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Phylogeny and maternal donor of Chinese Elymus (Triticeae: Poaceae) inferred from chloroplast trnH-psbA sequences
Biochemical Systematics and Ecology 68 (2016) 128e134
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Phylogeny and maternal donor of Chinese Elymus (Triticeae: Poaceae) inferred from chloroplast trnH-psbA sequences Gang Gao a, b, Jiabin Deng d, Yan Zhang b, Yangyi Li b, Weitian Li a, Yonghong Zhou c, Ruiwu Yang b, * a
Life Science and Food Engineering College, Yibin University, Yibin, 644000, Sichuan, China College of Life Science, Sichuan Agricultural University, Ya’an, 625014, Sichuan, China c Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, 611130, Sichuan, China d School of Geography and Tourism, Guizhou Normal College, Guiyang, 550018, Guizhou, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 May 2016 Received in revised form 29 June 2016 Accepted 10 July 2016
Hybridization and polyploidization can be major mechanisms for plant evolution and speciation. Thus, the process of polyploidization and evolutionary history of polyploids is of widespread interest. The chloroplast DNA regions trnH-psbA was used to analyze to phylogenetic relationships and maternal donor of Elymus species and their closely related species. The Neighbor-Joining phylogenetic reconstructions partitioned the Elymus species into two groups. All the Elymus species were related to species of Pseudoroegneria. These results indicated that (1) Pseudoroegneria (St genome) was the maternal donor of the polyploidy Elymus; (2) the St genome of Elymus had several origins and diverse species of Pseudoroegneria might have taken part in the formation of polyploid species of Elymus; (3) high degree genome differentiation exists among the Pseudoroegneria species. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Elymus trnH-psbA Phylogeny Maternal origin
1. Introduction Polyploidization is a major mechanism in plant evolution and speciation (Soltis et al., 2003; Otto, 2007). Recent studies using genetic markers in many genera suggested that multiple origins (including independent origin) of polyploid species are the rule rather than the exception (Soltis and Soltis, 2000; Symonds et al., 2010; Fan et al., 2012). Polyploidization and chromosome doubling can stimulate changes in genome size, cell size, genomic repatterning, gene expression, retrotransposon activation and epigenetic effect (Soltis et al., 2003; Otto, 2007). These changes may result in full fertility and stabilization of the hybrid condition and assist in establishing the phenotype in nature, which allows polyploids to adapt to new ecological niches or to be competitively superior to the parental donor (Otto, 2007; Fan et al., 2009; Yan and Sun, 2012). However, a clear and appropriate identification of phylogenetic relationships among taxa and genes, as well as genomic elements is needed (Yan et al., 2011). The wheat tribe (Poaceae: Triticeae), an important gene pool for genetic improvement of cereal crops, includes many autopolyploid and allopolyploid taxa (Liu et al., 2006). Data from extensive cytogenetic analyses have been used to illustrate systematic relationships of the tribe and to clarify the ancestry of many polyploidy species. One complex group of polyploids € ve (1984) based essentially on genomic within Triticeae is the genus Elymus, following the taxonomic delimitation by Lo
* Corresponding author. E-mail address: [email protected] (R. Yang). http://dx.doi.org/10.1016/j.bse.2016.07.008 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
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constitutions, includes approx 150 perennial species distributed in a wide range of ecological habitats over the temperate and subtropic regions. In Flora of China (Chinese version), Elymus include 12 species and 1 subspecies which widely distributed in € ve, 1984). Cytological studies north of China. Elymus has its origin through a typical allopolyploidy process (Dewey, 1984; Lo suggest that five basic genomes, namely, the St, Y, H, P and W in various combinations constitute Elymus species (Lu, 1994). The St genome is a fundamental genome that exists in all Elymus species and is donated by the genus Pseudoroegneria (Dewey, 1967). The H, P and W genomes are derived from the genera Hordeum, Agropyron and Australopyrum of Triticeae, respectively (Dewey, 1971; Jensen, 1990; Torabinejad and Mueller, 1993). However, the donor of the Y genome that is present in the majority of the Asiatic Elymus species has not yet been identified, although extensive investigations have been carried out (Lu, 1994; Gao et al., 2015). Molecular phylogenetic studies have provided a wealth of new insight into the dynamic process of polyploidy evolution (Wendel, 2000; Soltis et al., 2003; Fan et al., 2012). Nuclear, chloroplast sequences have all played a role in this work (Ge et al., 1999; Liu et al., 2006; Dong et al., 2015). Chloroplast DNA (cpDNA) differs from nuclear DNA because it is maternally inherited in most angiosperms, and it typically has a slower rate of evolution due to a highly conserved genome and an absence of recombination events. Therefore, cpDNA sequences are particularly useful to infer maternal events and subsequent divergence related to allopolyploid speciation. Evolutionary inferences based on cpDNA fragments, including matK, rbcL, trnH-psbA and trnL-F, have been reported in families or genera such as Elymus, Kengyilia and Pseudoroegneria (Liu et al., 2006; Zhang et al., 2009; Yu et al., 2010). Analysis of trnL-F sequences suggested that species of Pseudoroegneria (St-genome donor) served as the maternal donor of Elymus species (Liu et al., 2006). Redinbaugh et al. (2000) suggested that there was a strong preference for cpDNA inheritance from the St-containing parent in hybridizations between Triticeae species. The objectives of this study were to infer the maternal donor of Chinese Elymus species, and to reveal phylogenetic relationships between the species of Elymus and their closely related species based on cpDNA trnH-psbA sequences. 