... 3 (top) and examined with a binocular microscope. The box was constructed from a Petri dish p... more ... 3 (top) and examined with a binocular microscope. The box was constructed from a Petri dish painted matt black except for a 2-0 cm circle on the underside which coincided with the normal sub-stage light source on the microscope. ...
A pearl millet inbred germplasm association panel (PMiGAP) comprising 250 inbred lines, represent... more A pearl millet inbred germplasm association panel (PMiGAP) comprising 250 inbred lines, representative of cultivated germplasm from Africa and Asia, elite improved open-pollinated cultivars, hybrid parental inbreds and inbred mapping population parents, was recently established. This study presents the first report of genetic diversity in PMiGAP and its exploitation for association mapping of drought tolerance traits. For diversity and genetic structure analysis, PMiGAP was genotyped with 37 SSR and CISP markers representing all seven linkage groups. For association analysis, it was phenotyped for yield and yield components and morpho-physiological traits under both well-watered and drought conditions, and genotyped with SNPs and InDels from seventeen genes underlying a major validated drought tolerance (DT) QTL. The average gene diversity in PMiGAP was 0.54. The STRUCTURE analysis revealed six subpopulations within PMiGAP. Significant associations were obtained for 22 SNPs and 3 InDels from 13 genes under different treatments. Seven SNPs associations from 5 genes were common under irrigated and one of the drought stress treatments. Most significantly, an important SNP in putative acetyl CoA carboxylase gene showed constitutive association with grain yield, grain harvest index and panicle yield under all treatments. An InDel in putative chlorophyll a/b binding protein gene was significantly associated with both stay-green and grain yield traits under drought stress. This can be used as a functional marker for selecting high yielding genotypes with 'stay green' phenotype under drought stress. The present study identified useful marker-trait associations of important agronomics traits under irrigated and drought stress conditions with genes underlying a major validated DT-QTL in pearl millet. Results suggest that PMiGAP is a useful panel for
A promoter tagging program in the legume Lotus japonicus was initiated to identify plant genes in... more A promoter tagging program in the legume Lotus japonicus was initiated to identify plant genes involved in the nitrogen-fixing symbiosis between legumes and rhizobia. Seven transformed plant lines expressing the promoterless reporter gene uidA (β-glucuronidase; GUS) specifically in roots and/or nodules were identified. Four of these expressed GUS in the roots only after inoculation with nodule-forming Mesorhizobium loti. In one line (T90), GUS activity was found in the root epidermis, including root hairs. During seedling growth, GUS expression gradually became focused in developing nodules and disappeared from root tissue. No GUS activity was detected when a non-nodulating mutant of M. loti was used to inoculate the plants. The T-DNA insertion in this plant line was located 1.3 kb upstream of a putative coding sequence with strong homology to calcium-binding proteins. Four motifs were identified, which were very similar to the “EF hands” in calmodulin-related proteins, each binding...
This paper describes research on interactions between grassland plant species and soil microorgan... more This paper describes research on interactions between grassland plant species and soil microorganisms. Both parasitic and symbiotic microorganisms modify nutrient transfers between plants and soil. Experiments are described in which nematode infection of clover increased nitrogen transfer to companion ryegrass plants. Infection of clover enhanced activity of soil bacterial and fungal communities. Legume genotypes differing only in responses to symbionts (rhizobium and arbuscular mycorrhizal fungi) and pathogens are being developed for studies of gene expression during establishing and functional symbioses. Such plants can be used in experiments as defined perturbations that will provide information on the interactions and functions of symbiotic and pathogenic microorganisms. Such studies, related to field observations, may have value for defining biological attributes of sustainable grassland soil systems.
