The effect of B chromosomes on homoeologous pairing in species hybrids

The effect of B chromosomes on chromosome pairing at meiosis was investigated in the species hybrid Lolium temulentum x L. perenne at both the diploid and tetraploid level. The presence of B chromosomes drastically reduced association of homoeologous chromosomes in both the diploids and tetraploids. This was evident from the high frequency of univalents recorded in PMC's of diploid hybrids with B's and from the predominantly bivalent association of homologous chromosomes in tetraploids of this type. In the absence of B's homoeologous pairing was extensive giving a high frequency of bivalents in the diploids and multivalents as well as bivalents and univalents in the tetraploids.     

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.     

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.     

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.     

B-CHROMOSOMES,THEIR ORIGIN AND RELATION TO MEIOSIS IN INTERSPECIFIC LOLIUM HYBRIDS

The following Lolium species were shown to have 14 chromosomes: L. canariensis Steud. (= L. gracile Parl.), L. loliaceum (Bory and Chaub.) Hand.-Mazz., L. multiflorum Lam., L. perenne L., and L. temulentum L. B-chromosomes in addition to the normal complement were observed during metaphase I and anaphase I for L. persicum Boiss. and Hohen., L. remotum Schrank, L. rigidum Gaud., L. strictum Presl. (= L. rigidum?), and for several synthesized interspecific hybrids. General absence of B-chromosomes in diakinesis suggested that they originated by misdivision of A-chromosomes during prometaphase I. The B-chromosomes reached a maximum of eight but the number varied for microsporocytes of the same plant. B-chromosomes appeared spontaneously in progenies from sibbing of hybrid plants without supernumeraries but were also eliminated in the F₂ from other sibbings. Degree of chromosome pairing during meiosis was unrelated to the subsequent presence of B-chromosomes. Normal pairing of seven bivalents was typical for L. perenne × L. multiflorum, L. perenne × L. rigidum, L. multiflorum × L. loliaceum, and L. rigidum × L. loliaceum. Between five and seven bivalents were recorded for L. multiflorum × L. persicum, L. multflorum × L. remotum, L. multiflorum × L. strictum, L. rigidum × L. persicum, L. rigidum × L. remotum, L. rigidum × L. strictum, and L. rigidum × L. temulentum. The results indicated that these Lolium species have a common and generally undifferentiated genome suggesting relatively recent speciation.     

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.     

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 effect of B chromosomes on homoeologous pairing in species hybrids

The effect of B chromosomes on meiosis is described in the diploid and tetraploid interspecific hybrid Lolium multiflorum x Lolium perenne. Although the parental species are very closely related, the presence of B chromosomes in the diploid hybrid reduced both chiasma frequency and the number of bivalents at meiosis by a small but significant amount. However at the tetraploid level the presence of B chromosomes did not seem to alter the pairing pattern and chiasma frequency in any way. The use of B chromosomes to stabilize meiosis in amphiploids of this type between closely related outbreeding species is therefore ruled out.     

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

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. Received: 20 September 2000 / Accepted: 05 January 2001     

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.     

Chromosome pairing in a Lolium temulentum X Lolium perenne diploid hybrid with a low chiasma frequency

Summary Despite an average difference of about 50% in DNA amount, homoeologous chromosomes pair effectively at first metaphase in the diploid interspecific hybrid between Lolium temulentum and Lolium perenne. However, in the presence of accessory B chromosomes and diploidising genes pairing at metaphase I is severely reduced. Reconstruction of serial electron micrographs through pollen mother cell nuclei show that synaptonemal complexes are formed at pachytene between not only homoeologous but also non-homologous chromosome segments resulting in multivalent formation. These associations are largely ineffective in terms of chiasma formation and degenerate at late pachytene. It is highly probable that the pairing determinants exercise their control on chromosome pairing largely by prohibiting the siting of crossovers in homoeologously paired chromosome segments.     

Development and mapping of a public reference set of SSR markers in Lolium perenne L.

