Genes Genet. Syst. (1996) 71, p. 373–382 Identification and characterization of two tandem repeat sequences (TrsB and TrsC) and a retrotransposon (RIRE1) as genome-general sequences in rice Reiko Nakajima, Kenichi Noma, Hisako Ohtsubo and Eiichi Ohtsubo* Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan (Received 17 August 1996, accepted 27 December 1996) Three kinds of DNA sequences (here called TrsB, TrsC and RIRE1) have been previously reported to be those repeated in tandem specifically in the wild rice species with FF, CC or EE genome, respectively. To characterize these genome type-specific sequences, we carried out PCR using a pair of primers, which hybridize to a restricted region in the repeating unit sequence and prime DNA synthesis in both directions. Gel electrophoresis and DNA sequencing revealed that PCR using primers for TrsB (or TrsC) amplified the fragments with an integral series of a unit length not only from total DNA of the rice strain with FF (or CC) genome, but also from those of the rice strains with non-FF (or non-CC) genome. TrsB or TrsC was, however, found to be repeated in an extraordinary number of copies in the species with FF or CC genome, respectively, in which the TrsB (or TrsC) sequence has been originally identified. PCR using primers for RIRE1 produced various sizes of fragments from total DNA of the rice strains with EE genome. The fragments, however, showed no progression at interval of the unit length characteristic for tandem repeats. Nucleotide sequencing of the amplified fragments revealed that they were not the sequences repeated in tandem, but were those interspersed as an element having partial homology with the LTR sequences of retrotransposons, Wis- 2-1A in wheat and BARE-1 in barley. RIRE1 was present in the rice species with any types of genomes, but in the species with EE genome in an extraordinary number of copies. In rice species belonging to the genus Oryza, which have INTRODUCTION been classified into six diploid genome types (AA, BB, CC, Genomes of higher plants contain a large fraction of DD, EE and FF) and two tetraploid genome types (BBCC DNA sequences repeated in tandem or interspersed. and CCDD) (Chang, 1984), there exist genome-specific Besides the ribosomal RNA genes and microsatellite DNA, tandem repeats as well as genome-general tandem repeats various kinds of tandem repeat sequences have been found (Zhao et al., 1989; Wu and Wu, 1992). These tandem re- in Secale cereale (Bedbrook et al., 1980; Appels et al., peat sequences have been identified by partially digesting 1986), Arabidopsis thaliana (Martinez-Zapater et al., total genomic DNA with a restriction enzyme and carrying 1986), Avena sativa (Fabijanski et al., 1990), Oryza sativa out Southern hybridization using each sequence as a (Wu and Wu, 1987; Ohtsubo et al., 1991; De Kochko et al., probe, which demonstrates a unique ladder band pattern 1991), and Actinidia deliciosa (Crowhurst and Gardner, characteristic for the tandem repeat sequences. In this 1991). Interspersed sequences identified are various study, we employed a method based on polymerase chain kinds of elements, such as transposable DNA elements reaction (PCR) used to identify and characterize a tandem (Nevers et al., 1986; Peterson, 1987; Coen et al., 1989; repeat sequence in rice, named TrsA, which is repeated Fedoroff, 1989; Gierl and Saedler, 1992), retrotransposons even in a few copies (Ohtsubo and Ohtsubo, 1994). Using (Flavell et al., 1992; Moore et al., 1991; Voytas et al., 1992; this method, we demonstrated that the three kinds of ge- Hirochika and Hirochika, 1993) and retroposons nome-specific tandem repeat sequences previously found (Shepherd at al., 1984; Mochizuki et al., 1992; Yoshioka in rice, pOb1, pOo2 and pOa4 (Zhao et al., 1989) (here et al., 1993; Deragon et al., 1994). called TrsB, TrsC and RIRE1, respectively), were actually genome-general sequences, and that one of them is not a * Corresponding author. tandem repeat sequence, but a retrotransposon inter- 374 R. NAKAJIMA et al. spersed in rice genomes. [0.1% Ficoll (Pharmacia), 0.1% polyvinylpyrrolidon (Nakarai), 0.1% (w/v) bovine serum albumin (Seikagaku Kogyo)], 0.5% (w/v) sodium dodecyl sulfate (SDS), and 100 MATERIALS AND METHODS µg of sonicated salmon sperm DNA (Sigma)/ml. Then the Rice strains. Rice strains used are listed in Table 1. solution was exchanged for the hybridization solution Total genomic DNA of most of wild rice strains, which which contained the 32P-labeled oligonucleotide or cloned were isolated as described previously (Lichtenstein and DNA at 105 cpm/ml, and hybridization was carried out for Draper, 1985), was obtained from Y. Fukuta (Hokuriku 12 to 15 h at 65°C. The filter was washed sequentially in National Agricultural Experiment