Cytonuclear interplay in auto- and allopolyploids: a multifaceted perspective from the Festuca-Lolium complex.

Restoring cytonuclear stoichiometry is necessary after whole-genome duplication (WGD) and interspecific/intergeneric hybridization in plants. We investigated this phenomenon in auto- and allopolyploids of the Festuca-Lolium complex providing insights into the mechanisms governing cytonuclear interactions in early polyploid and hybrid generations. Our study examined the main processes potentially involved in restoring the cytonuclear balance after WGD comparing diploids and new and well-established autopolyploids. We uncovered that both the number of chloroplasts and the number of chloroplast genome copies were significantly higher in the newly established autopolyploids and grew further in more established autopolyploids. The increase in the copy number of the chloroplast genome exceeded the rise in the number of chloroplasts and fully compensated for the doubling of the nuclear genome. In addition, changes in nuclear and organelle gene expression were insignificant. Allopolyploid Festuca × Lolium hybrids displayed potential structural conflicts in parental protein variants within the cytonuclear complexes. While biased maternal allele expression has been observed in numerous hybrids, our results suggest that its role in cytonuclear stabilization in the Festuca × Lolium hybrids is limited. This study provides insights into the restoration of the cytonuclear stoichiometry, yet it emphasizes the need for future research to explore post-transcriptional regulation and its impact on cytonuclear gene expression stoichiometry. Our findings may enhance the understanding of polyploid plant evolution, with broader implications for the study of cytonuclear interactions in diverse biological contexts.

Both male and female meiosis contribute to non-Mendelian inheritance of parental chromosomes in interspecific plant hybrids (Lolium × Festuca).

Some interspecific plant hybrids show unequal transmission of chromosomes from parental genomes to the successive generations. It has been suggested that this is due to a differential behavior of parental chromosomes during meiosis. However, underlying mechanism is unknown. We analyzed chromosome composition of the F2 generation of Festuca × Lolium hybrids and reciprocal backcrosses to elucidate effects of male and female meiosis on the shift in parental genome composition. We studied male meiosis, including the attachment of chromosomes to the karyokinetic spindle and gene expression profiling of the kinetochore genes. We found that Lolium and Festuca homoeologues were transmitted differently to the F2 generation. Female meiosis led to the replacement of Festuca chromosomes by their Lolium counterparts. In male meiosis, Festuca univalents were attached less frequently to microtubules than Lolium univalents, lagged in divisions and formed micronuclei, which were subsequently eliminated. Genome sequence analysis revealed a number of non-synonymous mutations between copies of the kinetochore genes from Festuca and Lolium genomes. Furthermore, we found that outer kinetochore proteins NDC80 and NNF1 were exclusively expressed from the Lolium allele. We hypothesize that silencing of Festuca alleles results in improper attachment of Festuca chromosomes to karyokinetic spindle and subsequently their gradual elimination.

Reciprocal allopolyploid grasses (Festuca × Lolium) display stable patterns of genome dominance.

Allopolyploidization entailing the merger of two distinct genomes in a single hybrid organism, is an important process in plant evolution and a valuable tool in breeding programs. Newly established hybrids often experience massive genomic perturbations, including karyotype reshuffling and gene expression modifications. These phenomena may be asymmetric with respect to the two progenitors, with one of the parental genomes being "dominant." Such "genome dominance" can manifest in several ways, including biased homoeolog gene expression and expression level dominance. Here we employed a k-mer-based approach to study gene expression in reciprocal Festuca pratensis Huds. × Lolium multiflorum Lam. allopolyploid grasses. Our study revealed significantly more genes where expression mimicked that of the Lolium parent compared with the Festuca parent. This genome dominance was heritable to successive generation and its direction was only slightly modified by environmental conditions and plant age. Our results suggest that Lolium genome dominance was at least partially caused by its more efficient trans-acting gene expression regulatory factors. Unraveling the mechanisms responsible for propagation of parent-specific traits in hybrid crops contributes to our understanding of allopolyploid genome evolution and opens a way to targeted breeding strategies.

Cytogenetic and molecular genotyping in the allotetraploid Festuca pratensis × Lolium perenne hybrids.

