Genome balance in six successive generations of the allotetraploid Festuca pratensis × Lolium perenne

In the allotetraploid, Festuca pratensis Huds. (2n = 4x = 28) × Lolium perenne L. (2n = 4x = 28) the balance of chromatin, as determined by GISH, changes over successive generations of open pollination in favour of L. perenne. There is extensive recombination between chromosomes of the two parental genomes, as well as substitution of whole Festuca chromosomes by whole Lolium chromosomes. The total number of Lolium chromosomes increased from a mean 14.36 in the F₂ to 16.26 in the F₆, and the total number of Festuca chromosomes decreased correspondingly from a mean of 13.57 to a value of 11.56. The number of recombinant chromosomes and recombination breakpoints per genotype also increased from generation to generation, although the respective values of both characters were higher for Festuca (0.86–8.41 and 1.14–15.22) than for Lolium (0.68–4.59 and 0.68–6.0). The proportion of total genome length contributed by the L. perenne chromatin increased from about 50% in F₂ to 59.5% in F₆. The results are based on the sample of 134 plants studied (26–28 plants per generation), and are discussed in terms of the dominance of Lolium chromosomes over those of Festuca, and possible mechanisms underlying this phenomenon of chromatin substitution.     

Synaptonemal complex formation in hybrids of Lolium temulentum × Lolium perenne (L.)

Chromosome pairing and synaptonemal complex formation at zygotene and pachytene are described from serial section reconstructions of pollen mother cell nuclei in a triploid hybrid containing two haploid sets of Lolium perenne chromosomes, one of L. temulentum and two acces-sory B chromosomes. At pachytene the homologous L. perenne chromosomes form complete and continuous synaptonemal complexes while the L. temulentum chromosomes show extensive nonhomologous pairing both within and between themselves. At zygotene however, homoeologous pairing in the form of a trivalent and very little non-homologous pairing is observed. Evidently, there exists a mechanism that eliminates homoeologous association during zygotene to ensure strict bivalent formation between homologous chromosomes at pachytene. In Lolium this mechanism is under the influence of the B chromosomes and bears close similarity with that in allohexaploid wheat controlled by the Ph locus.     

Synaptonemal complex formation in hybrids of Lolium temulentum × Lolium perenne (L.)

The chromosomes of Lolium temulentum are longer and contain on average 50% more nuclear DNA than the chromosomes of L. perenne. In the hybrid, despite the difference in length and DNA content, pairing between the homoeologous chromosomes at pachytene is effective and the chiasma frequency at first metaphase in pollen mother cells is high, about 1.6 per bivalent, comparable to that in the L. perenne parent. Electron microscopic observations from reconstructed nuclei at pachytene show that synaptonemal complex (SC) formation in 40% of bivalents is perfect, complete and continuous from telomere to telomere. In others, SCs extend from telomere to telomere but incorporate lateral component loops in interstitial chromosome segments. Even in these bivalents, however, pairing is effective in the sense of chiasma formation. The capacity to form perfect SCs is achieved by an adjustment of chromosome length differences both before and during synapsis. Perfect pairing and SC formation is commoner within the larger bivalents of the complement. At zygotene, in contrast to pachytene, pairing is not confined to homoeologous chromosomes. On the contrary there is illegitimate pairing between non-homologous chromsomes resulting in multivalent formation. There must, therefore, be a mechanism operative between zygotene and pachytene that corrects and modifies associations in such a way as to restrict the pairing to bivalents comprised of strictly homoeologous chromosomes. Such a correction bears comparison with that known to apply in allopolyploids. In the hybrid and in the L. perenne parent also, certain specific nucleolar organisers are inactivated at meiosis.     

The genotypic control of homoeologous chromosome association in Lolium temulentum × Lolium perenne interspecific hybrids

Two contrasting genotypes of Lolium perenne and two inbred lines of L. temulentum were examined with regard to their effect on homoeologous chromosome pairing in interspecific hybrids derived from them. Substantial differences in chiasma frequency were observed between the hybrid progeny of the different parental types. The background genes involved were found to operate in the presence and in the absence of B chromosomes. The combination of A chromosome genes present in some of the 0B hybrids was found to result in a considerable suppression of chiasma formation at the diploid level, and the restriction of pairing to strict homologues at the tetraploid level. It appears, therefore, that genes are present within the diploid species of the genus Lolium which are capable of performing a function similar to that of the Ph locus in wheat.     

