New polymorphic microsatellite loci for different camel species

New microsatellite loci were screened and sequenced from the genomic DNA of male Camelus bactrianus. Among 32 loci, 23 were amplified in bactrian and dromedary species, 19 in llama and 20 in alpaca. The different species had similar fragment lengths per locus, with more striking similarities between bactrian and dromedary and between llama and alpaca, respectively. Seven loci had more than 10 alleles each, nine were monomorphic in all species, and one was monomorphic in Old World and polymorphic in New World camels. The results show that the informative microsatellite loci can be widely applied to several species.     

Variation of short tandem repeats within and between species belonging to the Canidae family

Frequency distribution and allele size in 20 canine microsatellite loci were analyzed in 33 flat-coated retrievers, 32 dachshunds, 10 red foxes, and 10 Arctic foxes. Overall, the major difference between the two dog breeds was the relative allele frequencies rather than the size ranges of alleles at the individual locus. The average heterozygosity within the two dog breeds was not significantly different. Since the average heterozygosity at several polymorphic loci is a relative measure of heterogeneity within the population, analysis of heterozygosity within microsatellite loci is suggested as a measure for the diversity of populations. Eighty percent (16 of 20) of the canine microsatellite primer pairs amplified corresponding loci in the two fox species. This reflects a very high sequence conservation within the Canidae family relative to findings in, for instance, the Muridae family. This indicates that it will be possible to utilize the well-characterized fox karyotype instead of the dog karyotype as a step towards physical mapping of the dog genome. Analysis of exclusion power and probabilities of genetic identity between unrelated animals by use of the seven most informative loci demonstrated that it will be possible to assemble a panel of microsatellite loci that is effective for parentage analysis in all breeds.     

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.     

Development, inheritance and cross-species amplification of microsatellite markers from Acacia mangium

Microsatellite markers were developed in Acacia mangium Willd. to provide highly variable co-dominant markers for linkage mapping and studies of the breeding system. After an enrichment procedure 40% of colonies contained microsatellites in contrast with less than 1% from a non-enriched library. The majority of microsatellite sequences were AC repeats. Co-dominant segregation of alleles in two full-sib crosses of A. mangium was demonstrated at 33 microsatellite loci. The markers were highly variable relative to restriction fragment lengths polymorphisms (RFLPs). In the two pedigrees 53% of microsatellite loci were fully informative compared with 15% of RFLPs. Based on alleles detected among four parental genotypes, the microsatellites consisting of dinucleotide repeats were more polymorphic than those with tri- and tetra-nucleotide repeats. The microsatellite markers were not as transferable across species in the genus Acacia as RFLPs. Two thirds of the primers developed in A. mangium (subgenus Phyllodineae, section Juliflorae) amplified DNA from other species within the same section but failed to amplify in species from the subgenus Acacia. The availability of multiallelic, PCR-based, co-dominant microsatellite loci makes possible efficient studies of gene flow and breeding systems in A. mangium, a species with low allozyme variation. Received: 30 December 1999 / Accepted: 10 May 2000     

Identification and mode of action of quantitative trait loci affecting seedling height and leaf area in Eucalyptus nitens

 Regions of the genome influencing height and leaf area in seedlings of a three-generation outbred pedigree of Eucalyptus nitens have been identified. Three QTLs affecting height and two QTLs affecting leaf area were located using single-factor analysis of variance. The three QTLs affecting height each explained between 10.3 and 14.7% of the phenotypic variance, while the two QTLs for leaf area each explained between 9.8 and 11.6% of the phenotypic variation. Analysis of fully informative marker loci linked to the QTLs enabled the mode of action of the QTLs to be investigated. For three loci the QTL effect segregated from only one parent, while for two loci the QTL showed multiple alleles and the effect segregated from both parents in the pedigree. The two QTLs affecting leaf area were located in the same regions as two of the QTLs affecting height. Analysis of these regions with fully informative markers showed that both QTLs were linked to the same markers, but one had a similar size of effects and a similar mode of action for both height and leaf area, whilst the other showed a different mode of action for the two traits. These regions may contain two closely linked genes or may involve a single gene with a pleiotrophic effect on both height and leaf area. The QTL with the greatest effect showed multiple alleles and an intra-locus interaction that reduced the size of the effect. Assessment for two of the QTLs in a second related family did not show an effect associated with the marker loci; however, this was consistent with the mode of action of these QTLs and the pattern of inheritance in the second family. Received: 1 August 1996 / Accepted: 25 October 1996     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Genetic diversity of Metrodorea nigra (Rutaceae) from a small forest remnant in Brazil assessed with microsatellite markers

