1181 Length and sequence heterogeneity in 55 rONA of Populus deltoides Madan S. Negi, Jyothi Rajagopal, Neeti Chauhan, Richard Cronn, and Malathi Lakshmikumaran Abstract: The 5S rRNA genes and their associated non-h·anscribed spacer (NTS) regions are present as repeat units ar­ ranged in tandem arrays in plant genomes. Length heterogeneity in SS rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The P CR-amplified products from the two accessions of P. deltoides (G3 and 048) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of SS rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (1t) estimates that are less than 0.15% of those obtained for class 2 spacers (1t 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides. = Key words: 5S rDNA, Populus, repetitive DNA, microsatellite, sequence heterogeneity. Resume : Les genes d' ADNr 5S et les espaceurs non-transcrits (NTS) qui y sont associes sont presents sous forme d'unites repetees en tandem chez les genomes vegetaux. L'heterogeneite quanta Ia taille des monomeres d'ADNr 5S a deja ete rapportee chez le Populus deltoides et a egalement ete observee au cours de ce travail. Des amorces ont ete con9ues pour amplifier des variants de taille au niveau de Ia region NTS de I' ADNr 5S du P. deltoides. Les amplicons obtenusa partir de deux accessions du P. deltoides (G3 et 048) ont indique Ia presence de variabilite quanta Ia taille des espaceurs tant au sein d'une accession qu'entre ces dernieres. La taille des amplicons variait entre 385 et 434 pb. Le sequenyage des espaceurs a revele !'existence de deux classes d' ADNr 5S au sein des accessions. Les espaceurs de classe 1 contiennent un microsatellite trinucleotidique (GAA) alors que Ia classe 2 est depourvue de sequences repe­ tees. L'espaceur de classe I montre de Ia variation quanta sa taille en raison de Ia presence d'un microsatellite : deux clones montrant 10 repetitions de GAA et un autre clone en montrant 16. Cependant, une analyse de distance montre que les espaceurs de classe 1 sont tres semblables entre eux, avec un indice de diversite nucleotidique (1t) inferieur de 0,15 par rapporta celui observe entre les espaceurs de classe 2 (1t 0,0183 vs. 0,1433, respectivement). La presence d'un microsatellite au sein de l'espaceur, laquelle eritralne une variation quanta Ia taille de l'espaceur, est rapportee est discutee pour Ia premiere fois chez le P. deltoides. = Mots ctes : ADNr 5S, Populus, ADN repetitif, microsatellite, heterogeneite de sequence. [Traduit par Ia Redaction] Introduction The genus Populus (2n 38; Salicaceae) is a model sys­ tem for basic forest biology (Dickmann and Stuart 1 983; Bradshaw et al. 2000). Populus comprises six distinct taxo­ nomical sections (Eckenwalder 1 996) consisting of nearly 30 species of worldwide distribution in the northern hemi= sphere. Poplars are deciduous, dioceous trees characterized by their remarkable potential for fast growth. They are one of the most intensively studied forest tree species because of their importance as a source of fiber and biofuel and as a model tree species (Stettler et al. 1 996). The DNA content of P opulus (IC 0.55 pg; Dhillon 1 987) is one of the low­ est among tree species, making it a model system for basic = Received 21 December 2001. Accepted 5 September 2002. Published on the NRC Research Press Web site at http://genome.nrc.ca on 6 November 2002. Corresponding Editor: G.J. Scoles. M.S. Negi, N. Chauhan, and M. Lal•shmil•umaran.1 Biotechnology and Bioresources Division, TERI, Darbari Seth Block, Habitat P lace, Lodhi Road, New Delhi 110003, India. J. Rajagopal. Department of Zoology & Genetics, Iowa State University, Ames, IA 50011, U.S.A. R. Cronn. Department of Botany, Bessey Hall, Iowa State University, Ames, IA 50011, U.S.A. 