Multiplexed microsatellite markers for the genetic analysis of Eucalyptus leucoxylon (Myrtaceae) and their utility for ecological and breeding studies in other eucalyptus species

Eucalyptus leucoxylon is a widespread woodland tree species found in southeastern Australia that has suffered from, and continues to be, threatened by the impacts of habitat clearance and degradation. Populations now consist predominantly of scattered individuals, and their conservation status is of increasing concern. We report the development and characterization of a set of eight highly polymorphic microsatellite loci for E. leucoxylon. The loci can be amplified in three PCR multiplexes and electrophoresed in a single lane, allowing rapid throughput of large numbers of samples. A total of 111 alleles were detected in 68 individuals with an average of 12.3 alleles per locus, a mean expected heterozygosity of 0.83, and a mean observed heterozygosity of 0.72. The combined probabilities of identity and probabilities of paternity exclusion allow an extremely precise level of individual identification, indicating that these microsatellite markers will be ideal for population genetic and parentage-type studies in E. leucoxylon. The markers also exhibited an average of 76% conservation within the subgenus Symphyomyrtus, to which E. leucoxylon belongs, and 53% conservation across other subgenera of Eucalyptus, demonstrating the potential of these markers in ecological and breeding studies in a wide range of Eucalyptus species.     

Conservation and synteny of SSR loci and QTLs for vegetative propagation in four Eucalyptus species

Conservation of microsatellite loci, heterozygous in Eucalyptus grandis, Eucalyptus urophylla, Eucalyptus tereticornis and Eucalyptus globulus, allowed us to propose homeologies among genetic linkage groups in these species, supported by at least three SSR loci in two different linkage groups. Marker-trait associations for sprouting and adventitious rooting ability were also compared in the four species. Putative quantitative trait loci (QTLs) influencing vegetative propagation traits were located on homeologous linkage groups. Our findings indicate high transferability of microsatellite markers between Eucalyptus species of the Symphyomyrtus subgenus and establish foundations for the use of synteny in the genetic analysis of this genus. Microsatellite markers should help integrate eucalypt genetic linkage maps from various sources. The availability of comparative linkage maps provides a basis of more-efficient use of genetic information for molecular breeding and evolutionary studies in Eucalyptus.     

Cross-species amplification of Eucalyptus microsatellite loci

This study examined the interspecific amplification of nuclear microsatellite loci developed mainly for eucalypts in the subgenus Symphyomyrtus across five species within the second most speciose subgenus, subgenus Eucalyptus. A set of eight to 10 loci, depending on taxon, have been identified that are highly variable and easily scored. The successful transfer of microsatellite loci to these eucalypt species sidesteps the expensive and time-consuming development of species-specific microsatellite libraries. This primer set will enable the examination and cross-species comparison of the genetic resources of commercially and ecologically important members of the subgenus Eucalyptus.     

A cladistic study of Arillastrum, Angophora and Eucalyptus (Myrtaceae)

A cladistic study of Anllastrum, Angophora and Eucalyptus (Myrtaceae). Transformed cladistic; character compatibility; branch and bound, and Farris-Wagner methods gave similar solutions in a cladistic study of Arillastrum, Angophora and Eucalyptus. These analyses, based on morphological characters, indicate that Eucalyptus is a monophyletic group and that its sister taxon is Angophora.
Within Eucalyptus , subgenera Blakella and Corymbia are sister taxa to all other groups; subgenera Monocalyptus, Idiogenes and Gaubaea form a monophyletic group with subgenus Monocalyptus sister to subgenera Idiogenes and Gaubaea ; subgenera Symphyomyrtus and Telocalyptus together also form a monophyletic group and, with Eucalyptus similis (subgenus Eudesmia group 4), are sister to the Monocalyptus group. Eucalyptus subgenus Telocalyptus (4 species), Eucalyptus subgenus Idiogenes (1 species) and Eucalyptus subgenus Gaubaea (2 species) should not be recognized as subgenera and some individual species need further examination. Eucalyptus subgenus Eudesmia is a paraphyletic group.
Some characters are identified as parallelisms, e.g. axillary inflorescences, sepaline operculum, bristle glands, and clustered anthers. A more congruent interpretation of the single operculum of Eucalyptus subgenus Monocalyptus as at least partly petaline rather than solely sepaline in origin is suggested.
The area relationships for the taxa are concordant with those derived from geological and climatological information. New Caledonia is sister area to Australia, and within Australia southwestern Australia is sister area to south-eastern and north-eastern Australia.     