2. Materials and methods 2.1. Plant material Twenty two species were sampled, including 10 Elymus species, twelve diploid species from 6 monogenomic genera in Triticeae and Bromus tectorum was used as the outgroup. The taxa names, accession numbers, genome constitution and GenBank accession numbers are listed in Table 1. All seed materials with PI were kindly provided by American National Plant Germplasm System (Pullman, Washington, USA) and the Triticeae Research Institute of Sichuan Agriculture University. The plants and voucher specimens of all the materials have been deposited at the perennial nursery and Herbarium of the Triticeae Research Institute, Sichuan Agriculture University, China (SAUTI). 2.2. DNA amplification and sequencing The total genomic DNA was extracted and purified from fresh leaf tissue of each accession followed a standard CTAB (cetyltrimethylammonium bromide) procedure (Doyle and Doyle, 1990). The chloroplast trnH-psbA gene was amplified with the universal primers: trnH-psbA forward primer (50 -CTTGGTATGGAAGTAATGCA-30 ) and trnH-psbA reverse primer (50 -ATCCACTTGGCTACATCCG-30 ). The PCR (Polymerase Chain Reaction) were conducted in 50 mL reaction volume, containing 2.0 mL template DNA at the concentration of 20 ng/mL, 25.0 mL 2 Taq PCRMasterMiX (4 mmol/L MgCl2, 0.4 mmol/L dNTPs of each nucleotide, 0.05 units/mL Taq DNA polymerase), 0.01 mmol/L primer1.5 mL and with an addition of ddH2O to the final volume. The PCR amplification protocols were performed with an initial denaturing step at 94 C for 4 min, followed by 35 cycles of 1 min denaturing at 94 C, 1 min annealing at 52 C, 1 min extension at 72 C, and a final extension step at 72 C for 10 min on BIO-RAD S1000™ Thermal cycler. PCR products were separated on a 1.5% agarose gel, the desired DNA band was excised and purified with the AxyPrep DNAkit (AXYGEN BIOSCIENCES, Hangzhou, China). Sequencing was conducted by BGI Company (Peking, China). 2.3. Data analysis DNA sequences were confirmed through BLAST nucleotide alignment on NCBI database and aligned through a multiple sequence alignment with the Clustal W algorithm (Thompson et al., 1994) and the alignments were refined manually, implemented by the software MegAlign (DNAStar Inc. USA). The homogeneity of the base composition with the Id-test, nucleotide substitutions, transition/transversion ratio, and variability in different regions of the sequences were calculated with MEGA software, version5.0 (Tamura et al., 2011). To assess the divergence and relationships among polyploids and its diploid progenitors, nucleotide diversity based on the average number of pairwise comparisons in a sample was estimated using haplotype diversity by Hd (Nei and Li, 1979), Tajima’s p (Tajima, 1989) and Watterson’s qw (Watterson, 1975). Testing of neutrality was also performed by Tajima’s and Fu and Li’s D statistic (Tajima, 1989; Fu and Li, 1993). Significance of D-values was estimated with the simulated distribution of random samples (1000 steps) using a coalescence algorithm assuming neutrality and population equilibrium (Hudson, 1990). These parameters were implemented by DnaSP version 5.10 (Rozas et al., 2003).
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Table 1 Elymus species and closely related species used in this study. No.
Species
Elymus L. 1 E. atratus (Nevski) Hand.-Mazz. 2 E. breviaristatus Keng ex P. C. Keng 3 E. breviaristatus Keng ex P. C. Keng 4 E. breviaristatus Keng ex P. C. Keng 5 E. breviaristatus Keng ex P. C. Keng 6 E. canadensis L. 7 E. canadensis L. 8 E. cylindricus (Franch.) Honda 9 E. cylindricus (Franch.) Honda 10 E. cylindricus (Franch.) Honda 11 E. cylindricus (Franch.) Honda 12 E. cylindricus (Franch.) Honda 13 E. dahuricus Trucz. 14 E. dahuricus Trucz. 15 E. dahuricus Trucz. 16 E. excelsus Trucz. 17 E. nutans Griseb. 18 E. nutans Griseb. 19 E. purpuraristatus C. P. Wang et H. L. Yang 20 E. sibiricus L. 21 E. sibiricus L. 22 E. sibiricus L. 23 E. tangutorum (Nevski) Hand.-Mazz. 24 E. tangutorum (Nevski) Hand.-Mazz. € ve Pseudoroegneria (Nevski) A. Lo 25 Pse. libanotica (Hackel) D. R. Dewey Lo € ve 26 Pse.spicata (Pursh) A. 27 Pse. stipifolia (Czern. ex Nevski) Lo € ve A. Lo €ve 28 Pse. strigosa (M. Bieb.) A. Hordeum L. 29 H. bogdanii L. 30 H. chilense Roem. & Schult. Agropyron Gaertner 31 Ag. cristatum (L.) Gaertn €ve Australopyrum (Tsvelev) A. Lo Lo € ve 32 Aus retrofractum (Vickery) A. Lo € ve Lophopyrum (Host) A. Lo €ve 33 Lo. elongatum (Host) A. Psathyrostachys (Boiss.) Nevski 34 Psa. huashanica Keng ex P.C.Kuo 35 Psa. juncea (Fisch.) Nevski 36 Bromus tectorum L.
