Clonal composition of the peach-potato aphid Myzus persicae (Homoptera: Aphididae) in France and Scotland: Comparative analysis with IGS fingerprinting and microsatellite markers

Fourteen colonies of the peach‐potato aphid, Myzus persicae, were taken either from French peach trees or weeds in 2001. Thirty five apomictic parthenogenetic lineages (APLs) were established. Ribosomal DNA intergenic spacer (IGS) fingerprinting was used to characterise these and 28 fingerprints were duly obtained. Those lineages with different fingerprints were considered different genotypes and those with the same fingerprint as the same. The genetic identity of APLs was further tested using four microsatellite loci. APLs that differed by IGS fingerprint had distinct microsatellite allele combinations and those that had the same IGS fingerprint had the same microsatellite allele combinations. The results confirmed that IGS types corresponded to different aphid genotypes. Independent APLs with identical IGS and microsatellite genotype were therefore considered different representatives of the same clone. APLs from M. persicae found on Scottish crops in 1995, 1996 and 2001, as well as a long‐term laboratory line were also examined by the same methods. Their IGS fingerprints were similar or identical suggesting that they all belonged to the same clone. Microsatellite markers also suggested that these lineages were derived from a single clone. Some field lineages exhibited slight modifications to their IGS fingerprints confirming that the IGS evolves more rapidly than these microsatellite alleles. Thus, IGS will continue to provide a useful marker for aphid fieldwork.     

Multilocus microsatellite typing shows three different genetic clusters of Leishmania major in Iran

Ten polymorphic microsatellite markers were used to analyse 25 strains of Leishmania major collected from cutaneous leishmaniasis cases in different endemic areas in Iran. Nine of the markers were polymorphic, revealing 21 different genotypes. The data displayed significant microsatellite polymorphism with rare allelic heterozygosity. Bayesian statistic and distance based analyses identified three genetic clusters among the 25 strains analysed. Cluster I represented mainly strains isolated in the west and south-west of Iran, with the exception of four strains originating from central Iran. Cluster II comprised strains from the central part of Iran, and cluster III included only strains from north Iran. The geographical distribution of L. major in Iran was supported by comparing the microsatellite profiles of the 25 Iranian strains to those of 105 strains collected in 19 Asian and African countries. The Iranian clusters I and II were separated from three previously described populations comprising strains from Africa, the Middle East and Central Asia whereas cluster III grouped together with the Central Asian population. The considerable genetic variability of L. major might be related to the existence of different populations of Phlebotomus papatasi and/or to differences in reservoir host abundance in different parts of Iran.     

An optimized microsatellite marker set for detection of Metarhizium anisopliae genotype diversity on field and regional scales

Thirty-three Metarhizium anisopliae isolates sampled across Switzerland as well as 35 and 36 M. anisopliae isolates sampled from two field sites were assembled in three isolate collections. All isolates were analyzed using 27 newly developed and 14 previously published microsatellite markers. The 41 markers allowed for detection of 25 genotypes in the Swiss collection while 30 and 11 genotypes were detected in the two field collections. This indicated high genetic diversity on a regional as well as on a field scale. In order to improve genotyping efficiency, an optimized marker set, which allows discrimination of a large number of genotypes with as few markers as possible was developed. The optimized marker set consisted of 16 common markers, which provided resolution close to maximal resolution in all three collections (91–93 %). The results demonstrated that optimized marker sets have to be validated before large scale application to previously unassessed collections in order to avoid suboptimal resolution. This genetic tool will be valuable for analyses of genetic population structure of M. anisopliae in different habitats on a regional as well as on a field scale.     

Patterns of introduction and adaptation during the invasion of Aegilops triuncialis (Poaceae) into Californian serpentine soils

Multiple introductions can play a prominent role in explaining the success of biological invasions. One often cited mechanism is that multiple introductions of invasive species prevent genetic bottlenecks by parallel introductions of several distinct genotypes that, in turn, provide heritable variation necessary for local adaptation. Here, we show that the invasion of Aegilops triuncialis into California, USA, involved multiple introductions that may have facilitated invasion into serpentine habitats. Using microsatellite markers, we compared the polymorphism and genetic structure of populations of Ae. triuncialis invading serpentine soils in California to that of accessions from its native range. In a glasshouse study, we also compared phenotypic variation in phenological and fitness traits between invasive and native populations grown on loam soil and under serpentine edaphic conditions. Molecular analysis of invasive populations revealed that Californian populations cluster into three independent introductions (i.e. invasive lineages). Our glasshouse common garden experiment found that all Californian populations exhibited higher fitness under serpentine conditions. However, the three invasive lineages appear to represent independent pathways of adaptation to serpentine soil. Our results suggest that the rapid invasion of serpentine habitats in California may have been facilitated by the existence of colonizing Eurasian genotypes pre‐adapted to serpentine soils.     

