Variations in Abundance of 2 Repetitive Sequences in Leymus and Psathyrostachys Species

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Variations in abundance of 2 repetitive sequences in Leymus and Psathyrostachys species

R.R.-C. Wang, J.-Y. Zhang, B.S. Lee, K.B. Jensen, M. Kishii, and H. Tsujimoto

Abstract: The Ns genome of the genus Psathyrostachys is a component of the polyploid genome in the genus Leymus. Using fluorescence in situ hybridization (FISH), the occurrence and abundance of 2 tandem repetitive sequences from Leymus racemosus (Lam.) Tzvelev, pLrTaiI-1 (TaiI family) and pLrPstI-1 (1 class of 350-bp family), were assayed in 4 species of the genera Psathyrostachys and Leymus. The pLrPstI-1 sequence was absent in all 4 Psathyrostachys species. While P. fragilis and P. huashanica did not have the pLrTaiI-1 sequence, 15 accessions of P. juncea and 2 accessions of P. lanuginosa had pLrTaiI-1 sites ranging in number from 7 to 16 and from 2 to 21, respectively. The numbers of pLrTaiI-1 and pLrPstI-1 sites were 1–24 and 0–30, respectively, in L. ramosus; 2–31 and 5–36 in L. racemosus; 0–4 and 0 in L. mollis; 2–9 and 24–27 in L. secalinus. The FISH assay on pLrTaiI-1 was successfully converted to a sequence-tagged-site polymerase chain reaction (STS-PCR) test using a primer pair designed from the sequence of this repetitive DNA. Seventy-three accessions representing 27 Leymus species were assayed for the abundance of pLrTaiI-1 by STS-PCR. With a few exceptions of uniformity in some accessions, nearly all Leymus species observed were heterogeneous for the abundance of pLrTaiI-1 sequence and no Leymus species was totally devoid of this repetitive sequence. These findings may have significance for the understanding of phylogeny, nature of polyploidy, adaptive ranges, and breeding potential of Leymus species. Key words: FISH, genome, polyploid, 350 bp family, pLrTaiI-1, STS-PCR. Résumé : Le génome Ns du genre Psathyrostachys est une composante du génome polyploïde chez le genre Leymus. Par hybridation in situ en fluorescence (FISH), la présence et l’abondance de 2 séquences répétées en tandem chez le Leymus racemosus (Lam.) Tzvelev, pLrTail-1 (famille des éléments Tail) et pLrPstI-1 (une classe des éléments de 350 pb) ont été examinées chez 4 espèces au sein des genres Psathyrostachys et Leymus. La séquence pLrPstI-1 était absente chez les 4 espèces du genre Psathyrostachys. Alors que le P. fragilis et le P. huashanica ne présentaient pas la séquence pLrTail-1, 15 accessions du P. juncea et 2 accessions du P. lanuginosa possédaient entre 7 et 16 ou entre 2 et 21 de ces séquences, respectivement. Le nombre de copies des séquences pLrTail-1 ou pLrPstI-1 variait entre 1 et 24 ou 0 et 30, respectivement, chez le L. ramosus ; entre 2 et 31 ou entre 5 et 36 chez les L. racemosus ;entre0et4ou 0 chez le L. mollis ; entre 2 et 9 ou 24 et 27 chez le L. secalinus. Une analyse FISH sur pLrTail-1 a été convertie en marqueur STS-PCR (« sequence-tagged site polymerase chain reaction ») en employant une paire d’amorces dérivée de la séquence de cet ADN répétitif. Soixante-treize accessions représentant 27 espèces du genre Leymus ont été exami- nées pour l’abondance de pLrTail-1 à l’aide de ce marqueur. A peu d’exceptions près, presque toutes les espèces du genre Leymus étaient hétérogènes pour l’abondance de pLrTail-1 et aucune espèce de Leymus n’était dépourvue de cette séquence répétée. Ces observations pourraient contribuer à une meilleure connaissance de la phylogénie, de la poly- ploïdie, de la gamme adaptative et du potentiel en sélection des espèces de Leymus. Mots clés : FISH, génome, polyploïde, éléments de 350 pb, pLrTail-1, STS-PCR. [Traduit par la Rédaction] Wang et al. 519

