Recovered frog populations coexist with endemic Batrachochytrium dendrobatidis despite load-dependent mortality.

Novel infectious diseases, particularly those caused by fungal pathogens, pose considerable risks to global biodiversity. The amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) has demonstrated the scale of the threat, having caused the greatest recorded loss of vertebrate biodiversity attributable to a pathogen. Despite catastrophic declines on several continents, many affected species have experienced population recoveries after epidemics. However, the potential ongoing threat of endemic Bd in these recovered or recovering populations is still poorly understood. We investigated the threat of endemic Bd to frog populations that recovered after initial precipitous declines, focusing on the endangered rainforest frog Mixophyes fleayi. We conducted extensive field surveys over 4 years at three independent sites in eastern Australia. First, we compared Bd infection prevalence and infection intensities within frog communities to reveal species-specific infection patterns. Then, we analyzed mark-recapture data of M. fleayi to estimate the impact of Bd infection intensity on apparent mortality rates and Bd infection dynamics. We found that M. fleayi had lower infection intensities than sympatric frogs across the three sites, and cleared infections at higher rates than they gained infections throughout the study period. By incorporating time-varying individual infection intensities, we show that healthy M. fleayi populations persist despite increased apparent mortality associated with infrequent high Bd loads. Infection dynamics were influenced by environmental conditions, with Bd prevalence, infection intensity, and rates of gaining infection associated with lower temperatures and increased rainfall. However, mortality remained constant year-round despite these fluctuations in Bd infections, suggesting major mortality events did not occur over the study period. Together, our results demonstrate that while Bd is still a potential threat to recovered populations of M. fleayi, high rates of clearing infections and generally low average infection loads likely minimize mortality caused by Bd. Our results are consistent with pathogen resistance contributing to the coexistence of M. fleayi with endemic Bd. We emphasize the importance of incorporating infection intensity into disease models rather than infection status alone. Similar population and infection dynamics likely exist within other recovered amphibian-Bd systems around the globe, promising longer-term persistence in the face of endemic chytridiomycosis.

Signatures of selection in recently domesticated macadamia.

Macadamia is a high value nut crop that is recently domesticated, ideal for testing the effect of artificial selection. Here, we sequence the genome of Hawaiian cultivar 'Kau' and assemble into 794 Mb in 14 pseudo-chromosomes with 37,728 genes. Genome analysis reveals a whole-genome duplication event, occurred 46.8 million years ago. Gene expansions occurred in gene families involves in fatty acid biosynthesis. Gene duplication of MADS-Box transcription factors in proanthocyanidin biosynthesis are relevant for seed coat development. Genome re-sequencing of 112 accessions reveals the origin of Hawaiian cultivars from Mount Bauple in southeast Queensland in Australia. Selective sweeps are detected in macadamia cultivars, including genes involved in fatty acid biosynthesis, seed coat development, and heat stress response. Such strong effects of artificial selection in few generations reveals the genomic basis for 'one-step operation' for clonal crop domestication. The knowledge gained could accelerate domestication of new crops from wild species.

The genome of the endangered Macadamia jansenii displays little diversity but represents an important genetic resource for plant breeding.

Macadamia, a recently domesticated expanding nut crop in the tropical and subtropical regions of the world, is one of the most economically important genera in the diverse and widely adapted Proteaceae family. All four species of Macadamia are rare in the wild with the most recently discovered, M. jansenii, being endangered. The M. jansenii genome has been used as a model for testing sequencing methods using a wide range of long read sequencing techniques. Here, we report a chromosome level genome assembly, generated using a combination of Pacific Biosciences sequencing and Hi-C, comprising 14 pseudo-molecules, with a N50 of 52 Mb and a total genome assembly size of 758 Mb of which 56% is repetitive. Completeness assessment revealed that the assembly covered -97.1% of the conserved single copy genes. Annotation predicted 31,591 protein coding genes and allowed the characterization of genes encoding biosynthesis of cyanogenic glycosides, fatty acid metabolism, and anti-microbial proteins. Re-sequencing of seven other genotypes confirmed low diversity and low heterozygosity within this endangered species. Important morphological characteristics of this species such as small tree size and high kernel recovery suggest that M. jansenii is an important source of these commercial traits for breeding. As a member of a small group of families that are sister to the core eudicots, this high-quality genome also provides a key resource for evolutionary and comparative genomics studies.

Immunological Aspects of Chytridiomycosis.

