Sandflies of the Phlebotomus perniciosus complex: mitochondrial introgression and a new sibling species of P. longicuspis in the Moroccan Rif

The bloodsucking adult females of Phlebotomus perniciosus Newstead and P. longicuspis Nitzulescu (Diptera: Psychodidae) are important vectors of the protozoan Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae) in western Mediterranean countries. The species status of the two phlebotomine sandflies was assessed, along with the epidemiological implications. Individual sandflies from three Moroccan Rif populations were characterized morphologically, isoenzymatically (by the isoelectrofocusing of alleles at the polymorphic enzyme loci of HK, GPI and PGM), and by comparative DNA sequence analysis of a fragment of mitochondrial Cytochrome b (mtDNA). By reference to the character profiles of specimens from other locations, including southern Spain and the type-locality countries, the Moroccan flies were placed in three lineages: first, the lineage of P. perniciosus, which contained two mtDNA sublineages, one (pnt) widely distributed and associated with the morphology of the male types from Malta, and the other (pna) associated with a P. longicuspis-like male morphology; second, the lineage of P. longicuspis sensu stricto, including typical forms from Tunisia; and third, a new sibling species of P. longicuspis. The mtDNA sublineage (pnt) of typical P. perniciosus was also found in some P. longicuspis from Morocco, indicating interspecific hybridization. The typical race of P. perniciosus occurs in Italy as well as in Malta, Tunisia and Morocco. It is replaced in southern Spain by the Iberian race (with the pni mtDNA sublineage). The discovery of interspecific gene introgression and a new sibling species mean that previous records of the two morphospecies do not necessarily reflect their true vectorial roles or geographical and ecological distributions.     

Development and characterization of eight polymorphic microsatellite loci from Pistacia lentiscus L. (Anacardiaceae)

We have developed a set of eight polymorphic nuclear microsatellite markers for the Mediterranean shrub Pistacia lentiscus by means of an enriched library method. Characterization for the eight loci was carried out on 42 individuals from two populations sampled in southern Spain. The overall number of alleles detected was 59, ranging from three to 13 per locus. Expected heterozygosity per locus and population ranged from 0.139 to 0.895. Two loci albeit only in one population (Seville) departed significantly from Hardy-Weinberg equilibrium expectations and no linkage disequilibrium between pairs of loci was detected. These markers will be used in studies of gene flow across a fragmented landscape.     

Isolation of 10 tetranucleotide microsatellite loci in the blackcap (Sylvia atricapilla)

We isolated 10 polymorphic microsatellite DNA loci from the blackcap (Sylvia atricapilla) and optimized them for future studies of population differentiation in populations with different migration strategies in southwestern Germany. The loci were screened for polymorphism using 178 individuals from two populations in Germany and Spain. The primers amplified highly variable loci characterized by two to 19 alleles per locus and their observed and expected heterozygosities range from 0.47 to 0.81 and from 0.50 to 0.91, respectively.     

Development of polymorphic simple sequence repeat markers for Pisolithus microcarpus

Six polymorphic simple sequence repeat (SSR) markers were developed for the ectomycorrhizal fungus Pisolithus microcarpus. A polymerase chain reaction (PCR)‐based technique was used in which random amplified polymorphic DNA (RAPD) fingerprints were probed with labelled SSR oligonucleotides by southern hybridization. The number of alleles per locus ranged from two to nine with expected heterozygosity values from 0.33 to 0.76. These loci will be potentially useful for genetic structure and gene flow studies of P. microcarpus populations. Cross‐species amplification with Pisolithus albus isolates at all loci was also observed.     

