• EST-derived SSR markers used as anchor loci for the construction of a consensus linkage map in ryegrass (Lolium spp.)

      Studer, Bruno; Kolliker, Roland; Muylle, Hilde; Asp, Torben; Frei, Ursula; Roldan-Ruiz, Isabel; Barre, Philippe; Tomaszewski, Celine; Meally, Helena; Barth, Susanne; et al. (Biomed Central, 16/08/2010)
      Background: Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm. Results: A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2. Conclusions: The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.
    • Exploring the potential of grass feedstock from marginal land in Ireland: Does marginal mean lower yield?

      Meehan, Peter; Burke, Brendan; Doyle, Deirdre; Barth, Susanne; Finnan, John; European Union; KBBE-2011-5-289461 (Elsevier, 2017-11-02)
      The production of biomass feedstock from marginal land has attracted much attention as a means of avoiding conflict between the production of food and fuel. Yield potentials from marginal lands have generally not been quantified although it is generally assumed that lower biomass yields can be expected from marginal lands. A three year study was conducted in Ireland in order to determine if grass yields of perennial rhizomatous grasses (cocksfoot, tall fescue, reed canary grass, festulolium) for anaerobic digestion from three marginal land sites (very wet site, very dry site, site prone to flooding) could match yields from better soils. Randomised complete block designs were established on each site in 2012 with two varieties of each grass species as treatments. Three grass harvests were taken from each site in 2013 and in 2014. There was no significant difference between yields from the control site and those from the very dry site and the site prone to flooding. Biomass yields from the very wet site were 85% of those from the control site. Highest yields were obtained from festulolium which were significantly higher than yields from perennial ryegrass. An energy analysis showed that maximising the production of grass from low lying mineral marginal grassland in Ireland could provide enough energy to meet the energy requirements of both the private car fleet and the heavy goods vehicle fleet while avoiding conflict with food production which could be concentrated on conventional land.
    • Genomic prediction of crown rust resistance in Lolium perenne

      Arojju, Sai Krishna; Conaghan, Patrick; Barth, Susanne; Milbourne, Dan; Casler, Michael D.; Hodkinson, Trevor R.; Michel, Thibauld; Byrne, Stephen; Department of Agriculture Food and the Marine; European Union; et al. (Springer Science and Business Media LLC, 2018-05-29)
      Background Genomic selection (GS) can accelerate genetic gains in breeding programmes by reducing the time it takes to complete a cycle of selection. Puccinia coronata f. sp lolli (crown rust) is one of the most widespread diseases of perennial ryegrass and can lead to reductions in yield, persistency and nutritional value. Here, we used a large perennial ryegrass population to assess the accuracy of using genome wide markers to predict crown rust resistance and to investigate the factors affecting predictive ability. Results Using these data, predictive ability for crown rust resistance in the complete population reached a maximum of 0.52. Much of the predictive ability resulted from the ability of markers to capture genetic relationships among families within the training set, and reducing the marker density had little impact on predictive ability. Using permutation based variable importance measure and genome wide association studies (GWAS) to identify and rank markers enabled the identification of a small subset of SNPs that could achieve predictive abilities close to those achieved using the complete marker set. Conclusion Using a GWAS to identify and rank markers enabled a small panel of markers to be identified that could achieve higher predictive ability than the same number of randomly selected markers, and predictive abilities close to those achieved with the entire marker set. This was particularly evident in a sub-population characterised by having on-average higher genome-wide linkage disequilibirum (LD). Higher predictive abilities with selected markers over random markers suggests they are in LD with QTL. Accuracy due to genetic relationships will decay rapidly over generations whereas accuracy due to LD will persist, which is advantageous for practical breeding applications.
    • Genomic prediction of crown rust resistance in Lolium perenne