Accession no.
Genome
Origin
GenBank no.
ZY3023 Y3063 Y2079 Y2493 ZY3063 PI531567 PI499412 Y0552 Y0735 Y0798 Y0660 ZY11060 per296 per97 PI531598 ZY11034 e ZY3060 ZY11039 PI598773 ZY3041 09001 Y1467 Y2078
StYH StYH StYH StYH StYH StH StH StYH StYH StYH StYH StYH StYH StYH StYH StYH StYH StYH StYH StH StH StH StYH StYH
Sichuan,China China China China Sichuan, China Canada Neimenggu, China Xinjiang, China Xinjiang, China Xinjiang, China China Neimenggu, China China China Pakistan Neimenggu, China Sichuan,China Gansu, China Neimenggu, China Kazakhstan Sichuan, China Qinghai, China Xinjiang, China Sichuan, China
KX181015 KX181016 KX181017 KX181018 KX181019 KX181020 KX181021 KX181022 KX181023 KX181024 KX181025 KX181026 KX181027 KX181028 KX181029 KX181030 KX181031 KX181032 KX181033 KX181034 KX181035 KX181036 KX181037 KX181038
PI228391
St
Iran
KX181044
PI506259 PI440095
St St
United States Russian
KX181045 KX181046
PI531752
St
Estonia
KX181047
Y1701 PI531781
H H
Hebei, China Rio Negro, Argentina
KX181039 KX181040
PI531539
P
Utah, United States
KX181012
PI531553
W
Australia
KX181013
PI574516
e
E
Former Soviet Union
KX181041
ZY3157 PI430871 PI221921
Ns Ns
Shaanxi, China Former Soviet Union Afghanistan
KX181042 KX181043 KX181014
The arrays of phylogenetic reconstruction and molecular evolutionary analyses were performed by the Neighbor-Joining (NJ) approach based on the Maximum Composite Likelihood model, using MEGA version 5.0 (Tamura et al., 2011). Topological robustness NJ analysis was assessed by bootstrap analysis with 1000 replicates. 3. Results 3.1. trnH-psbA sequences analysis The length of trnH-psbA sequences was 642 bp in the final aligned sequences of 36 accessions, 55 variable characters and 40 parsimony informative characters (Table 2). Meanwhile, The length of the of 36 trnH-psbA sequences varied from 607 bp to 630 bp and the average of G þ C content was 36.24%. In addition, some specific mutation loci were discovered among these genera (Fig. 1).
Table 2 Features of the matched data matrix.
trnH-psbA
Variable characters
Conserved characters
Informative characters
Identical pairs
Transitional pairs
Transversional pairs
55
572
40
608
3
4
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Fig. 1. The comparison of partial trnH-psbA sequences of Elymus and related diploid species.
The Agropyron cristatum-31 (P genome) had a 4 bp insertion (TCTT) at position 124e127 bp. However, Australopyrum retrofractum-32 with W genome had a 4 bp insertion (ATTT) at position 124e127 bp. Meanwhile, the Hordeum bogdanii and Hordeum chilense with H genome had a 8 bp deletion (CTGCTTT) at position 116e123 bp. Estimates of nucleotide diversity of trnH-psbA sequences including the total number of sites (n), the number of polymorphic sites (s), Haplotypediversity (Hd), the average pairwise diversity (p) and the diversity based on the number of segregating sites (qw). Neutrality tests such as Tajima’s D, Fu and Li’s D gave negative values for all accessions were 1.46284 (P > 0.10) and 0.59962 (P > 0.10) respectively (Table 3).
3.2. Phylogenetic analysis To reveal the phylogenetic relationship among Elymus and related diploid Triticeae species, all the 36 accessions were implemented by NJ phylogenetic reconstruction based on trnH-psbA sequences. NJ analysis of the trnH-psbA data yielded a strict consensus phylogenetic tree (Log likelihood ¼ 1245.619), with the following estimated NJ parameters: the assumed nucleotide frequencies A: 0.2928, C: 0.1704, G: 0.1920, T: 0.3448. The NJ phylogenetic tree was supported by the bootstrap test (50%) and bootstrap support (BS) above the branches (Fig. 2). The NJ tree was generally resolved, all the accessions of Elymus and their maternal donors were formed two clades, which corresponding to the St genome. The clade A consisted of 20 accessions of Elymus species, 3 accessions of Pseudoroegneria species and 1 accession of Lophopyrum species (68% BS value). Meanwhile, the clade B was comprised of 4 accessions of Elymus species and 1 accession of Pseudoroegneria species (63% BS
Table 3 Estimates of nucleotide diversity and test statistics for trnH-psbA data sets.