'Corresponding author (e-mail: malaks@teri.res.in). Genome 45: 1181-1188 (2002) DOl: I 0.1139/G02-094 © 2002 NRC Canada Genome Vol. 45, 2002 1182 research of tree genomes. The favorable ratio between ge­ netic length and physical length in Populus chromosomes makes the genus an attractive choice for genetic mapping and cloning of genes of special importance to forest trees (Bradshaw et al. 2000). In India, Populus ciliata grows natu­ rally in the Himalayas at elevations between 1 500 and 3000 m. Exotic poplar clones (mainly of Populus deltoides) were introduced in India in the 1 950s to meet the needs of plywood and matchwood industries. Originally, cuttings were obtained from the U.K. , the U. S. A., France, Germany, and Italy. A large number of species and clones were tried, but the majority of them could not withstand the high sum­ mer temperatures. In 1 970, two P. deltoides clones, namely G-3 and G-48, were introduced from Australia. These, along with D-1 00 and D-1 21 (obtained from Stoneville, Miss.), were found to perform well in field conditions and form the bulk of the poplars planted under irrigated conditions in agroforestry in northern India. The 5S rRNA genes are present as multiple copies ar­ ranged in tandem arrays in the nuclear genome of higher plants (Ganal et al. 1 988). Each 5S rDNA repeat unit con­ sists of a 1 20-bp coding sequence and a non-transcribed spacer (NTS), which is believed to play a role in the initia­ tion and termination of transcription (Scoles et al. 1 988). Al­ though the transcribed region is conserved in plant species, the non-transcribed spacer commonly shows length hetero­ geneity (Appels et al. 1 980; Khvyrleva et al. 1 988; Singh et al. 1 994; Udovicic et al. 1 995; Prado et al. 1 996) and exten­ sive sequence divergence (Baum and Bailey 1 997; Trontin et al. 1 999; Baker et al. 2000). The variation in the NTS region has been used in a number of plant species for studying intraspecific variation (Baum and Johnson 1 994), mapping 5S rDNA arrays (Kanazin et al. 1 993), genome evolution, and phylogenetic reconstruction (Kellogg and Appels 1 995; Udovicic et al. 1 995; Cronn et al. 1 996; Baum and Bailey 1 997; Baker et al. 2000). Apart from such gymnosperms as Pinus, Picea, and Larix (Cullis et al. 1 988; Moran et al. 1 992; Brown and Carlson 1 997; Trotin et al. 1 999), the 5S rDNA of forest trees has not been studied in great detail. Species such as Angophora (Udovicic et al. 1 995), Eucalyp­ tus (Udovicic et al. 1 995), oak (Quercus) (Bellarosa et al. 1 990), poplars (Populus) (Prado et al. 1 996), and rattans (Calamus) (Baker et al. 2000) are among the few angio­ sperm forest trees being investigated for 5S rDNA. Various marker systems including morphological markers and protein- and D NA-based markers have been used for· phylogenetic inferences in Populus (Eckenwalder 1 984; Hu et al. 1 985; Rajora and Zsuffa 1 990a, 1 990b; Smith and Sytsma 1 990; Rajora and Danick 1 992, 1 995a, 1 995b, 1 995c; Barrett et al. 1 993; Rahman et aL 2000). In addition, repetitive DNA elements, including the satellite DNA and 5S rDNA unit, have also been used for studying evolutionary affinities in the genus (Faivre-Rampant et al. 1 992; Prado et al. 1 996; Dayanandan et al. 1 998; Rajagopal et al. 1 999). Earlier studies indicate that 5S ribosomal DNA units of the members of this genus range from 450 to 600 bp, with the presence of two size classes in most of the species (Prado et al. 1 996; Gotlob-McHugh et al. 1 990). In this study, we have cloned the NTS of 5S