热锻炼对高羊茅(Festuca arundinacea)和多年生黑麦草(Lolium perenne)抗高温能力的影响

夏季高温胁迫已成为限制冷季型草坪草生长和发育的一个主要问题.以两种耐热性不同的冷季型草坪草高羊茅和多年生黑麦草(前者较耐热)为材料,经过3d 30℃的热锻炼预处理后,分别在38、42、46℃的高温下处理14h.在这些高温条件下,研究了经过热锻炼预处理的高羊茅和多年生黑麦草叶片膜脂过氧化、抗氧化剂含量以及叶绿体超微结构的变化.结果表明:(1)热锻炼提高了高羊茅和多年生黑麦草的耐热性,显著缓减了高温条件下两种草坪草叶片膜脂过氧化程度的加剧,降低了叶片过氧化氢(H2O2)和超氧阴离子(O2)的产生速率.(2)高温条件下,热锻炼使高羊茅和多年生黑麦草叶片中抗氧化剂抗坏血酸(AsA)和谷胱甘肽(GSH)的含量下降程度有所缓减.(3)热锻炼减轻了高温胁迫对高羊茅和多年生黑麦草叶片叶绿体超微结构的损伤.这些结果说明热锻炼能够减轻高温对草坪草叶绿体的伤害可能与其在高温胁迫下和对照相比具有较高的抗氧化剂含量有关,这也可能是冷季型草坪草对高温的适应机制之一.     

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.     

Reciprocal allopolyploid grasses (Festuca × Lolium) display stable patterns of genome dominance

Allopolyploidization entailing the merger of two distinct genomes in a single hybrid organism, is an important process in plant evolution and a valuable tool in breeding programs. Newly established hybrids often experience massive genomic perturbations, including karyotype reshuffling and gene expression modifications. These phenomena may be asymmetric with respect to the two progenitors, with one of the parental genomes being “dominant.” Such “genome dominance” can manifest in several ways, including biased homoeolog gene expression and expression level dominance. Here we employed a k-mer–based approach to study gene expression in reciprocal Festuca pratensis Huds. × Lolium multiflorum Lam. allopolyploid grasses. Our study revealed significantly more genes where expression mimicked that of the Lolium parent compared with the Festuca parent. This genome dominance was heritable to successive generation and its direction was only slightly modified by environmental conditions and plant age. Our results suggest that Lolium genome dominance was at least partially caused by its more efficient trans-acting gene expression regulatory factors. Unraveling the mechanisms responsible for propagation of parent-specific traits in hybrid crops contributes to our understanding of allopolyploid genome evolution and opens a way to targeted breeding strategies.     

Chromosome substitutions and recombination in the amphiploid Lolium perenne×Festuca pratensis cv Prior (2n=4x=28)

 The synthetic amphiploid cv Prior was created in the early 1970s at the Welsh Plant Breeding Station by crossing colchicine-induced autotetraploids of Lolium perenne (2n=14) and Festuca pratensis (2n=14). Meiosis in the early generations was characterized as stable, with frequent bivalent formation. In situ hybridization of a L. perenne total genomic DNA probe to mitotic chromosome spreads of 12 plants, from two extant populations of Prior, demonstrates extensive recombination between the two genomes. Recombination events occur along the whole length of chromosome arms but with a higher frequency in the medial portion. The species origins of chromosomes were assigned by the presence or absence of a fluorescent probe at the centromere. There has been a substitution of Festuca-origin chromosomes by those of Lolium-origin, resulting in a mean of 17.9 (15–21) Lolium and 9.7 (7–13) Festuca chromosomes per genotype. Mean chromatin length per genotype comprised 62.1% Lolium and 37.9% Festuca. On average 9.3 Lolium (51.1% of those present) and 3.5 Festuca (37.8%) chromosomes had no recombined segments. For chromosomes which did show recombination, fewer alien segments were observed in Lolium than in Festuca chromosomes. Festuca chromosomes in genotypes selected for drought resistance had undergone more recombination than in genotypes from an unselected population, though this difference was not statistically significant for the small sample examined. Received: 16 June 1998 / Accepted: 17 September 1998 RID="1" ID="1" <E5>Present address:</E5> Lithuanian Institute of Agriculture, 5051 Dotnuva-Akademija, Kedainiai, Lithuania RID=" ID=" Communicated by J. W. Snape RID=" ID=" <E5>Correspondence to</E5> P. H. Canter     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2) of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes. Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gyno- genetic progeny of allotetraploid hybrids of red crucian carp × common carp had important signifi- cances in both biological evolution and production application.     