      Arojju, Sai Krishna; Conaghan, Patrick; Barth, Susanne; Milbourne, Dan; Casler, M.D.; Hodkinson, Trevor R; Michel, Thibauld; Byrne, Stephen L.; Department of Agriculture, Food and the Marine, Ireland; Marie Sklodowska-Curie; et al. (Biomed Central, 29/05/2018)
      Background Genomic selection (GS) can accelerate genetic gains in breeding programmes by reducing the time it takes to complete a cycle of selection. Puccinia coronata f. sp lolli (crown rust) is one of the most widespread diseases of perennial ryegrass and can lead to reductions in yield, persistency and nutritional value. Here, we used a large perennial ryegrass population to assess the accuracy of using genome wide markers to predict crown rust resistance and to investigate the factors affecting predictive ability. Results Using these data, predictive ability for crown rust resistance in the complete population reached a maximum of 0.52. Much of the predictive ability resulted from the ability of markers to capture genetic relationships among families within the training set, and reducing the marker density had little impact on predictive ability. Using permutation based variable importance measure and genome wide association studies (GWAS) to identify and rank markers enabled the identification of a small subset of SNPs that could achieve predictive abilities close to those achieved using the complete marker set. Conclusion Using a GWAS to identify and rank markers enabled a small panel of markers to be identified that could achieve higher predictive ability than the same number of randomly selected markers, and predictive abilities close to those achieved with the entire marker set. This was particularly evident in a sub-population characterised by having on-average higher genome-wide linkage disequilibirum (LD). Higher predictive abilities with selected markers over random markers suggests they are in LD with QTL. Accuracy due to genetic relationships will decay rapidly over generations whereas accuracy due to LD will persist, which is advantageous for practical breeding applications.
    • Genotyping by Sequencing and Plastome Analysis Finds High Genetic Variability and Geographical Structure in Dactylis glomerata L. in Northwest Europe Despite Lack of Ploidy Variation

      Hodkinson, Trevor R.; Perdereau, Aude; Klaas, Manfred; Cormican, Paul; Barth, Susanne; European Union; 289461 (MDPI AG, 2019-06-28)
      Large collections of the forage and bioenergy grass Dactylis glomerata were made in northwest (NW) Europe along east to west and north to south clines for genetic resource conservation and to inform breeding programmes of genetic diversity, genepools, and ploidy. Leaves were sampled for genetic analysis and seed and rhizome for ex-situ conservation. Genotyping by sequencing (GBS) was used to assay nuclear DNA diversity and plastome single nucleotide polymorphism (SNP) discovery was undertaken using a long-read PCR and MiSeq approach. Nuclear and plastid SNPs were analysed by principal component analysis (PCA) to compare genotypes. Flow cytometry revealed that all samples were tetraploid, but some genome size variation was recorded. GBS detected an average of approximately 10,000 to 15,000 SNPs per country sampled. The highest average number of private SNPs was recorded in Poland (median ca. 2000). Plastid DNA variation was also high (1466 SNPs, 17 SNPs/kbp). GBS data, and to a lesser extent plastome data, also show that genetic variation is structured geographically in NW Europe with loose clustering matching the country of plant origin. The results reveal extensive genetic diversity and genetic structuring in this versatile allogamous species despite lack of ploidy variation and high levels of human mediated geneflow via planting.
    • Genotyping by Sequencing and Plastome Analysis Finds High Genetic Variability and Geographical Structure in Dactylis glomerata L. in Northwest Europe Despite Lack of Ploidy Variation

