Figures
Abstract
Origin and evolution of tetraploid Elymus fibrosus (Schrenk) Tzvelev were characterized using low-copy nuclear gene Rpb2 (the second largest subunit of RNA polymerase II), and chloroplast region trnL–trnF (spacer between the tRNA Leu (UAA) gene and the tRNA-Phe (GAA) gene). Ten accessions of E. fibrosus along with 19 Elymus species with StH genomic constitution and diploid species in the tribe Triticeae were analyzed. Chloroplast trnL–trnF sequence data suggested that Pseudoroegneria (St genome) was the maternal donor of E. fibrosus. Rpb2 data confirmed the presence of StH genomes in E. fibrosus, and suggested that St and H genomes in E. fibrosus each is more likely originated from single gene pool. Single origin of E. fibrosus might be one of the reasons causing genetic diversity in E. fibrosus lower than those in E. caninus and E. trachycaulus, which have similar ecological preferences and breeding systems with E. fibrosus, and each was originated from multiple sources. Convergent evolution of St and H copy Rpb2 sequences in some accessions of E. fibrosus might have occurred during the evolutionary history of this allotetraploid.
Citation: Wu D-C, He D-M, Gu H-L, Wu P-P, Yi X, Wang W-J, et al. (2016) Origin and Evolution of Allopolyploid Wheatgrass Elymus fibrosus (Schrenk) Tzvelev (Poaceae: Triticeae) Reveals the Effect of Its Origination on Genetic Diversity. PLoS ONE 11(12): e0167795. https://doi.org/10.1371/journal.pone.0167795
Editor: Wujun Ma, Murdoch University, AUSTRALIA
Received: July 29, 2016; Accepted: November 20, 2016; Published: December 9, 2016
Copyright: © 2016 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This project was supported in part by a Research Foundation for Talented Scholars from Anhui Agricultural University and the introduced leading talent research team for Universities in Anhui Province, a discovery development grants DDG-2015-00039 from the Natural Sciences and Engineering Research Council of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: This manuscript or substantial parts of it, submitted to the journal, has not been under consideration by any other journal. No material submitted as part of a manuscript infringes existing copyrights, or the rights of a third party. All authors have approved the manuscript. The authors have declared that no competing interests exist.
Introduction
The tribe Triticeae includes not only the world’s most economically important cereal crops and forage grasses, but also troublesome weeds distributed all over the world. Within this tribe, three quarters of the species are polyploids [1].
Elymus L. as delimited by Löve [2] is the largest genus with exclusively allopolyploids including approximately 150 species. Due to the abundance of polyploid species in Elymus and closely related diploid species from various taxa in Triticeae available, Elymus is an ideal model for examining the impact of polyploidization on speciation, and the role of alloployploidy as a driver of plant diversification [3]. Cytogenetically, five basic genomes (St, H, Y, P, and W) (genome symbols follow Wang et al., [4]) have been assigned to the species in this genus [5, 6]. The majorities of species are tetraploids and characterized by having either StH or the StY genomic combination. For StH genome species, the St genome was suggested maternally donated by Pseudoroegneria (Nevski) Á. Löve [7–13]. The H genome was derived from Hordeum L. The StH genome Elymus species have adapted to an enormously wide range of climates and habitats, making them of special interest for speciation and ecological research [3].
Elymus fibrosus (Schrenk) Tzvel. is one of StH genome species with a predominantly self-pollinating, distributed in Russia and northern Scandinavia [14]. It grows on wet meadows, riverside sand and pebbles, and among shrubs. It is usually found growing alone or sympatrically with E. caninus, E. sibiricus L., and E. repens (L.) Gould. It has been reported low genetic variation and deficiency in heterozygosity in E. fibrosus population, which might be caused by a potential bottleneck [15, 16].