We report on the characterization and mapping of 76 simple sequence repeat (SSR) markers for Lolium perenne. These markers are publicly available or obtained either from genomic libraries enriched for SSR motifs or L. perenne expressed sequence tag (EST) clones. Four L. perenne mapping populations were used to map the SSR markers. A consensus linkage map of the four mapping populations containing 65 of the SSR markers is presented, together with primer information and a quality score indicating the usefulness of the SSR marker in different populations. The SSR markers identified all seven L. perenne linkage groups.     

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.     

Genomic organization and phylogenetic relationships in the genus Dasypyrum analysed by Southern and in situ hybridization of total genomic and cloned DNA probes

Molecular cytogenetic methods have been used to study the controversial phylogenetic relationships between the species Dasypyrum villosum (L.) Candargy (2n=2x=14) and D. breviaristatum (Lindb. f.) Frederiksen (2n=4x=28). Using total genomic DNA from the two species as probes for in situ hybridization to chromosomes, we found that the pericentromeric regions of the chromosome arms of both species are similar, while distal regions show substantial differences. Two dispersed repetitive DNA sequences were isolated: pDbKB45 is distributed along the chromosomes but amplified in the subtelomeric regions of D. breviaristatum chromosomes, while pDbKB49, in both species, is less amplified in terminal regions. Size-separated restriction enzyme digests of DNA showed many repetitive fragments, but few in common between the two species. After probing Southern transfers with D. breviaristatum genomic DNA, all lanes showed similar hybridization patterns although one extra small band was evident in the D. breviaristatum lanes. In contrast, probing with D. villosum DNA showed very substantial differences between the two species. Genomic in situ hybridization to meiotic metaphases from an interspecific hybrid showed seven bivalents of D. breviaristatum origin and seven univalents from D. villosum. We also analysed the physical organization of 5S rDNA, 18S-25S rDNA and a tandemly repeated sequence from rye. Our data support an autotetraploid origin for D. breviaristatum, but its genome and that of D. villosum show extensive differences, so the tetraploid is unlikely to be directly derived from D. villosum. Received: 29 March 1996; in revised form: 28 December 1996 / Accepted: 2 February 1997     

Anther Culture of Lolium temulentum, Festuca pratensis and Lolium x Festuca Hybrids. I. Influence of Pretreatment, Culture Medium and Culture Incubation Conditions on Callus Production and Differentiation

This study was conducted with Lolium temulentum, Festuca pratensis,and the two hybrids L. multiflorum x F. pratensis ‘Elmet’and L. perenne x F. pratensis ‘Prior’. In a comparisonof various durations (7–42 d) of pretreatment at 4 or7 °C the highest yield of microspore-derived callus of L.temulentum was obtained after pretreatment of spikes at 7 °Cfor 28 d, conditions which also proved optimal for panicle pretreatmentwith F. pratensis. For ‘Elmet’, durations of 21–42d were optimal, and for ‘Prior’ the responses tendedto decline with increasing duration. In L. temulentum addition of charcoal (1–2 g l^–1)to medium containing 2, 4-D and KN wa     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Effect of genetic background on trisomy in ryegrass

Six primary trisomics of ryegrass, Lolium perenne L., were studied in perennial and perennial x annual hybrid backgrounds. Chromosome association at meiotic metaphase I and chiasma number per cell of the individual trisomes did not differ in the two genetic backgrounds. Hybrid trisomies showed wider variation in morphology, and had higher pollen fertility than the perennial trisomics and disomics. — It is concluded that the transfer of perennial ryegrass chromosomes and segments into annual ryegrass can be accomplished without any serious consequence on the cytological stability of the reconstituted genome.     

Morphological, yield, cytological and molecular characterization of a bread wheat X tritordeum F1 hybrid

