Genetic characterization of androgenic progeny derived from Lolium perenne x Festuca pratensis cultivars

Androgenesis and genetic characterization of androgenic progeny from Lolium perenne x Festuca pratensis cultivars (2n = 4x = 28) were investigated in order to develop a novel grass that combines favourable attributes of parent plants. A successful androgenesis was obtained using PG-96 medium. The green plant regeneration was 46%, 35% and 17% for Bx350, Bx351 and Prior, respectively, and over 800 green plants have been obtained. Androgenic progeny showed a large variation in freezing tolerance, 7% of 292 progeny exceeding that of freezing hardy F. pratensis despite containing chromosomes of L. perenne. More than 60% of 175 flowering progeny produced dehiscent anthers with pollens. Androgenic plants contained 14 or 28 chromosomes. There were 188 (56%), 204 (77%) and 114 (81%) dihaploids from Bx350, Bx351 and Prior, respectively. The nuclear DNA content varied significantly even between plants with the same chromosome number. High levels of chromosome pairing and recombination were observed by genomic in situ hybridization analysis because of close homology between two genomes. The results indicated that androgenic progeny of Festulolium showed high level of genetic variation, and provide potential for accelerating selection efficiency.     

Growth Studies on Natural Hybrids between Lolium perenne and Festuca pratensis

The growth-rates and chromosome numbers of plants taken froma natural population of hybrids between perennial rye grassand meadow fescue were determined. The growth-rates of diploidhybrids resembled that of rye grass while those of the triploidswere similar to that of meadow fescue. Experiments on plantsgrown singly and in competition showed that fescue was adverselyaffected by low temperature and rye grass by low light and alsoby the reduced nutrient supply arising from competition. The persistence of the sterile hybrids is explained by theirrecurrent formation coupled with greater longevity and vegetativespread, arising from growth characteristics intermediate betweenthose of their parents. The persistence of rye grass and thedisappearance of fescue when periodically grazed appear to resultfrom the earlier growth and more rapid rate of tillering ofthe former. However, rye grass has fewer and smaller meristemsavailable for regrowth if allowed to grow until anthesis, andunder these conditions fescue dominates. The intermediate growthcharacters of the hybrids allow them to survive spring grazingmore readily than meadow fescue and cutting for hay more readilythan rye grass.     

Physical and genetic mapping in the grasses Lolium perenne and Festuca pratensis

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. In this line recombination occurs throughout the length of the F. pratensis/L. perenne bivalent. The F. pratensis chromosome and recombinants between it and its L. perenne homeologue can be visualized using genomic in situ hybridization (GISH). GISH junctions represent the physical locations of sites of recombination, enabling a range of recombinant chromosomes to be used for physical mapping of the introgressed F. pratensis chromosome. The physical map, in conjunction with a genetic map composed of 104 F. pratensis-specific amplified fragment length polymorphisms (AFLPs), demonstrated: (1) the first large-scale analysis of the physical distribution of AFLPs; (2) variation in the relationship between genetic and physical distance from one part of the F. pratensis chromosome to another (e.g., variation was observed between and within chromosome arms); (3) that nucleolar organizer regions (NORs) and centromeres greatly reduce recombination; (4) that coding sequences are present close to the centromere and NORs in areas of low recombination in plant species with large genomes; and (5) apparent complete synteny between the F. pratensis chromosome and rice chromosome 1.     

A demonstration of a 1:1 correspondence between chiasma frequency and recombination using a Lolium perenne/Festuca pratensis substitution

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. The chromatin of F. pratensis and L. perenne can be distinguished by genomic in situ hybridization (GISH), and it is therefore possible to visualize the substituted F. pratensis chromosome in the L. perenne background and to study chiasma formation in a single marked bivalent. Recombination occurs freely in the F. pratensis/L. perenne bivalent, and chiasma frequency counts give a predicted map length for this bivalent of 76 cM. The substituted F. pratensis chromosome was also mapped with 104 EcoRI/Tru91 and HindIII/Tru91 amplified fragment length polymorphisms (AFLPs), generating a marker map of 81 cM. This map length is almost identical to the map length of 76 cM predicted from the chiasma frequency data. The work demonstrates a 1:1 correspondence between chiasma frequency and recombination and, in addition, the absence of chromatid interference across the Festuca and Lolium centromeres.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Neotyphodium Endophyte Changes Phytoextraction of Zinc in Festuca arundinacea and Lolium perenne

