Australian native Glycine clandestina seed microbiota hosts a more diverse bacterial community than the domesticated soybean Glycine max.

BACKGROUND: Plant microbiome composition has been demonstrated to change during the domestication of wild plants and it is suggested that this has resulted in loss of plant beneficial microbes. Recently, the seed microbiome of native plants was demonstrated to harbour a more diverse microbiota and shared a common core microbiome with modern cultivars. In this study the composition of the seed-associated bacteria of Glycine clandestina is compared to seed-associated bacteria of Glycine max (soybean). RESULTS: The seed microbiome of the native legume Glycine clandestina (crop wild relative; cwr) was more diverse than that of the domesticated Glycine max and was dominated by the bacterial class Gammaproteobacteria. Both the plant species (cwr vs domesticated) and individual seed accessions were identified as the main driver for this diversity and composition of the microbiota of all Glycine seed lots, with the effect of factor "plant species" exceeded that of "geographical location". A core microbiome was identified between the two Glycine species. A high percentage of the Glycine microbiome was unculturable [G. clandestina (80.8%) and G. max (75.5%)] with only bacteria of a high relative abundance being culturable under the conditions of this study. CONCLUSION: Our results provided novel insights into the structure and diversity of the native Glycine clandestina seed microbiome and how it compares to that of the domesticated crop Glycine max. Beyond that, it also increased our knowledge of the key microbial taxa associated with the core Glycine spp. microbiome, both wild and domesticated. The investigation of this commonality and diversity is a valuable and essential tool in understanding the use of native Glycine spp. for the discovery of new microbes that would be of benefit to domesticated Glycine max cultivars or any other economically important crops. This study has isolated microbes from a crop wild relative that are now available for testing in G. max for beneficial phenotypes.

Corrigendum: Implications of Seed Vault Storage Strategies for Conservation of Seed Bacterial Microbiomes.

[This corrects the article DOI: 10.3389/fmicb.2021.784796.].

The Role of Soil Microbial Diversity in the Conservation of Native Seed Bacterial Microbiomes.

Research into understanding the structure, composition and vertical transmission of crop seed microbiomes has intensified, although there is much less research into the seed microbiomes of crop wild relatives. Our previous study showed that the standard seed storage procedures (e.g., seed drying and storage temperature) can influence the seed microbiome of domesticated Glycine max. In this study, we characterized the seed microbiota of Glycine clandestina, a perennial wild relative of soybean (G. max (L.) Merr.) to expand our understanding about the effect of other storage procedures such as the periodic regeneration of seed stocks to bulk up seed numbers and secure viability on the seed microbiome of said seed. The G. clandestina microbiota was analysed from Generation 1 (G1) and Generation 2 (G2) seed and from mature plant organs grown in two different soil treatments T (treatment [native soil + potting mix]) and C (control [potting mix only]). Our dataset showed that soil microbiota had a strong influence on next generation seed microbiota, with an increased contribution of root microbiota by 90% and seed transmissibility by 36.3% in G2 (T) seed. Interestingly, the G2 seed microbiota primarily consisted of an initially low abundance of taxa present in G1 seed. Overall, our results indicate that seed regeneration can affect the seed microbiome composition and using native soil from the location of the source plant can enhance the conservation of the native seed microbiota.

Implications of Seed Vault Storage Strategies for Conservation of Seed Bacterial Microbiomes.

Global seed vaults are important, as they conserve plant genetic resources for future breeding to improve crop yield and quality and to overcome biotic and abiotic stresses. However, little is known about the impact of standard storage procedures, such as seed drying and cold storage on the seed bacterial community, and the ability to recover seed-associated bacteria after storage. In this study, soybean [Glycine max (L.) Merr.] seeds were analyzed to characterize changes in the bacterial community composition and culturability under varying storage conditions. The G. max bacterial microbiome was analyzed from undried seed, dried seed, and seed stored for 0, 3, 6, and 14months. Storage temperatures consisted of -20°C, 4°C, and room temperature (RT), with -20°C being commonly used in seed storage vaults globally. The seed microbiome of G. max was dominated by Gammaproteobacteria under all conditions. Undried seed was dominated by Pantoea (33.9%) and Pseudomonas (51.1%); however, following drying, the abundance of Pseudomonas declined significantly (0.9%), Pantoea increased significantly (73.6%), and four genera previously identified including Pajaroellobacter, Nesterenkonia, env.OPS_17, and Acidibacter were undetectable. Subsequent storage at RT, 4, or -20°C maintained high-abundance Genera at the majority of time points, although RT caused greater fluctuations in abundances. For many of the low-abundance Genera, storage at -20°C resulted in their gradual disappearance, whereas storage at 4°C or RT resulted in their more rapid disappearance. The changes in seed bacterial composition were reflected by cultured bacterial taxa obtained from the stored G. max seed. The main taxa were largely culturable and had similar relative abundance, while many, but not all, of the low-abundance taxa were also culturable. Overall, these results indicate that the initial seed drying affects the seed bacterial composition, suggesting that microbial isolation prior to seed drying is recommended to conserve these microbes. The standard seed storage condition of -20°C is most suitable for conservation of the bacterial seed microbiome, as this storage temperature slows down the loss of seed bacterial diversity over longer time periods, particularly low-abundance taxa.