Received 3 February 2005. Accepted 19 October 2005. Published on the NRC Research Press Web site at http://genome.nrc.ca on 10 May 2006. Corresponding Editor: G. Jenkins. R.R.-C. Wang,1 J.-Y. Zhang,3 B.S. Lee,2 and K.B. Jensen. USDA–ARS Forage and Range Research Laboratory, Logan, UT 84322-6300, USA. M. Kishii4 and H. Tsujimoto. Laboratory of Plant Genetics and Breeding Science, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan. 1Corresponding author (e-mail: [email protected]). 2Present address: Department of Biology, School of Natural Sciences, Jeonju University, Jeonju 560-759, Republic of Korea. 3Present address: Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401 USA. 4Present address: CIMMYT, Londres 40, Apdo. Postal 6-641, Delg. Cuauhtemoc 06600, Mexico.

Introduction Leymus (Tables 1 and 2) were studied by the 2-color fluorescence in situ hybridization (FISH) method of Kishii et al. Plant nuclear genomes encompass a wide range of varia- (1999). One to 4 plants per accession were assayed for the tion in size and nucleotide composition with diverse arrange- number of in situ hybridization sites of pLrTaiI-1 and ments of chromosomal segments, repetitive sequences and pLrPstI-1 repetitive sequences. pLrTaiI-1 and pLrPstI-1 were gene distribution (King 2002). The knowledge gained from labeled with digoxigenin-11-dUTP and biotin-16-dUTP studies on eukaryotic genome organization is important for (Roche Applied Science, Indianapolis, Ind.), respectively, us- understanding how genomes function and evolve, and it pro- ing PCR labeling. In situ hybridization sites were detected vides the basis for designing strategies for manipulating with anti-digoxigenin-rhodamine (Roche Applied Science) and genomes (Lapitan 1992). The chromosomes are generally FITC–avidin conjugated with biotinylated goat anti-avidin composed of heterochromatin and euchromatin regions. (Vector Laboratories, Burlingame, Calif.), respectively. Under Heterochromatin is defined as deeply staining chromosomal a fluorescence microscope (Zeiss, Jena, Germany), pLrTaiI-1 material that remains condensed in interphase, whereas and pLrPstI-1 sites appeared in red and green color, respec- euchromatin undergoes decondensation. Heterochromatin is tively, on the blue-colored chromosomes resulting from the found to occur near centromeres and telomeres, but intersti- counterstain, 4′,6-diamidino-2-phenylidone (DAPI), in the tial sites of heterochromatin are also common in plant Vectashield mounting medium (Vector Laboratories). Photo- genomes. Lippman et al. (2004) demonstrated that hetero- graphs were taken with a cooled CCD camera (AxioCam, chromatin in Arabidopsis is composed of transposable ele- Zeiss) on the microscope. ments and related tandem repeats. They also show that Southern hybridization of pLrTaiI-1 to genomic DNA was transposable elements can regulate genes epigenetically carried out with selected species of Psathyrostachys and when inserted within or very close to the structural genes. It Leymus. ScaI-digested genomic DNAs of P. juncea (acces- is now generally accepted that repetitive sequences are not sion HT17586), P. huashanica (HT17585), L. racemosus (PI all junk DNA, but that some are involved in regulation of 313965), L. mollis (MK10011), and L. secalinus (PI 499524) gene expression (Balakirev and Ayala 2003). were electrophoresed in a 1.5% agarose gel and transferred The Ns genome in the genus Psathyrostachys (Poaceae: to Hybond-N+ membranes (Amersham, Piscataway, N.J.). Triticeae) Nevski is a component of the polyploid genome in The probe (pLrPLrTaiI-1) was labeled by 32P-dCTP and the genus Leymus Hochst. The genome constitution of tetra- hybridized