Molecular Phylogeny of the Genus Hordeum Using Thioredoxin-Like Gene Sequences

Breeding Science 59: 595–601 (2009)

Molecular phylogeny of the genus Hordeum using thioredoxin-like gene sequences

Katsuyuki Kakeda*1), Shin Taketa2) and Takao Komatsuda3)

1) Graduate School of Bioresources, Mie University, 1577 Kurima-machiya, Tsu, Mie 514-8507, Japan 2) Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan 3) Plant Genome Research Unit, National Institute for Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan

Phylogenetic relationships within the genus Hordeum were investigated based on nucleotide sequences of the thioredoxin-like (HTL) gene. We analyzed amplified genomic DNA fragments of the HTL gene from 11 Hordeum species including 16 taxa (25 accessions), which cover mainly diploid accessions together with several tetraploid accessions. Phylogenetic analysis based on the HTL sequences demonstrated a clear divergence of the four basic genomes I (H. vulgare and H. bulbosum), Xa (H. marinum), Xu (H. murinum) and H (other species) in the genus. Phylogenetic clustering also led us to infer two separate clades, one containing the I and Xu, and the other containing the Xa and H genomes. In the diploid H-genome species, American species were confirmed to be closely related and divergent from Asian species. Analysis of the Xa-genome group suggested an alloploid origin of tetraploid H. marinum ssp. gussoneanum by hybridization between the diploid cytotypes of ssp. marinum and ssp. gussoneanum. Our data also supported the hypothesis that two tetraploid cytotypes of H. murinum (ssp. murinum and ssp. leporinum) have an alloploid origin, where one genome was presumably derived from the diploid cytotype ssp. glaucum and the other from an unknown Xu- genome species.

Key Words: Phylogeny, Hordeum, HTL gene, tetraploid cytotype, origin.

Introduction (ITS; Bustos et al. 2002, Blattner 2004). The chloroplast DNA sequences analyzed to date involve SSR regions The genus Hordeum (Poaceae) consists of 31 species and 45 (Provan et al. 1999), the rpoA gene (Petersen and Seberg taxa, including diploid (2n = 14), tetraploid (2n = 28) and 2003), the matK, atpB-rbcL and trnL-trnF regions (Nishikawa hexaploid (2n = 42) cytotypes with the basic chromosome et al. 2002), and the trnL-trnF region (Jakob and Blattner number x = 7. Earlier cytogenetic studies based on meiotic 2006). However, the results were not consistent among stud- chromosome pairing and karyotype analyses proposed the ies, especially between nuclear and chloroplast data. Several existence of four basic genomes (I, H, Xa and Xu) in the ge- uncertainties also remain regarding the origin of polyploid nus (Bothmer et al. 1995, Wang et al. 1996). The diploid taxa (or cytotypes). species are divided into the I-genome group (H. vulgare in- The reproductive system (i.e., inbreeding or outbreeding) cluding cultivated barley and H. bulbosum), the Xa-genome is highly variable among Hordeum species. Inbreeding group (H. marinum), the Xu-genome group (H. murinum) predominates in annual species, whereas perennial species and the H-genome group (all other species). The genome (the majority of the genus) have a versatile reproductive nomenclature used in this paper follows Wang et al. (1996) system (Bothmer et al. 1995). The perennial species in- (In another nomenclature system, swapping the genome clude two obligate outbreeding species, H. bulbosum and symbols H and I has been proposed (Taketa et al. 1999a, H. brevisubulatum, which possess a self-incompatibility (SI) 2001, Blattner 2009)). Molecular phylogenetic analyses of system. The SI system was shown to be controlled by Hordeum species based on nucleotide sequences have been two unlinked multiallelic loci, S and Z, in H. bulbosum carried out using various nuclear or chloroplast DNA se- (Lundqvist 1962), although neither gene has yet been cloned. quences. The former involve nucleotide sequences from The same