Research Article |
Corresponding author: Warren Mervyn Williams ( warren.williams@agresearch.co.nz ) Academic editor: Ekaterina Badaeva
© 2022 Helal Ahmad Ansari, Nicholas Ellison, Alan Vincent Stewart, Warren Mervyn Williams.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ansari HA, Ellison N, Stewart AV, Williams WM (2022) Distribution patterns of rDNA loci in the Schedonorus-Lolium complex (Poaceae). Comparative Cytogenetics 16(1): 39-54. https://doi.org/10.3897/compcytogen.v16.i1.79056
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The Schedonorus-Lolium complex of the subtribe Loliinae (Poaceae) includes several economically important forage and turf grasses. This complex encompasses Lolium Linnaeus, 1753, Festuca Linnaeus, 1753 subgenus Schedonorus (P. Beauvois, 1824) Petermann, 1849 and Micropyropsis Romero Zarco et Cabezudo, 1983. New FISH results of 5S and 18S–26S rDNA sequences are presented for three species and the results are interpreted in a review of distribution patterns of 5S and 18S–26S rDNA sequences among other species in the complex. Micropyropsis tuberosa Romero Zarco et Cabezudo, 1983 (2n = 2x = 14) displayed a distribution pattern of rDNA sequences identical to that of F. pratensis Hudson, 1762, supporting a close phylogenetic relationship at the bottom of the phylogenetic tree. “Lolium multiflorum” Lamarck, 1779 accessions sourced from Morocco showed a different pattern from European L. multiflorum and could be a unique and previously uncharacterised taxon. North African Festuca simensis Hochstetter ex A. Richard, 1851 had a marker pattern consistent with allotetraploidy and uniparental loss of one 18S–26S rDNA locus. This allotetraploid has previously been suggested to have originated from a hybrid with Festuca glaucescens (Festuca arundinacea var. glaucescens Boissier, 1844). However, the distribution patterns of the two rDNA sequences in this allotetraploid do not align with F. glaucescens, suggesting that its origin from this species is unlikely. Furthermore, comparisons with other higher alloploids in the complex indicate that F. simensis was a potential donor of two sub-genomes of allohexaploid Festuca gigantea (Linnaeus) Villars, 1787. In the overall complex, the proximal locations of both rDNA markers were conserved among the diploid species. Two types of synteny of the two markers could, to a considerable extent, distinguish allo- and autogamous Lolium species. The ancestral parentage of the three Festuca allotetraploids has not yet been determined, but all three appear to have been sub-genome donors to the higher allopolypoids of sub-genus Schedonorus. Terminal locations of both the markers were absent from the diploids but were very frequently observed in the polyploids.
Festuca, FISH, karyotype evolution, Lolium, rDNA locus evolution, species diversification
Ryegrasses of the genus Lolium Linnaeus, 1753 with ten diploid species and fescues of the genus Festuca Linnaeus, 1753 subgenus Schedonorus (P. Beauvois, 1824) Petermann, 1849 are closely related and, together with Micropyropsis Romero Zarco et Cabezudo, 1983, form the “Schedonorus-Lolium complex”, belonging to the family Poaceae Barnhart, 1895, subtribe Loliinae Dumortier, 1829 (Inda et al. 2013;
Since the last major taxonomic revision of the genus Lolium by
Several molecular genetic analyses involving DNA markers have been successfully carried out for the phylogenetic reconstruction of subtribe Loliinae. It has been shown that the Schedonorus-Lolium complex represents a monophyletic group, with Lolium clearly differentiated from Festuca (
Karyological differences featuring chromosome number, structure and morphology have long been used to infer the systematic status and the evolutionary history of species divergence. However, in some groups of species conventionally stained chromosome preparations do not clearly delineate structural differences among chromosomes or species karyotypes. Molecular cytogenetic mapping of specific DNA sequences through fluorescence in situ hybridization (FISH) can overcome such problems, and provide enhanced pictures of chromosome architecture, leading to clear karyotype and genome discrimination (
Species of the Schedonorus-Lolium complex all share x = 7 as the base chromosome number and all have very similar biarmed chromosome morphologies and symmetrical karyotypes. Therefore, conventional karyological information is of little value for evaluating evolutionary changes (
In this study, we have mapped the chromosomal dispositions of 5S and 18S rDNA loci in five taxa, three of which were previously unmapped, and have discussed the evolutionary implications of the new results. Following this we have drawn together all the available information from disparate sources and have framed a more complete picture of rDNA chromosome patterns within the whole of this economically important complex. This is the first time such information has been integrated across numerous studies.