Metrodorea nigra (Rutaceae) is an endemic Brazilian tree of great ecological importance, frequently found in the submontane regions of ombrophilous dense and semideciduous forests. This tree is useful for reforesting degraded areas and the wood can be employed in construction. We developed 12 microsatellite markers from a genomic library enriched for GA/CA repeats, for this species. Polymorphisms were assessed in 40 trees of a highly fragmented population found in Cravinhos, State of S?o Paulo, in southeastern Brazil. Among the 12 loci, 8 were polymorphic and only one had fixed alleles in this population. The number of alleles per locus and expected heterozygosity ranged from 2 to 11 and from 0.190 to 0.889, respectively. These results revealed moderate levels of genetic variation in M. nigra population when compared to other tropical species. Additionally, transferability of the 12 primers was tested in seven other Brazilian Rutaceae tree species (endemics: M. stipularis, Galipea jasminiflora, Esenbeckia leiocarpa and non-endemics: E. febrifuga, E. grandiflora, Balfourodendron riedelianum, Zanthoxylum riedelianum). Transferability ranged among species, but at least 8 loci (~67%) amplified in M. stipularis, demonstrating a high potential for transferring microsatellite markers between species of the same genus in the Rutaceae family.     

Isolation of microsatellites from Girella tricuspidata

Microsatellites were isolated from Girella tricuspidata (Girellidae) and screened for 64 individuals collected from coastal reefs in New South Wales, Australia. All seven loci tested were highly polymorphic, having seven to 42 alleles with average heterozygosities between 0.44 and 1.0. One locus (GT1N8) had a significant excess of homozygotes, probably due to the presence of null alleles. These microsatellite loci should be informative for examining population genetics of this species.     

Linkage of the equine serum esterase (Es) and mitochondrial glutamate oxaloacetate transaminase (GOTM) loci. A horse-mouse homology

Three previously described electrophoretic phenotypes of mitochondrial glutamate oxaloacetate transaminase (GOTM) in horse leukocytes are shown to be controlled by two codominant alleles at a single autosomal locus. The GOTM locus is linked to the serum esterase locus (Es), as no recombination between these loci was observed among 16 informative offspring in one sire family. The results assign GOTM to equine linkage group (LG) II. The hypothesis that a part of LG II (e-Es) shares homologies with mouse chromosome 8 is thus confirmed, as the murine homologue of GOTM is located within the cluster of esterase loci on chromosome 8. The assumed homology also involves rabbit LG VI, rat LG V, and human chromosome 16. The observation is a striking example of the conservation of linkage relationships between mammalian species.     

Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs)

Microsatellite repeat sequences were investigated as sequenced-tagged site (STS) DNA markers to determine the potential for genetic analysis of the grapevine genome. The PCR-generated markers detect codominant alleles at a single locus or site in the genome. The marker type is very informative detecting high heterozygosity (69%–88%) within individual grapevine cultivars and high genetic variation between cultivars, making it a useful marker type for plant genome mapping and genome typing. For five loci a screening of 26 V. vinifera cultivars found 13, 12, 8, 5, and 4 different length alleles respectively with some alleles more common than others. The genomic DNA sequences surrounding microsatellite sequences were conserved within the genus permitting STS primers to amplify STSs from other Vitis species. These Vitis species were found to have some unique alleles not present in V. vinifera.     

Internal genetic structure and outcrossing rate in a natural population of Araucaria angustifolia (Bert.) O. Kuntze

The internal genetic structure and outcrossing rate of a population of Araucaria angustifolia (Bert.) O. Kuntze were investigated using 16 allozyme loci. Estimates of the mean number of alleles per loci (1.6), percentage of polymorphic loci (43.8%), and expected genetic diversity (0.170) were similar to those obtained for other gymnosperms. The analysis of spatial autocorrelation demonstrated the presence of internal structure in the first distance classes (up to 70 m), suggesting the presence of family structure. The outcrossing rate was high (0.956), as expected for a dioecious species. However, it was different from unity, indicating outcrossings between related individuals and corroborating the presence of internal genetic structure. The results of this study have implications for the methodologies used in conservation collections and for the use or analysis of this forest species.     

