Microsatellite isolation, linkage group identification and determination of recombination frequency in the peach-potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)

Fifteen polymorphic microsatellite markers were used to establish linkage groups and relative rates of recombination in male and female Myzus persicae (Sulzer) (Hemiptera: Aphididae) (peach-potato aphid). We cloned nine markers from M. persicae and for these we report primer sequences and levels of allelic diversity and heterozygosity in four Australian M. persicae populations. Of the remaining six loci, four loci, also cloned from M. persicae, were obtained from G. Malarky (Natural History Museum, London) and two loci from Sitobion miscanthi were used. Additionally, the primer sequences of locus M77, a locus monomorphic in M. persicae but polymorphic in the closely related Myzus antirrhinii, are presented. Eleven of the 15 polymorphic markers were autosomal and four were X-linked. A linkage analysis was performed on a European pedigree of aphids containing five families with between seven and 11 offspring each. There was no linkage between any loci in females. In males, several pairwise comparisons yielded no recombinant offspring. With the exception of locus M40, these observations were supported in a linkage analysis performed on larger families produced from Australian M. persicae crosses. Locus M40 showed segregation consistent with involvement in a translocation between autosomes 1 and 3 in European samples but not in the Australian samples. From the Australian crosses we report an absence of recombination in males but high recombination rates in females. One X chromosome and four autosomal linkage groups were identified and tentatively assigned to chromosomes. The relevance of achiasmate meiosis to the evolution of sex is discussed.     

Environmentally related patterns of reproductive modes in the aphid Myzus persicae and the predominance of two 'superclones' in Victoria, Australia

Asexual organisms that naturally coexist with sexual relatives may hold the key to understanding the maintenance of sex and recombination, a long-standing problem in evolutionary biology. This situation applies to the peach-potato aphid, Myzus persicae, in southeastern Australia where cyclical parthenogens form mixed populations with obligate parthenogens. We collected M. persicae from several areas across Victoria, genotyped them at seven microsatellite loci and experimentally determined their reproductive mode. The geographic distribution of reproductive modes was correlated with two environmental variables that differentially affect obligate and cyclical parthenogens; obligate parthenogens were less frequent in areas with cold winters because they cannot produce frost-resistant eggs while cyclical parthenogens were limited by the availability of their primary host, peach, on which sexual reproduction takes place. Clonal diversity increased with the proportion of cyclical parthenogens in a sample because they tended to have unique microsatellite genotypes, whereas many obligate parthenogens were copies of the same genotype. Two obligately asexual genotypes stood out as being very abundant and widespread, one constituting 24% and the other 17.4% of the entire collection. Both of these highly successful genotypes were present in the majority of all collection sites. Genetic population structure was weak, albeit significant, with a multilocus FST of only 0.021 when samples were reduced to only one representative of each genotype. Interestingly, obligate parthenogens were, on average, more heterozygous and exhibited larger allele size differences between the two alleles at individual loci than cyclical parthenogens. This striking pattern could result from hybridization, for which we have no evidence, or may reflect the previously proposed model of biased mutational divergence of microsatellite alleles within asexual aphid lineages.     

基于微卫星标记的桃蚜种群寄主遗传分化

桃蚜Myzus persicae(Sulzer)是寄主范围最广、危害最大的蚜虫种类之一。为了探明桃蚜在不同寄主上的遗传分化特点,采用微卫星分子标记技术,对西兰花、桃树、辣椒上的桃蚜种群进行遗传多样性和遗传结构研究。结果表明,在所选用的5个微卫星位点上共检测到38个等位基因,平均每个位点的等位基因数达到7.6个,桃树种群遗传多样性最高,这可能是因为各种夏寄主上的桃蚜迁回桃树上越冬,从而使多种等位基因和基因型得以聚集的原因。等位基因频率差异分析显示西兰花种群、桃树种群和辣椒种群两两之间(除了桃树06种群和辣椒06种群之间没有遗传分化外)都出现了明显遗传分化,相比之下桃树种群和辣椒种群的分化程度要比桃树种群和西兰花种群的分化程度低,这可能预示着西兰花寄主上的桃蚜正在向远离桃树和辣椒种群的方向进化。     

