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Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers.

We have used a "two-way pseudo-testcross" mapping strategy in combination with the random amplified polymorphic DNA (RAPD) assay to construct two moderate density genetic linkage maps for species of Eucalyptus. In the cross between two heterozygous individuals many single-dose RAPD markers will be heterozygous in one parent, null in the other and therefore segregate 1:1 in their F1 progeny following a testcross configuration. Meiosis and gametic segregation in each individual can be directly and efficiently analyzed using RAPD markers. We screened 305 primers of arbitrary sequence, and selected 151 to amplify a total of 558 markers. These markers were grouped at LOD 5.0, theta = 0.25, resulting in the maternal Eucalyptus grandis map having a total of 240 markers into 14 linkage groups (1552 cM) and the paternal Eucalyptus urophylla map with 251 markers in 11 linkage groups (1101 cM) (n = 11 in Eucalyptus). Framework maps ordered with a likelihood support > or = 1000:1 were assembled covering 95% of the estimated genome size in both individuals. Characterization of genome complexity of a sample of 48 mapped random amplified polymorphic DNA (RAPD) markers indicate that 53% amplify from low copy regions. These are the first reported high coverage linkage maps for any species of Eucalyptus and among the first for any hardwood tree species. We propose the combined use of RAPD markers and the pseudo-testcross configuration as a general strategy for the construction of single individual genetic linkage maps in outbred forest trees as well as in any highly heterozygous sexually reproducing living organisms. A survey of the occurrence of RAPD markers in different individuals suggests that the pseudo-testcross/RAPD mapping strategy should also be efficient at the intraspecific level and increasingly so with crosses of genetically divergent individuals. The ability to quickly construct single-tree genetic linkage maps in any forest species opens the way for a shift from the paradigm of a species index map to the heterodox proposal of constructing several maps for individual trees of a population, therefore mitigating the problem of linkage equilibrium between marker and trait loci for the application of marker assisted strategies in tree breeding.

Base Sequence↗

The effects of Eucalyptus pollen on longevity and fecundity of Eucalyptus longhorned borers (Coleoptera: Cerambycidae).

The longevity and fecundity of adult Phoracantha recurva and Phoracantha semipunctata were strongly affected by diet. Female P. recurva fed a diet of Eucalyptus pollen and sucrose solution lived 34-56% longer than females fed diets containing other types of pollen, ground dog chow, or sucrose solution alone. Diet had no significant effect on longevity of P. recurva males. Similarly, longevities of P. semipunctata females were increased 48-71% on the Eucalyptus pollen diet compared with the other diets. Male P. semipunctata also lived longer on the Eucalyptus pollen diet than most of the other diets. Fecundity was dramatically affected by diet, with P. recurva females fed the Eucalyptus pollen diet laying approximately 4-8 times more eggs than females on the other diets. Eucalyptus pollen also increased the fecundity of P. semipunctata females approximately 3-5-fold. Diet resulted in only minor effects on egg size and percent egg hatch for both beetle species.

Animals↗

Antihyperglycemic actions of Eucalyptus globulus (Eucalyptus) are associated with pancreatic and extra-pancreatic effects in mice.

Eucalyptus globulus (eucalyptus) is used as a traditional treatment for diabetes. In this study, incorporation of eucalyptus in the diet (62.5 g/kg) and drinking water (2.5 g/L) reduced the hyperglycemia and associated weight loss of streptozotocin-treated mice. An aqueous extract of eucalyptus (AEE) (0.5 g/L) enhanced 2-deoxy-glucose transport by 50%, glucose oxidation by 60% and incorporation of glucose into glycogen by 90% in mouse abdominal muscle. In acute, 20 min incubations, 0.25-0.5 g AEE/L evoked a stepwise 70-160% enhancement of insulin secretion from the clonal pancreatic beta-cell line (BRIN-BD11). The stimulatory effect of 0.5 g/L AEE was unaltered by the presence of 400 micromol diazoxide/L and prior exposure to AEE did not alter subsequent insulin secretory response to L-alanine, thereby negating adetrimental effect on cell viability. The effect of AEE was not potentiated by glucose or demonstrable in cells exposed to a depolarizing concentration of KCl. Further study of the insulin-releasing effects of AEE revealed the activity to be heat stable, acetone insoluble, stable to acid, but abolished by exposure to alkali. Sequential extraction with solvents revealed activity in both methanol and water fractions, indicating the presence of more than one biologically active extract constituent. These data indicate that Eucalyptus globulus represents an effective antihyperglycemic dietary adjunct for the treatment of diabetes and a potential source for discovery of new orally active agent(s) for future therapy.