trnH-psbA
n
s
p
Hd
qw
Fu & Li’s D
Tajima’s D
595
47
0.011325
0.910
0.019049
0.59962 (P > 0.10)
1.46284 (P > 0.10)
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Fig. 2. Neighbor-Joining (NJ) tree of Elymus and its related diploid species inferred from the trnH-psbA sequences. Numbers at nodes indicate bootstrap values 50%.
value). In addition, the different populations of some Elymus species were not gathered in same clade. For example, E. sibiricus-22 was gathered in clade A, but E. sibiricus-20 and E. sibiricus-21 were grouped in clade B together with Pse. strigosa. 4. Discussion 4.1. The maternal donor to Elymus species cpDNA is generally uniparentally inherited in grass and mainly inherited from female parent in high plants, and thus can be used to identify the maternal genome donor of a given polyploid (Mason-Gamer et al., 2010; Liu et al., 2006). Previous
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studies based on cpDNA including ndhF (Redinbaugh et al., 2000), rpoA (Mason-Gamer et al., 2010), and trnL-F (Liu et al., 2006) gene data have suggested that species of Pseudoroegneria (St genome donor) served as the maternal donor of the species of Elymus L. sensu lato. In this study, The phylogenetic tree generated from the trnH-psbA showed that all the Chinese Elymus species and Pseudoroegneria species were grouped together. The result is in good agreement with the suggestion that Pseudoroegneria species might serve as the maternal donor of the Elymus species, but whether different Elymus species originated from different St donors remains an open question. In addition, the Lophopyrum elongatum-33 (Ee) was grouped in clade A, and sequences alignment results showed that there was no difference at position 116e137 bp between Lo. elongatum and St genome. So these results indicated that Lophopyrum (Ee) was closed to Pseudoroegneria (St) and the St and Ee genomes may have the same origin. Lots of cpDNA sequences were used to research the maternal donor of Triticeae perennial species, these results showed that St genome is more likely to act as female parent donor in the process of hybridization (Mason-Gamer et al., 2010; Liu et al., 2006). Meanwhile, the St genome exhibited strong dominant role, so the species which contain St genome had a lot of similarities in morphology (Yen and Yang, 2013). The reason leaded to morphological classification is very difficult in these species. This phenomenon is especially obvious in Elymus, such as the genome boundaries of E. sibiricus (StH genome) and E. nutans (StYH genome) is very clear, but morphological classification is difficult. It is likely to be due to the strong dominant role of the St and H genome, and the genes of Y genome are concealed, which exist, but not express (Yen and Yang, 2013). Previous studies and this study suggested that St genome is more likely to act as female parent donor in the allohexaploid with St, Y, H genome. However, different maternal donor was founded in related genus Kengyilia with StYP genome. Besides St genome, the P genome derived from Agropyron also can act as female parent donor, such as K. melanthera and K. thoroldiana (Luo et al., 2012; Gao et al., 2014). So the St genome act a variety of roles in polyploidization process of Triticeae allopolyploid. 4.2. Differentiation of St genome in polyploid Elymus species The St genome present in all species of Elymus, and is an important genome for this genus. The St genome donor genus is Pseudoroegneria which contains approximately 15 diploid (StSt) or tetraploid (StStStSt, or St1St1St2St2 or StStPP) species distributed in the Middle East, central Asia, northern China and western North America (Wang et al., 1986; Jensen and Chen, 1992). Considering the morphological differentiation of Pseudoroegneria, Pse. stipifolia has rough rachis densely covered by prickles; Pse. spicata has slender awns and unequal glumes; Pse. strigosa has long awns with equal glumes; but Pse. libanotica have no awns with unequal glumes (Dong et al., 2015). Cytological data suggested that genome differentiation exists among the Pseudoroegneria species (Wang et al., 1986). Mason-Gamer et al. (2010) have demonstrated that Pseudoroegneria may be paraphyletic. RPB2 gene data suggested that genetic material of St genome has changed among Pse. spicata, Pse. stipiflolia and Pse. libanotica (Sun et al., 2008). In this study, based on the trnH-psbA data, all the Elymus species were grouped with diploid Pseudoroegneria species, suggesting a high degree of similarity among the St genome in Pseudoroegneria species and the St genome in Elymus species. The phylogenetic tree suggests at least two phylogenetically distinct St genome donors to the Elymus species. Four Pseudoroegneria species weren’t grouped together, but formed sister group with different Elymus species. In addition, Elymus species were split into different St-groups. For instance, two accessions of hexaploid E. nutans were placed in separate Stgenome clade in the tree. E. nutans-18 was gathered in clade A, but E. nutans-17 was grouped in clade B together with Pse. strigosa. Such patterns indicated that differentiation of St genome existed in the species of Elymus at both the genus and species after polyploidization event based on the chloroplast trnH-psbA molecular data. The phylogenetic tree clearly shows St genome differentiation in Elymus species. Furthermore, the geographical origin of four Pseudoroegneria species were different, therefore geographical isolation may result in different evolution patterns. 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Phylogeny and maternal donor of Chinese Elymus (Triticeae: Poaceae) inferred from chloroplast trnH-psbA sequences Gang Gao a, b, Jiabin Deng d, Yan Zhang b, Yangyi Li b, Weitian Li a, Yonghong Zhou c, Ruiwu Yang b, * a