BACKGROUND: Species of the Festuca and Lolium genera, as well as intergeneric Festuca × Lolium (Festulolium) hybrids, are valuable fodder and turf grasses for agricultural and amenity purposes worldwide. Festulolium hybrids can merge in their genomes agronomically important characteristics. However, in polyploid plants, especially in allopolyploids, the hybridization of divergent genomes could contribute to various abnormalities, such as variability in chromosome number, structural rearrangements, and/or disorders in inheritance patterns. Here we studied these issues in allotetraploid Festuca pratensis × Lolium perenne hybrids. RESULTS: Cytogenetic procedures, including fluorescent in situ hybridization, genomic in situ hybridization, and molecular markers - inter-simple sequence repeats (ISSR) were exploited. This cytogenetic approach indicated the dynamics in the number and distribution of ribosomal RNA genes and structural rearrangements for both parental genomes (Festuca and Lolium) in hybrid karyotypes. The separate analysis of F. pratensis and L. perenne chromosomes in hybrid plants (F2-F3 generations of F. pratensis × L. perenne) revealed the asymmetrical level of rearrangements. Recognized structural changes were mainly located in the distal part of chromosome arms, and in chromosomes bearing ribosomal DNA, they were more frequently mapped in arms without this sequence. Based on the ISSR markers distribution, we found that the tetrasomic type of inheritance was characteristic for the majority of ISSR loci, but the disomic type was also observed. Nonetheless, no preference in the transmission of either Festuca or Lolium alleles to the following generations of allotetraploid F. pratensis × L. perenne hybrid was observed. CONCLUSION: Our study reports cytogenetic and molecular genotyping of the F. pratensis × L. perenne hybrid and its following F2-F3 progenies. The analysis of 137 allotetraploid F. pratensis × L. perenne hybrids revealed the higher level of recombination in chromosomes derived from F. pratensis genome. The results of ISSR markers indicated a mixed model of inheritance, which may be characteristic for these hybrids.

Karyotype reshufflings of Festuca pratensis × Lolium perenne hybrids.

Many different processes have an impact on the shape of plant karyotype. Recently, cytogenetic examination of Lolium species has revealed the occurrence of spontaneous fragile sites (FSs) associated with 35S rDNA regions. The FSs are defined as the chromosomal regions that are sensitive to forming gaps or breaks on chromosomes. The shape of karyotype can also be determined by interstitial telomeric sequences (ITSs), what was recognized for the first time in this paper in chromosomes of Festuca pratensis × Lolium perenne hybrids. Both FSs and ITSs can contribute to genome instabilities and chromosome rearrangements. To evaluate whether these cytogenetic phenomena have an impact on karyotype reshuffling observed in Festuca × Lolium hybrids, we examined F1 F. pratensis × L. perenne plants and generated F2-F9 progeny by fluorescent in situ hybridization (FISH) using rDNA sequences, telomere and centromere probes, as well as by genomic in situ hybridization (GISH). Analyses using a combination of FISH and GISH revealed that intergenomic rearrangements did not correspond to FSs but overlapped with ITSs for several analyzed genotypes. It suggests that internal telomeric repeats can affect the shape of F. pratensis × L. perenne karyotypes. However, other factors that are involved in rearrangements and have a more crucial impact could exist, but they are still unknown.

Orthology Guided Transcriptome Assembly of Italian Ryegrass and Meadow Fescue for Single-Nucleotide Polymorphism Discovery.

Single-nucleotide polymorphisms (SNPs) represent natural DNA sequence variation. They can be used for various applications including the construction of high-density genetic maps, analysis of genetic variability, genome-wide association studies, and map-based cloning. Here we report on transcriptome sequencing in the two forage grasses, meadow fescue ( Huds.) and Italian ryegrass ( Lam.), and identification of various classes of SNPs. Using the Orthology Guided Assembly (OGA) strategy, we assembled and annotated a total of 18,952 and 19,036 transcripts for Italian ryegrass and meadow fescue, respectively. In addition, we used transcriptome sequence data of perennial ryegrass ( L.) from a previous study to identify 16,613 transcripts shared across all three species. Large numbers of intraspecific SNPs were identified in all three species: 248,000 in meadow fescue, 715,000 in Italian ryegrass, and 529,000 in perennial ryegrass. Moreover, we identified almost 25,000 interspecific SNPs located in 5343 genes that can distinguish meadow fescue from Italian ryegrass and 15,000 SNPs located in 3976 genes that discriminate meadow fescue from both species. All identified SNPs were positioned in silico on the seven linkage groups (LGs) of using the GenomeZipper approach. With the identification and positioning of interspecific SNPs, our study provides a valuable resource for the grass research and breeding community and will enable detailed characterization of genomic composition and gene expression analysis in prospective × hybrids.