The effect of Neotyphodium endophytes on growth parameters and zinc (Zn) tolerance and uptake was studied in two grass species of Festuca arundinacea and Lolium perenne. Plants were grown under different Zn concentrations (control, 200, 400, 800, and 1800 mg kg^?1) in potted soil for 5 months. The results showed that the number of plant tillers was 85 and 51% greater in endophyte infected Festuca (FaEI) and Lolium (LpEI), respectively, compared to their endophyte free (EF) plants. Roots and shoots dry weights in infected Festuca were 87 and 9% greater than non-infected counterparts but in opposite, EF Lolium had 47 and 8% greater root and shoot dry weights than LpEI. Endophyte infected Festuca and Lolium improved chlorophyll fluorescence as Fv/Fm at high concentrations of Zn, showing their better chlorophyll functions and significant reduction of Zn stress in endophyte infected plants. Shoots of endophyte infectedFestuca had 82% greater concentration of Zn than EF Festuca when grown in soil containing 1800 mg kg^?1 Zn. Festuca and Lolium may tolerate high Zn concentration in soil without reduction in shoot and root growth. Endophyte infection in Festuca may help the grass accumulate and transport more Zn in aboveground parts under Zn-stress, thereby aiding phytoremediation of contaminated soils.     

Genomic in situ hybridization (GISH) reveals high chromosome pairing affinity between Lolium perenne and Festuca mairei.

Intergeneric hybridizations have been made between species of Lolium and Festuca. It has been demonstrated, largely through conventional cytogenetic analysis, that the genomes of the two genera are related, however, much information is lacking on exactly how closely related the genomes are between the two species. We applied genomic in situ hybridization (GISH) techniques to the F1 hybrids of tetraploid Festuca mairei with a genomic constitution of M1M1M2M2 and diploid Lolium perenne with a genomic constitution of LL. It was shown in the triploid hybrids (LM1M2) that the chromosomes of M1 and M2 from F. mairei could pair with each other, and it was further discovered that L chromosomes of L. perenne paired with M1 and M2 chromosomes. Our results showed that meiocytes of Lolium-Festuca are amenable to GISH analysis, and provided direct evidence for the hypothesis that the chromosomes of Lolium and Festuca may be genetically equivalent and that reciprocal mixing of the genomes may be possible.     

Meiosis and fertility of reciprocal triploid hybrids of Lolium multiflorum with Festuca pratensis

Diploid and tetraploid forms of Lolium multiflorum and Festuca pratensis were crossed under controlled conditions and after embryo rescue all four combinations of autoallotriploid hybrids were obtained. Male and female fertility and chromosome pairing at metaphase I of meiosis were studied in several plants from each hybrid combination. The hybrids with two genomes of L. multiflorum and one of F. pratensis (genomic formulae LmLmFp and FpLmLm) were male and female fertile while the hybrids with two genomes of F. pratensis and one of L. multiflorum had a reduced fertility (FpFpLm) or were completely sterile (LmFpFp). Chromosome pairing at metaphase I varied among hybrid combinations depending on their genomic composition. LmLmFp and FpLmLm hybrids had similar patterns of pairing (1.83I + 5.29II + 2.85III and 2.22I + 5.22II + 2.75III, respectively) but they differed from those of FpFpLm (3.65I + 4.65II + 2.68III) and especially from LmFpFp (4.78I + 5.87II + 1.49III). Conventional analysis of meiosis failed to explain the differences in chromosome behaviour and fertility/sterility levels between the autoallotriploid hybrids with two Lolium or two Festuca genomes.     