Structural investigations on the lignin–carbohydrate complexes of Lolium perenne

1. Lignin-carbohydrate complexes isolated from leaf blade, leaf sheath and stem tissue of ryegrass by extraction with dimethyl sulphoxide were examined by fractionation procedures. Although the complexes are heterogeneous, heterogeneity is shown only in the ratio of the individual monosaccharide residues and not in the ratio of lignin to carbohydrate. 2. The molecular weight of the complexes is high (>/=150000), but chemical modification by alkaline hydrolysis, borohydride reduction or lead tetra-acetate oxidation does not drastically decrease it. Low-molecular-weight fragments released by alkaline treatment were shown to contain acetic acid, ferulic acid and p-coumaric acid. 3. On the basis of the chemical stability of the complexes, it is postulated that at least three types of bonding may be present between lignin and carbohydrate, namely one cleaved on borohydride reduction, another cleaved by alkali and a linkage resistant to alkali. 4. The carbohydrate portion of the complexes is composed of beta-(1-->4)-linked d-glucose residues (cellulose) and beta-(1-->4)-linked chains of xylose residues. Side chains involving arabinose and galactose residues are linked to C-3 of some of the xylose residues. 5. How the components of the complexes are held together is not certain, but it is suggested that the phenolic acids may act as cross-linking agents.     

Hieracium × grofae — a rediscovered diploid hybrid from the Ukrainian Carpathians

Diploid hybrid plants (2n = 18) between sexual diploid cytotypes of Hieracium alpinum and H. umbellatum were found in the Ukrainian Eastern Carpathians. They were identified with H. × grofae Woł., originally used for the combination H. decipiens × H. umbellatum var. lactaris. As H. decipiens sensu Woł. (non Tausch) does not produce viable pollen grains and is most probably a polyploid apomict, it is unlikely to produce diploid hybrid plants with diploid H. umbellatum. Both parent species, Hieracium alpinum and H. umbellatum are also given by Wołoszczak from the original locality. Thus we conclude that H. × grofae is result of hybridization between H. alpinum and high mountain form of H. umbellatum. Hybrid plants are morphologically intermediate between the parent species, and moreover resemble closely or they are identical with the experimental hybrids of the same parent combination. Hybrids produce rather high amount of homogeneously sized pollen grains (values of standard deviation and coefficient of variation lower then upper limits for diploids — 3μm or 7.5%, respectively), and might probably serve as male parents in further crosses; on the other hand, they are fully seed-sterile. A lectotype of H. × grofae, a second proved nothotaxon in the genus Hieracium s.str., is designated. Localities of H. × grofae are located in subalpine belt of the Marmarosh Mts, the Svydovets’ Mts and the Horhany Mts (all in the Ukrainian Eastern Carpathians). Biotopes of hybrid plants usually represent secondary formed and disturbed pastures allowing close contact of altitudinally vicariant parent taxa.     

Sympatric Epichloë species and chemotypic profiles in natural populations of Lolium perenne

The distribution of different Epichloë species within eight natural populations of Lolium perenne was studied. In total, 40.2% of the asymptomatic plants were infected by Epichloë festucae var. lolii or by Epichloë typhina. Both species occurred in sympatry in seven grass populations, and some plants had dual infections by both taxa. No hybrid taxa such as Epichloë hybrida were detected. Epichloë festucae strains were classified into two morphotypes, M1 and M3, according to culture characters, both morphotypes occurred in sympatry in seven populations. Plants bearing stromata produced by Epichloë typhina were observed, but asymptomatic plants infected by this species also occurred in seven populations. The alkaloid profile of Lolium perenne plants was related to the morphotype of their infecting strains: most plants infected by M3-strains were characterized by lolitrem, and those with M1-strains contained either ergovaline or lolitrem. Plants infected by Epichloë typhina were characterized by high peramine content.     