Transcriptome Analyses of Barley Roots Inoculated with Novel Paenibacillus sp. and Erwinia gerundensis Strains Reveal Beneficial Early-Stage Plant-Bacteria Interactions.

Plant growth-promoting bacteria can improve host plant traits including nutrient uptake and metabolism and tolerance to biotic and abiotic stresses. Understanding the molecular basis of plant-bacteria interactions using dual RNA-seq analyses provides key knowledge of both host and bacteria simultaneously, leading to future enhancements of beneficial interactions. In this study, dual RNA-seq analyses were performed to provide insights into the early-stage interactions between barley seedlings and three novel bacterial strains (two Paenibacillus sp. strains and one Erwinia gerundensis strain) isolated from the perennial ryegrass seed microbiome. Differentially expressed bacterial and barley genes/transcripts involved in plant-bacteria interactions were identified, with varying species- and strain-specific responses. Overall, transcriptome profiles suggested that all three strains improved stress response, signal transduction, and nutrient uptake and metabolism of barley seedlings. Results also suggested potential improvements in seedling root growth via repressing ethylene biosynthesis in roots. Bacterial secondary metabolite gene clusters producing compounds that are potentially associated with interactions with the barley endophytic microbiome and associated with stress tolerance of plants under nutrient limiting conditions were also identified. The results of this study provided the molecular basis of plant growth-promoting activities of three novel bacterial strains in barley, laid a solid foundation for the future development of these three bacterial strains as biofertilisers, and identified key differences between bacterial strains of the same species in their responses to plants.

Transcriptomics differentiate two novel bioactive strains of Paenibacillus sp. isolated from the perennial ryegrass seed microbiome.

Paenibacillus species are Gram-positive bacteria that have been isolated from a diverse array of plant species and soils, with some species exhibiting plant growth-promoting (PGP) activities. Here we report two strains (S02 and S25) of a novel Paenibacillus sp. that were isolated from perennial ryegrass (Lolium perenne) seeds. Comparative genomics analyses showed this novel species was closely related to P. polymyxa. Genomic analyses revealed that strains S02 and S25 possess PGP genes associated with biological nitrogen fixation, phosphate solubilisation and assimilation, as well as auxin production and transportation. Moreover, secondary metabolite gene cluster analyses identified 13 clusters that are shared by both strains and three clusters unique to S25. In vitro assays demonstrated strong bioprotection activity against phytopathogens (Colletotrichum graminicola and Fusarium verticillioides), particularly for strain S02. A transcriptomics analysis evaluating nitrogen fixation activity showed both strains carry an expressed nif operon, but strain S02 was more active than strain S25 in nitrogen-free media. Another transcriptomics analysis evaluating the interaction of strains with F. verticillioides showed strain S02 had increased expression of core genes of secondary metabolite clusters (fusaricidin, paenilan, tridecaptin and polymyxin) when F. verticillioides was present and absent, compared to S25. Such bioactivities make strain S02 a promising candidate to be developed as a combined biofertiliser/bioprotectant.

Global Changes in Asexual Epichloë Transcriptomes during the Early Stages, from Seed to Seedling, of Symbiotum Establishment.

