Kathryn Michaela Guthridge

AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.)

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass.

High throughput whole rumen metagenome profiling using untargeted massively parallel sequencing

BackgroundVariation of microorganism communities in the rumen of cattle (Bos taurus) is of great interest because of possible links to economically or environmentally important traits, such as feed conversion efficiency or methane emission levels. The resolution of studies investigating this variation may be improved by utilizing untargeted massively parallel sequencing (MPS), that is, sequencing without targeted amplification of genes. The objective of this study was to develop a method which used MPS to generate “rumen metagenome profiles”, and to investigate if these profiles were repeatable among samples taken from the same cow. Given faecal samples are much easier to obtain than rumen fluid samples; we also investigated whether rumen metagenome profiles were predictive of faecal metagenome profiles.ResultsRather than focusing on individual organisms within the rumen, our method used MPS data to generate quantitative rumen micro-biome profiles, regardless of taxonomic classifications. The method requires a previously assembled reference metagenome. A number of such reference metagenomes were considered, including two rumen derived metagenomes, a human faecal microflora metagenome and a reference metagenome made up of publically available prokaryote sequences. Sequence reads from each test sample were aligned to these references. The “rumen metagenome profile” was generated from the number of the reads that aligned to each contig in the database. We used this method to test the hypothesis that rumen fluid microbial community profiles vary more between cows than within multiple samples from the same cow. Rumen fluid samples were taken from three cows, at three locations within the rumen. DNA from the samples was sequenced on the Illumina GAIIx. When the reads were aligned to a rumen metagenome reference, the rumen metagenome profiles were repeatable (P < 0.00001) by cow regardless of location of sampling rumen fluid. The repeatability was estimated at 9%, albeit with a high standard error, reflecting the small number of animals in the study. Finally, we compared rumen microbial profiles to faecal microbial profiles. Our hypothesis, that there would be a stronger correlation between faeces and rumen fluid from the same cow than between faeces and rumen fluid from different cows, was not supported by our data (with much greater significance of rumen versus faeces effect than animal effect in mixed linear model).ConclusionsWe have presented a simple and high throughput method of metagenome profiling to assess the similarity of whole metagenomes, and illustrated its use on two novel datasets. This method utilises widely used freeware. The method should be useful in the exploration and comparison of metagenomes.

Isolation and characterisation of a fructosyltransferase gene from perennial ryegrass (Lolium perenne)

Summary A fructosyltransferase ( LpFT1 ) cDNA and gene from perennial ryegrass was isolated and sequenced. Nucleotide sequence analysis revealed an open reading frame of 1947 bp encoding for a protein of 648 amino acids. LpFT1 is 69 % identical to barley 6-SFT, and contains plant fructosyltransferase functional domains. LpFT1 is present as a single copy gene and maps to the distal region of LG7 in perennial ryegrass. The expression pattern analysis of LpFT1 revealed transcript accumulation in young roots and shoots and in mature sheaths.

Development and implementation of molecular markers for forage crop improvement.

A number of plant species are used individually and in combination in pastures for the grazing of livestock. Temperate forages include grasses such as perennial ryegrass, tall fescue and phalaris and legumes such as white clover, red clover and alfalfa. Tropical forages include grasses such as buffelgrass and brachiaria and legumes such as cassia, siratro and stylo. Temperate and warm-season grass species are also important for turf and amenity purposes. In outbreeding forage species such as perennial ryegrass and white clover, natural and synthetic populations are genetically heterogeneous and show a high degree of developmental plasticity. Strategies for molecular marker development, genetic mapping, QTL detection and marker assisted selection must be designed in the context of these constraints. The current status of molecular marker technology with regard to genetic mapping, marker assisted selection, DNA profiling and molecular taxonomy is reviewed. The development of framework genetic maps based on highly informative co-dominant genetic markers (anchor RFLPs and SSRs) is described. Innovative strategies for implementation will be applied to these efficient marker systems in order to enhance forage breeding.

Isolation and characterisation of an invertase cDNA from perennial ryegrass (Lolium perenne)

An invertase (LpFT2) cDNA from perennial ryegrass was isolated and sequenced. Nucleotide sequence analysis revealed an ORF of 2016 bp encoding a protein of 671 amino acids. LpFT2 is 76% identical to sugarcane soluble acid invertase, and contains invertase and fructosyltransferase functional domains. LpFT2 is present as a single copy gene and maps to the distal region of LG6 in perennial ryegrass. The expression pattern analysis of LpFT2 revealed transcript accumulation in seedlings and in mature leaf sheaths. The LpFT2 recombinant protein expressed in yeast showed invertase and fructan exohydrolase-like activities with complete breakdown of sucrose, 1-kestose (DP3), 1,1-kestotetraose (DP4) and 1,1,1-kestopentaose (DP5) into glucose and fructose.