Metabolite profiling during cold acclimation of Lolium perenne genotypes distinct in the level of frost tolerance.

Abiotic stresses, including low temperature, can significantly reduce plant yielding. The knowledge on the molecular basis of stress tolerance could help to improve its level in species of relatively high importance to agriculture. Unfortunately, the complex research performed so far mainly on model species and also, to some extent, on cereals does not fully cover the demands of other agricultural plants of temperate climate, including forage grasses. Two Lolium perenne (perennial ryegrass) genotypes with contrasting levels of frost tolerance, the high frost tolerant (HFT) and the low frost tolerant (LFT) genotypes, were selected for comparative metabolomic research. The work focused on the analysis of leaf metabolite accumulation before and after seven separate time points of cold acclimation. Gas chromatography-mass spectrometry (GC/MS) was used to identify amino acids (alanine, proline, glycine, glutamic and aspartic acid, serine, lysine and asparagine), carbohydrates (fructose, glucose, sucrose, raffinose and trehalose) and their derivatives (mannitol, sorbitol and inositol) accumulated in leaves in low temperature. The observed differences in the level of frost tolerance between the analysed genotypes could be partially due to the time point of cold acclimation at which the accumulation level of crucial metabolite started to increase. In the HFT genotype, earlier accumulation was observed for proline and asparagine. The increased amounts of alanine, glutamic and aspartic acids, and asparagine during cold acclimation could be involved in the regulation of photosynthesis intensity in L. perenne. Among the analysed carbohydrates, only raffinose revealed a significant association with the acclimation process in this species.

Genome-dependent chromosome dynamics in three successive generations of the allotetraploid Festuca pratensis × Lolium perenne hybrid.

We focus on the identification of complete and recombined ribosomal DNA-bearing chromosomes, and the dynamics of chromosomal number and position of ribosomal DNA (rDNA) loci in the F2-F4 generations derived from the F1 hybrid of Festuca pratensis Huds. (2n = 4x = 28) × Lolium perenne L. (2n = 4x = 28). Lolium genomic DNA and rRNA genes were mapped by means of genomic and fluorescence in situ hybridization (GISH and FISH). The results revealed that plants of the three generations share various rDNA loci profiles with chromosome structural changes, possibly as a result of chromosomal inter- and intra-rearrangements. We observed an asymmetrical variation in the number of recombinant arms with and without rDNA loci between parental genomes. The Lolium genome was more affected by rearrangements in arms with rDNA loci, while Festuca was more affected in arms without them. Statistically significant differences between L. perenne and F. pratensis genomes concerned the number of recombined chromosomes without rDNA, and the number of recombined rDNA-bearing chromosomal arms of marked chromosomes, showing a tendency of F. pratensis genome-like chromosomes to be less stable, compared with L. perenne. We postulate a novel genome-dependent range and type of chromosome variation in plants of the F2-F4 generations derived from F. pratensis × L. perenne hybrid.

Differences in leaf proteome response to cold acclimation between Lolium perenne plants with distinct levels of frost tolerance.

Perennial ryegrass (Lolium perenne) is a high quality forage and turf grass mainly due to its excellent nutritive values and rapid establishment rate. However, this species has limited ability to perform in harsh winter climates. Though winter hardiness is a complex trait, it is commonly agreed that frost tolerance (FT) is its main component. Species growing in temperate regions can acquire FT through exposure to low, non-lethal temperatures, a phenomenon known as cold acclimation (CA). The research on molecular basis of FT has been performed on the model plants, but they are not well adapted to extreme winter climates. Thus, the mechanisms of cell response to low temperature in winter crops and agronomically important perennial grasses have yet to be revealed. Here, two L. perenne plants with contrasting levels of FT, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on analyses of leaf protein accumulation before and after 2, 8, 26 h, and 3, 5, 7, 14 and 21 days of CA, using a high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry (MS). Analyses of 580 protein profiles revealed a total of 42 (7.2%) spots that showed at a minimum of 1.5-fold differences in protein abundance, at a minimum of at one time point of CA between HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analyzed plants appeared most often on the 5th (18 proteins) and the 7th (19 proteins) day of CA. The proteins derived from 35 (83.3%) spots were successfully identified by the use of MS and chloroplast proteins were shown to be the major group selected as differentially accumulated during CA. The functions of the identified proteins and their probable influence on the level of FT in L. perenne are discussed.