      Hodkinson, Trevor R.; Perdereau, Aude; Klaas, Manfred; Cormican, Paul; Barth, Susanne; EU; 289461 (MDPI AG, 2019-06-28)
      Large collections of the forage and bioenergy grass Dactylis glomerata were made in northwest (NW) Europe along east to west and north to south clines for genetic resource conservation and to inform breeding programmes of genetic diversity, genepools, and ploidy. Leaves were sampled for genetic analysis and seed and rhizome for ex-situ conservation. Genotyping by sequencing (GBS) was used to assay nuclear DNA diversity and plastome single nucleotide polymorphism (SNP) discovery was undertaken using a long-read PCR and MiSeq approach. Nuclear and plastid SNPs were analysed by principal component analysis (PCA) to compare genotypes. Flow cytometry revealed that all samples were tetraploid, but some genome size variation was recorded. GBS detected an average of approximately 10,000 to 15,000 SNPs per country sampled. The highest average number of private SNPs was recorded in Poland (median ca. 2000). Plastid DNA variation was also high (1466 SNPs, 17 SNPs/kbp). GBS data, and to a lesser extent plastome data, also show that genetic variation is structured geographically in NW Europe with loose clustering matching the country of plant origin. The results reveal extensive genetic diversity and genetic structuring in this versatile allogamous species despite lack of ploidy variation and high levels of human mediated geneflow via planting.
    • The health status of Irish honeybee colonies in 2006

      Coffey, M. F.; Barth, Susanne; Hayes, K.; Breen, J.; European Agricultural Guidance and Guarantee Fund; Department of Agriculture, Food and the Marine, Ireland (Teagasc (Agriculture and Food Development Authority), Ireland, 2013)
      This study assessed the health status of Irish honeybee colonies and provides a snapshot of the incidence of a number of important colony parasites/pathogens including: the mite Varroa destructor; three associated viruses (deformed wing virus (DWV), acute bee paralysis virus (ABPV) and Kashmir virus (KBV)); the tracheal mite Acarapis woodi; the microsporidian Nosema spp., and the insect Braula coeca. During June/July 2006, 135 samples of adult bees were collected from productive colonies throughout Ireland and standard techniques were used to determine the presence and absence of the parasites and pathogens. Varroa destructor was positively identified in 72.6% of the samples and was widely distributed. Although the samples were analysed for three viruses, DWV, ABPV and KBV, only DWV was detected (frequency = 12.5%). Acarapis woodi and Nosema spp. occurred in approximately 11% and 22% of the samples, respectively, while B. coeca, a wingless dipteran that was once common in Irish honeybee colonies, was very rare (3.7%). Samples where all the pathogens/parasites were jointly absent were statistically under-represented in Leinster and DWV was statistically over-represented in Munster. In Ulster, there was over-representation of the categories where all parasites/pathogens were jointly absent and for A. woodi, and underrepresentation of V. destructor.
    • A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover

      Nagy, Istvan; Barth, Susanne; Mehenni-Ciz, Jeanne; Abberton, Michael T; Milbourne, Dan; Department of Agriculture, Food and the Marine, Ireland; RSF 07–566 (Biomed Central, 13/02/2013)
      Background: White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. Results: We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. Conclusions: In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a “core set” of ancestrally derived homeolog specific variants in white clover.
    • An Immortalized Genetic Mapping Population for Perennial Ryegrass: A Resource for Phenotyping and Complex Trait Mapping

      Velmurugan, Janaki; Milbourne, Dan; Connolly, Vincent; Heslop-Harrison, J. S.; Anhalt, Ulrike C. M.; Lynch, M. B.; Barth, Susanne; Teagasc Walsh Fellowship Programme (Frontiers, 2018-05-31)
      To address the lack of a truly portable, universal reference mapping population for perennial ryegrass, we have been developing a recombinant inbred line (RIL) mapping population of perennial ryegrass derived via single seed descent from a well-characterized F2 mapping population based on genetically distinct inbred parents in which the natural self-incompatibility (SI) system of perennial ryegrass has been overcome. We examined whether it is possible to create a genotyping by sequencing (GBS) based genetic linkage map in a small population of the F6 generation of this population. We used 41 F6 genotypes for GBS with PstI/MspI-based libraries. We successfully developed a genetic linkage map comprising 6074 SNP markers, placing a further 22080 presence and absence variation (PAV) markers on the map. We examined the resulting genetic map for general and RIL specific features. Overall segregation distortion levels were similar to those experienced in the F2 generation, but segregation distortion was reduced on linkage group 6 and increased on linkage group 7. Residual heterozygosity in the F6 generation was observed at a level of 5.4%. There was a high proportion of chromosomes (30%) exhibiting the intact haplotype of the original inbred parents of the F1 genotype from which the population is derived, pointing to a tendency for chromosomes to assort without recombining. This could affect the applicability of these lines and might make them more suitable for situations where repressed recombination is an advantage. Inter- and intra-chromosomal linkage disequilibrium (LD) analysis suggested that the map order was robust. We conclude that this RIL population, and subsequent F7 and F8 generations will be useful for genetic analysis and phenotyping of agronomic and biological important traits in perennial ryegrass.
    • An Immortalized Genetic Mapping Population for Perennial Ryegrass: A Resource for Phenotyping and Complex Trait Mapping

