Construction of microsatellite linkage maps for Corymbia

The genus Corymbia is closely related to the genus Eucalyptus, and like Eucalyptus contains tree species that are important for sub-tropical forestry. Corymbia's close relationship with Eucalyptus suggests genetic studies in Corymbia should benefit from transfer of genetic information from its more intensively studied relatives. Here we report a genetic map for Corymbia spp. based on microsatellite markers identified de novo in Corymbia sp or transferred from Eucalyptus. A framework consensus map was generated from an outbred F 2 population (n = 90) created by crossing two unrelated Corymbia torelliana x C. citriodora subsp. variegata F1 trees. The map had a total length of 367 cM (Kosambi) and was composed of 46 microsatellite markers distributed across 13 linkage groups (LOD 3). A high proportion of Eucalyptus microsatellites (90%) transferred to Corymbia. Comparative analysis between the Corymbia map and a published Eucalyptus map identified eight homeologous linkage groups in Corymbia with 13 markers mapping on one or both maps. Further comparative analysis was limited by low power to detect linkage due to low genome coverage in Corymbia, however, there was no convincing evidence for chromosomal structural differences because instances of non-synteny were associated with large distances on the Eucalyptus map. Segregation distortion was primarily restricted to a single linkage group and due to a deficit of hybrid genotypes, suggesting that hybrid inviability was one factor shaping the genetic composition of the F2 population in this inter-subgeneric hybrid. The conservation of microsatellite loci and synteny between Corymbia and Eucalyptus suggests there will be substantial value in exchanging information between the two groups.

Genetic variation within two sympatric spotted gum eucalypts exceeds between taxa variation

Population substructure and hybridization, among other factors, have the potential to cause erroneous associations in linkage disequilibrium (LD) mapping. Two closely related spotted gum eucalypts, Corymbia variegata and C. henryi (Myrtaceae) occur in sympatry in the east coast of Australia and potentially interbreed. They are morphologically similar but are distinguished as separate species based on capsule and foliage size. To determine whether they hybridize in nature and its implications for LD mapping, we investigated the level of molecular divergence between the two species at two sympatric locations separated by 300 kilometres. Very few individuals of intermediate morphology were identified, despite the two species occurring only metres apart. Analysis of genetic structure using 12 microsatellite loci showed that genetic differentiation between populations of the same species at different locations (FST = 0.07 for both species; p = 0.0001) was significantly higher than that observed between species at each location (mean FST = 0.02 and 0.04 for Cherry tree and Bunyaville respectively; p = 0.0001; all Mann-Whitney U-test p ≤ 0.01). No species-specific alleles or significant allele frequency differences were detected within a site, suggesting recurrent local gene flow between the two species. The lack of significant allele frequency differences implies no population stratification along taxonomic lines. This suggested that there is little concern for cryptic hybridization when sampling from sites of sympatry for LD mapping.

Comparative performance of Corymbia hybrids and parental species in subtropical Queensland and implications for breeding and deployment.

Eighty six full-sib Corymbia F1 hybrid families (crosses between C. torelliana and four spotted gum taxa: C. citriodora subsp. variegata, C. citriodora subsp. citriodora, C. henryi and C. maculata), were planted in six trials across six disparate sites in south-eastern Queensland to evaluate their productivity and determine their potential utility for plantation forestry. In each trial, the best-growing 20% of hybrid families grew significantly faster (P=0.05) than open-pollinated seedlots of the parent species Corymbia citriodora subsp. variegata, ranging from 107% to 181% and 127% to 287% of the height and diameter respectively. Relative performance of hybrid families growing on more than one site displayed consistency in ranking for growth across sites and analysis showed low genotype-by-environment interaction. Heritability estimates based on female and male parents across two sites at age six years for height and diameter at breast height, were high (0.62±0.28 to 0.64±0.35 and 0.31±0.21 to 0.69±0.37 respectively), and low to moderate (0.03±0.04 to 0.33±0.22) for stem straightness, branch size, incidence of ramicorns, and frost and disease resistance traits at ages one to three years. The proportion of dominance variance for height and diameter had reduced to zero by age six years. Based on these promising results, further breeding and pilot-scale family forestry and clonal forestry deployment is being undertaken. These results have also provided insights regarding the choice of a future hybrid breeding strategy.

Controlled pollination methods for creating Corymbia hybrids.

Inter-specific Corymbia hybrids are of increasing interest to plantation forestry, yet there is little knowledge of the most suitable controlled pollination methods for this genus. Inter-specific crosses were made between C. torelliana [CT(maternal parent)] and C. citriodora subsp. variegata (CCV), C. henryi (CH) and C. citriodora subsp. citriodora (CCC) using conventional pollination, one-stop pollination (OSP) and artificially-induced protogyny on yellow buds (AIP Y) pollination methods. Additional treatments included AIP on green buds (AIP G) and the use of exclusion bags for the OSP and AIP methods. Inter-specific hybrids (CT x CCV, CT x CH and CT x CCC) were successfully created using all three pollination methods. The AIP Y treatment provided the highest seed yields and achieved time savings of >41% over the conventional and OSP methods, resulting in up to five-fold increases in operator productivity. However, the AIP Y treatment also had the highest C. torelliana contamination levels (9.3–13.2%). The use of exclusion bags with the AIP method had minimal effect on contamination rates, indicating a high proportion of selfpollen contamination. Contamination rates varied between maternal parents, suggesting variation in selfcompatibility for C. torelliana individuals. AIP using semi-ripe green buds was not effective at reducing selfing and had low operator productivity. The AIP method is suitable for use in a large-scale hybrid breeding program for C. torelliana. When self-pollination effects are managed, it could greatly reduce the costs associated with the production of seed of elite family crosses for commercial forestry deployment.

Pollination methods, stigma receptivity and pollen tube growth in Eucalyptus argophloia

Eucalyptus argophloia Blakely (Western white gum) has shown potential as a commercial forestry timber species in marginal environments of north-eastern Australia. We measured early pollination success in Eucalyptus argophloia to compare pollination methods, determine the timing of stigma receptivity and compare fresh and stored pollen. Early pollination success was measured by counting pollen tubes in the style of E. argophloia 12 days after pollination. We compared the early pollination success of 1) Artificially Induced Protogyny (AIP), one-stop and three-stop methods of pollination; 2) flowers pollinated at 2 day intervals between 2 days before and 6 days after anthesis and 3) fresh pollen and pollen that had been stored for 9 months. Our results show significantly more pollen tubes from unpollinated AIP and AIP treatments than either the one-stop pollination or three-stop pollination treatments. This indicates that self-pollination occurs in the unpollinated AIP treatment. There was very little pollen tube growth in the one-stop method indicating that the three-stop method is the most suitable for this species. Stigma receptivity in E. argophloia commenced six days after anthesis and no pollen tube growth was observed prior to this. Fresh pollen resulted in pollen tube growth in the style whereas the stored pollen resulted in a total absence of pollen tube growth. We recommend that breeding programs incorporating E. argophloia as a female parent use the three-stop pollination method, and controlled pollination be carried out at least six days after anthesis using fresh pollen.