Multiple genetic divergences and population expansions of a Mediterranean sandfly,Phlebotomus ariasi,in Europe during the Pleistocene glacial cycles

Phlebotomus ariasi is one of the two sandflies transmitting the causative agent of zoonotic leishmaniasis, Leishmania infantum, in France and Iberia, and provides a rare case study of the postglacial re-colonization of France by a Mediterranean species. Four DNA sequences were analysed—mitochondrial cytochrome b (cyt b), nuclear elongation factor-1α (EF-1α) and two anonymous nuclear loci—for 14–15 French populations and single populations from northeast Spain, northwest Spain, Portugal and Morocco. The presence of cryptic sibling species was not revealed by phylogenetic analyses and testing for reproductive isolation between sympatric populations defined by the two most divergent cyt b haplogroups. No locus was shown to be under positive directional or balancing selection and, therefore, molecular variation was explained demographically. Each nuclear locus showed shallow isolation by distance from Portugal to the French Pyrenees, but for both cyt b and EF-1α there was then a step change to the upland Massif Central, where leading-edge populations showed low diversity at all loci. Multiple genetic divergences and population expansions were detected by analyses of cyt b and dated to the Pleistocene. Endemicity of one cyt b sub-lineage suggested the presence of a refuge north of the Pyrenees during the last glacial period. Monopolization of the Massif Central by genetically differentiated populations of P. ariasi might possibly hinder the northwards spread of leishmaniasis.     

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.     

Polymorphic tetranucleotide microsatellite DNA loci from the southern dusky salamander (Desmognathus auriculatus)

We describe polymerase chain reaction (PCR) primers and amplification conditions for seven tetranucleotide microsatellite DNA loci isolated from the southern dusky salamander (Desmognathus auriculatus). Primers were tested on 16 individuals from one population in Aiken County, South Carolina. We detected an average of 6.57 alleles per locus, an observed heterozygosity range of 0.44–0.94, and high polymorphic information contents (mean of 0.68).     

Restriction fragment length polymorphism among Israeli Holstein-Friesian dairy bulls

Israeli Holstein-Friesian dairy bulls were screened for restriction fragment length polymorphisms by hybridizing cloned DNA probes for bovine growth hormone, for chymosin, and for rat muscle beta-actin to restriction endonuclease-digested DNA immobilized on nitrocellulose filters. The population proved to be polymorphic at the growth hormone locus, with evidence consistent with the phenotypes being inherited in allelic fashion. A low level of polymorphism was also observed at one of the beta-actin gene family loci. The chymosin locus was monomorphic with the restriction enzymes utilized. The results illustrate the power of restriction fragment length polymorphism methodology in visualizing genetic variability in dairy cattle populations.     

Characterization of nineteen microsatellite markers and development of multiplex PCRs for the wedge clam Donax trunculus (Mollusca: Bivalvia)

The wedge clam Donax trunculus is an Atlantic-Mediterranean warm-temperate species found from Senegal to the northern coast of France, including the Mediterranean and Black Sea. It is commercially exploited in several European countries and constitutes an important fishing resource due to its high economical value. To contribute to its conservation and management, nineteen microsatellite markers were isolated from two enriched genomic libraries. These loci were characterized in 30 clams from a single population from northwest Spain. The number of alleles per locus ranged from 2 to 17 and observed and expected heterozygosity varied from zero to 0.714 and from 0.078 to 0.950, respectively. Linkage disequilibrium was not detected and nine loci were in agreement with Hardy–Weinberg equilibrium. Fifteen polymorphic markers were arranged into three multiplex PCR sets to reduce both time and cost of microsatellite genotyping. This is the first time that polymorphic microsatellite markers have been reported for D. trunculus. These new markers provide a valuable resource for future population genetics studies and management and culture of this species.     

Direct evidence for biased gene diversity estimates from dominant random amplified polymorphic DNA (RAPD) fingerprints

The relevance of using dominant random amplified polymorphic DNA (RAPD) fingerprints for estimating population differentiation was investigated when typically small population sample sizes were used. Haploid sexual tissues were first used to determine genotypes at RAPD loci for 75 eastern white pines ( Pinus strobus L.) representing five populations. Dominant RAPD fingerprints were then inferred from genotypic data for each individual at each locus, and gene diversity estimates from both sources of data were compared. Genotypic information at RAPD loci indicated little or no differentiation among populations, similar to allozyme loci. However, estimates of population differentiation derived from dominant RAPD fingerprints according to various common methods of analysis were generally inflated, especially when all fragments were considered. Simulations showed that an increase in loci sampling and population sample sizes did not significantly alleviate the biases observed.     