High-throughput identification of informative nuclear Loci for shallow-scale phylogenetics and phylogeography

One of the major challenges for researchers studying phylogeography and shallow-scale phylogenetics is the identification of highly variable and informative nuclear loci for the question of interest. Previous approaches to locus identification have generally required extensive testing of anonymous nuclear loci developed from genomic libraries of the target taxon, testing of loci of unknown utility from other systems, or identification of loci from the nearest model organism with genomic resources. Here, we present a fast and economical approach to generating thousands of variable, single-copy nuclear loci for any system using next-generation sequencing. We performed Illumina paired-end sequencing of three reduced-representation libraries (RRLs) in chorus frogs (Pseudacris) to identify orthologous, single-copy loci across libraries and to estimate sequence divergence at multiple taxonomic levels. We also conducted PCR testing of these loci across the genus Pseudacris and outgroups to determine whether loci developed for phylogeography can be extended to deeper phylogenetic levels. Prior to sequencing, we conducted in silico digestion of the most closely related reference genome (Xenopus tropicalis) to generate expectations for the number of loci and degree of coverage for a particular experimental design. Using the RRL approach, we: (i) identified more than 100,000 single-copy nuclear loci, 6339 of which were obtained for divergent conspecifics and 904 of which were obtained for heterospecifics; (ii) estimated average nuclear sequence divergence at 0.1% between alleles within an individual, 1.1% between conspecific individuals that represent two different clades, and 1.8% between species; and (iii) determined from PCR testing that 53% of the loci successfully amplify within-species and also many amplify to the genus-level and deeper in the phylogeny (16%). Our study effectively identified nuclear loci present in the genome that have levels of sequence divergence on par with mitochondrial loci commonly used in phylogeography. Specifically, we estimated that ～7% of loci in the chorus frog genome are >3% divergent within species; this translates to a prediction of approximately 50,000 single-copy loci in the genome with >3% divergence. Moreover, successful amplification of many loci at deeper phylogenetic levels indicates that the RRL approach represents an efficient method for rapid identification of informative loci for both phylogenetics and phylogeography. We conclude by making recommendations for minimizing the cost and maximizing the efficiency of locus identification for future studies in this field.     

Ten polymorphic microsatellite DNA markers for the platypus, Ornithorhynchus anatinus

We identified and optimized 10 microsatellite loci for the platypus, Ornithorhynchus anatinus (Monotremata: Ornithorhynchidae), and screened 21 individuals from the southern tablelands area of New South Wales, Australia. Each polymorphic locus possessed between two and 12 alleles with observed heterozygosities between 0.118 and 0.950. The intent of this effort was to provide informative loci for studies on the population genetics of this species.     

Additional microsatellites for Sparus aurata and cross‐species amplification within the Sparidae family

Six new microsatellite loci were isolated and characterized in 32 individuals from a farm population of gilthead seabream (Sparus aurata). Expected heterozygosity at all loci was high, ranging from 0.835 to 0.958 with between 10 and 27 alleles per locus. A multiplex polymerase chain reaction protocol was developed using four of the loci. Cross‐species amplification of the loci was tested in six species of the Sparidae family and four loci were successfully amplified in two or more related species.     

An electrophoretic investigation of populations of Atherina boyeri Risso, 1810 and A. presbyter Cuvier, 1829 (Teleostei: Atherinidae): genetic evidence in support of the two species

The data obtained using electrophoresis strongly support the specific status of Atherina boyeri Risso, 1810 and A. presbyfer Cuvier, 1829, and thuscontradict the recently proposedsynonymyof the two species. Four populations of A. boyeri and six populations of A. presbyter were assayed for 11 enzymes and general protein using muscle and liver extracts. Eight of the 11 enzymes were shown to be polymorphic at the 95% level. Sixteen loci, encoding 40 putative alleles were consistently resolved in all 10 populations.
The two species were fixed for different alleles at the EST-3 locus. At the G3PDH locus. with the exception of two heterozygotes, all individuals of each species were also homozygous for different alleles. At the PGM locus the common allele was unique to each species.
The mean Nei's genetic distance ( ), over all loci, calculated between populations of A. boyeri (= 0.10 ± 0.06); between populations of A. presbyter ( = 0.02 0.02) and between populations of A. boyeri and A. presbyter ( = 0.42 0.09) indicated the separateness of the two species.
UPGMA cluster analysis based on genetic distances produced a dendrogram whose principal dichotomy resulted in the formation of two clusters. The ordination of populations in the UPGMA cluster analysis strongly reflected the geographic distribution of populations in both species.     

