Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools

In a project on the biodiversity of chickens funded by the European Commission (EC), eight laboratories collaborated to assess the genetic variation within and between 52 populations from a wide range of chicken types. Twenty-two di-nucleotide microsatellite markers were used to genotype DNA pools of 50 birds from each population. The polymorphism measures for the average, the least polymorphic population (inbred C line) and the most polymorphic population (Gallus gallus spadiceus) were, respectively, as follows: number of alleles per locus, per population: 3.5, 1.3 and 5.2; average gene diversity across markers: 0.47, 0.05 and 0.64; and proportion of polymorphic markers: 0.91, 0.25 and 1.0. These were in good agreement with the breeding history of the populations. For instance, unselected populations were found to be more polymorphic than selected breeds such as layers. Thus DNA pools are effective in the preliminary assessment of genetic variation of populations and markers. Mean genetic distance indicates the extent to which a given population shares its genetic diversity with that of the whole tested gene pool and is a useful criterion for conservation of diversity. The distribution of population-specific (private) alleles and the amount of genetic variation shared among populations supports the hypothesis that the red jungle fowl is the main progenitor of the domesticated chicken.     

我国地方鸡品种保种群微卫星多态性分析与分子标记档案的建立

利用20个微卫星标记对国家家禽品种资源基因库中保存的11个地方鸡品种保种群进行了遗传检测,计算各群体的等位基因频率、平均基因杂合度、平均多态信息含量及各群体间的遗传距离,并用类平均法进行聚类分析。研究结果表明：20个微卫星标记在11个地方鸡品种保种群共检测到176个等位基因,平均为8.8个,基因频率分布在0.013～0.838之间。检测到等位基因中,有45个等位基因为11个地方鸡品种所共有;11个地方鸡品种平均杂合度在0.6800～0.7432之间。其中藏鸡最高,为0.7432;狼山鸡最低,为0.6800;平均多态信息含量在0.6329～0.7023之间,均大于0.5,表现为高度多态性;11 个鸡品种聚为4类。丝羽乌骨鸡、茶花鸡、仙居鸡、藏鸡、萧山鸡聚为一类,鹿苑鸡、狼山鸡聚为一类,固始鸡、北京油鸡、大骨鸡聚为一类,河南斗鸡单独聚为一类;通过利用20个微卫星基因座检测不同世代群体中等位基因及其频率、群体基因平均杂合度和多态信息含量,建立地方鸡品种保种群微卫星标记档案,并分析世代间的差异,预期可以达到监测保种效果的目的。     

Genetic characterization of Bhutanese native chickens based on an analysis of Red Junglefowl (Gallus gallus gallus and Gallus gallus spadecieus), domestic Southeast Asian and commercial chicken lines (Gallus gallus domesticus)

The genetic diversity of Bhutanese chickens needs to be understood in order to develop a suitable conservation strategy for these birds in Bhutan. In this, work, we used microsatellite markers to examine the genetic diversity of Bhutanese chickens. Four Bhutanese chicken varieties (Black plumage, Frizzle, Naked neck and Red Junglefowl-like, corresponding to Yuebjha Narp, Phulom, Khuilay and Seim, respectively), two subspecies of Red Junglefowl (Gallus gallus gallus and Gallus gallus spadecieus), two varieties of Thai native chickens (Pradhu Hang Dam and Chee; Gallus gallus domesticus) representing the Southeast Asian domestic chicken, and two commercial lines (Broiler and Single Comb White Leghorn) were genotyped with 18 microsatellites that included 16 loci recommended by the FAO/ISAG for investigations of genetic variability in chickens. All loci were polymorphic, with the number of alleles ranging from six (MCW0111) to 23 (MCW0183). Substantial genetic variation was observed in all populations, with the Bhutanese native chicken Yuebjha Narp (Black plumage chicken) showing the lowest genetic variability. Despite extensive intrapopulation variation, the genetic differentiation among 10 populations was moderate. A neighbor-joining tree revealed the genetic relationships involved while principal component analysis showed that Bhutanese native chickens should be given priority in conservation efforts because of their genetic distinctiveness. Chee chickens are especially valuable as a reservoir of predomestic diversity, as indicated by their greater genetic variation and their position in the phylogenetic tree.     

