Microsatellite loci from the northern house mosquito (Culex pipiens), a principal vector of West Nile virus in North America

Microsatellites were isolated and characterized in the northern house mosquito, Culex pipiens, a widespread pest species and important vector of diseases such as West Nile virus. An enrichment protocol yielded 150 positive clones. We designed primers to amplify 17 unique (GT)_n microsatellites, eight of which amplified cleanly and were polymorphic. A survey of 29 individuals showed that these loci are highly variable with the number of alleles ranging from seven to 19 and expected heterozygosity ranging from 0.66 to 0.93. These markers will be useful for studies of population structure and intraspecific variation in epidemiological characteristics of Cx. pipiens.     

Isolation of microsatellite loci in green abalone (Haliotis fulgens) and cross‐species amplification in two other North American red (Haliotis rufescens) and pink (Haliotis corrugata) abalones

Microsatellite loci were isolated in Haliotis fulgens using a (CT)_n enriched‐genomic library. From 33 sequenced clones, 21 microsatellites regions were identified, 15 with the expected (CT)_n. Eight microsatellite loci were amplified, six of which were polymorphic with a range of three to 20 alleles, and five cross‐amplified in two other species (Haliotis rufescens and Haliotis corrugata). These microsatellites will be useful as population genetic markers in the three species.     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

Microsatellites for three distantly related genera in the Brassicaceae

Microsatellites are important genetic markers both in population genetics and for delimitation of closely related species. However, to develop microsatellites for each target organism is expensive and time consuming. In this study, we have therefore developed 65 new microsatellite primers for the species Draba nivalis and tested cross-species and cross-genus transfer success of these primers for two other genera in the Brassicaceae; Cardamine and Smelowskia. Furthermore, 15 previously developed microsatellites were tested for amplification in these three genera. The microsatellite markers that amplify across these genera may be useful for other genera in the Brassicaceae as well.     

Isolation and Characterization of Highly Polymorphic Microsatellites from the Polychaete Pectinaria koreni

We report on the isolation of dinucleotide microsatellites from the polychaete Pectinaria koreni using (GT)₁₀ and (CT)₁₀ olignonucleotide probes. Compound and particularly imperfect microsatellites are predominant in this species. In most cases the associated element is (AT)_n leading to AT microsatellites being the most frequent class after GT repeats. Obtaining single-copy polymerase chain reaction products in the expected size range proved to be difficult, and only 24% of the primer pairs tested produced polymorphic single-locus markers. We obtained five highly variable loci with 16 to 41 alleles and an observed heterozygosity ranging from 0.29 to 0.81. Amplifications have also been obtained from the earliest postlarval stage. These highly informative markers will allow studies of the population structure of P. koreni at fine spatial and temporal scales. Received March 19, 1999; accepted September 10, 1999.     

Isolation and characterization of 10 polymorphic microsatellites in saltcedars (Tamarix chinensis and Tamarix ramosissima)

Tamarix ramosissima and Tamarix chinensis are invasive weed species in western North America. Previous studies based on single locus DNA sequence data revealed some information about the invasion process, but multilocus markers can provide additional information about levels of introgression and genotype origins. We have developed primers that amplify 10 polymorphic microsatellite loci from T. ramosissima; these primer pairs also successfully amplify polymorphic microsatellites from the closely related T. chinensis, a species that forms hybrids with T. ramosissima in the western USA.     

Chromosomal localization and molecular-marker tagging of the powdery mildew resistance gene (Lv) in tomato

We report the tagging of a powdery mildew [Leveillula taurica (Lév.) Arnaud.] resistance gene (Lv) in tomato using RAPD and RFLP markers. DNA from a resistant (cv Laurica) and a susceptible cultivar were screened with 300 random primers that were used to amplify DNA of resistant and susceptible plants. Four primers yielded fragments that were unique to the resistant line and linked to the resistance gene in an F₂ population. One of these amplified fragments, OP248, with a molecular weight of 0.7 kb, was subsequently mapped to chromosome 12, 1 cM away from CT134. Using RFLP markers located on chromosome 12, it was shown that approximately one half of chromosome 12 (about 42 cM), in the resistant variety is comprised of foreign DNA, presumably introgressed with the resistance gene from the wild species L. chilense. Further analysis of a backcross population revealed that the Lv gene lies in the 5.5-cM interval between RFLP markers, CT211 and CT219. As a prelude to map-based cloning of the Lv gene, we are currently enriching the density of markers in this region by a combination of RAPD primers and other techniques.     