Metabolomics analysis of the Lolium perenne–Neotyphodium lolii symbiosis: more than just alkaloids?

Above ground plant parts of Lolium perenne often harbour endophytic Neotyphodium lolii fungi. These occur both naturally and commercially, as variant strains are introduced to modify the grass metabolic profile. They reside in the apoplastic spaces and rarely cause visible symptoms of infection. The vast majority of literature has focussed on the biosynthesis, accumulation, and ecological relevance of a limited number of alkaloids produced by N. lolii which have been shown to negatively affect insect pests and vertebrate herbivores. Much less is known about the effects of other metabolites in these interactions or the role of resource supply on metabolic profiles, nor critically on the metabolic consequences of differences in the amount (concentration) of endophyte present. Here, we provide a synthesis of some of our recently published studies on effects of resource supply (nitrogen, carbohydrates) on concentrations of endophytes and endophyte specific metabolites in the L. perenne–N. lolii association. We present results of both quantitative PCR and targeted metabolomics studies, using contrasting endophyte strains in two perennial ryegrass cultivars. We also present and discuss a hypothetical schematic representation of possible links between plant and fungal metabolic networks. A multiple regression analysis of numerical insect responses and metabolic profiles indicates that effects of endophyte infection on insect population sizes could be predicted by concentrations of a range of metabolites other than alkaloids and depended on insect species, fungal strain, and nitrogen supply.     

High-affinity iron uptake is required for optimal Epichloë festucae colonization of Lolium perenne and seed transmission

Epichloë festucae uses a siderophore-mediated system to acquire iron, which is important to maintain endophyte–grass symbioses. Here we investigate the roles of the alternative iron acquisition system, reductive iron assimilation (RIA), via disruption of the fetC gene, which encodes a multicopper ferroxidase, either alone (i.e., ΔfetC) or in combination with disruption of the gene sidA, which encodes a siderophore biosynthesis enzyme (i.e., ΔfetC/ΔsidA). The phenotypic characteristics of these mutants were compared to ΔsidA and wild-type (WT) strains during growth under axenic culture conditions (in culture) and in symbiosis with the host grass, perennial ryegrass (in planta). Under iron deficiency, the colony growth rate of ΔfetC was slightly slower than that of WT, while the growth of ΔsidA and ΔfetC/ΔsidA mutants was severely suppressed. Siderophore analyses indicated that ΔfetC mutants hyperaccumulate ferriepichloënin A (FEA) at low iron concentrations and ferricrocin and FEA at higher iron concentrations. When compared to WT, all mutant strains displayed hyperbranching hyphal structures and a reduced ratio of Epichloë DNA to total DNA in planta. Furthermore, host colonization and vertical transmission through infection of the host seed were significantly reduced in the ΔfetC/ΔsidA mutants, confirming that high-affinity iron uptake is a critical process for Epichloë transmission. Thus, RIA and siderophore iron uptake are complementary systems required for the maintenance of iron metabolism, fungal growth, and symbiosis between E. festucae and perennial ryegrass.     

A DNA marker assay based on high-resolution melting curve analysis for distinguishing species of the Festuca–Lolium complex

The grass breeding industry is interested in a fast and cheap method of identifying contamination in seeds of Italian and perennial ryegrass (Lolium perenne L. and L. multiflorum Lam., respectively). This study shows that high-resolution melting curve analysis in combination with an unlabelled probe assay is an effective method of detecting single nucleotide polymorphisms (SNPs) in diverse Italian and perennial ryegrass backgrounds. This method proved efficient in differentiating ryegrass species and reducing the effect of additional DNA sequence polymorphisms close to the target SNP on the melting curve profiles. For the identification of contamination in Italian and perennial ryegrass seed production, high-resolution melting curve analysis shows great potential, as it is a single closed-tube PCR reaction with an easy workflow, providing results in <2 h after DNA extraction.