Separation of Myzus (Nectarosiphon) antirrhinii (Macchiati) from Myzus (N.) persicae (Sulzer) and related species in Europe (Homoptera: Aphididae)

Abstract. Multivariate morphometric analysis (canonical variates) was used to discriminate between closely related taxa within the Myzus persicae group. It is demonstrated that dark green, anholocyclic populations with a 2n=13 or 2n=14 karyotype in Europe, hitherto treated as a form or biotype of M.persicae, all conform to a discrete morphometric grouping and should therefore be treated as a separate taxonomic entity, permanently isolated from the M.persicae gene pool. It is suggested that this taxon was first described as Siphonophora antirrhinii by Macchiati in 1883. A discriminant function is provided to separate most individual apterae of antirrhinii from those of persicae. Myzus icelandicus Blackman sp.n., on Caryophyllaceae and other plants in Iceland, is distinguished from M.polaris H.R.L. and M.certus (Walker), and a key is given to apterous virginoparae of the species of the M.persicae group in Europe.     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers in Rhinanthus angustifolius and cross-species amplification

Fifteen polymorphic microsatellite loci were developed from an enriched genomic library of the annual plant Rhinanthus angustifolius and characterized using 36 individuals. These markers provided high polymorphism ranging from two to 15 alleles per locus. Four loci showed significant departure from Hardy-Weinberg equilibrium, probably because of the occurrence of null alleles. No significant linkage disequilibrium was detected between pairs of loci. Tests of cross-species transferability were performed on four congeners with a success rate of 100% in Rhinanthus minor, 93% in R. mediterraneus and R. glacialis, and 80% in R. alectorolophus. These microsatellite loci will be useful tools to study mating system, gene flow and hybridization in the genus Rhinanthus.     

犬MC1R基因R306ter与毛色性状相关性研究

目的　分析犬MC1R基因中R30 6ter位点多态性与犬毛色表型的相关性 ,为遗传育种 ,培育出更加优良的实验用犬奠定基础。方法　采用PCR SSCP技术 ,对MC1R基因R30 6ter位点进行基因多态性检测 ,分析位点多态性与毛色性状之间的相关性 ,并对该位点进行克隆测序。结果　PCR SSCP分析结果表明 ,R30 6ter位点序列具有多态性 ,表现为C、D二个等位基因和CC、CD及DD三种基因型。对R30 6ter多态性片段克隆测序发现 ,MC1R基因在编码第 30 6位氨基酸的密码子处存在一个由CGA到TGA的终止突变。结论　经统计分析结果表明在杂种犬中MC1R基因多态性与毛色性状不存在显著的相关性 ,这可能是由于外科手术学教学用犬是杂种犬 ,其遗传背景不同所致。由于MC1R基因的R30 6ter位点内存在碱基变异 ,因此在杂种犬中表现出明显的PCR SSCP多态性     

Isolation and characterisation of di and tri nucleotide microsatellite loci in Rosmarinus officinalis (Lamiaceae), using enriched genomic libraries

An enrichment protocol was used to isolate and characterise microsatellite loci in Rosmarinus officinalis, a Mediterranean chamephyte. Twelve microsatellite loci were characterised and amplified a total of 117 alleles in a sample of 30 individuals from one population, with an average of 9.75 alleles per locus. Observed heterozygosities ranged from 0.333 to 0.900. Cross-species transferability was also assayed in the two other species of the genus. The cumulated probabilities of exclusion for paternity and parentage of the 12 loci were of 0.999971 and 1, respectively, supporting the usefulness of these microsatellite loci for parentage analyses. Nine out of 12 microsatellite loci amplified in the two species and were polymorphic detecting a total of 49 and 45 in R. eriocalyx and R. tomentosus, respectively. Twenty-two alleles were exclusive of R. eriocalyx and 12 of R. tomentosus, additionally, three alleles were shared between these two species but were otherwise absent in the analysed individuals of R. officinalis. In total, this set of markers amplified 154 different microsatellite alleles, supporting their usefulness to conduct population genetic, reproductive biology and hybridisation studies in Rosmarinus.     