two-locus SI system is also observed in other nuclear genes of translation elongation factor-G (EF-G; grasses and is distributed within the subfamily Pooideae, Komatsuda et al. 1999, 2001, 2009), antifungal chitinase covering the tribes Triticeae, Aveneae and Poeae (Langridge (Bustos et al. 2002), disrupted meiotic cDNA 1 (DMC1; and Baumann 2008). In the course of mapping SI loci, a Petersen and Seberg 2003), RNA polymerase II (RPB2; Sun common DNA marker was found to be closely linked to the et al. 2009), and the rDNA internal transcribed spacer region S locus in H. bulbosum (Kakeda et al. 2000, 2008) and Phalaris coerulescens (Li et al. 1994, Bian et al. 2004). The Communicated by T. Terachi marker sequence was predicted to encode a thioredoxin h- Received July 21, 2009. Accepted October 23, 2009. like protein and was thus named HTL (Hordeum thioredoxin- *Corresponding author (e-mail: [email protected]) like) in H. bulbosum (Kakeda et al. 2000). Orthologous genes 596 Kakeda, Taketa and Komatsuda have also been isolated from another self-incompatible 50 μl TE buffer (pH 8.0) and used as the template for PCR. species, Secale cereale (rye), as well as from the self- Primers were designed based on the sequences conserved compatible cultivated barley (H. vulgare). Sequence com- between the HTL gene from H. bulbosum (Kakeda et al. parison of these orthologs showed that the coding region is 2000, 2008) and the homologous gene (Bm2) reported in highly conserved (93.9–98.4% nucleotide identity) among P. coerulescens (Li et al. 1994): Trx-F(5′-CGCRRAATATT the four species whereas the 5′-untranslated region (5′-UTR) CCACKTCCC-3′) and Trx-R(5′-YTGGTCCCAGTCCTCT is quite variable (48.4–85.8%) between the two Hordeum TTGG-3′). PCR was performed in a reaction mixture of 20 μl species and Secale or Phalaris (Kakeda et al. 2000). The containing approximately 100 ng of genomic DNA, 1x Pfx HTL gene was confirmed to be a single-copy gene exhibiting reaction buffer, 1 mM MgSO4, 0.3 mM dNTPs, 0.3 μM of each complete linkage to the S locus by a detailed mapping study primer and 0.5 unit of Pfx DNA polymerase (Invitrogen). using 626 individuals in H. bulbosum (Kakeda et al. 2008). The amplification program involved an initial denaturation These features of the HTL gene appear to be appropriate for of 95°C for 2 min, followed by 30 cycles of 95°C for 1 min, phylogenetic analysis, because a high level of interspecific 50°C for 1 min and 72°C for 1 min, and a final extension sequence variation could be present in the variable region of 72°C for 7 min. When a single fragment was produced while stable amplification from different species would be by PCR, the product was excised from an agarose gel, puri- expected using PCR primers designed for the conserved re- fied using a GFX purification kit (Amersham/GE Healthcare) gions. Moreover, it should be interesting to observe the or a GENECLEAN II kit (Qbiogene), and then directly se- molecular evolution of a gene that is closely linked to the SI quenced using Trx-F and Trx-R primers. When the PCR prod- locus, since it may provide insight into the diversification of ucts were multiple fragments of nearly the same size, the frag- the reproductive system in Hordeum. Thus, we analyzed ments were cloned into a Bluescript SK+ vector (Stratagene). HTL gene sequences of Hordeum species to investigate their The purified PCR product was treated with a T4 polynucleo- phylogenetic relationship. We demonstrate a clear differen- tide kinase (Takara), ethanol-precipitated and then ligated tiation of the four genome groups in Hordeum by analysis of into the blunt-ended vector digested with EcoRV. For each 11 diploid species. We also analyzed tetraploid cytotypes PCR fragment, more than three clones were sequenced using from the Xa- and Xu-genome groups to elucidate the origins M13 forward and reverse primers. The sequence was ana- of these polyploids. lyzed using a CEQ2000 DNA analysis