Crop Science 43:32-36 (2003)
© 2003 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
C-Banding and Localization of 18S-5.8S-26S rDNA in Tall Oatgrass Species
Crystal C. Mitchell,
Susan E. Parkinson,
Theron J. Baker and
Eric N. Jellen*
Brigham Young Univ., Dep. of Agronomy and Horticulture, 275 WIDB, Provo, UT 84602
* Corresponding author (enj{at}email.byu.edu)
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ABSTRACT
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The tall oatgrasses (Arrhenatherum spp., x = 7) are perennial diploid or autotetraploid forages of temperate grasslands. The most economically important species is common tall oatgrass, A. elatius (L.) P. Beauv. ex J. Presl & C. Presl. We were interested in cytogenetically characterizing this species due to its potential as a tertiary germplasm source for improving heat and cold tolerance in common oat (Avena sativa L.). We used C-banding and fluorescence in situ hybridization with an 18S-5.8S-26S (45S) ribosomal DNA probe to examine the degree of chromosomal variation among tetraploids of the tall oatgrass genus. C-banding karyotypes were analyzed for three geographically diverse accessions of A. elatius and one accession each of A. album (Vahl) Clayton, A. parlatorei (Woods) Potzt., and A. thorei Durieu [= A. longifolium (Thore) Dulac]. Tall oatgrass chromosomes were predominantly euchromatic and metacentric to submetacentric, with occasional subtelocentric chromosomes. The A. parlatorei karyotype was the most distinct of the six accessions, having a prominent telomeric C-band. Arrhenatherum album, A. elatius, and A. thorei had a single nucleolus organizer region (NOR) locus as evidenced by the appearance of four in situ hybridization sites on somatic metaphase chromosome preparations using the wheat 45S ribosomal DNA (rDNA) clone pTa71.
Abbreviations: FISH, fluorescence in situ hybridization • NOR, nucleolus organizer region • rDNA, ribosomal DNA • SSC, saline sodium citrate
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INTRODUCTION
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ARRHENATHERUM BEAUV. (tall oatgrass) has been assigned to the tribe Aveneae in the Poaceae family. Avena L. and Helictotrichon Besser. are the most closely related genera to Arrhenatherum within the tribe (Leggett, 1992). Arrhenatherum includes a series of diploid and polyploid bunchgrasses of primarily Eurasian origin with a base chromosome number of x = 7. Tall oatgrass, A. elatius, is an allogamous diploid or semiallogamous autotetraploid perennial forage (Petit et al., 1997; Katsiotis et al., 1996). Tall oatgrass is cultivated in the grasslands of the central and northern USA, western Eurasia, East Asia, and the Mediterranean basin.
In spite of its regional importance as a crop in its own right, there has been interest in A. elatius as a tertiary germplasm resource for improving oat. Whereas tall oatgrass is a perennial, capable of surviving winter temperatures well below 0°C, winter oat production is currently restricted to areas with mild winters due to the crop's susceptibility to freeze damage. In addition, tall oatgrass might be exploited as a source of heat tolerance to alleviate heat-induced sterility and yield reductions in spring oat. Although it has not been investigated, tall oatgrass might also prove a valuable source of resistance genes for common oat pathogens such as barley yellow dwarf virus. Reciprocal genetic exchanges could also potentially be exploited to improve forage quality of A. elatius.
Several reports hint at the close genetic relationship between Arrhenatherum and Avena. Gervais (1983) had limited success in obtaining putative hybrid embryos between Arrhenatherum and Avena. Katsiotis et al. (1996) reported that a cloned retrotransposon-like repetitive DNA sequence from a Av. vaviloviana (Malzev) Mordv. (AABB genomes) displayed greater homology to total genomic DNA of A. elatius than to DNA of C-genome Avena diploids in Southern hybridization experiments. Katsiotis et al. (2000) obtained similar results in hybridization experiments using dispersed, repetitive, nonretrotransposon sequences isolated from Av. strigosa Schreb. (AsAs genomes) and Av. sativa (AACCDD) as probes on a range of Avena species and A. elatius.