Amphibians are currently the most threatened vertebrate class, with the disease chytridiomycosis being a major contributor to their global declines. Chytridiomycosis is a frequently fatal skin disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). The severity and extent of the impact of the infection caused by these pathogens across modern Amphibia are unprecedented in the history of vertebrate infectious diseases. The immune system of amphibians is thought to be largely similar to that of other jawed vertebrates, such as mammals. However, amphibian hosts are both ectothermic and water-dependent, which are characteristics favouring fungal proliferation. Although amphibians possess robust constitutive host defences, Bd/Bsal replicate within host cells once these defences have been breached. Intracellular fungal localisation may contribute to evasion of the induced innate immune response. Increasing evidence suggests that once the innate defences are surpassed, fungal virulence factors suppress the targeted adaptive immune responses whilst promoting an ineffectual inflammatory cascade, resulting in immunopathology and systemic metabolic disruption. Thus, although infections are contained within the integument, crucial homeostatic processes become compromised, leading to mortality. In this paper, we present an integrated synthesis of amphibian post-metamorphic immunological responses and the corresponding outcomes of infection with Bd, focusing on recent developments within the field and highlighting future directions.

Chromosome-Scale Assembly and Annotation of the Macadamia Genome (Macadamia integrifolia HAES 741).

Macadamia integrifolia is a representative of the large basal eudicot family Proteaceae and the main progenitor species of the Australian native nut crop macadamia. Since its commercialisation in Hawaii fewer than 100 years ago, global production has expanded rapidly. However, genomic resources are limited in comparison to other horticultural crops. The first draft assembly of M. integrifolia had good coverage of the functional gene space but its high fragmentation has restricted its use in comparative genomics and association studies. Here we have generated an improved assembly of cultivar HAES 741 (4,094 scaffolds, 745 Mb, N50 413 kb) using a combination of Illumina paired and PacBio long read sequences. Scaffolds were anchored to 14 pseudo-chromosomes using seven genetic linkage maps. This assembly has improved contiguity and coverage, with >120 Gb of additional sequence. Following annotation, 34,274 protein-coding genes were predicted, representing 90% of the expected gene content. Our results indicate that the macadamia genome is repetitive and heterozygous. The total repeat content was 55% and genome-wide heterozygosity, estimated by read mapping, was 0.98% or an average of one SNP per 102 bp. This is the first chromosome-scale genome assembly for macadamia and the Proteaceae. It is expected to be a valuable resource for breeding, gene discovery, conservation and evolutionary genomics.

Maximising recombination across macadamia populations to generate linkage maps for genome anchoring.

The Proteaceae genus Macadamia has a recent history of domestication as a commercial nut crop. We aimed to establish the first sequence-based haploid-correlated reference genetic linkage maps for this primarily outcrossing perennial tree crop, with marker density suitable for genome anchoring. Four first generation populations were used to maximise the segregation patterns available within full-sib, biparental and self-pollinated progeny. This allowed us to combine segregation data from overlapping subsets of >4,000 informative sequence-tagged markers to increase the effective coverage of the karyotype represented by the recombinant crossover events detected. All maps had 14 linkage groups, corresponding to the Macadamia haploid chromosome number, and enabled the anchoring and orientation of sequence scaffolds to construct a pseudo-chromosomal genome assembly for macadamia. Comparison of individual maps indicated a high level of congruence, with minor discrepancies satisfactorily resolved within the integrated maps. The combined set of maps significantly improved marker density and the proportion (70%) of the genome sequence assembly anchored. Overall, increasing our understanding of the genetic landscape and genome for this nut crop represents a substantial advance in macadamia genetics and genomics. The set of maps, large number of sequence-based markers and the reconstructed genome provide a toolkit to underpin future breeding that should help to extend the macadamia industry as well as provide resources for the long term conservation of natural populations in eastern Australia of this unique genus.

Wild Origins of Macadamia Domestication Identified Through Intraspecific Chloroplast Genome Sequencing.

Identifying the geographic origins of crops is important for the conservation and utilization of novel genetic variation. Even so, the origins of many food crops remain elusive. The tree nut crop macadamia has a remarkable domestication history, from subtropical rain forests in Australia through Hawaii to global cultivation all within the last century. The industry is based primarily on Macadamia integrifolia and M. integrifolia-M. tetraphylla hybrid cultivars with Hawaiian cultivars the main contributors to world production. Sequence data from the chloroplast genome assembled using a genome skimming strategy was used to determine population structure among remnant populations of the main progenitor species, M. integrifolia. Phylogenetic analysis of a 506 bp chloroplast SNP alignment from 64 wild and cultivated accessions identified phylogeographic structure and deep divergences between clades providing evidence for historical barriers to seed dispersal. High levels of variation were detected among wild accessions. Most Hawaiian cultivars, however, shared a single chlorotype that was also present at two wild sites at Mooloo and Mt Bauple from the northernmost distribution of the species in south-east Queensland. Our results provide evidence for a maternal genetic bottleneck during early macadamia domestication, and pinpoint the likely source of seed used to develop the Hawaiian cultivars. The extensive variability and structuring of M. integrifolia chloroplast genomic variation detected in this study suggests much unexploited genetic diversity is available for improvement of this recently domesticated crop.