with the membrane in solution (6× SSC, 5× ploid Leymus species is still unresolved. Zhang and DvoÍák Denhardt, and 0.5% SDS) at 68 °C for 16 h, followed by (1991) and Anamthawat-Jónsson and Bödvarsdóttir (2001) washing in a 0.5× SSC, 0.1% SDS solution. Hybridization proposed a segmental autotetraploid (Ns1Ns1Ns2Ns2) and 32 Wang and Jensen (1994) proposed an allotetraploid was visualized by exposure of Kodak X-ray film to P. (NsNsXmXm; with the Xm coming from an unknown Based on the 566-bp DNA sequence of pLrTaiI-1 of source) structure for tetraploid Leymus species. Kishii et al. (1999), a pair of STS primers was designed to Kishii et al. (1999) reported the localization of 2 tandem amplify fragments of multiples of 550 bp. The forward ′ ′ repetitive sequences, pLrTaiI-1 (TaiI family, 570 bp/repeat primer was 5 -TTATTGACATTAGTCCCCCTGG-3 and the ′ ′ unit) and pLrPstI-1 (350 bp family, 380 bp /repeat unit), in reverse primer was 5 -TCCTTCAAAAGGAAGTGCCA-3 . subtelomeric heterochromatin regions of Leymus racemosus Bulked genomic DNA was extracted using the cetyltrimethy- (Lam.) Tzvelev chromosomes. These 2 repetitive sequences lammonium bromide (CTAB) method of Doyle and Doyle were not detected in Leymus mollis (Trin.) Pilger and (1987) from each accession of L. ramosus and L. racemosus P. huashanica Keng, and only pLrTaiI-1 was found in (Table 2) that had been assayed with the fluorescence in situ Psathyrostachys juncea (Fisch.) Nevski. Furthermore, they hybridization (FISH) method. These genomic DNA samples observed that even homologous chromosomes did not show were tested for amplification of pLrTaiI-1 by PCR. PCR was the same patterns of TaiI and 350 bp families. This observa- carried out in a reaction mixture (25 µL) containing 14.1 µL µ µ tion suggests that these 2 repetitive sequences may be nei- sterile ddH2O, 2.5 L of 10× buffer, 2 L of 8 mmol/L ther chromosome nor genome specific. dNTP, 1.5 µLof10µmol/L each of the designed primer, µ µ Variation in repetitive sequences has been used for study- 1.5 L of 50 mmol/L MgCl2, 0.4 L(2U)Taq polymerase ing germplasm diversity and relationships in Amaranthus (Gibco, Invitrogen, Carlsbad, Calif.), 0.5 µL of dimethyl (Sun et al. 1999). Variation in amplified fragment length sulfoxide (DMSO), and 1 µL template DNA (40 ng/µL). The polymorphism (AFLP) and restriction fragment length poly- GenAmp PCR System 9600 (Perkin Elmer, Wellesley, morphism (RFLP) of ribosomal genes has been observed be- Mass.) was programmed to run for 5 min at 95 °C, followed tween Leymus species and among populations of Leymus by 30 cycles of 45 s at 94 °C, 1 min at 58 °C, and 2 min at arenarius (L.) Hochst., and Leymus mollis (Anamthawat- 72 °C, and finally 7 min at 72 °C. Gel electrophoresis was Jónsson et al. 1999). In this paper, we surveyed the variation carried out in a 2% agarose gel containing 0.5 µg ethidium in the abundance of pLrTaiI-1 and pLrPstI-1 among acces- bromide in 1× Tris–borate EDTA (TBE) buffer. DNA frag- sions and species of Psathyrostachys and Leymus. The infor- ments were visualized and photographed under ultraviolet mation may be significant to the understanding of phylogeny, light; their sizes were estimated by comparing them with nature of polyploidy, adaptive ranges, and breeding potential 100 bp DNA size markers composed of 1500 bp, 1200 bp, of Leymus species. and from 1000–100 bp in increments of 100 bp (New Eng- land BioLabs, Ipswich, Mass.). Materials and Methods Twenty-seven Leymus species (Tables 2 and 3), repre- sented by 1–3 accessions each, were grown in the green- Forty accessions of 8 species of Psathyrostachys and house. DNA extracted from individual plants was stored as