rDNA of two accessions of P deltoides, namely 03 and 048. We report here the nucleo­ tide sequences and the presence of two classes within the NTS region of P. deltoides. The two NTS classes were · identified within both the accessions (03 and 048) under in­ vestigation. This is the first study in the genus characterizing the length and sequence heterogeneity in the NTS region of 5S rDNA. The molecular characterization of these units and the possible influence of primary sequence on spacer length variation in the P deltoides genome is discussed. Materials and methods Plant material and DNA isolation Populus deltoides clones were introduced to India in 1 952 to increase the availability of wood for match and plywood industries. Two accessions of P deltoides, namely 03 and 048, were introduced from Australia and are successfully grown in the northern plains of India. These two clones were thus selected for the initial study of poplar 5S rDNA NTS organization. Plant material used for the study was obtained from the germplasm maintained at Tata Energy Research In­ stitute (TERI) field station at Gurgaon, India (Gual Pahari; 28.3°N, 77.2°E) and Dr. Y.S. Parmar University of Horticul­ ture and Forestry, Solan, India (Shilly nursery; 31 °N, 77°E). Total genomic DNA from young leaves was isolated using the cetyltrimethylammonium bromide (CTAB) method as described by Kidwell and Osborn (1 992). Restriction, gel electrophoresis, and Southem blotting Total genomic DNA was digested with eight restriction enzymes, namely Alui, BamHI, EcoRI, EcoRV, Haeiii, Hindiii, Psti, and Rsai, following the instructions of the manufacturers (Boehringer Mannheim, Basel, Switzerland). Restricted genomic DNA was fractionated by gel electro­ phoresis on 1 % agarose gels in 0.5x TBE buffer (20 mM Tris-acetate (pH 8), 1 0 mM boric acid, 0.5 M EDTA) at 2 V /cm, and blotted onto Hybond-N membranes (Amersham Pharmacia, Piscataway, N. J.) by the capillary blotting method according to the manufacturer's instructions. The transferred DNA was covalently fixed to the membrane by UV crosslinking. Southern hybridization The 5S rDNA clone pBC5S-1 of Brassica campestris cloned in our laboratory (Bhatia et al. 1 993) was used as the probe and was labeled using the random primer method (Feinberg and Vogelstein 1 983) using [a-32P]dCTP. The ra­ dioactive [a-32P]dCTP was obtained from the Board of Radi­ ation and Isotope Technology (BRIT, Hyderabad, India). Southern hybridization was carried out as described by Lakshmikumaran and Negi (1 994). In brief, The UV­ crosslinked membrane was prehybridized with 5x sse buffer (1 X sse: 0. 1 5 M NaCl, 1 5 mM sodium citrate (pH 7.5)), 5x Denhardt's solution (lx: 0.02% w/v Ficoll, 0.02% w/v polyvinylpyrrolidone, 0.02% w/v bovine serum albumin), 0.1 mM Tris (pH 8.0), 0.2% w/v SDS, 0.1 mM EDTA, and 1 00 11g/mL sheared salmon sperm DNA at 55­ 600C for 1 6 h. The labeled probe was added and overnight hybridization was carried out at 65°C. The membrane filters were then washed twice in 2x SSC, 0. 1 % SDS; once with 0.5x SSC, 0. 1 % SDS; and once with 3 mM Tris. All washings were carried out for 20 min at room temperature. © 2002 NRC Canada Negi et al. The filters were exposed to X-rays for autoradiography (Sambrook et al. I989). Isolation and cloning of SS rDNA NTS Putative 5S rDNA NTS sequences were PCR amplified from the genomic DNA of P. deltoides accessions (G-3 and G-48) using primers F (5'-GTGCTGGTATGATCGCACCC-3') and R (5'-GGGAAGTCCTCGTGTTGC-3') obtained from Operon Technologies (Valencia, Calif.). The primers corre­ spond to the conserved coding region of the 5S rDNA and were designed from 5S rDNA sequence of