Microsatellite genetic variation in small and isolated populations of Magnolia sieboldii ssp. japonica

Magnolia sieboldii ssp. japonica, distributed mainly in western Japan, is restricted to high elevation areas (1000-2000 m above sea level) and usually forms small isolated populations. Four microsatellite loci were assayed for 19 populations from six regions spanning the range of distribution, and the levels and distribution of genetic variation were estimated. All four loci were variable, with a total of 39 alleles, but the overall level of microsatellite genetic variation was low, especially compared with a related species, M. obovata. Genetic structure in M. sieboldii was characterised by low intrapopulational genetic variation (A = 3.74 and H(o) = 0.366 on average) and high genetic differentiation even among regional populations. Highly significant isolation-by-distance (IBD) models at the short distance were detected. Genetic drift and limited gene flow was considered to be important in determining the genetic structure within regions. Total genetic differentiation was remarkably high (F(ST) = 0.488 and R(ST) = 0.538), suggesting genetic barriers among regions. Neighbour-joining dendrograms relating the 19 populations, and further analysis on the IBD models, revealed that a stepwise mutation model was more suited than an infinite allele model to explain the genetic differentiation among regions. It is suggested that mutation at microsatellite loci might be influential in generating the genetic differentiation among regions. These results showed the potential of hypervariable microsatellite loci to evaluate the effects of genetic drift and population isolation within regions, and to detect genetic distinctiveness, in spite of the loss of overall genetic variation in M. sieboldii.     

Microsatellite Loci Polymorphism of Apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.) Genotypes with Different Ploidy Level

In this paper, the microsatellite (SSR) loci analysis was used to study apple genotypes with different levels of ploidy. A total of 47 samples were studied (9 diploids, 21 triploids, and 17 tetraploids) for seven microsatellite loci (GD147, Hi02C07, CH02c11, CH04c07, CH03d07, CH02c09, and GD12). It was possible to refine the pedigrees for some forms. It was established that the tetraploidss 20-9-30 and 20-9-27, selected in a hybrid family from the crossing of Wealthy 4x and Antonovka Obyknovennaya, were probably obtained from the self-pollination of the maternal form, since in the most loci they did not inherit alleles from the paternal form. As a result of the alleles distribution analysis, the spontaneous triploid cultivars Nizkorosloe and Sinap Orlovsky were revealed to be formed from the merge of an unreduced ovum and haploid pollen, since in the heterozygous loci both alleles are inherited from the maternal form and only one from the paternal form. According to the obtained data, studied tetraploids may be divided into two groups, which also reflect the features of tetraploids origin. The first group includes tetraploids inherited alleles from one initial diploid form (including spontaneous and induced tetraploids, as well as forms from self-pollination of the tetraploid maternal form). These teraploids, like diploids, amplify 1–2 alleles per locus (on average, for all 7 loci, one genotype amplifies 13 alleles). The second group includes tetraploids carrying alleles from several initial diploid forms. Tetraploids of this group are highly heterozygous and amplify 3–4 alleles at most loci (the maximum number of alleles at all loci, 24 alleles, was identified in the form 30-47-88). Tetraploids of the second group have a greater potential for the genetic diversity of its offspring. Analysis of polymorphism of microsatellite loci can be used (1) as an alternative or additional method for identifying the triploid hybrids from heteroploid crosses of orthoploid forms, which is based on the analysis of the loci most polymorphic in parental forms, and (2) for the analysis of true hybridity (verification of pedigrees), including tetraploid forms. Moreover, we identified the most polymorphic loci suitable for the above purposes. The aspects of qualitative and quantitative interpretation of the fragment analysis of microsatellite loci results are considered. The possibilities and limitations of the SSR analysis for detection of apple ploidy level are discussed.     