Table 1. Variation in number of FISH signals for repetitive sequences pLrTaiI-1 and pLrPstI-1 in 4 diploid (2n = 14) Psathyrostachys species. Sites for Sites for Species Accession Origin pLrTaiI-1 pLrPstI-1 P. juncea PI 499675 People’s Republic of China 7–8 0–0 PI 499558 People’s Republic of China 12–15 0–0 PI 565045 Former USSR 10 0 PI 565049 Former USSR 10 0 PI 565048 Former USSR 10–14 0–0 PI 440624 Former USSR 11–12 0–0 PI 565047 Former USSR 12 0 PI 565079 Former USSR 12–12 0–0 PI 565043 Former USSR 12–16 0–0 PI 565044 Former USSR 14 0 PI 440625 Former USSR 14–14 0–0 PI 314521 Former USSR 16 0 PI 440627 Kazakhstan 12–14 0–0 PI 476299 USA, ‘Vinall’a 14–16 0–0 PI 206684 Turkey 16 0 P. lanuginose D-3376 People’s Republic of China 2–21 0–0 D2562 People’s Republic of China 2–12 0–0 P. fragilis PI 343190 Iran 0–0 0–0 PI 343192 Iran 0–0 0–0 PI 401392 Iran 0 0 PI 401397 Iran 0 0 KJ-127 Iran 0 0 P. huashanica PI 531823 People’s Republic of China 0–0 0–0 Note: A single number represents counts in 1 plant; numbers separated by a dash represent the range of counts in multiple (between 2 and 4) plants. a‘Vinall’ is a cultivar released in North Dakota, USA.

Table 2. Variation in number of FISH signals for repetitive sequences pLrTaiI-1 and pLrPstI-1 in 4 tetraploid (2n = 28) Leymus species. Sites for Sites for Species Accession Origin pLrTaiI-1 pLrPstI-1 L. ramosus DJ-4257 Former USSR 1–2 0–0 PI 499654 People’s Republic of China 2–2 0–0 PI 499653 People’s Republic of China 8–14 22–30 PI 440331 Kazakhstan 20–24 0–0 L. racemosus PI 502402 Former USSR 2–5 24–36 D-2949 Former USSR 12–13 22–26 PI 565037 Former USSR 18–20 5–8 PI 313965 Former USSR 30–31 16–28 PI 108491 Former USSR 8–12 26–30 PI 531811 Azerbaijan 5–12 26–30 PI 478832 Montana 8–11 29–32 PI 531812 Washington 8–15 24–26 D-3621 Washington 10–11 14–20 HT 15405 Bulgaria 28 8 L. mollis MK 10001 Japan 0–2 0–0 MK 10011 Japan 4 0 L. secalinus PI 499524 People’s Republic of China 2–9 24–27 Note: A single number represents counts in 1 plant; numbers separated by a dash represent the range of counts in multiple (between 2 and 4) plants. such. With a few exceptions, more than 5 plants were as- from 60 accessions of 25 species (Table 3) were assayed on sayed in each of the 60 accessions (Table 3) by the above- an individual basis and 13 accessions of 2 species described procedure of STS-PCR. A total of 73 accessions (L. ramosus and L. racemosus in Table 2) were assayed on a of 27 Leymus species were assayed by STS-PCR: 350 plants bulked-accession basis.