Perennial ryegrass (Lolium perenne L.) is the most important forage crop in temperate livestock a... more Perennial ryegrass (Lolium perenne L.) is the most important forage crop in temperate livestock agriculture. Its nutritional quality has significant impact on the quality of meat and milk for human consumption. Evidence suggests that higher energy content in forage can assist in reducing greenhouse gas emissions from ruminants. Increasing the fatty acid content (especially a-linolenic acid, an omega-3 fatty acid) may thus contribute to better forage, but little is known about the genetic basis of variation for this trait. To this end, quantitative trait loci (QTLs) were identified associated with major fatty acid content in perennial ryegrass using a population derived from a cross between the heterozygous and outbreeding high-sugar grass variety AberMagic and an older variety, Aurora. A genetic map with 434 restriction-associated DNA (RAD) and SSR markers was generated. Significant QTLs for the content of palmitic (C16:0) on linkage groups (LGs) 2 and 7; stearic (C18:0) on LGs 3, 4 and 7; linoleic (C18:2n-6) on LGs 2 and 5; and a-linolenic acids (C18:3n-3) on LG 1 were identified. Two candidate genes (a lipase and a beta-ketoacyl CoA synthase), both associated with C16:0, and separately with C18:2n-6 and C18:0 contents, were identified. The physical positions of these genes in rice and their genetic positions in perennial ryegrass were consistent with established syntenic relationships between these two species. Validation of these associations is required, but the utility of RAD markers for rapid generation of genetic maps and QTL analysis has been demonstrated for fatty acid composition in a global forage crop.
Background: Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can toler... more Background: Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. Results: Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing 'transcript tags'. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. Conclusions: This de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.
Clovers (genus Trifolium) are a large and widespread genus of legumes. A number of clovers are of... more Clovers (genus Trifolium) are a large and widespread genus of legumes. A number of clovers are of agricultural importance as forage crops in grassland agriculture, particularly temperate areas. White clover (Trifolium repens L.) is used in grazed pasture and red clover (T. pratense L.) is widely cut and conserved as a winter feed. For the diploid red clover, genetic and genomic tools and resources have developed rapidly over the last five years including genetic and physical maps, BAC (bacterial artificial chromosome) end sequence and transcriptome sequence information. This has paved the way for the use of genome wide selection and high throughput phenotyping in germplasm development. For the allotetraploid white clover progress has been slower although marker assisted selection is in use and relatively robust genetic maps and QTL (quantitative trait locus) information now exist. For both species the sequencing of the model legume Medicago truncatula gene space is an important development to aid genomic, biological and evolutionary studies. The first genetic maps of another species, subterranean clover (Trifolium subterraneum L.) have also been published and its comparative genomics with red clover and M. truncatula conducted. Next generation sequencing brings the potential to revolutionize clover genomics, but international consortia and effective use of germplasm, novel population structures and phenomics will be required to carry out effective translation into breeding. Another avenue for clover genomic and genetic improvement is interspecific hybridization. This approach has considerable potential with regard to crop improvement but also opens windows of opportunity for studies of biological and evolutionary processes.
Bacillus thuringiensis subsp, tenebrionis produces a 65 kilodalton polypeptide toxin which is let... more Bacillus thuringiensis subsp, tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the &endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.
The inheritance and expression of the reporter gene uidA, encoding β-glucuronidase (GUS), was pre... more The inheritance and expression of the reporter gene uidA, encoding β-glucuronidase (GUS), was previously analysed in the T1 generation of 25 independent transformed lines of Lotus corniculatus cv. Leo. In the work reported here, GUS activity in various tissues of seven of these lines was tested in the T2 generation. Four representative lines were chosen for more detailed study in
The Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) gene and its orthologs in other pla... more The Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) gene and its orthologs in other plant species (e.g. rice [Oryza sativa] OsFTL2/Hd3a) have an established role in the photoperiodic induction of flowering response. The genomic and phenotypic variations associated with the perennial ryegrass (Lolium perenne) ortholog of FT, designated LpFT3, was assessed in a diverse collection of nine European germplasm populations, which together constituted an association panel of 864 plants. Sequencing and genotyping of a series of amplicons derived from the nine populations, containing the complete exon and intron sequences as well as 5′ and 3′ noncoding sequences of LpFT3, identified a total of seven haplotypes. Genotyping assays designed to detect the genomic variation showed that three haplotypes were present in approximately equal proportions and represented 84% of the total, with a fourth representing a further 11%. Of the three major haplotypes, two were predicted to code for id...