Animals↗

Exacerbation of asthma related to Eucalyptus pollens and to herb infusion containing Eucalyptus.

Eucalyptus is known as a potential cause of cutaneous diseases due to contact with oil or pollens, and of respiratory allergic diseases due to exposure to pollens. We report the case of a 30-year-old woman with asthma and rhinoconjunctivitis in which symptoms appeared to be exacerbated by Eucalyptus pollens and by ingestion of an infusion containing Eucalyptus. Specific IgE were positive for Eucalyptus pollens and negative for common aeroallergens. Our report shows that Eucalyptus may elicit asthma and rhinitis with an IgE-mediated mechanism both by inhalation of pollens and by ingestion of herb infusions, and suggests that care should be taken in administering herbal medications in asthmatic subjects.

Adult↗

An efficient procedure to stably introduce genes into an economically important pulp tree (Eucalyptus grandis x Eucalyptus urophylla).

Regeneration problems are one of the main limitations preventing the wider application of genetic engineering strategies to the genus Eucalyptus. Seedlings from Eucalyptus grandis x Eucalyptus urophylla were selected according to their regeneration (adventitious organogenesis) and transformation capacity. After in vitro cloning, the best genotype of 250 tested was transformed via Agrobacterium tumefaciens. A cinnamyl alcohol dehydrogenase (CAD) antisense cDNA from Eucalyptus gunnii was transferred, under the control of the 35S CaMV promoter with a double enhancer sequence, into a selected genotype. According to kanamycin resistance and PCR verification, 120 transformants were generated. 58% were significantly inhibited for CAD activity, and nine exhibited the highest down-regulation, ranging from 69 to 78% (22% residual activity). Southern blot hybridisation showed a low transgene copy number, ranging from 1 to 4, depending on the transgenic line. Northern analyses on the 5-16 and 3-23 lines (respectively one and two insertion sites) demonstrated the antisense origin of CAD gene inhibition. With respectively 26 and 22% of residual CAD activity, these two lines were considered as the most interesting and transferred to the greenhouse for further analyses.

Base Sequence↗

Antimicrobial activities of eucalyptus leaf extracts and flavonoids from Eucalyptus maculata.

AIMS: We investigated the antimicrobial activities of eucalyptus leaf extracts to find effective antibacterial agents. METHODS AND RESULTS: The antimicrobial activities of leaf extracts from 26 species of eucalyptus were measured. Extracts of Eucalyptus globulus, E. maculata and E. viminalis significantly inhibited the growth of six Gram-positive bacteria (Staphylococcus aureus, MRSA, Bacillus cereus, Enterococcus faecalis, Alicyclobacillus acidoterrestris, Propionibacterium acnes), and of a fungus (Trichophyton mentagrophytes), but they did not show strong antibacterial activity against Gram-negative bacteria (Escherichia coli, Pseudomonas putida). 2',6'-dihydroxy-3'-methyl-4'-methoxy-dihydrochalcone, eucalyptin and 8-desmethyl-eucalyptin, isolated from E. maculata extracts, exhibited potent antimicrobial activities against seven micro-organisms with minimum inhibitory concentrations (MIC) ranging from 1.0 to 31 mg l(-1). CONCLUSIONS: The eucalyptus extracts and three compounds from E. maculata were found to be effective against micro-organisms that cause food poisoning, acne and athlete's foot. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows potential uses of extracts from E. globulus, E. maculata and E. viminalis, and antimicrobial compounds isolated from E. maculata.

Anti-Bacterial Agents↗

Changes in essential oil during enzyme-assisted ensiling of lemongrass (Cymbopogon citratus Stapf.) and lemon eucalyptus (Eucalyptus citriodora Hook).

