G. F. Moran

Polymorphisms in Cinnamoyl CoA Reductase ( CCR ) Are Associated With Variation in Microfibril Angle in Eucalyptus spp.

Linkage disequilibrium (LD) mapping using natural populations results in higher resolution of marker-trait associations compared to family-based quantitative trait locus (QTL) studies. Depending on the extent of LD, it is possible to identify alleles within candidate genes associated with a trait. Analysis of a natural mutant in Arabidopsis has shown that mutations in cinnamoyl CoA reductase (CCR), a key lignin gene, affect physical properties of the secondary cell wall such as stiffness and strength. Using this gene, we tested whether LD mapping could identify alleles associated with microfibril angle (MFA), a wood quality trait affecting stiffness and strength of wood. We identified 25 common single-nucleotide polymorphism (SNP) markers in the CCR gene in Eucalyptus nitens. Using single-marker and haplotype analyses in 290 trees from a E. nitens natural population, two haplotypes significantly associated with MFA were found. These results were confirmed in two full-sib families of E. nitens and Eucalyptus globulus. In an effort to understand the functional significance of the SNP markers, we sequenced the cDNA clones and identified an alternatively spliced variant from the significant haplotype region. This study demonstrates that LD mapping can be used to identify alleles associated with wood quality traits in natural populations of trees.

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 1∶1 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.

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

Abstract 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.

An integrated genetic linkage map for eucalypts using RFLP, RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.

Population divergence in the chloroplast genome of Eucalyptus nitens

Variation in the chloroplast genome of Eucalyptus nitens was assessed for ten individuals from each of eight populations covering the natural range of the species. Twenty-five mutations were detected which were distributed over 13 haplotypes. The mutations were present both within and between populations. The level of nucleotide diversity within the species was high and the majority of the variation was distributed between populations and regions. The level and distribution of haplotype diversity were similar to those of the nucleotide diversity. The interpopulational variation may be related to the large range and disjunct populations that characterize the species, due to the effects of isolation and genetic drift. There were three mutations which were unique and fixed in one small population. Individuals in the species could be distinguished into two cpDNA groups. The distribution of these groups was not consistent with any geographical pattern. The distribution of the cpDNA groups suggests that they are of ancient origin and predate the isolation of the regions. The pattern of cpDNA variation is not consistent with patterns of variation in morphological traits.

A genetic linkage map for Eucalyptus globulus with candidate loci for wood, fibre, and floral traits

Abstract A genetic linkage map containing potential candidate loci for wood, fibre and floral traits has been constructed for Eucalyptus globulus (Labill.) based on the segregation of 249 codominant loci in an outbred F1 population of 148 individuals. The map contains 204 RFLP loci, including 31 cambium-specific expressed sequence tags (ESTs) and 14 known function genes, and 40 microsatellite and five isozyme loci. Independent male and female maps were constructed, and the 98 loci (39%) that segregated in both parents were used to combine the parental maps into an integrated map. The 249 loci mapped to 11 major linkage groups (n=11 in eucalypts) and a 12th small linkage group containing three loci that segregated in the male parent only. Total map distance is 1375 cM with an average interval of 6 cM. Forty one of the mapped loci identify known proteins (five isozymes) or sequences with known function (14 genes and 22 ESTs). The mapped genes include enzymes involved in lignin and cell-wall polysaccharide biosynthesis, and floral-development genes. This map will be used to locate quantitative trait loci for wood, fibre, and other traits in Eucalyptus.

Gene expression in Eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G‐layers

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.

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

Abstract 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.

Temporal heterogeneity of outcrossing rates in alpine ash (Eucalyptus delegatensis R.T. Bak.)

SummaryThree seed crops of a Eucalyptus delegatensis population were assayed for their allozyme genotype at three loci to determine estimates of mating system parameters. In the pollen the allelic frequencies at each of the three loci were similar to those in the parents and the progeny. Overall there was a significant amount of inbreeding (23%) in the population. The levels of outcrossing in each crop were significantly different from each other indicating apparent temporal variation in outcrossing rates. The outcrossing rate was greatest in the oldest crop (85%) and lowest in the most recent crop (66%). Mean heterozygosity in the progeny of all three crops was less than the heterozygosity in the parents indicating that selection favours heterozygotes during the life cycle. The implications of a ‘balanced’ mixed mating system for a eucalypt breeding program are discussed.

Mapping of quantitative trait loci influencing frost tolerance in Eucalyptus nitens

Abstract Regions of the genome influencing frost tolerance in an outbred family of Eucalyptus nitens have been identified. Two QTLs present on the same linkage group, but located 40 cM apart, were identified using single-factor analysis of variance. The QTLs explained between 7.7 and 10.8% of the phenotypic variation for frost tolerance in this family. Analysis of marker loci linked to the QTLs showed one of them to have a simple mode of action with the effect segregating from the male parent in the family. For the other QTL multiple alleles were identified. This QTL showed segregation from the female parent which gave a positive effect on frost tolerance; however, an allele segregating from the male parent was identified which showed a negative interaction with the allele for increased frost tolerance.