Brassica campestris (Bhatia et al. I993). The primers were designed in such a manner that they amplified the NTS region. The PCR thermocycling parameters were as follows: one cycle of 2 min at 94°C, I min at 50°C, and I min at 72°C; followed by 35 cycles of I min at 94°C, I min at 50°C, and 1 min at 72°C. A final extension was carried out for 7 min at 72°C. The amplified products were purified using the Wizard™ PCR purification system from Promega (Madison, Wis.). The purified amplification products were cloned into pGEM­ T vectors (Promega) and the recombinant plasmids were transformed into Escherichia coli DH5a. Positive recombinants were identified by colony PCR using universal primers (T7 and SP6) obtained from Promega. Sequencing and sequence data analysis Clones containing 5S rDNA inserts (three each from ac­ cessions G3 and G48) were selected from the resulting transformants. Recombinant plasmids were sequenced in an ABI 377 automated sequencer (PE Applied Biosystems, Foster City, Calif.) using a fluorescent dye terminator with SP6/T7 primers. Sequences for the six 5S rDNA clones were aligned using the ClustalW (Higgins et al. I994) program, with the final alignment corrected by eye at a few positions. Computations for raw pairwise (p) distances and corrected distances for multiple substitution (Jukes-Cantor distances (Jukes and Cantor I969)) were aided by the computer pro­ gram PAUP* 4.0 (Swofford 2000). A distance phenogram was constructed using Jukes-Cantor distances and the mini­ mum evolution criterion of reconstruction (PAUP* 4.0). Measurements of nucleotide diversity (n; Nei I987) were calculated using the computer program DnaSP 3.0 (Rozas and Rozas I999). For calculating all the distance and poly­ morphism parameters, the invariant 5S gene nucleotides (aligned positions I-23 and 479-498), and the regions con­ taining large alignment gaps (positions 24I-29I, 328-338, and 413-431 ), were removed. The sequences are deposited in the European Molecular Biology Laboratory (EMBL) da­ tabase under the accession numbers AJ292052-AJ292057. Results and discussions In P. deltoides, Southern hybridization studies using 5S ri­ bosomal units by Gottlob-McHugh et al. (I990) have indi­ cated the presence of two length variants corresponding to 543 bp and 634 bp. To investigate whether such length vari­ ants of 5S rDNA exist in the accessions G3 and G48 of P. deltoides, Southern hybridization studies were undertaken. Genomic DNA of P. deltoides (G3 accession) was digested with eight different restriction endonucleases and probed with a homologous 5S rDNA cloned from B. campestris 1183 Fig. 1. Populus deltoides (G3 accession) genomic DNA was di­ gested with different restriction endonucleases (as indicated) and Southern blotted. The blot was hybridized to 5S rDNA fragment obtained from Brassica campestris. kb 1.5 __.. 1.0 + 0.5 + (Bhatia et al. I993) to screen for the length heterogeneity of 5S rDNA repeats. A distinctive ladder pattern, characteristic of tandem repeats, was obtained with BamHI and Haeiii genomic DNA digests (Fig. I). The other enzymes tested produced a strong signal in the high molecular weight re­ gion, indicating that 5S repeats lacked these restriction sites. The monomeric unit of the ladder for BamHl genomic di­ gests was approximately 500 bp (Fig. I). A prominent band of similar size was obtained with Haeiii, although minor bands were identified between the major 500-bp increments. These "shadow bands" reveal heterogeneity in the Haeiii sites in P. deltoides. Similar results were obtained with the G48 accession of P. deltoides. Interestingly, the two discrete size variants reported in 