Sequence of 18S rDNA of actinorhizal Alnus glutinosa (Betulaceae)

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  • Plant Molecular Biology 16: 725-728, 1991. 1991 Kluwer Academic Publishers. Printed in Belgium.

    Plant Molecular Biology Update

    Sequence of 18S rDNA of actinorhizal Alnus glutinosa (Betulaceae)

    725

    L. Savard and M. Lalonde Centre de Recherche en Biologie Forestibre, D@artement des Sciences Forestidres, Facultd de Foresterie et de G~omatique, UniversitO Laval, Qudbec, Canada GIK 7P4

    Received 16 November 1990; accepted 20 November 1990

    Key words: alder, angiosperm, PCR amplification, SSU, sequence comparison, nitrogen fixation, Frankia

    Abstract

    The small subunit ribosomal DNA for a woody actinorhizal, Alnus glutinosa, was isolated by the PCR method. Amplification products were cloned into the Bluescript SK - vector. Full sequence, 1698 bp, was obtained with NS 1 to NS8 primers. Sequence alignments were made by UWGCG sequence data analysis computer programs. 18S rDNA sequence ofA. glutinosa was compared to analogous segments of four other angiosperms, tomato, rice, maize and soybean. Sequence homologies are discussed and application for the technique is suggested.

    The ribosomal RNA gene (rDNA) occurs, in eukaryotic nuclear genomes, as multiple copies of tandemly repeated units. Each transcription unit consists of a 18S, 5.8S and 28S rRNA gene sepa- rated by non-coding intergenic spacers (IGS) [12]. rRNA coding sequences have been highly conserved during evolution, indicating a strong positive selection pressure [11]. Thus the sequence encoding the small subunit (18S) of ribosomal RNA has been used extensively in phylogenetic studies, particularly for the Protista [ 11 ]. At present, little information exists on 18S rDNA sequences of plants. Sequences have been established for four angioperms, namely maize [10], rice [13], tomato [6] and soybean [5] and a gymnosperm, the cyclad Zamia pumila [ 11 ].

    We report here the first sequence of the 18S rDNA of a perennial angiosperm, Alnus glutinosa, which forms nitrogen-fixing actinorhizae when

    symbiotically associated with the actinomycete Frankia alni [8]. The 18S rDNA was amplified by PCR (polymerase chain reaction) using two primers, SS38 and SS1492, as described by Bousquet et al. [ 1 ]. High molecular weight DNA was isolated, by the CTAB method [2], from leaf material from in vitro propagated 3-year-old trees (clone Ag8) [14]. DNA concentration was measured using a fluorometric assay [3] and adjusted to approximately 250 ng/ml in sterile dis- tilled water. Fifty /~1 of the DNA sample was added to 50/zl of 20mM Tris-HC1 pH 8.4, 100mM KCI, 3mM MgC12, 0.02~o gelatin, 100 mM of each dNTP, 0.625 units of Taq poly- merase (Cetus Corporation, USA) and 1.25 pmol of each primer. The PCR mixture was covered with 50 #1 of paraffin oil (Sigma, USA). Amplifi- cation was performed in the thermal cycler (Perkin-Elmer, USA) for 35 cycles [1, 7]. The

    The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X54984.

  • 726 1 Ag8 ACTGTGAAAC TGCGAATGGC TCATTAAATC AGTTATAGTT TGTTTGATGG TATCTGCTAC TCGGATAACC GTAGTAATTC TAGAGCTAAT ACGTGCAACA

    Tom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ag8 AACCCCGACT TCTGGAAGGG ACGCATTTAT TAGATAAAAG GTCGACGACG GCTCTGCGT- TTGCTCTGAT GATTCATGAT AACTCGACGG ATCGCACGGC

    Tom . . . . . . . . . . . . . . . . . . . . . T . . . . A . . . . . . . . . . . . . . . . . . . . CG . . . . . . . . - - - - . . . .GC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . A . .ACA . . . . . . . . C .G . . . . . T . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . .

    Ric . . . . . . . . . . . . C . .G . . . . G . . . . . . . . . . . . . . . . . . . . CT . . . . CG . . . . . . . . CCG C . .A . .C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ma i . . . . . . . . . . . . C . .G . . . . G . . . . . . . . . . . . . . . . . . . . CT . . . . CG . . . . . . . . CCG CC.A . .C . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . .

    Ag8 CATCGTGCCG GCGACGCATC ATTCAAATTT CTGCCCTATC AACTTTCGAT GGTAGGATAG AGGCCTACTA TGGTGGTGAC GGGTGACGGA GAATTAGGGT

    Tom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy .T .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TG . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic .C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma l .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ag8 TCGATTCCGG AGAGGGAGCC TGAGAAACGG CTACCACATC CAAGGAAGGC AGCAGGCGCG CAAATTACCC AATCCTGACA CGGGGAGGTA GTGACAATAA

    Tom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ag8 ATAACAATAC CGGGCTCTTA TGAGTCTGGT AATTGGAATG AGTACAATCT AAATCCCTTA ACGA-GGATC CATTGGAGGG CAAGTCTGGT GCCAGCAGCC

    Tom . . . . . . . . . . . . . . . . C - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . . . . . . . . . . . CA . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R lc . . . . . . . . . . . . . . . GC. .T A .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . . . . . . . . . . . . G .G .T A .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    501 Ag8 GCGGTAATTC CAGCTCCAAT AGCGTATATT TAAGTTGTTG CAGTTAAAAA GCTCGTAGTT GGATCTTGGG TTGGGCAGAT C-GGTCCGCC CCTGGTGTG-

    Tom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CT . . . . . A . . . . . C .GC . . . . . . . . . . . TA . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . TC . . . . . . . . . . . . . T .C . . . . . . .

    R ic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . CGC. . .C .GG .C . . . . . . . . T .AC.GCA.G

    Ma i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . CC . . . . C .GG TGCCG . . . . . GTAC.G.CAG

    Ag8 CACCGG- -G- CTCGTCCCTT CTACCGGCGA TACGCTCCTG GTCTTAATTG GCCGGGT-CG TGCCTCCGGT GCTGTTACTT TGAAGAAATT AGAGTGCTCA

    Tom . . . . . . TC .T . . . . . . . . . . . . GT . . . . . . . G . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . TC .G . . . . . . . . . . . . G . . . . . . . . G . . . . . . . . TC . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic . . . . . ACCTG . . . . A . . . . . . . G . . . . . . . . G . . . . . . . . . C . . . . . C . . . . . . . . . T . . . . . . . . . . . C . .C . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i A . . . . ACC.G . . . . A . . . . . . . G . . . . . . . . G . . . . . . . . . C . . . . . C . . . . . . . . . . . . . . . . . . . . . - . .C . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ag8 AAGCAAGCCT ACGCTCTGTA TACATTAGCA TGGGATAACA TCATAGGATT TCGGTCCTAT TCTCTTGCCC TTCGGGATCG GAGTAATGAT TAACAGGAAC

    Tom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . ACG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . .C . . . . . . CT .A . . . . . . . G .G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . .

    R ic . . . . . . . . . A T . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . G .G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . .G . .

    Ma i . . . . . . . . . A T . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . G .G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . .G . .

    Ag8 AGTCGGGGGC ATTCGTATTT CATACTGAGA GGTGAAATTC TTGGATTTAT GAAAGACGAA CAACTGCTGA AAGCATTTGC CAAGGAPGTT TTCATTAATC

    Tom . . . . . . . . . . . . . . . . . . . . . . . . G .C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . . G .C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic . . . . . . . . . . . . . . . . . . . . . . . . G .C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . . . . . . . . . . . . . . . . . . . . . G .C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ag8 AAGAACGAAA GTTGGGGGCT CGAAGACGAT CAGATACCGT CCTAGTCTCA ACCATAAACG ATGCCGACCA GGGATCGGCG GATGTTGATT TCAGGACTCC

    Tom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . .T . . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . C . . .T . . . . . . . .

    R ic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . AT . . . . . . . .

    Ma l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . - . . . . . C .A AT . . . . . . . .

    i001 Ag8 GCCGGC.ACC TTATGAGAAA TCAAAGTTCT TTGGGTTCCG GGGGGAGTAT GGTCGCAAGG CTGAAACTTA AAAGAATTGA CGGAAGGGCA CCACCAGGAG

    Tom . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . .T . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ric . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . C . Ma i . .T . . .C . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . C .

    Ag8 TGGACG-TGC GGCTTAATTT GACTCAACAC GGGGAAACTT ACCAGGTCCA GACATAGTAA GGATTGACAG ACTGAGAGCT CTTTCTTGAT TCTATGGGTG

    Tom . . . . GCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . GCC . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic . . .GGCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . GCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ag8 GTGGTGCATG GCCGTTCTTA GTTGGTGGAG CGATTTGTCT GGTTAATTCC GTTAACGAAC GAGACCTCAG CCTGTTAACT AGCTAT-CGG AGGTGACCCT

    Tom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . G . . . . . . . AT . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . C . . .A . . . . . . . GT . . . . . . A . . . . .