Microsatellite DNA marker inheritance indicates preferential pairing between two highly homologous genomes in polyploid and hemisexual dog-roses, Rosa L. Sect. Caninae DC

According to previous cytological evidence, the hemisexual dog-rose species, Rosa sect. Caninae, transmit only seven chromosomes (derived from seven bivalents) through their pollen grains, whereas egg cells contain 21, 28 or 35 chromosomes (derived from seven bivalents and 14, 21 or 28 univalents) depending on ploidy level. Two sets of reciprocal pairwise interspecific crosses involving the pentaploid species pair R. dumalis and R. rubiginosa, and the pentaploid/tetraploid species pair R. sherardii and R. villosa, were analysed for 13 and 12 microsatellite DNA loci, respectively. Single loci were represented by a maximum of three simultaneously occurring alleles in R. villosa, and four alleles in the other three parental plants. In the experimentally derived offspring, the theoretical maximum of five alleles was found for only one locus in the pentaploid progenies. Microsatellite DNA allele composition was identical with that of the maternal parent in 10 offspring plants, which were probably derived through apomixis. Almost all microsatellite DNA alleles were shared with the maternal parent also in the remaining offspring, but 1-4 alleles shared only with the paternal parent, indicating sexual seed formation. Analysis of quantitative peak differences allowed a tentative estimation of allelic configuration in the individual plants, and suggested that bivalent formation preferentially takes place between chromosomes that consistently share the same microsatellite alleles and therefore appear to be highly homologous. Moreover, alleles that were shared between the species in each cross combination comparatively often appear to reside on the bivalent-forming chromosomes, whereas species-specific alleles instead occur comparatively often on the univalent-forming chromosomes and are therefore inherited through the maternal parent only. Recombination then takes place between very similar genomes also in interspecific crosses, resulting in a reproduction system that is essentially a mixture between apomixis and selfing.     

Linked vs unlinked markers: multilocus microsatellite haplotype-sharing as a tool to estimate gene flow and introgression

We have explored the use of multilocus microsatellite haplotypes to study introgression from cultivated (Malus domestica) into wild apple (Malus sylvestris), and to study gene flow among remnant populations of M. sylvestris. A haplotype consisted of alleles at microsatellite loci along one chromosome. As destruction of haplotypes through recombination occurs much faster than loss of alleles due to genetic drift, the lifespan of a multilocus haplotype is much shorter than that of the underlying alleles. When different populations share the same haplotype, this may indicate recent gene flow between populations. Similarly, haplotypes shared between two species would be a strong signal for introgression. As the expected lifespan of a haplotype depends on the strength of the linkage, the length [in centiMorgans (cM)] of the haplotype shared contains information on the number of generations passed. This application of shared haplotypes is distinct from using haplotype-sharing to detect association between markers and a certain trait. We inferred haplotypes for four to eight microsatellite loci on Linkage Group 10 of apple from genotype data using the program phase, and then identified those haplotypes shared between populations and species. Compared with a Bayesian analysis of unlinked microsatellite loci using the program structure, haplotype-sharing detected a partially different set of putative hybrids. Cultivated haplotypes present in M. sylvestris were short (< 1.5 cM), indicating that introgression had taken place many generations ago, except for two Belgian plants that contained a haplotype of 47.1 cM, indicating recent introgression. In the estimation of gene flow, F(ST) based on unlinked loci indicated small (0.032-0.058) but statistically significant differentiation between some populations only. However, various M. sylvestris haplotypes were shared in nearly all pairwise comparisons of populations, and their length indicated recent gene flow. Hence, all Dutch populations should be considered as one conservation unit. The added value of using sharing of multilocus microsatellite haplotypes as a source of population genetic information is discussed.     

Microsatellite loci in the Japanese subterranean termite,Reticulitermes speratus

Reticulitermes termites have such a cryptic life style and complex colony structure that polymorphic microsatellite markers are desired to investigate their population and colony structures. We successfully isolated seven microsatellite loci in R. speratus , the Japanese subterranean termite, five of which were polymorphic. These polymorphic loci had 2–8 alleles per locus and their observed heterozygosities ranged from 0.059 to 0.438. These five loci were also polymorphic in R . kanmonensis , distributed sympatrically with R. speratus in the Kanmon Region, western Japan; the number of alleles per locus was 2–5, and observed heterozygosity was 0.176–0.625.     

Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers.

