Molecular breeding of transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered fructan biosynthesis through the expression of fructosyltransferases

Perennial ryegrass is the most important forage grass used in temperate agriculture. Transgenic perennial ryegrass events with altered fructan biosynthesis have the potential not only to increase animal production by improving digestibility of the grasses, but also to increase pasture intake by the animal due to lower neutral detergent fibre (NDF) concentrations. Transgenic perennial ryegrass plants were shown to have increased (P?<?0.05) in fructan concentrations of leaf blades in transgenic T₀ events and have thus an increase in water-soluble carbohydrate and in vivo dry matter digestibility concentrations as well as a decrease in the NDF concentration within the plant in spring and summer. These changes in nutritive value have led to an increase in metabolisable energy of up to 1.7 MJ ME·kg DM^?1 in selected T₀ events compared to FLp418-20 during spring and summer, with no differences in autumn or winter. The field evaluation of these events and the further development of these events using molecular breeding technologies are described in this paper.     

An SSR-based genetic linkage map for perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

A simple sequence repeat (SSR)-based linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross reference population. A total of 309 unique perennial ryegrass SSR (LPSSR) primer pairs showing efficient amplification were evaluated for genetic polymorphism, with 31% detecting segregating alleles. Ninety-three loci have been assigned to positions on seven linkage groups. The majority of the mapped loci are derived from cloned sequences containing (CA)_n-type dinucleotide SSR arrays. A small number (7%) of primer pairs amplified fragments that mapped to more than one locus. The SSR locus data has been integrated with selected data for RFLP, AFLP and other loci mapped in the same population to produce a composite map containing 258 loci. The SSR loci cover 54% of the genetic map and show significant clustering around putative centromeric regions. BLASTN and BLASTX analysis of the sequences flanking mapped SSRs indicated that a majority (84%) are derived from non-genic sequences, with a small proportion corresponding to either known repetitive DNA sequence families or predicted genes. The mapped LPSSR loci provide the basis for linkage group assignment across multiple mapping populations.     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Genetic characterisation of seed yield and fertility traits in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Seed yield is a trait of major interest for the key grassland species Lolium perenne L. An F2 mapping population of perennial ryegrass (VrnA), recently characterised for vernalisation response, was assessed in a glasshouse for traits related to seed yield based on a lattice design with four replications over 2 years. The traits heading date, plant height, length of panicles, number of panicles per plant, seed yield per panicle, flag leaf length, flag leaf width and seed yield per plant revealed repeatabilities ranging from 41 to 76% and a considerable amount of genetic variation in the VrnA population. Path analysis partitioned the direct and indirect effects of seed yield components on seed yield per plant. Seed yield per panicle showed the highest effect on total seed yield. The adjusted mean values of each trait and a genetic linkage map consisting of 97 anonymous and 85 gene associated DNA markers were used for quantitative trait loci (QTL) analysis. Of particular interest were two QTL on linkage group (LG) 1 and LG 2, explaining 41 and 18%, respectively, of the observed phenotypic variation for the trait seed yield per panicle. Both QTL co-located with two major QTL for total seed yield per plant possibly representing the S and Z loci of the gametophytic self incompatibility (SI) system of perennial ryegrass. The diversity of SI alleles in mapping parents and the degree of heterozygosity at SI loci in the full sib progeny determines the interference of self incompatibility with seed production.     

Synteny between a major heading-date QTL in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) and the<Emphasis Type="Italic"> Hd3</Emphasis> heading-date locus in rice

The genetic control of induction to flowering has been studied extensively in both model and crop species because of its fundamental biological and economic significance. An ultimate aim of many of these studies has been the application of the understanding of control of flowering that can be gained from the study of model species, to the improvement of crop species. The present study identifies a region of genetic synteny between rice and Lolium perenne, which contains the Hd3 heading-date QTL in rice and a major QTL, accounting for up to 70% of the variance associated with heading date in L. perenne. The identification of synteny between rice and L. perenne in this region demonstrates the direct applicability of the rice genome to the understanding of biological processes in other species. Specifically, this syntenic relationship will greatly facilitate the genetic dissection of aspects of heading-date induction by enabling the magnitude of the genetic component of the heading-date QTL in L. perenne to be combined with the sequencing and annotation information from the rice genome.Communicated by Q. Zhang     

Inheritance patterns of the response to in vitro doubled haploid induction in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