    R ic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . G . . . . . . . AT . . . .

    Ma i . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . G . . . . . . . AT . . . .

    A~8 CCGCGGCCAG CTTCTTAGAG GGACTA-TGG CCGCTTAGCA - -CGG-AGTT TGAGGCAATA ACAGG.CTGT GATTGCCCTA GATGTTCTGG GCG-CACTCG

    Tom T . . . . . . . . . . . . . . . . . . . . . . . . . GCCT T - - - . . . .GC CG. . .A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . G . .

    Soy . .AC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GC CA. . .A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CG. . .G . .

    R ic . . . . A . .T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GC CA. . .A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . .

    Ma i . . .TA .TT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GC CG. .A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CG. . .G . .

    Ag8 CGCTACACTG ATGTATTCAA CGAGTTTATA GCCTTGGCCG ACAGGCCCGG GTAATCTTTG -AAATTTCAT -CGTGATGGG GATAGATCAT TGCAATTGTT

    Tom . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic . . . . . . . . . . . . . . . . C . . . . . . . . A . . . . . . . . G .T . . . . . . . . . . . . . . . . . . . . . G . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . . . . . . . . . . . . . C . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . G . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    1501

    Ag8 GGTCTTAAAC GAGGAATTCC TAGTAAGCGC GAGTCATCAG CTCGCGTTGA CTACGTCCCT GCCCTTTGTA CACACCGCCC GTCGCTCCTA CCGATTGAAT

    Tom . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ag8 GGTCCGGTGA AGTGTTCGGA - -TCGCGGCG ACGTGGGCGG TTCGCTGCCG GCGAC-GTC- GC-AGAAAGT CCACTGAACC TTATCATTTA GAGGAAGGAG

    Tom .A . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . GAG . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . A . . . . . . . . . . . . . C G . . . . . . . T . .T .GAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    R ic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G .G . . . . . . . . . C . . .C C . . . . . . . . G - . .GAG . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . . . . . . . . . . . . . . . . . GC. . .GCCGC . .C - . . - - - - . . . . . C . . .C C . . . . C . . .G . .CGAG . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . .

    Ag8 AAGTCGTAAC AAGGT-TCGCG 1720

    Tom . . . . . . . . . . . . . . . . . . . . .

    Soy . . . . . . . . . . . . . . . . . . . . .

    R ic . . . . . . . . . . . . . . . . . . . . .

    Ma i . . . . . . . . . . . . . . . . . . . . .

    Fig. 1. Sequence of 18S rRNA gene ofAlnus glutinosa (Ag8) aligned with that of tomato (Tom), soybean (Soy), rice (Ric) and maize (Mai). Variable regions are underlined.

  • first ten cycles included a denaturation step at 93 C for 1 min, an annealing step at 50 C for 1 min and an extension step at 72 C for .1.5 min. For each of the last 25 cycles, the extension step was increased by 10 s every cycle. The last cycle was followed by a 10 min incubation at 72 C. Amplification products were resolved on a 1 ~o agarose/2)'o Nusieve (Mandel Scientific)gel and visualized by ethidium bromide fluorescence. Protruding 3' tails on the PCR-amplified product was removed by T4 DNA polymerase treatment before cloning into the Bluescript SK- vector (Stratagene, USA). Approximately 200ng of amplified DNA was diluted in 38 #1 of sterile water with 10 #1 of enzyme buffer (250 mM Tris- HC1 pH 8.8, 25mM MgCI2, 25mM DTT, 25 mg/ml BSA) and 5U of T4 DNA polymerase (BRL). The mixture was incubed 2 min at 11 C. One microlitre of a 5 mM mixture of each dNTP was added and the incubation was continued for 20 min at 11 C. DNA was then purified on an agarose gel. One microgram of Bluescript vector SK + was digested with Eco RV for 2 hours at 37 C and then ligated with 18S rDNA as described in Maniatis etal. [9] for 6 hours at 22 C. Before transformation into XL 1 blue com- petent cells (Stratagene, USA), DNA was digested with Eco RV to reduce background. The nucleotide sequence of the CsCl-purified super- coiled DNA was determined by the dideoxy chain termination method according to a double- stranded DNA protocol (Sequenase kit). The nucleotide mixture and the T7 polymerase enzyme of the Sequenase kit were used for sequencing. Two clones were sequenced using primers NS 1 to NS8 as described by Innis et al. [7]. The sequence of 1698 bp ofAlnus glutinosa 18S rDNA unit is shown in Fig. 1. The sequence was compared with the other plant species sequences by the computer programs Bestfit and GAP of the UWGCG sequence data analysis package [4].

