Assessing success in gene transfer betweenLolium multiflorum andFestuca arundinacea

Summary AFestuca arundinacea (2n=6x-42) plant with three PGI/2 homoeoalleles marking three homoeologous chromosomes was crossed with aLolium multiflorum plant (2n=4x=28) with a different PGI/2 phenotype to give a pentaploid hybrid (2n=5x=35) with five chromosomes each marked by a different PGI/2 allele. This hybrid plant was backcrossed twice with diploidL. multiflorum (2n=2x=14) with a different PGI/2 phenotype. Numbers of interspecific recombinants involving chromosomes marked by PGI/2 were then determined in both backcross generations. In the BC1, recombinants involving only one PGI/2 allele were found but in the BC2, all three homoeologousF. arundinacea chromosomes carrying the PGI/2 locus recombined withLolium with one in greater frequency and the others in equal but lower frequency. The evidence supports claims made forF pratensis (2n=2x=14) andF. arundinacea var.glaucescens (2n=4x=28) being progenitors forF. arundinacea.     

Breeding for hard red winter wheat cultivars adapted to conventional-till and no-till systems in northern latitudes

Summary Monosomic alien addition lines combining individual F. drymeja chromosomes and the L. multiflorum complement were isolated from the cross between the triploid hybrid L. multiflorum (4x) × F. drymeja (2x) and diploid L. multiflorum (2x). Chromosome pairing in the addition lines was studied at metaphase 1 of meiosis and the relationship between single F. drymeja chromosomes and the corresponding homologous pair in L. multiflorum is discussed. Trivalent frequency in the addition lines was higher than expected from observations of chromosome pairing in the triploid hybrid and there were differences between lines in the number of trivalent associations formed. There is some evidence to suggest that trivalent frequency is not entirely dependent on chromosome length and that transmission of the alien chromosome in the female is dependent on the size of the added chromosome. Morphological studies were made to assess the phenotypic effects of the addition of single F. drymeja chromosomes to the L. multiflorum complement. Two plants (2n=14) with recombination between a L. multiflorum and a F. drymeja chromosome were identified.     

Cytogenetic Studies of Hybrids between Festuca gigantea Vill. and Lolium multiflorum Lam.

The meiotic behaviour of the hybrid between Festuca gigantea (2n – 6x = 42) and Festuca gigantea (2n = 6x = 42) indicates distinct structural differences between the two species. These differences are also apparent in the degree of chromosome pairing observed in Lolium multiflorum×F. gigantea compared with previous reports on the L. multiflorum×F. arundinacea hybrids. Although the L. multiflorum×F. gigantea 8x amphiploid com Dines the complementary characters of the two species and is agronomically interesting, there are some irregularities in meiotic behaviour that could affect the stability of the amphiploid. Seed fertility in the amphiploid is high and preliminary studies show evidence of a high degree of self-fertility. The pentaploid hybrid between autotetraploid L. multiflorum×F. gigantea is sufficiently fertile to be used as the pollen plant in crosses with diploid L. multiflorum (BC₁). Backcrossing the BC₃ hybrid to L. multiflorum results in mainly diploid progeny. The possibilities, of using this crossing scheme to introgress F. gigantea characters into L. multiflorum is discussed as an alternative approach to amphiploid breeding as a means o: combining specific complementary characters of the two species.     

Cytogenetic Studies of Crosses between Lolium multiflorum Lam. and Festuca arundinacea Schreb. III. The Generations C1, C2 and C3

Meiotic observations of 56-chromosome plants of the C₁, C₂ and C₃ generations of the amphidiploid Lolium multiflorum × Festuca arundinacea showed that most of the chromosomes paired as bivalents. However, uni- and multivalents were frequently observed. No difference in meiotic regularity and stability could be detected between the different generations. The average chromosome number and; the percentage of 56-chromosome plants decreased with increasing generations. The direct use of the 56-chromosome amphidiploids of L. multiflorum and F. arundinacea in a breeding programme appears to be quite difficult, but these plants can be used in a backcross programme with Lolium and Festuca.     