Life Science and Food Engineering College, Yibin University, Yibin, 644000, Sichuan, China College of Life Science, Sichuan Agricultural University, Ya’an, 625014, Sichuan, China c Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, 611130, Sichuan, China d School of Geography and Tourism, Guizhou Normal College, Guiyang, 550018, Guizhou, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 May 2016 Received in revised form 29 June 2016 Accepted 10 July 2016
Hybridization and polyploidization can be major mechanisms for plant evolution and speciation. Thus, the process of polyploidization and evolutionary history of polyploids is of widespread interest. The chloroplast DNA regions trnH-psbA was used to analyze to phylogenetic relationships and maternal donor of Elymus species and their closely related species. The Neighbor-Joining phylogenetic reconstructions partitioned the Elymus species into two groups. All the Elymus species were related to species of Pseudoroegneria. These results indicated that (1) Pseudoroegneria (St genome) was the maternal donor of the polyploidy Elymus; (2) the St genome of Elymus had several origins and diverse species of Pseudoroegneria might have taken part in the formation of polyploid species of Elymus; (3) high degree genome differentiation exists among the Pseudoroegneria species. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Elymus trnH-psbA Phylogeny Maternal origin
1. Introduction Polyploidization is a major mechanism in plant evolution and speciation (Soltis et al., 2003; Otto, 2007). Recent studies using genetic markers in many genera suggested that multiple origins (including independent origin) of polyploid species are the rule rather than the exception (Soltis and Soltis, 2000; Symonds et al., 2010; Fan et al., 2012). Polyploidization and chromosome doubling can stimulate changes in genome size, cell size, genomic repatterning, gene expression, retrotransposon activation and epigenetic effect (Soltis et al., 2003; Otto, 2007). These changes may result in full fertility and stabilization of the hybrid condition and assist in establishing the phenotype in nature, which allows polyploids to adapt to new ecological niches or to be competitively superior to the parental donor (Otto, 2007; Fan et al., 2009; Yan and Sun, 2012). However, a clear and appropriate identification of phylogenetic relationships among taxa and genes, as well as genomic elements is needed (Yan et al., 2011). The wheat tribe (Poaceae: Triticeae), an important gene pool for genetic improvement of cereal crops, includes many autopolyploid and allopolyploid taxa (Liu et al., 2006). Data from extensive cytogenetic analyses have been used to illustrate systematic relationships of the tribe and to clarify the ancestry of many polyploidy species. One complex group of polyploids € ve (1984) based essentially on genomic within Triticeae is the genus Elymus, following the taxonomic delimitation by Lo
* Corresponding author. E-mail address: [email protected] (R. Yang). http://dx.doi.org/10.1016/j.bse.2016.07.008 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
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constitutions, includes approx 150 perennial species distributed in a wide range of ecological habitats over the temperate and subtropic regions. In Flora of China (Chinese version), Elymus include 12 species and 1 subspecies which widely distributed in € ve, 1984). Cytological studies north of China. Elymus has its origin through a typical allopolyploidy process (Dewey, 1984; Lo suggest that five basic genomes, namely, the St, Y, H, P and W in various combinations constitute Elymus species (Lu, 1994). The St genome is a fundamental genome that exists in all Elymus species and is donated by the genus Pseudoroegneria (Dewey, 1967). The H, P and W genomes are derived from the genera Hordeum, Agropyron and Australopyrum of Triticeae, respectively (Dewey, 1971; Jensen, 1990; Torabinejad and Mueller, 1993). However, the donor of the Y genome that is present in the majority of the Asiatic Elymus species has not yet been identified, although extensive investigations have been carried out (Lu, 1994; Gao et al., 2015). Molecular phylogenetic studies have provided a wealth of new insight into the dynamic process of polyploidy evolution (Wendel, 2000; Soltis et al., 2003; Fan et al., 2012). Nuclear, chloroplast sequences have all played a role in this work (Ge et al., 1999; Liu et al., 2006; Dong et al., 2015). Chloroplast DNA (cpDNA) differs from nuclear DNA because it is maternally inherited in most angiosperms, and it typically has a slower rate of evolution due to a highly conserved genome and an absence of recombination events. Therefore, cpDNA sequences are particularly useful to infer maternal events and subsequent divergence related to allopolyploid speciation. Evolutionary inferences based on cpDNA fragments, including matK, rbcL, trnH-psbA and trnL-F, have been reported in families or genera such as Elymus, Kengyilia and Pseudoroegneria (Liu et al., 2006; Zhang et al., 2009; Yu et al., 2010). Analysis of trnL-F sequences suggested that species of Pseudoroegneria (St-genome donor) served as the maternal donor of Elymus species (Liu et al., 2006). Redinbaugh et al. (2000) suggested that there was a strong preference for cpDNA inheritance from the St-containing parent in hybridizations between Triticeae species. The objectives of this study were to infer the maternal donor of Chinese Elymus species, and to reveal phylogenetic relationships between the species of Elymus and their closely related species based on cpDNA trnH-psbA sequences. 