Correction to "Whole genome shotgun sequences for microsatellite discovery and application in cultivated and wild Macadamia (Proteaceae)".

[This corrects the article DOI: 10.3732/apps.1300089.].

Molecular Detection of Diverse Leifsonia Strains Associated With Sugarcane.

Leifsonia xyli subsp. xyli, causal agent of ratoon stunting disease (RSD) of sugarcane (Saccharum interspecific hybrids), is the most well-known member of the Microbacteriaceae genus Leifsonia. However, the presence of other Leifsonia strains associated with sugarcane has not been reported. A total of 697 Australian and 40 Indonesian sugarcane fields were screened by leaf sheath biopsy (LSB) PCR using primers specific for L. xyli subsp. xyli, in addition to primers designed to amplify DNA from other members of the genus Leifsonia. While L. xyli subsp. xyli was detected in 126 fields, a total of 37 distinct and novel Leifsonia and non-Leifsonia strains were detected in 116 fields. Representatives of these strains were also detected in multiple samples of expressed xylem sap. Sequencing and phylogenetic analyses demonstrated the presence of a broad complex of novel Leifsonia strains, in addition to strains closely related to the recently erected Cnuibacter genus. Attempts to isolate Leifsonia strains were unsuccessful; however, one strain related to Cnuibacter was recovered from expressed xylem sap. Among the genetically diverse lineages discovered, identical genotypes were present in multiple sugarcane varieties growing in disparate regions in different years, strongly suggesting an ongoing association with sugarcane. The epidemiological significance of these strains is unknown, but there is evidence that they can interfere with serological and microscopic RSD diagnostics, and there is the potential that they may represent new and distinct pathologies of sugarcane.

Genome and transcriptome sequencing characterises the gene space of Macadamia integrifolia (Proteaceae).

BACKGROUND: The large Gondwanan plant family Proteaceae is an early-diverging eudicot lineage renowned for its morphological, taxonomic and ecological diversity. Macadamia is the most economically important Proteaceae crop and represents an ancient rainforest-restricted lineage. The family is a focus for studies of adaptive radiation due to remarkable species diversification in Mediterranean-climate biodiversity hotspots, and numerous evolutionary transitions between biomes. Despite a long history of research, comparative analyses in the Proteaceae and macadamia breeding programs are restricted by a paucity of genetic information. To address this, we sequenced the genome and transcriptome of the widely grown Macadamia integrifolia cultivar 741. RESULTS: Over 95 gigabases of DNA and RNA-seq sequence data were de novo assembled and annotated. The draft assembly has a total length of 518 Mb and spans approximately 79% of the estimated genome size. Following annotation, 35,337 protein-coding genes were predicted of which over 90% were expressed in at least one of the leaf, shoot or flower tissues examined. Gene family comparisons with five other eudicot species revealed 13,689 clusters containing macadamia genes and 1005 macadamia-specific clusters, and provides evidence for linage-specific expansion of gene families involved in pathogen recognition, plant defense and monoterpene synthesis. Cyanogenesis is an important defense strategy in the Proteaceae, and a detailed analysis of macadamia gene homologues potentially involved in cyanogenic glycoside biosynthesis revealed several highly expressed candidate genes. CONCLUSIONS: The gene space of macadamia provides a foundation for comparative genomics, gene discovery and the acceleration of molecular-assisted breeding. This study presents the first available genomic resources for the large basal eudicot family Proteaceae, access to most macadamia genes and opportunities to uncover the genetic basis of traits of importance for adaptation and crop improvement.

Efficient Diagnosis of Ratoon Stunting Disease of Sugarcane by Quantitative PCR on Pooled Leaf Sheath Biopsies.

Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli, is arguably one of the most devastating diseases of sugarcane. Four diagnostic techniques were compared for 100 fields of sugarcane (Saccharum interspecific hybrids) of unknown infection status. These were quantitative polymerase chain reaction on pooled leaf sheath biopsies (LSB-qPCR), conventional PCR on the same templates (LSB-PCR), evaporative-binding enzyme immunoassay (EB-EIA) coupled with phase contrast microscopy (PCM) on expressed xylem sap from the same fields, and conventional PCR on the same xylem sap samples. LSB-qPCR and LSB-PCR detected the causal agent in 27 and 18 fields, respectively, whereas, from samples of expressed xylem sap from the same fields, conventional PCR identified 12 infections and EB-EIA/PCM detected L. xyli subsp. xyli in 3 fields. The sensitivities of qPCR and PCR were approximately 103 and 104 CFU ml-1, respectively, determined from plate counts of a dilution series. Tests were conducted on a further 139 LSB samples from across the Australian industry, with qPCR and PCR diagnosing RSD in 31 and 25 fields, respectively. Using qPCR and PCR on LSB samples, RSD was diagnosed in a range of cultivars throughout the year, and qPCR and PCR could detect L. xyli subsp. xyli in sugarcane ranging from 3 months to greater than 1 year old.

Complete chloroplast genome of Macadamia integrifolia confirms the position of the Gondwanan early-diverging eudicot family Proteaceae.

BACKGROUND: Sequence data from the chloroplast genome have played a central role in elucidating the evolutionary history of flowering plants, Angiospermae. In the past decade, the number of complete chloroplast genomes has burgeoned, leading to well-supported angiosperm phylogenies. However, some relationships, particulary among early-diverging lineages, remain unresolved. The diverse Southern Hemisphere plant family Proteaceae arose on the ancient supercontinent Gondwana early in angiosperm history and is a model group for adaptive radiation in response to changing climatic conditions. Genomic resources for the family are limited, and until now it is one of the few early-diverging 'basal eudicot' lineages not represented in chloroplast phylogenomic analyses. RESULTS: The chloroplast genome of the Australian nut crop tree Macadamia integrifolia was assembled de novo from Illumina paired-end sequence reads. Three contigs, corresponding to a collapsed inverted repeat, a large and a small single copy region were identified, and used for genome reconstruction. The complete genome is 159,714 bp in length and was assembled at deep coverage (3.29 million reads; ~2000 x). Phylogenetic analyses based on 83-gene and inverted repeat region alignments, the largest sequence-rich datasets to include the basal eudicot family Proteaceae, provide strong support for a Proteales clade that includes Macadamia, Platanus and Nelumbo. Genome structure and content followed the ancestral angiosperm pattern and were highly conserved in the Proteales, whilst size differences were largely explained by the relative contraction of the single copy regions and expansion of the inverted repeats in Macadamia. CONCLUSIONS: The Macadamia chloroplast genome presented here is the first in the Proteaceae, and confirms the placement of this family with the morphologically divergent Plantanaceae (plane tree family) and Nelumbonaceae (sacred lotus family) in the basal eudicot order Proteales. It provides a high-quality reference genome for future evolutionary studies and will be of benefit for taxon-rich phylogenomic analyses aimed at resolving relationships among early-diverging angiosperms, and more broadly across the plant tree of life.

Whole genome shotgun sequences for microsatellite discovery and application in cultivated and wild Macadamia (Proteaceae).

PREMISE OF THE STUDY: Next-generation sequencing (NGS) data are widely used for single-nucleotide polymorphism discovery and genetic marker development in species with limited available genome information. We developed microsatellite primers for the Proteaceae nut crop species Macadamia integrifolia and assessed cross-species transferability in all congeners to investigate genetic identification of cultivars and gene flow. • METHODS AND RESULTS: Primers were designed from both raw and assembled Illumina NGS paired-end reads. The final 12 microsatellite markers selected were polymorphic among wild individuals of all four Macadamia species-M. integrifolia, M. tetraphylla, M. ternifolia, and M. jansenii-and in commercial macadamia cultivars including hybrids. • CONCLUSIONS: We demonstrate the utility of raw and assembled Illumina NGS reads from total genomic DNA for the rapid development of microsatellites in Macadamia. These primers will facilitate future studies of population structure, hybridization, parentage, and cultivar identification in cultivated and wild Macadamia populations.

Next-generation technologies to determine plastid genome sequences.

Sequencing of chloroplast genomes is a key tool for analysis of chloroplasts and the impact of manipulation of chloroplast genomes by biotechnology. Advances in genome sequencing allow the complete sequencing of the chloroplast genome and assessment of variation in the chloroplast genome sequences within a plant. Isolation of chloroplast DNA has been a traditional starting point in these analyses, but the capacity of current sequencing technologies allows effective analysis of the chloroplast genome sequence by shotgun sequencing of a preparation of total DNA from the plant. Chloroplast insertions in the nuclear genome can be distinguished by their much lower copy number. Short-read sequences are best assembled by alignment with a reference chloroplast genome.

From local to global-fifty years of research on Salvia divinorum.