The ability to produce doubled haploid (DH) plants has found broad application in research and breeding. For major crop species such as maize (Zea mays L.) and barley (Hordeum vulgare L.), routine large-scale production of DHs has enabled the acceleration of breeding processes, for example through efficient generation of homozygous lines. However, in forage crops such as perennial ryegrass (Lolium perenne L.), low and genotype-specific responses to in vitro anther culture (AC) still limit wide-spread use of DHs. Here, we report the responses of nine bi-parental populations, segregating for microspore embryogenesis and plant regeneration capacity, to an effective AC protocol. Genotypes of exceptionally high androgenic ability, producing over 200 green plants per 100 anthers cultured, could be selected. Continuous and distinctly shaped distributions for the evaluated traits were indicative of quantitative polygenic control and the presence of different alleles in each population. An insignificant association of embryo production with plant regeneration, as well as a low correlation between green and albino plant yield (ρ?=?0.20), suggested that different genes influence these traits. The populations evaluated here provide a rich source of alleles needed for the introgression of high levels of androgenic capacity into recalcitrant material. Moreover, this germplasm is ideally suited for use in future genotyping and mapping studies so that the genetic control of androgenic capacity in perennial ryegrass can be elucidated. Ultimately, our results will help to realize the potential of DH induction in one of the world’s most important forage crop species.     

Regeneration from leaf-base explants of <Emphasis Type="Italic">Lolium perenne</Emphasis> L. and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> L.

The regenerability of Lolium perenne and Lolium multiflorum was investigated using leaf-base sections excised from plantlets grown in vitro. Young leaf bases were cut into three sections each 2 mm in length, the lowest 0–2 mm section adjacent to, but not including, the apical meristem. Callus development followed by embryogenesis occurred only in the lowest 2 mm leaf-base sections of L. perenne cv. Limes, whereas L. multiflorum leaf bases showed an embryogenic response along the entire 6 mm section. Rooted, green plantlets were obtained from 100% of the 0–2 mm sections of L. perenne cv. Limes and from 91%, 65% and 26% of leaf-base sections at 0–2, 2–4 and 4–6 mm respectively from L. multiflorum. Microprojectile bombardment of leaf-base explants resulted in transient expression of the -glucuronidase gene.     

Fine-scale comparative genetic and physical mapping supports map-based cloning strategies for the self-incompatibility loci of perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Perennial ryegrass is an obligate outbreeding pasture grass of the Poaceae family, with a two-locus (S and Z) gametophytic self-incompatibility (SI) mechanism. This system has provided a major obstacle to targeted varietal development, and enhanced knowledge is expected to support more efficient breeding strategies. Comparative genetics and physical mapping approaches have been developed to permit molecular cloning of the SI genes. SI gene-linked genetic markers based on heterologous cDNA restriction fragment length polymorphisms (RFLPs) and homologous genomic DNA-derived simple sequence repeats (SSRs) were converted to single nucleotide polymorphism (SNP) format for efficient genotyping. Genetic mapping identified the location of SI loci and demonstrated macrosynteny between related grass species. S- and Z-linked bacterial artificial chromosome (BAC) clones were sequenced using massively parallel pyrosequencing technology to provide the first physical mapping data for Poaceae SI loci. The sequence assembly process suggested a lower prevalence of middle repetitive sequences in the Z locus region and hence precedence for positional cloning strategy. In silico mapping using data from rice, Brachypodium distachyon and Sorghum revealed high sequence conservation in the vicinity of the Z locus region between SI and self-compatible (SC) grass species. Physical mapping identified a total of nine genes encoded in the Z locus region. Expression profiling and nucleotide diversity assessment identified two Z-linked genes, LpTC116908 and LpDUF247, as plausible candidates for the male and female determinants of the S-Z SI system.     

Quantitative Trait Locus (QTL) meta-analysis and comparative genomics for candidate gene prediction in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Background

In crop species, QTL analysis is commonly used for identification of factors contributing to variation of agronomically important traits. As an important pasture species, a large number of QTLs have been reported for perennial ryegrass based on analysis of biparental mapping populations. Further characterisation of those QTLs is, however, essential for utilisation in varietal improvement programs.

Results

A bibliographic survey of perennial ryegrass trait-dissection studies identified a total of 560 QTLs from previously published papers, of which 189, 270 and 101 were classified as morphology-, physiology- and resistance/tolerance-related loci, respectively. The collected dataset permitted a subsequent meta-QTL study and implementation of a cross-species candidate gene identification approach. A meta-QTL analysis based on use of the BioMercator software was performed to identify two consensus regions for pathogen resistance traits. Genes that are candidates for causal polymorphism underpinning perennial ryegrass QTLs were identified through in silico comparative mapping using rice databases, and 7 genes were assigned to the p150/112 reference map. Markers linked to the Lp DGL1, Lp Ph1 and Lp PIPK1 genes were located close to plant size, leaf extension time and heading date-related QTLs, respectively, suggesting that these genes may be functionally associated with important agronomic traits in perennial ryegrass.