Changes in essential oil during ensiling of lemongrass and lemon eucalyptus were studied. Wilted lemongrass and eucalyptus leaves were ensiled in 0.25-L anaerobic jars. Samples consisted of a control (no additives) and a treated sample (0.5% glucose and lactic acid bacteria and 1% cellulase plus 1% hemicellulase plus pectinase). Three jars per treatment were sampled on days 2, 6, 10, and 36 for analysis of essential oil. Essential oil was obtained by extraction and by hydrodistillation. Extraction efficacy of essential oil from the lemongrass was improved by the enzyme treatment, but it was much lower than the amount obtained by distillation. The major components of the essential oil were neral and geranial. In the eucalyptus, total essential oils obtained by distillation decreased during ensiling, and the amount was similar to the amount obtained by extraction. Citronellal, which was the major component of the essential oil in the fresh eucalyptus leaves, decreased, whereas isopulegol and 3,8-terpinolhydrate increased during ensiling.

Anti-Bacterial Agents↗

Comparative genetic linkage maps of Eucalyptus grandis, Eucalyptus globulus and their F1 hybrid based on a double pseudo-backcross mapping approach.

Comparative genetic mapping in interspecific pedigrees presents a powerful approach to study genetic differentiation, genome evolution and reproductive isolation in diverging species. We used this approach for genetic analysis of an F(1) hybrid of two Eucalyptus tree species, Eucalyptus grandis (W. Hill ex Maiden.) and Eucalyptus globulus (Labill.). This wide interspecific cross is characterized by hybrid inviability and hybrid abnormality. Approximately 20% of loci in the genome of the F(1) hybrid are expected to be hemizygous due to a difference in genome size between E. grandis (640 Mbp) and E. globulus (530 Mbp). We investigated the extent of colinearity between the two genomes and the distribution of hemizygous loci in the F(1) hybrid using high-throughput, semi-automated AFLP marker analysis. Two pseudo-backcross families (backcrosses of an F(1) individual to non-parental individuals of the parental species) were each genotyped with more than 800 AFLP markers. This allowed construction of de novo comparative genetic linkage maps of the F(1) hybrid and the two backcross parents. All shared AFLP marker loci in the three single-tree parental maps were found to be colinear and little evidence was found for gross chromosomal rearrangements. Our results suggest that hemizygous AFLP loci are dispersed throughout the E. grandis chromosomes of the F(1) hybrid.

Chromosome Mapping↗

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.

Australia↗

Leaf water relations of Eucalyptus cloeziana and Eucalyptus argophloia in response to water deficit.

Leaf water relations responses to limited water supply were determined in 7-month-old plants of a dry inland provenance of Eucalyptus argophloia Blakely and in a humid coastal provenance (Gympie) and a dry inland provenance (Hungry Hills) of Eucalyptus cloeziana F. Muell. Each provenance of E. cloeziana exhibited a lower relative water content at the turgor loss point, a lower apoplastic water content, a smaller ratio of dry mass to turgid mass and a lower bulk modulus of elasticity than the single provenance of E. argophloia. Osmotic potential at full turgor and water potential at the turgor loss point were significantly lower in E. argophloia and the inland provenance of E. cloeziana than in the coastal provenance of E. cloeziana. There was limited osmotic adjustment in response to soil drying in E. cloeziana, but not in E. argophloia. Between-species differences in water relations parameters were larger than those between the E. cloeziana provenances. Both E. cloeziana provenances maintained turgor under moderate water stress through a combination of osmotic and elastic adjustments. Eucalyptus argophloia had more rigid cell walls and reached lower water potentials with less reduction in relative water content than either of the E. cloeziana provenances, thereby enabling it to extract water from dryer soils.

Dehydration↗

Comparison of classical and ultrasound-assisted isolation procedures of cellulose from kenaf (Hibiscus cannabinus L.) and eucalyptus (Eucalyptus rodustrus Sm.).

A comparative study of classical and ultrasound-assisted extraction and purification of cellulose from kenaf (Hibiscus cannabinus L.) and eucalyptus (Eucalyptus rodustrus Sm.), has been conducted. The isolated cellulose samples were studied by diffuse reflectance infrared Fourier transform spectroscopy and 13C nuclear magnetic resonance (13C-NMR) spectroscopy and the crystallinity was also determined. The use of ultrasound decreased the total time of treatment, in addition the purity of the obtained cellulose was very high.

