Molecular tagging of a senescence gene by introgression mapping of a stay-green mutation from Festuca pratensis

* Intergeneric hybrids between Lolium multiflorum and Festuca pratensis (Lm/Fp) and their derivatives exhibit a unique combination of genetic and cytogenetic characteristics: chromosomes undergo a high frequency of homoeologous recombination at meiosis; the chromosomes of the two species can easily be discriminated by genomic in situ hybridization (GISH); recombination occurs along the entire length of homoeologous bivalents; a high frequency of marker polymorphism is observed between the two species. * This combination of characters has been used to transfer and isolate a F. pratensis chromosome segment carrying a mutant 'stay-green' gene conferring a disrupted leaf senescence phenotype into L. multiflorum. * The genetic location within the introgressed F. pratensis segment of the senescence gene has been mapped using amplified fragment length polymorphisms (AFLPs), and F. pratensis-specific AFLP markers closely flanking the green gene have been cloned. * The use of these cloned sequences as markers for the stay-green locus in marker-assisted selection programmes has been tested. The potential application of Lm/Fp introgressions as a tool for the map-based cloning of introgressed Fp genes is discussed.     

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.     

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

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.     

Genetic regulation of diploid-like chromosome pairing in the hexaploid species,Festuca arundinacea Schreb. and F. rubra L. (Gramineae)

The basis of diploid-like chromosome pairing in hexaploid (2n=6x=42) Festuca arundinacea Schreb. and hexaploid F. rubra L. has been investigated. On the combined evidence derived from chromosome pairing in some euploid (2n=42) and monosomic (2n=41) hybrids from a diallel set of crosses between ten geographically diverse ecotypes of tall fescue, intergeneric hybrids involving tall fescue as well as red fescue, and euploid (2n=56) and aneuploid (2n=52, 53, 54, 55) amphiploids between Lolium multiflorum and F. arundinacea, it is concluded that diploid-like meiosis in these hexaploid species as well as in other natural polyploid species of Festuca is under genetic control. It is further inferred that this diploidizing gene(s) system must at least be disomic in dosage to be effective in suppressing homoeologous pairing and, therefore, had no influence upon pairing in haploid complements of the hybrids, i.e., it is haplo-insufficient or hemizygous-ineffective. — It has also been shown that sterility in hybrids between some geographically isolated ecotypes of tall fescue results from irregular meiosis due to the breakdown of the regulatory mechanism, rather than from chromosomal differentiation of the parental ecotypes as widely believed so far. The evolutionary significance of such a gene-repressing effect of certain genotypes or genes is indicated. — It is further suggested that the hemizygous ineffectiveness of the genetic control of bivalent pairing is of evolutionary significance and could have major implications on the cytogenetic relationships and the breeding of the entire Lolium-Festuca complex.     

Rapd fingerprinting of diploid Lolium perenne × hexaploid Festuca arundinacea hybrid genomes

We tested the application of RAPD technology for identification of hybrid genomes originated from a maternal clone of Lolium perenne L. (2n = 2x = 14) bearing cytoplasmic male sterility, which was pollinated separately by five clones of Festuca arundinacea Schreb. cv. Barocco (2n = 6x = 42). Six classes of RAPD markers were recognized, specific to: 1) Festuca genome and inherited into F1 hybrid genomes, 2) Lolium genome inherited into F1 hybrid genomes, 3) Lolium-specific bands not found in F1 progeny, 4) Festuca-specific bands not found in F1 progeny, 5) new bands found only in F1 hybrid profiles, 6) bands common to all parental and F1 hybrid genotypes. RAPD data were shown to have full potential a) to serve as an unequivocal proof of genome recombination in perennial ryegrass × tall fescue hybrids, b) to identify hybrid genomes, c) to reveal phenetic relationships of the accessions from crossing families, d) to enhance, by fingerprinting, the selection of superior hybrid material for further breeding. RAPD data were found to be consistent with the festucoid phenotype of F1 hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of Ploidy on Quality of Tall Fescue, Italian Ryegrass x Tall Fescue and Tall Fescue x Giant Fescue Hybrids