Asexual Epichloë fungi are strictly seed-transmitted endophytic symbionts of cool-season grasses and spend their entire life cycle within the host plant. Endophyte infection can confer protective benefits to its host through the production of bioprotective compounds. Inversely, plants provide nourishment and shelter to the resident endophyte in return. Current understanding of the changes in global gene expression of asexual Epichloë endophytes during the early stages of host-endophyte symbiotum is limited. A time-course study using a deep RNA-sequencing approach was performed at six stages of germination, using seeds infected with one of three endophyte strains belonging to different representative taxa. Analysis of the most abundantly expressed endophyte genes identified that most were predicted to have a role in stress and defence responses. The number of differentially expressed genes observed at early time points was greater than those detected at later time points, suggesting an active transcriptional reprogramming of endophytes at the onset of seed germination. Gene ontology enrichment analysis revealed dynamic changes in global gene expression consistent with the developmental processes of symbiotic relationships. Expression of pathway genes for biosynthesis of key secondary metabolites was studied comprehensively and fuzzy clustering identified some unique expression patterns. Furthermore, comparisons of the transcriptomes from three endophyte strains in planta identified genes unique to each strain, including genes predicted to be associated with secondary metabolism. Findings from this study highlight the importance of better understanding the unique properties of individual endophyte strains and will serve as an excellent resource for future studies of host-endophyte interactions.

Profiling, isolation and characterisation of beneficial microbes from the seed microbiomes of drought tolerant wheat.

Climate change is predicted to increase the incidence and severity of drought conditions, posing a significant challenge for agriculture globally. Plant microbiomes have been demonstrated to aid crop species in the mitigation of drought stress. The study investigated the differences between the seed microbiomes of drought tolerant and drought susceptible wheat lines. Furthermore, it highlighted and quantified the degree of drought tolerance conferred by specific microbes isolated from drought tolerant wheat seed microbiomes. Metagenomic and culture-based methods were used to profile and characterise the seed microbiome composition of drought tolerant and drought susceptible wheat lines under rainfed and drought conditions. Isolates from certain genera were enriched by drought tolerant wheat lines when placed under drought stress. Wheat inoculated with isolates from these targeted genera, such as Curtobacterium flaccumfaciens (Cf D3-25) and Arthrobacter sp. (Ar sp. D4-14) demonstrated the ability to promote growth under drought conditions. This study indicates seed microbiomes from genetically distinct wheat lines enrich for beneficial bacteria in ways that are both line-specific and responsive to environmental stress. As such, seed from stress-phenotyped lines represent an invaluable resource for the identification of beneficial microbes with plant growth promoting activity that could improve commercial crop production.

Fungus-originated genes in the genomes of cereal and pasture grasses acquired through ancient lateral transfer.

Evidence for ancestral gene transfer between Epichloë fungal endophyte ancestors and their host grass species is described. From genomes of cool-season grasses (the Poeae tribe), two Epichloë-originated genes were identified through DNA sequence similarity analysis. The two genes showed 96% and 85% DNA sequence identities between the corresponding Epichloë genes. One of the genes was specific to the Loliinae sub-tribe. The other gene was more widely conserved in the Poeae and Triticeae tribes, including wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). The genes were independently transferred during the last 39 million years. The transferred genes were expressed in plant tissues, presumably retaining molecular functions. Multiple gene transfer events between the specific plant and fungal lineages are unique. A range of cereal crops is included in the Poeae and Triticeae tribes, and the Loliinae sub-tribe is consisted of economically important pasture and forage crops. Identification and characterisation of the 'natural' adaptation transgenes in the genomes of cereals, and pasture and forage grasses, that worldwide underpin the production of major foods, such as bread, meat, and milk, may change the 'unnatural' perception status of transgenic and gene-edited plants.

Novel Xanthomonas Species From the Perennial Ryegrass Seed Microbiome - Assessing the Bioprotection Activity of Non-pathogenic Relatives of Pathogens.

The productivity of the Australian dairy industry is underpinned by pasture grasses, and importantly perennial ryegrass. The performance of these pasture grasses is supported by the fungal endophyte Epichloë spp. that has bioprotection activities, however, the broader microbiome is not well characterized. In this study, we characterized a novel bioprotectant Xanthomonas species isolated from perennial ryegrass (Lolium perenne L. cv. Alto). In vitro and in planta bioassays against key fungal pathogens of grasses (Sclerotium rolfsii, Drechslera brizae and Microdochium nivale) indicated strong bioprotection activities. A complete circular chromosome of ∼5.2 Mb was generated for three strains of the novel Xanthomonas sp. Based on the 16S ribosomal RNA gene, the strains were closely related to the plant pathogen Xanthomonas translucens, however, comparative genomics of 22 closely related xanthomonad strains indicated that these strains were a novel species. The comparative genomics analysis also identified two unique gene clusters associated with the production of bioprotectant secondary metabolites including one associated with a novel nonribosomal peptide synthetase and another with a siderophore. The analysis also identified genes associated with an endophytic lifestyle (e.g., Type VI secretion system), while no genes associated with pathogenicity were identified (e.g., Type III secretion system and effectors). Overall, these results indicate that these strains represent a novel, bioactive, non-pathogenic species of the genus Xanthomonas. Strain GW was the designated type strain of this novel Xanthomonas sp.

Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event.

Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (Lolium perenne L.)-associated Epichloë endophyte strains using A. tumefaciens-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.

Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis.

Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

Horizontal transfer of a ß-1,6-glucanase gene from an ancestral species of fungal endophyte to a cool-season grass host.

Molecular characterisation has convincingly demonstrated some types of horizontal gene transfer in eukaryotes, but nuclear gene transfer between distantly related eukaryotic groups appears to have been rare. For angiosperms (flowering plants), nuclear gene transfer events identified to date have been confined to genes originating from prokaryotes or other plant species. In this report, evidence for ancient horizontal transfer of a fungal nuclear gene, encoding a ß-1,6-glucanase enzyme for fungal cell wall degradation, into an angiosperm lineage is presented for the first time. The gene was identified from de novo sequencing and assembly of the genome and transcriptome of perennial ryegrass, a cool-season grass species. Molecular analysis confirmed the presence of the complete gene in the genome of perennial ryegrass. No corresponding sequence was found in other plant species, apart from members of the Poeae sub-tribes Loliinae and Dactylidinae. Evidence suggests that a common ancestor of the two sub-tribes acquired the gene from a species ancestral to contemporary grass-associated fungal endophytes around 9-13 million years ago. This first report of horizontal transfer of a nuclear gene from a taxonomically distant eukaryote to modern flowering plants provides evidence for a novel adaptation mechanism in angiosperms.

Genomic and metabolic characterisation of alkaloid biosynthesis by asexual Epichloë fungal endophytes of tall fescue pasture grasses.

Symbiotic associations between tall fescue grasses and asexual Epichloë fungal endophytes exhibit biosynthesis of alkaloid compounds causing both beneficial and detrimental effects. Candidate novel endophytes with favourable chemotypic profiles have been identified in germplasm collections by screening for genetic diversity, followed by metabolite profile analysis in endogenous genetic backgrounds. A subset of candidates was subjected to genome survey sequencing to detect the presence or absence and structural status of known genes for biosynthesis of the major alkaloid classes. The capacity to produce specific metabolites was directly predictable from metabolic data. In addition, study of duplicated gene structure in heteroploid genomic constitutions provided further evidence for the origin of such endophytes. Selected strains were inoculated into meristem-derived callus cultures from specific tall fescue genotypes to perform isogenic comparisons of alkaloid profile in different host backgrounds, revealing evidence for host-specific quantitative control of metabolite production, consistent with previous studies. Certain strains were capable of both inoculation and formation of longer-term associations with a nonhost species, perennial ryegrass (Lolium perenne L.). Discovery and primary characterisation of novel endophytes by DNA analysis, followed by confirmatory metabolic studies, offers improvements of speed and efficiency and hence accelerated deployment in pasture grass improvement programs.

De novo assembly and characterisation of the field pea transcriptome using RNA-Seq.

BACKGROUND: Field pea (Pisum sativum L.) is a cool-season grain legume that is cultivated world-wide for both human consumption and stock-feed purposes. Enhancement of genetic and genomic resources for field pea will permit improved understanding of the control of traits relevant to crop productivity and quality. Advances in second-generation sequencing and associated bioinformatics analysis now provide unprecedented opportunities for the development of such resources. The objective of this study was to perform transcriptome sequencing and characterisation from two genotypes of field pea that differ in terms of seed and plant morphological characteristics. RESULTS: Transcriptome sequencing was performed with RNA templates from multiple tissues of the field pea genotypes Kaspa and Parafield. Tissue samples were collected at various growth stages, and a total of 23 cDNA libraries were sequenced using Illumina high-throughput sequencing platforms. A total of 407 and 352 million paired-end reads from the Kaspa and Parafield transcriptomes, respectively were assembled into 129,282 and 149,272 contigs, which were filtered on the basis of known gene annotations, presence of open reading frames (ORFs), reciprocal matches and degree of coverage. Totals of 126,335 contigs from Kaspa and 145,730 from Parafield were subsequently selected as the reference set. Reciprocal sequence analysis revealed that c. 87% of contigs were expressed in both cultivars, while a small proportion were unique to each genotype. Reads from different libraries were aligned to the genotype-specific assemblies in order to identify and characterise expression of contigs on a tissue-specific basis, of which 87% were expressed in more than one tissue, while others showed distinct expression patterns in specific tissues, providing unique transcriptome signatures. CONCLUSION: This study provided a comprehensive assembled and annotated transcriptome set for field pea that can be used for development of genetic markers, in order to assess genetic diversity, construct linkage maps, perform trait-dissection and implement whole-genome selection strategies in varietal improvement programs, as well to identify target genes for genetic modification approaches on the basis of annotation and expression analysis. In addition, the reference field pea transcriptome will prove highly valuable for comparative genomics studies and construction of a finalised genome sequence.