Conclusions

Functional markers are valuable for QTL meta-analysis and comparative genomics. Enrichment of such genetic markers may permit further detailed characterisation of QTLs. The outcomes of QTL meta-analysis and comparative genomics studies may be useful for accelerated development of novel perennial ryegrass cultivars with desirable traits.

     

Use of a gnotobiotic plant assay for assessing root colonization and mineral phosphate solubilization by <Emphasis Type="Italic">Paraburkholderia bryophila</Emphasis> Ha185 in association with perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Aims

The mechanisms by which rhizosphere bacteria increase the availability of mineral P precipitates for plant use are understudied. However, Paraburkholderia bryophila Ha185 is known to solubilize inorganic phosphate in vitro via a novel process. Therefore, this study aimed to demonstrate P solubilization by Ha185 in association with roots of perennial ryegrass (Lolium perenne L.).

Methods

We developed a gnotobiotic plant assay to assess P solubilization by Ha185 on ryegrass roots under various nutrient conditions. A green fluorescent protein (GFP)-tagged derivative of Ha185 was used in conjunction with fluorescent microscopy and confocal microscopy to visualize colonization of ryegrass roots.

Results

Ha185 solubilized mineral P (hydroxyapatite) in association with ryegrass roots and increased ryegrass growth by 20% under P-limited conditions. The GFP-tagged Ha185 strain colonized the rhizoplane and penetrated the primary root of ryegrass, possibly through “crack entry” at the point of lateral root emergence, but also by entering the epidermal cells via root hairs.

Conclusions

Ha185 supported ryegrass growth under P-limited conditions, indicating this strain may improve availability of soil P for uptake by ryegrass. Tools developed in this study have broad application in the study of rhizobacteria-plant interactions.

     

Cloning,characterization and expression analysis of tonoplast intrinsic proteins and glutamine synthetase in ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Perennial ryegrass (Lolium perenne L.) is the most important turf and forage grass species of the temperate regions. It requires substantial input of nitrogen fertilizer for optimum yield. Improved nitrogen use efficiency (NUE) is therefore one of the main breeding targets. However, limited knowledge is currently available on the genes controlling NUE in perennial ryegrass. The aim of the present study was to isolate genes involved in ammonium transport and assimilation. In silico screening of a Lolium EST-library using known sequences of tonoplast intrinsic proteins (TIPs) and cytosolic glutamine synthetase (GS1) revealed a number of homologous sequences. Using these sequences, primers were designed to obtain the full-length sequences by RACE-PCR. Three TIP genes (LpTIP1;1, LpTIP1;2 and LpTIP2;1) and two GS genes (LpGS1a and LpGS1b) were isolated. Characterization in S. cerevisiae confirmed a function in ammonium transport for LpTIP1;1 and LpTIP2;1 and in synthesis of glutamine for LpGS1a and LpGS1b. Cytoimmunochemical studies showed that GS protein was present in the chloroplasts and cytosol of leaf cells, while TIP1 proteins localized to the tonoplast. At the expression level, Lolium GS1 genes responded to N starvation and re-supply in a manner consistent with functions in primary N assimilation and N remobilization. Similarly, the expression of LpTIPs complied with a role in vacuolar ammonium storage. Together, the reported results provide new understanding of the genetic basis for N assimilation and storage in ryegrass.     

Discovery and genetic mapping of single nucleotide polymorphisms in candidate genes for pathogen defence response in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Susceptibility to foliar pathogens commonly causes significant reductions in productivity of the important temperate forage perennial ryegrass. Breeding for durable disease resistance involves not only the deployment of major genes but also the additive effects of minor genes. An approach based on in vitro single nucleotide polymorphism (SNP) discovery in candidate defence response (DR) genes has been used to develop potential diagnostic genetic markers. SNPs were predicted, validated and mapped for representatives of the pathogenesis-related (PR) protein-encoding and reactive oxygen species (ROS)-generating gene classes. The F(1)(NA(6) x AU(6)) two-way pseudo-test cross population was used for SNP genetic mapping and detection of quantitative trait loci (QTLs) in response to a crown rust field infection. Novel resistance QTLs were coincident with mapped DR gene SNPs. QTLs on LG3 and LG7 also coincided with both herbage quality QTLs and candidate genes for lignin biosynthesis. Multiple DR gene SNP loci additionally co-located with QTLs for grey leaf spot, bacterial wilt and crown rust resistance from other published studies. Further functional validation of DR gene SNP loci using methods such as fine-mapping and association genetics will improve the efficiency of parental selection based on superior allele content.     