Range‐wide population structure of European sea bass Dicentrarchus labrax

The euryhaline European sea bass Dicentrarchus labrax L., inhabiting the coasts of the eastern Atlantic Ocean and Mediterranean Sea, has had many opportunities for differentiation throughout its large natural range. However, evidence for this has been incompletely documented geographically and with an insufficient number of markers. Therefore, its full range was sampled at 22 sites and individuals were genotyped with a suite of mapped markers, including 14 microsatellite loci (N = 536) and 46 neutral or gene‐linked single nucleotide polymorphisms (SNPs; N = 644). We confirm that the Atlantic and Mediterranean basins harbour two distinct lineages. Within the Atlantic Ocean no pattern was obvious based on the microsatellite and SNP genotypes, except for a subtle difference between South‐eastern and North‐eastern Atlantic sea bass attributed to limited introgression of alleles of Mediterranean origin. SNP genotypes of the Mediterranean lineage differentiated into three groups, probably under the influence of geographical isolation. The Western Mediterranean group showed genetic homogeneity without evidence for outlier loci. The Adriatic group appeared as a distinct unit. The Eastern Mediterranean group showed a longitudinal gradient of genotypes and most interestingly an outlier locus linked to the somatolactin gene. Overall, the spatial pattern fits those observed with other taxa of between‐basin segregation and within‐basin connectivity, which concurs well with the swimming capabilities of European sea bass. Evidence from a few outlier loci in this and other studies encourages further exploration of its regional connectivity and adaptive evolution.     

Microsatellite markers for Sclerotinia subarctica nom. prov., a new vegetable pathogen of the High North

Eight polymorphic microsatellite loci were isolated from the ascomycete fungus Sclerotinia subarctica nom. prov. In Alaska, this pathogen causes white mold vegetable diseases sympatrically with the cosmopolitan and closely related Sclerotinia sclerotiorum. Eighteen alleles were observed across the 41 isolates tested and ranged from two to three alleles per locus. Together, the alleles from the eight polymorphic loci yielded only four haploid multilocus genotypes and exhibited significant linkage disequilibrium, reflecting extreme selfing and clonal vegetative reproduction.     

Rdr3, a novel locus conferring black spot disease resistance in tetraploid rose: genetic analysis, LRR profiling, and SCAR marker development

Black spot disease of rose, incited by the fungus Diplocarpon rosae, is found worldwide and is the most important disease of garden roses. A gene-for-gene interaction in this pathosystem is evidenced by the presence of pathogenic races of D. rosae and the previous discovery of a dominant resistance allele at the Rdr1 locus in the diploid Rosa multiflora. The objective of the present study was to genetically analyze resistances to North American black spot races 3, 8, and 9 previously reported in tetraploid roses. Resistance to North American races 3 and 8 segregated 1:1 in multiple F₁ populations, indicating that both are conferred by dominant alleles at single loci and are present in simplex (Rrrr) configuration. Gene pyramiding was demonstrated by combining both resistances into single genotypes. Resistance to race 9 was partial and segregated in a quantitative fashion. Analysis of these populations with microsatellite markers previously developed for Rdr1 revealed that the gene conferring race 3 resistance resides within the same R gene cluster as Rdr1. Race 8 resistance segregated independently and is, therefore, a novel locus for black spot resistance in rose which we have named Rdr3. NBS and LRR profiling were used in a bulked segregant analysis to identify a marker 9.1 cM from Rdr3, which was converted to a SCAR marker form for marker-assisted breeding.     