    Nucleotide sequence alignment revealed a con- siderable homology between alder and tomato (96~o), rice (94~o), soybean (96~o) and maize (94 ~o). Though A. glutinosa showed more sequence homology with dicots (tomato and soybean) than with monocots (rice and maize).

    727

    Thus, the 18S rDNA of Alnus has a sequence which is very similar to sequences of other angio- sperms. The sequence alignment, also, allowed to corroborate a previous report by Shimdt-Puchta et al. [12] who proposed four variable regions in the 18S rDNA. These variable regions are under- lined in Fig. 1 (shown at position 140 bp, 570 bp, 1400 bp and 1660 bp).

    We have demonstrated that the polymerase chain reaction can rapidly obtain DNA sequences for woody plants. Presence of phenols and tan- nins in the leaves made DNA extraction [ 10] and DNA digestion difficult, but did not interfere dur- ing PCR amplification of the DNA. The same protocol can be used with DNA extracted from as little as 1 mg of leaf, seed, embryo, cell sus- pension, or rootlet [1]. This technique is now applicable for obtaining DNA sequences of SSU (small ribosomal subunit) from other species of Alnus and Betula (members of the Betulaceae family). A comparison between these sequences will be made to obtain specific regions that can be used as molecular probes to identify, in field, some species of alder.

    Acknowledgements

    We would like to thank Dr Louis Bemier (Univer- sit6 Laval) for very stimulating discussions, and Dr. Thomas Bruns (Berkeley, USA) for providing primers NS1 to NS8. This work was supported by Natural Sciences and Engineering Research Council of Canada operating grant (No A-2920) to M.L. and La Fondation de l'Universit6 Laval scholarship to L.S.

    References

    1. Bousquet J, Simon L, Lalonde M: DNA amplification from vegetative and sexual tissues of trees using poly- merase chain reaction. Can J For 20:254-257 (1990).

    2. Bousquet J, Girouard E, Strobeck C, Dancik BP, Lalonde M: Restriction fragment polymorphisms in the rDNA region among seven species of Alnus and Betula papyrifera. Plant Soil 118:231-240 (1989).

    3. Cesarone CF, Bolognesi C, Santi L. Improved micro-

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    fluorometric DNA determination in biological material using 33258Hoechst. Anal Biochem 100:188-197 (1979).

    4. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12:387-395 (1984).

    5. Eckenrode VK, Arnold J, Meagher RB: Comparison of the nucleotide sequence of soybean 18S rRNA with the sequence of other small subunit rRNAs. J Mol Evol 21: 259-269 (1985).

    6. Kiss T, Szkukalek A, Solymosy F: Nucleotide sequence of 17S (18S) rRNA gene from tomato: Nucl Acids Re 17: 2127 (1989).

    7. Innis MA, Gelfang DH, Sninsky JJ, White TJ: PCR protocols, a guide to methods and application: Academic Press, San Diego (1990).

    8. Lalonde M, Simon L, Bousquet J, Sdguin A: Advances in the taxonomy of Frankia: Recognition of species alni and elaeagni and novel subspecies pommerii and vanddijkii. In: Bothe, de Bruijn, Newton (eds) Nitrogen Fixation: Hundred Years After. Gustav Fisher, New York (1988).

    9. Maniatis TE, Fritsch EF, Sambrook J: Molecular Clon- ing: A Laboratory Manual. Cold Spring Harbor Labora- tory, Cold Spring Harbor, NY (1982).

    10. Messing J, Carlson J, Hagen G, Rubenstein I, Oleson A: Cloning and sequencing of the ribosomal RNA genes in maize: The 17S region: DNA 3:31-40 (1984).

    11. Nairn CJ, Ferl RJ: The complete nucleotide sequence of the small-subunit ribosomal RNA coding region for the cycad Zamia pumila: phylogenetic implications: J Mol Evol 27:133-141 (1988).

    12. Schmidt-Puchta W, Kiitemeier G, G0nther I, Haas B, Slinger HL: Cloning and sequence analysis of the 18S ribosomal RNA gene of tomato and secondary structure model for the 18 S rRNA of angiosperms. Mol Gen Genet 219:17-25 (1989).

    13. Takaiwa F, Oono K, Sugiura M: The complete nucle- otide sequence of a rice 17S rRNA gene. Nucl Acids Res 12:5441-5448 (1984).

    14. Tremblay F, Lalonde M: Requirements for in vitro propa- gation of seven nitrogen-fixing Alnus species: Plant Cell Tissue Organ Culture 189-199 (1984).

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