A novel genome‐wide microsatellite resource for species of Eucalyptus with linkage‐to‐physical correspondence on the reference genome sequence

Keystone species in their native ranges, eucalypts, are ecologically and genetically very diverse, growing naturally along extensive latitudinal and altitudinal ranges and variable environments. Besides their ecological importance, eucalypts are also the most widely planted trees for sustainable forestry in the world. We report the development of a novel collection of 535 microsatellites for species of Eucalyptus, 494 designed from ESTs and 41 from genomic libraries. A selected subset of 223 was evaluated for individual identification, parentage testing, and ancestral information content in the two most extensively studied species, Eucalyptus grandis and Eucalyptus globulus. Microsatellites showed high transferability and overlapping allele size range, suggesting they have arisen still in their common ancestor and confirming the extensive genome conservation between these two species. A consensus linkage map with 437 microsatellites, the most comprehensive microsatellite‐only genetic map for Eucalyptus, was built by assembling segregation data from three mapping populations and anchored to the Eucalyptus genome. An overall colinearity between recombination‐based and physical positioning of 84% of the mapped microsatellites was observed, with some ordering discrepancies and sporadic locus duplications, consistent with the recently described whole genome duplication events in Eucalyptus. The linkage map covered 95.2% of the 605.8‐Mbp assembled genome sequence, placing one microsatellite every 1.55 Mbp on average, and an overall estimate of physical to recombination distance of 618 kbp/cM. The genetic parameters estimates together with linkage and physical position data for this large set of microsatellites should assist marker choice for genome‐wide population genetics and comparative mapping in Eucalyptus.     

Mycorrhizal status of <Emphasis Type="Italic">Eucalyptus</Emphasis> plantations in south China and implications for management

The aim of this study is to assess the mycorrhizal status of Eucalyptus plantations in south China and to determine the need for inoculation. In four provinces in south China, 155 plantations were sampled for sporocarps of ectomycorrhizal (ECM) fungi, spores of arbuscular mycorrhizal (AM) fungi, and mycorrhizas over 2 years. This study revealed a low above-ground diversity of ECM fungi consisting of 15 taxa fruiting beneath Eucalyptus plantations. The most common ECM genera were Scleroderma and Pisolithus, but they were infrequent. A total of 21 AM fungi, mostly Glomus species, were recognized from spores collected from eucalypt plantations. Four Glomus species were frequently present in soils, but spore density and relative abundance of AM fungi were generally low. Eucalypt roots from all plantation sites were poorly colonized by either ECM fungi or AM fungi. A bioassay with E. urophylla as a bait host, using soils collected from 11 eucalypt plantations, confirmed low levels of inoculum of both ECM and AM fungi in field soil. This is the first integrated study on the mycorrhizal status of eucalypt plantations in China. Findings from this research can be used to encourage adoption of mycorrhizal technology by eucalypt nurseries in the region. The potential of using spores of compatible ECM fungi or collections for forest nurseries is discussed.     

High-density genetic linkage maps with over 2,400 sequence-anchored DArT markers for genetic dissection in an F2 pseudo-backcross of Eucalyptus grandis × E. urophylla

Traits that differentiate cross-fertile plant species can be dissected by genetic linkage analysis in interspecific hybrids. Such studies have been greatly facilitated in Eucalyptus tree species by the recent development of Diversity Arrays Technology (DArT) markers. DArT is an affordable, high-throughput marker technology for the construction of high-density genetic linkage maps. Eucalyptus grandis and Eucalyptus urophylla are commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions. We analysed 7,680 DArT markers in an F2 pseudo-backcross mapping pedigree based on an F1 hybrid clone of E. grandis and E. urophylla. A total of 2,440 markers (31.7%) were polymorphic and could be placed in linkage maps of the F1 hybrid and two pure-species backcross parents. An integrated genetic linkage map was constructed for the pedigree resulting in 11 linkage groups (n = 11) with 2,290 high-confidence (LOD ≥ 3.0) markers and a total map length of 1,107.6 cM. DNA sequence analysis of the mapped DArT marker fragments revealed that 43% were located in protein coding regions and 90% could be placed in the recently completed draft genome assembly of E. grandis. Together with the anchored genomic sequence information, this linkage map will allow detailed genetic dissection of quantitative traits and hybrid fitness characters segregating in the F2 progeny and will facilitate the development of markers for molecular breeding in Eucalyptus.     