Utilization of flow cytometry for festulolium breeding (Lolium multiflorum (2x) × Festuca arundinacea (6x))

Festulolium is a hybrid between Festuca and Lolium species that has valuable agronomic traits from both grass species. The purpose of our breeding program is to produce hexaploid festulolium that introduces tolerance to summer depression into Italian ryegrass (Lolium multiflorum) by crossing it with tall fescue (Festuca arundinacea). However, we found the DNA ploidy of hexaploids was not stable and was reduced in successive generations. We aimed to find out how to obtain stable high-ploidy festulolium. F1 hybrids of L. multiflorum and F. arundinacea were produced. The F3 generation was produced from putative hexaploid F2 individuals by open pollination. The F4 to F6 generations were obtained by polycrossing. The DNA ploidy levels of F2 to F6 individuals were estimated by flow cytometry. Cytological characteristics of the F5 and F6 individuals were investigated by FISH and GISH. The DNA ploidy level of hexaploid festulolium was reduced and stabilized at almost the same level as a tetraploid. Seed fertility was inversely correlated with an increase in ploidy level. GISH revealed no preferential Lolium transmission. FISH with a telomere probe revealed that counting the exact number of chromosomes in festulolium was difficult. DNA ploidy level was strongly correlated with the number of chromosomes.     

Androgenesis as a means of dissecting complex genetic and physiological controls: selecting useful gene combinations for breeding freezing tolerant grasses

The advantages of androgenesis from Lolium × Festuca hybrids as a means towards enhanced gene expression and capture of rare genetic variation are reviewed. New evidence is presented for the technique’s use in combination with introgression-mapping for targeting Festuca-derived genes for enhanced freezing-tolerance. As a starting point, a dihaploid genotype derived by androgenesis from a Lolium multiflorum × Festuca pratensis amphiploid (2n = 4x = 28) hybrid cultivar is used as female parent in a backcross breeding programme with L. multiflorum (2n = 2x = 14). A derivative of the backcross␣breeding programme was a genotype of L.␣multiflorum (2n = 2x = 14) incorporating a F.␣pratensis introgression on chromosome 4 that was more freezing-tolerant than Lolium. New evidence of the importance in Lolium and Festuca species of the adaptive capabilities of Photosystem II (PSII) in relation to subsequent freezing-tolerance, is presented. Non-photochemical quenching (NPQ) mechanisms for expulsion of excess light energy during cold acclimation are found in F. pratensis but not in L. multiflorum. Screens of a backcross population derived from an initial dihaploid genotype (n + n = 14) produced by androgenesis from a L. multiflorum × F.␣pratensis amphiploid, indicate a direct relationship between cold acclimation induced increases in NPQ and freezing-tolerance. Preliminary evidence of a role for genes found on chromosome 4 of F. pratensis for increased NPQ expression, is presented.     

Improved Drought Resistance in Introgression Lines Derived from Lolium multiflorum × Festuca arundinacea Hybrids

In a back-crossing programme involving Lolium multiflorum (the recurrent parent) and Festuca arundinacea, the diploid L. multiflorum phenotype was rapidly recovered with the inclusion, in some progeny, of a small number of genes from the fescue parent. In field drought trials derivatives of these backcross populations were on average less drought resistant than the L. multiflorum parental populations, but 3 % of individuals were as drought resistant as F. arundinacea. After only one cycle of selection and polycrossing these drought-resistant Lolium-like plants, the mean drought resistance of most progeny lines was significantly improved, in some cases to near that of F. arundinacea. Available evidence strongly indicates that this improved drought resistance was due to the transfer of genetic material from Festuca into Lolium. These populations will contribute to variety improvement and to our understanding of the genetics and physiology of drought resistance.     

Cytoplasmic male sterility in ryegrasses (Lolium SPP.) detected after intergeneric hybridization

Summary Among diploid derivatives of a tetraploid intergeneric hybrid originating from a cross between a diploid F₄ hybrid of Lolium perenne x L. multiflorum and an autotetraploid plant of Festuca pratensis as female and male parent, respectively, a great number of male-sterile Lolium plants have been found. The male sterility appears to be based on an interaction of one or two recessive nuclear genes and sterilizing cytoplasm. The data available indicate a high frequency of B types (O types) in perennial ryegrass.     

Intergeneric symmetric and asymmetric somatic hybridization in Festuca and Lolium