Selected quality parameters were measured for forage leaf tissuefrom a spaced-plant nursery. The genotypes used were Ky 31 tallfescue and hybrids of Italian ryegrass (Lolium multiflorum Lam.)x tall fescue (Festuca arundinacea Schreb.) and tall fescuex giant fescue [Fescue gigantea (L ) Vill.]. Hybrid ploidy rangedfrom 2n = 28 to 84 chromosomes. Forage quality was characterizedby neutral detergent fibre (NDF), acid detergent fibre (ADF),total soluble carbohydrates (TSC) nutritive value index (NVI),hemicellulose, and in vitro dry matter disappearance (DMD). Quality of tall fescue, as measured by increased DMD, was improvedby hybridization with giant fescue. Improved DMD and NVI correlatedwith lower NDF and ADF in the hybrids. A few hybrids of Italianryegrass x tall fescue (2n = 28) were higher in some qualityparameters than Ky 31. Tall fescue x giant fescue hybrids (2n= 80 to 84), as a group, had significant quality improvementover Ky 31 in higher DMD and NVI and lower NDF and ADF. Whilesome individual hybrids within each group were significantlyhigher in quality, only the 2n = 80 to 84 chromosome group wasconsistently higher than Ky 31. Prediction equations for DMD,NDF, and ADF were established based on solvent extraction withnear-infrared reflectance spectroscopy (NIRS). Linear correlationcoefficients between chemical measurement and NIRS for eachquality parameter were 0–95 or higher. Acid detergent fibre, neutral detergent fibre, dry matter disappearance, hemicellulose, nutritive value index, Festuca arundinacea, Festuca gigantea, Lolium multiflorum     

Phylogeny of the festucoid grasses of subtribe Loliinae and allies (Poeae, Pooideae) inferred from ITS and trnL-F sequences

Analyses of ribosomal ITS and chloroplast trnL-F sequences provide phylogenetic reconstruction for the festucoids (Poeae: Loliinae), a group of temperate grasses with morphological and molecular affinities to the large genus Festuca. Parsimony and Bayesian analyses of the combined ITS/trnL-F dataset show Loliinae to be monophyletic but unresolved for a weakly supported clade of 'broad-leaved Festuca,' a well-supported clade of 'fine-leaved Festuca,' and Castellia. The first group includes subgenera Schenodorus, Drymanthele, Leucopoa, and Subulatae, and sections Subbulbosae, Scariosa, and Pseudoscariosa of Festuca, plus Lolium and Micropyropsis. The second group includes sections Festuca, Aulaxyper, Eskia, and Amphigenes of Festuca, plus Vulpia, Ctenopsis, Psilurus, Wangenheimia, Cutandia, Narduroides, and Micropyrum. Subtribes Dactylidinae and Cynosurinae/Parapholiinae are sister clades and are the closest relatives of Loliinae. Vulpia is polyphyletic within the 'fine-leaved' fescues as revealed by the two genome analyses. Lolium is resolved as monophyletic in the ITS and combined analyses, but unresolved in the trnL-F based tree. Conflict between the ITS and the trnL-F trees in the placement of several taxa suggests the possibility of past reticulation events, although lineage sorting and possible ITS paralogy cannot be ruled out.     

Microsporogenesis in sexual Brachiaria hybrids (Poaceae)

Three sexual interspecific hybrids of Brachiaria (HBGC076, HBGC009, and HBGC014) resulting from crosses between B. ruziziensis (female genitor) and B. decumbens and B. brizantha (male genitors) produced by Embrapa Beef Cattle in the 1980s were cytologically analyzed by conventional methods for meiotic studies. The cytogenetic analysis showed the occurrence of common meiotic abnormalities among them. The most frequent abnormalities were those related to irregular chromosome segregation due to polyploidy. Other abnormalities, such as chromosome stickiness, absence of cytokinesis, irregular cytokinesis, abnormal spindle orientation, and abnormal nucleolus disintegration, were found in the three hybrids, while, chromosome disintegration was detected only in HBGC014. All the abnormalities, except for abnormal nucleolus disintegration, can cause unbalanced gamete formation, leading to pollen sterility. Multivalent chromosome association at diakinesis revealed genome affinity between the two parental species in the hybrids, suggesting some possibility for gene introgression. Presently, the Brachiaria breeding program has the objective of releasing, primarily, apomictic hybrids as new cultivars since they do not segregate but preserve the genetic makeup indefinitely. Besides, they result in homogeneous pastures which are easier to manage. The sexual hybrids, however, are paramount in the breeding program: they work as 'bridges' to introgress traits of interest into the apomictic genotypes. The cytogenetic analyses of these three hybrids substantiate their maintenance in the breeding program due to low frequency of meiotic abnormalities, complemented by interesting agronomic traits. They may be used in crosses to generate new cultivars in the future.     