Use of a microsatellite-based pedigree in estimation of heritabilities for economic traits in Australian blue mussel, Mytilus galloprovincialis

This study aimed to estimate the heritabilities of three economically important traits (total weight, shell shape and meat yield) in Australian blue mussels. The estimates were derived using a pedigree reconstructed from a suite of both published and newly developed microsatellite markers. A total of 135 microsatellite loci were tested, of which 10 loci produced consistent PCR amplification and reliable results across all samples (74 full-sibling families including 74 pairs of parents and 2536 offspring). Lack of polymorphism at the non-repetitive region of the adhesive protein gene confirmed that the broodstock were derived from a single species. A total of 1538 progenies (62.5%) could be assigned to single parent pairs, and the remainder were assigned to two families or more, so were discarded from further analysis. Heritabilities for total weight, shell shape and meat yield were low (0.051 ± 0.027, 0.085 ± 0.038 and 0.049 ± 0.028, respectively) but reflected large environmental variation rather than limited genetic variation, suggesting a family-based breeding programme could improve these traits. The genetic correlation between weight and meat yield, expressed as percentage of total mussel which was not shell, was negative, while the genetic correlation between meat yield and shell shape was weakly positive.

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

BackgroundPerennial 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.ResultsA 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 β-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.ConclusionsUnique 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.

Sequence Analysis of SSR-Flanking Regions Identifies Genome Affinities between Pasture Grass Fungal Endophyte Taxa

Fungal species of the Neotyphodium and Epichloë genera are endophytes of pasture grasses showing complex differences of life-cycle and genetic architecture. Simple sequence repeat (SSR) markers have been developed from endophyte-derived expressed sequence tag (EST) collections. Although SSR array size polymorphisms are appropriate for phenetic analysis to distinguish between taxa, the capacity to resolve phylogenetic relationships is limited by both homoplasy and heteroploidy effects. In contrast, nonrepetitive sequence regions that flank SSRs have been effectively implemented in this study to demonstrate a common evolutionary origin of grass fungal endophytes. Consistent patterns of relationships between specific taxa were apparent across multiple target loci, confirming previous studies of genome evolution based on variation of individual genes. Evidence was obtained for the definition of endophyte taxa not only through genomic affinities but also by relative gene content. Results were compatible with the current view that some asexual Neotyphodium species arose following interspecific hybridisation between sexual Epichloë ancestors. Phylogenetic analysis of SSR-flanking regions, in combination with the results of previous studies with other EST-derived SSR markers, further permitted characterisation of Neotyphodium isolates that could not be assigned to known taxa on the basis of morphological characteristics.

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

BackgroundTall 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.ResultsMultiple copies of the translation elongation factor 1-a (tefA) and β-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.ConclusionsComplex 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.

Characterisation of novel perennial ryegrass host–Neotyphodium endophyte associations

Abstract. The temperate pasture grass Lolium perenne L. is commonly found in symbiotic association with the asexual fungal endophyte Neotyphodium lolii. Levels of endophyte colonisation and alkaloid content were evaluated in associations formed by plant genotypes from cv. Bronsyn with the standard endophyte (SE) and five distinct commercial endophyte strains. Bronsyn–SE produced all of the measured alkaloids (lolitrem B, peramine, and ergovaline). Bronsyn–AR1 produced only peramine, while Bronsyn–AR37 produced none of the tested alkaloids. Bronsyn–NEA2, Bronsyn–NEA3, and Bronsyn–NEA6 produced both ergovaline and peramine. Both endophyte strain and host genotype exerted significant effects on alkaloid production. Analysis of endophyte colonisation using qPCR revealed differences between each association. With the exception of Bronsyn–AR1 and Bronsyn–NEA3, host genotype also significantly affected colonisation levels. Phenotypic performance of each association was also assessed, based on measurement of morphological traits under glasshouse conditions in hydroponic culture. Significant variation due to different endophyte and host genotypes was observed. Collectively, these studies confirm that differences in both endophyte and host genotypes contribute to host–endophyte performance in a complex interactive manner.