HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Jones, T.A. Articles by Zhang, Y. Search for Related Content PubMed Articles by Jones, T.A. Articles by Zhang, Y. Agricola Articles by Jones, T.A. Articles by Zhang, Y.

CROP BREEDING, GENETICS & CYTOLOGY

The Western Wheatgrass Chloroplast Genome Originates in Pseudoroegneria

^a USDA-ARS, Forage and Range Research Lab., Utah State Univ., Logan, UT 84322-6300 USA
^b USDA-ARS, Corn and Soybean Research, OARDC, 1680 Madison Ave., Wooster, OH 44691-4096 USA
^c Utah State Univ., Logan, UT 84322-6300 USA

tomjones{at}cc.usu.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
The octoploid Pascopyrum smithii (Rydb.) A. Löve (StHNsXm) arose from hybrization among four diploid genera, three of which are known from genome analysis, but the diploid origin of the Pascopyrum chloroplast genome remains unknown. Identification of the maternal parents in the hybridizations leading to Pascopyrum will guide efforts to reconstruct allopolyploid germplasm from ancestral taxa. We compared the DNA sequences of a 762-base pair (bp) segment of the ndhF (nicotinamide adenine dinucleotide dehydrogenase subunit F) chloroplast gene of western wheatgrass (Pascopyrum smithii; StHNsXm) with that of its putative allotetraploid, i.e., Elymus (StH) and Leymus (NsXm), and diploid, i.e., Pseudoroegneria (St), Hordeum (H), Psathyrostachys (Ns), ancestors. To ascertain the 2x to 8x chloroplast phylogeny, the gene sequences were aligned and a phylogenetic tree was constructed by the neighbor-joining method. Pascopyrum smithii differed by 0 to 2 and 6 to 9 bp from its two 4x ancestors, Elymus and Leymus, respectively. Elymus differed by 2 and 10 to 13 bp from its two 2x ancestors, Pseudoroegneria and Hordeum, respectively. Pascopyrum, Elymus, and Pseudoroegneria taxa clustered together, but separately from Leymus and Hordeum taxa. The Pascopyrum chloroplast genome appears to have originated from the diploid Pseudoroegneria through the tetraploid Elymus. De novo synthesis of Pascopyrum germplasm from its tetraploid ancestors should be conducted with cognizance of the preference for Pseudoroegneria cytoplasm found in nature. Leymus differed from its diploid ancestor, Psathyrostachys, by 14 to 15 bp, indicating that the second unknown diploid ancestor of Leymus may have contributed its chloroplast DNA.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
SPECIES of Pascopyrum, Elymus, Leymus, and Pseudoroegneria are important range grasses in western North America. Western wheatgrass (Pascopyrum smithii [Rydb.] A. Löve), the sole member of the genus (Löve, 1980), is octoploid (Gillet and Senn, 1960), although aneuploids are frequent in this species because it may propagate by rhizomes (Dewey, 1975). North American Elymus (StH) and most Leymus (NsXm) are tetraploid, while most North American Pseudoroegneria (St) are diploid. Dewey (1975) maintained that western wheatgrass is an allooctoploid hybrid between Elymus and Leymus tetraploids. Elymus has been designated as the genus to include all StH tetraploid species (Dewey, 1984). Investigators agree that one of the Leymus genomes originates from Psathyrostachys (Ns), but delineation of the second has been problematic (Zhang and Dvoák, 1991). Wang and Hsiao (1984) accepted J (= E^b of Thinopyrum bessarabicum) as the second Leymus genome on the basis of cytological (Petrova, 1970) and morphological (Löve, 1984) considerations, but more recent DNA hybridization (Zhang and Dvo1.gif" BORDER="0">ák, 1991; Wang and Jensen, 1994) and genomic (Wang and Jensen, 1994) data have eliminated it as a possible Leymus genome. Zhang and Dvoák (1991) presented repeated-sequence hybridization data which indicated that the second genome of Leymus is also derived from Psathyrostachys. Wang and Jensen (1994), however, found insufficient numbers of trivalents in Leymus x Psathyrostachys triploid hybrids to justify this conclusion and contended that the identity of the second genome remains unknown. This genome has been designated as Xm, making Leymus NsXm and Pascopyrum StHNsXm (Wang et al., 1995).