system (Beckman Coulter). Materials and Methods Data analysis Plant materials Since the amplified PCR fragments were around 1 kb Twenty-five accessions from 16 Hordeum taxa (cyto- long, the nucleotide sequences adjacent to both primers types) were used (Table 1). The taxonomy follows Bothmer (Trx-F and Trx-R) could not be determined by direct se- et al. (1995). All are diploids except for three tetraploid quencing from either end. Thus, we used only the internal cytotypes: H. marinum ssp. gussoneanum 4x, H. murinum sequence (949–1270 bp) for each fragment by removing ca. ssp. murinum 4x and H. murinum ssp. leporinum 4x. One 30–40 bp from both end sequences. Multiple sequence align- accession each was analyzed for two H. murinum 4x cyto- ments were made using ClustalW with gap open penalty 5, types (ssp. murinum and leporinum) and five diploid species gap extension penalty 3, and gap distance 8. Phylogenetic (H. bogdanii, H. roshevitzii, H. chilense, H. pubiflorum and analysis was performed using PAUP* 4.0b10 software H. pusillum), while two accessions were analyzed for each (Swofford 2002). Maximum parsimony (MP) trees were of the remaining taxa. Psathyrostachys juncea was used as produced using the heuristic search option and TBR branch an outgroup. swapping, and a strict consensus tree was computed from all trees obtained. Alignment gaps were treated as missing data. Extraction of DNA, PCR amplification and sequencing The neighbor-joining (NJ) method (Saitou and Nei 1987) Genomic DNA was extracted by the following method. was also used to construct a tree, where pairwise distances Leaf samples (0.05–0.1 g) were put into a 1.5-ml tube, frozen were calculated with uncorrected (“p”) setting. Bootstrap with a liquid N2, homogenized in 400 μl extraction buffer analysis was performed to assess the reliability of groups by (200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA- 1000 resamples. 2Na, 0.5% SDS) and incubated for 1 h at room temperature. The supernatant obtained after centrifugation (15000 rpm for Results 5 min) was extracted with an equal volume of phenol/chloro- form/isoamyl alcohol (25 : 24 : 1), precipitated with isopro- Genomic DNA fragments homologous to the HTL gene panol and resuspended with 200 μl TE buffer (pH 8.0). The were successfully amplified from all 26 accessions (Table 1) DNA solution was treated with 10 μg/ml RNase A for 30 min by PCR using the primers (Trx-F and Trx-R) designed to the at 37°C, followed by the same series of steps described above cDNA regions (Fig. 1). A single PCR product of approxi- with an additional extraction step using diethylether before mately 1 kb was seen on gels for each accession except for precipitation. Finally, the DNA solution was dissolved in tetraploid (4x) cytotypes of H. murinum. In two accessions Molecular phylogeny of the genus Hordeum 597

Table 1. List of species and sequences used for phylogenetic analysis Genome Length DDBJ Species Ploidy Accession Origin Sequence name group (bp) accession no. H. vulgare ssp. vulgare 2x Bonus I Sweden vulgare vulg. (1) 949 AB509227 Chikurin-ibaraki 1 I Japan vulgare vulg. (2) 949 AB509228 ssp. spontaneum 2x OUH620 I Mexico vulgare spon. (1) 949 AB509229 OUH779 I Afghanistan vulgare spon. (2) 949 AB509230 H. bulbosum 2x 710-17 I Morocco bulbosum (1) 1016 AB509231 IB5-14 I Morocco bulbosum (2) 1019 AB509232 H. marinum ssp. marinum 2x H41 Xa Turkey marinum mar. 2x(1) 987 AB509233 H515 Xa Spain marinum mar. 2x(2) 996 AB509234 ssp. gussoneanum 2x H28 Xa Hungary marinum guss. 2x(1) 969 AB509235 H539 Xa Spain marinum guss. 2x(2) 969 AB509236 ssp. gussoneanum 4x H819 Xa Turkey marinum guss. 4x(1)-A 988 AB509239 marinum guss. 4x(1)-B 969 AB509237 H2303 Xa USA marinum guss. 4x(2)-A 988 AB509240 marinum guss. 4x(2)-B 969 AB509238 H. murinum ssp. glaucum 2x JIC line 71 Xu Unknown murinum glauc. 2x(1) 976 AB509241 H10289 Xu Tajikistan murinum glauc. 