5S rDNA of P. deltoides by Gotlob­ McHugh et al. (I990) were not identified in this study; only a single, broad band of -500 bp was observed in the ladder (Fig. I), suggesting the presence of size variants in acces­ sions G3 and G48 that are very close to 500 bp. To screen for variants at the sequence level, the NTS re­ gion of the 5S rDNA of six clones was cloned. Primers F and R were designed based on the conserved sequences of the 5S rRNA gene to be amplified. Primers F and R ampli­ fied the NTS region, as well as a part of the 5S rDNA se­ © 2002 NRC Canada Genome Vol. 45, 2002 1184 Fig. 2. Diagrammatic representation of the positions of primers F and R used to amplify the non-transcribed spacer (NTS) region. The boxed area represents the 5S rRNA gene (coding region), with the spacer present between adj acent coding regions. The di­ agram is not to the scale. Bamm --1 Gene .... BamHI R Spacer I I I Gene R !1!:: 1- F a R !PCR D o PCR lilication Spacer .... · product F quences (Fig. 2). Three unique SS rDNA clones from each of the two P. deltoides accessions (03 and 048) were se­ lected based on the length variation. It was expected that products of dissimilar length may represent different "classes" of the SS rDNA repeat elements. The amplification products ranged from 428 bp in clone 048-20 to 476 bp in clone 048- 1 9 (Fig. 3). We predict that the length of SS units (gene + spacer) in P. deltoides accessions 03 and 048 ranges from 505 (clone 048-20) to 553 bp (clone 048-1 9). These values are in accordance with Southern hybridization data (Fig. 1 ). The SS gene-coding region (shown in bold in Fig. 3) and the non-transcribed spacer were inferred from other published sequence data (Singh et a!. 1 994). Immedi­ ately downstream of the gene was the T-rich region (Fig. 3, nucleotide position 24-31 bp), which is responsible for the termination of transcription. A TATA box was identified 29 bp upstream of the start signal (Fig. 3), which was based on sequence comparison with published data from other plant species (Ellis et a!. 1 988; Bhatia et a!. 1 993; Scoles et a!. 1 988). The length of the non-transcribed spacer region ranged from 385 bp in clone 048-20 to 433 bp in clone 048- 1 9. Se­ quence analysis of the six clones indicated the presence of two classes of SS rDNA elements. The main difference be­ tween the two classes is the presence or absence of a microsatellite (OAA) in the spacer region (represented as in­ sertion-deletion (indel) 1 , Fig. 3). The class of NTS display­ ing the microsatellite repeat element has been designated class 1 and is represented by three clones (03- 1 , 03-5, and 048-1 9). Spacers lacking the element have been referred to as class 2 and are also represented by three clones (048-20, 03-8, and 048-1 7). Class 1 repeats differed in the number of copies of the trinucleotide OAA sequence (indel 1 , Fig. 3): clones 03-1 and 03-5 had 1 0 copies of the trinucleotide repeat, whereas clone 048- 1 9 had 1 6 copies (Fig. 3). Clone 048-1 9 exhibited two variant repeats, GAAA and OOAA. These repeats were identified at the 1 4th (GAAA) and 1 5th (OGAA) repeat positions (underlined in Fig. 3). Clone 03-5 also showed a variant repeat, OAAA, at the 9th repeat position (underlined in Fig. 3). A comparison of the sequences at the NTS regions of class 1 and class 2 clones revealed an interesting feature. The region corre­ sponding to the microsatellite (OAA)11 sequences, identified in class 1 clones, was marked by an A-rich track in class 2 clones. It is possible that an A to 0 transition in the A-rich region gave rise to the OAA motif, which subsequently ex­ panded via unequal crossing over