Phylogenomics of asexual Epichloë fungal endophytes forming associations with perennial ryegrass.

BACKGROUND: Perennial ryegrass (Lolium perenne L.) is one of the most important species for temperate pastoral agriculture, forming associations with genetically diverse groups of mutualistic fungal endophytes. However, only two taxonomic groups (E. festucae var. lolii and LpTG-2) have so far been described. In addition to these two well-characterised taxa, a third distinct group of previously unclassified perennial ryegrass-associated endophytes was identified as belonging to a putative novel taxon (or taxa) (PNT) in a previous analysis based on simple sequence repeat (SSR) marker diversity. As well as genotypic differences, distinctive alkaloid production profiles were observed for members of the PNT group. RESULTS: A detailed phylogenetic analysis of perennial ryegrass-associated endophytes using components of whole genome sequence data was performed using complete sequences of 7 nuclear protein-encoding genes. Three independently selected genes (encoding a DEAD/DEAH box helicase [Sbp4], a glycosyl hydrolase [family 92 protein] and a MEAB protein), none of which have been previously used for taxonomic studies of endophytes, were selected together with the frequently used 'house-keeping' genes tefA and tubB (encoding translation elongation factor 1-α and ß-tubulin, respectively). In addition, an endophyte-specific gene (perA for peramine biosynthesis) and the fungal-specific MT genes for mating-type control were included. The results supported previous phylogenomic inferences for the known species, but revealed distinctive patterns of diversity for the previously unclassified endophyte strains, which were further proposed to belong to not one but two distinct novel taxa. Potential progenitor genomes for the asexual endophytes among contemporary teleomorphic (sexual Epichloë) species were also identified from the phylogenetic analysis. CONCLUSIONS: Unique taxonomic status for the PNT was confirmed through comparison of multiple nuclear gene sequences, and also supported by evidence from chemotypic diversity. Analysis of MT gene idiomorphs further supported a predicted independent origin of two distinct perennial ryegrass-associated novel taxa, designated LpTG-3 and LpTG-4, from different members of a similar founder population related to contemporary E. festucae. The analysis also provided higher resolution to the known progenitor contributions of previously characterised perennial ryegrass-associated endophyte taxa.

Phylogenomics of fescue grass-derived fungal endophytes based on selected nuclear genes and the mitochondrial gene complement.

BACKGROUND: Tall fescue and meadow fescue are important as temperate pasture grasses, forming mutualistic associations with asexual Neotyphodium endophytes. The most frequently identified endophyte of Continental allohexaploid tall fescue is Neotyphodium coenophialum, while representatives of two other taxa (FaTG-2 and FaTG-3) have been described as colonising decaploid and Mediterranean hexaploid tall fescue, respectively. In addition, a recent study identified two other putatively novel endophyte taxa from Mediterranean hexaploid and decaploid tall fescue accessions, which were designated as uncharacterised Neotyphodium species (UNS) and FaTG-3-like respectively. In contrast, diploid meadow fescue mainly forms associations with the endophyte taxon Neotyphodium uncinatum, although a second endophyte taxon, termed N. siegelii, has also been described. RESULTS: Multiple copies of the translation elongation factor 1-a (tefA) and ß-tubulin (tub2) 'house-keeping' genes, as well as the endophyte-specific perA gene, were identified for each fescue-derived endophyte taxon from whole genome sequence data. The assembled gene sequences were used to reconstruct evolutionary relationships between the heteroploid fescue-derived endophytes and putative ancestral sub-genomes derived from known sexual Epichloë species. In addition to the nuclear genome-derived genes, the complete mitochondrial genome (mt genome) sequence was obtained for each of the sequenced endophyte, and phylogenetic relationships between the mt genome protein coding gene complements were also reconstructed. CONCLUSIONS: Complex and highly reticulated evolutionary relationships between Epichloë-Neotyphodium endophytes have been predicted on the basis of multiple nuclear genes and entire mitochondrial protein-coding gene complements, derived from independent assembly of whole genome sequence reads. The results are consistent with previous studies while also providing novel phylogenetic insights, particularly through inclusion of data from the endophyte lineage-specific gene, as well as affording evidence for the origin of cytoplasmic genomes. In particular, the results obtained from the present study imply the possible occurrence of at least two distinct E. typhina progenitors for heteropoid taxa, as well the ancestral contribution of an endophyte species distinct from (although related to) contemporary E. baconii to the extant hybrid species. Furthermore, the present study confirmed the distinct taxonomic status of the newly identified fescue endophyte taxa, FaTG-3-like and UNS, which are consequently proposed to be renamed FaTG4 and FaTG5, respectively.

