Isolation and characterization of microsatellites in Pachygrapsus marmoratus (Grapsidae; Decapoda; Brachyura)

Seven polymorphic microsatellite loci were isolated and characterized from the grapsid crab Pachygrapsus marmoratus using FIASCO (fast isolation by AFLP of sequences containing repeats) protocol. Twenty‐seven primer pairs were designed, 14 of which worked well in polymerase chain reaction (PCR), amplifying a fragment of the expected size. Variability was tested in 18 specimens collected along the coast of Tuscany (Italy). Five loci were discarded due to stutter products during their amplification, and two resulted to be monomorphic. The remaining seven loci, showed a number of alleles ranging from two to 14 and an observed heterozygosity ranging from 0.12 to 0.67.     

Development and characterization of polymorphic markers for the sap-stain fungus Ophiostoma quercus

Eight polymorphic markers were developed from South African isolates of Ophiostoma quercus. The genome was screened for repeat regions using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol and 20 de novo primer pairs flanking putative microsatellite regions were designed. Eight loci were optimized and their polymorphisms evaluated by sequencing. The repeat and flanking regions were highly polymorphic containing both indels and base-pair substitutions revealing a total of 46 alleles in 14 isolates and an average heterozygosity of 0.68. Substantial sequence variability makes these markers useful for genotyping populations in order to calculate diversity and monitor global movement of O. quercus.     

Sixteen polymorphic microsatellites in bighead carp (Aristichthys nobilis) and cross-amplification in silver carp (Hypophthalmichthys molitrix)

A (GT)(n) enriched partial genomic library of bighead carp (Aristichthys nobilis) was constructed by employing the (fast isolation by AFLP of sequences containing repeats) FIASCO protocol. Sixteen loci exhibited polymorphism with two to seven alleles/locus (mean 3.263) in a test population and the observed heterozygosity ranging from 0.100 to 0.690 (mean 0.392). Eleven of the 16 bighead carp microsatellites were found to be also polymorphic in silver carp. These polymorphic loci should provide sufficient level of genetic diversity to evaluate population structure of bighead carp.     

High-throughput microsatellite marker development in two sparid species and verification of their transferability in the family Sparidae

Recently, 454 sequencing has emerged as a popular method for isolating microsatellites owing to cost-effectiveness and time saving. In this study, repeat-enriched libraries from two southern African endemic sparids (Pachymetopon blochii and Lithognathus lithognathus) were 454 GS-FLX sequenced. From these, 7370 sequences containing repeats (SCRs) were identified. A brief survey of 23 studies showed a significant difference between the number of SCRs when enrichment was performed first before 454 sequencing. We designed primers for 302 unique fragments containing more than five repeat units and suitable flanking regions. A fraction (<11%) of these loci were characterized with 18 polymorphic microsatellite loci (nine in each of the focal species) being described. Sanger sequencing of alleles confirmed that size variation was because of differences in the number of tandem repeats. However, a case of homoplasy and sequencing errors in the 454 sequencing were identified. These newly developed and four previously isolated loci were successfully used to identify polymorphic markers in nine other economically important species, representative of sparid diversity. The combination of newly developed markers with data from previous sparid cross-species studies showed a significant negative correlation between genetic divergence to focal species and microsatellite transferability. The high level of transferability we described (48% amplification success and 32% polymorphism) suggests that the 302 microsatellite loci identified represent an excellent resource for future studies on sparids. Microsatellite marker development should commonly include tests of transferability to reduce costs and increase feasibility of population genetics studies in nonmodel organisms.     

FMR1 in global populations.

Fragile X syndrome, a frequent form of inherited mental retardation, results from the unstable expansion of a cryptic CGG repeat within the 5' UTR region of the FMR1 gene. The CGG repeat is normally polymorphic in length, and the content is frequently interrupted by AGG triplets. These interruptions are believed to stabilize the repeat, and their absence, leading to long tracts of perfect CGG repeats, may give rise to predisposed alleles. In order to examine the stability of normal FMR1 alleles, the repeat length of 345 chromosomes from nine global populations was examined with the content also determined from 114 chromosomes as assessed by automated DNA sequencing. The FMR1 alleles, defined by the CGG repeat, as well as by the haplotypes of nearby polymorphic loci, were very heterogeneous, although the level of variation correlated with the age and/or genetic history of a particular population. Native American alleles, interrupted by three AGG repeats, exhibited marked stability over 7,000 years. However, in older African populations, parsimony analysis predicts the occasional loss of an AGG, leading to more perfect CGG repeats. These data therefore support the suggestion that AGG interruptions enhance the stability of the FMR1 repeat and indicate that the rare loss of these interruptions leads to alleles with longer perfect CGG-repeat tracts.     