Table 3. Abundance of pLrTaiI-1 sequence in Leymus species assayed by STS-PCR amplification using the primer set 5′-TTATTGACATTAGTCCCCCTGG-3′ and 5′-TCCTTCAAAAGGAAG- TGCCA-3′. No. of plants with varying abundance of pLrTaiI-1 Species Accession Origin High Low Absent L. mollis (2n = 28) D-4086 Former USSR 0 0 7 DJ-4100 Former USSR 0 0 1 L. secalinus (2n = 28) PI 499658 People’s Republic of China 2 3 2 PI 499528 People’s Republic of China 3 3 1 JA-123 Kazakhstan 3 0 3 L. paboanus (2n = 56) PI 531808 Former USSR 2 4 0 KJ-226 Mongolia 2 3 1 L. angustus (2n = 84) PI 499650 People’s Republic of China 3 2 1 (2n = 84) D-3695 People’s Republic of China 5 1 0 (2n = 70/84) D-3740 People’s Republic of China 4 0 2 L. arenarius (2n = 56) PI 294582 Sweden 1 1 4 PI 531799 Estonia 0 0 6 D-3567 Netherlands 3 0 3 L. chinensis (2n = 28) PI 499514 People’s Republic of China 4 1 1 PI 499516 People’s Republic of China 6 0 0 PI 499518 People’s Republic of China 3 0 3 L. condensatus (2n = 28) Atkins 102a California 0 4 2 PI 531804b California 0 2 4 L. flavescens (2n = 28) Hassell-Harris Idaho 0 5 1 KJ-39 Idaho 0 5 0 KJ-36 Idaho 0 6 0 L. flexilis (2n = 42/56) DJ-4110 Former USSR 1 1 3 L. innovatus (2n = 28) PI 531805 Canada 6 0 0 PI 236820 Canada 5 0 1 L. karataviensis (2n = 28) PI 314667 Former USSR 5 1 0 PI 314677 Former USSR 6 0 0 PI 314678 Former USSR 6 0 0 L. karelinii (2n = 56/70) PI 430791 Former USSR 6 0 0 (2n = 56) PI 430838 Former USSR 6 0 0 (2n = 56) PI 430842 Former USSR 3 2 1 L. mojavensis (2n = 42) D-3610 California 1 1 4 (2n = 28) D-3620 California 0 0 3 L. multiflorus (2n = 56) Atkins 89 California 0 0 6 (2n = 28) D-3366 California 3 2 1 L. multicaulis (2n = 28) PI 314666 Former USSR 1 5 0 PI 440321 Kazakhstan 1 5 0 PI 531806 Kyrgyzstan 1 4 1 L. paeboelus (2n = 84) D-4258 4 2 0 L. pseudoracemosus PI 531810 People’s Republic of China 6 0 0 (2n = 28) PI 531809 People’s Republic of China 1 4 1 L. sabulosus (2n = 28) D-3488 Former USSR 6 0 0 PI 531814 Ukraine 6 0 0 PI 531813 Ukraine 6 0 0 L. salinus (2n = 28) KJ-41 Utah 3 0 3 (2n = 28) PI 565038 Utah 1 0 4 (2n = 56) PI 531815 Wyoming 0 0 6 L. triticoides (2n = 28) PI 531822 Nevada 2 1 3 Asay M-9 Wyoming 4 0 2 PI 610977 Utah 0 0 6 L. akmolinensis (2n = 56) PI 531794 Germany 6 0 0 (2n = 28) PI 440306 Former USSR 6 0 0 (2n = 28) DJ-3815 Former USSR 5 1 0

Table 3 (concluded). No.ofplantswith varying abundance of pLrTaiI-1 Species Accession Origin High Low Absent L. ambiguous (2n = 28) PI 531796 Colorado 0 0 6 PI 565019 Colorado 0 0 6 KJ-47 Colorado 6 0 0 L. salmonis (2n = 28) KJ-31 Idaho 6 0 0 L. simplex (2n = 28) Dewey Wyoming 2 1 3 L. cinereus (2n = 28) ‘Trailhead’b Montana 0 0 6 (2n = 56) ‘Magnar’b Saskatchewan 0 0 6 (2n = 56) PI 516191 Oregon 2 0 2 aAtkins 102 and PI 531804 are the same. b‘Trailhead’ and ‘Magnar’ are cultivar names for PI 478831 and PI 469229, respectively. All others are accession codes; those with the prefix of PI are accessions in the USDA Germplasm Resources Information Net- work (GRIN) system (http://www.ars-grin.gov/).