Phylogenetic analyses have demonstrated multiple origins of the H and St haplome in the StH tetraploid species of Elymus [17–22] and reticulate evolution in the Elymus [17, 18, 20–22]. The studies on genetic diversity of E. caninus [23, 24], E. fibrosus [15, 16], E. alaskanus [25–28], and E. trachycaulus complex [29, 30] have shown that despite some of these four species having similar ecological preferences and breeding systems, their population structure and genetic variation deviated highly, and each species possesses a unique pattern of genetic variation. Multiple origins are often considered as a potential source for increasing genetic variation in polyploids [31]. Our previous studies have shown multiple origins of allopolyploid wheatgrass E. caninus [21] and E. trachycaulus [22]. Elymus fibrosus is a tetraploid containing genomes of a Pseudoroegneria species (St genome) and a wild Hordeum species (H genome) [1], but its origin was rarely explored at molecular level.
In this study, single copy nuclear gene, the second largest subunit of RNA polymerase II (Rpb2), and chloroplast DNA TrnL–trnF region (spacer between the tRNA-Leu (UAA) gene and the tRNA-Phe (GAA) gene) were used to explore the origin of tetraploid E. fibrosus. The effect of origination on genetic diversity of this species was discussed.
Materials and Methods
Plant materials and DNA extraction
DNA from ten accessions of E. fibrosus species was extracted from fresh young leaf tissues using the method of Junghans and Metzlaff [32], and amplified using low copy nuclear gene Rpb2 and chloroplast TrnL-F sequences. Rpb2 and TrnL-F sequences from 19 Elymus StH genome species and some diploid Triticeae species representing the St, H, I, Xa, Xu, W, P, E and V genomes included in the analyses were downloaded from GenBank (NCBI) or obtained from the published data [17, 21, 33]. Accession number, genomic constitution, geographical origin, and GenBank identification number of these materials are listed in Table 1. The voucher specimens of E. fibrosus were deposited at Anhui Agricultural University.
DNA amplification and sequencing
The single and low copy nuclear gene Rpb2 and cpDNA gene TrnL-F sequences were amplified by polymerase chain reaction (PCR) using the primers P6F and P6FR [33], and TrnL and TrnF [9], respectively. The sequences of Rpb2 and TrnL-F region were amplified in a 20 μl reaction containing 20 ng template DNA, 0.25 mM dNTP, 2.0 mM MgCl2, 0.25 μM of each primer and 2.0 U Taq polymerase (TransGen, Beijing, China). The amplification profiles for the Rpb2 gene and TrnL-F were described in Zuo et al. [22]. PCR products were purified using the EasyPure Quick Gel Extraction Kit (TransGen, Beijing, China) according to manufacturer’s instruction.
The amplified PCR products of Rpb2 gene were cloned into the pGEM-easy T vector (Promega Corporation, Madison, Wis., USA), and transformed into E. coli competent cell DH5α according to the manufacturer’s instruction (TransGen, Beijing, China). At least 10 clones from each accession were screened and sequenced. Both the PCR products and positive colonies were commercially sequenced by the Shanghai Sangon Biological Engineering & Technology Service Ltd (Shanghai, China). Each PCR product amplified by cpDNA primer TrnL-F was independently amplified twice in order to avoid any error which would be induced by Taq DNA polymerase during PCR amplification, since Taq error that cause substitution is mainly random and it is unlikely that any two sequences would share identical Taq errors to create a false synapomorphy.
Data analysis
The chromatograph of each automated sequence was visually checked. Multiple sequences were aligned using Clustal X [34] with default parameters, and additional manual editing to minimize gaps using GeneDoc program. Maximum-parsimony (MP) analysis was performed using the computer program PAUP ver. 4 beta 10 [35]. All characters were specified as unweighted and unordered, and gaps were not included in the analysis. A heuristic search using the Tree Bisection-Reconnection (TBR) option with MulTrees on, and ten replications of random addition sequences with the stepwise addition option were used to generate most-parsimonious trees. A strict consensus tree was generated from the obtained multiple parsimonious trees. Overall character congruence was estimated by the consistency index (CI), and the retention index (RI). The robustness of clades was inferred using bootstrap values calculated with 1000 replications [36].