Development of single sequence repeat markers for the ant Aphaenogaster senilis and cross-species amplification in A. iberica, A . gibbosa, A. subterranea and Messor maroccanus

Eight polymorphic microsatellite loci were isolated and characterized for Aphaenogaster senilis, a common ant species distributed in the Western Mediterranean. Characterization of 15 individuals form southern Spain showed moderate to high allelic diversity ranging from 2 to 9 alleles per locus. Cross-species tests on 10 individuals of A. iberica, A. gibbosa, A. subterranea and Messor maroccanus revealed successful amplification for most loci. This set of markers can be useful for population genetic studies and might even prove useful in other phylogenetically close species of the subfamily Myrmicinae.     

Development and characterization of 11 polymorphic compound tetranucleotide microsatellite loci for the Leisler’s bat, Nyctalus leisleri (Vespertilionidae, Chiroptera)

Eleven polymorphic microsatellite marker loci were developed from a Leisler’s bat (Nyctalus leisleri) genomic enriched library. Assessment of the usefulness of these markers for population genetics studies of Leisler’s bats was carried out by screening 100 specimens sampled from eight locations in Ireland and two in Northeastern France. Both moderately and highly polymorphic marker loci were identified. Five to 28 alleles were found to be segregating per locus with observed heterozygosities values ranging from 28.4 to 94%. Initial evaluation indicates that these microsatellites will be useful for genetic based studies aiming, for instance, at parentage and population structure of Leisler’s bats.     

Eleven new microsatellite markers in jack mackerel (Trachurus japonicus) derived from an enriched genomic library

Jack mackerel (Trachurus japonicus, Carangidae) are a commercially important fisheries resource in Korea. To understand patterns of genetic variation for conservation and management efforts, we developed microsatellite DNA markers fromT. japonicus. We report the isolation and characterization of eleven microsatellite loci isolated using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 50 individuals from a naturalT. japonicus population in southern Korea were genotyped. All loci except one, KTJ38, were polymorphic with an average of 14 alleles per locus (range 6–23). The mean observed and expected heterozygosities were 0.70 (range 0.46–0.92) and 0.81 (range 0.49–1.00), respectively. Significant deviation from Hardy-Weinberg equilibrium was observed at three loci, KTj3, KTJ20 and KTJ28. Such high variability indicates that these microsatellites are useful markers for high-resolution analysis for population gemetic studies.     

Genetic polymorphism study at four minisatellite loci (D1S80, D17S5, D19S20, and APOB) among five Indian population groups

The present study reports the genetic variation observed among five anthropologically distinct population groups of India, using four highly polymorphic minisatellite loci (D1S80, D17S5, D19S20, and APOB 3' VNTR) in order to examine the effect of geographical and linguistic affiliations on the genetic affinities among these groups. Random individuals from five ethnic groups were studied; the sample size ranged from 235 to 364. The population groups belong to two geographically separated regions of India, the state of Maharashtra (western India) and the state of Kerala (southern India). The two Maharashtrian groups (Konkanastha Brahmins and Marathas) speak "Marathi," an Indo-European language, whereas the three Kerala population groups (Nairs, Ezhavas, and Muslims) speak "Malayalam," an Indo-Dravidian language. Genomic DNA was extracted from peripheral blood samples and analyzed using amplified fragment length polymorphism (Amp-FLP) technique. All four loci displayed high heterozygosity with average heterozygosity in the range of 0.82 to 0.84. The Polymorphic Information Content and Power of Discrimination were > or = 0.75 and > or = 0.80, respectively. The coefficient of gene differentiation was found to be low (average G(ST) = 1.2%; range between 0.6% at D1S80 locus to 1.6% at APOB 3' VNTR locus) across the loci, indicating close affinity among the population groups. The neighbor-joining tree revealed two clear clusters, one for the two Maharashtrian population groups and the other for the three Kerala population groups. The results obtained are in conformity with the geographical and linguistic backgrounds of the studied populations.     