      Velmurugan, Janaki; Milbourne, Dan; Connolly, Vincent; Heslop-Harrison, J. S.; Anhalt, Ulrike C. M.; Lynch, M. B.; Barth, Susanne; Teagasc Walsh Fellowship Programme; Teagasc (Frontiers, 2018-05-31)
      To address the lack of a truly portable, universal reference mapping population for perennial ryegrass, we have been developing a recombinant inbred line (RIL) mapping population of perennial ryegrass derived via single seed descent from a well-characterized F2 mapping population based on genetically distinct inbred parents in which the natural self-incompatibility (SI) system of perennial ryegrass has been overcome. We examined whether it is possible to create a genotyping by sequencing (GBS) based genetic linkage map in a small population of the F6 generation of this population. We used 41 F6 genotypes for GBS with PstI/MspI-based libraries. We successfully developed a genetic linkage map comprising 6074 SNP markers, placing a further 22080 presence and absence variation (PAV) markers on the map. We examined the resulting genetic map for general and RIL specific features. Overall segregation distortion levels were similar to those experienced in the F2 generation, but segregation distortion was reduced on linkage group 6 and increased on linkage group 7. Residual heterozygosity in the F6 generation was observed at a level of 5.4%. There was a high proportion of chromosomes (30%) exhibiting the intact haplotype of the original inbred parents of the F1 genotype from which the population is derived, pointing to a tendency for chromosomes to assort without recombining. This could affect the applicability of these lines and might make them more suitable for situations where repressed recombination is an advantage. Inter- and intra-chromosomal linkage disequilibrium (LD) analysis suggested that the map order was robust. We conclude that this RIL population, and subsequent F7 and F8 generations will be useful for genetic analysis and phenotyping of agronomic and biological important traits in perennial ryegrass.
    • Markers associated with heading and aftermath heading in perennial ryegrass full-sib families

      Arojju, Sai Krishna; Barth, Susanne; Milbourne, Dan; Conaghan, Patrick; Velmurugan, Janaki; Hodkinson, Trevor R; Byrne, Stephen L.; Department of Agriculture, Food and the Marine, Ireland; Teagasc Walsh Fellowship Programme; EU Marie-Sklodowska-Curie Fellowship; et al. (Biomed Central, 16/07/2016)
      Background Heading and aftermath heading are important traits in perennial ryegrass because they impact forage quality. So far, genome-wide association analyses in this major forage species have only identified a small number of genetic variants associated with heading date that overall explained little of the variation. Some possible reasons include rare alleles with large phenotypic affects, allelic heterogeneity, or insufficient marker density. We established a genome-wide association panel with multiple genotypes from multiple full-sib families. This ensured alleles were present at the frequency needed to have sufficient statistical power to identify associations. We genotyped the panel via partial genome sequencing and performed genome-wide association analyses with multi-year phenotype data collected for heading date, and aftermath heading. Results Genome wide association using a mixed linear model failed to identify any variants significantly associated with heading date or aftermath heading. Our failure to identify associations for these traits is likely due to the extremely low linkage disequilibrium we observed in this population. However, using single marker analysis within each full-sib family we could identify markers and genomic regions associated with heading and aftermath heading. Using the ryegrass genome we identified putative orthologs of key heading genes, some of which were located in regions of marker-trait associations. Conclusion Given the very low levels of LD, genome wide association studies in perennial ryegrass populations are going to require very high SNP densities. Single marker analysis within full-sibs enabled us to identify significant marker-trait associations. One of these markers anchored proximal to a putative ortholog of TFL1, homologues of which have been shown to play a key role in continuous heading of some members of the rose family, Rosaceae.
    • A Novel Multivariate Approach to Phenotyping and Association Mapping of Multi-Locus Gametophytic Self-Incompatibility Reveals S, Z, and Other Loci in a Perennial Ryegrass (Poaceae) Population