Results low, and absent. Generally, plants of L. ramosus and L. racemosus having fewer than 5 FISH pLrTaiI-1 sites Fluorescence in situ hybridization would produce low amplification intensity from this STS- Variation in number of FISH sites for pLrTaiI-1 sequence PCR assay. Both P. fragilis and P. huashanica that did not was observed both within and among accessions of show FISH TaiI sites would not amplify STS-PCR products P. juncea, P. lanuginosa, L. ramosus, and L. racemosus (Ta- of TaiI family either (data not shown). Variability of the bles 1 and 2; Figs. 1 and 2). No variation was detected abundance of TaiI family repetitive sequence was observed among different cells within a plant. When 2–4 plants of an both within and among accessions of all Leymus species accession were studied, variation among different plants was (Table 3). Taking both Tables 2 and 3 into consideration, ev- observed in most accessions (Tables 1 and 2). Psathyro- ery Leymus species had at least 2 plants containing the TaiI stachys fragilis and P. huashanica did not reveal FISH sites family repetitive sequence. for pLrTaiI-1. Although pLrPstI-1 was not detectable in 4 Psathyro- stachys species (Table 1; Fig. 1), it was present in all acces- Discussion sions of L. racemosus and 1 accession each of L. secalinus The genus Psathyrostachys is a small genus with no more and L. ramosus (Table 2; Fig. 2). This repetitive sequence than 10 species (Löve 1984). Species are mainly diploid was not detected in L. mollis or in 3 out of 4 accessions (2n = 14); however, tetraploid cytotypes have been reported of L. ramosus. in P. lanuginosa from China (Linde-Laursen and Baden 1994a), P. fragilis subsp. secaliformis from Turkey (Linde- Southern hybridization Laursen and Baden 1994b), and P. juncea from Kazakhstan Southern hybridization of Hybond-N+ membrane contain- (Asay et al. 1996). The genome symbol for Psathyrostachys ing ScaI-digested genomic DNA of P. juncea, P. huas- has been changed from J (Dewey 1970) to N (Dewey 1984; hanica, L. racemosus, L. mollis, and L. secalinus revealed Löve 1984), and then to Ns (Wang et al. 1994). Karyotype patterns and intensities of hybridization bands (Fig. 3) con- differences between P. juncea, P. lanuginose, P. fragilis, and sistent with the FISH results (Tables 1 and 2; Figs. 1 and 2). P. huashanica were reported by Hsiao et al. (1986). The type Psathyrostachys huashanica did not show hybridization sig- species of Psathyrostachys is P. lanuginosa, which has nals, whereas L. mollis, L. secalinus, P. juncea, and pLrTaiI-1 sites ranging in number from 2 to 21 (Table 1). L. racemosus gave hybridization signals with increasing in- Psathyrostachys juncea exhibited between 7 and 16 pLrTaiI-1 tensities. sites (Table 1). All Leymus species studied have plants show- ing at least 2 pLrTaiI-1 sites (Tables 2 and 3), and P. fragilis STS-PCR and P. huashanica lack the pLrTaiI-1 sequence (Table 1). STS-PCR using the primer pair designed for amplification Therefore, P. fragilis and P. huashanica are unlikely to be of the TaiI family sequences produced fragments of 550 and the donor species of the Ns genome in the Leymus species. 1100 bp and longer (Fig. 4). Reproducibility of this STS- Based on the presence and frequency of pLrTaiI-1 sites, PCR was confirmed in 2 replicate assays. The smear appear- P. lanuginosa and P. juncea are likely candidates as possible ance of amplification products is due to variable lengths of ancestral parents of Leymus species. TaiI family sequences evidenced by the varying sizes of The variation in abundance of TaiI family sequence in FISH signals in different Leymus chromosomes within the Psathyrostachys species may be related to the geographic cells (Fig. 2). The same cause also resulted in 2 amplified ranges of these species. Psathyrosatchys fragilis is distrib- fragments of approximately 550 bp and 600 bp in some spe- uted in northern and southern parts of Iran; P. huashanica cies (Fig. 4b). Overall amplification intensities, including the has a restricted distribution in the Shanxi province of China; smear, could be arbitrarily grouped into 3 classes — high, P. juncea is widely distributed in the former USSR (Euro-

