391 A Y genome specific STS marker in Pseudoroegneria and Elymus species (Triticeae: Gramineae)1 Pungu Okito, Ivan W. Mott, Yajun Wu, and Richard R.-C. Wang Abstract: The tribe Triticeae Dumortier in the grass family (Poaceae) includes the most important cereal crops (e.g., wheat, barley, and rye) and some economically important forage grasses. Elymus L. is the largest and most complex genus in the Triticeae tribe with approximately 150 species occurring worldwide. The genomic constitutions of ~40% of Elymus species are unknown and some have unverified genomic combinations. Of those known for genome constitutions, Elymus species have a genomic formula of StH, StP, StY, StStY, StHY, StPY, or StWY. However, the origin of the Y genome is unknown because no diploid species have been identified as the Y genome donor. A putative Y genome specific random amplified polymorphic DNA (RAPD) marker was converted to a sequence tagged site (STS) marker. The reliability of this STS marker for confirming the presence of the Y genome was demonstrated using 42 accessions of Elymus. The STS- PCR for the Y genome marker was then assayed on 43 accessions of diploid Pseudoroegneria (Nevski) A. Lo¨ve species having the St genome to identify possible donors of the Y genome. A rare accession of Pseudoroegneria spicata (Pursh) A. Lo¨ve was found to possess sequences that most closely related to those from the tetraploid Elymus longearistatus (Boiss.) Tzvelev (StStYY), making P. spicata the most likely donor of the Y genome, although Pseudoroegneria libano- tica (Heck.) D.R. Dewey or other Pseudoroegneria species could not be excluded. Our findings support the hypothesis that the Y genome in some Elymus species shares a progenitor genome (designated StY) with the St genome of Pseudor- oegneria. Key words: PCR, polyploidization, repetitive sequence, speciation, wheat tribe. Re´sume´ : La tribu des Tritice´es Dumortier au sein de la famille des Gramine´es (Poace´es) comprend les plus importantes espe`ces ce´re´alie`res (par ex. le ble´, l’orge et le seigle) ainsi que certaines gramine´es fourrage`res d’importance e´conomique. Le genre Elymus L. est le plus grand et le plus complexe au sein des Tritice´es puisqu’il compte environ 150 espe`ces a` l’e´chelle mondiale. Les constitutions ge´nomiques d’environ 40 % des espe`ces d’Elymus sont inconnues et certaines ont des combinaisons de ge´nomes qui n’ont pas e´te´ ve´rifie´es. Parmi celles dont la constitution ge´nomique est connue, les espe`ces d’Elymus pre´sentent des formules StH, StP, StY, StStY, StYH, StYP ou StWY. Cependant, l’origine du ge´nome Y est in- connue car aucune espe`ce diploı¨de n’a e´te´ identifie´e comme e´tant l’espe`ce donatrice de ce ge´nome. Un marqueur RAPD (polymorphisme d?ADN amplifiO˜ au hasard) possiblement spe´cifique du ge´nome Y a e´te´ converti en marqueur spe´cifique de site (STS ou « sequence tagged site »). La fiabilite´ de ce marqueur STS dans la de´tection du ge´nome Y a e´te´ de´montre´e sur 42 accessions d’Elymus. Le STS-PCR pour le ge´nome Y a ensuite e´te´ employe´ sur 43 accessions d’espe`ces diploı¨des du genre Pseudroegneria (Nevski) A. Lo¨ve posse´dant le ge´nome St afin d’identifier de possibles donneurs du ge´nome Y. Une accession rare du Pseudroegneria spicata (Pursh) A. Lo¨ve s’est ave´re´e posse´der les se´quences ressemblant le plus a` celles de l’Elymus longearistatus (Boiss.) Tzvelev (StStYY), faisant ainsi du P. spicata le donateur le plus probable du ge´- nome Y, bien qu’il n’ait pas e´te´ possible d’exclure des alternatives comme le Pseudroegneria libanotica (Heck.) D.R. De- wey ou d’autres espe`ces de Pseudoroegneria. Les re´sultats de ces travaux supportent l’hypothe`se que le ge´nome Y chez certaines espe`ces d’Elymus partagerait un ge´nome ancestral (de´signe´ StY) avec le ge´nome St du genre Pseudoroegneria. Mots-cle´s:PCR, polyploı¨disation, se´quence re´pe´te´e, spe´ciation, tribu du ble´. Received 18 September 2008. Accepted 15 February 2009. Published on the NRC Research Press Web site at genome.nrc.ca on 3 April 2009. Corresponding Editor: P. Gustafson. P. Okito and Y. Wu. Department of Plants, Soils, and Climate, Utah State University, Logan, UT 84322-4820, USA. I.W. Mott and R.R.-C. Wang.2 United States Department of Agriculture, Agricultural Research Service, Forage and Range Research Laboratory, Utah State University, Logan, UT 84322-6300, USA. 1Parts of this paper were derived from the thesis research that partially fulfilled the requirements of an M.S. degree earned by Mr. Pungu Okito at the Graduate School, Utah State University. This research was supported in part by the Utah Agricultural Experiment Station, Utah State University, Logan, UT 84322-4810, USA. Approved as Utah Agricultural Experiment Station journal paper No. 7973. 