Seeds from five populations (Table
Taxon | Identity and source of seed |
---|---|
Festuca simensis Hochstetter ex A. Richard, 1851 | BL 2043, Margot Forde Forage Germplasm Centre |
Lolium perenne Linnaeus, 1753 | Cv Impact, Margot Forde Forage Germplasm Centre |
Lolium multiflorum Lamarck, 1779 | B 3380, Margot Forde Forage Germplasm Centre |
Lolium multiflorum MRCN | Cv. Barberia, PGG Wrightson Seeds |
Micropyropsis tuberosa Romero Zarco et Cabezudo, 1983 | BZ 8319, Margot Forde Forage Germplasm Centre |
The DNA probes used for FISH were pTr18S (GenBank accession number AF071069), a 1.8 kb fragment from Trifolium repens Linnaeus, 1753 containing almost the entire 18S rDNA sequence representing the 35S rDNA and pTr5S (GenBank accession number AF072692), a 596 bp DNA fragment encoding the T. repens 5S rRNA. 35S and 5S rDNA probes were directly labelled with fluorochromes Fluor-X-dCTP and Cy-3-dCTP (GE Healthcare, NZ), respectively by nick translation according to manufacturer’s specifications. Double target FISH using the above DNA probes, post-hybridisation washing and counterstaining of somatic chromosomes with DAPI were carried out as described earlier (
Results of double colour FISH mapping using 35S and 5S rDNA sequences as probes on pro-metaphase or metaphase chromosomes of Lolium perenne Linnaeus, 1753 (2n = 2x = 14) are given in Fig.
DAPI stained (grey scale) metaphase cells in the left column and the same cells in the right column displaying FISH mapping of 5S (red signals) and 35S rDNA sequences (green signals) in a, b L. perenne c, d L. multiflorum, European origin e, f L. multiflorum MRCN Moroccan origin g, h M. tuberosa i, j F. simensis. Dotted lines in a, c, e, g, and i denote decondensed 35S rDNA chromatin.
In contrast to L. perenne and L. multiflorum of north European origin, L. multiflorum (2n = 2x = 14) of Moroccan origin displayed only two pairs of NORs (Fig.
Micropyropsis tuberosa, 2n = 2x = 14, with a symmetrical karyotype, displayed one 5S and one 35S rDNA locus, each on separate chromosome pairs, and located proximally on the short arms (Fig.
Festuca simensis, 2n = 4x = 28, displayed all biarmed chromosomes and a symmetrical karyotype. The eight FISH signals were distributed on separate chromosomes (Fig.
We have mapped the diversity in the chromosomal locations of the two rDNA sequences for five taxa of the Schedonorus-Lolium complex. Three of these, M. tuberosa, L. multiflorum MRCN and F. simensis, were previously unmapped. The results for L. perenne and N European L. multiflorum agree with previous studies (
Micropyropsis tuberosa exhibited single 5S and 35S rDNA loci positioned proximally on separate chromosomes as was also the case for F. pratensis (
The “L. multiflorum” of Moroccan origin is typical of the main Lolium lineage in having more than one 35S rDNA locus. One of these 35S loci has a syntenic 5S locus on the opposite chromosome arm, in common with L. perenne and L. multiflorum of Eurasian origin. However, compared with Eurasian L. multiflorum the Moroccan taxon has one fewer 35S locus. The Moroccan “L. multiflorum” could be a new and unique N African taxon that has chromosomal affinities with the allogamous Eurasian Lolium species.