The work of Gervais (1983) and Katsiotis et al. (1996) indicated that intergeneric hybridization between Arrhenatherum and Avena might hold promise for gene exchange between the two genera. Our objective was to produce C-banding karyotypes of Arrhenatherum accessions to compare chromosome morphology with previously C-banded Avena species (Jellen et al., 1993a,b; Jellen and Gill, 1996; Jellen and Ladizinsky, 2000) and to determine the number of NOR ribosomal DNA (45S rDNA) sites using fluorescence in situ hybridization (FISH). In addition to providing basic information regarding the genomic organization of tall oatgrass tetraploids, these karyotypes could also be used in future intergeneric breeding experiments to identify alien Arrhenatherum chromosomes introgressed into oat.
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MATERIALS AND METHODS
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Plant Materials
Six accessions of Arrhenatherum were included in the C-banding portion of this study (Table 1). Of these, two were A. elatius, one was A. elatius ssp. bulbosum, one was A. album PI 254870, one was A. thorei, and one was A. parlatorei. Arrhentherum thorei and A. parlatorei were obtained from the Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK)–Genbank, Gatersleben, Germany. All other accessions were obtained from the USDA, ARS, NPGS, WPRIS in Pullman, WA, USA. In addition, Av. sativa cv. Ogle was used as a control and A. album PI 283189 was included for the FISH experiments. A. parlatorei was not included in the FISH experiments due to an insufficient quantity of viable seed.
Somatic Metaphase Chromosome Preparations
Dehulled seeds from all accessions were placed on filter paper in Petri plates and pretreated with 100 mg L-1 solution of GA3 (Sigma, St. Louis, MO) at 4°C for 5 to 14 d to break dormancy. Petri plates were then moved to a 23°C incubator, or to 
23°C in a lab drawer until emergent roots reached 0.5 to 5.0 cm long. We used a cell synchronization protocol based on Arumuganathan and Earle (1991) and Lee et al. (1997). A 1.25-mM solution of hydroxyurea was added to the Petri plates containing germinating seeds at room temperature for 21 to 26 h, after which the seeds were rinsed once with distilled water. Fresh distilled water was then added and the seeds incubated at room temperature for 2 to 3 h. The water was removed and 1 to 3 µM trifluralin (Treflan, Dow AgroSciences, Indianapolis, IN) solution was added for 5 to 6 h at 23°C. Root tips were fixed in a 3:1 mixture of ethanol:glacial acetic acid.
Squashing proceeded generally as described in Jellen et al. (1993a). For in situ hybridization, slide preparations were made in 40% glacial acetic acid and were not heated over an alcohol flame as part of the squashing process. Immediately following inspection under phase-contrast, preparations for C-banding were placed on a block of dry ice for several minutes, the cover slips were removed with a razor blade, and the slides were immersed in 100% ethanol overnight. For in situ hybridization, slides containing good chromosome spreads were frozen on dry ice, the cover slips were removed, and the slides were stored at -20°C until use.
C-Banding Procedure
The C-banding protocol followed that of Jellen et al. (1993a) with the exception that slides were placed in a 0.2-M solution of HCl at 60°C for 2.5 min and the final staining solution consisted of 30 mL of 0.6 M KH2PO4, 42 mL of 0.6 M Na2HPO4, and 4 mL Giemsa liquid stain (Sigma, St. Louis, MO).
Fluorescence In Situ Hybridization Procedure
Wheat 45S rDNA probe pTa71 (Gerlach and Bedbrook, 1979) was graciously provided by B.S. Gill and labeled with Alexafluor 488-5-dUTP direct-label fluorophore (Molecular Probes, Eugene, OR) using standard nick translation procedures, according to manufacturer's recommendations. The labeled probe was then purified by centrifugation through a Sephadex G-50 column (Millipore, Bedford, MA). Slides were pretreated with 100 µg mL-1 RNase A and pepsin according to the protocol of Schmidt et al. (1994). The DNA on the chromosome preparations was denatured according to the method of Jiang et al. (1995). Our hybridization solution consisted of a mixture of 2 to 4 ng µL-1 labeled DNA probe, 20% (w/v) formamide, 10% dextran sulfate, 0.15% SDS (sodium dodecyl sulfate), and 30 ng µL-1 sheared herring sperm DNA in 5 x SSC (saline sodium citrate: 0.3 M NaCl, 0.3 M Na-citrate). The solution was denatured by incubating at 70°C for 10 min, then placed on ice for 5 to 15 min. A total of 30 µL of hybridization solution was added to each slide, which was covered with a plastic cover slip, and the slides were incubated in a humid chamber at 37°C for 16 to 20 h. Following hybridization, slides were washed in 2 x SSC for 5 min at room temperature, in 2 x SSC for 10 min at 37°C, in 2 x SSC at room temperature for 5 min, and then in 1 x PBS (phosphate-buffered saline) at room temperature for 5 min, according to the modified protocol of Jiang et al. (1995). Slides were counterstained with a solution of 5 µg mL-1 propidium iodide.
