Isolation and characterization of polymorphic microsatellite loci for the study of paternity and population structure in the little penguin Eudyptula minor

We isolated and characterized eight novel microsatellite loci in the little penguin Eudyptula minor, using nonradioactive polymerase chain reaction‐based techniques to screen GA and GAAA repeats from enriched genomic DNA libraries. All eight loci were polymorphic and seven were variable in our main study population (mean H_E = 0.613, mean N_A = 7.14). Cross‐amplification using a microsatellite primer developed in Spheniscus demersus (African penguin) yielded one additional polymorphic locus. This locus combined with six of the little penguin loci is suitable for paternity assignment in little penguins (exclusion probability for seven unlinked loci = 0.993).     

Twenty-three polymorphic microsatellite markers for the Caribbean endemic Zenaida dove, Zenaida aurita, and its conservation in related Zenaida species

Twenty-three polymorphic microsatellite loci, six dinucleotidic loci and 17 tetranucleotidic loci, were developed for the Zenaida dove (Zenaida aurita), a bird species endemic to the Caribbean Islands. From a set of 30 individuals captured at one single location in Barbados, we obtained 20 loci that did not deviate from Hardy–Weinberg Equilibrium. Number of alleles per locus ranged between 2 and 11 (average 7.05) and the expected heterozygosity per locus, He ranged between 0.321 and 0.881 (average 0.712). This gives an exclusionary power for parental analysis of 0.9999 and 1.0000, knowing the genotype of one social parent, or both, respectively. Such results indicate that these 20 loci will be useful for both studying population genetics and mate choice patterns in Z. aurita. All 20 loci amplified in four other Zenaida species, the Galápagos dove, Z. galapagoensis, the eared dove, Z. auriculata, the mourning dove, Z. macroura, the Pacific dove, Z. meloda, with 30–96% being polymorphic.     

Isolation and characterization of eight microsatellite loci from the endangered plant species Hypochaeris salzmanniana (Asteraceae)

We report the isolation and characterization of eight microsatellite loci for Hypochaeris salzmanniana, an endangered species endemic to the southwestern coast of Spain and the Atlantic coast of Morocco. A total of 32 alleles were detected across a sample of 45 individuals, with an average number of 4.0 alleles per locus. The average polymorphic information content (PIC) was 0.533 and the observed (H _O) and expected (H _E) heterozygosity values varied from 0.022 to 0.978 and from 0.434 to 0.759, respectively. Five loci exhibited significant deviation from Hardy–Weinberg equilibrium (P ≤ 0.001) and three pairs of loci showed significant linkage disequilibrium (P ≤ 0.01). The eight loci were tested for transferability in three others species (H. arachnoidea, H. glabra, and H. radicata) belonging to the same section of H. salzmanniana. With the exception of locus Hsalz7, all loci successfully amplified in the three species. These preliminary data confirm the usefulness of microsatellite markers for assessing the ecology and genetic structure of H. salzmanniana and to understand the evolution of species within the section Hypochaeris.     

Ten new microsatellite loci for analysis of genetic diversity in isolated populations of the Alpine land snail <Emphasis Type="Italic">Cylindrus obtusus</Emphasis>

In this study we characterized 10 polymorphic microsatellite markers for the land snail Cylindrus obtusus, an endemism of the Austrian Alps with a distribution in isolated populations above approximately 1,600 m. The microsatellite loci were analyzed in 44 individuals from two populations. Number of alleles per locus ranged between two and eight. Observed heterozygosity ranged between 0.00 and 1.00, and expected heterozygosity between 0.09 and 0.72. No significant linkage disequilibrium was found between pairs of loci. One of the sampled populations (Dachstein) showed no deviation from Hardy–Weinberg equilibrium and no presence of null alleles, whereas the other one (Schneeberg) did. These diverging results probably reflect differences in population structure rather than characteristics of the microsatellite loci and underline the usefulness of these markers for studying genetic diversity, population structure and differentiation in C. obtusus.     

Characterization of eight microsatellite loci in Sea Otter, Enhydra lutris, and cross-species amplification in other Mustelidae

Herein we describe the development of eight microsatellite markers for the northern sea otter, Enhydra lutris kenyoni. A total of 45 primer pairs were developed and screened from enriched AAAT, CATC, TACA, and TAGA libraries derived from genomic DNA of E. lutris kenyoni. Of these, eight amplified successfully. The average observed heterozygosity, expected heterozygosity, and number of alleles per locus was 0.506, 0.537, and 3.4, respectively. These eight loci were tested across three additional genra; Vulpes lagopus, Martes americana, and Mustela nivalis. Based on the success of our results these loci will be useful for future studies across all sub-species of E. lutris.     

Isolation and characterization of microsatellite markers in the southern emu‐wren (Stipiturus malachurus: Aves)

We isolated and characterized eight novel microsatellite loci in the southern emu‐wren (Stipiturus malachurus). We used nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic DNA library. Based on genotypes from a single population, six loci showed no evidence of null alleles and were polymorphic (allele range = 2–9, mean heterozygosity = 0.57), and one locus was sex‐linked (N_A = 4). These loci were variable and had different allele size ranges in three other populations of southern emu‐wrens, and are therefore useful for determining levels of genetic diversity within and between populations of the species.     