不同生境三裂叶豚草种群的遗传结构

采用水平切片淀粉凝胶电泳测定了东北地区分布的不同生态环境条件下7个三裂叶豚草种群的遗传结构。统计分析了10个酶系统的10个位点,结果表明:三裂叶豚草种群内存在着丰富的遗传变异。多态位点百分率为80.00%,等位基因平均数为1.80,期望杂合度为0.375,遗传一致度和遗传距离为0.959和0.042。     

Genetic distances and taxonomic analysis of human populations of the Volga-Ural region based on data on DNA polymorphism]

DNA polymorphism was studied in the human diallelic loci MET and D7S23 linked to the cystic fibrosis gene, diallelic locus PAH (the phenylketonuria gene), polyallelic locus ApoB, and hypervariable DNA sequences identified by means of DNA fingerprinting with phage M13 DNA as a probe. The obtained data were used to calculate genetic distances and perform taxonomic analysis of populations of the Volga-Ural region (Turkic and Finno-Ugric ethnic groups). The DNA polymorphic systems studied were demonstrated to be highly informative; their advantages and disadvantages were revealed. According to the data obtained, the genetic distances that were calculated from DNA fingerprints more adequately reflected the genetic relationships between the populations studied than the distances calculated from the allelic frequencies of four DNA loci. It was also found that, in population studies, it would suffice to analyze only the 3.5-6 kb fingerprint fragment that is most suitable for reading, rather than the entire fingerprint obtained.     

Development of microsatellite markers for the critically endangered Limonium dufourii (Girard) Kuntze (Plumbaginaceae)

Limonium dufourii is an endemic plant from the eastern Mediterranean coast of Spain with a triploid chromosome number and apomictic reproduction. We have isolated and characterized 13 polymorphic microsatellite loci from an enriched library in order to investigate its population genetic structure. Simple sequence repeat (SSR) loci were screened in 120 individuals from the six extant populations of this species. They show an average of 5.76 alleles per locus, ranging from 2 to 18, with seven loci exhibiting heterozygosities larger than 0.60. Three loci present one single allele in each individual, whereas one locus presents three alleles in every individual analysed.     

Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers.

Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.     

Isolation and characterization of polymorphic tetranucleotide microsatellite loci in the pelagic perciform fish Pomatomus saltatrix (Linnaeus, 1766) from South Africa

Eight polymorphic microsatellite loci, containing simple tetranucleotide repeats, were isolated de novo from a Pomatomus saltatrix partial genomic library using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. These loci were further characterized in 100 individuals from two putative populations off the South African east coast. The loci are highly polymorphic with 18-37 alleles (on average 24 alleles/locus) and the observed heterozygosity in both populations was high (0.79). These loci will be used to assess population structuring in P. saltatrix along the southern African coast with consideration of implications for future management of this important linefish species.     

Isolation and characterization of microsatellite loci from the orchid Serapias vomeracea (Orchidaceae) and cross‐priming to other Serapias species

In this paper we describe the isolation and characterization of six polymorphic microsatellite loci from the orchid Serapias vomeracea. This species is widely distributed in the Mediterranean region. Microsatellite loci were isolated from an enriched library and primer pairs were designed for 18 loci. Primer pairs for six loci amplified well and were tested on samples from southern Italy. Levels of genetic variability detected at these six loci are high, with numbers of alleles per locus ranging from 3 to 6, and observed heterozygosity (H_O) ranging from 0.35 to 0.86. All primer pairs tested amplified DNA from four other Serapias species, indicating that the primers are useful for population genetic studies throughout the genus.