Development and assessment of microarray-based DNA fingerprinting in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis. In contrast, recently developed microarray technology holds the promise of parallel analysis of thousands of markers in plant genomes. The aim of this study was to develop a DNA fingerprinting chip for Eucalyptus grandis and to investigate its usefulness for fingerprinting of eucalypt trees. A prototype chip was prepared using a partial genomic library from total genomic DNA of 23 E. grandis trees, of which 22 were full siblings. A total of 384 cloned genomic fragments were individually amplified and arrayed onto glass slides. DNA fingerprints were obtained for 17 individuals by hybridizing labeled genome representations of the individual trees to the 384-element chip. Polymorphic DNA fragments were identified by evaluating the binary distribution of their background-corrected signal intensities across full-sib individuals. Among 384 DNA fragments on the chip, 104 (27%) were found to be polymorphic. Hybridization of these polymorphic fragments was highly repeatable (R²>0.91) within the E. grandis individuals, and they allowed us to identify all 17 full-sib individuals. Our results suggest that DNA microarrays can be used to effectively fingerprint large numbers of closely related Eucalyptus trees.     

Genomic Characterization of DArT Markers Based on High-Density Linkage Analysis and Physical Mapping to the Eucalyptus Genome

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference genome is yet available to allow such detailed characterization.     

Australian biogeographical connections and the phylogeny of large genera in the plant family Myrtaceae

Aim To compare the phylogeny of the eucalypt and melaleuca groups with geological events and ages of fossils to discover the time frame of clade divergences. Location Australia, New Caledonia, New Guinea, Indonesian Archipelago. Methods We compare published molecular phylogenies of the eucalypt and melaleuca groups of the plant family Myrtaceae with geological history and known fossil records from the Cretaceous and Cenozoic. Results The Australasian eucalypt group includes seven genera, of which some are relictual rain forest taxa of restricted distribution and others are species‐rich and widespread in drier environments. Based on molecular and morphological data, phylogenetic analyses of the eucalypt group have identified two major clades. The monotypic Arillastrum endemic to New Caledonia is related in one clade to the more species‐rich Angophora, Corymbia and Eucalyptus that dominate the sclerophyll vegetation of Australia. Based on the time of rifting of New Caledonia from eastern Gondwana and the age of fossil eucalypt pollen, we argue that this clade extends back to the Late Cretaceous. The second clade includes three relictual rain forest taxa, with Allosyncarpia from Arnhem Land the sister taxon to Eucalyptopsis of New Guinea and the eastern Indonesian archipelago, and Stockwellia from the Atherton Tableland in north‐east Queensland. As monsoonal, drier conditions evolved in northern Australia, Arnhem Land was isolated from the wet tropics to the east and north during the Oligocene, segregating ancestral rain forest biota. It is argued also that the distribution of species in Eucalyptopsis and Eucalyptus subgenus Symphyomyrtus endemic in areas north of the stable edge of the Australian continent, as far as Sulawesi and the southern Philippines, is related to the geological history of south‐east Asia‐Australasia. Colonization (dispersal) may have been aided by rafting on micro‐continental fragments, by accretion of arc terranes onto New Guinea and by land brought into closer proximity during periods of low sea‐level, from the Late Miocene and Pliocene. The phylogenetic position of the few northern, non‐Australian species of Eucalyptus subgenus Symphyomyrtus suggests rapid radiation in the large Australian sister group(s) during this time frame. A similar pattern, connecting Australia and New Caledonia, is emerging from phylogenetic analysis of the Melaleuca group (Beaufortia suballiance) within Myrtaceae, with Melaleuca being polyphyletic. Main conclusion The eucalypt group is an old lineage extending back to the Late Cretaceous. Differentiation of clades is related to major geological and climatic events, including rifting of New Caledonia from eastern Gondwana, development of monsoonal and drier climates, collision of the northern edge of the Australian craton with island arcs and periods of low sea level. Vicariance events involve dispersal of biota.     