A previous cytological analysis of the tropical African broad-leaved fescue, F. simensis, showed it to be tetraploid (2n = 4x = 28) and AFLP fingerprinting revealed a close phylogenetic relationship with European broad-leaved fescues, especially with hexaploid F. gigantea, (
Based on a low-copy nuclear gene analysis,
Schematic representation of the putative evolutionary lineages for chromosomes carrying 5S and 35S rDNA loci in the Schedonorus-Lolium complex. The numbers of marker and non-marker chromosomes are given inside the boxes. Red and black double circles represent 5S and 35S rDNA loci, respectively. *species in solid boxes were investigated during the present study; †synonym for L. rigidum var. rottbollioides; ††synonym for F. arundinacea subsp. fenas (Lagasca y Segura) Bornmüller, 1928 (
All Lolium species, along with M. tuberosa and F. pratensis are natural diploids. The Lolium species, are evolutionarily more recent than the Festuca species based on DNA sequence phylogenies (
A single 5S rDNA locus (two FISH signals per cell) consistently occurred in all Lolium species. The number of 35S loci displayed has previously been noted as a distinguishing feature between F. pratensis (one locus) and Lolium species (more than one locus) (
The two types of rDNA loci can be located on the same chromosome (syntenic) or on separate chromosomes (non-syntenic) (
The data presented in Fig.
The numbers of 5S loci range from two in the tetraploids, F. mairei St. Yves, 1922 and F. glaucescens to eight in decaploid F. letourneuxiana (Festuca arundinacea var. letourneuxiana (St. Yves) Torrecilla et Catalán, 2002) while 35S numbers ranged from one in tetraploid F. simensis to six in F. letourneuxiana (Fig.
Seven of the eight Festuca polyploids had the 5S rDNA loci in the proximal region, either exclusively or in addition to other regions (Fig.
Two allotetraploids, F. mairei and F. glaucescens have been suggested as the ancestral parents of allo-octoploid F. atlantigena (Festuca arundinacea subsp. atlantigena (St. Yves) Auquier, 1976) based on the formation of fertile interspecific hybrids between the two suggested ancestral parental species (
All four Festuca higher polyploids with putative parents reveal additivity of numbers of 5S loci, but, in three cases, losses of 35S loci, (Fig.
The three allotetraploids (F. simensis, F. mairei and F. glaucescens), as the putative sub-genome donors to the allohexaploid and octoploid species, provide a novel example of sequential allopolyploidisation. The putative progenitors of all three allotetraploids remain unknown. However, nuclear and chloroplast DNA sequence analyses (
The variations in numbers of 35S sites in Lolium and the post-polyploidisation changes in the Festuca species have apparently occurred without any obvious changes in the symmetrical bi-armed karyotype that is a consistent feature of the Schedonorus-Lolium complex. Such lability in the absence of obvious structural changes might be attributable to paracentric chromosome rearrangements and/or the activity of transposable elements (
This report has extended the distributional data on the rDNA sequences to seven of the ten known Lolium species and has added F. simensis to the list of seven polyploid fescue species already characterised. It has also explored the distribution patterns of rDNA loci within the Schedonorus-Lolium complex and considers some possible evolutionary trends. While these patterns can be used to deduce relationships among the higher polyploid Festuca species, the diploid progenitors of the allotetraploid species remain unidentified and enigmatic.
HAA designed the study with AVS and WMW. HAA performed the experiment, analysed the data and wrote the manuscript with co-writing from WMW. NWE isolated the DNA and labelled all the probes for FISH. AVS and NWE provided significant help in improving the manuscript. All authors read and approved the final manuscript.
We thank Joy Dick and Jaspreet Singh Sidhu for their help in formatting the manuscript for this journal. The work was supported by the New Zealand Ministry of Business, Innovation and Employment (contract no. C10X1202).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Warren Mervyn Williams https://orcid.org/0000-0002-3156-3821