Alien introgression in the grasses Lolium perenne (perennial ryegrass) and Festuca pratensis (meadow fescue): the development of seven monosomic substitution lines and their molecular and cytological characterization

Background and Aims

To address the issues associated with food security, environmental change and bioenergy in the context of crop plants, the production, identification and evaluation of novel plant phenotypes is fundamental. One of the major routes to this end will be wide hybridization and introgression breeding. The transfer of chromosomes and chromosome segments between related species (chromosome engineering or alien introgression) also provides an important resource for determining the genetic control of target traits. However, the realization of the full potential of chromosome engineering has previously been hampered by the inability to identify and characterize interspecific introgressions accurately.

Methods

Seven monosomic substitution lines have been generated comprising Festuca pratensis as the donor species and Lolium perenne as the recipient. Each of the seven lines has a different L. perenne chromosome replaced by the homoeologous F. pratensis chromosome (13 L. perenne + 1 F. pratensis chromosome). Molecular markers and genomic in situ hybridization (GISH) were used to assign the F. pratensis chromosomes introgressed in each of the monosomic substitutions to a specific linkage group. Cytological observations were also carried out on metaphase I of meiosis in each of the substitution lines.

Results

A significant level of synteny was found at the macro-level between L. perenne and F. pratensis. The observations at metaphase I revealed the presence of a low level of interspecific chromosomal translocations between these species.

Discussion

The isolation of the seven monosomic substitution lines provides a resource for dissecting the genetic control of important traits and for gene isolation. Parallels between the L. perenne/F. pratensis system and the Pooideae cereals such as wheat, barley, rye, oats and the model grass Brachypodium distachyon present opportunities for a comparison across the species in terms of genotype and phenotype.     

The genetics of meiotic chromosome pairing in Lolium temulentum x Lolium perenne tetraploids

Summary The degree of preferential pairing of homologous chromosomes was estimated in a series of tetraploid hybrids of Lolium temulentum x Lolium perenne by means of cytological and genetic analyses. The correlations between the frequency of bivalents at first metaphase of meiosis in the hybrid tetraploids and the degree of preferential pairing calculated from the segregation pattern of isozyme alleles in a test cross was extremely high. The results showed clearly that suppression of heterogenetic pairing in these Lolium tetraploids is achieved by a genetic system involving the A chromosomes as well as the B chromosome system which has been known for some time. Certain similarities with the genetic system controlling pairing in polyploid wheats are discussed.     

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.     

A decade of "chromosome painting" in Lolium and Festuca

GISH has been a particularly useful technique for studying the Lolium-Festuca species complex of forage grasses. The reason for this utility is two-fold: (i) the complex is unique amongst crop plants in which fertile hybrids, and backcross progenies, can be produced which recombine genomes and promiscuously exchange their genes through homoeologous recombination; (ii) dispersed repetitive DNAs differ between species, and this allows tracking of the identity of chromosomes and chromosome segments. This tracking property has enabled several fruitful lines of research to produce a harvest of new information for both fundamental and practical purposes. We review this first decade of GISH (genomic in situ hybridization) in Lolium-Festuca, and discuss and summarize the achievements which have accrued.     

Anther Culture of Lolium temulentum, Festuca pratensis and Lolium x Festuca Hybrids. II. Anther and Pollen Development In Vivo and In Vitro

The various pathways of pollen development were investigatedin cultured anthers of Lolium temulentum, Festuca pratensisand the L. multiflorum x F. pratensis hybrid ‘Elmet’.In all three, development from the vegetative cell was the predominantpathway of pollen callus development. However, there were characteristicdifferences in the behaviour of the generative cell. In L. temulentumit remained attached to the pollen wall and degenerated, whereasin F. pratensis it divided. In ‘Elmet’ it detachedfrom the pollen wall and remained undivided. Both polarizedand unpolarized partitioned calluses were observed. Developmentof the fusion product of the vegetative and generative nucleiwere also observed in anthers of L. temulentum. Anomalous grainswere not found to be major source of pollen calluses. Sections of anthers of L. temulentum were used to investigatethe origin of S pollen grains, the small pale-staining grainswhich denote pollen dimorphism. Such grains form out of contactwith the tapetum and are therefore determined before or duringmeiosis (i.e. before harvest of anthers for culture). Sectionswere also used to demonstrate the influence of the durationof pretreatment on the development of the middle layer of theanther wall. Festuca pratensis, Lolium temulentum, Lolium x Festuca, anther culture, haploid, microspore, pollen     

