Copyright (c) 2010 All rights reserved. http://works.bepress.com/mervyn_shepherd Recent documents in Mervyn Shepherd en-us Tue, 21 Dec 2010 01:31:12 PST 3600 http://works.bepress.com/mervyn_shepherd/201 http://works.bepress.com/mervyn_shepherd/201 Sun, 19 Dec 2010 16:31:32 PST Mervyn Shepherd http://works.bepress.com/mervyn_shepherd/200 http://works.bepress.com/mervyn_shepherd/200 Sun, 19 Dec 2010 16:31:28 PST It is difficult to extract pure high-quality DNA from trees, which may not be amenable to advances in extraction methods suitable for other plants. A new commercial high-throughput DNA extraction system, using a silica binding matrix for purification and a multisample mixer mill for tissue disruption, was evaluated for its suitability with Eucalyptus spp., Pinus spp., and Araucaria cunninghamii (hoop pine). DNA suitable for a range of molecular biology applications was successfully extracted from all genera. The method was highly reliable when tested in more than 500 preparations and could be adapted to different tree species with relatively minor modifications. Mervyn Shepherd http://works.bepress.com/mervyn_shepherd/198 http://works.bepress.com/mervyn_shepherd/198 Thu, 16 Dec 2010 17:14:11 PST Breeding for wood quality is limited by the long generation times and the delay before wood quality can be measured reliably. Association studies allow links between phenotype and genotype to be made, and are a prelude to accelerated domestication of trees by molecular breeding approaches. This study uses association genetics to identify DNA polymorphisms that correlate with solid wood properties of Eucalyptus pilularis Smith (blackbutt). We undertook extensive phenotyping of dimensional stability, growth and structural wood properties on a nine-year-old progeny trial established by Forests NSW at Hannam Vale, near Port Macquarie in NSW. A subset of 372 phenotyped individuals representing 284 families collected from 37 provenances was used as the association population for genotypic assessment. Fifty-two out of 127 novel DNA polymorphisms were surveyed within four candidate genes, CCR, CAD, MYB1 and MYB2. Several putative associations between wood quality traits and selected DNA polymorphisms are reported, along with the likely mechanism of action on wood quality. Association studies such as this will facilitate non-destructive DNA tests for heritable wood properties that can be used to enrich breeding populations at any developmental stage with desirable alleles. Keywords: wood properties; phenotypic variation; genetic variation; molecular markers; selection; marker-aided selection; Eucalyptus pilularis Timothy R. Sexton http://works.bepress.com/mervyn_shepherd/197 http://works.bepress.com/mervyn_shepherd/197 Thu, 16 Dec 2010 17:14:06 PST Genetic control of vegetative propagation traits was described for a second-generation, outbred, intersectional hybrid family (N = 208) derived from two species, Corymbia torelliana (F. Muell.) K.D. Hill & L.A.S. Johnson and Corymbia variegata (F. Muell.) K.D. Hill & L.A.S. Johnson, which contrast for propagation characteristics and in their capacity to develop lignotubers. Large phenotypic variances were evident for rooting and most other propagation traits, with significant proportions attributable to differences between clones (broad-sense heritabilities 0.2–0.5). Bare root assessment of rooting rate and root quality parameters tended to have the highest heritabilities, whereas rooting percentage based on root emergence from pots and shoot production were intermediate. Root biomass and root initiation had the lowest heritabilities. Strong favourable genetic correlations were found between rooting percentage and root quality traits such as root biomass, volume, and length. Lignotuber development on a seedling was associated with low rooting and a tendency to poor root quality in cuttings and was in accord with the persistence of species parent types due to gametic phase disequilibrium. On average, nodal cuttings rooted more frequently and with higher quality root systems, but significant cutting type × genotype interaction indicated that for some clones, higher rooting rates were obtained from tips. Low germination, survival of seedlings, and rooting rates suggested strong hybrid breakdown in this