ETHNOPHARMACOLOGICAL RELEVANCE: In 1962 ethnopharmacologists, Hofmann and Wasson, undertook an expedition to Oaxaca, Mexico. These two researchers were the first scientists to collect a flowering specimen of Salvia divinorum allowing the identification of this species. While the species' traditional use is confined to a very small region of Mexico, since Hofmann and Wasson's expedition 50 years ago, Salvia divinorum has become globally recognized for its main active constituent, the diterpene salvinorin A, which has a unique effect on human physiology. Salvinorin A is a kappa-opioid agonist and the first reported psychoactive diterpene. METHODS: This review concentrates on the investigation of Salvia divinorum over the last 50 years including ethnobotany, ethnopharmacology, taxonomy, systematics, genetics, chemistry and pharmacodynamic and pharmacokinetic research. For the purpose of this review, online search engines were used to find relevant research. Searches were conducted between October 2011 and September 2013 using the search term "Salvia divinorum". Papers were excluded if they described synthetic chemical synthesis of salvinorin A or analogues. RESULTS: Ethnobotanically there is a comprehensive body of research describing the traditional Mazatec use of the plant, however, the modern ethnobotanical use of this plant is not well documented. There are a limited number of botanical investigations into this plant and there are still several aspects of the botany of Salvia divinorum which need further investigation. One study has investigated the phylogenetic relationship of Salvia divinorum to other species in the genus. To date the main focus of chemistry research on Salvia divinorum has been salvinorin A, the main active compound in Salvia divinorum, and other related diterpenoids. Finally, the effects of salvinorin A, a KOR agonist, have primarily been investigated using animal models. CONCLUSIONS: As Salvia divinorum use increases worldwide, the emerging cultural use patterns will warrant more research. More botanical information is also needed to better understand this species, including germination, pollination vector and a better understanding of the endemic environment of Salvia divinorum. As well there is a gap in the genetic knowledge of this species and very little is known about its intra-species genetics. The terpenes in Salvia divinorum are very well documented, however, other classes of constituents in this species warrant further investigation and identification. To date, the majority of the pharmacology research on Salvia divinorum has focused on the effects of salvinorin A using animal models. Published human studies have not reported any harmful effects when salvinorin A is administered within the dose range of 0.375-21µg/kg but what are the implications when applied to a larger population? More data on the toxicology and safety of Salvia divinorum are needed before larger scale clinical trials of the potential therapeutic effects of Salvia divinorum and salvinorin A are undertaken.

Application of large-scale sequencing to marker discovery in plants.

Advances in DNA sequencing provide tools for efficient large-scale discovery of markers for use in plants. Discovery options include large-scale amplicon sequencing, transcriptome sequencing, gene-enriched genome sequencing and whole genome sequencing. Examples of each of these approaches and their potential to generate molecular markers for specific applications have been described. Sequencing the whole genome of parents identifies all the polymorphisms available for analysis in their progeny. Sequencing PCR amplicons of sets of candidate genes from DNA bulks can be used to define the available variation in these genes that might be exploited in a population or germplasm collection. Sequencing of the transcriptomes of genotypes varying for the trait of interest may identify genes with patterns of expression that could explain the phenotypic variation. Sequencing genomic DNA enriched for genes by hybridization with probes for all or some of the known genes simplifies sequencing and analysis of differences in gene sequences between large numbers of genotypes and genes especially when working with complex genomes. Examples of application of the above-mentioned techniques have been described.

Chloroplast genome sequence confirms distinctness of Australian and Asian wild rice.

Cultivated rice (Oryza sativa) is an AA genome Oryza species that was most likely domesticated from wild populations of O. rufipogon in Asia. O. rufipogon and O. meridionalis are the only AA genome species found within Australia and occur as widespread populations across northern Australia. The chloroplast genome sequence of O. rufipogon from Asia and Australia and O. meridionalis and O. australiensis (an Australian member of the genus very distant from O. sativa) was obtained by massively parallel sequencing and compared with the chloroplast genome sequence of domesticated O. sativa. Oryza australiensis differed in more than 850 sites single nucleotide polymorphism or indel from each of the other samples. The other wild rice species had only around 100 differences relative to cultivated rice. The chloroplast genomes of Australian O. rufipogon and O. meridionalis were closely related with only 32 differences. The Asian O. rufipogon chloroplast genome (with only 68 differences) was closer to O. sativa than the Australian taxa (both with more than 100 differences). The chloroplast sequences emphasize the genetic distinctness of the Australian populations and their potential as a source of novel rice germplasm. The Australian O. rufipogon may be a perennial form of O. meridionalis.

Chloroplast genome sequences from total DNA for plant identification.

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost-effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non-trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost-effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single-locus DNA barcode for plants.