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.     

Genetic relationships within and among Iberian fescues (Festuca L.) based on PCR-amplified markers.

The genus Festuca comprises approximately 450 species and is widely distributed around the world. The Iberian Penninsula, with more than 100 taxa colonizing very diverse habitats, is one of its main centers of diversification. This study was conducted to assess molecular genetic variation and genetic relatedness among 91 populations of 31 taxa of Iberian fescues, based on several molecular markers (random amplified polymorphic DNA, amplified fragment length polymorphisms, and trnL sequences). The analyses showed the paraphyletic origin of the broad-leaved (subgenus Festuca, sections Scariosae and Subbulbosae, and subgenus Schedonorus) and the fine-leaved fescues (subgenus Festuca, sections Aulaxyper, Eskia, and Festuca). Schedonorus showed a weak relationship with Lolium rigidum and appeared to be the most recent of the broad-leaved clade. Section Eskia was the most ancient and Festuca the most recent of the fine-leaved clade. Festuca and Aulaxyper were the most related sections, in concordance with their taxonomic affinities. All taxa grouped into their sections, except F. ampla and F. capillifolia (section Festuca), which appeared to be more closely related to Aulaxyper and to a new independent section, respectively. Most populations clustered at the species level, but some subspecies and varieties mixed their populations. This study demonstrated the value in combining different molecular markers to uncover hidden genetic relationships between populations of Festuca.     

Parental genome composition and genetic classifications of derivatives from intergeneric crosses of Festuca mairei and Lolium perenne

Intergeneric hybridization between Festuca and Lolium has been a long-term goal of forage and turfgrass breeders to generate improved cultivars by combining stress tolerance of Festuca and rapid establishment of Lolium. However, wide-distance hybridizations usually result in the wild genome being eliminated from the hybrid due to incomplete chromosome pairing and crossovers. In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to detect the parental genome composition of F₁ hybrids and backcross, generated from crosses between Festuca mairei St. Yves (Fm) and Lolium perenne L. (Lp). Each of the hybrids exhibited integration of Fm and Lp genomes with varying levels of Fm/Lp genome ratios. However, cluster and principle component analyses of the progeny consistently revealed four groups depending on the amount of genome introgression from both parents. The parental genome composition and classifications of intergeneric progeny would be useful for breeding material selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chromosome pairing in triploid intergeneric hybrids ofFestuca pratensis withLolium multiflorum, revealed by GISH

Genomic in situ hybridisation (GISH) was used to reveal chromosome pairing in two partly fertile, triploid (2n = 3x = 21) hybrids obtained by crossing the diploid (2n = 2x = 14) Festuca pratensis Huds. (designated FpFp), used as a female parent, with the autotetraploid (2n = 4x = 28) Lolium multiflorum Lam. (designated LmLmLmLm), used as a male parent. The pattern of chromosome pairing calculated on the basis of the mean values of chromosome configurations identified in all 100 PMCs analysed, was: 0.71I Lm + 2.24I Fp + 2.18II Lm/Lm + 0.54II Lm/Fp + 4.18III Lm/Lm/Fp. A relatively high number of Lm/Lm bivalents and Fp univalents, and a low number of Lm/Fp bivalents and Lm univalents indicated that the pairing was preferential between L. multiflorum chromosomes. Other observations regarding chromosome pairing within the Lm/Lm/Fp trivalents also confirmed this preferential pairing in the analysed triploids, as the Fp chromosome was not randomly located in the chain- and frying-pan-shaped trivalents. The similarities and differences in chromosome pairing at metaphase I and the level of preferential pairing between Lolium chromosomes in the different triploid Lolium-Festuca hybrids are discussed.     