family. Mervyn Shepherd http://works.bepress.com/mervyn_shepherd/195 http://works.bepress.com/mervyn_shepherd/195 Thu, 16 Dec 2010 17:13:58 PST M Abasolo http://works.bepress.com/mervyn_shepherd/194 http://works.bepress.com/mervyn_shepherd/194 Thu, 16 Dec 2010 17:13:54 PST Timothy Sexton http://works.bepress.com/mervyn_shepherd/193 http://works.bepress.com/mervyn_shepherd/193 Tue, 21 Sep 2010 20:06:51 PDT The red mahogany group (Eucalyptus ser. Annulares Blakely) includes some of the most important commercial species (i.e. Eucalyptus urophylla S.T.Blake) worldwide for forestry in the subtropics and tropics. However, the taxonomic status of some species in this group is unclear and the relationship among and genetic structuring within some species is unresolved. The present study examined genetic variation at 13 microsatellite loci in E. pellita F.Muell., E. resinifera Smith and E. scias L.Johnson & K.Hill. Despite close geographical proximity and natural hybridisation in northern Queensland, E. resinifera and E. pellita remain genetically distinct as taxa. Within E. pellita, two genetic groups were clearly resolved, one from New Guinea and one from Queensland (Cape York Peninsula populations were not sampled). Geographic structuring was also evident in E. resinifera, with northern Queensland populations separating from those from Fraser Island southwards. Ecological factors and species disjunctions were implicated in the genetic substructuring of these two taxa because patterns of geographic variation aligned with biogeographical regions. E. scias was indistinguishable from southern E. resinifera and its three subspecies could not be resolved. Son Le http://works.bepress.com/mervyn_shepherd/192 http://works.bepress.com/mervyn_shepherd/192 Sun, 19 Sep 2010 12:28:05 PDT Microsatellites or SSR (simple sequence repeats) are usually single locus, highly polymorphic and robust markers. Microsatellites are increasingly the marker of choice for genome analysis, mapping and diversity studies in plants (Gupta et al. 1996, Westman and Kresovicch 1997) and forest trees (for example Bradshaw et al. 1994, Brondani et al. 1998, Devey et al. 1996). The isolation and development of microsatellite markers, however, is often costly and problematic, especially in organisms with large genomes (Fisher and Bachmann 1998), such as conifers. For example, from our own experience, enriched microsatellite libraries for seven angiosperms (Eucalyptus pilularus, E. globulus, E. grandis, E. cloeziana, Grevillia robusta, Corymbia variegata, Toona ciliata) and three gymnosperm (Araucaria cunninghamii, Pinus elliottii, P. caribaea), using the method of Edwards et al. 1996, yielded enrichment of up to 50% in the angiosperm species but below 10% for all the gymnosperm species. Here we outline a library screening method which recovered high levels of microsatellite containing clones from libraries of coniferous species. Leon J. Scott http://works.bepress.com/mervyn_shepherd/191 http://works.bepress.com/mervyn_shepherd/191 Sun, 11 Jul 2010 18:05:23 PDT Geographically distributed genetic variation is expected in species like Eucalyptus pilularis that have wide latitudinal and habitat ranges. Coastal and inland ecotypes of this tall forest tree have been distinguished in genecological studies, but patterns of regionally distributed quantitative variation are weak. At the coarsest level, variation at microsatellite markers (n=12) divided a range-wide sample of 424 E. pilularis trees into two zones, the region to the south of Sydney forming one zone distinct from regions to the north. Genetic structuring did not correspond with ecotypes but rather with a biogeographic division suggesting an imprint of historical isolation. Typical and uniform levels of genetic diversity (He = 0.78 (0.02) mean (SE)) were found across 10 geographic regions. Structuring into regions (PhiRT = 3%), localities within regions (PhiPT = 2%), between coastal and inland provenances (PhiPT = 2%), or due to isolation by distance, was subtle. These factors, along with the lack of evidence for bottlenecks, suggested cohesion within zones due to gene flow and large population sizes historically. Recent colonisation and adaptation better explained the low levels of diversity and poor growth performance of