Analysis of genetic diversity and phylogenetic relationships among red jungle fowls and Chinese domestic fowls

Genetic diversity and phylogenetic relationships among 568 individuals of two red jungle fowl subspe- cies (Gallus gallus spadiceus in China and Gallus gallus gallus in Thailand) and 14 Chinese domestic chicken breeds were evaluated with 29 microstaellite loci, the genetic variability within population and genetic differentiation among population were estimated, and then genetic diversity and phylogenetic relationships were analyzed among red jungle fowls and Chinese domestic fowls. A total of 286 alleles were detected in 16 population with 29 microsatellite markers and the average number of the alleles observed in 29 microsatellite loci was 9.86±6.36. The overall expected heterozygosity of all population was 0.6708±0.0251, and the number of population deviated from Hardy-Weinberg equilibrium per locus ranged from 0 to 7. In the whole population, the average of genetic differentiation among population, measured as FST value, was 16.7% (P<0.001), and all loci contributed significantly (P<0.001) to this differentiation. It can also be seen that the deficit of heterozygotes was very high (0.015) (P<0.01). Reynolds' distance values varied between 0.036 (Xiaoshan chicken-Luyuan chicken pair) and 0.330 (G. gallus gallus-Gushi chicken pair). The Nm value ranged from 0.533 (between G. gallus gallus and Gushi chicken) to 5.833 (between Xiaoshan chicken and Luyuan chicken). An unrooted consensus tree was constructed using the neighbour-joining method and the Reynolds' genetic distance. The heavy-body sized chicken breeds, Luyuan chicken, Xiaoshan chicken, Beijing Fatty chicken, Henan Game chicken, Huainan Partridge and Langshan chicken formed one branch, and it had a close genetic relationship between Xiaoshan chicken-Luyuan chicken pair and Chahua chicken-Tibetan chicken pair. Chahua chicken and Tibetan chicken had closer genetic relationship with these two subspecies of red jungle fowl than other domestic chicken breeds. G. gallus spadiceus showed closer phylogenetic relationship with Chinese domestic chicken breeds than G. gallus gallus. All 29 microstaellite loci in this study showed high levels of polymorphism and significant genetic differentiation was observed among two subspecies of red jungle fowl and 14 Chinese domestic chicken breeds. The evolutional dendrogram is as follows: evolutional breeds→primitive breeds (Chahua chicken and Tibetan)→red jungle fowl in China (G. gallus spadiceus)→red jungle fowl in Thailand (G. gallus gallus). The results supported the theory that the domestic fowls might originate from different subspecies of red jungle fowl and Chinese domestic fowls had independent origin.     

Development of microsatellite markers in Myotis sodalis and cross-species amplification in M. gricescens, M. leibii, M. lucifugus, and M. septentrionalis

The Indiana bat (Myotis sodalis) is a highly endangered vespertilionid bat whose distribution is associated with limestone caves in the eastern United States. We present nine new polymorphic nuclear microsatellite markers for Myotis sodalis developed using an enriched library method. A total of 62 M. sodalis from two populations were used to estimate genetic diversity parameters. In M. sodalis, the number of alleles observed for each locus ranged from 17 to 48 alleles and expected heterozygosity ranged from 0.894 to 0.973. The 9 microsatellite markers were also tested on M. gricescens, M. leibii, M. lucifugus, and M. septentrionalis. These polymorphic microsatellite markers provide a valuable tool for investigating the population genetics of these species and will provide important genetic data useful for the conservation and recovery of the endangered Indiana bat.     

Microsatellite markers for the driver ant Dorylus (Anomma) molestus

We report primer sequences for five novel polymorphic microsatellite loci that were developed for the African driver ant Dorylus (Anomma) molestus. The number of alleles in the studied population ranged from three to 10 with observed heterozygosities between 0.458 and 0.806. These microsatellite markers will be useful for the study of mating system evolution and the genetic structure of colonies and populations of army ants.     

A genome-wide set of 106 microsatellite markers for the blue tit (Cyanistes caeruleus)

We have characterized a set of 106 microsatellite markers in 26-127 individual blue tits (Cyanistes caeruleus), and assigned their location on the zebra finch (Taeniopygia guttata) and on the chicken (Gallus gallus) genome on the basis of sequence homology. Thirty-one markers are newly designed from zebra finch EST (expressed sequence tags) sequences, 22 markers were developed by others from EST sequences using different methods and the remaining 53 loci were previously designed or modified passerine markers. The 106 microsatellite markers are distributed over 26 and 24 chromosomes in the zebra finch and in the chicken genome respectively and the number of alleles varies between 2 and 49. Eight loci deviate significantly from Hardy-Weinberg equilibrium and show a high frequency of null alleles, and three pairs of markers located in the same chromosome appear to be in linkage disequilibrium. With the exception of these few loci, the polymorphic microsatellite markers presented here provide a useful genome-wide resource for population and evolutionary genetic studies of the blue tit, in addition to their potential utility in other passerine birds.     