      Thorogood, Daniel; Yates, Steven; Manzanares, Chloé; Skot, Leif; Hegarty, Matthew; Blackmore, Tina; Barth, Susanne; Studer, Bruno; Biotechnology and Biological Sciences Research Council; Swiss National Science Foundation; et al. (Frontiers, 2017-08-02)
      Self-incompatibility (SI) is a mechanism that many flowering plants employ to prevent fertilisation by self- and self-like pollen ensuring heterozygosity and hybrid vigour. Although a number of single locus mechanisms have been characterised in detail, no multi-locus systems have been fully elucidated. Historically, examples of the genetic analysis of multi-locus SI, to make analysis tractable, are either made on the progeny of bi-parental crosses, where the number of alleles at each locus is restricted, or on crosses prepared in such a way that only one of the SI loci segregates. Perennial ryegrass (Lolium perenne L.) possesses a well-documented two locus (S and Z) gametophytic incompatibility system. A more universal, realistic proof of principle study was conducted in a perennial ryegrass population in which allelic and non-allelic diversity was not artificially restricted. A complex pattern of pollinations from a diallel cross was revealed which could not possibly be interpreted easily per se, even with an already established genetic model. Instead, pollination scores were distilled into principal component scores described as Compatibility Components (CC1-CC3). These were then subjected to a conventional genome-wide association analysis. CC1 associated with markers on linkage groups (LGs) 1, 2, 3, and 6, CC2 exclusively with markers in a genomic region on LG 2, and CC3 with markers on LG 1. BLAST alignment with the Brachypodium physical map revealed highly significantly associated markers with peak associations with genes adjacent and four genes away from the chromosomal locations of candidate SI genes, S- and Z-DUF247, respectively. Further significant associations were found in a Brachypodium distachyon chromosome 3 region, having shared synteny with Lolium LG 1, suggesting further SI loci linked to S or extensive micro-re-arrangement of the genome between B. distachyon and L. perenne. Significant associations with gene sequences aligning with marker sequences on Lolium LGs 3 and 6 were also identified. We therefore demonstrate the power of a novel association genetics approach to identify the genes controlling multi-locus gametophytic SI systems and to identify novel loci potentially involved in already established SI systems.
    • A Novel Multivariate Approach to Phenotyping and Association Mapping of Multi-Locus Gametophytic Self-Incompatibility Reveals S, Z, and Other Loci in a Perennial Ryegrass (Poaceae) Population