In addition to maximum parsimony analysis, Bayesian analysis was also performed. Models of sequence evolution were tested for each data set using PhyML 3.0 program [37]. The general time-reversible (GTR) [38] substitution model led to a largest ML score for both Rpb2 and TrnL-F compared to the other 7 substitution models (JC69, K80, F81, F84, HKY85, TN93 and custom), and was chosen in the Bayesian analysis using MrBayes 3.1 [39].
MrBayes 3.1 was run with the program’s standard setting of two analyses in parallel, each with four chains, and estimates convergence of results by calculating standard deviation of split frequencies between analyses. When 571,000 generations for Rpb2 data and 1,788,000 generations for TrnL-F were reached, the standard deviation of split frequencies fell below 0.01. Samples were taken every 1000 generations under the GTR model with gamma-distributed rate variation across sites and a proportion of invariable sites. The first 25% of samples from each run were discarded as burn-in to ensure the stationarity of the chains. Bayesian posterior probability (PP) values were calculated from a majority rule consensus tree generated from the remaining sampled trees.
Result
Rpb2 sequence and phylogeny analysis
The amplified patterns from tetraploid E. fibrosus species showed two bands with sizes of approximately 900 bp and 1000 bp, respectively, which corresponded well with previous report by Sun et al. [17]. Extensive sequence variations were detected between the sequences from the St and H genomes. Sequence alignment with the St, H, W and E genomic species indicated that the 900 bp and 1000 bp amplified in tetraploid E. fibrosus corresponded to the size of sequences from H and St genome, respectively.
A total of 58 sequences were used for phylogenetic analysis, which included 18 sequences from 10 accessions of E. fibrosus, 22 sequences from 16 other Elymus species with StH genome, and 17 sequences from diploid species in the tribe Triticeae with H, E, P, St and W genomes, and one sequence from Bromus sterilis that was used as an outgroup.
Total of 732 characters were used for phylogenetic analysis, including 385 constant, 122 parsimony uninformative and 225 parsimony informative characters. Maximum parsimony analysis of these 58 Rpb2 sequences generated 544 equally most parsimonious trees with CI = 0.807 and RI = 0.929. The separated Bayesian analyses using GTR model resulted in identical trees with arithmetic mean log-likelihood values of -4011.69 and -4014.63. The tree topology generated by Bayesian analyses using the GTR model is similar to those generated by maximum parsimony. Consensus strict tree generated from maximum parsimonious trees with Bayesian PP and maximum parsimony bootstrap (1000 replicates) value is shown in Fig 1.
Numbers above and below branches are bootstrap values from MP and Bayesian posterior probability (PP) values, respectively. Bromus sterilis was used as an outgroup. Consistency index (CI) = 0.807, retention index (RI) = 0.929.
Two distinct copies of sequences were obtained for 8 out of 10 tetraploid E. fibrosus accessions that were amplified and sequenced (Table 1). Phylogenetic analysis well separated the two copies of sequences from each accession into two different clades, one in the H genome clade, another in the St genome clade with exception of two sequences from the accession PI 564933 (Fig 1). Of two copies of sequences from accession PI 564933 recovered, one was grouped into the H clade in 93% bootstrap support and 1.00 PP, another was sister to the H clade in 89% bootstrap support and 1.00 PP (Fig 1). Only one copy of sequence each from accession H10339 and PI 345585 was recovered. The sequences from the accession H10339 was placed into the H clade, while the sequence from PI 345585 was grouped into the St clade.
Within the St clade, all sequences from E. fibrosus were grouped together with the sequences from E. canadensis, E. confusus, E. trachycaulus, E. transbaicalensis and E. wawawaiensis in a support of 0.64 PP. The sequences from other species formed a subclade in 89% bootstrap and 0.99 PP support, included in which are the sequences from diploid St genome species and tetraploid E. dentatus, E. sibiricus, E. virescens and E. wiegandii. Within the H clade, all sequences of E. fibrosus accessions except the one from accession PI 406448 were placed together in 55% bootstrap and 0.97 PP support. The sequences from E. caninus and E. dentatus are sister to the E. fibrosus group in 67% bootstrap support and 0.95 PP.
