391 A Y genome specific STS marker in Pseudoroegneria and species (: Gramineae)1

Pungu Okito, Ivan W. Mott, Yajun Wu, and Richard R.-C. Wang

Abstract: The tribe Triticeae Dumortier in the grass family () 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 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 , 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 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 J. Gaertn., and W from marker (GenBank accession BV679236) derived from E. Australopyrum (Tzvelev) A. Lo¨ve. The letters X and Y rectisetus using the Primer3 program (Rozen and Skaletsky have been assigned by various authors to denote the uniden- 1996–1997): OPB14F1 (5’-TCCGCTCTGGGATGTGAC-3’) tified genomes in Triticeae species (Wang et al. 1995). The and OPB14R1 (5’-TCCTGAAGGTAAAACTTTCTGTTT- Y genome is found in many polyploidy species of Elymus TT-3’). The STS-PCR product from various Triticeae spe- from Central Asia eastward to Japan (Dewey 1980). About cies using these primers is hereafter named B14(F+R). 30 StY genome Elymus species are found restrictedly in The PCR mixture was composed of 1Â buffer, 2 mmol/L temperate Asia (Liu et al. 2006). Torabinejad and Mueller MgCl2, 0.20 mmol/L dNTP, 1 U of Taq polymerase, (1993) reported that Elymus rectisetus (Nees in Lehm.) A. 1.6 mmol/L each primer, and 20–60 ng of template DNA Lo¨ve & Connor and Elymus scabrous (R. Br.) A. Lo¨ve, (20 ng for diploid species, 40 ng for tetraploid, and 60 ng which are endemic to New Zealand and Australia, share the for hexaploid) in a final volume of 25 mL. PCR was per- three genomes: St, Y, and W. Among Elymus species, some formed in an Applied BioSystems 9700 thermocycler (Ap- still have questionable genomic constitutions and ~40% still plied BioSystems, Foster City, California) programmed to have unknown genome constitutions (Assadi and Runemark run at 95 8C for 2 min followed by 30 cycles of a denatur- 1995; Svitashev et al. 1996). ing step of 95 8C for 30 s, an annealing step of 55 8C for Chromosome pairing studies suggest that all the North 30 s, and an extension step of 72 8C for 45 s followed by a American species of Elymus (e.g., Elymus canadensis L.) final extension at 72 8C for 5 min.

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Table 1. Plant materials used in this study.