Twelve novel microsatellite loci from an endangered marine fish species golden pompano Trachinotus blochii

The golden pompano (Trachinotus blochii) is a marine fish species in tropical regions. No information about genetic variation and population structure of wild populations is available. A first set of 12 polymorphic microsatellites isolated from this species were characterized. The number of alleles ranged from 5 to 14 with an average of 8.0 alleles per locus. All 12 markers conformed to HWE and were in linkage equilibrium. These 12 microsatellite markers supply the first set of co-dominant DNA markers for studying population genetics of the species T. blochii.     

Application of microsatellite markers developed for arvicoline species in a population genetic study of the root vole <Emphasis Type="Italic">Microtus oeconomus</Emphasis>

Using a root vole Microtus oeconomus (Pallas, 1776) population in NE Poland we applied 31 microsatellite markers previously developed for root voles and closely related species, with the aim to improve the population genetic tools in this species. Here we present 16 polymorphic microsatellite markers grouped into four sets suitable for simultaneous amplification and genetically sex identification in M. oeconomus. The number of alleles per locus in 227 individuals varied from 7 to 26 with a low frequency of null alleles, expected heterozygosity ranged from 0.758 to 0.927, and observed heterozygosity from 0.722 to 0.947. Two loci showed significant deviation from Hardy-Weinberg equilibrium (p<0.05) and all loci showed independent inheritance. We expect these markers to be useful for studies of genetic population structure and kinship of M. oeconomus populations.     

Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken

Background  

Domestication of chicken is believed to have occurred in Southeast Asia, especially in Indus valley. However, non-inclusion of Indian red jungle fowl (RJF), Gallus gallus murghi in previous studies has left a big gap in understanding the relationship of this major group of birds. In the present study, we addressed this issue by analyzing 76 Indian birds that included 56 G. g. murghi (RJF), 16 G. g. domesticus (domestic chicken) and 4 G. sonneratii (Grey JF) using both microsatellite markers and mitochondrial D-loop sequences. We also compared the D-loop sequences of Indian birds with those of 779 birds obtained from GenBank.     

Isolation and characterization of microsatellite loci from the endangered highlands scrub hypericum (Hypericum cumulicola)

We report the isolation of 19 primer pairs for amplification of polymorphic microsatellite loci for Hypericum cumulicola. These markers were evaluated in 24 individuals from one population; two to four alleles were detected per locus, and observed heterozygosity ranged from 0 to 0.5. Two loci demonstrated significant heterozygote deficiencies, possibly due to null alleles, and significant linkage disequilibrium was found between six pairs of loci. The remaining microsatellite loci will help determine if genetic differentiation is responsible for life‐history differences between natural and anthropogenically disturbed populations of H. cumulicola.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

Genome-wide cross-amplification of domestic sheep microsatellites in bighorn sheep and mountain goats

We tested for cross‐species amplification of microsatellite loci located throughout the domestic sheep (Ovis aries) genome in two north American mountain ungulates (bighorn sheep, Ovis canadensis, and mountain goats, Oreamnos americanus). We identified 247 new polymorphic markers in bighorn sheep (≥ 3 alleles in one of two study populations) and 149 in mountain goats (≥ 2 alleles in a single study population) using 648 and 576 primer pairs, respectively. Our efforts increased the number of available polymorphic microsatellite markers to 327 for bighorn sheep and 180 for mountain goats. The average distance between successive polymorphic bighorn sheep and mountain goat markers inferred from the Australian domestic sheep genome linkage map (mean ± 1 SD) was 11.9 ± 9.2 and 15.8 ± 13.8 centimorgans, respectively. The development of genomic resources in these wildlife species enables future studies of the genetic architecture of trait variation.     

Isolation and characterization of highly polymorphic microsatellite markers in Hypochaeris radicata (Asteraceae)

We developed five highly polymorphic dinucleotide microsatellite loci for the grassland species Hypochaeris radicata (Asteraceae). Polymorphism of these markers was examined in six populations in the Netherlands. All loci were polymorphic in all populations. The number of alleles per locus varied between 18 and 43. Expected heterozygosity was between 0.86 and 0.91. Cross‐species amplification was tested in six Hypochaeris species and was successful for three different loci in four species. These microsatellites are a useful tool in population genetic, dispersal and metapopulation studies or in testing levels of inbreeding.     