Identification of leaf proteins differentially accumulated during cold acclimation between Festuca pratensis plants with distinct levels of frost tolerance.

Festuca pratensis (meadow fescue) as the most frost-tolerant species within the Lolium-Festuca complex was used as a model for research aimed at identifying the cellular components involved in the cold acclimation (CA) of forage grasses. The work presented here also comprises the first comprehensive proteomic research on CA in a group of monocotyledonous species which are able to withstand winter conditions. Individual F. pratensis plants with contrasting levels of frost tolerance, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on the analysis of leaf protein accumulation before and after 2, 8, and 26 h, and 3, 5, 7, 14, and 21 d of CA, using high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry. The analyses of approximately 800 protein profiles revealed a total of 41 (5.1%) proteins that showed a minimum of a 1.5-fold difference in abundance, at a minimum of one time point of CA for HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analysed plants appeared relatively early during cold acclimation, most often after 26 h (on the 2nd day) of CA and one-half of the differentially accumulated proteins were all parts of the photosynthetic apparatus. Several proteins identified here have been reported to be differentially accumulated during cold conditions for the first time in this paper. The functions of the selected proteins in plant cells and their probable influence on the level of frost tolerance in F. pratensis, are discussed.

Chromosome pairing in allotetraploid hybrids of Festuca pratensis x Lolium perenne revealed by genomic in situ hybridization (GISH).

Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F(1) hybrid plants of the allotetraploid Festuca pratensis x Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents - Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents - Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift.

Chromosome pairing in triploid intergeneric hybrids of Festuca pratensis with Lolium 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.

Genome balance in six successive generations of the allotetraploid Festuca pratensis x Lolium perenne.

In the allotetraploid, Festuca pratensis Huds. (2n = 4x = 28) x Lolium perenne L. (2n = 4x = 28) the balance of chromatin, as determined by GISH, changes over successive generations of open pollination in favour of L. perenne. There is extensive recombination between chromosomes of the two parental genomes, as well as substitution of whole Festuca chromosomes by whole Lolium chromosomes. The total number of Lolium chromosomes increased from a mean 14.36 in the F(2) to 16.26 in the F(6), and the total number of Festuca chromosomes decreased correspondingly from a mean of 13.57 to a value of 11.56. The number of recombinant chromosomes and recombination breakpoints per genotype also increased from generation to generation, although the respective values of both characters were higher for Festuca (0.86-8.41 and 1.14-15.22) than for Lolium (0.68-4.59 and 0.68-6.0). The proportion of total genome length contributed by the L. perenne chromatin increased from about 50% in F(2) to 59.5% in F(6). The results are based on the sample of 134 plants studied (26-28 plants per generation), and are discussed in terms of the dominance of Lolium chromosomes over those of Festuca, and possible mechanisms underlying this phenomenon of chromatin substitution.

Application of GISH and AFLP techniques for identification of Lolium-Festuca introgressions.

At present, breeding programmes aimed at combining advantageous traits within the Lolium-Festuca complex, are mainly focused on introgression procedures. One principal objective, is the transfer of genes conferring resistance to abiotic stresses from Festuca species (F. pratensis, F. arundinacea and F. glaucescens) into Lolium multiflorum and L. perenne germplasm. In our experiments, two different hybrids: triploid - L. multiflorum (4x) x F. pratensis (2x) and pentaploid - F. arundinacea (6x) x L. multiflorum (4x) were backcrossed twice onto L. multiflorum cultivars, and numerous BC2 progeny generated. BC2 plants from both combinations were tested in field and/or simulated conditions for winter hardiness and drought resistance. GISH (genomic in situ hybridisation) analyses were then performed on the most winter hardy and drought resistant plants to locate putative genes for stress resistance. Using resistant L. multiflorum genotypes with a single Festuca chromatin segment, it was possible to allocate AFLP (amplified fragment length polymorphism) markers specific to that segment. Markers associated with genes conferring stress resistance facilitate marker-assisted selection programmes to obtain new, more persistent grass cultivars. Preliminary results of GISH analysis, to identify Festuca chromosome segments in L. multiflorum introgression lines and to find segment-specific AFLP markers, are presented