On the basis of morphological traits, ecological adaptation, germination characteristics, geographical distribution, and reproductive biology, Dewey (1975) suggested that the most likely tetraploid progenitors of western wheatgrass were thickspike wheatgrass [E. lanceolatus (Scribn. & J.G. Smith) Gould] and beardless wildrye [L. triticoides (Buckl.) Pilger]. However, the thickspike wheatgrass x beardless wildrye hybrid was never made by Dewey, and its genetic similarity to western wheatgrass remains undemonstrated. Instead, amphiploids of the genomically similar E. canadensis x E. dasystachys (= L. secalinus) hybrid were crossed with western wheatgrass, producing progeny when the amphiploid served as the pollen parent (Dewey, 1975). All of these octoploid hybrids (western wheatgrass x amphiploid), however, exhibited meiotic abnormalities and were completely sterile.

The chloroplast genome is maternally inherited in grasses and provides a mechanism to determine the direction of hybridization in polyploid evolution. Here, we sequenced a 762-bp segment from the ndhF chloroplast gene of octoploid western wheatgrass (StHNsXm) from putative tetraploid (Elymus [StH], Leymus [NsXm]) and diploid (Pseudoroegneria [St], Hordeum [H], Psathyrostachys [Ns]) ancestors. Our objective was to identify the diploid origin of the chloroplast genome of Pascopyrum, Elymus, and Leymus polyploids. This information will guide efforts to reconstruct allopolyploid germplasm from ancestral taxa.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Seedlings of 17 accessions (Genbank accession numbers beginning with AF) representing the genera Pascopyrum (8x), Elymus (4x), Pseudoroegneria (2x), Hordeum (2x), Leymus (4x), and Psathyrostachys (2x) were included here (Table 1) . Young leaves from two greenhouse-grown plants per accession were harvested and immersed in liquid nitrogen immediately prior to DNA isolation. The two plants from each accession served as replicates. Total DNA was isolated by a modification of the CTAB extraction method (Lassner et al., 1989; Williams et al., 1993). The 3' end of the chloroplast ndhF gene (830 bp) was amplified with 1318F and 2110R primers (Olmstead and Sweere, 1994). DNA was amplified in 100-µL reactions containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% (v/v) Trition X-100, 0.25 mM dNTP, 0.86 pM ndhF 2110R primer, 0.42 pM ndhF 1318F primer, 2 mM MgCl₂, and 50 ng template DNA. Samples were preheated at 93°C for 3 min, and 2.5 U Taq DNA polymerase (Promega, Madison, WI) was added. Thirty cycles (35 s at 93°C, 35 s at 51°C, 2.5 min at 72°C) of amplification were followed by a 10-min incubation at 72°C. Size and purity of the amplified DNA was verified by agarose gel electrophoresis (Sambrook et al., 1989). The PCR products were purified with Promega Wizard PCR Preps DNA Purification System according to the manufacturer's instructions. DNA concentration was determined on a TKO 100 mini-fluorometer using Hoescht 33258 according to manufacturer's instructions (Hoefer, San Francisco, CA).

DNA sequences were aligned by the PILEUP and LINEUP functions of the Wisconsin Package (Devereux, 1994). Phylogenetic trees were generated by the neighbor-joining distance method (Saitou and Nei, 1987) of the MEGA program (Kumar et al., 1993). Bootstrap values (based on 500 replications) were generated by MEGA. Distances among nucleotide sequences were generated by the Jukes-Cantor distance method (Jukes and Cantor, 1969). The method gives the maximum likelihood estimate of the number of nucleotide substitutions between two sequences. Our sequences met the criteria for the Jukes-Cantor method, where the number of nucleotide substitutions per site (d ) was 0.3 and the transition/transversion ratio was < 2 (Nei, 1991; Kumar et al., 1993). We used the p parameter (Kumar et al., 1993), the proportion of amino acid sites at which sequences of two accessions differ, to estimate the genetic distance between their deduced amino acid sequences. Oat (Avena sativa L.), Kentucky bluegrass (Poa pratensis L.), and ricegrass [Piptatherum racemosum (Smith) Barkw.] served as outgroups. Sequences for these accessions as well as Hordeum vulgare L. were obtained from Genbank (Genbank accession numbers beginning with U) (Table 1).