Visualization and Analysis of Images
Images of C-banded and FISH cells were viewed under a Zeiss Axioplan 2 epifluorescence microscope and images captured using a SenSys cooled CCD camera (Photometrics, Tucson, AZ) under x630 magnification. Sharpening, contrast enhancement, and chromosome arm measurements were performed using Zeiss Image 3.2 software (Carl Zeiss, Thornwood, NY) with measurements in pixels. Karyotypes were arranged and, when necessary, images enhanced or pseudocolorized using Photoshop 5.0 and Illustrator 7.0 (Adobe, San Jose, CA) software programs. Lengths, arm ratios, and calculations of standard deviations were based on measurements of at least one cell from three plants per accession.
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RESULTS AND DISCUSSION
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C-Banding Karyotypes
The C-banding karyotypes of each of the four Arrhenatherum species are presented in Fig. 1. All of the chromosomes in the Arrhenatherum accessions examined were generally euchromatic, with most telomeres and secondary constrictions having C-bands. Not all seven of the chromosome groups could be clearly identified in these accessions due to a general lack of chromosome-specific banding patterns and relatively uniform chromosome morphology. In addition, the karyotypes revealed relatively few C-banding polymorphisms or likely rearrangements among the different accessions, with the notable exception of A. parlatorei (Fig. 1a). Chromosomes were aligned and grouped as much as possible according to centromere position, arm length, and C-bands. Five of the six C-banded accessions had similar karyotypes, with one pair of satellited chromosomes and most or all chromosomes having heterochromatic centromeres. Most of the telomeres were heterochromatic (C-bands) and centromere positions ranged from metacentric to submetacentric. Chromosome arm ratio groupings are defined as follows: 1 to 1.29, metacentric; 1.3 to 1.84, submetacentric; 1.85 to 3.0, subtelocentric. The seven chromosomes in each accession could generally be aligned in groups of four, as expected in autotetraploids.
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Fig. 1. C-banding karyotypes of (a) Arrhenatherum parlatorei GRA 633/93, (b) A. thorei GRA 579/94, (c) A. album PI 254870 (Iraq) and (d) A. elatius PI 251946 (Austria).
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The A. parlatorei 633/93 karyotype was the most divergent from the rest of the accessions. Alone among the accessions, its chromosomes had euchromatic centromeres and prominent heterochromatic C-bands at both telomeres of chromosomes 2, 4, 6, and 7, while chromosomes 1, 3, and 5 have one prominent telomeric C-band (Fig. 1a).
The other seven accessions had similar banding, though these bands were very faint in several of the chromosome groups. The A. thorei 579/94 karyotype (Fig. 1b) was generally similar to A. elatius and A. album karyotypes. Arrhenatherum album PI 254870 (Fig. 1c) contained only minor C-band intensity differences when compared with other A. album accessions (not shown), A. thorei, and A. elatius.
Arrhenatherum elatius karyotypes possessed a few subtle morphological and C-band polymorphisms. For example, PI 302853 had the only subtelocentric chromosome among the accessions examined (Table 1). The A. elatius accessions had a heterochromatic C-band in the long arm of the satellited chromosome (chromosome 4), although this band was very faint in PI 251946 (Fig. 1d).
Not all seven of the chromosome groups could be clearly identified in these accessions, as evidenced by large standard errors in chromosome measurements (Table 2). We attribute this to inherent heterogeneity within each partially allogamous accession. Another possible explanation is that minor chromosomal rearrangements are responsible for morphological divergences in these karyotypes as part of the evolutionary process.