TrnL-F analysis
Maximum parsimonious analysis of 63 TrnL-F sequences was performed using B. tectorum as an outgroup. Total of 838 characters were used for phylogenetic analysis, of which 739 were constant, and 42 were parsimony informative. Maximum parsimonious analysis generated 124 most parsimonious trees with a CI = 0.903 (excluding uninformative characters) and RI = 0.951. The Bayesian analyses using GTR model resulted in identical trees with arithmetic mean log-likelihood values of -2232.85 and -2235.11. One of the maximum parsimonious tree is shown in Fig 2 with BS values from maximum parsimonious analysis and PP value from Bayesian analysis.
Numbers above and below branches are bootstrap values from MP and Bayesian posterior probability (PP) values, respectively. Bromus tectorum was used as an outgroup. Consistency index (CI) = 0.903, retention index (RI) = 0.951.
The phylogenetic tree showed several obvious groups. All sequences from Hordeum species were grouped together with the sequences from Psathyrostachys. The sequences from H genome were well separated from the sequences from St genome species. All sequences from E. fibrosus were grouped together with diploid Pseudoroegneria species (St genome), Dasypyrum villosum (V). The sequences also included in this group were from tetraploid Elymus species and Thinopyrum intermedium.
Discussion
Cytologically, Elymus fibrosus was considered as a tetraploid containing the genomes of a Pseudoroegneria species (St genome) and a wild Hordeum species (H genome) [1]. The phylogenetic analysis based on TrnL-F data here grouped all sequences from E. fibrosus with the sequences from diploid Pseudoroegneria species (St genome), Dasypyrum villosum (V), other Elymus species and Thinopyrum intermedium together (Fig 2). This grouping is not exceptional. A study using combined cpDNA restriction sites, rpoA sequences, and tRNA spacer sequences also grouped several North American Elymus species with Pseudoroegneria (St) and Dasypyrum (V) [9]. It has been suggested maternal genome of Elymus species might be donated by three possible donors: Pseudoroegneria, Dasypyrum or Thinopyrum [40]. The study on the genomic constitution and evolution of Thinopyrum intermedium using TrnL-F placed the sequences from Pseudoroegneria (St), Dasypyrum (V) and Thinopyrum intermedium in a clade [41]. Our phylogenetic analysis of E. trachycaulus (StH) also grouped the TrnL-F sequences from E. trachycaulus with the sequences from Pseudoroegneria (St), Dasypyrum (V) and Thinopyrum (E) together [22]. cpDNA data on diploid species in Triticeae revealed a close relationship among Pseudoroegneria (St), Dasypyrum (V) and Thinopyrum (E) [42]. The TrnL-F sequence from Thinopyrum intermedium chloroplast that was placed in this clade (Fig 2) was downloaded from Mahelka et al. [43], and was from the St genome since Th. intermedium is hexaploid species with the St genome donated by Pseudoroegneria (St) [41]. The chloroplast sequences from Hordeum species were well separated from the sequences from E. fibrosus (Fig 2). The presence of Pseudoroegneria-derived chloroplast sequences is consistent with the nuclear gene Rpb2 sequence data, in which the two distinct copies of Rpb2 sequences from each E. fibrosus were well separated into Pseudoroegneria and Hordeum clades (Fig 1). Taking all into consideration, we suggested Pseudoroegneria as the likeliest maternal progenitor of E. fibrosus. It was documented that Pseudoroegneria (St) was the maternal parent of polypoids containing the St nuclear genome in combination with other genomes [7], which has been reported in numerous studies [8–13, 21]. However, a study also suggested that not only Pseudoroegneria (St) but also Agropyron (P) are the likely maternal genome donors to Kengyilia (StYP) species [44]. The Pseudoroegneria as the likeliest maternal progenitor of E. fibrosus should be verified by additional chloroplast sequences.