or slipped-strand mispairing. Owing to the commonly observed high rate of microsatellite evolution, we predict that a more exhaustive analysis of clones may lead to the identification of addi­ tional trinucleotide repeat variants in this region. Additional length variation is evident in the form of class­ specific indels, which have been identified as indel 2 and indel 3 (Fig. 3). The indel 2 region is OC rich, 1 1 bp long, and is present in class 1 spacers. Because an outgroup se­ quence was not included. in this analysis, it is not possible to determine whether this region has been deleted from class 2 types of SS rDNA or whether it arose as an insertion in class 1 types of SS rDNA. The third indel (indel 3, Fig. 3) is a e­ rich, 1 9-bp sequence present in class 2 spacers and absent in class 1 spacers. . The analysis of the SS rDNA sequences data in P. deltoides clearly indicates the presence of two classes of spacer. Similarly in rattan, two classes of SS rDNA were identified based on sequence variation. Comparison of SS rDNA from the same palm genome revealed a variable level of intra-genome divergence in this region (Baker et a!. 2000). The major difference between the spacers identified in P. deltoides accessions is the presence or absence of microsatellite repeat sequences. Identification of repeat ele­ ments within the NTS region has been reported in several plant genera (Udovicic et a!. 1 995; Ellis et a!. 1 988; Oolds­ brough et al. 1 982). However, the presence of a micro­ satellite element within the NTS region has been reported only in the genus Hordeum (Baum and Johnson 1 994). Inter­ estingly, the microsatellite repeat elements present in P. deltoides have not been identified from other species of this genus (P. ciliata, P. lasiocarpa, P. maximowiczii, P. nigra, and P. euphratica; M. Lakshmikumaran et al., un­ published data). Raw pairwise (p) distances and corrected distances for multiple substitutions (Jukes-Cantor distances; Jukes and Cantor 1 969) of the SS rDNA spacers are shown in Table 1 . Pairwise Jukes-Cantor distances (Jukes and Cantor 1 969) between SS spacers ranged from 0.01 09 (between 03-5 and 048-19; Table 1 ), to 0.1 799 (between 03-1 and 048-1 7; Ta­ ble 1 ). Distance analysis showed that these six sequences fall into two general clusters (Fig. 4) that correspond to the classes marked by the presence or absence of microsatellites and simple indels, though these positions were not used in distance calculations. To quantify the diversity of SS rDNA NTS sequences, we calculated the nucleotide diversity pa­ rameter (n; Nei 1 987), which is the average number of base differences per site for two homologous sequences randomly selected from a population. Nucleotide diversity among members of these two classes ranged from 0.01 83 (standard deviation (s.d) 0.0061 ) for class 1 spacers to 0.1 433 (s.d. 0.0357) for class 2 spacers. This finding highlights the ex­ tent of variation within different classes of spacers, as poly­ morphism in class 2 repeats is significantly higher than that in class 1 repeats, which is evident in Fig. 4. = = © 2002 NRC Canada Negi et al. 1185 Fig. 3. Aligned nucleotide sequences of SS rDNA spacers from Populus de!toides accessions G-3 and G-48. The flanking SS coding region, TATA box region, and GAA trinucleotide repeats are indicated in bold print (the variant repeats are underlined). The deletions have been indicated as indels I, 2, and 3. Asterisks below the alignment indicate positions of sequence identity. G3-5 G3-1 G48-19 G3-8 G48-17 G48-20 GGGAAGTCCTCGTGTTGCACCCCTCCTTTTTGCCCGCTTCTCCATCCGGCCAGCCCCTCTGTCTTCCTTTCTTCTTTTTC