Isolation and characterization of microsatellites in Chinese soft‐shelled turtle,Pelodiscus sinensis

Fifteen polymorphic microsatellite loci were developed for the Chinese soft‐shelled turtle (Pelodiscus sinensis) from the (GT)_n microsatellite‐enriched genomic library, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The polymorphism of all 15 loci ranged from two to seven alleles with observed heterozygosities ranging from 0.03 to 0.98 (mean 0.43) in one population of 40 individuals. These novel loci will be helpful for understanding the population structure at genetic level and marker‐assisted breeding of this vulnerable species.     

花鲈微卫星标记分离及其多态性分析

该文利用FIASCO法(fast isolation by AFLP of sequences containing repeats)和GenBank数据库搜索法开发花鲈微卫星标记,并对筛选的标记进行多态性检测.两种方法共获得54条能够设计引物的序列,扩增结果显示15对引物具有多态性,多态性微卫星位点的等位基因数为2～10个.15个多态性位点中,4个位点偏离了Hardy-Weinberg平衡;各位点间没有连锁不平衡现象;仅位点SP52可能存在无效等位基因;除SP17和SP468外,其余引物的P1C值均在0.5以上,可用于花鲈群体遗传分析等研究.     

Instability of interstitial telomeric sequences in the human genome

The length variability of four human interstitial telomeric sequences (ITs) is described. Three of the ITs contain short telomeric stretches ranging between 53 and 84 bp and are localized in 21q22, 2q31, and 7q36; the fourth IT derives from the subtelomeric domain of chromosome 6p and contains a tract of a few hundred basepairs of exact and degenerate repeats. Using primers flanking the repeats, we amplified the genomic DNA from unrelated individuals and from family members, and we found that all the loci are polymorphic. At the 21q22 IT locus, two equally frequent alleles were found, while the number of alleles at the 2q31, 7q36, and 6pter IT loci was 8, 6, and 4, respectively. Sequence analysis revealed that in the three loci containing short ITs the alleles differ from one another for multiples of the hexanucleotide; it is likely that the mechanism leading to the polymorphism is DNA polymerase slippage. These loci were also unstable in gastric tumor cells characterized by microsatellite instability. At the 6pter IT locus, the four alleles range in length from about 500 to about 700 bp; this variability is probably due to unequal exchange or gene conversion. Our data indicate that stretches of exact internal telomeric repeats can be highly unstable, like microsatellites with shorter units, and that they can be useful polymorphic markers for linkage analysis, for forensic applications, and for the detection of genetic instability in tumors.     

Development and characterization of 11 microsatellite markers in the rock sparrow,Petronia petronia

Eleven polymorphic microsatellite loci were isolated in the passeriform Petronia petronia using the enrichment protocol FIASCO (fast isolation by AFLP of sequences containing repeats). We detected three to 13 alleles per locus in 25 specimens collected from an Italian population. The level of expected heterozygosity ranged from 0.439 to 0.856. One locus is sex linked to the Z chromosome. The total exclusionary probabilities using these loci for the first and the second parent were 0.978 and 0.999, respectively. These are the first microsatellite loci characterized from the rock sparrow that can be used for estimating population structure and for large‐scale parentage analysis.     

Isolation and characterization of six polymorphic microsatellite markers in the freshwater turtle Mauremys rivulata (Testudines: Geoemydidae)

Six polymorphic microsatellite loci containing dinucleotide repeats were developed for the freshwater turtle Mauremys rivulata. The number of alleles ranged from five to 18. The observed and expected heterozygosities ranged from 0.19 to 0.79 and 0.46 to 0.90, respectively. These markers may serve as a valuable tool for population genetics analyses and provide information on the evolutionary history of the species.     

The C1167T polymorphism of the catalase gene and polymorphic markers D11S907 and D11S2008 located in its vicinity are associated with diabetes mellitus type 2

To study the contribution of the catalase (CAT) gene in diabetes mellitus (DM) type 2, the allele and genotype frequencies of internal (polymorphism C1167T) and two neighboring (minisatellites D11S907 and D11S2008) polymorphic markers were studied in 132 healthy individuals and 154 patients from Moscow. Allele C and genotype CC of the C1167T polymorphism proved associated with a higher risk of DM type 2. Seven D11S907 alleles containing 14 to 20 dinucleotide repeats were found. The frequencies of alleles 15 and 16 and genotype 18/20 were significantly higher and those of allele 18 and genotypes 17/18 and 18/19 were lower in patients than in controls. Eight D11S2008 alleles containing 15 to 22 tetranucleotide repeats were found. The frequencies of alleles 17 and 18 and genotype 18/20 in patients were significantly higher than in controls. An association of the three polymorphic loci and DM type 2 was suggested.     