Genome Genus Species Accession No. Origin ID No. symbol Ploidy Agropyron cristatum PI 499389 People’s Republic of China 7260 P 2x Australopyron retrofractum PI 531553 Australia 5434 W 2x Dasypyrum villosum D-2990 Greece 5272 V 2x Elymus alatavicus PI 531709 Estonia 7261 StPY 6x Elymus alatavicus W6 14441 8118 StPY 6x Elymus arizonicus PI 531558 United States 6749 StH 4x Elymus batalinii PI 314623 (Former) USSR 6745 StPY 6x Elymus canadenisis PI 531565 United States 6750 StH 4x Elymus caninus PI 547306 USSR 6746 StH 4x Elymus caucasicus PI 531572 Estonia 5026 StY 4x Elymus cylindricus Jinfeng People’s Republic of China 1984 StHY 6x Elymus dahuricus T 216 3254 StHY 6x Elymus dahuricus PI 531587 Pakistan 6759 StHY 6x Elymus drobovii PI 314196 USSR 6747 StHY 6x Elymus elymoides TAJ 90401 United States 2702 StH 4x Elymus excelsus W 94039 People’s Republic of China 6767 StHY 6x Elymus fibrosus PI 547320 USSR 6753 StH 4x Elymus glaucus PI 232265 United States 3991 StH 4x Elymus gmelinii AJC 266 USSR 6754 StY 4x Elymus kengii PI 504457 People’s Republic of China 6756 StPY 6x Elymus kengii KJ-329 People’s Republic of China 8128 StPY 6x Elymus lanceolatus PI 469235 United States 2703 StH 4x Elymus longearistatus PI 401282 5992 StY 4x Elymus longearistatus PI 401278 Iran 7302 StY 4x Elymus macrochaetus T 211 Tajikistan 3249 StY 4x Elymus nevski H-10215 People’s Republic of China 6758 StY 4x Elymus praeruptus T 217 Tajikistan 3255 StY 4x Elymus rectisetus PI 533028 Australia 6112 StWY 6x Elymus scabrus PI 533217 Australia 4967 StWY 6x Elymus sibiricus PI 499464 People’s Republic of China 6763 StH 4x Elymus sibiricus T 215 Tajikistan 3249 StH 4x Elymus tangutorus CPI 11975 People’s Republic of China 1280 StHY 6x Elymus trachycaulus PI 636525 United States 6764 StH 4x Elymus tschimganicus PI 564998 Kazakhstan 6767 StStY 6x Elymus tsukushiensis PI 499624 People’s Republic of China 5464 StHY 6x Elymus villifer KJ-174 People’s Republic of China 6766 StHY 6x Hordeum bogdanii PI 269406 7312 H 2x Hordeum bulbosum PI 318649 Greece 5181 I 2x Hordeum marinum NGB 90249.2 Greece 8279 XaXa 4x Hordeum murinum PI 206686 8140 XuXu 4x Psathyrostachys juncea PI 314521 USSR 5187 Ns 2x Pseudoroegneria aegilopoides PI 499637 People’s Republic of China 8355 St 2x Pseudoroegneria aegilopoides PI 565082 People’s Republic of China 8359 St 2x Pseudoroegneria aegilopoides PI 531754 People’s Republic of China 8357 St 2x Pseudoroegneria aegilopoides W6 14037 People’s Republic of China 8360 St 2x Pseudoroegneria aegilopoides PI 499638 People’s Republic of China 8356 St 2x Pseudoroegneria cognata W6 14018 Pakistan 7313 St 2x Pseudoroegneria cognata T 219 Tajikistan 3257 St 2x Pseudoroegneria libanotica PI 380649 Iran 8369 St 2x Pseudoroegneria libanotica PI 228391 Iran 8365 St 2x Pseudoroegneria libanotica PI 228392 Iran 8366 St 2x Pseudoroegneria libanotica PI 401326 Iran 8376 St 2x Pseudoroegneria libanotica PI 380652 Iran 8372 St 2x Pseudoroegneria libanotica PI 401322 Iran 8374 St 2x Pseudoroegneria libanotica PI 401325 Iran 8375 St 2x Pseudoroegneria libanotica PI 401336 Iran 8379 St 2x Pseudoroegneria libanotica PI 401339 Iran 8381 St 2x

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Table 1 (concluded).

Genome Genus Species Accession No. Origin ID No. symbol Ploidy Pseudoroegneria libanotica PI 401319 Iran 8373 St 2x Pseudoroegneria libanotica PI 380644 Iran 8368 St 2x Pseudoroegneria libanotica PI 380650 Iran 8370 St 2x Pseudoroegneria libanotica PI 401327 Iran 8377 St 2x Pseudoroegneria libanotica PI 380651 Iran 8371 St 2x Pseudoroegneria libanotica PI 401331 Iran 8378 St 2x Pseudoroegneria spicata PI 610986 United States 6784 St 2x Pseudoroegneria spicata D-2837 United States 8383 St 2x Pseudoroegneria spicata D-2838 United States 8384 St 2x Pseudoroegneria spicata D-2844 United States 8385 St 2x Pseudoroegneria spicata P-739 United States 8387 St 2x Pseudoroegneria spicata PI 232140 United States 8390 St 2x Pseudoroegneria spicata PI 236668 Canada 4938 St 2x Pseudoroegneria spicata PI 236681 Canada 8391 St 2x Pseudoroegneria spicata PI 619629 United States 8386 St 2x Pseudoroegneria spicata P-5B United States 2730 St 2x Pseudoroegneria spicata PI 232127 United States 8388 St 2x Pseudoroegneria spicata PI 232134 United States 8389 St 2x Pseudoroegneria spicata MB-36-51-60 United States 2048 St 2x Pseudoroegneria spicata PI 372641 United States 8392 St 2x Pseudoroegneria spicata PI 421022 United States 8396 St 2x Pseudoroegneria spicata PI 236670 Canada 5176 St 2x Pseudoroegneria stipifolia PI 313960 USSR 5994 St 2x Pseudoroegneria strigosa W6 14033 People’s Republic of China 8362 St 2x Secale montanum PI 531835 Iran 7308 R 2x Thinopyrum bessarabicum PI 531710 Ukraine 7293 Eb 2x Thinopyrum elongatum PI 531718 Tunisia 5040 Ee 2x Triticum aestivum Chinese Spring People’s Republic of China 5271 ABD 6x