A serious canker disease of Eucalyptus in South Africa caused by a new species of Coniothyrium

Eucalyptus spp. are being propagated extensively as exotics in plantations in South Africa, and many other parts of the world. In South Africa, a number of diseases result in serious losses to this resource. This paper describes a new and very damaging stem canker disease, which has recently appeared on plantation-grown eucalyptus in South Africa. The disease, first noted in an isolated location in Zululand is now common in other parts of the country, and is typified by discrete necrotic lesions on stems. These lesions coalesce to form large, gum-impregnated cankers and malformed stems. The causal agent of the disease, as inferred from pathogenicity tests, is a new species of Coniothyrium described here as C. zuluense. This fungus is a serious impediment to eucalypt propagation in South Africa, and is most likely a threat to similar forest industries elsewhere in the world.     

Molecular evolution and genome divergence at <Emphasis Type="Italic">RPB2</Emphasis> gene of the St and H genome in <Emphasis Type="Italic">Elymus</Emphasis> species

Molecular evolution of the second largest subunit of low copy nuclear RNA polymerase II (RPB2) in allotetrploid StH genomic species of Elymus is characterized here. Our study first reported a 39-bp MITE stowaway element insertion in the genic region of RPB2 gene for all tetraploid Elymus St genome and diploid Pseudoroegneria spicata and P. stipifolia St genome. The sequences on 3′-end are highly conserved, with AGTA in all sequences but H10339 (E. fibrosis), in which the AGTA was replaced with AGAA. All 12 Stowaway-containing sequences encompassed a 9 bp conserved TIRs (GAGGGAGTA). Interestingly, the 5′-end sequence of GGTA which was changed to AGTA or deleted resulted in Stowaway excision in the H genome of Elymus sepcies, in which Stowaway excision did not leave footprint. Another two large insertions in all St genome sequences are also transposable-like elements detected in the genic region of RPB2 gene. Our results indicated that these three transposable element indels have occurred prior to polyploidization, and shaped the homoeologous RPB2 loci in St and H genome of Eymus species. Nucleotide diversity analysis suggested that the RPB2 sequence may evolve faster in the polyploid species than in the diploids. Higher level of polymorphism and genome-specific amplicons generated by this gene indicated that RPB2 is an excellent tool for investigating the phylogeny and evolutionary dynamics of speciation, and the mode of polyploidy formation in Elymus species.     

Benefits and risks of biotic exchange between Eucalyptus plantations and native Australian forests

Australia is unique in having two highly diverse plant genera, Eucalyptus and Acacia, that dominate the vegetation on a continent‐wide scale. The recent shift in plantation forestry away from exotic Pinus radiata to native Eucalyptus species has resulted in much more extensive exchange of biota between native forest and plantation ecosystems than exchange in the past with plantations of exotic species. Growing numbers of hectares are being planted to Eucalyptus globulus across Australia, and plantations are providing resources and corridors for native biota. The present paper focuses on both the benefits and risks of having large‐scale forestry plantations of native species that are closely related to dominant native taxa in local forests. At least 85 species of insects have been recorded as pests of Eucalyptus plantations around Australia; the vast majority of these have been insects using the same host species, or closely related taxa, in native forests. Plantations of native species may also benefit from closely related local forests through the presence of: (i) the diverse array of ectomycorrhizal fungi favourable for tree growth; (ii) natural enemies harboured in native habitats; and (iii) recruitment of other important mutualists, such as pollinators. Exchanges work in two directions: plantations are also likely to influence native forests through the large amount of insect biomass production that occurs in outbreak situations, or through the introduction or facilitation of movements for insects that are not native to all parts of Australia. Finally, older plantations in which trees flower may exchange genes with surrounding forest species, given the high degree of hybridization exhibited by many Eucalyptus species. This is an aspect of exchange for which few data have been recorded. In summary, because of Australia’s unique biogeography, plantation forestry using eucalypt species entails exchanges with natural habitats that are unparalleled in scale and diversity in any other part of the world. More exchanges are likely as plantations occupy greater area, and as the time under cultivation increases.     

Analysis of saccharification in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> stems under mild conditions of hydrolysis

Background  

Brachypodium distachyon constitutes an excellent model species for grasses. It is a small, easily propagated, temperate grass with a rapid life cycle and a small genome. It is a self-fertile plant that can be transformed with high efficiency using Agrobacteria and callus derived from immature embryos. In addition, considerable genetic and genomic resources are becoming available for this species in the form of mapping populations, large expressed sequence tag collections, T-DNA insertion lines and, in the near future, the complete genome sequence. The development of Brachypodium as a model species is of particular value in the areas of cell wall and biomass research, where differences between dicots and grasses are greatest. Here we explore the effect of mild conditions of pretreatment and hydrolysis in Brachypodium stem segments as a contribution for the establishment of sensitive screening of the saccharification properties in different genetic materials.     