Genomes,multiple origins,and lineage recombination in the Glycine tomentella (Leguminosae) polyploid complex: histone H3-D gene sequences

Relationships among the various diploid and polyploid taxa that comprise Glycine tomentella have been hypothesized from crossing studies, isozyme data, and repeat length variation for the 5S nuclear ribosomal gene loci. However, several key questions have persisted, and detailed phylogenetic evidence from homoeologous nuclear genes has been lacking. The histone H3-D locus is single copy in diploid Glycine species and has been used to elucidate relationships among diploid races of G. tomentella, providing a framework for testing genome origins in the polyploid complex. For all six G. tomentella polyploid races (T1-T6), alleles at two homoeologous histone H3-D loci were isolated and analyzed phylogenetically with alleles from diploid Glycine species, permitting the identification of all of the homoeologous genomes of the complex. Allele networks were constructed to subdivide groups of homoeologous alleles further, and two-locus genotypes were constructed using these allele classes. Results suggest that some races have more than one origin and that interfertility within races has led to lineage recombination. Most alleles in polyploids are identical or closely related to alleles in diploids, suggesting recency of polyploid origins and spread beyond Australia. These features parallel the other component of the Glycine subgenus Glycine polyploid complex, G. tabacina, one of whose races shares a diploid genome with a G. tomentella polyploid race.     

Genetic variation in Glossina brevipalpis, G.longipennis and G.pallidipes, and the phenetic relationships of Glossina species

Glossina brevipalpis Newstead, G.longipennis Corti, and G.pallidipes Austen maintained at ILRAD, Nairobi, Kenya, were examined for genetic variation of fourteen enzyme loci, using polyacrylamide gel electrophoresis. G.brevipalpis had six polymorphic loci, an average of 1.46 effective alleles per locus and a mean heterozygosity per locus of 20.0 +/- 7.1%. The figures for the same parameters in G.longipennis were 3, 1.16 and 8.2 +/- 4.9%, and for G.pallidipes the figures were 7, 1.40 and 22.3 +/- 6.3%. Seven rare alleles were lost from the G.brevipalpis colony during a 1-year period, but no statistically significant changes were observed in the genetics of the colony during this period. Using allele frequency data for ten of the enzymes studied, and frequencies for these enzymes in other taxa, a phenogram was constructed that indicated that the subgenus Austenina (i.e. the fusca group) is the oldest of the three subgenera within the genus Glossina, and that the subgenus Glossina s.str. (i.e. the morsitans group) may be paraphyletic.     

Microsatellite primers in the white proteas (Protea section Exsertae, Proteaceae), a rapidly radiating lineage

? Premise of the study: Microsatellite primers were developed in the South African sclerophyllous shrub Protea punctata to investigate the degree of population differentiation within and between P. punctata and closely related species. ? Methods and Results: 10 primer pairs were identified from three individuals of Protea punctata. The primers amplified di- and tri-nucleotide repeats. Across all P. punctata samples, the loci have 8-49 alleles. All primers also amplified in Protea section Exsertae (P. aurea, P. aurea subsp. potbergensis, P. mundii, P. venusta, P. lacticolor, and P. subvestita). The loci had 14-69 alleles across the subgenus. ? Conclusions: These results show the broad utility of microsatellite loci for future studies of population genetics in the white proteas and their potential utility across the entire genus.     

Genetic tags applied to the European hake, Merluccius merluccius (L.)

Three biochemical gene markers test the hypothesis that the European hake, Merluccius merluccius (L.), along the west European continental shelf are one race. The three polymorphic loci were serum transferrin (Tf), eye vitreous fluid butyric esterase (Es) and liver superoxide dismutase (Sod). Five transferrin alleles, three esterase alleles and two superoxide dismutase alleles were identified. Heterogeneity tests on genotype frequency distribution for twelve areas ranging from Norway to Biscay revealed no significant variation. The results using these genetic tags are consistent with the unit race hypothesis for hake throughout the sea areas sampled.     

Development of tetranucleotide microsatellite markers for the cushion star, Asterina gibbosa, and cross-species amplification

We describe nine polymorphic tetranucleotide microsatellite loci from the starfish, Asterina gibbosa. Loci were isolated from a partial genomic library that had been enriched for AAAC repeat sequences. Number of alleles per locus ranged from two to 14 in a sample of 85 individuals from three populations (two from Spain and one from the UK). Observed and expected heterozygosities per population ranged from 0.000 to 0.400 and from 0.040 to 0.784, respectively. All loci presented significant heterozygote deficits in one or more populations. Eight of these loci were amplified and variable in A. pancerii and A. phylactica. These loci will be used to study population structure in A. gibbosa.     

Genetic tags applied to the European hake, Merluccius merluccius (L.)