Cellulose↗

Composition and protein quality of honeybee-collected pollen of Eucalyptus marginata and Eucalyptus calophylla.

The composition and protein quality of the two most important Western Australian export-quality pollens were investigated. Crude pollen protein content was 20.6% and 27.9% for Jarrah (Eucalyptus marginata) and Marri (Eucalyptus calophylla), respectively. Lysine was the limiting amino acid relative to the FAO protein scoring pattern (Food and Agriculture Organization of the United Nations), and the amino acid scores were 0.73 and 0.66 for Jarrah and Marri pollen, respectively. Apparent biological value (BV) was 61.7 for Jarrah pollen, 66.9 for Marri pollen and 71.4 for the casein controls. Adjusted protein efficiency ratio (PER) values were 2.5, 1.2 and 1.1 for casein and Jarrah and Marri pollens, respectively. Apparent net protein utilization (NPU) was significantly reduced for both pollens (32.8 for Jarrah and 39.5 for Marri) compared to casein (63.6). The low apparent NPU values result from the relatively low digestibility of pollens. Apparent digestibility was 52 and 59% for Jarrah and Marri pollen compared to 89% for casein. Although both Jarrah and Marri pollen are relatively high in protein and have favorable amino acid patterns, their relatively low digestibility will be a limiting factor in their usefulness as a food for humans and monogastric animals. The proximate analysis and mineral content of the pollens are also presented.

Amino Acids↗

Distinct effects of auxin and light on adventitious root development in Eucalyptus saligna and Eucalyptus globulus.

Adventitious rooting is essential for vegetative propagation of woody species. We studied the effects of auxin and light on the development of adventitious roots in cuttings obtained from seedlings of Eucalyptus saligna Smith and E. globulus Labill in an attempt to characterize the adventitious rooting process and identify factors controlling rhizogenesis. Root development was scored as rooting percentage, root density (roots per rooted cutting), mean rooting time and root length. In both species, rooting time was reduced in the presence of auxin. Cuttings from 2-month-old E. saligna seedlings were responsive to lower auxin concentrations than comparable cuttings from E. globulus seedlings. Cuttings from 3-month-old E. saligna seedlings rooted promptly and rooting was not significantly affected by light conditions. In contrast, rooting of cuttings from 3-month-old E. globulus seedlings exhibited recalcitrant behavior and no roots were formed if illuminated during the root formation phase. Effective root regeneration of E. globulus cuttings was obtained by a 4-day exposure to 10 mg l(-1) IBA and culture in darkness during the root formation step. Loss of rooting capacity with seedling age was more pronounced in E. globulus than in E. saligna. The possibility of switching adventitious rooting off and on by manipulating light regime and exogenous auxin supply in E. globulus, and the constitutive nature of rooting in E. saligna may provide useful models for examining the rooting process at the biochemical and molecular levels in Eucalyptus.

Eucalyptus↗

Physiology and anatomy of lenticel-like structures on leaves of Eucalyptus nitens and Eucalyptus globulus seedlings.

Intumescences or abnormal, non-pathogenic, blister-like protuberant growths, form on Eucalyptus globulus Labill. and, to a much lesser extent, Eucalyptus nitens (Deane and Maiden) Maiden leaves when plants are grown in a high relative humidity environment. We examined the histology of intumescences and their effects on leaf photosynthetic processes. Intumescences were induced by placing E. globulus and E. nitens seedlings in a relative humidity of 80% in a greenhouse for 5 days. Symptomatic and asymptomatic leaves of plants with intumescence development were compared with leaves of control plants. Light-saturated carbon dioxide (CO(2)) assimilation (A(max)) and responses of CO(2) assimilation (A) to varying intercellular CO(2) partial pressure (C(i)) were measured. Symptomatic and asymptomatic leaf samples were fixed and sectioned and cellular structure was examined. Intumescences greatly reduced the photosynthetic capacity of E. globulus leaves and were associated with reduced electron transport rate and ribulose bisphosphate (RuBP) regeneration capacity. Tissue necrotization and cellular collapse of the palisade mesophyll and deposition of phenolic compounds in the affected areas, probably reduced light penetration to photosynthesizing cells as well as reducing the amount of photosynthesizing tissue. Photosynthetic capacity of E. nitens was unaffected. The intumescences resembled simple lenticels, both morphologically and developmentally. To our knowledge, this is the first time that lenticel-like structures developed in response to environmental conditions have been described on leaves.

