Hereditas 148: 8–27 (2011) Polymorphic nuclear gene sequences indicate a novel genome donor in the polyploid genus Thinopyrum MATT ARTERBURN 1 , ANDRIS KLEINHOFS 2 , TIMOTHY MURRAY3 and STEPHEN JONES 2 1 Department of Biology, Washburn University, Topeka, KS, USA 2 Department of Crop and Soil Sciences, Washington State University, Pullman, WA, USA 3 Department of Plant Pathology, Washington State University, Pullman, WA, USA Arterburn, M., Kleinhofs, A., Murray, T. and Jones, S. 2010. Polymorphic nuclear gene sequences indicate a novel genome donor in the polyploid genus Thinopyrum . – Hereditas 148 : 8–27. Lund, Sweden. eISSN 1601-5223. Received 16 June 2008. Accepted 15 November 2010. For decades, the wheatgrass genus Thinopyrum has been of interest to plant breeders as a source of genes that confer competitive traits. This genus has been a considerable challenge to plant systematists because of the impacts of polyploidization on the evolution of this group. This study was aimed to augment existing cytogenetic data with a sequence-based investigation of the genomes of these species. Sequences of the internal transcribed spacer 1 (ITS1), introns 9 through 11 of the granule-bound starch synthase (GBSSI) gene and intron III of the beta-amylase gene ( Bmy1 ) were isolated from the genomes of polyploid Thinopyrum species by PCR, cloning and sequencing and the evolutionary distances between these species and putative diploid ancestors were estimated with Kimura ’ s two-parameter method. Phylogenetic analysis of these sequences largely agrees with what has been established through cytogenetic means for the Th. caespitosum (Koch) Liu & Wang and Ps geniculata (Trin.) Á . L ö ve, and suggests a contribu- tion of the St genome of Ps. spicata (Pursh) Á . L ö ve to the tetraploids Th. scirpeum (Presl) Dewey and Th. junceiforme ( Á . L ö ve & D. L ö ve) Á . L ö ve. A unique Bmy1 allele, divergent from other Triticeae but shared between Th. caespitosum , Th. intermedium (Host) Barkworth & Dewey, Th. junceum (L.) Á . L ö ve and Th. ponticum Barkworth & Dewey, implies a connection between these species. Distinct oligonucleotide polymorphisms and distance calculations based on the three loci implicate Crithopsis delileana (Schult.) Roshev. and Taeniatherum caput-medusae (L.) Nevski in the evolution of the hexaploid Th. intermedium and the decaploid Th. ponticum and also suggest a potential connection of these polyploids with Elytrigia repens (L.) Desv. ex Nevski. None of these species have been previously associated with the Thinopyrum genus. Allele-specifi c PCR was employed to detect the putative Crithopsis allele of ITS1 in a number of accessions. Real-time PCR indicates that two of six genomes of the hexaploid Th. interme- dium have the Crithopsis -type ITS1 allele and that all ITS1 loci in the decaploid Th. ponticum are of this type. These results are supportive of the hypothesis that concerted evolution has homogenized the rDNA of Th. ponticum to the allele derived from the Crithopsis or Taeniatherum ancestor. Discovery of these novel alleles, with close homology to Ta. caput-medusae , may represent a fundamental change in the view of the evolution of Th. intermedium and Th. ponticum . Matt Arterburn, Department of Biology, Washburn University, 1700 SW College Ave, Topeka, 66621 KS, USA . E-mail: matt. [email protected] The wheatgrass genus Thinopyrum harbors species Members of the genus Thinopyrum have, at different which range in ploidy from diploid to decaploid and times, been included in the genera Elymus , Elytrigia , thus exhibit considerable evolutionary complexity at the Lophopyrum, Pseudoroegneria and Agropyron . Because genome level. By contrast, the anatomical features of their robust capacity for wide hybridization, classifi ca- of these species are often so similar that historically col- tion of these species has been diffi cult, although a consid- lectors of these wild grasses have sometimes grouped erable amount of effort has been invested in doing so. accessions of different ploidy levels into the same spe- Current classifi cation of members in the Thinopyrum cies. Although all these species are perennial, some genus is largely based on anatomical and karyotypic are rhizomatous while others have caespitose, bunch- homology, and the pairing behaviors of their chromo- like growth habit. Although frequently employed by somes in interspecifi c hybrids and/or amphiploids. The plant breeders in wide hybridizations, and the object polyploid members of this group are believed to have of a considerable amount of cytogenetic scrutiny, the originated from polyploidization events involving three