Plant Genetic Resources: Characterization and Utilization, 2008
The genetic structure of marram grass populations at coastal and inland locations, 200 m apart, w... more The genetic structure of marram grass populations at coastal and inland locations, 200 m apart, was investigated at three sites by means of amplified fragment length polymorphism (AFLP) DNA markers. We expected a genetic differentiation between coastal and inland populations and more genetic variation in the coastal areas as a result of different events of colonization by different plant materials. An assignment test showed that the sampledAmmophila arenariacould be assigned to two groups based on AFLP data. The spatial distribution of the two AFLP types ofA. arenariavaried with sampling location. In two of the three locations, mainly one type (1) was found in the newly formed dunes. This type did also occur further landward, but the second type (2) was preferentially found in inland populations. Genetic diversity was very low and of similar value in both coastal and inland populations. For each site, outlier loci with respect toFSTvalue were identified, which may be indicative of d...
Mycoflora of pea seeds and the effectiveness of Rhizobium leguminosarum on important seed-borne p... more Mycoflora of pea seeds and the effectiveness of Rhizobium leguminosarum on important seed-borne pathogens were determined. Mycoflora were changed according to seed groups with or without surface sterilization. The most common isolated fungi were Fusarium spp., Alternaria spp., Macrophomina phaseolina, Phytophthora megasperma, Rhizoctonia solani and Sclerotium rolfsii. R. leguminosarum showed antagonistic effect on Fusarium oxysporum and P. megasperma by 66.3 and 62.1%, respectively, in dual culture test in vitro. R. leguminosarum application reduced disease severity of P. megasperma, R. solani, F. oxysporum and S. rolfsii by 52.9, 50, 67.9 and 52%, respectively. Results indicated that R. leguminosarum showed effects on plant pathogenic fungi and could be used as biocontrol agent.
Development of molecular tools for the analysis of the plant genetic contribution to rhizobial an... more Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For fa...
Resistance and susceptibility in barley to the powdery mildew fungus (Blumeria graminis f. sp. ho... more Resistance and susceptibility in barley to the powdery mildew fungus (Blumeria graminis f. sp. hordei) is determined at the single-cell level. Even in genetically compatible interactions, attacked plant epidermal cells defend themselves against attempted fungal penetration by localized responses leading to papilla deposition and reinforcement of their cell wall. This conveys a race-nonspecific form of resistance. However, this defense is not complete, and a proportion of penetration attempts succeed in infection. The resultant mixture of infected and uninfected leaf cells makes it impossible to relate powdery mildew-induced gene expression in whole leaves or even dissected epidermal tissues to resistance or susceptibility. A method for generating transcript profiles from individual barley epidermal cells was established and proven useful for analyzing resistant and successfully infected cells separately. Contents of single epidermal cells (resistant, infected, and unattacked control...
growth conditions. It was concluded that SPS overexpression causes increased foliar sucrose/starc... more growth conditions. It was concluded that SPS overexpression causes increased foliar sucrose/starch Arabidopsis thaliana ecotype Columbia was transratios in Arabidopsis leaves and favours decreased formed with a maize sucrose phosphate synthase (SPS) foliar carbohydrate contents when plants are grown cDNA under the control of the promoter for the small for long periods with CO 2 enrichment. subunit of ribulose-1,5-bisphosphate carboxylase from tobacco (rbcS). The effects of SPS over-expression Key words: Arabidopsis, sucrose, starch, photosynthesis, were compared in plants of the T 2 and T 3 generacarbon partitioning, CO 2 enrichment. tions grown either in air or with CO 2 enrichment (700 ml l−1) for either 4 or 10 weeks. Maximal extractable foliar SPS activities were three times those of the
We describe a candidate gene approach for associating SNPs with variation in flowering time and w... more We describe a candidate gene approach for associating SNPs with variation in flowering time and water-soluble carbohydrate (WSC) content and other quality traits in the temperate forage grass species Lolium perenne. Three analysis methods were used, which took the significant population structure into account. First, a linear mixed model was used enabling a structured association analysis to be incorporated with the nine populations identified in the structure analysis as random variables. Second, a within-population analysis of variance was performed. Third, a tree-scanning method was used, in which haplotype trees were associated with phenotypes on the basis of inferred haplotypes. Analysis of variance within populations identified several associations between WSC, nitrogen (N), and dry matter digestibility with allelic variants within an alkaline invertase candidate gene LpcAI. These associations were only detected in material harvested in one of the two years. By contrast, consi...