Fig. 1. Fluorescence in situ hybridization (FISH) sites of the pLrTaiI-1 (red) and pLrPstI-1 (green) repetitive sequences in somatic chromosomes of Psathyrostachys juncea accessions PI 499675 (a), PI 440627 (b), and PI 476299 (c); P. lanuginosa accessions D-3376 (d), and D-2562 (e); and P. fragilis accession PI 343190 (f). Arrows indicate centromeric sites. Bars represent 10 µm.

Fig. 2. Fluorescence in situ hybridization (FISH) sites of the pLrTaiI-1 (red) and pLrPstI-1 (green) repetitive sequences in somatic chromosomes of Leymus racemosus accessions PI 313965 (a) and PI 531811 (b); L. mollis accession MK 10011 (c); L. secalinus accession PI 499524 (d); L. ramosus accessions PI 440331 (e) and PI 499653 (f). Arrows indicate centromeric sites. Bars represent 10 µm.

Fig. 3. Southern hybridization of pLrTaiI-1 probe to ScaI- situ telomeric locations of chromosomes in both C. bogdanii digested genomic DNA of Psathyrostachys juncea (lane 1), and Pseudoroegneria spicata (Pursh) Á. Löve (Appels et al. P. huashanica (2), Leymus racemosus (3), L. mollis (4), 1989). However, the pLrPstI probe of L. racemosus does not and L. secalinus (5). hybridize to the terminal heterochromatic sequences of Psathyrostachys huashanica chromosomes, indicating its species-specific nature. Species-specific sequences could appear by independent amplification events during the evolution of the species (Appels et al. 1989). In contrast, the DNA sequences in the TaiI family are less variable. All the repeats in this family occurring in Psathyrostachys, Leymus, and Triticum could be detected by a single probe cloned from L. racemosus. Evidence for the involvement of Psathyrostachys juncea in the speciation of Leymus species was obtained in meiotic studies of hybrids of P. juncea with Leymus species, including L. cinereus (Scribner & Merr.) Á. Löve, L. triticoides (Buckl.) Pilger, L. innovatus (Beal) Pilger, L. salinus (M.E. Jones) Á Löve, L. multicaulis (Kar. & Kir.) Tzve- lev, L. racemosus, L. secalinus (Dewey 1970, 1972, 1976) and L. mollis (Wang and Hsiao 1984). It was also substanti- ated in molecular studies of Leymus species (Jensen and Wang 1997; Svitashev et al. 1998; Hole et al. 1999). The genome symbol for Leymus species was originally designated JN (Dewey 1984; Löve 1984), with the J coming from a Thinopyrum species and the N from Psathyrostachys.How- ever, the absence of the J genome in Leymus was shown both in meiotic pairing patterns of the hybrids Thinopyrum elongatum (Host) D. Dewey × L. mollis (Petrova 1970) and Th. elongatum × L. salinus subsp. salmonis (C. L. Hitch- cock) R. Atkins (Wang and Jensen 1994), and in molecular hybridization (Wang and Jensen 1994). Therefore, the genome symbol for Leymus species was changed to NsXm (Wang et al. 1994), where Xm stands for the genome of an unknown source. However, Zhang and DvoÍák (1991) and Anamthawat-Jónsson and Bödvarsdóttir (2001) advocated pean part, eastern and western Siberia, central Asia), Mon- that tetraploid Leymus species are segmental autotetraploids golia, and the northwestern part of China (the Xinjiang and with the Ns1Ns1Ns2Ns2 genome formula. A test for the for- Gansu provinces). Variation within an accession of a mer hypothesis would be facilitated by a useful STS-PCR Psathyrostachys species could be attributed to heterogeneity protocol for the pLrPstI-1 sequence. Although we tried to owing to obligate cross pollination (Jensen et al. 1990). develop a workable STS-PCR for assaying pLrPstI-1, the de- Kishii et al. (1999) observed all pLrTaiI-1 and pLrPstI-1 signed primers 5′-CTGCAGAAATATGCGCCTTG-3′ and 5′- sites exclusively in subtelomeric heterochromatin regions. CAGACTATACGGATGAGTTGGC-3′ amplified non-target This sequence is located also in the centromeres of wheat fragments from template DNA of species that did not have (Kishii and Tsujimoto 2002). In this study, a pair of the FISH pLrPstI-1 sites. Different primers must be designed centromeric pLrTaiI-1 sites were observed in P. juncea ac- and tested. A working protocol would allow easy identifica- cession PI 476299 (Fig. 1c) and in L. lanuginosa accession tion of species that may be candidate ancestral sources of D-3376 (Fig. 1d), but a single site was observed the Xm genome. The latter hypothesis requires carrying out in L. racemosus accession PI 531811 (Fig. 2b). The occur- the FISH study of other Psathyrostachys species that have rence of an odd number of pLrTaiI-1 and pLrPstI-1 sites in not been included in this study for the presence of pLrPstI-1 Psathyrostachys and Leymus plants (Tables 1 and 2) indi- sequence to determine the source of the second Ns genome cates that these FISH features could not be used as land- in tetraploid Leymus species. marks for identifying homologous chromosomes. This was The fact that these 2 repetitive sequences could vary from confirmed earlier in a study by Kishii et al. (2003) that total absence to a high number of sites in the genome of showed the pairing of homologous chromosomes having dif- Leymus species suggests that they are neither chromosome ferent subtelomeric repeat sequences. nor genome specific. Variation in AFLP profiles between old The 350 bp family to which pLrPstI-1 belongs is com- and new populations of an Alaskan Leymus mollis accession monly present in Triticeae species including Secale in Iceland was attributed to genotype changes resulting from cereale L., Elymus trachycaulus (Link) Gould ex Shinners, changes in environment or ecosystem and migration and col- Agropyron cristatum (L.) J. Gaertner, P. huashanica, and onization of the species in a new environment, i.e., from others. The 350 bp family sequence probe pCb4.14 from Alaska to southern Iceland (Anamthawat-Jónsson et al. Critesion bogdanii (Wilensky) Á. Löve could hybridize in 1999). Similarly, variation in the abundance of repetitive se-