Quantitative PCR (qPCR) the B14(F+R) STS marker was y = –1.23 and r2 = 0.953. qPCR was carried out to determine the relative copy num- The normalized values for B14(F+R)N were determined by ber of the Y genome marker B14(F+R) in three tetraploids dividing their average copy value by the average actin value. (Elymus longearistatus (Boiss.) Tzvelev, ID No. 5992; E. The standard deviation quotient was calculated from the canadensis, 6750; Elymus fibrosus (Schrenk) Tzvel., 6753) standard deviations of B14(F+R) and the actin values ac- 2 2 –2 and three diploids (P. spicata, 6748 and 8389; Pseudoroeg- cording to the formula cv = (cv1 +cv2) , where cv = stand- neria libanotica (Heck.) D.R. Dewey, 8376). The qPCR ard deviation/mean value. Amplified PCR product (8 mL) mixture (25 mL) contained 20 ng of genomic DNA, 1Â reac- was also electrophoresed in ethidium bromide stained 2% tion buffer, 1.5 mmol/L MgCl2, 0.4 mmol/L each of agarose gels to confirm the integrity of the amplified DNA. OPB14F1 and OPB14R1 primers, 0.25 mmol/L dNTPs, 0.2Â SYBR Green 1, and 1.25 U of Taq DNA polymerase Gel electrophoresis (Promega, Madison, Wisconsin). Thermal cycling was car- The PCR product was mixed with 10Â loading dye solu- ried out in an Engine Opticon2 System (MJ Research, Wal- tion by using 7 mL of the total 25 mL PCR product and 3 mL tham, Massachusetts) as follows: 95 8C for 2 min followed of loading dye and then analyzed by electrophoresis on a by 35 cycles of 95 8C for 30 s, 55 8C for 30 s, and 72 8C 2% agarose gel to confirm the presence of STS marker for 45 s. Actin was amplified as the internal control using DNA. DNA profiles were recorded using a UVP 2UV primers Actin-qF 5’-CTTTCCCTCTATGCAAGTGGTC-3’ Transilluminator imaging system. The size of the fragments and Actin-qR 5’-TTCATAAGGGAGTCCGTGAGAT-3’. was estimated using 100 bp ladders. All reactions were per- Opticon Monitor 2 version 2.02.24 software (MJ Research) formed in triplicate, and only the positive bands were con- was used to analyze the data. Four replicated PCRs were sidered for this study. used for each genomic DNA – primer combination. Sample DNA concentrations were diluted to 5 ng/mL and checked Cloning and sequencing of the STS Y marker with a ND-1000 Spectrophotometer (NanoDrop Technolo- PCRs producing the Y genome STS marker B14(F+R) gies) to ensure equal loading of 4 mL (20 ng) of genomic were purified using the QIAquick spin columns (Qiagen, DNA. Standard curves for each primer pair were established Chatsworth, California). Purified fragments were ligated and by pooling all genomic DNAs to determine the amplification cloned into pSC-A vector (Stratagene, La Jolla, California) efficiency of each primer–DNA combination. The standard and transformed into Escherichia coli (StraClone Solopack curve for actin was y = –1.55 and r2 = 0.979 and that for competent cells). LB–ampicilin plates were prepared by

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Fig. 1. The Y genome specific STS marker B14(F+R) (arrowed) amplified from Pseudoroegneria spicata (St haplome), Hordeum bogdanii (H), Agropyron cristatum (P), Australopyrum retrofractum (W), Elymus longearistatus (StY), Elymus rectisetus (StWY), Thinopyrum bes- sarabicum (Eb), Thinopyrum elongatum (Ee), Secale montanum (R), Psathyrostachys juncea (Ns), Hordeum bulbosum (I), Dasypyrum villo- sum (V), Triticum aestivum (ABD), and negative control (Ck). M contains the 100 bp DNA ladders as size markers.

Fig. 2. Amplification of the Y genome STS marker B14(F+R) (arrowed) in species of Pseudoroegneria (St genome), Hordeum (H, Xa, Xu), Agropyron (P), Australopyrum (W), and Elymus (StH, StY, StHY, StPY, StWY). Template DNA was normalized according to ploidy levels so that each genome was approximately 20 ng. Three DNA concentrations of diploid Pseudoroegneria libanotica (8376) were used to show that 30 cycles of PCR amplification was appropriate for the test. Note the similar band intensity between Elymus fibrosus (lane 11) and another accession (8389) of Pseudoroegneria spicata (lane 15). Lanes M (1 and 20) contain the 100 bp DNA ladders as size markers.