2Corresponding author (e-mail: [email protected]). Genome 52: 391–400 (2009) doi:10.1139/G09-015 Published by NRC Research Press 392 Genome Vol. 52, 2009 Introduction are StStHH allotetraploids (Mason-Gamer et al. 2002). Anal- ysis of chromosome pairing confirmed the presence of the St The genus Elymus L. (Triticeae, Poaceae) is composed of and Y genomes in Elymus borianus (Melderis) A. Lo¨ve and approximately 150 perennial species; thus, it is the largest suggested that the genomic formula of this species should be and most morphologically diverse taxon in the Triticeae StYX, with X and Y symbolizing the unknown genomes (Dewey 1984; Wang and Jensen 2009). Elymus is also the (Svitashev et al. 1998). From cytological analysis of artifi- most widely distributed (Dewey 1984; Jensen and Asay cial hybrids among StY species, evidence suggested that the 1996), occurring from the Arctic to temperate and subtropi- degree of chromosome pairing in the hybrids gradually de- cal regions. Approximately 80 of the known Elymus species creases with increases in geographical distance between the originated in Asia, many of which have never been thor- localities of their parental species (Lu 1993; Lu and Salo- oughly studied. North America has the second largest num- mon 1993). No diploid species containing the Y genome ber of endemic Elymus species (McMillan and Sun 2004). has been identified; thus, the donor species of the Y genome The genus extends from North America into Europe, South in Elymus is still unknown (Dewey 1984; Wang et al. 1986; America, and Australia (Barkworth and Dewey 1985; Wang McMillan and Sun 2004; Xu and Ban 2004; Yen et al. 1992; Jensen and Asay 1996; Lewis et al. 1996). 2005). Nevertheless, based on nuclear ribosomal internal The genus Elymus is a complex group of allopolyploids transcribed spacer and chloroplast trnl-f sequences, it has containing multiple copies of different genomes (Liu et al. been hypothesized that the St and Y genomes originated 2006). It has been reported by Stebbins and Ayala (1985) from a common ancestor genome (Liu et al. 2006). Thus, a that more than 80% of the Gramineae family have under- putative Y genome specific sequence tagged site (STS) gone polyploidization during their speciation. The number marker was tested on Elymus species for its reliability in de- of chromosomes in Elymus is between 2n =4x = 28 and 2n tecting the Y genome. It was then used to screen accessions =8x = 56 (Jensen and Salomon 1995; Jensen and Asay of Pseudoroegneria to identify possible Y genome donors. 1996; Ellneskog-Staam et al. 2007). The occurrence of poly- ploidy in Elymus may contribute to the facts that they are Materials and methods more resistant to cold, heat, and drought and are better adapted to new environmental conditions than their diploid Plant materials with accession number, country of origin, progenitors. According to Stebbins and Antero (1954), the identification number, genomic constitution, and ploidy ancestors of Pseudoroegneria spicata (Pursh) A. Lo¨ve and level used herein are presented in Table 1. Hordeum L. migrated from Asia to North America, hybri- dized, and gave rise to some North America polyploidy spe- DNA extraction and quantification cies and then migrated to South America. Therefore, the Approximately 100 mg of fresh leaf tissue was collected genus Elymus is a model for studying morphological varia- from each seedling for DNA extraction using the CTAB (ce- bility, phenotypical plasticity, and natural hybridization. tyltrimethylammonium bromide) method (Rogers and Bend- The genus also provides excellent plant materials for cytoge- ich 1988). Genomic DNA was quantified with a Nanodrop netics, molecular genetics, and phylogeny investigations Spectrophotometer ND-1000 (NanoDrop Technologies, Wil- (Dı´az et al. 1999). mington, Delaware) at a wavelength of 260 nm. The ge- Dewey (1980) initially described the genomic constitution nomic DNA was adjusted and normalized to 40 ng/mL and of the Central Asian hexaploid (2n = 42). The genome com- then evaluated by using 2% agarose gel stained with ethi- binations for Elymus species include StH, StY, StP, StStH, dium bromide (5 mg/mL) and recorded using a 2UV Trans- StHY, StPY, and StWY (Dewey 1984; Baum et al. 1991; illuminator imaging system (UVP, Inc., Upland, California). Wang 1992; Wang et al. 1995; Larson et al. 2003). Thus, all of the Elymus species share a common St genome origi- PCR nated from the genus Pseudoroegneria (Nevski) A. Lo¨ve A pair of STS primers was designed for the Y genome (Dewey 1980; Wang 1992), while the H genome originated specific random amplified polymorphic DNA (RAPD) from Hordeum, P from Agropyron J. Gaertn., and W from marker (GenBank accession BV679236) derived from E.