2. Materials and methods 2.1. Plant material Twenty two species were sampled, including 10 Elymus species, twelve diploid species from 6 monogenomic genera in Triticeae and Bromus tectorum was used as the outgroup. The taxa names, accession numbers, genome constitution and GenBank accession numbers are listed in Table 1. All seed materials with PI were kindly provided by American National Plant Germplasm System (Pullman, Washington, USA) and the Triticeae Research Institute of Sichuan Agriculture University. The plants and voucher specimens of all the materials have been deposited at the perennial nursery and Herbarium of the Triticeae Research Institute, Sichuan Agriculture University, China (SAUTI). 2.2. DNA amplification and sequencing The total genomic DNA was extracted and purified from fresh leaf tissue of each accession followed a standard CTAB (cetyltrimethylammonium bromide) procedure (Doyle and Doyle, 1990). The chloroplast trnH-psbA gene was amplified with the universal primers: trnH-psbA forward primer (50 -CTTGGTATGGAAGTAATGCA-30 ) and trnH-psbA reverse primer (50 -ATCCACTTGGCTACATCCG-30 ). The PCR (Polymerase Chain Reaction) were conducted in 50 mL reaction volume, containing 2.0 mL template DNA at the concentration of 20 ng/mL, 25.0 mL 2 Taq PCRMasterMiX (4 mmol/L MgCl2, 0.4 mmol/L dNTPs of each nucleotide, 0.05 units/mL Taq DNA polymerase), 0.01 mmol/L primer1.5 mL and with an addition of ddH2O to the final volume. The PCR amplification protocols were performed with an initial denaturing step at 94 C for 4 min, followed by 35 cycles of 1 min denaturing at 94 C, 1 min annealing at 52 C, 1 min extension at 72 C, and a final extension step at 72 C for 10 min on BIO-RAD S1000™ Thermal cycler. PCR products were separated on a 1.5% agarose gel, the desired DNA band was excised and purified with the AxyPrep DNAkit (AXYGEN BIOSCIENCES, Hangzhou, China). Sequencing was conducted by BGI Company (Peking, China). 2.3. Data analysis DNA sequences were confirmed through BLAST nucleotide alignment on NCBI database and aligned through a multiple sequence alignment with the Clustal W algorithm (Thompson et al., 1994) and the alignments were refined manually, implemented by the software MegAlign (DNAStar Inc. USA). The homogeneity of the base composition with the Id-test, nucleotide substitutions, transition/transversion ratio, and variability in different regions of the sequences were calculated with MEGA software, version5.0 (Tamura et al., 2011). To assess the divergence and relationships among polyploids and its diploid progenitors, nucleotide diversity based on the average number of pairwise comparisons in a sample was estimated using haplotype diversity by Hd (Nei and Li, 1979), Tajima’s p (Tajima, 1989) and Watterson’s qw (Watterson, 1975). Testing of neutrality was also performed by Tajima’s and Fu and Li’s D statistic (Tajima, 1989; Fu and Li, 1993). Significance of D-values was estimated with the simulated distribution of random samples (1000 steps) using a coalescence algorithm assuming neutrality and population equilibrium (Hudson, 1990). These parameters were implemented by DnaSP version 5.10 (Rozas et al., 2003).
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Table 1 Elymus species and closely related species used in this study. No.
Species
Elymus L. 1 E. atratus (Nevski) Hand.-Mazz. 2 E. breviaristatus Keng ex P. C. Keng 3 E. breviaristatus Keng ex P. C. Keng 4 E. breviaristatus Keng ex P. C. Keng 5 E. breviaristatus Keng ex P. C. Keng 6 E. canadensis L. 7 E. canadensis L. 8 E. cylindricus (Franch.) Honda 9 E. cylindricus (Franch.) Honda 10 E. cylindricus (Franch.) Honda 11 E. cylindricus (Franch.) Honda 12 E. cylindricus (Franch.) Honda 13 E. dahuricus Trucz. 14 E. dahuricus Trucz. 15 E. dahuricus Trucz. 16 E. excelsus Trucz. 17 E. nutans Griseb. 18 E. nutans Griseb. 19 E. purpuraristatus C. P. Wang et H. L. Yang 20 E. sibiricus L. 21 E. sibiricus L. 22 E. sibiricus L. 23 E. tangutorum (Nevski) Hand.-Mazz. 24 E. tangutorum (Nevski) Hand.-Mazz. € ve Pseudoroegneria (Nevski) A. Lo 25 Pse. libanotica (Hackel) D. R. Dewey Lo € ve 26 Pse.spicata (Pursh) A. 27 Pse. stipifolia (Czern. ex Nevski) Lo € ve A. Lo €ve 28 Pse. strigosa (M. Bieb.) A. Hordeum L. 29 H. bogdanii L. 30 H. chilense Roem. & Schult. Agropyron Gaertner 31 Ag. cristatum (L.) Gaertn €ve Australopyrum (Tsvelev) A. Lo Lo € ve 32 Aus retrofractum (Vickery) A. Lo € ve Lophopyrum (Host) A. Lo €ve 33 Lo. elongatum (Host) A. Psathyrostachys (Boiss.) Nevski 34 Psa. huashanica Keng ex P.C.Kuo 35 Psa. juncea (Fisch.) Nevski 36 Bromus tectorum L.