      Thorogood, Daniel; Yates, Steven; Manzanares, Chloé; Skot, Leif; Hegarty, Matthew; Blackmore, Tina; Barth, Susanne; Studer, Bruno; Biotechnology and Biological Sciences Research Council; Swiss National Science Foundation; et al. (Frontiers, 2017-08-02)
      Self-incompatibility (SI) is a mechanism that many flowering plants employ to prevent fertilisation by self- and self-like pollen ensuring heterozygosity and hybrid vigour. Although a number of single locus mechanisms have been characterised in detail, no multi-locus systems have been fully elucidated. Historically, examples of the genetic analysis of multi-locus SI, to make analysis tractable, are either made on the progeny of bi-parental crosses, where the number of alleles at each locus is restricted, or on crosses prepared in such a way that only one of the SI loci segregates. Perennial ryegrass (Lolium perenne L.) possesses a well-documented two locus (S and Z) gametophytic incompatibility system. A more universal, realistic proof of principle study was conducted in a perennial ryegrass population in which allelic and non-allelic diversity was not artificially restricted. A complex pattern of pollinations from a diallel cross was revealed which could not possibly be interpreted easily per se, even with an already established genetic model. Instead, pollination scores were distilled into principal component scores described as Compatibility Components (CC1-CC3). These were then subjected to a conventional genome-wide association analysis. CC1 associated with markers on linkage groups (LGs) 1, 2, 3, and 6, CC2 exclusively with markers in a genomic region on LG 2, and CC3 with markers on LG 1. BLAST alignment with the Brachypodium physical map revealed highly significantly associated markers with peak associations with genes adjacent and four genes away from the chromosomal locations of candidate SI genes, S- and Z-DUF247, respectively. Further significant associations were found in a Brachypodium distachyon chromosome 3 region, having shared synteny with Lolium LG 1, suggesting further SI loci linked to S or extensive micro-re-arrangement of the genome between B. distachyon and L. perenne. Significant associations with gene sequences aligning with marker sequences on Lolium LGs 3 and 6 were also identified. We therefore demonstrate the power of a novel association genetics approach to identify the genes controlling multi-locus gametophytic SI systems and to identify novel loci potentially involved in already established SI systems.
    • An Optimized Chloroplast DNA Extraction Protocol for Grasses (Poaceae) Proves Suitable for Whole Plastid Genome Sequencing and SNP Detection

      Diekmann, Kerstin; Hodkinson, Trevor R; Fricke, Evelyn; Barth, Susanne; Teagasc Walsh Fellowship Programme (PLoS, 30/07/2008)
      Background Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering) and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses. Methodology/Principal Findings We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge. Conclusions/Significance The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus×giganteus, Panicoideae). The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank.
    • Plastid genome sequencing reveals biogeographical structure and extensive population genetic variation in wild populations of Phalaris arundinacea L. in north‐western Europe

      Perdereau, Aude; Klass, Manfred; Barth, Susanne; Hodkinson, Trevor R.; European Union; 289461 (Wiley, 2016-03-31)
      New and comprehensive collections of the perennial rhizomatous reed canary grass (Phalaris arundinacea) were made in NW Europe along north‐to‐south and east‐to‐west clines from Denmark, Germany, Ireland, Poland, Sweden and the United Kingdom. Rhizome, seed and leaf samples were taken for analysis and genetic resource conservation. A subsample covering the geographical range was characterized using plastid genome sequencing and SNP discovery generated using a long‐read PCR and MiSeq sequencing approach. Samples were also subject to flow cytometry and all found to be tetraploid. New sequences were assembled against a Lolium perenne (perennial ryegrass) reference genome, and an average of approximately 60% of each genome was aligned (81 064 bp). Genetic variation was high among the 48 sequenced genotypes with a total of 1793 SNPs, equating to 23 SNPs per kbp. SNPs were subject to principal coordinate and Structure analyses to detect population genetic groupings and to examine phylogeographical pattern. Results indicate substantial genetic variation and population genetic structuring of this allogamous species at a broad geographical scale in NW Europe with plastid genetic diversity organized more across an east‐to‐west than a north‐to‐south cline.
    • Quantitative trait loci associated with different polar metabolites in perennial ryegrass - providing scope for breeding towards increasing certain polar metabolites