The morphological, yield, cytological and molecular characteristics of bread wheat X tritordeum F₁ hybrids (2n =6x = 42; AABBDH^ch) and their parents were analysed. Morphologically, these hybrids resembled the wheat parent. They were slightly bigger than both parents, had more spikelets per spike, and tillered more profusely. The hybrids are self-fertile but a reduction of average values of yield parameters was observed. For the cytological approach we used a double-target fluorescencein situ hybridization performed with total genomic DNA fromHordeum chilense L. and the ribosomal sequence pTa71. This technique allowed us to confirm the hybrid nature and to analyse chromosome pairing in this material. Our results showed that the expected complete homologous pairing (14 bivalents plus 14 univalents) was only observed in 9.59% of the pollen mother cells (PMCs) analysed. Some PMCs presented autosyndetic pairing of Hch and A, B or D chromosomes. The average number of univalents was higher in the wheat genome (6.8) than in the Hch genome (5.4). The maximum number of univalents per PMC was 20. We only observed wheat multivalents (one per PMC) but the frequency of trivalents (0.08) was higher than that of quadrivalents (0.058). We amplified 50 RAPD bands polymorphic between the F₁ hybrid and one of its parents, and 31 ISSR polymorphic bands. Both sets of markers proved to be reliable for DNA fingerprinting. The complementary use of morphological and yield analysis, molecular cytogenetic techniques and molecular markers allowed a more accurate evaluation and characterization of the hybrids analysed here.     

A novel intergeneric hybrid in the Triticeae: Triticum aestivum x Psathyrost achys juncea

Summary Two hybrid embryos of intergeneric origin between Triticum aestivum cv Fukuho (2n=6x=42, AABBDD) and Psathyrostachys juncea (2n=2x=14, NN) were successfully rescued. One hybrid plant had the expected chromosome number of 28 (ABDN), whereas the second plant had 35 chromosomes. The average meiotic chromosome pairing in the 35-chromosome hybrid was 21.87 univalents + 6.38 bivalents + 0.11 trivalents + 0.009 quadrivalents, which indicates that two copies of the N genome were present. Chromosome pairing in the 28-chromosome hybrid was low (1.35 bivalents), and pointed out the lack of homology between the wheat genomes and the P. juncea genome. These new hybrids showed some necrosis and chlorosis, which caused severe floral abortion in the plant that had 35 chromosomes. These problems became gradually less severe after 18 months.Contrib. no. 372     

Indeterminate meiotic restitution (IMR): a novel type of meiotic nuclear restitution mechanism detected in interspecific lily hybrids by GISH

A detailed analysis of microsporogenesis was carried out in three diploid lily cultivars (2n=2x=24) and three diploid interspecific hybrids (2n=2x=24) using DNA in situ hybridisation methods (GISH and FISH). In cvs. Gelria (Lilium longiflorum; L genome), Connecticut King and Mont Blanc (both Asiatic hybrids; Agenome) meiosis was regular and only haploid gametes were formed while the three interspecific hybrids between L. longiflorum×Asiatic hybrid (LA) showed a variable frequency of meiotic nuclear restitution and stainable 2n-pollen formation ranging from 3% to 30%. An analysis of meiotic chromosome behaviour of the LA hybrids through GISH and FISH revealed that: (1) the parental chromosomes could be clearly discriminated into univalents, half-bivalents and bivalents in the PMCs; (2) in some of the PMCs the entire complement was present either as univalents or half-bivalents which had the potential to divide equationally (following centromere division) during the first division leading to first division restitution (FDR) gametes; (3) more frequently, however, in one and the same PMC the univalents and half-bivalents divided equationally whereas the bivalents disjoined reductionally at the same time giving rise to 2n-gametes that could vary from the well-known FDR or SDR 2n-gametes. We indicate this novel type of restitution mechanism as Indeterminate Meiotic Restitution (IMR). In order to confirm the occurrence of IMR gametes, the chromosome constitutions of eight triploid BC₁ progenies derived from backcrossing the 2n-gamete producing the LAhybrids to the Asiatic hybrid parents were analysed through in situ hybridisation. The results indicated that there were seven BC₁ plants in which FDR 2n-gametes, with or without homoeologous recombinations, were functional, whereas in one case the 2n-gamete resulting from IMR was functional. In the latter, there was evidence for the occurrence of genetic recombination through homoeologous crossing-over as well as through the assortment of homoeologous chromosomes. A singular feature of the IMR 2n-gamete was that although it transmitted a euploid number of 24 chromosomes to the BC₁ progeny, the number of chromosomes transmitted from the two parental species was dissimilar: 9 L-genome chromosomes and 15 A-genome chromosomes instead of 12 of each. Received: 15 May 2000 / Accepted: 4 December 2000