The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis

The complete nucleotide sequence of mulberry (Morus indica cv. K2) chloroplast genome (158,484 bp) has been determined using a combination of long PCR and shotgun-based approaches. This is the third angiosperm tree species whose plastome sequence has been completely deciphered. The circular double-stranded molecule comprises of two identical inverted repeats (25,678 bp each) separating a large and a small single-copy region of 87,386 bp and 19,742 bp, respectively. A total of 83 protein-coding genes including five genes duplicated in the inverted repeat regions, eight ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids, were assigned on the basis of homology to predicted genes from other chloroplast genomes. The mulberry plastome lacks the genes infA, sprA, and rpl21 and contains two pseudogenes ycf15 and ycf68. Comparative analysis, based on sequence similarity, both at the gene and genome level, indicates Morus to be closer to Cucumis and Lotus, phylogenetically. However, at genome level, inclusion of non-coding regions brings it closer to Eucalyptus, followed by Cucumis. This may reflect differential selection pressure operating on the genic and intergenic regions of the chloroplast genome.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Communicated by Y. Tsumura     

桉属植物非挥发性化学成分和药理活性研究进展

桉属(Eucalyptus L. Herit)是桃金娘科(Myrtaceae)的大属,该属约600余种,主要分布于世界各地热带亚热带地区。我国引入品种较多,主要分布于华南地区,其中广东和广西为桉树的主要种植基地。桉属植物具有较多的工业价值,其木材、叶、果实等是化学工业、香料、医药领域的重要原料,可用作开发高性能桉木重组材、竹桉复合材料、造浆与造纸等。桉属植物作为民间药材被使用,具有抑菌消炎、疏风解热、防腐止痒等功效,其药理研究表明,桉属植物具有良好的抗氧化、抗炎、抗菌、抗病毒、抗肿瘤、抗心血管疾病等药理活性。该研究通过查阅近三十年桉属植物相关的国内外文献报道,对桉属植物不同部位的421个非挥发性化学成分及其药理活性等进行了较详细的分类阐述,其中黄酮类化合物共73个、有机酸化合物共61个、萜类化合物共45个、多酚类化合物共229个、脂肪醇类化合物共13个,药理活性多集中在抗氧化、抗菌、抗病毒、抗肿瘤等,但相关机制仍需进一步阐明。该文重点关注桉属植物的药用部位,充分发掘其药用价值,开展临床转化和新药研究工作,为今后桉属植物的进一步研究、开发和利用提供科学依据。     

Genome Size of Three <Emphasis Type="Italic">Miscanthus</Emphasis> Species

Environmental and economic factors have stimulated research in the area of bioenergy crops. While many plants have been identified as potential energy crops, one species in particular, Miscanthus x giganteus, appears to have the most promise. As researchers attempt to exploit and improve M. x giganteus, genome information is critical. In this study, the genome size of M. x giganteus and its two progenitor species were examined by flow cytometry and stomatal cell analyses. M. x giganteus was found to have genome size of 7.0 pg while Miscanthus sinensis and Miscanthus sacchariflorus were observed to have genome sizes of 5.5 and 4.5 pg respectively with stomatal size correlating with genome size. Upon computing the two tetraploid × diploid hybrids theoretical genome sizes, the data presented in this paper supports the hypothesis of the union of a 2x M. sacchariflorus and a 1x M. sinensis gamete for the formation of the allotriploid, M. x giganteus. Such genomic information provides basic knowledge that is important in M. x giganteus plant improvement.     