Allelopathic interference of alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) genotypes to annual ryegrass (<Emphasis Type="Italic">Lolium rigidum</Emphasis>)

Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for allelopathic impact using annual ryegrass (Lolium rigidum) as the target species in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and 40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM). Alfalfa genotypes displayed a range of allelopathic interference in ryegrass seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased in response to increasing density of alfalfa seedlings. At the lowest density, Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index was calculated to rank each alfalfa genotype. Reduction in seed germination of annual ryegrass occurred in the presence of several alfalfa genotypes including Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes. Variation in chemical compounds was found between alfalfa root extracts and exudates and also between genotypes. Further individual compound assessments and quantitative study at greater chemical concentrations are needed to clarify the allelopathic activity. Considerable genetic variation exists among alfalfa genotypes for allelopathic activity creating the opportunity for its use in weed suppression through selection.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Festuca arundinacea</Emphasis> (Schreb.) and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> (Lam.)

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of shoot apices of Kentucky bluegrass (<Emphasis Type="Italic">Poa pratensis</Emphasis> L.) and production of transgenic plants carrying a <Emphasis Type="Italic">betA</Emphasis> gene

Apical meristems of multiple shoots produced from axenic seedlings of Kentucky bluegrass (Poa pratensis L.) were used for Agrobacterium tumefaciens-mediated transformation. Transformation parameters were optimized for concentration of bacterial cells, duration of infection, and vacuum infiltration. The highest transformation frequency (1.42%) was obtained by infection with Agrobacterium suspension of OD₆₀₀ = 0.6 for 5 min, under a negative pressure of 0.5 × 10⁵ Pa. After co-cultivation, the herbicide-resistant plants were rooted and transplanted into flowerpots. Transgenic plants were confirmed by polymerase chain reaction (PCR) assay and Southern blot analysis. Using this transformation system, the betA gene encoding choline dehydrogenase and mutant als gene encoding the enzyme acetolactate synthase were introduced into three Kentucky bluegrass cultivars.     

The impact of phosphorus on interactions of the hemiparasitic angiosperm <Emphasis Type="Italic">Rhinanthus minor</Emphasis> and its host <Emphasis Type="Italic">Lolium perenne</Emphasis>

The effects of phosphorus supply on the outcome of interactions between the hemiparasitic angiosperm Rhinanthus minor L. with its host species Lolium perenne L. were investigated in a glasshouse experiment. Host plants were grown in 3-l pots in the presence and absence of R. minor at limiting (0.13 mm P) and optimal (0.65 mm P) concentrations of phosphorus for the growth of the host species. Phosphorus was supplied at 2-day intervals in the form of half-strength Long Ashton nitrate-based solution with phosphorus concentrations adjusted accordingly. Parasitism by R. minor significantly suppressed host growth, with final biomass losses ranging between 32% and 44%. Phosphorus supply had a marked impact on the outcome of the host-parasite interaction. By the end of the growing period, parasite biomass at 0.65 mm P was 90% lower than that achieved at 0.13 mm P. In contrast, host biomass at 0.65 mm P was 74% higher than achieved at 0.13 mm P, indicting that the negative impact of parasitism on the host species was reduced when phosphorus supply was increased. The effects of phosphorus on the host-parasite association appeared to be mediated by changes in both the morphological characteristics of the host roots and the relative sink strengths of the host and parasite. Received: 29 May 1999 / Accepted: 20 December 1999     

Influence of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> strain on the production of transgenic peas (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

We compared the efficiency of two Agrobacterium tumefaciens strains, AGL 1 and KYRT1, for producing transgenic pea plants. KYRT1 is a disarmed strain of Chry5 that has been shown to be highly tumourigenic on soybean. The efficacies of the strains were compared using cotyledon explants from three pea genotypes and two plasmids. The peas were sourced from field-grown plants over three Southern Hemisphere summer seasons. Overall, KYRT1 was found to be on average threefold more efficient than AGL 1 for producing transgenic plants. We suggest that KYRT1 is sensitive to cocultivation temperature as the expected increase in efficiency was not achieved at high laboratory temperatures.Communicated by P. Debergh     

Transfer and expression of <Emphasis Type="Italic">npt</Emphasis>II and <Emphasis Type="Italic">bar</Emphasis> genes in cucumber (<Emphasis Type="Italic">Cucumis satavus</Emphasis> L.)

Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l⁻¹ phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2–6 mg l⁻¹). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l⁻¹) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.