Three biochemical gene markers test the hypothesis that the European hake, Merluccius merluccius (L.), along the west European continental shelf are one race. The three polymorphic loci were serum transferrin (Tf), eye vitreous fluid butyric esterase (Es) and liver superoxide dismutase (Sod). Five transferrin alleles, three esterase alleles and two superoxide dismutase alleles were identified. Heterogeneity tests on genotype frequency distribution for twelve areas ranging from Norway to Biscay revealed no significant variation. The results using these genetic tags are consistent with the unit race hypothesis for hake throughout the sea areas sampled.     

Localization of oleuropeyl glucose esters and a flavanone to secretory cavities of myrtaceae

We report the widespread occurrence of structurally diverse oleuropeyl glucose esters, including the new diester eucaglobulin B, localized specifically to the essential oil secretory cavities of myrtaceous species. Clear taxonomic patterns in the composition of cavity extracts within the genus Eucalyptus are shown with species from subgenus Symphyomyrtus dominated by oleuropeyl glucose esters and species from subgenus Eucalyptus dominated instead by the flavanone, pinocembrin. We also examined the intra-species occurrence of oleuropeyl glucose esters by quantifying the abundant constituents cuniloside B and froggattiside A in trees from two populations of Eucalyptus polybractea R.T. Baker. All trees contained both compounds, which were positively correlated with total essential oil concentration. This apparent ubiquity of oleuropeyl glucose esters at both intra- and inter-specific levels in Eucalyptus is indicative of important physiological or ecological functions. The significance of their prevalence and the sequestration of these esters and also pinocembrin to the extracellular domain of secretory cavities is discussed in light of their potential biological activities and our findings that they are spatially segregated to the exterior of cavity lumina. The localization of oleuropeyl glucose esters to a specific and isolatable tissue type has the potential to aid in future elucidation of function and biosynthesis.     

Species discrimination and population differentiation in ants using microsatellites

This study was conducted to establish the regional scale of population differentiation of ants in the wheat belt of central western New South Wales. Microsatellite variation was surveyed at five loci in two morphologically similar ant species (designated "A" and "B") from the Camponotus ephippium complex. Three of the five scored microsatellite loci were highly variable with totals, in the two species, of 11, 13 and 42 alleles. The other loci had two and three alleles. The mean number of alleles per locus per sample ranged from 2.0 to 4.6 for species A and from 1.4 to 3.8 in species B. Mean observed heterozygosity was 0.385 for species A and 0.363 for species B. The geographic distribution of genotypes was significantly non-random for all tested loci in both species. Eight of 47 alleles in species A and 15 of 28 in species B were restricted to a single site. Allelic accumulation percentages were calculated for several orderings of samples - level of heterozygosity, sample size and geographic position. In all orderings three or more samples must be included for more than three-quarters of alleles to be represented.     

Genetics of four plasma protein loci in Equus przewalskii: new alleles at the prealbumin, postalbumin and transferrin loci

This paper reports genetic variation at the prealbumin (Pr), postalbumin (Pa) and transferrin (Tf) loci in Equus przewalskii found using thin layer isoelectric focusing and an amphoteric separator. The method resolves all three loci plus serum esterase (Es) on a single gel, and typing of all four loci is readily achieved. In addition to the esterase alleles previously reported by Fisher & Scott (1979), five alleles were found at the Pr locus, three at the Pa locus and six at the Tf locus. Analysis of several mating types confirms inheritance is autosomal and codominant for all four loci.     

Genetics of four plasma protein loci in Equus przewalskii: new alleles at the prealbumin, postalbumin and transferrin loci

This paper reports genetic variation at the prealbumin ( Pr ), postalbumin ( Pa ) and transferrin ( Tf ) loci in Equus przewalskii found using thin layer isoelectric focusing and an amphoteric separator. The method resolves all three loci plus serum esterase ( Es ) on a single gel, and typing of all four loci is readily achieved. In addition to the esterase alleles previously reported by Fisher & Scott (1979), five alleles were found at the Pr locus. three at the Pa locus and six at the Tf locus. Analysis of several mating types confirms inheritance is autosomal and codominant for all four loci.     

A Two-Locus Neutrality Test: Applications to Humans, E. COLI and Lodgepole Pine

The expected disequilibrium between two loci with k alleles at one locus and l alleles at the other is given for a sample of size n drawn from a population under neutrality equilibrium. Three different measures of disequilibrium with 95% intervals are tabulated for combinations of n, k, l and 4Nc, where N is the effective population size and c is the amount of recombination between the loci. The extent and pattern of disequilibrium are strongly dependent upon 4Nc and are somewhat dependent on n, k and l. The 95% intervals are large, particularly for low numbers of alleles and low values of 4Nc. As examples, observed disequilibrium from histocompatibility loci in humans (HLA) and electrophoretic data in E. coli and lodgepole pine were compared to these theoretical values. Using information about recombination rates, the HLA data showed more disequilibrium than neutrality expectations, whereas electrophoretic data from E. coli and lodgepole pine had somewhat less disequilibrium than neutrality expectations.     