Accession no.
Genome
Origin
GenBank no.
ZY3023 Y3063 Y2079 Y2493 ZY3063 PI531567 PI499412 Y0552 Y0735 Y0798 Y0660 ZY11060 per296 per97 PI531598 ZY11034 e ZY3060 ZY11039 PI598773 ZY3041 09001 Y1467 Y2078
StYH StYH StYH StYH StYH StH StH StYH StYH StYH StYH StYH StYH StYH StYH StYH StYH StYH StYH StH StH StH StYH StYH
Sichuan,China China China China Sichuan, China Canada Neimenggu, China Xinjiang, China Xinjiang, China Xinjiang, China China Neimenggu, China China China Pakistan Neimenggu, China Sichuan,China Gansu, China Neimenggu, China Kazakhstan Sichuan, China Qinghai, China Xinjiang, China Sichuan, China
KX181015 KX181016 KX181017 KX181018 KX181019 KX181020 KX181021 KX181022 KX181023 KX181024 KX181025 KX181026 KX181027 KX181028 KX181029 KX181030 KX181031 KX181032 KX181033 KX181034 KX181035 KX181036 KX181037 KX181038
PI228391
St
Iran
KX181044
PI506259 PI440095
St St
United States Russian
KX181045 KX181046
PI531752
St
Estonia
KX181047
Y1701 PI531781
H H
Hebei, China Rio Negro, Argentina
KX181039 KX181040
PI531539
P
Utah, United States
KX181012
PI531553
W
Australia
KX181013
PI574516
e
E
Former Soviet Union
KX181041
ZY3157 PI430871 PI221921
Ns Ns
Shaanxi, China Former Soviet Union Afghanistan
KX181042 KX181043 KX181014
The arrays of phylogenetic reconstruction and molecular evolutionary analyses were performed by the Neighbor-Joining (NJ) approach based on the Maximum Composite Likelihood model, using MEGA version 5.0 (Tamura et al., 2011). Topological robustness NJ analysis was assessed by bootstrap analysis with 1000 replicates. 3. Results 3.1. trnH-psbA sequences analysis The length of trnH-psbA sequences was 642 bp in the final aligned sequences of 36 accessions, 55 variable characters and 40 parsimony informative characters (Table 2). Meanwhile, The length of the of 36 trnH-psbA sequences varied from 607 bp to 630 bp and the average of G þ C content was 36.24%. In addition, some specific mutation loci were discovered among these genera (Fig. 1).
Table 2 Features of the matched data matrix.
trnH-psbA
Variable characters
Conserved characters
Informative characters
Identical pairs
Transitional pairs
Transversional pairs
55
572
40
608
3
4
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Fig. 1. The comparison of partial trnH-psbA sequences of Elymus and related diploid species.
The Agropyron cristatum-31 (P genome) had a 4 bp insertion (TCTT) at position 124e127 bp. However, Australopyrum retrofractum-32 with W genome had a 4 bp insertion (ATTT) at position 124e127 bp. Meanwhile, the Hordeum bogdanii and Hordeum chilense with H genome had a 8 bp deletion (CTGCTTT) at position 116e123 bp. Estimates of nucleotide diversity of trnH-psbA sequences including the total number of sites (n), the number of polymorphic sites (s), Haplotypediversity (Hd), the average pairwise diversity (p) and the diversity based on the number of segregating sites (qw). Neutrality tests such as Tajima’s D, Fu and Li’s D gave negative values for all accessions were 1.46284 (P > 0.10) and 0.59962 (P > 0.10) respectively (Table 3).
3.2. Phylogenetic analysis To reveal the phylogenetic relationship among Elymus and related diploid Triticeae species, all the 36 accessions were implemented by NJ phylogenetic reconstruction based on trnH-psbA sequences. NJ analysis of the trnH-psbA data yielded a strict consensus phylogenetic tree (Log likelihood ¼ 1245.619), with the following estimated NJ parameters: the assumed nucleotide frequencies A: 0.2928, C: 0.1704, G: 0.1920, T: 0.3448. The NJ phylogenetic tree was supported by the bootstrap test (50%) and bootstrap support (BS) above the branches (Fig. 2). The NJ tree was generally resolved, all the accessions of Elymus and their maternal donors were formed two clades, which corresponding to the St genome. The clade A consisted of 20 accessions of Elymus species, 3 accessions of Pseudoroegneria species and 1 accession of Lophopyrum species (68% BS value). Meanwhile, the clade B was comprised of 4 accessions of Elymus species and 1 accession of Pseudoroegneria species (63% BS
Table 3 Estimates of nucleotide diversity and test statistics for trnH-psbA data sets.