The evolution of genotypic diversity with population age remains poorly explored in clonal plant ... more The evolution of genotypic diversity with population age remains poorly explored in clonal plant populations despite the potential for important shifts to occur through the course of time. Woody sprouting species are particularly under-represented in studies investigating intraspecific variations in levels of clonality from one locality to the next and through time. In this study we sought to determine the incidence and frequency of replicate genotypes in natural Populus nigra L. (Salicaceae) stands of different ages. Ten stands of this woody riparian sprouting species were selected in each of three distinct age groups ('young', 'middle-aged' and 'old') along a 30 km stretch of the River Garonne (south-west France). Leaf samples were collected from 15 neighbouring trees in each stand (450 samples in total) and replicate genotypes were identified using five SSR markers. Replicate genotypes were identified in two-thirds of all stands sampled (i.e. 50% of young stands, 100% of middle-aged stands and 50% of old stands). Young stands had significantly fewer replicated genotypes than middle-aged or old stands, while middle-aged stands had the greatest number of replicated genotypes. Replicate genotypes were most often found to occur as nearest neighbours and formed relatively small, discrete units (i.e. 2-4 trees growing in close proximity to one another). This suggests that asexual regeneration frequently occurs through flood-training in this species, although asexual regeneration from translocated fragments also evidently occurs as evidenced by 11 cases of replicate genotypes occurring in widely separated stands (up to 19 km apart). The results of this study highlight the need for a hierarchical sampling strategy in space and across age groups for an accureate understanding of the genotypic structure of woody sprouting species populations. Conservation and management of effective population sizes will benefit from better insight into not only spatial, but also temporal variations in levels of genotypic diversity.
The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vec... more The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of δ-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The δ-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the δ-endotoxin expression was not affected by the promoter (rhi) used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.
Background: Genetic markers and linkage mapping are basic prerequisites for marker-assisted selec... more Background: Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and mapbased 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.
... 3 (top) and examined with a binocular microscope. The box was constructed from a Petri dish p... more ... 3 (top) and examined with a binocular microscope. The box was constructed from a Petri dish painted matt black except for a 2-0 cm circle on the underside which coincided with the normal sub-stage light source on the microscope. ...
A pearl millet inbred germplasm association panel (PMiGAP) comprising 250 inbred lines, represent... more A pearl millet inbred germplasm association panel (PMiGAP) comprising 250 inbred lines, representative of cultivated germplasm from Africa and Asia, elite improved open-pollinated cultivars, hybrid parental inbreds and inbred mapping population parents, was recently established. This study presents the first report of genetic diversity in PMiGAP and its exploitation for association mapping of drought tolerance traits. For diversity and genetic structure analysis, PMiGAP was genotyped with 37 SSR and CISP markers representing all seven linkage groups. For association analysis, it was phenotyped for yield and yield components and morpho-physiological traits under both well-watered and drought conditions, and genotyped with SNPs and InDels from seventeen genes underlying a major validated drought tolerance (DT) QTL. The average gene diversity in PMiGAP was 0.54. The STRUCTURE analysis revealed six subpopulations within PMiGAP. Significant associations were obtained for 22 SNPs and 3 InDels from 13 genes under different treatments. Seven SNPs associations from 5 genes were common under irrigated and one of the drought stress treatments. Most significantly, an important SNP in putative acetyl CoA carboxylase gene showed constitutive association with grain yield, grain harvest index and panicle yield under all treatments. An InDel in putative chlorophyll a/b binding protein gene was significantly associated with both stay-green and grain yield traits under drought stress. This can be used as a functional marker for selecting high yielding genotypes with 'stay green' phenotype under drought stress. The present study identified useful marker-trait associations of important agronomics traits under irrigated and drought stress conditions with genes underlying a major validated DT-QTL in pearl millet. Results suggest that PMiGAP is a useful panel for
A promoter tagging program in the legume Lotus japonicus was initiated to identify plant genes in... more A promoter tagging program in the legume Lotus japonicus was initiated to identify plant genes involved in the nitrogen-fixing symbiosis between legumes and rhizobia. Seven transformed plant lines expressing the promoterless reporter gene uidA (β-glucuronidase; GUS) specifically in roots and/or nodules were identified. Four of these expressed GUS in the roots only after inoculation with nodule-forming Mesorhizobium loti. In one line (T90), GUS activity was found in the root epidermis, including root hairs. During seedling growth, GUS expression gradually became focused in developing nodules and disappeared from root tissue. No GUS activity was detected when a non-nodulating mutant of M. loti was used to inoculate the plants. The T-DNA insertion in this plant line was located 1.3 kb upstream of a putative coding sequence with strong homology to calcium-binding proteins. Four motifs were identified, which were very similar to the “EF hands” in calmodulin-related proteins, each binding...