Phylogenetic distribution and genetic mapping of a (GGC)n microsatellite from rice (Oryza sativa L.)

DNA microsatellites are ubiquitously present in eukaryotic genomes [30] and represent a vast source of highly informative markers [30, 33, 34, 2]. We describe in this article a (GGC)_n microsatellite which is widely distributed in eukaryotic genomes. Using polymerase chain reaction (PCR) techniques and DNA sequencing, we demonstrated for the first time in plant species that a (GGC)_n microsatellite locus is moderately polymorphic. Six alleles are present at this locus in rice and length polymorphisms are caused by variation in the number of tandem GGC repeats. By scoring a backcross mapping population, we were able to demonstrate that this locus is stably inherited and does not link to any known RFLP markers on the rice RFLP map. Our results suggest that DNA microsatellites should be useful in plants for construction of genetic linkage maps, extension of the existing genetic linkage maps, linkage analysis of disease and pest resistance genes, and the study of population genetics.     

Microsatellites from the burbot (Lota lota), a freshwater gadoid fish (Teleostei)

We developed 21 polymorphic dinucleotide microsatellite loci, (CA)_n and (CT)_n, for the Holarctic freshwater fish, Lota lota, using an enriched genomic library protocol. The species has an interesting life history because winter‐spawning adults migrate over long distances to form spawning aggregations, a behaviour which should maintain genetic homogeneity across large spatial scales. Availability of the reported microsatellites will facilitate the investigation of population genetic structure with regard to postglacial colonization history and conservation strategies. The primers were screened on 30 individuals from a natural population (Lake Constance, southern Germany), revealing three to 24 alleles per locus with expected heterozygosities ranging from 0.48 to 0.93.     

Isolation of polymorphic microsatellite markers from <Emphasis Type="Italic">Przewalskia tangutica</Emphasis> (Solanaceae)

Microsatellite-containing regions were isolated and characterized in Przewalskia tangutica Maxim (Solanaceae), an endemic and endangered species to the Qinghai-Tibetan Plateau of China. An enrichment protocol yielded 200 positive clones. We designed primers to amplify 29 unique microsatellites, 12 of which amplified cleanly and were polymorphic. A survey of 17 individuals showed that these loci are highly variable with the number of alleles ranging from 3 to 12, and expected heterozygosity ranged from 0.2929 to 0.4947. Those markers will be useful for studies of population structure and intraspecific variation in P. tangutica.     

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.     

Tetranucleotide microsatellite loci from the black bear (Ursus americanus)

We describe primers and polymerase chain reaction conditions to amplify 21 tetranucleotide microsatellite DNA loci in black bears (Ursus americanus). We tested primers using individuals from two populations, one each in Georgia and Florida. Among individuals from Georgia (n = 29), primer pairs yielded an average of 2.9 alleles (range, one to four) and an average observed heterozygosity (H_O) of 0.50 (range, 0.00 to 0.79). Among individuals from Florida (n = 19), primer pairs yielded an average of 5.7 alleles (range, one to 14) and an H_O of 0.55 (range, 0.00 to 1.00). A comparison of previously developed markers with individuals from Georgia suggests that bear populations in Georgia and Florida have reduced allelic diversity relative to other populations.     

Characterization of microsatellite markers for the apicultural pest Varroa destructor (Acari: Varroidae) and its relatives

Sixteen microsatellite loci were isolated and characterized for Varroa destructor using two procedures to screen genomic libraries. Together with those previously designed, they provide useful markers for the study of this harmful apicultural pest whose populations on Apis mellifera are poorly variable. Observed variability has been expressed as the number of alleles because heterozygosity is only rarely present. The defined primers have been assayed on another species of the same genus (V. jacobsoni) and almost half of them successfully cross‐amplified and revealed polymorphism. These results suggest that the microsatellites isolated here should prove useful for population studies in different Varroa species.     