Chloroplast DNA variation among five species ofRanunculaceae: Structure,sequence divergence,and phylogenetic relationships

A restriction site map of the chloroplast genome ofCaltha palustris L. (Ranunculaceae) has been constructed for 13 restriction endonucleases using filter hybridization with cloned tobacco chloroplast DNA fragments. A size of 153.8 kb has been estimated for theCaltha chloroplast genome. Forty-six chloroplast genes and four open reading frames have been mapped using small tobacco chloroplast gene probes. Chloroplast DNA sequence divergence has been estimated for all pairs of five species ofRanunculaceae, Caltha palustris, Ranunculus bulbosus, R. fascicularis, R. recurvatus, andTrollius ledebourii, and ranges between 0.2% and 9.6% for the total genome. Divergence values are much higher in the small and large single copy regions than in the inverted repeat. Phylogenetic relationships between the five species have been hypothesized using chloroplast DNA restriction site mapping. One hundred and six informative restriction site mutations have been detected using eleven restriction endonucleases. Cladistic analyses of the restriction site mutations have been performed using Wagner and Dollo parsimony algorithms, and confidence intervals have been calculated for the resulting monophyletic groups using bootstrapping. It is demonstrated that restriction site comparisons are applicable to theRanunculaceae on intergeneric level, with the exception of groups having extensive genomic rearrangements. Moreover, sequence divergence is low enough at the interspecific level to allow phylogenetic analyses within genera such asRanunculus.     

Complete nucleotide sequence of the <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species

Background  

The recent determination of complete chloroplast (cp) genomic sequences of various plant species has enabled numerous comparative analyses as well as advances in plant and genome evolutionary studies. In angiosperms, the complete cp genome sequences of about 70 species have been determined, whereas those of only three gymnosperm species, Cycas taitungensis, Pinus thunbergii, and Pinus koraiensis have been established. The lack of information regarding the gene content and genomic structure of gymnosperm cp genomes may severely hamper further progress of plant and cp genome evolutionary studies. To address this need, we report here the complete nucleotide sequence of the cp genome of Cryptomeria japonica, the first in the Cupressaceae sensu lato of gymnosperms, and provide a comparative analysis of their gene content and genomic structure that illustrates the unique genomic features of gymnosperms.     

SSR标记在桉树树种精准鉴定中的应用分析

为精准鉴定桉树种质,利用4个在种内完全保守的SSR位点对28种桉树(Eucalyptus)进行鉴定。根据这4个SSR位点的微卫星重复次数和侧翼序列特异核苷酸组合,构建了28种桉树种间种质资源的鉴定条码,能够精准鉴定9种桉树。这为桉树杂交育种工作提供了生物学依据。     

A repeated sequence probe for the C genome in Avena (Oats)

Summary The genus Avena consists of at least 23 species composed of three ploidy levels. Cytogenetic analysis has characterised four distinct karyotypes. These are the A, B, C and D genomes. We have isolated a repeated sequence clone that can be used for the detection of the C genome in Avena by filter hybridization techniques. This clone, termed RS-1, is a genomic DNA clone containing at least one highly repeated sequence that is abundant in Avena species containing the C genome. This sequence or a related sequence is also present, but at much reduced levels, in species that do not contain the C genome. Because of its abundance and the characteristic Southern blot pattern, we have termed this clone a C genome specific clone. We have also done similar analysis of the Avena genus using a rDNA clone from wheat. The results of these experiments demonstrate that clearly definable C genome-specific markers can be identified with both probes. These molecular probes can be useful in studying the genomic relationships of Avena and can provide some clues as to the origin of the cultivated Avena species. These results can, therefore, provide breeders with directions for the efficient transfer of desirable traits of wild Avena species into commencal varieties.     

Colonization of eucalypts in New Zealand by Australian insects

Since the 1860s, Australian insects have steadily colonized eucalypts in New Zealand. The rate of colonization has increased markedly over the last two decades. This increase may be related to increasing trade between the two countries. Currently there are 26 specialist eucalypt insect species and approximately 31 polyphagous insect species that can feed on Eucalyptus in New Zealand. The specialist eucalypt insects endemic to Australia have generally caused more damage than polyphagous or native insects. Eucalypt‐specific insects are dominated by sap sucking bugs, particularly psyllids, and defoliating Coleoptera and Lepidoptera. In some cases the major insect pest species have been those that are only occasional pests in Australia, for example Gonipterus scutellatus, Ctenarytaina eucalypti, Eriococcus coriaceus and Phylacteophaga froggatti. Some important insect pests have been rare, or even not described from Australia, prior to their appearance as a pest in New Zealand, for example Paropsis charybdis and Ophelimus eucalypti. Invading eucalypt insects are more likely to establish in the Auckland region than anywhere else in New Zealand.