W. G. Morgan

Discriminating the ancestral progenitors of hexaploid Festuca arundinacea using genomic in situ hybridization

The phylogeny of Festuca arundinacea Schreb. (2n = 6x = 42) was determined using GISH. Total genomic DNA of putative ancestral species was labelled with rhodamine and hybridized to chromosome preparations of hybrids involving these species and F. arundinacea. The degree of hybridization to chromosomes known to be homologous to the probe DNA was compared with that found simultaneously on chromosomes of the genome of F. arundinacea. It was concluded that the tetraploid species Festuca arundinacea var. glaucescens contributed two genomes and the diploid species Festuca pratensis one, to create the allohexaploid species F. arundinacea.

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.

Designing grasses with a future - combining the attributes of Lolium and Festuca

Lolium species (considered the ideal grasses for European agriculture) are not sufficiently robust to meet many of the environmental challenges that face extensive agriculture in less favoured areas. Fortunately, adaptations to abiotic and biotic stresses exist amongst Festuca species related closely to Lolium. The complex of species has an enormous wealth of genetic variability and potentiality for genetic exchange, thus offering unique opportunities for the production of versatile hybrid varieties with new combinations of useful characters suited to modern grassland farming. The attributes of Lolium and Festuca can be combined into a single genotype by amphiploidy or alternatively, a limited number of characters can be selectively introgressed from Festucainto Lolium or vice versa. Androgenesis of the interspecific hybrids can generate genotypes combining characters that may not be recovered by sexual backcrossing. Genomic in situ hybridization(GISH) can differentially ‘paint’ the chromosomes of Lolium and Festuca and identify Lolium-Festuca recombinant chromosomes. GISH is valuable in the analysis of amphiploids, introgressions and androgenic genotypes and can be used to physically map introgressed traits. Introgression mapping is a powerful new approach to the mapping of traits and arises from a fusion of physical and genetic mapping. For example, in a diploidLolium introgression genotype with only one introgressed Festucasegment, the gene(s) for any Festucaderived trait expressed by the plant must be located within the segment. Using GISH and molecular markers, a dense but highly localised map of the Festuca segment is made in isolation of the Loliumgenome – this may simplify QTL analysis.

Comparison of ribosomal DNA sites inLolium species by fluorescencein situ hybridization

The position of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of sevenLolium taxa. 18S-5.8S-26S sites were seen on two pairs of chromosomes in the inbreeding taxa. In the outbreeding taxa six sites were found in theL. multiflorum, seven inL. perenne and nine inL. rigidum var.rigidum. Two 5S sites were found in each of the taxa. In the inbreeders, the 5S sites were found adjacent to the 18S-5.8S-26S sites on chromosome 2. InL. multiflorum andL. perenne the 5S sites were on the short arm of chromosome 3. However, inL. rigidum var.rigidum the 5S rDNA site was found in either of the two positions.

Expression of the stay-green character introgressed into Lolium temulentum Ceres from a senescence mutant of Festuca pratensis

Abstract A mutant allele at the nuclear locus sid confers indefinite greenness on senescing leaves of the pasture grass Festuca pratensis. Via the bridging species Lolium multiflorum and a programme of backcrossing and selfing, the mutant allele (designated sidy) was introgressed into Lolium temulentum Ceres. The latter is a fast-growing, annual, inbreeding model grass with many advantages over the slower, perennial, genetically heterogeneous outbreeder F. pratensis. Analyses of photosynthetic pigments, total leaf proteins and individual plastid polypeptide components in senescing attached and detached leaves of yy, yY and YY plants confirmed that the stay-green phenotype of yy F. pratensis had been successfully introduced into the L. temulentum background.

Gross chromosome rearrangements are occurring in an accession of the grass Lolium rigidum.

Chromosome structure was analysed at mitosis in root tip meristems of eight genotypes of Lolium rigidum. FISH revealed changed positions in the rDNA sites indicating extensive chromosome rearrangements; indeed no two genotypes were the same. In one genotype, there were differences between cells within individual root tips. The changed positions of the rDNA sites appear to be reflections of chromosome translocations and this was confirmed by the presence of quadrivalents at metaphase I of meiosis. Possible mechanisms are discussed for this exceptional level of chromosome instability.

Introgression mapping in the grasses. II. Meiotic analysis of the Lolium perenne/ Festuca pratensis triploid hybrid

Lolium perenne/Lolium perenne/Festuca pratensis triploid hybrids are a potentially important source of material for determining the genetic control of agronomically and scientifically important characters and determining how genetic maps relate to physical maps. The evolutionary relationship between L. perenne (Lp) and F. pratensis (Fp) is, however, unclear. In order to determine the genomic relationships between Lp and Fp, genomic in situ hybridization (GISH) and the Chapman and Kimber mathematical model (1992) were used to analyse metaphase I in meiocytes of an LpLpFp triploid hybrid. Both analyses clearly demonstrated that recombination occurs preferentially between the homologous Lp genomes in the LpLpFp triploid, indicating that the genomes of the two species have diverged. Direct analysis of homologous vs. homoeologous chromosome association, as measured by GISH, was in broad agreement with the mathematical analysis. It is therefore concluded that the Chapman and Kimber model (1992) is a valid means of assessing chromosome pairing in these triploid hybrids. The significance of the data for elucidating the closeness of the relationship of L. perenne and F. pratensis is discussed.

Introgression of chromosomes of Festuca arundinacea var. glaucescens into Lolium multiflorum revealed by genomic in situ hybridisation (GISH)

Abstract An F1 hybrid (n=4x=28) between the tetraploid species Festuca arundinacea var. glaucescens (GGG′G′) and a synthetic tetraploid Lolium multiflorum (LmLmLmLm) was backcrossed to diploid L. multiflorum to produce triploid (2n=3x=21) BC1 hybrids (LmLmG). At metaphase I of meiosis the triploids had a preponderance of ring bivalents and univalents with some linear and frying-pan trivalents. Genomic in situ hybridisation (GISH) differentiated the Festuca chromosomes from Lolium and revealed that the bivalents were exclusively between Lolium homologues, while the univalents were Festuca. Despite the limited amount of homoeologous chiasmata pairing in the triploids, some recombinant chromosomes were recovered in the second backcross when the hybrids were further crossed to diploid L. multiflorum. The progeny from the second backcross was predominantly diploid. Genotypes with recombinant chromosomes and chromosome additions involving an extra Festuca chromosome were identified using GISH. Changes in plant phenotype were related to the presence of Festuca chromatin.

Intergenomic translocations and the genomic composition of Avena maroccana Gdgr. revealed by FISH

Fluorescencein situ hybridization (FISH) using total genomic DNA from putative diploid progenitors was used to confirm the presence of the A and C genomes inAvena maroccana. These results confirm cytological data that intergenomic translocations are present inA. maroccana.

Genetic and physical analysis of a single Festuca pratensis chromosome segment substitution in Lolium perenne

Abstract. Molecular marker analysis and genomic in situ hybridisation (GISH) were used to examine the process of chromosome segment introgression in BC2 diploid hybrids (2n=2x=14) between Lolium perenne and Festuca pratensis. Two genotypes having what appeared to be the same, single, introgressed chromosome segment of F. pratensis in the L. perenne background were crossed with diploid L. perenne to produce a recombinant series for the introgressed region. Physical and genetic analysis of this series showed that, while recombination seemed to be possible at all points along the chromosome arm, the rate of recombination varied depending on relative position: more recombination was detected in the interstitial region as compared with the centromeric or telomeric regions. The implications of these results for the use of GISH and molecular marker analysis in the measurement of linkage drag in backcross breeding programmes is discussed.