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Several plastid DNA sequences have been used to develop phylogenies in plants including the coding regions of the chloroplast rbcL (gene for ribulose bisphosphate carboxylase–oxygenase large subunit) and ndhF genes and the intron and intergenic spacer regions of the trnL (gene for leucine tRNA) gene (Taberlet et al., 1991; Olmstead and Sweere, 1994; Clark et al., 1995). The ndhF coding region is more variable than the rbcL (Olmstead and Sweere, 1994; Clark et al., 1995), due in part to the greater length of ndhF (>2000 bp vs. 1400 for the rbcL). Among the Poaceae, Clark et al. (1995) found 60% of the variability in the 3' third of the ndhF sequence. Because Triticeae species being examined were closely related, we focused our sequence analysis on the 3' third of the ndhF chloroplast gene. As in other species from the Poaceae family, the most variable region of the Triticeae ndhF was at the 3' end of the gene, especially from bp 500 to 600 (corresponding to bp 1860–1960 of the tobacco gene). Although insertions or deletions of up to 1 to 5 codons were identified among Poaceae ndhF sequences (Clark et al., 1995), none were found among the sequences we examined.

Sequences for the two individuals of each of the 18 Triticeae accessions were identical, indicating that the accessions were largely uniform for this sequence and that no point mutations were introduced during amplification. However, length differences have been found between ndhF genes of Triticeae grasses and non-Triticeae grasses, e.g., rice (Oryza sativa L.) (Clark et al., 1995), and dicots, e.g., tobacco (Olmstead and Sweere, 1994). The 762-bp sequence analyzed here codes for 253 amino acids, with the open reading frame beginning at bp 2. The sequence is A-T rich. The `Rodan' western wheatgrass sequence, for example, is 69% AT.

Jukes-Cantor distances, for nucleic acid sequences, and p-distances, for deduced amino acid sequences, i.e., the number of differences per number of sites analyzed, were calculated for the 21 accessions. Nucleotide substitutions were found at 30 of the 762 positions (27 of the 253 codons) among the 18 Triticeae accessions. These 30 mutations consisted of 23 single-base, two double-base (bp 548–550; 587–589), and one triple-base (bp 575–577) substitution. Multiple nucleotide substitutions were found at one position within the 18 Triticeae accessions and at six additional positions among the Triticeae accessions and the outgroups. Transitions (substitutions of a purine for a purine or a pyrimidine for a pyrimidine) accounted for 46% of all nucleotide substitutions among the 18 Triticeae accessions, thus numbers of transitions and transversions (substitutions of a purine for a pyrimidine or a pyrimidine for a purine) were about equal. When all 21 accessions (including outgroups) were considered, nucleotide substitutions were found at 129 (17%) of all positions in 98 codons. Single, double, and triple-base substitutions were found in 71, 23, and 4 codons, respectively, in the 21-accession data set.

Among the 18 Triticeae accessions, nonsynonymous nucleotide substitutions resulted in 14 (6%) amino acid substitutions. Nonsynonymous substitutions occurred at 64 (25%) additional positions between the Triticeae accessions and the outgroups. Multiple amino acid substitutions were found at one position within the 18 Triticeae accessions and at eight additional positions between the Triticeae accessions and the outgroups.

Three different sequences were found among the 10 western wheatgrass accessions examined and were assigned to three different groups accordingly. Group 1 includes cultivars Flintlock (origin: central and southwestern Nebraska and northwestern Kansas), Barton (origin: Barton Co., KS), Arriba (origin: Flagler, CO), Walsh (origin: southern Alberta and southwestern Saskatchewan), Rodan (origin: Morton Co., ND), and the accessions Atkins-172 (origin: Utah) and R-9-1—5 (origin: Green River, WY); Group 2 includes the cultivar Rosana (origin: Forsyth, MT) and the accession Atkins-142 (origin: Arizona); Group 3 includes only the accession EPC-8 (origin: Vernal, UT). Groups 1 and 2 both include materials from each side of the Continental Divide. Groups 1 and 2 differed by two base pairs, Groups 2 and 3 by one base pair, and Groups 1 and 3 by three base pairs. The sequence of Group 2 was identical to the sequence of E. lanceolatus/wawawaiensis. Deduced amino acid sequences of Group 1 differed by two moieties from sequences of Groups 2 and 3 and the two Elymus accessions. Thus, 2 of the 3 base pair differences found within western wheatgrass were reflected in the deduced amino acid sequence.