Fig. 4. Agarose gel images of ethidium bromide stained PCR products from genomic DNA of Leymus species using the primer pair for the pLrTaiI-1 sequence. The STS-PCR products are multiples of 550 bp (arrowed for the 550 and 1100 bp fragments). (a) Leymus ramosus accessions DJ-4257 (lane 1), PI 499654 (2), PI 499653 (3), and PI 440331 (4); L. racemosus accessions PI 502402 (5), PI 531811 (6), PI 478832 (7), PI 108491 (8), PI 531812 (9), D-3621 (10), D-2949 (11), PI 565037 (12), and PI 313965 (13); L. mollis accessions D-4086 (lanes 14– 20), and DJ-4100 (21); L. secalinus accessions PI 499658 (lanes 22 – 28), and PI 499528 (lanes 29–35). For lanes 1–13, each template DNA sample was a bulked DNA from 6 or 7 plants in each accession. Single plant DNA was used as template for accessions of L. mollis and L. secalinus (lanes 14–35). (b) Leymus arenarius accessions PI 294582 (lanes 1–6), PI 531799 (lanes 7–12), and D-3567 (lanes 13–18); L. chinensis accessions PI 499514 (lanes 19–24), PI 499516 (lanes 25–30), and PI 499518 (lanes 31–36). Each accession was assayed with 6 plants using single plant DNA samples as templates. M indicates the bands for (top to bottom) 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp size markers.

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