The genetic legacy of polyploid Bolivian Daphnia: the tropical Andes as a source for the North and South American D. pulicaria complex

We investigated genetic variation in asexual polyploid members of the water flea Daphnia pulex complex from a set of 12 Bolivian high-altitude lakes. We used nuclear microsatellite markers to study genetic relationships among all encountered multilocus genotypes, and combined this with a phylogenetic approach using DNA sequence data of three mitochondrial genes. Analyses of mitochondrial gene sequence divergence showed the presence of three very distinct clades that likely represent cryptic undescribed species. Our phylogenetic results suggest that the Daphnia pulicaria group, a complex of predominantly North American species that has diversified rapidly since the Pleistocene, has its origin in South America, as specific tests of topology indicated that all three South American lineages are ancestral to the North American members of this species group. A comparison between variation of nuclear and mitochondrial markers revealed that closely related polyploid nuclear genotypes sometimes belonged to very divergent mitochondrial lineages, while distantly related nuclear genotypes often belonged to the same mitochondrial lineage. This discrepancy suggests that these South American water fleas originated through reciprocal hybridization between different endemic, sexually reproducing parental lineages. It is also likely that polyploidy of the investigated lineages resulted from this hybridization. Nevertheless, no putative diploid parental lineages were found in the studied region.     

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     

Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene

Genetic studies of the protozoan parasite Toxoplasma gondii have identified three main distinct types according to virulence in some hosts. Several methods have been developed to differentiate genotypes currently dominated by microsatellite markers targeting single-copy loci. We analyzed the possibility of using the 35-fold repetitive B1 gene via high-resolution melting (HRM) curve analysis. Sequencing of the B1 gene of 14 reference strains (four Type I, six Type II, and four Type III strains) identified 18 single nucleotide polymorphisms (SNP). Primers were designed to amplify eight of them for HRM analysis and for relative quantification of each nucleotide variation using SNaPshot mini-sequencing. Genotyping with five microsatellite markers was performed for comparison. Two to four HRM profiles were obtained depending on the SNP tested. The differences observed relied on the different ratios of nucleotides at the SNP locus as evidenced via SNaPshot mini-sequencing. The three main lineages could be distinguished by using several HRM profiles. Some HRM profiles proved more informative than the analysis based on five microsatellite markers, showing additional differences in Type I and Type II strains. Using HRM analysis, we obtained at least an equally good discrimination of the main lineages than that based on five microsatellite markers.     

Genetic diversity, clonality and sexuality in Toxoplasma gondii

The majority of Toxoplasma gondii strains from a variety of human and animal sources have been grouped into three highly clonal but closely related lineages. The low occurrence of nucleotide differences among the three predominant lineages and their unusual dimorphic allelic composition suggest that they have arisen from a recent common ancestry. Less than 1% of the previously studied strains contain unique genotypes and high divergence of DNA sequence, and therefore are considered 'exotic' or 'atypical' strains. The seemingly low genetic diversity in T. gondii may have been underestimated because most parasite strains in previous studies were collected from human patients and domestic animals in North America and Europe. To investigate the genetic diversity of T. gondii, we analysed parasite strains isolated from remote geographical regions by multilocus microsatellite sequencing and phylogenetic analysis. The genetic diversity indices, the molecular analysis of microsatellite genotypes and the constructed phylogram considered together suggest that the global T. gondii population is highly diversified and not characteristic of a clonal organism. The most parsimonious hypothesis is that T. gondii presents a complex population structure with a mix of clonal and sexual propagation as a function of the environmental conditions. The comparison between domestic strains data on one hand and wild strains data on the other hand is in favour of more frequent sexual recombinations in wild environment even though Toxoplasma subpopulation in human and domestic animals is largely clonal.     

Transferability and Level of Heterozygosity of Microsatellite Markers in <Emphasis Type="Italic">Citrus</Emphasis> Species

Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.     

Inheritance and development of molecular markers linked to angular leaf spot resistance genes in the common bean accession G10909

Angular leaf spot (ALS) is one of the major diseases of the common bean (Phaseolus vulgaris L.). Different sources of resistance have been identified but few have been characterized. Studies were conducted to elucidate the inheritance of ALS resistance in the bean accession G10909 and to identify molecular markers linked to these genes. Evaluation of parental genotypes, F₁, F₂ and backcross to susceptible parent (Sprite) populations revealed that two dominant and complementary genes conditioned ALS resistance. Allelism tests showed that the ALS resistance genes in G10909 were different from those in the Mesoamerican cultivars Mexico 54, MAR 2, G10474 and Cornell 49-242. Three sequence-characterized amplified region (SCAR) markers, PF13₃₁₀, PF9₂₆₀ and OPE04₇₀₉, and a microsatellite, Pv-gaat001, segregated in coupling with the resistance genes in G10909. Pairwise segregation analysis revealed that markers PF13₃₁₀, PF9₂₆₀ and OPE04₇₀₉ were linked, while Pv-gaat001 segregated in a 9:3:3:1 ratio from all markers. Markers PF13₃₁₀, PF9₂₆₀ and OPE04₇₀₉ were mapped to linkage group B08, and segregated with resistance gene Phg _G10909B at 4.9, 7.4 and 9.9 cM, respectively. Pv-gaat001, previously mapped to linkage group B04, segregated with resistance gene Phg _G10909A at 13 cM. The potential utility of these markers to aid breeding for ALS resistance is discussed.     