Two distinct copies of Rpb2 sequences each from 8 out of ten accessions of E. fibrosus were discovered. Phylogenetic analysis well separated the two distinct copies of sequences from each accession into St and H clades except the two distinct copies of sequences from the accession PI 564933, confirming that seven accessions (PI 439999, PI 564930, PI 598465, PI 406467, PI 531609, PI 406448, and PI 564932) of E. fibrosus have the StH genomic constitution, and supporting the cytological evidence on the genomic constitution of this species [1]. However, one copy from the accession PI 564933 was placed into the H clade, while another copy is sister to the H clade in phylogenetic analysis (Fig 1). This is unexpected, but has also been reported in other Elymus species. In E. trachycaulus species, two/three H-like Pepc sequences from some accessions were obtained without St copy sequence from these accessions. Phylogenetic analysis clearly separated the two distinct copies of sequences from each accession into H1 and H2 clades [22]. In some accessions of StStHH allotetraploids E. lanceolatus and E. wawawaiensis, each has two different copies of sequences from the St genome, including one P. spicata–like sequence and another P. strigosa–like sequence [18]. As what has been discussed in Zuo et al [22], gene introgression from Hordeum into E. fibrosus following polyploidization; incomplete concerted evolution which incompletely homogenized St copy of Rpb2 toward second H copy of Rpb2, and concerted evolution even it is very common for highly repetitive nuclear sequences [45, 46] could occur for some low-copy nuclear genes, are the more likely reasons causing two distinct H-like copies of sequences present in the accession PI 564933 of allotetraploid E. fibrosus. It cannot be excluded that homoeologous rearrangements in Brassica napus [47, 48] and exchange among homoeologous chromosomes [49] might promote convergent evolution after polyploidization, which could result in two original distinct copies of sequences similar to each other. Only one copy of sequence each from accession H10339 and PI 345585 was recovered. If no bias in cloning or PCR amplification, there is 99.9% chance of obtaining at least one copy of each of the two ancestral allelic types for the allotetraploid [50]. This might be due to mutation in the primers region causing failure of amplification of the “missing” gene copy. Another possibility might be genome convergent evolution in allopolyploids as discussed above.Allozyme, RAPD and microsatellite studies on E. fibrosus indicated that although there are differences in the amount of genetic diversity detected by allozyme, RAPD and microsatellite analyses, this species possesses a very low amount of genetic variation in its populations [15, 16]. However, E. caninus, E. alaskanus, and E. trachycaulus with StH genomic constitution have similar ecological preferences and breeding systems with E. fibrosus, genetic diversity of E. caninus [23, 24], E. alaskanus [25–28], and E. trachycaulus complex [29, 30] showed much higher level than that in E. fibrosus. Genetic diversity within plant species is complex results of many factors such as abiotic ecological forces, biotic agents, and species characteristics including population size, breeding system, migration and dispersal [51, 52]. Multiple origins are often considered as one of potential sources for increasing genetic variation in polyploids [31]. Our previous studies have shown that St genome in E. caninus has two distinct origins from either St1 and/or St2, and that P. spicata and P. stipifolia are the most likely donors of the St2 genome copies. The sequences data also indicated multiple origins of the H genome in E. caninus [21]. Zou et al. [22] indicated that the St genome in E. trachycaulus was originated from either P. strigosa, P. stipifolia, P. spicata or P. geniculate, and that the H genome in E. trachycaulus was contributed by multiple sources. However, phylogenetic analysis of Rpb2 sequences here revealed that all St copies of sequences from E. fibrosus were grouped together, and all H copy sequences of E. fibrosus accessions except the accession PI 406448 were also placed together, suggesting that St and H genome in E. fibrosus each was more likely originated from single gene pool, which might be one of the reasons causing genetic diversity in E. fibrosus lower than those in E. caninus and E. trachycaulus.