Microsatellite loci from the trematode Lecithochirium fusiforme,a parasite of the European conger eel

Seven polymorphic loci containing dinucleotide repeats and one trinucleotide microsatellite were developed for the hemiurid fluke Lecithochirium fusiforme, a parasite of the European conger eel Conger conger. All parasites that were collected from a single individual host (a total of 54 specimens) were genotyped. The number of alleles ranged from two to eight. The observed and expected heterozygosities ranged from 0.057 to 0.736 and from 0.091 to 0.794, respectively. Hardy–Weinberg deviations were statistically significant for two loci. These markers will be useful for study of parasite transmission patterns and population genetic structure.     

Isolation and characterization of polymorphic microsatellite markers in the wall lizard Podarcis erhardii (Squamata: Lacertidae)

Five polymorphic microsatellite loci (containing di‐ and penta‐nucleotide repeats) were developed for the Erhard's wall lizard, Podarcis erhardii, an endemic species of southern Balkan Peninsula. The number of alleles ranged from five to 17. Observed and expected heterozygosities ranged from 0.28 to 0.92 and 0.57 to 0.91, respectively. These markers will provide a valuable tool for population genetics analyses and can contribute to the evaluation of conservation programs.     

Microsatellite markers for the roman, Chrysoblephus laticeps (Teleostei: Sparidae), an overexploited seabream from South Africa

Eleven polymorphic microsatellite loci were developed from an enrichment library of the roman, Chrysoblephus laticeps, and characterized for 40 individuals collected in Africa's largest and oldest Marine Protected Area (MPA), the Tsitsikamma National Park. The number of alleles per locus ranged from three to 19, and heterozygosities ranged from 0.20 to 0.85. A significant departure from Hardy-Weinberg equilibrium was detected for one locus, and linkage disequilibrium was identified among three pairs of loci. The markers will be useful to detect whether populations resident in MPAs along the South African coast are genetically connected, and whether there is spillover of recruits into adjacent exploited areas.     

Eighteen new tetranucleotide microsatellite DNA markers for Coregonus lavaretus cloned from an alpine lake population

We developed 18 polymorphic microsatellite markers for Coregonus lavaretus from genomic libraries enriched for (GACA)(n) and (GATA)(n) repeat sequences. Emphasis was placed on developing highly polymorphic, perfect repeats. These loci were screened in 69 individuals from two alpine populations in Austria. Allelic variation was high with nine to 37 alleles per locus and expected heterozygosities ranging from 0.37 to 0.95. The high level of polymorphism revealed by these loci will be relevant for population studies in context to the evolutionary history of this species.     

Isolation of highly polymorphic microsatellite markers from the intertidal barnacle Chthamalus montagui Southward

This study reports the isolation and characterization of seven highly polymorphic microsatellite loci in Chthamalus montagui (Crustacea, Cirripedia). The loci were isolated from a library constructed from genomic DNA enriched for CA repeats. The markers yielded three to 43 alleles per locus (mean 16.7) in samples averaging 49 individuals. Observed heterozygosity ranged from 0.08 to 0.58 (mean 0.39). These microsatellite loci will be valuable tools for population genetic studies of this species.     

Polymorphic dinucleotide microsatellite loci in the clonal ascidian Diplosoma listerianum: predominance of compound and highly interrupted imperfect repeats

We report the isolation of the first dinucleotide microsatellite loci from the clonal ascidian, Diplosoma listerianum. Most repeats were compound and highly interrupted, with flanking sequences containing many small interspersed repetitive regions. Consequently, most primers detected polymerase chain reaction products outside the expected size range, and only five out of 15 primers detected polymorphic single‐locus markers. Characterization of five variable loci from two UK populations revealed three to seven alleles per locus, with observed heterozygosity of 0.00–0.86 and expected heterozygosity of 0.10–0.66. Three loci showed significant heterozygote deficits either because of inbreeding, population substructure or the presence of null alleles.     

Microsatellite loci for behavioural studies of rainbow bee‐eaters (Merops ornatus: Aves)

A set of polymorphic microsatellite loci was identified for population and behavioural studies of the rainbow bee‐eater (Merops ornatus). Eight loci were newly isolated from a rainbow bee‐eater genomic library enriched for GA‐containing repeats, and six loci from those previously reported for the European bee‐eater (Merops apiaster). From these two sources, the seven most variable loci that amplified reliably were unlinked and in Hardy–Weinberg equilibrium, had five to 13 alleles (mean 7.7) and an expected heterozygosity of 0.60–0.86 (mean 0.73).