Summary Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10–500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.Species-specific sequences from F. arundinacea and L. multiflorum being dispersed and evenly-represented in the corresponding genomes were isolated and used for the molecular characterization of the nuclear make-up of the intergeneric, somatic Festulolium plants recovered. The irradiation of Italian ryegrass protoplasts with 250 Gy X-rays prior to fusogenic treatment favoured the unidirectional elimination of most or part of the donor chromosomes. Irradiation of L. multiflorum protoplasts with 500 Gy produced highly asymmetric (over 80% donor genome elimination) nuclear hybrids and clones showing a complete loss of donor chromosomes.The RFLP analysis of the organellar composition in symmetric and asymmetric tall fescue (+) Italian ryegrass regenerants confirmed their somatic hybrid character and revealed a bias towards recipient-type organelles when extensive donor nuclear genome elimination had occurred.Approaches aimed at improving persistence of ryegrasses based on asymmetric somatic hybridization with largely sexually-incompatible grass species (F. rubra and Alopecurus pratensis), and at transferring the cytoplasmic male sterility trait by intra- and inter-specific hybridization in L. multiflorum and L. perenne, have been undertaken.Abbreviations cpDNA chloroplast DNA - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxy-acetic acid - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Androgenesis in a Lolium multiflorum × Festuca arundinacea hybrid to generate genotypic variation for drought resistance

The pentaploid hybrid of Lolium multiflorum and Festuca arundinacea (2n = 5x = 35) combines the high growth rate of L. multiforum with the drought resistance and freezing-tolerance of F. arundinacea. Unfortunately, it also displays the deleterious traits associated with Festuca, namely those associated with high leaf fibre content giving rise to poor palatability and digestibility. To access different combinations of these characters, anther cultures were initiated and regenerated into single embryo derived plants. The anther culture method was very productive since out of a total of 2349 androgenic plants derived from the same parent plant, 57% were green plantlets, although only 507 (22%) subsequently established into plants following transfer to soil. Chromosome counts of randomly selected lines showed that plants with euploid chromosome numbers (14, 21, and 28) would appear to have selective advantage during regeneration. There was wide variation between mature androgenic plants grown under field conditions in plant height, leaf length, leaf width, tiller number and herbage dry matter. The variation between genotypes in response to drought stress was assessed by placing replicate clones under rain-out shelters or under irrigated control conditions in the field. Herbage dry matter under drought was higher in a number of androgenic lines than either parents, but not higher than the pentaploid hybrid. Androgenesis was shown to be a highly effective procedure to expose diverse phenotypic variation all derived from the same Lolium × Festuca hybrid genotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Androgenesis from a Lolium multiflorum × Festuca arundinacea hybrid to generate extreme variation for freezing-tolerance

New opportunities for plant breeding using androgenesis in Lolium × Festuca hybrids have been identified. Plants derived by anther culture from a Lolium multiflorum × Festuca arundinacea (5x) hybrid were screened for freezing-tolerance, and their post-freezing recovery compared. The androgenic population showed extreme diversity in freezing-tolerance. While the majority of androgenic plants had inferior freezing-tolerance compared with the freezing-sensitive L. multiflorum parent, 6% of the population were more freezing resistant than the freezing-tolerant Festuca parent. Novel Lolium and Festuca gene combinations resulting from rare meiotic events were recovered within the androgenic population. The two most freezing-tolerant androgenic plants carried virtually the entire F. pratensis subgenome of F. arundinacea. F. pratensis is known to carry genes for freezing-tolerance and would be expected to be the primary source of genes governing this trait within the F. arundinacea genome. The most freezing-tolerant androgenic plants were more freezing-tolerant than the hybrid plant from which they were derived. Consequently, androgenesis was also effective in removing factor(s) reducing the expression of freezing-tolerance within the L. multiflorum × F. arundinacea (5x) hybrid.     

Genomic constitution of Festulolium cultivars released in the Czech Republic

Genomic in situ hybridization (GISH) was used to characterize the chromosome constitutions of individual plants from a set of tetraploid and hexaploid cultivars of Festulolium developed and released in the Czech Republic from hybrids of Lolium multiflorum with Festuca pratensis and F. arundinacea. A simplified GISH protocol readily discriminated parental genomes in the hybrids and facilitated the screening of large numbers of plants per accession. The contribution of parental genomes in the cultivars tested ranged from predominance of chromatin from one of the parents to a more balanced contribution from both parents. However, in none of the cultivars were equal proportions of chromatin from both parents present. The parental contribution to the hybrids was both in the form of complete chromosomes or as chromosome translocations. In hexaploid cultivars from (L. multiflorum × F. arundinacea) × F. arundinacea hybrids the average numbers of complete L. multiflorum chromosomes ranged from 4.95 to 7.5 and the numbers of translocations from 6.33 to 10.21. Two tetraploid cultivars from (L. multiflorum × F. arundinacea) × L. multiflorum hybrids showed a strong prevalence of L. multiflorum chromatin and intergeneric translocations were rare. In the tetraploid cultivar ‘Perun’ of the L. multiflorum × F. pratensis hybrid there were 11.7 chromosomes of L. multiflorum and 14.7 recombined chromosomes on average. Reasons for the domination of one of the parental genomes in hybrid cultivars are not clear and are only partially explained by breeding history. Recombination rates of individual genomes in hybrids involving F. arundinacea were evaluated in double hybridization experiments. The results indicated a strong affinity of the L. multiflorum genome for the F. pratensis genome present in F. arundinacea and little affinity for the F. glaucescens genome. This suggests that introgressions from F. arundinacea into L. multiflorum are primarily limited to the F. pratensis genome which can be more readily accessed in L. multiflorum × F. pratensis hybrids.     