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Table 2. Relative copy number of B14(F+R) and actin genes in Pseudoroegneria spicata (6784, St genome), Elymus longearista- tus (5992, StY), Elymus canadensis (6750, StH), Elymus fibrosus (6753, StH), Pseudoroegneria libanotica (8376, St), and Pseu- doroegneria spicata (8389, St) as determined by real-time quantitative PCR.

Arbitrary actin Arbitrary B14(F+R) B14(F+R) relative B14(F+R)N normalized B14(F+R)N relative Germplasm copy number copy number copy number* to actin{ copy number{ 6748 33.9±8.8 0.010±0.007 0.4 b 0.000±0.798 0.3 5992 37.1±12.2 360.408±65.872 13133.5 a 1.775±0.377 2901.1 6750 46.5±12.6 0.000±0.000 0.0 c 0.000±0.720 0.0 6753 35.3±6.0 0.131±0.055 4.8 d 0.002±0.456 2.6 8376 42.4±10.5 0.027±0.026 1.0 bce 0.001±0.979 1.0 8389 33.3±6.4 0.099±0.051 3.6 de 0.002±0.546 2.5 *Arbitrary B14(F+R) copy number not normalized to actin but only shown relative to P. libanotica (8376) B14(F+R). Values that share same letters are not significantly different (p < 0.05). {B14(F+R) values were determined by dividing the average copy value by the average actin value. { Relative copy numbers were determined by dividing the B14(F+R)N values by that of P. libanotica (8376). spreading 40 mL of 2% X-gal on each plate. Twenty-five to Fig. 3. A phylogram, generated by ClustalW2 multiple alignment 350 mL of the transformation mixture was plated on the LB– analysis, depicting relationships among 39 Y genome specific ampicillin X-gal plates and incubated overnight at 37 8C. B14(F+R) STS marker sequences amplified from Elymus longear- The formation of blue or white colonies was observed the istatus (ID number 5992, Y of StY, GenBank accessions GF099431 following day. to GF099440, except GF099454 for 5992-3), Pseudoroegneria spi- The white or light blue colonies were selected for PCR cata (8389, St, GF099441 to GF099448), Pseudoroegneria cognata using M13 forward and reverse primers and the colonies (3257, St, GF099453), and Pseudoroegneria libanotica (8376, St, that contained the correct size of DNA were cultured over- GF099449 to GF099452) in relation to the RAPD marker from night at 37 8C. Plasmid DNA preparation was performed us- Elymus rectisetus (Y of StWY, GenBank accession BV679236). ing a QIAprep Miniprep kit (Qiagen). The plasmid DNA Sequences are named as plant material ID number - clone number - was quantified and then sequenced on an Applied BioSys- genome symbol - number of identical sequences. tems 3730 sequencer (Applied BioSystems) at the Center for Integrated BioSystems, Utah State University.

Analyses of DNA sequences DNAstar (Lasergene 7 Software, Madison, Wisconsin) was used to edit the sequences. Multiple-sequence alignment analysis was performed to generate a phylogram using the ClustalW2 program at the European Molecular Biology Lab- oratory Web site. The homology between B14(F+R) and DNA sequences in the National Center for Biotechnology Information databases was checked using the WU-blastn program available at www.ncbi.nlm.nih.gov/.

Results Specificity of RAPD-converted Y genome STS marker The primers B14F1 and B14R1, designed from the 271 bp RAPD marker BV679236 and expected to produce a frag- cessions that are known to have the Y genome. The follow- ment of 269 bp, was used for testing the presence of the Y ing Elymus species were negative in all three replicated PCR genome. The initial test showed that the STS marker was tests for the Y genome: Elymus arizonicus (Scribner & present only in Elymus species having the Y genome Smith) Gould, E. canadensis, Elymus caninus (L.) L., Ely- (Fig. 1). mus lanceolatus (Scribner & Smith) Gould, Elymus mutabi- Subsequently, 33 accessions of Elymus were tested in this lis (Drobov) Tzvelev, Elymus trachycaulus (Link) Gould ex study for the presence or absence of the Y genome STS Shinners, Elymus sibiricus L., Elymus breviaristatus subsp. marker. Agropyron and Australopyrum species possessing scabrifolius (Doll) A. Lo¨ve, and Elymus glaucus Buckl. the P and W genomes, respectively, were used as negative These species contain St and H genomes; thus, the negative controls. Elymus longearistatus (PI 401282, StY) (Jensen result was expected. Only the StH tetraploid E. fibrosus and Wang 1991) was used as the positive control. yielded the unexpected positive result, even though at a As expected, the STS-PCR test was negative for Hordeum weak band intensity (Fig. 2). bogdanii Wilenski (H genome), Agropyron cristatum (L.) J. Gaertner (P), and Australopyrum retrofractum (J.W. Vick- Y genome STS marker in Pseudoroegneria species ery) A. Lo¨ve (W). The primer pair B14 (F+R) yielded one Forty accessions of Pseudoroegneria were tested for the fragment of the expected size, 269 bp, in all 23 Elymus ac- Y genome STS markers. The Y genome marker band pro-