Carbon Dioxide↗

The rare silver gum, Eucalyptus cordata, is leaving its trace in the organellar gene pool of Eucalyptus globulus.

The process of genetic assimilation of rare species by hybridizing congeners has been documented in a number of plant genera. This raises the possibility that some of the genetic diversity found in phylogeographical studies of widespread species has been acquired through hybridization with species that are now rare or extinct. In this fine-scale phylogeographical analysis, we show that a rare eucalypt species is leaving its trace in the chloroplast genome of a more abundant congener. The heart-leafed silver gum, Eucalyptus cordata, is a rare endemic of south-eastern Tasmania. Its populations are scattered amidst populations of more abundant related species, including the Tasmanian blue gum, Eucalyptus globulus. Using 339 samples from across the full range of both species, we compared chloroplast (cp) DNA haplotype phylogeography in E. globulus and E. cordata. The genealogy and distribution of chloroplast haplotypes suggest that E. globulus has acquired cpDNA from E. cordata in at least four different mixed populations. Shared haplotypes are highest in E. globulus sampled within 2 km of known E. cordata populations and drop to zero at a distance of 25 km from the nearest known E. cordata population. Localized haplotype sharing occurs in the absence of obvious hybrid zones or locally shared nuclear ribosomal DNA sequences. Given that the future loss of E. cordata from some mixed populations is likely, these findings indicate that phylogeographical analyses of organellar DNA should consider the possibility of introgression, even from species that have been eliminated from the sites of interest.

Base Sequence↗

Genetics of postzygotic isolation in Eucalyptus: whole-genome analysis of barriers to introgression in a wide interspecific cross of Eucalyptus grandis and E. globulus.

The genetic architecture of hybrid fitness characters can provide valuable insights into the nature and evolution of postzygotic reproductive barriers in diverged species. We determined the genome-wide distribution of barriers to introgression in an F(1) hybrid of two Eucalyptus tree species, Eucalyptus grandis (W. Hill ex Maiden.) and E. globulus (Labill.). Two interspecific backcross families (N = 186) were used to construct comparative, single-tree, genetic linkage maps of an F(1) hybrid individual and two backcross parents. A total of 1354 testcross AFLP marker loci were evaluated in the three parental maps and a substantial proportion (27.7% average) exhibited transmission ratio distortion (alpha = 0.05). The distorted markers were located in distinct regions of the parental maps and marker alleles within each region were all biased toward either of the two parental species. We used a Bayesian approach to estimate the position and effect of transmission ratio distorting loci (TRDLs) in the distorted regions of each parental linkage map. The relative viability of TRDL alleles ranged from 0.20 to 0.72. Contrary to expectation, heterospecific (donor) alleles of TRDLs were favored as often as recurrent alleles in both backcrosses, suggesting that positive and negative heterospecific interactions affect introgression rates in this wide interspecific pedigree.

Alleles↗

Cryopreservation of axillary buds of a Eucalyptus grandis x eucalyptus camaldulensis hybrid.

This paper presents preliminary results from a study to develop methodology for the cryopreservation of axillary buds from an in vitro hybrid Eucalyptus grandis (W. Hill ex Maiden.) x Eucalyptus camaldulensis (Dehnnh.), maintained for use in a genetic modification programme. Axillary buds were encapsulated in an alginate gel, precultured on media containing elevated levels of sucrose, or a combination of sucrose and glycerol. Encapsulated buds were then dehydrated by evaporation prior to a two-step freezing process in liquid nitrogen. Eighteen percent of shoot explants survived freezing when sucrose alone was used as a protectant against dehydration and cryopreservation. Significantly higher survival (49%) was obtained with the incorporation of glycerol into the protocol. Following cryopreservation, shoots appeared to develop normally, with no evidence of adventitious meristems.

Cryopreservation↗