2x(2) 981 AB509242 ssp. murinum 4x H614 Xu Greece murinum mur. 4x-A 1270 AB509245 murinum mur. 4x-B 974 AB509243 ssp. leporinum 4x H509 Xu Spain murinum lep. 4x-A 1270 AB509246 murinum lep. 4x-B 974 AB509244 H. brevisubulatum ssp. violaceum 2x H304 H Turkey brevisubulatum (1) 979 AB509247 H316 H Iran brevisubulatum (2) 982 AB509248 H. bogdanii 2x H4014 H Pakistan bogdanii 996 AB509249 H. roshevitzii 2x H9152 H China roshevitzii 996 AB509250 H. brachyantherum ssp. californicum 2x H3317 H USA brachyantherum (1) 992 AB509251 H1954 H USA brachyantherum (2) 991 AB509252 H. chilense 2x Camb.line 1 H Unknown chilense 988 AB509253 H. pubiflorum 2x H1296 H Argentina pubiflorum 991 AB509254 H. pusillum 2x H2038 H USA pusillum 979 AB509255 Psathyrostachys juncea 2x H10108 — Russia P. juncea 1007 AB509256

(H614 and H509) of the 4x subspecies murinum and informative. All characters were equally weighted and all leporinum, two fragments of different sizes (ca. 1.0 and gaps were treated as missing. 1.3kb) were amplified and each fragment was purified and Phylogenetic analysis based on the HTL sequences was analyzed. In addition, we found that two different sequences carried out using the MP and NJ methods. A strict consensus of nearly the same size were amplified in two accessions tree from the two most parsimonious trees (tree length = 297, (H819 and H2303) of the 4x cytotype of H. marinum consistency index = 0.8889, retention index = 0.9481 for ssp. gussoneanum. By sequence analysis of all these PCR each) is shown in Fig. 2, the NJ is shown in Fig. 3. Both the fragments, a total of 30 nucleotide sequences were obtained MP and NJ analyses resulted in highly similar tree topolo- from the 26 accessions examined. Each tetraploid accession gies that demonstrate a clear divergence of four major clades had two different sequences, which were named type A corresponding to the four basic genomes. Phylogenetic clus- and type B (see Table 1 for all sequence names). The size tering also allowed us to infer two separate clades, one con- of sequences varied from 949 bp (H. vulgare) to 1270 bp taining the I- and Xu-genome species (bootstrap values = (H. murinum 4x subspecies murinum and leporinum (4x-A)) 85% (MP) and 83% (NJ)) and the other containing the Xa- (Table 1). On comparison with the cDNA sequences deter- and H-genome species (bootstrap values = 98% (MP) and mined for H. vulgare and H. bulbosum (Kakeda et al. 2000), 99% (NJ)). H. bulbosum was confirmed to be the species the amplified genomic sequences were shown to include the phylogenetically closest to cultivated barley (H. vulgare). gene region of three exons and two introns (Fig. 1). All se- In the diploid H-genome species, four American quences from other species were also predicted to have the species (H. brachyantherum, H. chilense, H. pubiflorum, same gene structure. Multiple alignment of all 30 sequences H. pusillum) were confirmed to be closely related and diver- was constructed using ClustalW and is shown in the supple- gent from Asian species (H. brevisubulatum, H. bogdanii, mentary figure (ESM 1). Of 1426 total characters, 1188 H. roshevitzii). H. brevisubulatum, a self-incompatible characters were constant, 77 variable characters were parsi- species, was further separated from the other H-genome mony uninformative, and 161 characters were parsimony species, although this split got only low bootstrap support 598 Kakeda, Taketa and Komatsuda

Fig. 1. Schematic representation of the amplified region of the HTL gene used for sequence analysis. Regions of the putative open read- ing frame (ORF) are shaded. Arrowheads indicate positions of PCR primers (Trx-F and Trx-R) used for amplification of genomic DNA fragments. Gene structures of the amplified regions from H. vulgare (ssp. vulgare, ‘Bonus’) and H. bulbosum (accession 710-17) are shown. Exons and introns are indicated with boxes and lines, respectively, with sizes in basepairs. Nucleotide sequence identity (%) of each exon and intron is also shown.