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Fig. 4. Alignment of BV679236, the Y genome RAPD marker from Elymus rectisetus (genome StWY), with 39 Y genome B14(F+R) STS markers amplified from Elymus longearistatus (ID number 5992, Y of StY, GenBank accessions GF099431 to GF099440, except GF099454 for 5992-3), Pseudoroegneria spicata (8389, St, GF099441 to GF099448), Pseudoroegneria cognata (3257, St, GF099453), and Pseudor- oegneria libanotica (8376, St, GF099449 to GF099452). Sequences are named as plant material ID number - clone number - genome sym- bol - number of identical sequences.

duced by 3 out of 40 accessions of Pseudoroegneria species known to have the Y genome than that in species having (P. libanotica PI 401326, P. spicata PI 232134, and Pseu- other genomes. doroegneria cognata (Hackel) A. Lo¨ve T 219) (Fig. 2, lanes 12, 15, and 16, respectively)) was at a lower intensity than qPCR that from Y-containing Elymus species (Fig. 2, lanes 6–9). To accurately determine the copy number of the To verify the results of Y genome marker amplification, B14(F+R) STS marker in E. longearistatus (5992), E. cana- template DNA concentration was normalized according to densis (6750), E. fibrosus (6753), P. spicata (6748 and ploidy levels of tested species. The three diploid Pseudor- 8389), and P. libanotica (8376), a qPCR test was conducted oegneria species and two tetraploid Hordeum species that (Table 2). There were almost 3000 copies of B14(F+R) in previously tested positive were included in this test along the StY genome of E. longearistatus, 3 copies each in E. fi- with species known to have or lack the Y genome. The in- brosus and P. spicata 8389, 1 copy in P. libanotica, and tensity of the marker band was much stronger in species none in either P. spicata 6748 or E. canadensis. These re-

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Fig. 4 (continued).

sults are totally in agreement with those shown in Fig. 2 for ClustalW2 (Figs. 3 and 4). BV679236 shared 92%–95% these samples. identities with clones from P. spicata (8389) and the posi- tive control E. longearistatus (5992), with the exception of Cloning and sequencing the STS marker from clone 5992-3, which had only 71% homology. BV679236 Pseudoroegneria shared lower identities (91%–92%) with all clones of P. li- The amplified B14(F+R) products from the genomic DNA banotica and P. cognata. The 14 sequences from P. spicata of suspected Y genome donor species were cloned and se- (8389) shared 91%–99% homology, whereas those from P. quenced. Fourteen clones of plasmid DNA sequence were cognata (3257) and P. libanotica (8376) shared 92%–95% isolated from P. spicata (8389, St), 5 from P. cognata and 92%–97% homology, respectively, with sequences from (3257, St), 9 from P. libanotica (8376, St), and 11 from the E. longearistatus (5992) except 5992-3. The phylogram positive control E. longearistatus (5992, StY). While all 5 (Fig. 3) also shows that 5 of 14 sequences from P. spicata sequences from 3257 were identical, there are 11 variant (8389) were much more similar to all of those, except types of sequences for 5992, 8 for 8389, and 4 for 8376. 5992-3, from E. longearistatus (5992) and E. rectisetus Forty sequences ranging from 269 to 270 bp, including (BV679236) than all others. However, one of the P. libano- the the Y genome’s RAPD marker (BV679236) that was tica (8376-6) sequences was grouped with one from E. lon- amplified from E. rectisetus (StWY), were analyzed using gearistatus (5992-4).