Identification and characterization of microsatellite loci in Betula maximowicziana Regel

The sequences of eight primer pairs of microsatellite loci screened from a genomic library of Betula maximowicziana are reported in this paper. Seven loci showed polymorphism in 44 individuals of the Mitomi population in the central area of Japan and the observed and expected heterozygosities ranged from 0.045 to 0.659 (mean = 0.412) and from 0.044 to 0.642 (mean = 0.397), respectively. Although the one remaining locus is not polymorphic there, it showed variation in other populations. Thus, these eight microsatellite markers might be useful tools for studying the population and ecological genetics of B. maximowicziana.     

A multiplex panel of microsatellite markers for widespread sub‐Saharan rodents of the genus Mastomys

We isolated and characterized 12 polymorphic microsatellite loci in the sub‐Saharan rodent Mastomys huberti. We tested cross‐species amplification of all these loci in three closely related Mastomys species: M. coucha, M. erythroleucus and M. natalensis. Multiplex panels comprising 11 loci were developed and their application to a set of individuals in each species allowed clear and easy characterization of allele sizes. Statistics from 31 M. huberti coming from one locality in Mali showed no deviation from Hardy–Weinberg equilibrium except for one locus, and no significant linkage disequilibria between loci.     

Microsatellite markers for the common vole (<Emphasis Type="Italic">Microtus arvalis</Emphasis>) and their cross-species utility

Fragmentation of natural habitats of the common vole (Microtus arvalis) provides an excellent model system to study the consequences of restricted gene flow and small population sizes for isolated populations. Here we describe the isolation and characteristics of 10 autosomal and one X-linked microsatellite marker. These new markers were tested in 24 voles from a natural population in eastern Germany. Loci were highly polymorphic with numbers of alleles per locus ranging from three to 26 and expected heterozygosities from 0.51 to 0.97. All loci except for the X-linked locus Mar105 followed Hardy–Weinberg expectations. Cross-species amplifications revealed that most loci were polymorphic as well in M. agrestis, M. thomasi, and M. pennsylvanicus.     

Characterization of microsatellite markers for the endemic sand dune lizard,Sceloporus arenicolus

Here we characterize and report on the genetic variability of eight microsatellite loci for the endemic sand dune lizard, Sceloporus arenicolus, that amplified consistently for individuals throughout the species’ range. The number of alleles per locus was high (mean = 13.25) and observed heterozygosities ranged from 0.354 to 0.808. With the exception of a single locus, the loci did not deviate from Hardy–Weinberg expectations. This set of markers is currently being used to examine population structure and landscape genetics in S. arenicolus, a habitat specialist with a restricted range and patchy distribution.     

Characterization of microsatellite loci in the parasitoid,Cotesia congregata (Say) (Hymenoptera Braconidae)

Nine microsatellite loci were developed and assayed for polymorphism in four groups of the insect parasitoid, Cotesia congregata (Say) (Hymenoptera). One locus was monomorphic and eight were polymorphic in C. congregata. Allelic distributions differed significantly between groups of C. congregata collected from solanaceous and catalpa host food plants (P = 0). Loci were tested for cross‐reactivity to six congeners: C. acuminata (Reinhard), C. bignellii (Marshall), C. cynthiae (Nixon), C. euphydryidis (Musebeck), C. koebelei (Riley) and C. melitaearum (Wilkinson). In the congeners, eight loci were polymorphic, four of which yielded allele sizes that differed substantially from sizes of amplification products observed for C. congregata.     

Characterization of microsatellite markers for snouted treefrogs in the Scinax perpusillus species group (Anura, Hylidae)

We characterized eight microsatellite loci for snouted treefrogs in the Scinax perpusillus species group, a group of hylid frogs endemic to the Atlantic Coastal Forest of Brazil, and tested their utility in mainland and island species of the complex. All eight loci were polymorphic in one population of S. perpusillus; four of the loci showed excess homozygosity and three of those deviated from Hardy–Weinberg expectations, possibly due to null alleles, inbreeding, or population structure in sampled individuals. Six loci amplified and were polymorphic in S. arduous, S. argyreornatus, and S. faivovichi, but only one in S. alcatraz. These markers will be useful for quantifying effects of habitat fragmentation on population genetic diversity and connectivity in coastal and island populations of this threatened species group.     