      Foito, Alexandre; Hackett, Christine A; Stewart, Derek; Velmurugan, Janaki; Milbourne, Dan; Byrne, Stephen L.; Barth, Susanne; Department of Agriculture, Food and the Marine, Ireland; RSF 06–346 (Biomed Central, 10/10/2017)
      Background Recent advances in the mapping of biochemical traits have been reported in Lolium perenne. Although the mapped traits, including individual sugars and fatty acids, contribute greatly towards ruminant productivity, organic acids and amino acids have been largely understudied despite their influence on the ruminal microbiome. Results In this study, we used a targeted gas-chromatography mass spectrometry (GC-MS) approach to profile the levels of 25 polar metabolites from different classes (sugars, amino acids, phenolic acids, organic acids and other nitrogen-containing compounds) present in a L. perenne F2 population consisting of 325 individuals. A quantitative trait (QTL) mapping approach was applied and successfully identified QTLs regulating seven of those polar metabolites (L-serine, L-leucine, glucose, fructose, myo-inositol, citric acid and 2, 3-hydroxypropanoic acid).Two QTL mapping approaches were carried out using SNP markers on about half of the population only and an imputation approach using SNP and DArT markers on the entire population. The imputation approach confirmed the four QTLs found in the SNP-only analysis and identified a further seven QTLs. Conclusions These results highlight the potential of utilising molecular assisted breeding in perennial ryegrass to modulate a range of biochemical quality traits with downstream effects in livestock productivity and ruminal digestion.
    • Transcriptome sequencing of Festulolium accessions under salt stress

      Teshome, A.; Byrne, Stephen L.; Didion, T.; De Vega, J.; Jensen, C. S; Klaas, M.; Barth, Susanne; European Union; Marie Sklodowska-Curie Actions; FP7-KBBE-2011-5-289461; et al. (Biomed Central, 2019-05-31)
      Objectives The objective of this study was to establish transcriptome assemblies of Festulolium hybrids under salt stress, and identify genes regulated across the hybrids in response to salt stress. The development of transcriptome assemblies for Festulolium hybrids and cataloguing of genes regulated under salt stress will facilitate further downstream studies. Results Plants were grown at three salt concentrations (0.5%, 1% and 1.5%) and phenotypic and transcriptomic data was collected. Salt stress was confirmed by progressive loss of green leaves as salt concentration increased from 0 to 1.5%. We generated de-novo transcriptome assemblies for two Festulolium pabulare festucoid genotypes, for a single Festulolium braunii genotype, and a single F. pabulare loloid genotype. We also identified 1555 transcripts that were up regulated and 1264 transcripts that were down regulated in response to salt stress in the Festulolium hybrids. Some of the identified transcripts showed significant sequence similarity with genes known to be regulated during salt and other abiotic stresses.
    • Transcriptomic response of maize primary roots to low temperatures at seedling emergence

      Di Fenza, Mauro; Hogg, Bridget; Grant, Jim; Barth, Susanne; Department of Agriculture, Food and the Marine; RSF 07 501 (PeerJ, 2017-01-05)
      Background Maize (Zea mays) is a C4 tropical cereal and its adaptation to temperate climates can be problematic due to low soil temperatures at early stages of establishment. Methods In the current study we have firstly investigated the physiological response of twelve maize varieties, from a chilling condition adapted gene pool, to sub-optimal growth temperature during seedling emergence. To identify transcriptomic markers of cold tolerance in already adapted maize genotypes, temperature conditions were set below the optimal growth range in both control and low temperature groups. The conditions were as follows; control (18 °C for 16 h and 12 °C for 8 h) and low temperature (12 °C for 16 h and 6 °C for 8 h). Four genotypes were identified from the condition adapted gene pool with significant contrasting chilling tolerance. Results Picker and PR39B29 were the more cold-tolerant lines and Fergus and Codisco were the less cold-tolerant lines. These four varieties were subjected to microarray analysis to identify differentially expressed genes under chilling conditions. Exposure to low temperature during establishment in the maize varieties Picker, PR39B29, Fergus and Codisco, was reflected at the transcriptomic level in the varieties Picker and PR39B29. No significant changes in expression were observed in Fergus and Codisco following chilling stress. A total number of 64 genes were differentially expressed in the two chilling tolerant varieties. These two varieties exhibited contrasting transcriptomic profiles, in which only four genes overlapped. Discussion We observed that maize varieties possessing an enhanced root growth ratio under low temperature were more tolerant, which could be an early and inexpensive measure for germplasm screening under controlled conditions. We have identified novel cold inducible genes in an already adapted maize breeding gene pool. This illustrates that further varietal selection for enhanced chilling tolerance is possible in an already preselected gene pool.
    • Transcriptomic response of maize primary roots to low temperatures at seedling emergence