Supporting Information
S1 File. RPB2 sequences used in phylogenetic analysis.
https://doi.org/10.1371/journal.pone.0167795.s001
(PDF)
S2 File. TrnL–trnF sequences used for phylogenetic analysis.
https://doi.org/10.1371/journal.pone.0167795.s002
(PDF)
Author Contributions
- Conceptualization: GS DXW.
- Data curation: GS.
- Formal analysis: GS PPW.
- Funding acquisition: GS.
- Investigation: DCW DMH HLG PPW XY WJW HFS.
- Methodology: DCW DXW GS.
- Project administration: DXW GS.
- Resources: GS DXW.
- Supervision: DXW GS.
- Validation: DCW DXW GS.
- Visualization: DCW DXW GS.
- Writing – original draft: GS DCW.
- Writing – review & editing: GS.
References
- 1.
Dewey DR (1984) The genomic system of classification as a guide to intergeneric hybridization with the perennial Triticeae. In: Gustafson JP, ed. Gene manipulation in Plant Improvement. New York: Columbia University Press. pp. 209–280.
- 2. Löve A (1984) Conspectus of the Triticeae. Feddes Report 95: 425–521.
- 3. Sun GL (2014) Molecular phylogeny revealed complex evolutionary process in Elymus species. J Syst Evol 52(6): 706–711.
- 4.
Wang RR– C, von Bothmer R, Dvorak J, Fedak G, Linde-Laursen I, Muramatsu M (1994) Genome symbols in the Triticeae. In: Wang RRC, Jensen KB, Jaussi C, eds. Proceeding of the 2nd International Triticeae Symposium. Utah: Logan Press. pp. 29–34.
- 5. Jensen KB, Salomon B (1995) Cytogenetics and morphology of Elymus panormitanus var. heterophyllus (Keng) Á. Löve and its relationship to Elymus panormitanus (Poaceae: Triticeae). Int J Plant Sci 156:31–739.
- 6. Torabinejad J, Mueller RJ (1993) Genome Constitution of the Australian hexaploid grass Elymus scabrus (Poaceae: Triticeae). Genome 36: 147–151. pmid:18469977
- 7. Redinbaugh M G, Jones T A, Zhang Y (2000) Ubiquity of the St chloroplast genome in St-containing Triticeae polyploids. Genome 43: 846–852. pmid:11081975
- 8. Mason-Gamer RJ (2001) Origin of North America Elymus (Poaceae: Triticeae) allotetraploids based on granule-bound starch synthase gene sequences. Syst Bot 26: 757–758.
- 9. Mason-Gamer RJ, Orme NL, Anderson CM (2002) Phylogenetic analysis of North American Elymus and the monogenomic Triticeae (Poaceae) using three chloroplast DNA data sets. Genome 45: 991–1002. pmid:12502243
- 10. McMillan E, Sun GL (2004) Genetic relationships of tetraploid Elymus species and their genomic donor species inferred from polymerase chain reaction–restriction length polymorphism analysis of chloroplast gene regions. Theor Appl Genet 108: 535–542. pmid:14513222
- 11. Xu DH, Ban T (2004) Phylogenetic and evolutionary relationships between Elymus humidus and other Elymus species based on sequencing of non-coding regions of cpDNA and AFLP of unclear DNA. Theor Appl Genet 108: 1443–1448. pmid:14963652
- 12. Liu Q, Ge S, Tang H, Zhang X, Zhu G, et al (2006) Phylogenetic relationships in Elymus (Poaceae: Triticeae) based on the nuclear ribosomal internal transcribed spacer and chloroplast trnL-F sequences. New Phytol 170: 411–420. pmid:16608465
- 13. Ni Y, Asamoah-Odei N, Sun GL (2011) Maternal origin, genome constitution and evolutionary relationships of polyploid Elymus species and Hordelymus europaeus. Biol Plant 55: 68–74.
- 14. Tzvelev NN (1989) The system of grasses (Poaceae) and their evolution. Bot Rev 55: 141–204.
- 15. Sun GL, Díaz O, Salomon B, Bothmer von R (1999) Microsatellite variation and its comparison with isozyme and RAPD variation in Elymus fibrosus. Hereditas 129: 275–282.