the Fraser Island ecotype rather than genetic isolation as there was no evidence of inbreeding. Mervyn Shepherd http://works.bepress.com/mervyn_shepherd/190 http://works.bepress.com/mervyn_shepherd/190 Tue, 23 Feb 2010 22:05:09 PST Timothy Sexton http://works.bepress.com/mervyn_shepherd/189 http://works.bepress.com/mervyn_shepherd/189 Tue, 23 Feb 2010 22:05:08 PST Mervyn Shepherd http://works.bepress.com/mervyn_shepherd/188 http://works.bepress.com/mervyn_shepherd/188 Tue, 23 Feb 2010 22:05:07 PST Corymbia variegata (spotted gum) is an important commercial hardwood timber species in Australia. Fourteen polymorphic microsatellite loci were isolated from C. variegata, with 3–5 alleles amplified in three individuals examined. Cross-species amplification in Corymbia was successful for all primer pairs, while 10 loci (71%) were successfully transferred to at least one species in the closely related genus Eucalyptus. Megan E. Jones http://works.bepress.com/mervyn_shepherd/187 http://works.bepress.com/mervyn_shepherd/187 Tue, 23 Feb 2010 22:05:06 PST Robert J. Henry http://works.bepress.com/mervyn_shepherd/186 http://works.bepress.com/mervyn_shepherd/186 Tue, 23 Feb 2010 22:05:05 PST Large fruited spotted gum eucalypt Corymbia henryi occurs sympatrically with small fruited spotted gum Corymbia citriodora subspecies variegata over a large portion of its range on the east coast of Australia. The two taxa are interfertile, have overlapping flowering times and share a common set of insect and vertebrate pollinators. Previous genetic analysis of both taxa from two geographically remote sites suggested that the two were morphotypes rather than genetically distinct species. In this study we further explore this hypothesis of genic species by expanding sampling broadly through their sympatric locations and examine local-scale spatial genetic structure in stands that differ in species and age composition. Delineation of populations at five microsatellite loci, using an individual-based approach and Bayesian modelling, as well as clustering of individuals based on allele frequencies showed the two species to be molecularly homogeneous. Genetic structure aligned largely with geographic areas of origin, and followed an isolation-by-distance model, where proximal populations were generally less differentiated than more distant ones. At the stand level, spotted gums also generally showed little structure consistent with the high levels of gene flow inferred across the species range. Disturbances in the uniformity of structuring were detected, however, and attributed to localised events giving rise to even aged stands, probably due to regeneration from a few individuals following fire. Joel W. Ochieng http://works.bepress.com/mervyn_shepherd/185 http://works.bepress.com/mervyn_shepherd/185 Tue, 23 Feb 2010 22:05:04 PST Mis-priming associated with uncharacterised single nucleotide polymorphisms (SNPs) may lead to failure of PCR for genotyping. This is particularly troublesome in high-throughput SNP genotyping applications relying on multiplex PCR (2–40-plex) generating many short amplicons (80–120 bp) of similar size, an approach best suited for whole genome scans. However, if the target SNPs are clustered within a few target genes one option to ameliorate this is to increase the amplicon length, effectively reducing the potential for primer/template interactions and mis-priming. We tested this approach in a diverse population of 372 Eucalyptus pilularis individuals (π = 8.11 × 10−3, H e = 0.75) using a modified Sequenom iPLEX gold assay. Four candidate genes (MYB1, MYB2, CAD and CCR) were amplified in a single long range multiplex capture PCR generating 6 long amplicons ranging in size from 907 to 2,225 bp. This contrasts with the standard approach which would have required the amplification of 98 short amplicons in 4 multiplex reactions. These 6 long amplicons provided the assay template for 98 assays (87 SNP and 11 InDel) within the 4 candidate genes. Reaction results indicated that longer amplicons could provide a suitable template for genotyping assays, with 90.8% of assays functional and 84.3% of assays suitable for downstream analysis. Additional advantages of this approach were the capacity for troubleshooting using gel electrophoresis and savings