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Fig. 4 (concluded). marker are all tetraploids (Fig. 2). The Y genome has higher copy numbers of the repetitive STS marker than St, Xu, and Xa (Fig. 2). Furthermore, qPCR results (Table 2) confirmed the reliability of regular PCR that used adjusted template DNA concentrations based on ploidy levels of tested species (Fig. 2). The Xu and Xa genomes would have had less than one copy of B14(F+R). Therefore, the Xa and Xu genomes in Hordeum were less likely the donor of the Y genome in the Elymus species than the St genome of Pseudoroegneria. The B14(F+R) STS marker is useful in identifying the Y genome. Our results confirmed that P. libanotica (PI 401326 = 8376), P. spicata (PI 232134 = 8389), and P. cognata (T 219 = 3257) are all diploids possessing the Y ge- nome marker; thus, they are potential donors of the Y ge- nome. They had also been confirmed to be diploid Pseudoroegneria species using three STS-PCR tests for the St genome (data not shown). Our results added evidence supporting the hypothesis that the St and Y genomes may have originated from a common ancestor (Yen et al. 2005; Liu et al. 2006). Y genome specific STS sequences amplified from P. spi- cata (8389) showed a higher level of similarity to those from E. longearistatus (5992) than the other two Pseudor- Results from homology search using the WU-blastn pro- oegneria species, P. cognata (3257) and P. libanotica gram revealed that B14(F+R) sequences had only a partial (8376) (Fig. 3). Additionally, the amplified sequences from homology (63%–80%) over several spans (141–252 bp) P. spicata had the second highest variability (14 sequences with bacterial artificial chromosome (BAC) clone (such as classified to eight types), only lower than those from E. lon- TA3B95C9 and 925M15) sequences (AM932684, gearistatus (11 sequences that were all different). Sequence 246 833 bp; AM050695, 180 914 bp) of the 3B and an un- variability was lower for those in P. libanotica (nine sequen- known chromosome in wheat (Triticum aestivum L.). They ces belonging to four types) and P. cognata (all five sequen- are dispersed in five and two regions, respectively, along a ces were identical). The internal transcribed spacer part of the chromosomes covered by these BAC clones. sequences of Liu et al. (2006) also showed that E. longear- istatus was slightly closer to P. spicata than to P. libanotica. Discussion Based on these results, it is clear that P. spicata contains se- quences that are most closely related to the Y genome STS To validate the reliability of the Y genome STS marker, a marker in both E. rectisetus (StWY) and E. longearistatus total of 33 accessions representing 28 species of Elymus (StY). Therefore, P. spicata is the prime candidate as donor were tested. They were classified into two groups: (I) 23 ac- of the Y genome to E. longearistatus (StY). However, this cessions known to have the Y genome in their genomic con- does not exclude the possibility that P. libanotica and P. stitution and (II) 10 accessions known to lack the Y genome. cognata identified in this study, or other existing accessions All accessions in group I amplified the Y genome STS of Pseudoroegneria species carrying the Y genome specific marker, making the STS-PCR assay extremely reliable. repetitive sequence, may be the Y genome donor in other In group II, accessions known not to carry the Y genome, Elymus species of the StY, StPY, StWY, or StHY genome 1 out of 10 accessions tested positive for the Y genome constitution. marker and 9 other accessions were confirmed to lack the Y We may designate the progenitor of St and Y genomes as genome marker. Elymus fibrosus (PI 547320), previously (St-Y) and the St genome with the Y genome STS marker classified as an StStHH tetraploid species, was tested posi- sequence as StY. Then, we postulate that (St-Y) diverged tive for the Y genome marker. However, with further scru- into St and StY in many Pseudoroegneria species, such as tiny based on the intensity of the marker fragment (Fig. 2, P. spicata, P. libanotica, and P. cognata identified in this lane 11) and qPCR, this positive result could be attributed study. The StY eventually evolved, when it was combined to the presence of about three copies of the Y genome se- with St in an amphidiploid having the StStStYStY genome quence in its St genome rather than to the presence of a composition, into the present-day Y genome by amplifica- true present-day Y genome that contains about 3000 copies tion and transposition of the repetitive sequence (Bennetzen of this repetitive sequence (Table 2). Therefore, E. fibrosus 2002) into most, if not all, of the seven chromosomes of the should still be designated as an StH genome tetraploid. genome (similar to a postulation in Li et al. 2007). Thus, Y Y Tests on accessions of Hordeum marinum Hudson (Xa StStSt St tetraploid individuals of a Pseudoroegneria spe- haplome) and Hordeum murinum L. (Xu) with the primers cies or interspecific hybrid could be progenitors of StStYY B14F1 and B14R1 demonstrated that there is no diploid species. This hypothesis remains to be tested. Hordeum species that could be the source of the Y genome (data not shown). The few (7 out of 46) accessions of H. References murinum that tested weakly positive for the Y genome Assadi, M., and Runemark, H. 1995. Hybridization, genomic con-

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