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Table 2. Chromosome lengths and arm ratios for six Arrhenatherum accessions. Measurements were based on chromosomes in three complete cells.
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In Situ Hybridization with pTa71
We were able to hybridize wheat probe pTa71, containing a cloned monomer of the 45S rDNA nucleolus organizer rDNA repeat (NOR rDNA) labeled with Alexafluor 488-5-dUTP, to somatic metaphase and interphase chromosomes of A. elatius accessions PI 251946, A. album PI 283189, and A. thorei GRA 579/94, as well as to Av. sativa cv. Ogle (not shown). Whereas Av. sativa cv. Ogle control slides showed the expected six NOR rDNA hybridization sites (Jellen et al., 1994), the Arrhenatherum accessions had four major hybridization sites on somatic metaphase preparations at the location of the secondary constriction (Fig. 2). Since we had previously observed quadrivalent pairing in meiotic prophase in A. elatius (unpublished data, 1998), the presence of four hybridization sites was indicative of a single NOR rRNA locus on a set of four homologous chromosomes in these autotetraploid accessions. This result indicates that the diploid Arrhenatherum ancestors of A. album, A. elatius, and A. thorei likely possessed a single NOR rRNA locus. This is an unusual finding given that related Avena diploids (x = 7) have two (A-genome) or three (C-genome) loci (Linares et al., 1996), and that Avena tetraploids Av. agadiriana B.R. Baum & Fedak, Av. barbata Pott ex Link, Av. maroccana Gand. (syn. Av. magna H.C. Murphy & Terrill), Av. murphyi Ladiz., and Av. vaviloviana harbor between three and five loci (Fominaya et al., 1995; Hayasaki et al., 2001; Irigoyen et al., 2001).
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Fig. 2. In situ hybridization of wheat NOR rDNA probe pTa71 to somatic prometaphase or metaphase chromosomes of (a) Arrhenatherum album PI 283189, (b) A. thorei GRA 579/94 and (c) A. elatius PI 251946. The probe was direct-labeled with Alexafluor 488-5-dUTP and cells were counterstained with propidium iodide. Hybridization sites are indicated with arrows.
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As a general observation, the primarily euchromatic, telomere-banded chromosomes and symmetrical karyotypes of Arrhenatherum were reminiscent of published C-banding karyotypes of diploid A-genome Avena species such as Av. atlantica B.R. Baum & Fedak, Av. longiglumis Durieu, and Av. strigosa, but were markedly different from the heterochromatic karyotypes of C-genome Av. clauda Durieu, Av. eriantha Durieu, and Av. ventricosa Balansa ex Coss (Fominaya et al., 1988; Jellen and Gill, 1996). This observation further supports the molecular data of Katsiotis et al. (1996)(and 2000), which indicated a closer relationship between the Arrhenatherum genome and the Avena A genome than between the A and C genomes of Avena. This also lends hope to further efforts at realizing sexual hybridization between species of the two genera.
The karyotypes of these accessions of tall oatgrass should be useful in future intergeneric hybridization experiments, in evolutionary studies, in physical and genetic mapping, and in further genetic studies of this genus. The C-banded karyotypes also provide reference tools for studying chromosomal inheritance in future intergeneric hybridization experiments with Avena species. Future physical mapping efforts will focus on identifying 5S rDNA sites and on mapping repetitive sequences like retrotransposons and similar elements having homology to cloned Avena DNA probes.
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ACKNOWLEDGMENTS
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We appreciate the assistance of the IPK-Gatersleben and the USDA, ARS, NPGS in providing germplasm used in this study. We are also grateful to Dr. B. Friebe and Dr. B.S. Gill, Kansas State University, for providing pTa71 and for their helpful suggestions. This work fulfilled portions of M.S. thesis projects of C.C. Mitchell and S.E. Parkinson. The former was funded by a Brigham Young University Graduate Fellowship, while the latter was a National Science Foundation Graduate Fellow. The experiments comply with the current laws of the country in which they were performed.
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NOTES
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In partial fulfillment of the requirements for the M.S. degree, C.C. Mitchell and S.E. Parkinson.
Received for publication January 31, 2002.
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ABSTRACT
INTRODUCTION