Development of microsatellite markers for Aquilegia buergeriana var. oxysepala (Ranunculaceae), a vulnerable Japanese herb

Aquilegia buergeriana var. oxysepala is a vulnerable herb, for which 20 microsatellite markers were developed. The applicability of these markers was confirmed by genotyping of 66 individuals collected from four natural populations in Japan. Of the 20 loci, 15 showed polymorphism. For the 15 polymorphic markers, the number of alleles per locus ranged from 2 to 12, and the expected and observed heterozygosities ranged from 0.13 to 0.77 and 0.02 to 0.65, respectively. The markers described here are sufficiently polymorphic and informative to investigate the genetic diversity and population genetic structure of A. buergeriana var. oxysepala.     

Isolation and characterization of microsatellite markers from Brown Eared-pheasant (Crossoptilon mantchuricum)

The Brown Eared-pheasant (Crossoptilon mantchuricum) is an endangered species endemic to China. Here we developed eight microsatellite loci using a modified biotin-capture method. In the analyses of 28 individuals sampled, the number of alleles per locus ranged from four to nine. The expected (H _E) and observed (H _O) heterozygosities were 0.466–0.825 and 0.619–0.847, respectively. Results that eight microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as genetic diversity and population genetic structure of C. mantchuricum.     

Isolation and characterization of EST‐derived microsatellite loci from the fungal wheat pathogen Phaeosphaeria nodorum

Eleven polymorphic microsatellite loci and one minisatellite locus originating from expressed sequence tag (EST) libraries of Phaeosphaeria (syn. Stagonospora) nodorum were isolated and characterized. The satellite markers were used to genotype isolates from field populations collected in China, North America and South Africa. The number of alleles per locus ranged from two to 15. Genotype diversity ranged from 87.5 to 95.3 and gene diversity from 0.1 to 0.8. The variable levels of polymorphism within and among populations of P. nodorum renders these 12 satellite loci ideal markers for population genetic analysis of P. nodorum.     

Development and characterization of microsatellite loci in endangered Astrophytum asterias (Cactaceae)

We report the development of polymorphic microsatellite markers for the endangered North American cactus Astrophytum asterias (Cactaceae). Six loci, averaging 8.5 alleles per locus, were found to amplify genomic DNA consistently in 94 individuals from four geographically defined demes in South Texas. These markers will permit the generation of appropriate data for estimating population genetic parameters, population structure and the degree of inbreeding in the small, fragmented populations of A. asterias that currently exist. These are the first microsatellites reported for the genus Astrophytum and for the tribe Cacteae.     

Genetic analysis of salt-marsh sedge Carex scabrifolia Steud. populations using newly developed microsatellite markers

Nine microsatellite loci were isolated and characterized from the clonal salt-marsh sedge Carex scabrifolia, and genetic diversities within four populations were analyzed. The number of alleles per locus ranged from 2 to 7, with an average of 4.7. The observed and expected heterozygosities ranged from 0.000 to 1.000 and 0.000 to 0.679, respectively. In two populations, almost all polymorphic loci showed a significant excess of heterozygotes due to their high clonal reproduction and low number of genotypes (genets) composing the populations. Thus, the number of identical genets varied greatly among populations and ranged from 1 to 28, irrespective of population size (i.e., population area and ramet number). When attempting to conserve and restore clonal plants such as C. scabrifolia, it is important to preserve sufficient genetic diversity within a population, which can be assessed using genetic markers such as the simple sequence repeat markers described here.     

Development of microsatellite markers for southern catfish (Silurus meriaionalis) and cross‐species amplification in northern sheatfish (Silurus soldatovi)

Forty microsatellite markers were developed from a (CA)_n enrichment library created from the DNA of southern catfish (Silurus meriaionalis). Also, the population structure of northern sheatfish (Silurus soldatovi) was examined by 24 microsatellite loci. They are polymorphic in at least one of the two geographically distant populations sampled from the Heilongjiang River and the Songhuajiang River in North China, respectively. Unbiased expected heterozygosity levels varied from 0.435 to 0.946 and number of alleles per locus varied from three to 20. Results indicated that these microsatellite loci were highly polymorphic and could be used as genetic markers.