Evolutionary relationships among 12 species belonging to three genera of the family Microhylidae in Papua New Guinea revealed by allozyme analysis

To elucidate the potential of electrophoretic analysis for understanding relationships among microhylid frogs in Papua New Guinea, an allozyme analysis was conducted. A total of 119 individuals from nine species of Cophixalus, two species of Sphenophryne and one species of Barygenys, all of which belong to the family Microhylidae, were studied. Fourteen enzymes extracted from skeletal muscles and livers were analyzed by starch-gel electrophoresis. These enzymes were encoded by genes at 20 loci. There were 2-15 phenotypes produced by 2-12 alleles at these loci. The mean proportion of heterozygous loci per individual, mean proportion of polymorphic loci per population, and mean number of alleles per locus in 12 species were 6.1%, 17.1% and 1.17a on average, respectively. The NJ and ML trees constructed from Nei's genetic distances showed that the genus Sphenophryne can be distinguished biochemically from Cophixalus and Barygenys, and that the species groups of Cophixalus, which are similar in external morphology, can be divided biochemically into several species.     

Parental assignment in fish using microsatellite genetic markers with finite numbers of parents and offspring

Deterministic predictions for the proportion of offspring assigned to different numbers of parent-pairs are developed in order to investigate the power of microsatellite loci for parental assignment in fish species. Comparisons with stochastic simulation results show that predictions based on exclusion probabilities are accurate, provided that the number of parents involved in the crosses is large. Accounting for sampling of parents gave very accurate predictions for a small number of parents and a single biallelic locus. For large numbers of loci or large numbers of alleles per locus stochastic simulations are, however, the only available method to predict the power of assignment of a particular set of loci when the number of parents is small. Nine 5-allele loci or six 10-allele loci with equifrequent alleles, are sufficient for assigning, with certainty, parents to 99% of the fish resulting from either 100 or 400 crosses. Results simulating a set of highly polymorphic microsatellites developed for Atlantic salmon show that the four most informative loci are sufficient to assign at least 99% of the offspring to the correct pair with 100 crosses involving 100 males and 100 females. An additional locus is required for correctly assigning 99% of the offspring when the 100 crosses are produced with 10 males and 10 females.     

Isolation of highly polymorphic microsatellite loci from the temperate damselfish Parma microlepis

Microsatellites were isolated from the damselfish Parma microlepis (Günther 1862) (Pomacentridae) and screened for 100 individuals. Seven of the eight loci tested were highly polymorphic, having 14–43 alleles with average heterozygosities between 0.86 and 0.97. These loci should be informative for studies on population genetics of this species.     

Mutability of microsatellites developed for the ant Camponotus consobrinus

Five highly polymorphic (GA)n microsatellite loci are reported for the formicine ant Camponotus consobrinus. The occurrence of many nests with a simple family structure enabled a search for new mutations, 11 of which were found from 3055 informative typings. These mutations were not randomly distributed across loci, 10 of them occurring at the locus Ccon70. The spectrum of mutations across alleles at Ccon70 was also nonrandom, with all of them occurring in alleles in the upper half of the allele size distribution. Six of the Ccon70 mutations decreased allele size. The mutations observed fit the stepwise mutation model well, i.e. mutations could always be assigned to an allele which differed in size from them by one repeat unit. The parental origins of the Ccon70 mutations were established and appear more female biased than vertebrate mutations, significantly so compared with human haemophilia A and primate intron mutations. This result may indicate that the lack of meiosis in males (which are haploid in ants) reduces the mutation rate in that sex relative to species in which both sexes are diploid.     

Characterization of Spanish grapevine cultivar diversity using sequence-tagged microsatellite site markers.

A broad germplasm bank collection containing most of the autochthonous Spanish grapevine cultivars was analyzed using six sequence-tagged microsatellite site (STMS) loci: VVS2, VVMD5, VVMD7, ssrVrZAG47, ssrVrZAG62, and ssrVrZAG79. The number of alleles obtained ranged from 9 in ssrVrZAG47 to 13 in VVS2, and the observed genotypes per locus varied between 24 (ssrVrZAG47) and 41 (VVSS2). A total of 57 unique genotypes were obtained considering all 6 loci, and 40 varieties presented at least 1 of these specific genotypes. The genotypic combinations for the 6 loci have generated 163 different profiles in the 176 cultivars. Ten pairs of accessions and one group of four Garnacha's cultivars can not be differentiated. The observed heterozygosity varied between 75.6 (VVMD7) and 90.9% (VVMD5), without significant differences from the expected values for any loci. The VVMD5 locus was the most informative, and also showed the highest discrimination power. The cumulative discrimination power for all six loci was practically 1; however, in fact, these STMS loci have differentiated only about 93% of the accessions, probably owing to high relatedness of the plant material. Usefulness of this STMS set for characterization of a Spanish grapevine collection is emphasized, as well as the elaboration of databases with these molecular markers.