- 16. Díaz O, Sun GL, Salomon B, Bothmer von R (2000) Level and distribution of allozyme and RAPD variation in populations of Elymus fibrosus (Poaceae). Genet Resour Crop Evol 47: 11–24.
- 17. Sun G, Ni Y, Daley T (2008) Molecular phylogeny of RPB2 gene reveals multiple origin, geographic differentiation of H genome, and the relationship of the Y genome to genomes in Elymus species. Mol Phylogenet Evol 46: 897–907. pmid:18262439
- 18. Mason-Gamer RJ, Burns MM, Naum M (2010) Reticulate evolutionary history of a complex group of grasses: phylogeny of Elymus StStHH allotetraploids based on three nuclear genes. PLoS ONE 5, e10989. pmid:20543952
- 19. Sun GL, Komatsuda T (2010) Origin of the Y genome in Elymus and its relationship to other genomes in Triticeae based on evidence from elongation factor G (EF-G) gene sequences. Mol Phylogenet Evol 56: 727–733. pmid:20363342
- 20. Sun GL, Zhang X (2011) Origin of H genome in StH-genome Elymus species based on single copy nuclear gene DMC1. Genome 54: 655–662. pmid:21848405
- 21. Yan C, Sun GL (2012) Multiple origins of allopolyploid wheatgrass Elymus caninus revealed by RPB2, PepC and TrnD/T genes. Mol Phylogenet Evol 64: 441–451. pmid:22617317
- 22. Zou HW, Wu PP, Wu DX, Sun GL (2015) Origin and Reticulate Evolutionary Process of Wheatgrass Elymus trachycaulus (Triticeae: Poaceae). PLoS ONE 10(5): e0125417. pmid:25946188
- 23. Díaz O, Salomon B, Bothmer von R (1999) Genetic diversity and structure in populations of Elymus caninus (L.) L. (Poaceae). Hereditas 131: 63–74.
- 24. Sun GL, Díaz O, Salomon B, Bothmer von R (1999) Genetic diversity in Elymus caninus as revealed by isozyme, RAPD and microsatellite markers. Genome 42: 420–431. pmid:10382290
- 25. Díaz O, Salomon B, Bothmer von R (1999) Genetic variation and differentiation in Nordic populations of Elymus alaskanus (Scrib. ex Merr.) Löve (Poaceae). Theor Appl Genet 99: 210–217.
- 26. Sun GL, Salomon B, Bothmer von R (2002) Microsatellite polymorphism and genetic differentiation in three Norwegian populations of Elymus alaskanus (Poaceae). Plant Syst Evol 234: 101–110.
- 27. Zhang XQ, Salomon B, von Bothmer R (2002) Application of random amplified polymorphic DNA markers to evaluate intraspecific genetic variation in the Elymus alaskanus complex (Poaceae). Genet Resour Crop Evol 49: 397–407.
- 28. Sun GL, Salomon B (2003) Microsatellite variability and the heterozygote deficiency in arctic-alpine species Elymus alaskanus complex. Genome 46: 729–737. pmid:14608389
- 29. Gaudett M, Salomon B, Sun GL (2005) Molecular variation and population structure in Elymus trachycaulus and comparison with its morphologically similar E.alaskanus. Plant Syst Evol 250: 81–91.
- 30. Sun GL, Li WB (2006) Molecular diversity of Elymus trachycaulus complex species and their relationships to non-North American taxa. Plant Syst Evol 256: 179–191.
- 31. Symonds VV, Soltis PS, Soltis DE (2010) Dynamics of polyploidy formation in Tragopogon (Asteraceae): recurrent formation, gene flow, and population structure. Evolution 64: 1984–2003. pmid:20199558
- 32. Junghans H, Metzlaff M (1990) A simple and rapid method for the preparation of total plant DNA. Biotechniques 8: 176. pmid:2317373
- 33. Sun G, Daley T, Ni Y (2007) Molecular evolution and genome divergence at RPB2 gene of the St and H genome in Elymus species. Plant Mol Biol 64: 645–655. pmid:17551673
- 34. Thompson JD, Gibson TJ, Plewniak F, Jeanmouguin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25: 4876–4882. pmid:9396791
- 35.