of 94% in capture primer synthesis costs. This approach will have the greatest relevance for candidate gene approaches for association testing in uncharacterised populations of organisms with high sequence diversity. Timothy R. Sexton http://works.bepress.com/mervyn_shepherd/184 http://works.bepress.com/mervyn_shepherd/184 Tue, 23 Feb 2010 22:05:03 PST Nicole F. Rice http://works.bepress.com/mervyn_shepherd/183 http://works.bepress.com/mervyn_shepherd/183 Tue, 23 Feb 2010 22:05:02 PST Mervyn Shepherd http://works.bepress.com/mervyn_shepherd/182 http://works.bepress.com/mervyn_shepherd/182 Tue, 23 Feb 2010 22:05:01 PST Single Nucleotide Polymorphisms (SNPs) code for much of the morphological variation observed between individuals. The aim of this experiment was to identify SNPs conserved among three species of interest Eucalyptus globulus, Eucalyptus pyrocarpa and Eucalyptus pilularis. Encompassing the Eucalyptus and Symphyomyrtus subgenera, conserved SNPs are of particular interest as they will transfer between many of the ~700 Eucalyptus species and are valuable candidates for genotyping in association studies. They also provide a valuable insight into the evolution and divergence of the species examined. Genomic DNA from 30 diverse individuals from each species was pooled. A total of 44 candidate genes for wood quality were amplified by PCR, with amplicons ranging in size from 400bp to 9kb. At equimolar concentrations amplicons were then pooled back into their respective species groups. The pooled PCR products were sheared, before preparation of paired-end libraries. Deep sequencing of each gene was achieved up to a level of 10,000X coverage using both 75bp paired end reads and 36bp single reads from the Illumina Genome Analyser IIx. Using this approach several thousand SNPs were identified. Relationships of conserved SNPs will be reported along with findings from association studies. Timothy R. Sexton http://works.bepress.com/mervyn_shepherd/181 http://works.bepress.com/mervyn_shepherd/181 Tue, 23 Feb 2010 22:05:00 PST Genetic control of foliar oil composition was investigated amongst half-sib progeny of an interspecific eucalypt hybrid. The oil was found to be largely composed of the monoterpenes, limonene, α−pinene, γ−terpinene, 1,8 cineole and p-cymene. Due to difficulties in the interpretation of the compositional data based on raw proportions, further analysis was conducted using log-ratio variables. A high degree of intercorrelation amongst log-ratios was thought to be a consequence of commonality in the biosynthetic origins of the monoterpenes. Quantitative trait locus (QTL) analysis of log-ratio variables indicated that a significant (68–81%) proportion of the variation in four out of the ten possible log-ratios were controlled by a single genomic region of the maternal Eucalyptus grandis parent. The impact of this genomic region upon oil composition was thought to be a consequence of a gene, or genes, controlling the production of limonene, as limonene was the predominant oil constituent in many hybrid individuals and was common to all log-ratios associated with the identified genomic region. Mervyn Shepherd http://works.bepress.com/mervyn_shepherd/180 http://works.bepress.com/mervyn_shepherd/180 Tue, 23 Feb 2010 22:04:59 PST Natural populations of eucalypts exhibit variation to defoliation by adult Christmas beetles at the species, subspecies and intra-tree levels. In this study we examine experimental populations of eucalypts in order to establish the origins of variability and the importance of genetic factors in host-insect interactions. A simple laboratory bio-assay was developed to overcome difficulties with measurement of defoliation in the field. Leaf area consumed (LAC) by beetles was used to estimate variation within and between several families of interspecific hybrid eucalypts. Extreme susceptibility to defoliation resulted from interspecific hybridisation where individuals from non-adapted parental species introduced susceptibility into a family. Host genotype, at the family and individual level were found to be significant in determining the amount of foliage consumed in bio-assays. Ranking of resistance classes in bio-assays was shown to correspond with field assessments of defoliation. Mervyn Shepherd