(73% (MP) and 68% (NJ)). We analyzed relationships between diploid and tetraploid cytotypes in the Xa genome group (H. marinum). The tetraploid cytotype (ssp. gussoneanum 4x) had two distinct sequences (types A and B), and these were commonly obtained from the two tetraploid accessions (H819 and H2303) collected in Turkey and the USA (Table 1). The type A sequences from these two accessions were clustered with sequences from ssp. marinum (Fig. 2 and Fig. 3). This clade was highly supported (bootstrap values = 99%) by both phylogenetic trees. On the other hand, the type B sequence was Fig. 2. Phylogenetic tree of Hordeum based on HTL gene sequences identical to sequences obtained from ssp. gussoneanum 2x. obtained by the maximum parsimony (MP) method. A strict consensus = This clade was also highly supported (bootstrap values tree was calculated from the two most parsimonious trees (length = 100%) in both phylogenetic trees. These results suggest an 297). Numbers above branches indicate bootstrap values (%) from alloploid origin of ssp. gussoneanum 4x. 1000 bootstrap replications. Details of the taxa (cytotypes) and se- We also analyzed two tetraploid cytotypes (ssp. murinum quence names are shown in Table 1. and ssp. leporinum) together with a diploid cytotype (ssp. glaucum) in the Xu-genome group (H. murinum). Both tetraploid cytotypes shared two distinct sequences (types A Discussion and B) of different sizes (Table 1). The type A sequence (1270 bp) was longer than the type B sequence (974 bp) due Phylogenetic analysis of Hordeum based on nuclear HTL to a 289-bp insertion in the first exon (5′-UTR) of a miniature gene sequences inverted-repeat terminal element (MITE) highly similar to Phylogenetic analysis based on the HTL gene sequences ‘Talisker’ (ESM 1). Despite this insertion, the type A se- demonstrated that the four basic genomes of Hordeum are quence was grouped in a highly supported clade (bootstrap unambiguously distinguishable and further subdivided into values = 100%) together with sequences from the diploid two sister groups, one containing the I and Xu genomes and ssp. glaucum that carries no insertion (Fig. 2 and Fig. 3). On the other containing the Xa and H genomes (Fig. 2 and the other hand, the type B sequence without the insertion Fig. 3). These results agree with the phylogeny based on EF- was unique to the tetraploid cytotypes and distinctly sepa- G sequences (Komatsuda et al. 1999) and on other nuclear rated from the clade including the diploid cytotype. DNA sequences reported recently. Blattner (2004) reported Molecular phylogeny of the genus Hordeum 599

DMC1 and EF-G); although the results based on individual gene data sets varied in details, the phylogeny of all diploid species and subspecies resulting from the combined analysis is consistent with that obtained in the present analysis. Therefore, the phylogenetic relationship of the four basic genomes presented in this study can be considered to represent the conclusive phylogeny based on nuclear gene sequences. Petersen and Seberg (2003) claimed that there were some discrepancies between the results based on plastid and nuclear data regarding the phylogeny of diploid species of Hordeum. They pointed out one major conflict concerning the two subspecies of H. marinum: the nuclear data strongly favored their monophyly while the plastid data (based on rbcL sequences) supported ssp. marinum as a sister to H. brevisubulatum and ssp. gussoneanum placed in an unre- solved clade containing the other H-genome diploid species. Another phylogenetic analysis, based on three chloroplast DNA sequences, also revealed the presence of different plastid types in the two subspecies, suggesting potential polyphyly of H. marinum (Nishikawa et al. 2002). However, none of the nuclear data have supported this polyphyly, including the present analysis and the extensive phylogenetic analyses based on the nuclear gene (EF-G) sequences using more than 100 accessions of H. marinum (Komatsuda et al. 2001). The incongruence has been explained by the plastid data perhaps being misleading due to incomplete lineage sorting (Petersen