Swofford DL (2003) PAUP. Phylogenetic analysis using parsimony (and other methods). Version 4. Sinauer Associates, Sunderland, MA, USA.
- 36. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.
- 37. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696–704. pmid:14530136
- 38. Lanave C, Preparata G, Saccone C, Serio G (1984) A new method for calculating evolutionary substitution rates. J Mol Evol 20: 86–93. pmid:6429346
- 39.
Ronquist F, Huelsenbeck JP (2005) Bayesian analysis of molecular evolution using MrBayes. In: Nielsen R. (Ed.), Statistical methods in molecular evolution. Springer-Verlag Press, Berlin, pp. 183–232.
- 40. Mason-Gamer RJ (2007) Allopolyploids of the genus Elymus (Triticeae, Poaceae): a phylogenetic perspective. Aliso 23: 372–379.
- 41. Mahelka V, Kopecký D, Paštová L (2011) On the genome constitution and evolution of intermediate wheatgrass (Thinopyrum intermedium: Poaceae, Triticeae). BMC Evol Biol 11: 127. pmid:21592357
- 42.
Mason-Gamer RJ, Kellogg EA (1995) Chloroplast DNA analysis of the monogenomic Triticeae: phylogenetic implications and genome-specific markers. In: Jauhar P.P., ed. Methods of genome analysis in plants: their merits and pitfalls. Boca Raton, Florida: CRC press.
- 43. Mahelka V, Fehrer J, Krahulec F, Jarolímová V (2007). Recent Natural Hybridization between Two Allopolyploid Wheatgrasses (Elytrigia, Poaceae): Ecological and Evolutionary Implications. Ann Bot 100(2), 249–260. pmid:17562679
- 44. Zhang C, Fan X, Yu HQ, Zhang L, Wang XL, Zhou YH (2009) Different maternal genome donor to Kengyilia species infeered from chloroplast trnL-F sequences. Biol Plant 53: 759–763.
- 45. Clegg MT, Cummings MP, Durbin ML (1997) The evolution of plant nuclear genes. Proc Natl Acad Sci USA 94: 7791–7798. pmid:9223265
- 46. Small RL, Cronn RC, Wendel JF (2004) Use of nuclear genes for phylogeny reconstruction in plants. Aust Syst Bot 17: 145–170.
- 47. Udall JA, Quijada PA, Osborn TC (2005) Detection of chromosomal rearrangements derived from homoeologous recombination in four mapping populations of Brassica napus L. Genetics 169: 967–979. pmid:15520255
- 48. Leflon M, Eber F, Letanneur JC, Chelysheva L, Coriton O, Huteau V, et al. (2006) Pairing and recombination at meiosis of Brassica rapa (AA) x Brassica napus (AACC) hybrids. Theor Appl Genet 113: 1467–1480. pmid:16983552
- 49. Gaeta RT, Pires JC, Iniguez-Luy F, Leon E, Osborn TC (2007) Genomic changes in resynthesized Brassica napus and their effect on gene expression and phenotype. Plant Cell 19: 3403–3417. pmid:18024568
- 50. Gaudett M, Salomon B, Sun GL. 2005. Molecular variation and population structure in Elymus trachycaulus and its relationship with E. alaskanus. Plant Syst Evol 250: 81–91.
- 51. Jakobsson M, Hagenblad J, Tavaré S, Säll T, Halldén C, Lind-Halldén C, et al. (2006) A unique recent origin of the allotetraploid species Arabidopsis suecica: Evidence from nuclear DNA markers. Mol Biol Evol 23: 1217–1231. pmid:16549398
- 52.
Frankel OH, Brown AHD, Burdon JJ. 1995. The conservation of plant biodiversity. Cambridge University Press.