Microsatellites have many desirable marker properties and have been increasingly used in crop plants in genetic diversity studies. Here we report on the characterisation of microsatellite markers and on their use for the determination of genetic identities and the assessment of genetic variability among accessions from a germplasm collection of hop. Thirty-two polymorphic alleles were found in the 55 diploid genotypes, with an average number of eight alleles (3.4 effective alleles) for four microsatellite loci. Calculated polymorphic information content values classified three loci as informative markers and two loci as suitable for mapping. The average observed heterozygosity was 0.7 and the common probability of identical genotypes was 3.271 x 10(-4). An additional locus, amplified by one primer pair, was confirmed by segregation analysis of two crosses. The locus discovered was heterozygous, with a null allele in the segregating population. The same range of alleles was detected in nine triploid and five tetraploid hop genotypes. Cultivar heterozygosity varied among all 69 accessions, with only one cultivar being homozygous at four loci. Microsatellite allele polymorphisms distinguished 81% of all genotypes; the same allelic profile was found mainly in clonally selected cultivars. Cultivar-specific alleles were found in some genotypes, as well as a specific distribution of alleles in geographically distinct hop germplasms. The genetic relationship among 41 hop accessions was compared on the basis of microsatellite and AFLP polymorphisms. Genetic similarity dendrograms showed low correlation between the two marker systems. The microsatellite dendrogram grouped genetically related accessions reasonably well, while the AFLP dendrogram showed good clustering of closely related accessions and, additionally, separated two geographically distinct hop germplasms. The results of microsatellite and AFLP analysis are discussed from the point of view of the applicability of the two marker systems for different aspects of germplasm evaluation.     

Does insecticide resistance alone account for the low genetic variability of asexually reproducing populations of the peach-potato aphid Myzus persicae?

The typical life cycle of aphids includes several parthenogenetic generations and a single sexual generation (cyclical parthenogenesis), but some species or populations are totally asexual (obligate parthenogenesis). Genetic variability is generally low in these asexually reproducing populations, that is, few genotypes are spread over large geographic areas. Both genetic drift and natural selection are often invoked to account for this low genetic variability. The peach-potato aphid, Myzus persicae, which encompasses both cyclical and obligate parthenogens, has developed several insecticide resistance mechanisms as a consequence of intense insecticide use since the 1950s. We collected asexually reproducing M. persicae from oilseed rape and examined genetic variability at eight microsatellite loci and three insecticide resistance genes to determine whether their genetic structure was driven by drift and/or selection. We identified only 16 multilocus microsatellite genotypes among 255 individuals. One clone, which combined two insecticide resistance mechanisms, was frequently detected in all populations whatever their location over a large geographical area (the northern half of France). These unexpected findings suggest that drift is not the unique cause of this low variability. Instead, the intensification of both insecticide treatments and oilseed rape cultivation may have favored a few genotypes. Thus, we propose that selective pressures resulting from human activities have considerably modified the genetic structure of M. persicae populations in northern France in a relatively short period of time.     

Polymorphic microsatellite markers for the human oomycete pathogen Pythium insidiosum

We describe the development and characterization of microsatellite loci from the human oomycete pathogen Pythium insidiosum. Nine of 15 microsatellite loci were shown to be appropriate for population genetic study. All loci were polymorphic with observed heterozygosities ranging from 0.241 to 0.912 and from five to 18 alleles per locus among 65 individuals in Thailand. These markers are being used to ascertain multilocus genotypes for molecular epidemiological and population genetic analyses of this little known human pathogen.     

The use of microsatellite markers for detection of genetic diversity in barley populations

 A barley lambda-phage library was screened with (GA)n and (GT)n probes for developing microsatellite markers. The number of repeats ranged from 2 to 58 for GA and from 2 to 24 for GT. Fifteen selected microsatellite markers were highly polymorphic for barley. These microsatellite markers were used to estimate the genetic diversity among 163 barley genotypes chosen from the collection of the IPK Genebank, Germany. A total of 130 alleles were detected by 15 barley microsatellite markers. The number of alleles per microsatellite marker varied from 5 to 15. On average 8.6 alleles per locus were observed. Except for GMS004 all other barley microsatellite markers showed on average a high value of gene diversity ranging from 0.64 to 0.88. The mean value of gene diversity in the wild forms and landraces was 0.74, and even among the cultivars the gene diversity ranged from 0.30 to 0.86 with a mean of 0.72. No significant differences in polymorphism were detected by the GA and GT microsatellite markers. The estimated genetic distances revealed by the microsatellite markers were, on average , 0.75 for the wild forms, 0.72 for landraces and 0.70 among cultivars. The microsatellite markers were able to distinguish between different barley genotypes. The high degree of polymorphisms of microsatellite markers allows a rapid and efficient identification of barley genotypes. Received: 26 November 1997 / Accepted: 19 January 1998     