      Di Fenza, Mauro; Hogg, Bridget; Grant, Jim; Barth, Susanne; Irish Department of Agriculture, Food and the Marine; RSF 07 501 (PeerJ, 2017-01-05)
      Background. Maize (Zea mays) is a C4 tropical cereal and its adaptation to temperate climates can be problematic due to low soil temperatures at early stages of establishment. Methods. In the current study we have firstly investigated the physiological response of twelve maize varieties, from a chilling condition adapted gene pool, to sub-optimal growth temperature during seedling emergence. To identify transcriptomic markers of cold tolerance in already adapted maize genotypes, temperature conditions were set below the optimal growth range in both control and low temperature groups. The conditions were as follows; control (18 ◦C for 16 h and 12 ◦C for 8 h) and low temperature (12 ◦C for 16 h and 6 ◦C for 8 h). Four genotypes were identified from the condition adapted gene pool with significant contrasting chilling tolerance. Results. Picker and PR39B29 were the more cold-tolerant lines and Fergus and Codisco were the less cold-tolerant lines. These four varieties were subjected to microarray analysis to identify differentially expressed genes under chilling conditions. Exposure to low temperature during establishment in the maize varieties Picker, PR39B29, Fergus and Codisco, was reflected at the transcriptomic level in the varieties Picker and PR39B29. No significant changes in expression were observed in Fergus and Codisco following chilling stress. A total number of 64 genes were differentially expressed in the two chilling tolerant varieties. These two varieties exhibited contrasting transcriptomic profiles, in which only four genes overlapped. Discussion. We observed that maize varieties possessing an enhanced root growth ratio under low temperature were more tolerant, which could be an early and inexpensive measure for germplasm screening under controlled conditions. We have identified novel cold inducible genes in an already adapted maize breeding gene pool. This illustrates that further varietal selection for enhanced chilling tolerance is possible in an already preselected gene pool.
    • Using variable importance measures to identify a small set of SNPs to predict heading date in perennial ryegrass.

      Byrne, Stephen; Conaghan, Patrick; Barth, Susanne; Arojju, Sai Krishna; Casler, Michael; Michel, Thibauld; Velmurugan, Janaki; Milbourne, Dan; E.U. Marie Skłodowska-Curie Fellowship; Teagasc Walsh Fellowship Programme; et al. (Nature, 2017-06-15)
      Prior knowledge on heading date enables the selection of parents of synthetic cultivars that are well matched with respect to time of heading, which is essential to ensure plants put together will cross pollinate. Heading date of individual plants can be determined via direct phenotyping, which has a time and labour cost. It can also be inferred from family means, although the spread in days to heading within families demands roguing in first generation synthetics. Another option is to predict heading date from molecular markers. In this study we used a large training population consisting of individual plants to develop equations to predict heading date from marker genotypes. Using permutation-based variable selection measures we reduced the marker set from 217,563 to 50 without impacting the predictive ability. Opportunities exist to develop a cheap assay to sequence a small number of regions in linkage disequilibrium with heading date QTL in thousands of samples. Simultaneous use of these markers in non-linkage based marker-assisted selection approaches, such as paternity testing, should enhance the utility of such an approach.