and Seberg 2003, Jacob and Blattner 2006). The HTL gene is closely linked to the S locus (at 0 cM), one of the two self-incompatibility loci, in H. bulbosum (Kakeda et al. 2000, 2008). The homologous gene (bm2) in P. coerulescens (Aveneae) was also confirmed for complete linkage to the S locus (Li et al. 1994, Bian et al. 2004). The conserved linkage relationship between these two loci in different tribes allows us to speculate about possible co- evolution of these two genes. The two HTL alleles from H. bulbosum analyzed in this study are from S1 and S3 homozygotes. Two highly homologous sequences were ob- Fig. 3. Neighbor-joining (NJ) tree of Hordeum based on HTL gene se- tained from two accessions of diploid H. brevisubulatum quences. Numbers above branches indicate bootstrap values (%) from (ssp. violaceum), another self-incompatible species. Phylo- 1000 bootstrap replications. Details of the taxa (cytotypes) and se- genetic analysis gave no indication that the sequences from quence names are shown in Table 1. these two self-incompatible species are related but are separated from those of the other species (Fig. 2 and Fig. 3). Thus, it seems unlikely that the molecular evolution of the phylogeny based on rDNA ITS sequences using 91 HTL gene is correlated with the diversification of reproduc- Hordeum accessions. The data strongly supported the sister tive systems in Hordeum. However, since the S gene in the group relationship between H. marinum (Xa) and the H- grasses has yet to be cloned, both the genomic organization genome group in addition to the monophyly of H. marinum. in the vicinity of the S locus and the differences in the corre- Another sister group relationship between H. murinum (Xu) sponding genomic regions between the self-incompatible and the I-genome group was only weakly confirmed with and self-compatible species remain to be clarified. Further low bootstrap support (57%), suggesting that the split be- information on the relative locations of these genes in the tween these groups is the oldest and beyond safe resolution genome will be needed to interpret these results. of the ITS region. Phylogenetic analysis of RPB2 sequences also demonstrated that the four genomes are subdivided into Analyses of tetraploid cytotypes in H. marinum and H. murinum two groups (Sun et al. 2009). Furthermore, Petersen and Our phylogenetic analysis also has provided new infor- Seberg (2009) reported a combined analysis of nucleotide mation on the origins of tetraploid cytotypes in H. marinum sequence data from five nuclear genes (XYL, BLZ1, NUC, (the Xa-genome group) and H. murinum (the Xu-genome 600 Kakeda, Taketa and Komatsuda group). Basically, two types of sequences were obtained H. murinum comprises diploid (ssp. glaucum), tetraploid from each tetraploid cytotype and these sequences were used (ssp. murinum and ssp. leporinum) and hexaploid to infer their diploid donor taxa. (ssp. leporinum) cytotypes. The polyploid cytotypes have H. marinum consists of two morphologically distinct been considered either autoploid or segmental alloploid taxa, ssp. marinum and ssp. gussoneanum (Several authors (Bothmer et al. 1995). An alloploid origin of the tetraploid have treated them as two species, i.e., as H. marinum and cytotype ssp. murinum 4x has been suggested by several H. gussoneanum (2x and 4x), see Jakob et al. 2007). The cytological studies based on its C-banding pattern (Linde- former has only a diploid cytotype while the latter comprises Laursen et al. 1989) and rDNA sites (Bustos et al. 1996, a diploid cytotype (gussoneanum 2x) and a tetraploid cyto- Taketa et al. 1999b). Moreover, in situ hybridization analyses type (gussoneanum 4x). The gussoneanum 4x cytotype is using repetitive DNA sequences as probes suggested that morphologically indistinguishable from the 2x cytotype and ssp. murinum 4x is an alloploid involving the diploid cyto- is thought to be autotetraploid. Jakob et al. (2007) reported type ssp. glaucum 2x and an unidentified diploid species chloroplast data that strongly support a clear distinction be- (Taketa et al. 2000b). Our analysis revealed the