Sugarcane genome architecture decrypted with chromosome‐specific oligo probes

Sugarcane (Saccharum spp.) is probably the crop with the most complex genome. Modern cultivars (2n = 100–120) are highly polyploids and aneuploids derived from interspecific hybridization between Saccharum officinarum (2n = 80) and Saccharum spontaneum (2n = 40–128). Chromosome‐specific oligonucleotide probes were used in combination with genomic in situ hybridization to analyze the genome architecture of modern cultivars and representatives of their parental species. The results validated a basic chromosome number of x = 10 for S. officinarum. In S. spontaneum, rearrangements occurred from a basic chromosome of x = 10, probably in the Northern part of India, in two steps leading to x = 9 and then x = 8. Each step involved three chromosomes that were rearranged into two. Further polyploidization led to the wide geographical extension of clones with x = 8. We showed that the S. spontaneum contribution to modern cultivars originated from cytotypes with x = 8 and varied in proportion between cultivars (13–20%). Modern cultivars had mainly 12 copies for each of the first four basic chromosomes, and a more variable number for those basic chromosomes whose structure differs between the two parental species. One?four of these copies corresponded to entire S. spontaneum chromosomes or interspecific recombinant chromosomes. In addition, a few inter‐chromosome translocations were revealed. The new information and cytogenetic tools described in this study substantially improve our understanding of the extreme level of complexity of modern sugarcane cultivar genomes.     

Variability of ribosomal DNA sites inFestuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH

This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploidFestuca pratensis andLolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploidF. pratensis ×L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) withL. perenne genomicDNAas a probe, andF. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. InF. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standardF. pratensis karyotypes. Losses of 45S rDNA loci were more frequent inL. perenne cultivars and intergeneric hybrids. Comparison of theF. pratensis andL. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location inL. perenne. A greater instability ofF. pratensis-genome-like andL. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 inF. pratensis and on chromosome 3 inL. perenne are useful markers for these chromosomes in intergenericFestuca ×Lolium hybrids.     

GISH/FISH mapping of genes for freezing tolerance transferred from Festuca pratensis to Lolium multiflorum

The first backcross breeding programme for the transfer of freezing-tolerance genes from winter hardy Festuca pratensis to winter-sensitive Lolium multiflorum is described. A partly fertile, triploid F(1) hybrid F. pratensis (2n=2x=14) x L. multiflorum (2n=4x=28) was employed initially, and after two backcrosses to L. multiflorum (2x) a total of 242 backcross two (BC(2)) plants were generated. Genomic in situ hybridisation (GISH) was performed on 61 BC(2) plants selected for their good growth and winter survival characters in the spring following one Polish winter (2000-2001). Among the winter survivors, diploid chromosome numbers were present in 80% of plants. An appropriate single Festuca introgression in an otherwise undisturbed Lolium genome could provide increased freezing tolerance without compromise to the good growth and plant vigour found in Lolium. Among all the diploids, a total of 20 individuals were identified, each with a single F. pratensis chromosome segment. Another diploid plant contained 13 Lolium chromosomes and a large metacentric F. pratensis chromosome, identified as chromosome 4, with two large distal Lolium introgressions on each chromosome arm. Three of the diploid BC(2), including the genotype with Festuca chromosome 4 DNA sequences, were found to have freezing tolerance in excess of that of L. multiflorum, and in one case in excess of the F. pratensis used as control. A detailed cytological analysis combining GISH and fluorescence in situ hybridisation analyses with rDNA probes revealed that the other two freezing-tolerant genotypes carried a Festuca chromosome segment at the same terminal location on the non-satellite arm of Lolium chromosome 2.     

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.     