The neighbor-joining tree for nucleotide sequence (Fig. 1) grouped the sequences for Secar (E. wawawaiensis) and Critana (E. lanceolatus), which were identical, with sequences of 10 western wheatgrass accessions (bootstrap value of 89%). This means that these 12 accessions clustered together and apart from all other accessions in 89% of the 500 trees constructed from random samples of the data. Jukes-Cantor distances among the 12 accessions ranged from d = 0.000 to 0.004 (Table 2) . These data support the contention that the western wheatgrass chloroplast was inherited from the E. lanceolatus tetraploid.

View larger version (18K): [in this window] [in a new window]   Fig. 1 Neighbor-joining phylogenetic tree for ndhF DNA sequences. Poa pratensis, Avena sativa, and Piptatherum racemosum served as outgroups. Numbers at the branch points give the bootstrap confidence values for 500 replications. Values greater than 90 indicate statistically significant groupings of accessions at that branch node (Kumar et al., 1993). Genetic distances calculated according to Jukes and Cantor (1969)

Leymus cinereus and L. triticoides accessions clustered separately from the Elymus, Pascopyrum, and Pseudoroegneria accessions, indicating that a tetraploid Leymus species likely did not donate its chloroplast to Pascopyrum (Fig. 1). Leymus accessions grouped with H. bogdanii (H) and H. vulgare (I). Jukes-Cantor distances between the Leymus/Hordeum cluster and the Elymus/Pascopyrum/Pseudoroegneria cluster were d = 0.005 to 0.020 (Table 2). Jukes-Cantor distances between Leymus and Psathyrostachys accessions were d = 0.019 to 0.020. Psathyrostachys juncea clustered separately from all other Triticeae species (Fig. 1).

The neighbor-joining method for deduced amino acid sequence generated a tree similar to the nucleotide-sequence tree (not shown). Pascopyrum and Elymus clustered together with a bootstrap value of 93%. p-Distances among these 12 accessions were 0.000 to 0.008 (Table 2). These 12 accessions clustered together with Pseudoroegneria (bootstrap value = 87%). Variation among the 18 Triticeae accessions was found at nine of 64 amino acid positions in the deduced sequence.

Empirical studies and computer simulations have shown that the neighbor-joining method is a highly efficient method for recovering the correct topology with a relatively small number of data points (Nei, 1991; Kumar et al., 1993). Branch-and-bound and heuristic maximum parsimony trees (not shown) were essentially similar to the neighbor-joining tree in that Pa. smithii clustered with Elymus and Pseudoroegneria spicata. The neighbor-joining tree was also topologically identical to a tree generated by UPGMA (unweighted pair group method using arithmetic averages). The similarity of these trees suggest that the chloroplast genomes of Pascopyrum, Elymus, and Pseudoroegneria are closely related. The chloroplast genome of Leymus appears to be distantly related to these three. As such, the Leymus chloroplast is not likely ancestral to the Pascopyrum chloroplast. While Psathyrostachys (2x) is considered a direct nuclear progenitor of Leymus (4x), their chloroplasts are less closely related than Pseudoroegneria (2x) is to Pascopyrum (8x). The Leymus chloroplast may have originated from the unknown ancestor carrying the Xm genome rather than from Psathyrostachys. Indeed, H. bogdanii (H) and H. vulgare (I) are more similar to Leymus (d = 0.007–0.012) than Leymus is to Psathyrostachys (d = 0.019–0.020) (Table 2). However, fluorescent in situ hybridization data indicate that the identity of the unknown Xm genome is neither H nor I (R. R-C. Wang, USDA-ARS, Logan, UT, 1998, personal communication).

Our data indicate that the Pseudoroegneria chloroplast genome was preferred both in speciation of Elymus upon hybridization of its diploid ancestors and in speciation of Pa. smithii upon hybridization of its tetraploid ancestors. Therefore, revised genomic designations for Pa. smithii and E. lanceolatus/wawawaiensis are StHNsXm and StH, respectively, where the underline indicates the genome of cytoplasmic origin. While the sequences of the 10 western wheatgrass accessions surveyed were not identical, they appear to have originated from Pseudoroegneria in all cases. While this preference cannot be attributed to the sequence itself, the sequence has allowed us to document a preferred pattern of inheritance of the chloroplast genome in the polyploid evolution of western wheatgrass. De novo synthesis of Pascopyrum germplasm from its tetraploid ancestors should be conducted with cognizance of the preference for Pseudoroegneria cytoplasm found in nature.Gillett Senn 1960

	ACKNOWLEDGMENTS

 
We thank Yeelan Wang (Utah State University Biotechnology Center) for providing accurate and timely DNA sequence analysis.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Contribution of the Utah Agric. Exp. Stn. Paper no. 7030.

Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.

Received for publication September 16, 1998.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 

• Clark L.G., Zhang W.P., Wendel J.F. A phylogeny of the grass family (Poaceae) based on ndhF sequence data. System. Bot. 1995;20:436-460.
• Devereux J.R. GCG, sequence analysis software package, version 8. Madison, WI: University Research Park, 1994.
• Dewey D.R. The origin of Agropyron smithii. Am. J. Bot. 1975;62:524-530.
• Dewey D.R. The genomic system of classification as a guide to intergeneric hybridization with the perennial Triticeae. In: Gustafson J.P., ed. Gene manipulation in plant improvement. New York: Plenum Press, 1984:209-279.
• Gillett J.M., Senn H.A. Cytotaxonomy and infraspecific variation of Agropyron smithii Rydb. Can. J. Bot. 1960;38:747-760.
• Jukes T.H., Cantor C.R. Evolution of protein molecules. In: Munro H.N., ed. Mammalian protetin metabolism. New York: Academic Press, 1969:21-32.
• Kumar, S.K., K. Tamura, and M. Nei. 1993. Molecular evolutionary genetics analysis. Version 1.0. Institute of Molecular Evolutionary Genetics, The Pennsylvania State University, University Park.
• Lassner M.W., Peterson P., Yoder J.I. Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny. Plant Mol. Biol. Report. 1989;7:116-128.
• Löve A. IOPB chromosome number reports. LXVII. Poaceae-Triticeae-Americanae. Taxon 1980;29:163-169.
• Löve A. Conspectus of the Triticeae. Feddes Repertonium 1984;95:425-521.
• Nei M. Relative efficiencies of different tree making methods for molecular data. In: Miyamoto M.M., Cracraft J.L., eds. Recent advances in phylogenetic studies of DNA sequences. Oxford, UK: Oxford University Press, 1991:90-128.
• Olmstead R.G., Sweere J.A. Combining data in phylogenetic systematics: An empirical approach using three molecular data sets in the Solanaceae. Syst. Biol. 1994;43:467-481.[ISI]
• Olmstead R.G., Sweere J.A., Wolfe K.H. Ninety extra nucleotides in ndhF gene of tobacco chloroplast DNA: A summary of revisions to the 1986 genome sequence. Plant Mol. Biol. 1993;22:1191-1193.[ISI][Medline]
• Petrova, K.A. 1970. Morphology and cytological investigation of Agropyron elongatum (Host.) P.B. 2 n = 14 x Elymus mollis Trin. 2 n = 28; F₁ hybrids and amphidiploids. p. 158–176. In Otaldalen. gibridiz. i poliploidiya. Nauka, Moscow.
• Saitou N., Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987;4:406-425.[Abstract]
• Sambrook J., Fritsch E.F., Maniatis T. Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.
• Taberlet P., Gielly L., Patou G., Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol. 1991;17:1105-1109.[ISI][Medline]
• Wang, R. R-C., R. von Bothmer, J. Dvorak, G. Fedak, I. Linde-Laursen, and M. Muramatsu. 1995. Genome symbols in the Triticeae (Poaceae). p. 29–34. In R. R-C. Wang et al. (ed.) Proc. 2nd Int. Triticeae Symp., Logan, UT. 20–24 June 1994. Utah State University Publication Design and Production, Utah State University, Logan.
• Wang R.R.-C., Hsiao C. Morphology and cytology of interspecific hybrids of Leymus mollis. J. Heredity 1984;75:488-492.[Abstract/Free Full Text]
• Wang R.R.-C., Jensen K.B. Absence of the J genome in Leymus species (Poaceae: Triticeae): Evidence from DNA hybridization and meiotic pairing. Genome 1994;37:231-235.
• Williams J.G.K., Hanafey M.K., Rafalski J.A., Tingey S.V. Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol. 1993;218:704-740.[ISI][Medline]
• Zhang H.-B., Dvoák J. The genome origin of tetraploid species of Leymus (Poaceae: Triticeae) inferred from variation in repeated nucleotide sequences. Am. J. Bot. 1991;78:871-884.

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Jones, T.A. Articles by Zhang, Y. Search for Related Content PubMed Articles by Jones, T.A. Articles by Zhang, Y. Agricola Articles by Jones, T.A. Articles by Zhang, Y.