Culture of embryos and shoot tips for chromosome doubling in Lolium perenne and sterile hybrids between Lolium and Festuca

An improved method is reported for polyploid induction in Lolium (ryegrass), and in sterile F₁ hybrids between Lolium and Festuca (fescue). Two factors greatly increased the survival rate of colchicine‐treated embryos of Lolium perenne (perennial ryegrass) germinated and cultured in vitro (1) a high concentration of sucrose (100 g/1) in the germination medium and (1), maintenance at a low temperature of 10°C for 2 weeks after treatment. The maximum success rate for chromosome doubling among survivors of perennial ryegrass was 79.1%, and for L. perenne×Festuca arundinacea F₁ hybrid embryos it exceeded 40%. The same doubling treatment also works with shoot tip culture in non‐ flowering genotypes obtained by anther culture of L. multiflorum×F. arundinacea hybrids.     

Cytological and Isozyme Evaluation of Tall Fescue × Italian Ryegrass Hybrids

Development of tall fescue (Festuca arundinacea Schreb., 2n = 6x - 42) × Italian rye-grass (Lolium multiflorum Lam., 2n = 2 ×= 14) hybrids would enhance efforts to improve the quality of tall fescue. Two ‘Kentucky 31’ tall fescue בLemtal Italian ryegrass hybrids were obtained via embryo rescue on MS media containing casein hydrolysate, ascorbic acid and sucrose. Chromosome pairing at metaphase I had an average of more than 12 bivalents per cell. Since Festuca-Lolium pairing can account only for seven of the paired chromosomes, intergenomic as well as interspecific chromosome pairing is indicated. There was no cytoplasmic effect on chromosome pairing. To determine if enzymes could be used as genetic markers for distinguishing hybrids from self-contaminants in crosses, zymograms of PGI, 6-PGD, MDH, GOT and ACPH were obtained from parents and hybrids using starch gel etectrophoresis. PGI, 6-PGD and MDH had fewer bands in the diploid ryegrass, as compared with the hexaploid tall fescue and the tetraploid hybrid.     

Introgression of crown rust resistance from Festuca spp. into Lolium multiflorum

Crown rust, Puccinia coronata Corda, causes one of the most damaging foliar diseases in Italian ryegrass, Lolium multiflorum Lam. For introgression of crown rust resistance, highly resistant hybrids of the crosses Festuca arundinacea (2n = 6x = 42) ×L. multiflorum (2n = 4x = 28) and reciprocally, L. multiflorum (2n = 2x = 14) ×Festuca pratensis (2n = 4x = 28) and subsequently resistant recombinant individuals were used as female parents and susceptible cultivars of Italian ryegrass as male parents in three successive backcrosses. The BC₃ plants were selfed and crossed mutually. Uredospores of seven different crown rust isolates collected from plants of L. multiflorum, Lolium perenne, F. pratensis, F. arundinacea and L. multiflorum × F. pratensis hybrids were applied to identify the resistance or susceptibility of the parental species, backcrossed, selfed and intercrossed progenies. The various crown rust populations revealed a species-specific capability to infect plants of the Lolium-Festuca complex corresponding to the host species from which the spores originated. Selected BC⁹ plants, however, were found to be completely resistant to all crown rust populations tested. Successful introgression of the resistance was achieved from F. arundinacea as well as from F. pratensis. The resistance represents a dominant character, apparently based on a strong heterologous incompatibility between host and pathogen. In phenotype, bivalent formation during meiosis and in fertility, the novel germplasms are comparable with the L. multiflorum cultivars.     