Station). Total 2 × SSC, 1 × SSC and 0.1 × SSC, each containing 0.1% SDS, genomic DNA of several wild rice strains was obtained at the temperature calculated from each of the probe se- from H. Hirochika (National Institute of Agrobiological quences in Table 2 as described by Meikoth and Wahl Resources), and Y. Sano (National Institute of Genetics). (1984). Cloning and DNA sequencing of the PCR-amplified Slot-blot hybridization. The copy number of a se- fragments. PCR was carried out by the standard quence was estimated by slot-blot hybridization described method (Saiki et al., 1988) using 2.5 units of AmpliTaq previously (Tenzen et al., 1994) using the DNA of plasmid DNA polymerase (Perkin Elmer), 0.5 µg total rice DNA, pRN43 carrying the TrsB sequence, pRN59 carrying the and 1 µM of each pair of primers (Table 2). Thirty cycles TrsC sequence, or pRN28 carrying a portion of the RIRE1 of amplification were carried out under the following con- sequence. For the copy number calculation, the sizes of dition; denaturation for 1 min at 93°C, annealing for 1 min the haploid genome of O. sativa and O. brachyantha were at 55°C, and DNA synthesis for 2 min at 72°C. The PCR- taken as 430 Mb, and the sizes of the haploid genome of O. amplified fragments were ligated to a linear pCRII vector officinalis and O. australiensis were taken as 550 and 960 in a TA cloning kit (Invitrogen) as recommended by the Mb, respectively (Martinez et al., 1994). supplier. DNA sequencing was carried out by the dideoxynucleotide chain termination method (Messing, Accession numbers. The nucleotide sequence data re- 1983; Sanger et al., 1977) using Sequenase Ver. 2.0 ported appear in the DDBJ, EMBL and GenBank nucle- (United States Biochemicals). The frequency of muta- otide sequence databases under the accession numbers, tions induced by PCR under the condition described above D85598~85604. is 1 per 642 bp (Tenzen et al., 1994). RESULTS Southern hybridization. The PCR-amplified frag- ments were electrophoresed in a 1.8% agarose gel and Amplification of the repeated sequences in rice by stained with ethidium bromide. DNA was transferred to PCR. Three kinds of DNA sequences, pOb1 (159 bp) in a nylon membrane (Hybond-N+; Amersham) by alkaline O. brachyantha with FF genome, pOo2 (366 bp) in O. blotting under the condition recommended by the supplier. officinalis with CC genome and pOa4 (511 bp) in O. The filter was preincubated at 65°C for 1 h in 5 ml/100 cm2 australiensis with EE genome, have been reported as ge- of the hybridization solution containing 6 × SSC (0.9 M nome-specific tandem repeats (Zhao et al., 1989). To NaCl, 0.09 M trisodium citrate), 5 × Denhardt’s solution identify and characterize these sequences, we carried out Table 1. Rice strains used Strain Species Genome type Source or reference Nipponbare O. sativa AA Mochizuki et al. (1993) T65 O. sativa AA Mochizuki et al. (1993) W1581 O. barthii AA Y. Fukuta W1192 O. glumaepatula AA Y. Fukuta W1625 O. meridionalis AA Y. Fukuta W1577 O. punctata BB Y. Sano W1582 O. punctata BB Y. Fukuta W0002 O. officinalis CC Y. Fukuta W1521 O. eichingeri CC Y. Fukuta W1538 O. australiensis EE Y. Fukuta W0008 O. australiensis EE Y. Sano W1401 O. brachyantha FF Y. Fukuta WM5 O. alta CCDD H. Hirochika W0046 O. latifolia CCDD H. Hirochika Tandem repeat sequences and retrotransposon in rice 375 Table 2. Oligonucleotides used Primer Sequenceb Origin Positionc or probea Ob1f 5'-gagctcTGACCTTGGATTCATTCCAT-3' pOb1 (=TrsB) 1- 20 Ob1b 5'-gagctcCCCTTCCAGCCCGAGAGTTT-3' pOb1 (=TrsB) 158-149 Oo2f 5'-ggatccGGATATGCGATGCGTTTTAG-3' pOo2 (=TrsC) 1- 20 Oo2b 5'-ggatccAAAGGAATGTGCCAACCATG-3' pOo2 (=TrsC) 366-357 Oa4f 5'-ggatccATCCAACAATGTGTTCTCTA-3' pOa4 (=RIRE1) 316-297 Oa4b 5'-ggatccAGACCCACCATGAGACACTA-3' pOa4 (=RIRE1) 317-337 F1 5'-TCCGTCATCAACTTCACC-3' TrsB 16- 33 F1' 5'-GAGCACGAAGTGTAAACC-3' TrsB 119-102 F2 5'-AGATTCGACTATTAACTTC-3' TrsB 53- 71 C1 5'-GGGCAATGTCCTTAAAGT-3' TrsC 20- 37 C1' 5'-AACAAGGTGAGGAATGTG-3' TrsC 353-336 C2 5'-GAACGACATTGGACGGGCTA-3' TrsC 143-162 E1 5'-ATCCAACAATGTGTTCTCT-3' RIRE1 316-298 E5' 5'-TGTGCCATTAGTTGGTCTCA-3' RIRE1 347-366 E6 5'-ATCCCTTTGCCTCACGAT-3' RIRE1 725-709 E6' 5'-TGTTAGTGATATGCCCT-3' RIRE1 1- 16 E4 5'-TCTCCATGTTGCCTCTAGGG-3' RIRE1 33- 14 E4' 5'-CGAGTGAATGACTAAGCT-3' RIRE1 650-667 a A pair of primers Ob1f/Ob1b were used to amplify the fragments containing the pOb1 (= TrsB) sequence. Primers Oo2f/Oo2b and Oa4f/Oa4b were used to amplify the pOo2 (= TrsC) and pOa4 (= RIRE1) sequences, respectively. Primers F1/F1' and C1/C1' were de- signed on the consensus sequences of TrsB and TrsC and used to amplify each Trs sequence from total DNA of the various rice strains. Primers E1/E6' and E5'/E6 were used to amplify the DNA fragments of the two regions, 1-316 and 349-727, within LTR of RIRE1 (see Figs.