Thinopyrum group is less well-characterized in terms of putative progenitors of the genus: Th. elongatum Dewey molecular sequence data. A growing body of sequence (2n ϭ 2x ϭ 14, E e E e ), Th. bessarabicum (Savul & Rayss) data should help illuminate new aspects of the evolution- Á . L ö ve. (2n ϭ 2x ϭ 14, E b E b ), and either Pseudoroegne- ary history and the dynamics of polyploidization that ria strigosa (Bieb) Á . L ö ve (2n ϭ 2x ϭ 14, StSt) or the have occurred in this genus, an understanding which will closely-related Ps. spicata (Pursh) Á . L ö ve (2n ϭ 2x ϭ 14, aid crop improvement. StSt) ( DEWEY 1984). The genomes of Th. elongatum and © 2011 The Authors. This is an Open Access article. DOI: 10.1111/j.1601-5223.2010.02084.x Hereditas 148 (2011) Polymorphic sequences indicate a novel ancestor of Thinopyrum 9 Th. bessarabicum have previously been denoted E and J hybrids (2n ϭ 4x ϭ E e E e E b E b ) ( LIU and WANG 1992, respectively, although these are now frequently referred to 1993). On the basis of GISH analysis, R EFOUFI et al. (2001) as the E e and E b genomes, based on the work of WANG suggested that Th. junceiforme is instead an autotetraploid (1992) which suggests that chromosome pairing patterns of the E e genome. Two other species, Th. nodosum (Boiss. in interspecifi c hybrids of these two is such that a single & Heldr.) Á . L ö ve and Th. caespitosum (Koch) Liu & letter designation should be used to indicate their close Wang are thought to be allotetraploids of the E b and St evolutionary relationship. We will use the E e and E b desig- genomes (2n ϭ 4x ϭ E b E b StSt) ( LIU and WANG 1989, nation for these genomes throughout this study, for clarity. 1992). Th. scirpeum (Presl) Dewey is considered an auto- Designation of the various genomes present in Thinopy- tetraploid of the E e genome ( LIU and WANG 1993). rum polyploids has been based largely on the ability to The higher ploidy Thinopyrum species are of particular generate interspecifi c hybrids, the ability of chromosomes interest because they are frequently used in wide crosses to pair in these hybrids or in amphiploids and on the rela- to introgress agronomically useful genes into cereal crops tive intensity of signal produced via genomic in situ and for production of perennial grains for sustainable hybridization (GISH) when using genomic DNA of puta- agricultural systems ( CHEN et al. 1998a; COX et al. 2002; tive ancestors as a probe ( WANG 1989; ZHANG et al 1996a; Z HANG et al 1996b; SCHEINHOST et al. 2001). Th. junceum C HEN et al. 1998b). (L.) Á . L ö ve is described as an allohexaploid combining The current evolutionary view of the genus Thinopy- the E e and E b genomes ( LIU and WANG 1993). LIU rum defi nes the polyploid members as either autopoly- and WANG (1993) described Th. intermedium (Host) ploids or allopolyploids of the E e , E b and St genomes, in Barkworth & Dewey as an allohexaploid of the E e and various combinations (Table 1). Of the eight tetraploids in St genomes (2n ϭ 6x ϭ 42, E e E e E e E e StSt) on the basis this genus, four, Th. distichum (Thunb.) L ö ve, Th. curvi- of chromosome pairing and C-banding in interspecifi c folium (Lange) D.R. Dewey, Th. sartorii (Boiss. 7 Heldr.) hybrids. CHEN et al. (1998b), based on GISH analysis, Á . L ö ve and Th. junceiforme ( Á . L ö ve & D. L ö ve) suggested that this species is an allohexaploid of the Á . L ö ve, are considered allotetraploids of the E e and E b Eb genome, St genome and J s genomes (2n ϭ 6x ϭ 42, genomes based on chromosome pairing in interspecifi c Eb E b J S J S StSt). The J S designation is based on their Table 1. Summary of ITS1, Bmy1 and GBSSI sequence analysis. Sequenced alleles are matched to the closest putative diploid ancestor. Evolutionary distance values from closest ancestor alleles are given in parentheses; multipliers indicate number of clones isolated of each allelic type. Hypothetical genome constitutions are given in the rightmost column . Sequence Current genome based Species (accesion) designations ITS results Bmy1 results GBSSI results constitution Th. elongatum (PI547313) E e E e E e E e E e Th. bessarabicum (PI531711) E b E b E b E b E b Ps. strigosa (PI499493) St St St St St Cr. delileana (01C4200003) K K K K K Ta. caput-medusae (PI222048) Ta Ta Ta Ta Ta Th. scirpeum (PI531749) E e E e * E e (0.034 x5) St (0.008 x2) ND E e St Th. junceiforme (PI531731) E e E b * E e (0.021 x5) E e (0.000 x1) ND E e St E e E e ∗ ∗ ∗ St (0.011 x1) Ps. geniculata (PI565009 ) E e St ∗ ∗ St (0.021 x5) E e (0.019 x2) ND E e St St (0.008 x2) Th. caespitosum (PI228276) E e St ∗ ∗ St (0.021 x4) E b (0.014 x3) ND E b X or E b St X (0.272 x1) Th.