Genetic segregation in relation to chromosome pairing in tetraploid hybrids between Lolium perenne and L. multiflorum

Summary Segregation at one of the loci controlling tiller-base pigmentation was studied to determine the mode of inheritance in tetraploid hybrids between Lolium perenne and L. multiflorum. The results could be explained by tetrasomic inheritance and thus did not support previous reports of a degree of preferential chromosome pairing in this material. However, double reduction and aneuploidy may to some extent have masked any tendency to disomic segregation brought about by preferential pairing. Moreover, there was significant heterogeneity between families in the segregation ratios which may indicate genetically controlled differences in pairing behaviour. The results are related to previous cytological and genetic studies.     

Genetic control of androgenetic response in Lolium perenne L.

In a study of androgenesis in 90 Norwegian genotypes of perennial ryegrass (Lolium perenne L.), heritabilities ranged from h _b ² =0.46 to 0.80. Very high or completely positive genotypic correlations were found between most characters of androgenetic response (e.g. embryo-like structures per 100 anthers, plants per 100 embryo-like structures, albino plants per 100 anthers, green plants per 100 anthers). Three genotypes, 2 Norwegian (7-5 and 9-5) and 1 Danish (245), which had significantly different androgenetic responses were selected to study the genetic control of the processes. Genotypes 7-5 and 9-5 were highly embryogenie, 7–5 and 245 were relatively high producers of green plants, while 9-5 was unable to produce green plants. The six possible reciprocal crosses between these three genotypes were made, and 10 or 11 F₁ plants from each cross were used for anther culture experiments. The cross 7-5 x 245 showed average superiority over both parents for total plant regeneration and green plant production, results not previously reported. The phenotypic correlations estimated among progenies from the crosses ranged from r=-0.99^*** to 0.81^***. These considerable changes, relative to the results of the screening experiment, are most likely the result of changed allele frequencies caused by the strong selection of parents in these crosses, and a relatively simple genetical control. This is also inferred from the large transgressive segregation observed.Abbreviations ANT anthers - ELS embryo-like structures - ALB albino plants - GRP green plants - DH doubled haploid plants     

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

 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 sid ^y ) 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. Received: 21 September 1998 / Accepted: 19 November 1998     

QTL mapping of protein content in rice using single chromosome segment substitution lines

Protein content (PC) is an important component of rice nutritional quality. In order to better understand the genetic basis of this trait and increase related breeding efficiency, 21 single chromosome segment substitution (SCSS) lines grown in four sites over two growing seasons (regarded as eight environments) were used to associate PC with particular chromosome segments. Segments from 15 chromosomes were found to contain quantitative trait loci (QTLs) for PC in at least one environment. These included segments from chromosome 3 and 8, in which QTLs for PC had not previously been identified. The segment of chromosome 8 in CSSL-48 had the largest positive effect across all environments. The interaction between substitution and environment was highly significant. Some substitutions had large effects in one environment, but no effect in another (i.e. CSSL-08 and CSSL-17), while some substitutions significantly increased PC in one environment but decreased it in another (i.e. CSSL-41 and CSSL-43). By biplot and clustering analysis, the eight environments were grouped into two contrasting environment types, that is, Hainan and Jiangsu. The segment of chromosome 8 in CSSL-48 had PC-enhancing QTLs in both of the environment types. The segments in CSSL-34 had QTLs which increase PC in the Jiangsu environment but have no effect in the Hainan environment. For enhancing PC, CSSL-48 could be explored in breeding for wide adaptation across all environments, while CSSL-12, CSSL-14, CSSL-17, CSSL-41 and CSSL-43, and that in CSSL-34 could be explored in breeding for specific adaptation to the Hainan and Jiangsu environments, respectively. Near isogenic lines are under development to validate the QTLs with large effects in a range of genetic backgrounds relevant to Jiangsu and Hainan breeding programs. Secondary mapping populations are also being developed for further localising the responsible QTLs in CSSL-14, CSSL-34 and CSSL-48.