This paper describes research on interactions between grassland plant species and soil microorgan... more This paper describes research on interactions between grassland plant species and soil microorganisms. Both parasitic and symbiotic microorganisms modify nutrient transfers between plants and soil. Experiments are described in which nematode infection of clover increased nitrogen transfer to companion ryegrass plants. Infection of clover enhanced activity of soil bacterial and fungal communities. Legume genotypes differing only in responses to symbionts (rhizobium and arbuscular mycorrhizal fungi) and pathogens are being developed for studies of gene expression during establishing and functional symbioses. Such plants can be used in experiments as defined perturbations that will provide information on the interactions and functions of symbiotic and pathogenic microorganisms. Such studies, related to field observations, may have value for defining biological attributes of sustainable grassland soil systems.
Perennial ryegrass (Lolium perenne L.) is the most important forage crop in temperate livestock a... more Perennial ryegrass (Lolium perenne L.) is the most important forage crop in temperate livestock agriculture. Its nutritional quality has significant impact on the quality of meat and milk for human consumption. Evidence suggests that higher energy content in forage can assist in reducing greenhouse gas emissions from ruminants. Increasing the fatty acid content (especially a-linolenic acid, an omega-3 fatty acid) may thus contribute to better forage, but little is known about the genetic basis of variation for this trait. To this end, quantitative trait loci (QTLs) were identified associated with major fatty acid content in perennial ryegrass using a population derived from a cross between the heterozygous and outbreeding high-sugar grass variety AberMagic and an older variety, Aurora. A genetic map with 434 restriction-associated DNA (RAD) and SSR markers was generated. Significant QTLs for the content of palmitic (C16:0) on linkage groups (LGs) 2 and 7; stearic (C18:0) on LGs 3, 4 and 7; linoleic (C18:2n-6) on LGs 2 and 5; and a-linolenic acids (C18:3n-3) on LG 1 were identified. Two candidate genes (a lipase and a beta-ketoacyl CoA synthase), both associated with C16:0, and separately with C18:2n-6 and C18:0 contents, were identified. The physical positions of these genes in rice and their genetic positions in perennial ryegrass were consistent with established syntenic relationships between these two species. Validation of these associations is required, but the utility of RAD markers for rapid generation of genetic maps and QTL analysis has been demonstrated for fatty acid composition in a global forage crop.
Background: Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can toler... more Background: Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. Results: Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing 'transcript tags'. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. Conclusions: This de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.
Clovers (genus Trifolium) are a large and widespread genus of legumes. A number of clovers are of... more Clovers (genus Trifolium) are a large and widespread genus of legumes. A number of clovers are of agricultural importance as forage crops in grassland agriculture, particularly temperate areas. White clover (Trifolium repens L.) is used in grazed pasture and red clover (T. pratense L.) is widely cut and conserved as a winter feed. For the diploid red clover, genetic and genomic tools and resources have developed rapidly over the last five years including genetic and physical maps, BAC (bacterial artificial chromosome) end sequence and transcriptome sequence information. This has paved the way for the use of genome wide selection and high throughput phenotyping in germplasm development. For the allotetraploid white clover progress has been slower although marker assisted selection is in use and relatively robust genetic maps and QTL (quantitative trait locus) information now exist. For both species the sequencing of the model legume Medicago truncatula gene space is an important development to aid genomic, biological and evolutionary studies. The first genetic maps of another species, subterranean clover (Trifolium subterraneum L.) have also been published and its comparative genomics with red clover and M. truncatula conducted. Next generation sequencing brings the potential to revolutionize clover genomics, but international consortia and effective use of germplasm, novel population structures and phenomics will be required to carry out effective translation into breeding. Another avenue for clover genomic and genetic improvement is interspecific hybridization. This approach has considerable potential with regard to crop improvement but also opens windows of opportunity for studies of biological and evolutionary processes.