Development and validation of a TaqMan PCR assay for the Australian abalone herpes-like virus.

The recent emergence of a herpes-like virus in both farmed and wild populations of abalone in Victoria, Australia, has been associated with high mortality rates in animals of all ages. Based on viral genome sequence information, a virus-specific real-time TaqMan assay was developed for detection and identification of the abalone herpes-like virus (AbHV). The assay was shown to be specific as it did not detect other viruses from either the Herpesvirales or the Iridovirales orders which have genome sequence similarities. However, the TaqMan assay was able to detect DNA from the Taiwanese abalone herpes-like virus, suggesting a relationship between the Taiwanese and Australian viruses. In addition, the assay detected < 300 copies of recombinant plasmid DNA per reaction. Performance characteristics for the AbHV TaqMan assay were established using 1673 samples from different abalone populations in Victoria and Tasmania. The highest diagnostic sensitivity and specificity were 96.7 (95% CI: 82.7 to 99.4) and 99.7 (95% CI: 99.3 to 99.9), respectively, at a threshold cycle (C(T)) value of 35.8. The results from 2 separate laboratories indicated good repeatability and reproducibility. This molecular assay has already proven useful in confirming presumptive diagnosis (based on the presence of ganglioneuritis) of diseased abalone in Victorian waters as well as being a tool for surveillance of wild abalone stocks in other parts of Australia.

Comparison of homoeolocus organisation in paired BAC clones from white clover (Trifolium repens L.) and microcolinearity with model legume species.

BACKGROUND: White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised. RESULTS: A total of four paired homoeologous BACs were selected and sequenced to generate 173 kb of overlapping sequence between the O and P' sub-genomes. Equivalent gene content was generally observed, apart from small-scale deletions, in contrast to conservation of intergenic sequences, which varied between the four selected regions. Measurement of the number of synonymous substitutions between homoeologous genes led to estimation of a 4.2 million year divergence time between the two sub-genomes. Microsynteny was observed between the genomes of white clover and L. japonicus for all four targeted regions, but corresponding M. truncatula genomic regions were only identified for two BAC pairs. CONCLUSIONS: This study describes the first analysis of sub-genome structural conservation across selected genomic regions in white clover. Although the high levels of sequence conservation between the O and P' sub-genomes would complicate efforts for whole genome sequence assembly, the conserved microsynteny with model legume genomes, especially that of L. japonicus, will be highly valuable for the future of white clover genomics and molecular breeding.

Molecular characterisation and genetic mapping of candidate genes for qualitative disease resistance in perennial ryegrass (Lolium perenne L.).

BACKGROUND: Qualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site--leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database. RESULTS: Amplification of R gene sequences was performed using template EST data and information from orthologous candidate using a degenerate consensus PCR approach. A total of 102 unique partial R genes were cloned, sequenced and functionally annotated. Analysis of motif structure and R gene phylogeny demonstrated that Lolium R genes cluster with putative ortholoci, and evolved from common ancestral origins. Single nucleotide polymorphisms (SNPs) predicted through resequencing of amplicons from the parental genotypes of a genetic mapping family were validated, and 26 distinct R gene loci were assigned to multiple genetic maps. Clusters of largely non-related NBS-LRR genes were located at multiple distinct genomic locations and were commonly found in close proximity to previously mapped defence response (DR) genes. A comparative genomics analysis revealed the co-location of several candidate R genes with disease resistance quantitative trait loci (QTLs). CONCLUSION: This study is the most comprehensive analysis to date of qualitative disease resistance candidate genes in perennial ryegrass. SNPs identified within candidate genes provide a valuable resource for mapping in various ryegrass pair cross-derived populations and further germplasm analysis using association genetics. In parallel with the use of specific pathogen virulence races, such resources provide the means to identify gene-for-gene mechanisms for multiple host pathogen-interactions and ultimately to obtain durable field-based resistance.