers over 100 AFLPs. Cluster analysis based on standard genetic distance between breeds indicates that Sarda is the most distant population, while Bionda, Frisa, Verzaschese and Val di Livo seem to be highly related populations. Interestingly, Saanen is closer than Orobica to the other four goat populations of the Lombardy Alps. Principal co-ordinates analysis based on Dice similarities confirms these observations. Genetic diversity of the goat populations investigated confirms what is expected on the basis of their geographical location. Results from Orobica are not correlated with geographical distances and may reflect undocumented migrations and gene flows and identify an original genetic resource.     

Genetic diversity studies of Kherigarh cattle based on microsatellite markers

We report a genetic diversity study of Kherigarh cattle, a utility draught-purpose breed of India, currently declining at a startling rate, by use of microsatellite markers recommended by the Food and Agriculture Organization. Microsatellite genotypes were derived, and allelic and genotypic frequencies, heterozygosities and gene diversity were estimated. A total of 131 alleles were distinguished by the 21 microsatellite markers used. All the microsatellites were highly polymorphic, with mean (±s.e.) allelic number of 6.24 ±1.7, ranging 4–10 per locus. The observed heterozygosity in the population ranged between 0.261 and 0.809, with mean (±s.e.) of 0.574 ±0.131, indicating considerable genetic variation in this population. Genetic bottleneck hypotheses were also explored. Our data suggest that the Kherigarh breed has not experienced a genetic bottleneck in the recent past.     

利用微卫星标记分析我国13个地方灰羽鹅品种的遗传多样性

我国地方鹅品种具有很多优良性状,但长期以来由于没有形成科学的选育制度和培育方法,品种的遗传多样性没有得到完整保存。为了深入了解我国地方鹅品种的遗传结构,使之得到更好的保护和利用,作者选用31个多态性较高的微卫星标记(其中19个是首次用磁珠富集法从AFLP片段中分离),检测了我国13个地方灰羽鹅(An-sercygnnoides)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)和群体间的遗传距离(DA)。结果表明:13个地方灰羽鹅品种中,平均多态信息含量为0.323–0.398,平均杂合度为0.4985–0.6727,各品种的杂合度都较高,最高的是狮头鹅(0.6727),最低的是雁鹅(0.4985)。用UPGMA法进行聚类分析的结果显示,13个品种被聚为4类:丰城灰鹅、武冈铜鹅、兴国灰鹅、狮头鹅、乌棕鹅、阳江鹅、马冈鹅、钢鹅、雁鹅聚为第1类;伊犁鹅自聚为第2类;长乐鹅、右江鹅聚为第3类;永康灰鹅自聚为第4类。本研究为鹅遗传育种提供了参考资料。     

DNA遗传标记在山羊遗传多样性研究上的应用

综述DNA遗传标记在山羊的遗传多样性分析上的应用,并分别叙述了RFLP、线粒体DNA多态性、RAID、微卫星标记、AFLP等几个方面目前在山羊上的研究。