Identification and characterization of 10 polymorphic microsatellite loci in the German cockroach,Blattella germanica

Primer sequences and initial characterization are presented for 10 microsatellite loci isolated from the German cockroach, Blattella germanica. In a sample of 30 individuals from a single population sample, all loci were polymorphic with two to 12 alleles segregating per locus and levels of observed heterozygosity ranging from 0.27 to 0.92. One locus showed a deficit of heterozygotes. Experimental conditions are described for polymerase chain reaction multiplexing, which enables the genotyping of eight loci in three electrophoretic runs consisting of one set of three and two sets of two markers. Seven primer sets cross‐amplify in the related Blattella asahinai.     

New polymorphic microsatellite loci for population studies in the European anchovy, Engraulis encrasicolus (L.)

Eleven microsatellite loci were developed in the European anchovy, Engraulis encrasicolus, and tested in samples from two geographically distant populations (Atlantic and Mediterranean Sea). Number of alleles ranged from eight to 28 and observed heterozygosity from 0.440 to 0.920. There was no evidence of linkage disequilibrium, although two loci are indeed linked. All loci were in Hardy-Weinberg equilibrium, except for one locus in the Atlantic and two loci in the Mediterranean sample. These three loci plus two more showed evidence for null alleles.     

Rapid expansion of microsatellite sequences in pines

Microsatellite persistence time and evolutionary change was studied among five species of pines, which included a pair of closely related species (Pinus sylvestris and Pinus resinosa) in the subgenus Pinus, their relative Pinus radiata, and another closely related species pair (Pinus strobus and Pinus lambertiana) in the subgenus Strobus. The effective population sizes of these species are known to have ranged from the very small bottlenecks of P. resinosa to vast populations of P. sylvestris. This background allowed us to place the microsatellite evolution in a well-defined phylogenetic setting. Of 30 loci originating from P. strobus and P. radiata, we were able to consistently amplify 4 in most of the these pine species. These priming sites had been conserved for over 100 Myr. The four microsatellites were sequenced in the five species. Flanking sequences were compared to establish that the loci were orthologous. All microsatellites had persisted in these species, despite very different population sizes. We found a recent microsatellite duplication: a closely related pair of loci in P. strobus, where the other four species had just one locus. On two independent occasions, the repeat area of this same microsatellite (locus RPS 105a/b) had grown from a very low repeat number to 15 or 17 in the last 10-25 Myr. Other parts of the same compound microsatellite had remained virtually unchanged. Locus PR 4.6 is known to be polymorphic in both P. radiata and P. sylvestris, but the polymorphism in the two species is due to different motifs. The very large pine genomes are highly repetitive, and microsatellite loci also occur as gene families.     

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     

Reconstructing species phylogeny of the carabid beetles Ohomopterus using multiple nuclear DNA sequences: heterogeneous information content and the performance of simultaneous analyses

We attempted a phylogenetic reconstruction for the carabid subgenus Ohomopterus (genus Carabus), a notable case of radiation with mitochondrial introgression across species. Sequence data from five nuclear single copy loci were used, including wingless (Wg), phosphoenolpyruvate carboxykinase (PepCK), cytochrome c (Cytc), elongation factor-1alpha (EF-1alpha), and an anonymous single copy locus (Carab1). Sequences of Cytc, EF-1alpha, and Carab1 included intron or intron-like parts with length variation. The analysis of individual loci resulted in low resolution of the phylogenetic relationships, and the monophyly of several morphologically recognized species for which multiple specimens were analyzed was not revealed. Several specimens were heterozygous, with non-monophyletic alleles observed in three of the five loci at which alleles in heterozygotes were separated. In a simultaneous analysis of the five loci with ambiguously aligned parts eliminated and heterozygotic sites treated as missing, the resulting tree was well resolved, but the branch support was generally weak because of conflicting phylogenetic signals from different loci. We also attempted to incorporate allelic sequence data plus the ambiguously aligned parts in the analysis, by using all possible combinations of alleles from different loci in heterozygotic individuals, but the resultant tree was not supported more strongly. Nonetheless, these simultaneous analyses provided support for the monophyly of several species and species groups, and revealed the basic evolutionary trend of OHOMOPTERUS: initial widespread groups with simpler genitalia and the origination of exaggerated genitalia in a derived clade. This study exemplifies problems inherent in the phylogenetic reconstruction of closely related organisms where low levels of variation limit the information content from each locus, while heterozygosity, different phylogenetic history of multiple loci, and alignment ambiguity further hamper phylogenetic reconstruction unless several loci converge on a uniform signal.