Characterization of the genetic resources of redcurrant (Ribes rubrum: subg. Ribesia) using anchored microsatellite markers

 Redcurrant, Ribes rubrum L., germplasm was screened for molecular polymorphism using anchored microsatellite primers in polymerase chain reactions (PCRs). Eighty markers were detected using only three primers and 16 redcurrant genotypes were fingerprinted using these markers. The genetic relatedness of these genotypes, as determined by the anchored microsatellite markers, and the implications for redcurrant breeding programmes are examined. Received: 18 October 1997 / Accepted: 1 December 1997     

Development and characterization of microsatellite markers in Eucommia ulmoides Oliver (Eucommiaceae)

Twenty‐three microsatellite markers were developed from an AC‐enriched genomic library of Eucommia ulmoides, an economically important tree species for both herbal medicine and organic chemical industry in China. Nineteen microsatellite loci were found polymorphic by testing 36 individuals from 10 populations, with two to 14 alleles per locus. The expected heterozygosity ranged from 0.054 to 0.874. This set of microsatellite markers has provided a useful tool for the ongoing efforts in studying population genetic structure of E. ulmoides.     

Mapping and tagging of seed coat colour and the identification of microsatellite markers for marker-assisted manipulation of the trait in Brassica juncea

Microsatellite marker technology in combination with three doubled haploid mapping populations of Brassica juncea were used to map and tag two independent loci controlling seed coat colour in B. juncea. One of the populations, derived from a cross between a brown-seeded Indian cultivar, Varuna, and a Canadian yellow-seeded line, Heera, segregated for two genes coding for seed coat colour; the other two populations segregated for one gene each. Microsatellite markers were obtained from related Brassica species. Three microsatellite markers (Ra2-A11, Na10-A08 and Ni4-F11) showing strong association with seed coat colour were identified through bulk segregant analysis. Subsequent mapping placed Ra2-A11 and Na10-A08 on linkage group (LG) 1 at an interval of 0.6 cM from each other and marker Ni4-F11 on LG 2 of the linkage map of B. juncea published previously (Pradhan et al., Theor Appl Genet 106:607–614, 2003). The two seed coat colour genes were placed with markers Ra2-A11 and Na10-A08 on LG 1 and Ni4-F11 on LG 2 based on marker genotyping data derived from the two mapping populations segregating for one gene each. One of the genes (BjSC1) co-segregated with marker Na10-A08 in LG 1 and the other gene (BjSC2) with Ni4-F11 in LG 2, without any recombination in the respective mapping populations of 130 and 103 segregating plants. The identified microsatellite markers were studied for their length polymorphism in a number of yellow-seeded eastern European and brown-seeded Indian germplasm of B. juncea and were found to be useful for the diversification of yellow seed coat colour from a variety of sources into Indian germplasm.     

Hybridization between two sympatrically breeding species of fur seal at Iles Crozet revealed by genetic analysis

Introgressive hybridization occurs when closely related taxa overlap in distribution and is often associated with historically isolated populations coming into contact as a result of anthropogenic disturbance. There is evolutionary and conservation interest in detecting hybridization to determine its implications on future species composition, especially for threatened and recovering taxa such as subantarctic (Arctocephalus tropicalis) and Antarctic (A. gazella) fur seals, which were driven to the brink of extinction by human exploitation. Hybridization between these species has been reported at two locations and they breed sympatrically at a third site, Iles Crozet. While hybrid individuals have previously been identified based on phenotype, individuals can be difficult to classify based on these characteristics alone. Genotypic hybrid identification has been successful in several species, including fur seals. In this study we conducted an assignment test using microsatellite data to identify hybrids and to measure the frequency of hybridization at Iles Crozet. Samples were collected from 372 individuals and screened with 6 polymorphic microsatellite markers. MtDNA genotypes were also determined for individuals identified as hybrids or backcrosses based on microsatellite genotype. Phenotype, microsatellite and mtDNA genotype were then compared in order to identify hybrids. The results indicate that 1% of the population have hybrid genotypes and at a minimum, 2.4% of the population are backcrossed to parental species. We found that the two species are genetically distinct from one another and given the low rate of hybridization it is unlikely that they will fuse. These results suggest that there is a mechanism for species recognition that acts as a barrier to hybridization. It therefore seems unlikely that fur seals are threatened by significant introgression. Further investigation of fur seal mating systems would provide valuable insight into the mechanisms governing hybridization and species recognition in mate choice.     