Ataxia and pancytopenia caused by a mutation in TINF2

Disseminated superficial actinic porokeratosis (DSAP) is a chronic autosomal dominant cutaneous disorder with high genetic heterogeneity. Two genetic loci for DSAP were identified, but no specific genes were reported to date. The pathogenic mechanism of this disorder remains to be elucidated. In this study, a large, five-generation Chinese family with DSAP was genetically characterized. Two known DSAP loci, DSAP1 and DSAP2, two DSAP candidate genes (SART3 and SSH1), one DSP-linked locus and one PPPD-linked locus were first excluded in the family. The family was then characterized by genome-wide linkage analysis and a new DSAP locus was identified on chromosome 1p31.3–p31.1 with a maximum two-point LOD score of 5.09 with marker D1S2897 (θ = 0). Fine mapping showed that the disease gene was located within an 8.2 cM or 11.9 Mb region between markers D1S438 and D1S464. This is the third locus identified for DSAP (DSAP3). Eight candidate genes including GNG12, IL12RB2, ITGB3BP, DNAJ6, PIN1L, GADD45A, RPE65 and NEGR1 were sequenced, but found to be negative for functional sequence variants. Further mutational analysis of the candidate genes in the region will identify the specific gene for DSAP, which will provide insights into the pathogenesis of DSAP.     

Microsatellite development for the endangered Yangtze sturgeon (Acipenser dabryanus Duméril, 1869) using 454 sequencing

The Yangtze sturgeon (Acipenser dabryanus) is an endemic species in China. Using 454 sequencing, eight polymorphic tri‐, tetra‐, penta‐, and hexanucleotide microsatellite loci were isolated in this study. The raw sequence data from a one‐eighth run of 454 sequencings were 38.0 Mbp containing 94 222 reads/sequences. Of 80 microsatellite loci, only eight loci were polymorphic in a population of 30 individuals. The number of alleles per locus ranged from 4 to 14 (mean 7.62), and the observed heterozygosities varied between 0.46 and 0.88 (mean 0.74). Cross amplification was tested in congeneric species Acipenser sturio and Acipenser sinensis. These new microsatellite markers will be useful for further studies on genetic variation, parentage analysis, and conservation management for this critically endangered species.     

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.     

Eight SSR loci from Oyster Crassostrea gigas EST database and cross-species amplification in C. plicatula

Oyster (Crassostrea plicatula) is widely distributed in coastal areas of China. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Crassostrea gigas and to amplify EST-SSR in C. plicatula. Characteristics of eight EST-SSR loci were investigated using 37 wild-type C. plicatula individuals. The number of alleles per locus ranged from four to six. The observed heterozygosity (H _O) ranged from 0.1892 to 0.7027 (0.3919 on average) and the expected heterozygosity (H _E) ranged from 0.6068 to 0.7656 (0.7039 on average) (P < 0.05). The average observed heterozygosity was much lower than the average expected heterozygosity. All loci except C15 departed from Hardy-Weinberg equilibrium (P < 0.05) significantly. Contribution of the eight EST-SSR primers presented here will provide necessary and powerful molecular tools for management and conservation studies on the species of oysters in the future.     

Identification and characterization of nine polymorphic microsatellite loci in the North American pika,Ochotona princeps

Nine polymorphic microsatellite loci were developed for the North American pika (Ochotona princeps) from di‐ and tetranucleotide repeat‐enriched genomic libraries. Polymorphism was assessed for 165 individuals from eight geographical locations in the western United States. All loci were polymorphic. The number of alleles per locus ranged from three to 14, with observed heterozygosity between 0.189 and 0.822. All loci were in Hardy–Weinberg equilibrium (< 0.05). Regional differences were evident with unique alleles at multiple loci in six of eight populations.     

Polymorphic microsatellite markers isolated from partially enriched genomic library of Chitala chitala

A total eight polymorphic microsatellite loci were obtained from genomic library of Indian feather back, Chitala chitala (order Osteoglossiformes, family Notopteridae) and the 46 samples were analysed to determine genetic variation. The mean number of alleles per locus ranged from 4.50 to 5.25, and expected heterozygosity ranged from 0.124 to 0.852. Deviation from Hardy–Weinberg equilibrium expectations (P < 0.002) was observed at loci Cch2, Cch9 (Bhaghirathi) and Cch9 (Brahmaputra). The identified microsatellite loci were found promising for population genetics studies of C. chitala and related species Notopterus notopterus (family Notopteridae).     

Isolation and characterization of eight polymorphic microsatellite loci in the rainforest canopy tree,Syzygium sayeri (Myrtaceae)

Eight polymorphic microsatellite loci were isolated from the rainforest canopy tree, Syzygium sayeri, in order to study parentage and subsequently pollen dispersal among individuals in wild populations. Screening of one natural population (n = 64) mapped in a 500 × 500 m area at Cape Tribulation, north Queensland, Australia, yielded two to 11 alleles per locus with observed levels of heterozygosity ranging from 0.07 to 0.70. One locus was significantly out of Hardy–Weinberg equilibrium after Bonferroni correction for multiple tests. These loci should provide a useful tool in further understanding the dispersal patterns of this species.     

Characterization of microsatellite markers for the tanoak tree,Lithocarpus densiflorus

We describe the development of 19 primers for amplification of microsatellite loci for tanoak, Lithocarpus densiflorus (Hook. & Arn.). A population from Soquel State Demonstration Forest, Santa Cruz County, California (n = 20), and eight individuals from four additional coastal populations and one interior (Sierra Nevada) population were used to investigate polymorphism. The total number of alleles per locus ranged from two to 10. Observed heterozygosity in the Soquel population ranged from 0.05 to 0.70, with two loci being fixed.