Estimation of introgression rate of a fescue isozymic marker into tetraploid Italian ryegrass at early generations of backcross

The potential for introgression of Festuca glaucescens into tetraploid ryegrass Lolium multiflorum was assessed using a simplex homeoallele at the phosphoglucoisomerase isoenzyme locus (Pgi-2). Overall, the F. glaucescens species-specific marker was found to be transmitted into Italian ryegrass at a rate close to 0.5 suggesting a high rate of homeologous pairing in BC1 and of intergeneric recombination in BC2. Significant variations in transmission rate among progenies in advanced generations indicated that recombination could result from various chromosome rearrangements possibly involving more than one fescue chromosome with some selection against duplex-like introgressed genotypes. Hence, introgressing F. glaucescens genes into tetraploid ryegrass appeared as further procedure to exploit versatility in fescue species, mostly restricted so far to introgression from F.arundinacea or F. pratensis into diploid ryegrass. Implications in terms of breeding strategy and marker-assisted selection are briefly discussed in respect to the genetic control of traits of interest to be introgressed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Disturbed Mendelian segregations at isozyme marker loci in early backcrosses of Lolium multiflorum × Festuca pratensis hybrids to L. multiflorum

Summary The segregation of interspecific recombinant Festuca pratensis (Fp) alleles, introgressed into the germolasm of Lolium multiflorum (Lm), at four loci (PGI/2, AcP/2, GOT/3 and BAP) is described. Heterozygous (Lm/Fp) plants were backcrossed to L. multiflorum (2n=2×=14) and subsequent BC2 Lm/Fp sibling genotypes intercrossed.In crosses between BC1 heterozygous plants (Lm/Fp) used as males and L. multiflorum, there was a reduced transmission of the F. pratensis (Donor Parent) derived alleles in the populations with PGI/2 and AcP/2 marker loci compared to the reciprocal cross but the reduction was not significantly different in those with GOT/3 and BAP markers.Two PGI/2 marked BC2 half-sib families in particular exhibited a more extreme deficiency of Fp/Fp progeny plants than anticipated from the BC2 segregations indicating possible linkage to zygotic lethals. Deficiencies of F. pratensis alleles were, in most cases, less marked in BC2 half-sib families indicating that a further round of recombination had reduced the size of the introgressed chromosome segment or that deleterious linkages had been broken. A tendency towards heterozygote advantage was found in one BAP marked halfsib family.The significance for forage grass breeding of reduced transmission rates of Donor Parent alleles in early back-cross generations especially through the male gametes is discussed.     

Occurrence of numerically unreduced (2n) gametes in Alstroemeria interspecific hybrids and their significance for sexual polyploidisation

The F1 hybrids of seven diploid Alstroemeria species (2n=2x=16) were investigated for the production of numerically unreduced (2n) gametes and their mode of origin. Based on a survey of 17 interspecific hybrid combinations,consisting of 119 genotypes, it was found that the F1 hybrids of Chilean-Brazilian species mostly produced first division restitution (FDR) 2n gametes. These F1 hybrids were self-pollinated in order to obtain F2 seeds, which was an indication that the F1 plants also produced 2neggs simultaneously. All the F2 progeny plants were typical allotetraploids, most of which formed 16 bivalents and a small proportion formed multivalents during metaphase I stages of meiosis. Through genomic in situ hybridisation (GISH) it was proved that multivalent formation in F2plants, derived from A. inodora ×A. pelegrina hybrid, was due to homoeologous recombination but not from reciprocal translocations. In order to test the segregation pattern of the recombinant chromosomes, an F3 population from one genotype, P6C49-6, was investigated. The recombinant chromosomes assorted independently from each other supporting the hypothesis that the segregation of chromosomes in ring quadrivalents did not behave like those in translocation heterozygotes. It was concluded that in allopolyploids of Alstroemeria,bilateral sexual polyploidisation could accomplish genetic recombination by both homoeologous crossing-over as well as through the assortment of chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of Lolium. I. Identification of linkage groups and the establishment of a genetic map

A genetic map of Lolium has been produced using isozyme, restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers applied to a segregating family derived from an F₁ hybrid plant of L. perenne × L. multiflorum provenance, crossed on to a doubled haploid L. perenne. A total of 106 markers, out of a total of 160 polymorphic loci analysed, have been ascribed to seven linkage groups covering a map distance of 692cM, Two of these groups may be allocated to chromosomes 2 and 6 of the Lolium genome. The remaining unallocated markers, the majority of which showed severe segregation distortion, could be associated into small groups of two or three markers which showed no linkage with the main groups at a LOD of 2.8 or, if associated, could not be mapped in a satisfactory manner. This high incidence of disturbed segregations could be accounted for by the use of an interspecific hybrid between two species of differing genome size, with consequent cytological imbalance.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.