presence tween ssp. marinum and ssp. gussoneanum (2x, 4x). The two of two distinct sequences common to both 4x cytotypes, cytotypes of ssp. gussoneanum showed little variation in chlo- ssp. murinum and ssp. leporinum. One (type A) contained roplast haplotypes, demonstrating that the 2x cytotype is the a 289-bp insertion of MITE and formed a clade with se- maternal donor of the 4x cytotype. However, ssp. marinum quences from the 2x cytotype (ssp. glaucum), whereas the had remarkable variation in chloroplast haplotypes. other (type B) was unique to the 4x cytotypes (Fig. 2 and Komatsuda et al. (2001) performed large-scale phylogenetic Fig. 3). The results support the alloploid origin of both 4x analysis of H. marinum based on the nuclear DNA (EF-G) cytotypes suggested by Taketa et al. (2000b). One hypoth- sequences using more than 100 accessions of the three cyto- esis regarding the diploid donors of the two genomes is types. The analysis detected two distinct types of sequences that one genome was derived from ssp. glaucum 2x and the in the gussoneanum 4x cytotype. One (915 bp) was shared by other from an unknown Xu-genome species, possibly an ex- the gussoneanum 2x cytotype, whereas another (931 bp) was tinct species. A similar assumption was also made in the not found in any of the 23 accessions examined for the 2x cy- study using rDNA ITS sequences (Blattner 2004). Further totype. Based on this result, they suggested that the 4x cyto- support for the hypothesis has also been provided from re- type consists of two genomes from different lineages, one of cent phylogenetic studies using different nuclear gene se- which must be from gussoneanum 2x. In ssp. marinum, al- quences (Jakob and Blattner 2009, Tannno et al. 2009). though two distinct types of sequences (932 bp each) were The findings obtained from our analyses of tetraploid cy- also found, neither was supported to form a clade with the totypes in H. marinum and H. murinum have strengthened 931-bp sequence from gussoneanum 4x, possibly due to an in- the view that most Hordeum polyploids are of alloploid or at sufficient number of variable sites (Komatsuda et al. 2001). least segmental alloploid origin, apart from the polyploid cy- Taketa et al. (2000a) also suggested, based on comparison totypes of H. bulbosum (4x) and H. brevisubulatum (4x, 6x) of rDNA sites on the chromosomes, that gussoneanum (Blattner 2004). These self-incompatible taxa must have 4x may not be a simple autotetraploid of its 2x cytotype. generated only autopolyploids due to strict control of inter- In the present analysis, we detected two distinct clades in specific pollination by their incompatibility system, and the H. marinum (Fig. 2 and Fig. 3). One is represented by an polyploid cytotypes that are more adaptive in certain envi- identical sequence shared by gussoneanum 2x and 4x, which ronments than diploid cytotypes could have evolved owing is consistent with the results of Komatsuda et al. (2001). An- to their perennial growth habits. other clade involves a different type (type A) of sequences from gussoneanum 4x, which is clustered with those from Acknowledgments ssp. marinum with high bootstrap support (99% for both MP and NJ trees). On the basis of this result, we propose that the We are grateful to Prof. R. von Bothmer and Prof. J.S. tetraploid cytotype has an alloploid origin; i.e., not only the Heslop-Harrison for providing seeds of Hordeum acces- gussoneanum 2x cytotype, but also ssp. marinum was in- sions. This work was supported by a grant from the Ministry volved in the formation of the gussoneanum 4x cytotype. of Agriculture, Forestry and Fisheries of Japan (Genomics This hypothesis should be tested with more accessions from for Agricultural Innovation, TRC1004). different geographical origins to confirm the consistency of the geographical distributions of the diploid donors, in Literature Cited particular ssp. marinum. We analyzed two accessions of Bian, X.Y., A. Friedrich, J-R. Bai, U. Baumann, D.L. Hayman, S.J. ssp. marinum, H41 from Turkey and H515 from the Iberian Barker and P. 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