Bacillus thuringiensis subsp, tenebrionis produces a 65 kilodalton polypeptide toxin which is let... more Bacillus thuringiensis subsp, tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the &endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.
The inheritance and expression of the reporter gene uidA, encoding β-glucuronidase (GUS), was pre... more The inheritance and expression of the reporter gene uidA, encoding β-glucuronidase (GUS), was previously analysed in the T1 generation of 25 independent transformed lines of Lotus corniculatus cv. Leo. In the work reported here, GUS activity in various tissues of seven of these lines was tested in the T2 generation. Four representative lines were chosen for more detailed study in
The Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) gene and its orthologs in other pla... more The Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) gene and its orthologs in other plant species (e.g. rice [Oryza sativa] OsFTL2/Hd3a) have an established role in the photoperiodic induction of flowering response. The genomic and phenotypic variations associated with the perennial ryegrass (Lolium perenne) ortholog of FT, designated LpFT3, was assessed in a diverse collection of nine European germplasm populations, which together constituted an association panel of 864 plants. Sequencing and genotyping of a series of amplicons derived from the nine populations, containing the complete exon and intron sequences as well as 5′ and 3′ noncoding sequences of LpFT3, identified a total of seven haplotypes. Genotyping assays designed to detect the genomic variation showed that three haplotypes were present in approximately equal proportions and represented 84% of the total, with a fourth representing a further 11%. Of the three major haplotypes, two were predicted to code for id...
Plant Genetic Resources: Characterization and Utilization, 2008
The genetic structure of marram grass populations at coastal and inland locations, 200 m apart, w... more The genetic structure of marram grass populations at coastal and inland locations, 200 m apart, was investigated at three sites by means of amplified fragment length polymorphism (AFLP) DNA markers. We expected a genetic differentiation between coastal and inland populations and more genetic variation in the coastal areas as a result of different events of colonization by different plant materials. An assignment test showed that the sampledAmmophila arenariacould be assigned to two groups based on AFLP data. The spatial distribution of the two AFLP types ofA. arenariavaried with sampling location. In two of the three locations, mainly one type (1) was found in the newly formed dunes. This type did also occur further landward, but the second type (2) was preferentially found in inland populations. Genetic diversity was very low and of similar value in both coastal and inland populations. For each site, outlier loci with respect toFSTvalue were identified, which may be indicative of d...
Mycoflora of pea seeds and the effectiveness of Rhizobium leguminosarum on important seed-borne p... more Mycoflora of pea seeds and the effectiveness of Rhizobium leguminosarum on important seed-borne pathogens were determined. Mycoflora were changed according to seed groups with or without surface sterilization. The most common isolated fungi were Fusarium spp., Alternaria spp., Macrophomina phaseolina, Phytophthora megasperma, Rhizoctonia solani and Sclerotium rolfsii. R. leguminosarum showed antagonistic effect on Fusarium oxysporum and P. megasperma by 66.3 and 62.1%, respectively, in dual culture test in vitro. R. leguminosarum application reduced disease severity of P. megasperma, R. solani, F. oxysporum and S. rolfsii by 52.9, 50, 67.9 and 52%, respectively. Results indicated that R. leguminosarum showed effects on plant pathogenic fungi and could be used as biocontrol agent.
Development of molecular tools for the analysis of the plant genetic contribution to rhizobial an... more Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For fa...