Ecological-genomic diversity of microsatellites in wild barley,Hordeum spontaneum,populations in Jordan

We analyzed the ecological-genomic diversity of microsatellites of wild barley, Hordeum spontaneum (C. Koch) Thell., at 18 loci in 306 individuals of 16 populations from Jordan across a southward transect of increasing aridity. The 18 microsatellites revealed a total of 249 alleles, with an average of 13.8 alleles per locus (range 3-29), with nonrandom distribution. The proportion of polymorphic loci per population averaged 0.91 (range 0.83-1.00); gene diversity, He, averaged 0.512 (range 0.38-0.651). We compared the number of alleles of the 18 loci to those found in Israel populations by Turpeinen et al. Out of the 280 alleles, 138 (49.3%) were unique (i.e. occurred in only one of the countries). The percentage of unique alleles in Jordan and Israel populations was 43.0% and 17.9%, respectively, suggesting that Jordan is an important center of origin and diversity of wild barley. Estimates of mean gene diversity were highest in the populations collected near the Golan Heights, such as Shuni North, Shuni South and Jarash. Sixty nine percent of the microsatellite variation was partitioned within populations and 31% between populations. Associations between ecogeographical values and gene diversity were established for eight microsatellite loci. The cluster produced by simple sequence repeat (SSR) data is mostly coincidence with the result of the dendrogram of the Spalax ehrenbergi superspecies of subterranean mole rats in Jordan based on allozyme gene loci. The major soil type in the wild barley habitat of each ecological group was different. Stepwise multiple regression analysis indicated that the variance of gene diversity was explained by altitude (R(2) = 0.362**). These observations suggest that microsatellites are at least partly adaptive and subject to natural selection.     

中国龙虾微卫星标记的筛选及遗传多样性分析

文章以M13通用引物和重复序列(CT)₁₅、(AT)₁₅引物, 利用PCR法对中国龙虾(Panulirus stimpsoni Hoehuis)部分基因组DNA文库进行筛选。共获得78个微卫星序列, 分别分布于55个阳性重组克隆中, 其中完美型(perfect)共50个, 占64%; 非完美型(imperfect)3个, 占3.8%; 混合完美型(compound perfect)6个, 占7.7%; 混合非完美型(compound imperfect)19个, 占24.5%。根据微卫星序列, 设计并筛选出15对微卫星多态性引物, 对中国龙虾的群体进行了遗传多样性分析。获得3~12个等位基因, 等位基因大小在78~425 bp之间, 基本符合引物设计的理论长度。期望杂合度范围为0.48~0.87, 平均值为0.71, 表明中国龙虾基因组微卫星具有较高的杂合度与遗传多样性。15个微卫星位点的PIC值从0.44到0.84, 平均值为0.60, 说明这些微卫星位点在中国龙虾基因组中包含丰富的遗传信息, 合适用于中国龙虾的各种分子标记及遗传学分析和应用。     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

Development and Characterization of 15 Polymorphic Micro satellite Markers for a Highly Adaptive and Wide-range Frog （Microhyla fissipes）

In order to analyze population genetic structure at multiple spatial scales, microsatellite loci were developed for the ornamented pygmy flog （Microhylafissipes）, and 15 polymorphic microsatellite loci were successfully screened from 105 individuals, of which 82 from four populations distributed in the Sichuan Basin and 23 from the Sangzhi population in western Hunan. Five loci were found to deviate significantly from Hardy-Weinberg equilibrium in one to three popu lations, probably due to small sample size or null alleles. The average number of alleles in all loci was 8.5, ranging from 4 to 13, and the observed and expected heterozygosity ranged from 0.26 to 0.90 and 0.63 to 0.90, respectively. The Sangzhi population and the remaining four populations can be clearly separated using Bayesian clustering methods, showing that the genetic structure of M. fissipes was probably affected by the topography, especially mountain barriers. These polymorphic microsatellite loci could be used for further study on the landscape genetics of this highly adaptive and widely distributed species.