Two Mitochondrial Barcodes for one Biological Species: The Case of European Kuhl's Pipistrelles (Chiroptera)

The Kuhl’s pipistrelle (Pipistrellus kuhlii) is a Western Palaearctic species of bat that exhibits several deeply divergent mitochondrial lineages across its range. These lineages could represent cryptic species or merely ancient polymorphism, but no nuclear markers have been studied so far to properly assess the taxonomic status of these lineages. We examined here two lineages occurring in Western Europe, and used both mitochondrial and nuclear markers to measure degrees of genetic isolation between bats carrying them. The sampling focused on an area of strict lineage sympatry in Switzerland but also included bats from further south, in North Africa. All individuals were barcoded for the COI gene to identify their mitochondrial lineages and five highly polymorphic microsatellite loci were used to cluster them according to their nuclear genotypes. Despite this low number of nuclear markers, all North African nuclear genotypes were grouped in a highly distinct subpopulation when compared with European samples sharing the same mitochondrial barcodes. The reverse situation prevailed in Switzerland where bats carrying distinct barcodes had similar nuclear genotypes. There was a weak east/west nuclear structure of populations, but this was independent of mitochondrial lineages as bats carrying either variant were completely admixed. Thus, the divergent mitochondrial barcodes present in Western Europe do not represent cryptic species, but are part of a single biological species. We argue that these distinct barcodes evolved in allopatry and came recently into secondary contact in an area of admixture north of the Alps. Historical records from this area and molecular dating support such a recent bipolar spatial expansion. These results also highlight the need for using appropriate markers before claiming the existence of cryptic species based on highly divergent barcodes.     

Isolation and characterization of polymorphic DNA microsatellite markers from Schistosoma japonicum

The ability of microsatellite loci to reveal genetic diversity within the trematode Schistosoma japonicum is demonstrated. Eleven novel microsatellite markers were isolated, assessing variability within 80 S. japonicum individuals from three Chinese and one Philippine population. Novel primers were also tested upon S. mansoni and S. haematobium. Eight primers showed polymorphic amplification from all S. japonicum groups, three amplified S. mansoni DNA, but none amplified S. haematobium DNA. Only six of 27 previously isolated S. mansoni‐specific primers amplified S. japonicum DNA. Allelic diversity and observed heterozygosity ranged from 4 to 21 and 0.05 to 0.82, respectively, suggesting high variability.     

Widespread co‐occurrence of divergent mitochondrial haplotype lineages in a Central American species of poison frog (Oophaga pumilio)

Aim To analyse the phylogeographic structure of the strawberry poison frog, Oophaga pumilio (Dendrobatidae), across a large part of its range using a combination of mitochondrial and nuclear markers. Location Costa Rica and Panama. Methods Sequence analyses of a mitochondrial (cytochrome b) and a nuclear (RAG‐1) gene fragment as well as analyses of seven microsatellite loci were carried out on 269 individuals of O. pumilio sampled from 24 localities and on two individuals of O. vicentei. Results Two main mitochondrial haplotype lineages, corresponding to a northern (north Costa Rica) and a southern (south Costa Rica and eastern Panama) lineage, were identified. They differed by up to 7% uncorrected distance. We observed co‐occurrence of both lineages in seven populations in Costa Rica and Panama, indicating a pattern of extensive admixture. The two main mitochondrial lineages of O. pumilio roughly corresponded to a previously described phylogeographic pattern. Microsatellites indicate admixture spanning over a wide geographic area, but significant variation between the northern and southern groups was also found with microsatellite data. While microsatellite data reconstructed a separation south of an assumed Caribbean valley barrier, mitochondrial haplotypes of the ‘southern lineage’ shifted this barrier towards the north. Main conclusions Despite admixture, all three markers showed significant variation between the northern and southern groups. Phylogeographical breaks known from other anuran species in the study region could not be verified for O. pumilio. The unexpected clustering of the population from Escudo de Veraguas and the individuals of O. vincentei with the northern O. pumilio lineage indicates the need for a fundamental and careful taxonomic revision, including an interspecific phylogeography of the entire genus.