Chloroplast DNA markers (cpSSRs,SNPs) for <Emphasis Type="Italic">Miscanthus,Saccharum</Emphasis> and related grasses (Panicoideae,Poaceae)

Miscanthus and Saccharum are closely related perennial C₄ grasses. Miscanthus has recently attracted interest as a non-food crop for energy and fibre production. However, molecular genetic tools for the selection of new Miscanthus genotypes and study of its genetic resources are limited. We have identified six chloroplast (plastid) marker loci,containing both microsatellites (cpSSRs) and single nucleotide polymorphisms (SNPs) and developed primers to amplify and sequence these regions. The primers were designed using the complete chloroplast genome sequence of sugarcane and were tested on a collection of 164 Miscanthus genotypes and 14 related species of the subfamily Panicoideae. The cpSSR markers were highly polymorphic, with the number of alleles ranging from 10 to 16 per locus. Within the six cpSSR marker loci, the hybrid M. ×giganteus exhibits virtually no cpDNA variation compared with its putative parents M. sinensis and M. sacchariflorus. These SNP markers enable the differentiation of most Miscanthus species and detect infraspecific variation suitable for defining cytoplasmic genepools of Miscanthus for breeding purposes.     

Isolation and characterization of microsatellite markers in the solitary hunting wasp Ancistrocerus adiabatus (Vespidae: Eumeninae) and their cross utility in other species of Ancistrocerus

We have developed seven microsatellite primers for the solitary wasp Ancistrocerus adiabatus. The number of alleles per locus ranged from three to 14. The markers were developed to study population structure, inbreeding and sex determination of A. adiabatus. For each locus, we genotyped 30 female wasps collected from Southwest Michigan. The observed heterozygosity ranges from 0.067 to 0.933. We have also determined that primers for these microsatellite loci amplify in three other species in the genus: Ancistrocerus antilope, Ancistrocerus catskill, and Ancistrocerus campesterus.     

Hypervariable microsatellite loci in the Japanese macaque (Macaca fuscata) conserved in related species

We describe seven polymorphic microsatellites isolated from a Japanese macaque (Macaca fuscata) genomic library selected for (GT)_n content. The primer sets amplified from four to 11 different alleles in a sample of 14 Japanese macaques from nine different sites along the central and southern distribution of the species. These heterologous primers also detected variability in four other cercopithecine species. Am. J. Primatol. 43:357–360, 1997. © 1997 Wiley-Liss, Inc.     

Isolation of polymorphic microsatellite markers from the malaria vector Anopheles marajoara (Diptera: Culicidae)

Microsatellite‐containing regions were isolated and characterized in Anopheles (Nyssorhynchus) marajoara, a primary vector of malaria parasites in northeastern Amazonia, Brazil. An enrichment protocol yielded 500 positive clones. We designed primers to amplify 40 unique microsatellites, 11 of which amplified cleanly and were polymorphic. A survey of 323 individuals showed that these loci are highly variable with the number of alleles ranging from 11 to 52, and expected heterozygosity ranging from 0.64 to 0.95. These markers will be useful for studies of population structure and intraspecific variation in A. marajoara.     

Polymorphic microsatellites in a mangrove species,Rhizophora mangle L. (Rhizophoraceae)

Twenty‐six microsatellite loci were isolated and characterized from the mangrove species Rhizophora mangle using (GT)_n and (CT)_n repeats. Eighty‐four per cent of the clones contained microsatellite sequences; the most common dinucleotides were the (GA/CT) and (CA/GT) repeats. Ten primers were selected to investigate the polymorphism among individuals of R. mangle from two natural populations of the Colombian Pacific Coast. The observed heterozygosity per locus varied from 0.20 to 0.80, the power of discrimination was 0.32–0.84 and the power of exclusion was 0.03–0.75. This set of microsatellites offers an efficient tool for population genetics studies on this species.     

Identification and characterization of microsatellite loci in the common fig (Ficus carica L.) and representative species of the genus Ficus

We developed microsatellites in fig (Ficus carica L.). A TC and TG‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Eight primer pairs produced amplification products that were both interpretable and polymorphic in 14 fig cultivars and two French wild‐growing populations of F. carica (n₁ = 9 and n₂ = 10). Number of alleles per locus ranged from three to six. Except for one microsatellite locus, the observed heterozygosity was higher than the expected value. The F. carica microsatellites gave amplification products in 17 other Ficus species in 86% of the cases.