Resistance and susceptibility in barley to the powdery mildew fungus (Blumeria graminis f. sp. ho... more Resistance and susceptibility in barley to the powdery mildew fungus (Blumeria graminis f. sp. hordei) is determined at the single-cell level. Even in genetically compatible interactions, attacked plant epidermal cells defend themselves against attempted fungal penetration by localized responses leading to papilla deposition and reinforcement of their cell wall. This conveys a race-nonspecific form of resistance. However, this defense is not complete, and a proportion of penetration attempts succeed in infection. The resultant mixture of infected and uninfected leaf cells makes it impossible to relate powdery mildew-induced gene expression in whole leaves or even dissected epidermal tissues to resistance or susceptibility. A method for generating transcript profiles from individual barley epidermal cells was established and proven useful for analyzing resistant and successfully infected cells separately. Contents of single epidermal cells (resistant, infected, and unattacked control...
growth conditions. It was concluded that SPS overexpression causes increased foliar sucrose/starc... more growth conditions. It was concluded that SPS overexpression causes increased foliar sucrose/starch Arabidopsis thaliana ecotype Columbia was transratios in Arabidopsis leaves and favours decreased formed with a maize sucrose phosphate synthase (SPS) foliar carbohydrate contents when plants are grown cDNA under the control of the promoter for the small for long periods with CO 2 enrichment. subunit of ribulose-1,5-bisphosphate carboxylase from tobacco (rbcS). The effects of SPS over-expression Key words: Arabidopsis, sucrose, starch, photosynthesis, were compared in plants of the T 2 and T 3 generacarbon partitioning, CO 2 enrichment. tions grown either in air or with CO 2 enrichment (700 ml l−1) for either 4 or 10 weeks. Maximal extractable foliar SPS activities were three times those of the
We describe a candidate gene approach for associating SNPs with variation in flowering time and w... more We describe a candidate gene approach for associating SNPs with variation in flowering time and water-soluble carbohydrate (WSC) content and other quality traits in the temperate forage grass species Lolium perenne. Three analysis methods were used, which took the significant population structure into account. First, a linear mixed model was used enabling a structured association analysis to be incorporated with the nine populations identified in the structure analysis as random variables. Second, a within-population analysis of variance was performed. Third, a tree-scanning method was used, in which haplotype trees were associated with phenotypes on the basis of inferred haplotypes. Analysis of variance within populations identified several associations between WSC, nitrogen (N), and dry matter digestibility with allelic variants within an alkaline invertase candidate gene LpcAI. These associations were only detected in material harvested in one of the two years. By contrast, consi...
The evolution of genotypic diversity with population age remains poorly explored in clonal plant ... more The evolution of genotypic diversity with population age remains poorly explored in clonal plant populations despite the potential for important shifts to occur through the course of time. Woody sprouting species are particularly under-represented in studies investigating intraspecific variations in levels of clonality from one locality to the next and through time. In this study we sought to determine the incidence and frequency of replicate genotypes in natural Populus nigra L. (Salicaceae) stands of different ages. Ten stands of this woody riparian sprouting species were selected in each of three distinct age groups ('young', 'middle-aged' and 'old') along a 30 km stretch of the River Garonne (south-west France). Leaf samples were collected from 15 neighbouring trees in each stand (450 samples in total) and replicate genotypes were identified using five SSR markers. Replicate genotypes were identified in two-thirds of all stands sampled (i.e. 50% of young stands, 100% of middle-aged stands and 50% of old stands). Young stands had significantly fewer replicated genotypes than middle-aged or old stands, while middle-aged stands had the greatest number of replicated genotypes. Replicate genotypes were most often found to occur as nearest neighbours and formed relatively small, discrete units (i.e. 2-4 trees growing in close proximity to one another). This suggests that asexual regeneration frequently occurs through flood-training in this species, although asexual regeneration from translocated fragments also evidently occurs as evidenced by 11 cases of replicate genotypes occurring in widely separated stands (up to 19 km apart). The results of this study highlight the need for a hierarchical sampling strategy in space and across age groups for an accureate understanding of the genotypic structure of woody sprouting species populations. Conservation and management of effective population sizes will benefit from better insight into not only spatial, but also temporal variations in levels of genotypic diversity.
The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vec... more The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of δ-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The δ-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the δ-endotoxin expression was not affected by the promoter (rhi) used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.
Background: Genetic markers and linkage mapping are basic prerequisites for marker-assisted selec... more Background: Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and mapbased 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.
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Papers by Leif Skøt