trnH-psbA
n
s
p
Hd
qw
Fu & Li’s D
Tajima’s D
595
47
0.011325
0.910
0.019049
0.59962 (P > 0.10)
1.46284 (P > 0.10)
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Fig. 2. Neighbor-Joining (NJ) tree of Elymus and its related diploid species inferred from the trnH-psbA sequences. Numbers at nodes indicate bootstrap values 50%.
value). In addition, the different populations of some Elymus species were not gathered in same clade. For example, E. sibiricus-22 was gathered in clade A, but E. sibiricus-20 and E. sibiricus-21 were grouped in clade B together with Pse. strigosa. 4. Discussion 4.1. The maternal donor to Elymus species cpDNA is generally uniparentally inherited in grass and mainly inherited from female parent in high plants, and thus can be used to identify the maternal genome donor of a given polyploid (Mason-Gamer et al., 2010; Liu et al., 2006). Previous
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studies based on cpDNA including ndhF (Redinbaugh et al., 2000), rpoA (Mason-Gamer et al., 2010), and trnL-F (Liu et al., 2006) gene data have suggested that species of Pseudoroegneria (St genome donor) served as the maternal donor of the species of Elymus L. sensu lato. In this study, The phylogenetic tree generated from the trnH-psbA showed that all the Chinese Elymus species and Pseudoroegneria species were grouped together. The result is in good agreement with the suggestion that Pseudoroegneria species might serve as the maternal donor of the Elymus species, but whether different Elymus species originated from different St donors remains an open question. In addition, the Lophopyrum elongatum-33 (Ee) was grouped in clade A, and sequences alignment results showed that there was no difference at position 116e137 bp between Lo. elongatum and St genome. So these results indicated that Lophopyrum (Ee) was closed to Pseudoroegneria (St) and the St and Ee genomes may have the same origin. Lots of cpDNA sequences were used to research the maternal donor of Triticeae perennial species, these results showed that St genome is more likely to act as female parent donor in the process of hybridization (Mason-Gamer et al., 2010; Liu et al., 2006). Meanwhile, the St genome exhibited strong dominant role, so the species which contain St genome had a lot of similarities in morphology (Yen and Yang, 2013). The reason leaded to morphological classification is very difficult in these species. This phenomenon is especially obvious in Elymus, such as the genome boundaries of E. sibiricus (StH genome) and E. nutans (StYH genome) is very clear, but morphological classification is difficult. It is likely to be due to the strong dominant role of the St and H genome, and the genes of Y genome are concealed, which exist, but not express (Yen and Yang, 2013). Previous studies and this study suggested that St genome is more likely to act as female parent donor in the allohexaploid with St, Y, H genome. However, different maternal donor was founded in related genus Kengyilia with StYP genome. Besides St genome, the P genome derived from Agropyron also can act as female parent donor, such as K. melanthera and K. thoroldiana (Luo et al., 2012; Gao et al., 2014). So the St genome act a variety of roles in polyploidization process of Triticeae allopolyploid. 4.2. Differentiation of St genome in polyploid Elymus species The St genome present in all species of Elymus, and is an important genome for this genus. The St genome donor genus is Pseudoroegneria which contains approximately 15 diploid (StSt) or tetraploid (StStStSt, or St1St1St2St2 or StStPP) species distributed in the Middle East, central Asia, northern China and western North America (Wang et al., 1986; Jensen and Chen, 1992). Considering the morphological differentiation of Pseudoroegneria, Pse. stipifolia has rough rachis densely covered by prickles; Pse. spicata has slender awns and unequal glumes; Pse. strigosa has long awns with equal glumes; but Pse. libanotica have no awns with unequal glumes (Dong et al., 2015). Cytological data suggested that genome differentiation exists among the Pseudoroegneria species (Wang et al., 1986). Mason-Gamer et al. (2010) have demonstrated that Pseudoroegneria may be paraphyletic. RPB2 gene data suggested that genetic material of St genome has changed among Pse. spicata, Pse. stipiflolia and Pse. libanotica (Sun et al., 2008). In this study, based on the trnH-psbA data, all the Elymus species were grouped with diploid Pseudoroegneria species, suggesting a high degree of similarity among the St genome in Pseudoroegneria species and the St genome in Elymus species. The phylogenetic tree suggests at least two phylogenetically distinct St genome donors to the Elymus species. Four Pseudoroegneria species weren’t grouped together, but formed sister group with different Elymus species. In addition, Elymus species were split into different St-groups. For instance, two accessions of hexaploid E. nutans were placed in separate Stgenome clade in the tree. E. nutans-18 was gathered in clade A, but E. nutans-17 was grouped in clade B together with Pse. strigosa. Such patterns indicated that differentiation of St genome existed in the species of Elymus at both the genus and species after polyploidization event based on the chloroplast trnH-psbA molecular data. The phylogenetic tree clearly shows St genome differentiation in Elymus species. Furthermore, the geographical origin of four Pseudoroegneria species were different, therefore geographical isolation may result in different evolution patterns. 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