Cytogenetic analysis of reciprocal hybrids and their parents between <Emphasis Type="Italic">Larix leptolepis</Emphasis> and <Emphasis Type="Italic">Larix gmelinii</Emphasis>: implications for identifying hybrids

We studied the parental taxa and the interspecific reciprocal hybrids between Larix leptolepis with Larix gmelinii, using classical cytogenetic methods, as well as fluorescence in situ hybridization (FISH) and genomic in situ hybridization. A high frequency (>90%) of complete bivalent formation was observed in reciprocal hybrids. Less than 10% of pollen mother cells exhibited abnormalities. The most frequent abnormalities were bridges. Multivalent chromosome associations were also observed in both reciprocal hybrids, suggesting that some chromosome interchange events did occur, and introgressions from one to the other species were possible. Intergenomic recombination indicates that genes might be readily introgressed into one species from the other in the genus Larix. Interspecific hybridization may be a potential method for genetic improvement in larch. FISH markers documented that the recombinant genomes of reciprocal hybrids were strictly additive and stable, indicating that FISH also might be a useful tool in Larix breeding.     

Comparison of tall fescue (Cyperales: Gramineae) to other cool-season turfgrasses for tolerance to European chafer (Coleoptera: Scarabaeidae)

Three cultivars of tall fescue, Festuca arundinacea Schreb., were compared with three cultivars each of fine fescue (Festuca spp.), Kentucky bluegrass (Poa pratensis L.), and perennial ryegrass (Lolium perenne L.) to evaluate tolerance to root-feeding by European chafer grubs, Rhizotrogus majalis (Razoumowsky). Potted turfgrasses were infested with initial densities equivalent to 33 or 66 grubs per 0.1 m2 on 19 August 2000. More grubs were added in late September and October, bringing the total to 66 or 143 grubs per 0.1 m2. Plant growth, root loss, weight gain, and survival of grubs were measured. The experiment was repeated in fall of 2001 with an initial density of 66 grubs per 0.1 m2. The proportion of root mass lost as a result of grub feeding was a function of turf species, root growth, grub survival, and grub growth during the test. Grubs gained the most weight and consumed the most roots when feeding on fine fescue. Fine fescue suffered the greatest percentage of root loss in 2000, despite having the most rapid root growth and largest mass in control pots. Cultivars of tall fescue appeared to be the most tolerant of grub feeding, having the smallest reduction in root mass in both years. Data from fine fescue, Kentucky bluegrass, and perennial ryegrass cultivars were not as consistent as tall fescue, because for some cultivars root growth and grub survival were different between years. We also found that grubs increased in mass by 20% when the mass of available roots was doubled.     

Introgression mapping of genes for winter hardiness and frost tolerance transferred from Festuca arundinacea into Lolium multiflorum

Genes for winter hardiness and frost tolerance were introgressed from Festuca arundinacea into winter-sensitive Lolium multiflorum. Two partly fertile, pentaploid (2n = 5x = 35) F(1) hybrids F. arundinacea (2n = 6x = 42) x L. multiflorum (2n = 4x = 28) were generated and backcrossed twice onto L. multiflorum (2x). The backcross 1 (BC(1)) and backcross 2 (BC(2)) plants were preselected for high vigor and good fertility, and subsequently, a total of 83 BC(2) plants were selected for winter hardiness after 2 Polish winters and by simulated freezing tests. Genomic in situ hybridization (GISH) was performed on 6 winter-hardy plants selected after the first winter and shown to be significantly (P < 0.05) more frost tolerant than the L. multiflorum control. Among the analyzed BC(2) winter survivors, only diploid (2n = 2x = 14) plants were found. Five plants carried 13 intact L. multiflorum chromosomes and 1 L. multiflorum chromosome with a single introgressed F. arundinacea terminal chromosome segment. The sixth BC(2) winter survivor appeared to be Lolium without any Festuca introgression capable of detection by GISH. A combined GISH and fluorescence in situ hybridization analysis with rDNA probes of the most winter-hardy (after 2 winters) and frost-tolerant BC(2) plant revealed the location of an F. arundinacea introgression on the nonsatellite arm of L. multiflorum chromosome 2, the same chromosome location reported previously as a site for frost tolerance genes in the diploid and winter-hardy species Festuca pratensis.