GGGAAGTCCTCGTGTTGCACCCCTC-TTTTTGCCCGCTTCTTCATACGGCCAGCCTCTCTGTCTTCCTTTCTTCTTTTTC GGGAAGTCCTCGTGTTGCACCCCTCCTTTTTGCC-GCTTCTCCATCCGGCCAGCCCCTCTGTCTTCCTTTCTTCTTTTTC GGGAAGTCCTCGTGTTGCACCCCTTCTTTTTGCCCACTTCTCAATCCGGCCAGCCCATTTGTCTTTCTTTCTTCCTTTTC GGGAAGTCCTCGTGTTGCACCCCTTCTTTGTGCTCGCTTCTCCATCTTGTCAGCCCCTTTGTCTTTCTTTCTTCCTTTTC GGGAAGTCCTCGTGTTGCACCCCTCCTTTTTGGCCGCTTCTCCATCCGG-CAGCC--TCTGTCTTCCTTTCTTCTTTTTC 80 79 79 80 80 77 G3-5 G3-1 G48-19 G3-8 G48-17 G48-20 TCTCCCCCGCCTCGCTTTCCTCCCCCGCTATCGAGAGGGCGGGGCTAGAGGCCGAAGGCTACTAGTCTTGTTCAGAATGT TCTCCCCCGCCTCGCTTTCCTCCCGCGCTATCGAGAGGGCGGG-CTAGAGGCCGAAG-CTACTAGTCTTGTTCAGAATGT TCTCCCCCGCCTCGCTTTCCTCCCCCGCTACCGATAGGGCGGGGCTAGAGGCCGAAGGCTACTAGTCTTGTTCAGAATGT TCTACCCCGCCTCCCCTTCCT-CCCCAGAATCTATAGGGCTGGGTTAGCGGCCGGAGGCCCCTAGTCTTGTTCAGAATGT TCTCCCCCGCCTCCCCTTCCTGCCCCGCAATCGATAGGGCGGGGCTAGAGGCCGGAGGCCATTAGTCTTGTTCAGAATAT TCTCCCCCGCCTCGCTTTCCTCCCCCGCTACCGATAGGCGGGG--TAGAGGCCGAAGGCTACTAGTCTTGTTCAGAATGT 160 157 159 159 160 155 G3-5 G3-1 G48-19 G3-8 G48-17 G48-20 CGTTTCGCTTCAAGAAACGAAAGGTCGACACCTAATTCCACCAAGAATTTGGATTTGAACAGTAAAAGTCCGCTTTTTACGTTTCGCTTCAAGAAACGAAAGGTCGACACCTAATTCCACCAAGAATTTGGATTTGAACAGTAAAAGTCCGCTTTT-ACGTTTCGCTTCAAGAAACGAAAGGTCGACACCTAATTCCACCAAGAATTTGGATTTGAACAGTAAAAGTCTACTTTTTACGTTTCGCTTCAAGAAAGGAAAGGTCAACACCTAATTCCACAAAGAATTTGGTTATGAACAGTAAAAACCTATTTTTTTC CGTTTCGCTTCAAGAAACGAAAGGTCGACACCTAATTCCACCAAGAATTTTGTTATGAACAGCAAAAACCTATTTTTTTA CGTTTCGCTTCAAGAAACGAAAGGTCGACAGCTAATTCCACCAAGAATTTGGTTATGAACAATAAAAACCTATTTT---- 239 235 238 239 240 231 G3-5 G3-1 G48-19 G3-8 G48-17 G48-20 GAAGAAGAAGAAGAAGAAGAAGAAGAAAGAA-------------------AACAAAAGAAACAACAATCAAGGCGAATAA GAAGAAGAAGAAGAAGAAGAAGAAGAAGAA---------------------AC-AAAGAAACAACAATCAAGGCGAATAA GAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAAGGAAGAAGACAAAAGAAACAACAATCAAGGCGAATAA TTAAAAAA------------------------------------------CAAAATAAAGAATAACCAAAGCGAATAA AAAA------------------AAA -----------------------CCAAATTAAGTAACAAACAAAACGAATAA AAAAAA--.-------------------------------------- -C-AATTAAATACCAACCAAAGCAAATAA * ** ** * ** *** * ***** indel 1 300 293 318 277 278 266 G3-5 G3-1 G48-19 G3-8 G48-17 G48-20 AAATCCGGGCGCGATCCCACGGTCGGATCGCGGGCTACGGCCCCTTTGAGCC-GCCGCCC-TTTGCGGCGGCGGTGCCTG AAATCCGGGCGCGATCCCACGGTCGGATCGCGG-CTACGGCCCCTTTGAGCCCGTCGCCC-TTTGCGGCGGCGGTGCCTG AAATCCGGGCGCGATCCCACGGTCGGATCGCGGGCTACGGCCCCTTTGAGCC-GCCGCCCCTTTGCGGCGGCGGTGCCTG AAATCCA-----------ACAGTCGGATCACAGGTTACAGCCCTCTTGAGCC-CCCGCCC---CGTCGCGGCGGTGCCTG AAATCCA-----------ACGGTTGGATCGTACCTTACGGCCCTCTTGAGCC-GCT---------TCGCGTCGGTGCCTG AA-TCCA 4ij ·------.- ACGGTCTAATCGCAGGTTACGACCCTATTGAGCC-GCTGCCC---CTTCGCGGTGGTGCCTG *** ******** ** * *** *** ******* Indel 2 ** ** ** *** 378 371 397 342 337 330 G3-5 G3-1 G48-19 G3-8 G48-17 G48-20 AACCGC-GGGGA-------------------TCCTCCTCTTCGAAGCGCTTATAGCAATTGCGCGTGCATGGACTAAC AACCGCCGGGGA-------------------TCCTCCTCTTCGAAGCGCTTATAGCAATTGCGCGTGCATGGACTAAC AACCGCCGGGGA-------------------TCCTCCTCTTCGAAGCGCTTATAGCAATTGCGCGTGCATGGACTAAC AACCGCCGGGGATCCTCCCATCGCTCCCCGATCCTCCTCTTCGAAGTGCTTATAGCAATTGCGCGTGCATGGACTAAC AACTGCCTGGGAGCCTCCCATTGCTCCCCGATCCTCCTCTTCGAAGCGCTTATAGCAATTGCGTGAGCATGGACTAAC AACCACCGGGGA CTCCCATCACTCCCC TCTTCCTCTTAGAAGCGCTTATAGCAATTGCGCGTGCATAGATTAAC *** * **** ** ******* **** **************** * **** ** **** indel 3 G3-5 G3-1 G48-19 G3-8 G48-17 G48-20 GGGTGCGATCATACCAGCAC GGGTGCGATCATACCAGCAC GGGTGCGATCATACCAGCAC GGGTGCGATCATACCAGCAC GGGTGCGATCATACCAGCAC GGGTGCGATCATACCAGCAC '------' ************************ ********************* **** *** ** ***** * * * *** ** ** * ***** * ****** *** ***** ** * ******** ****************** '------'***************** *********************** ******** * * ****** ***** ******************** ***** * *** 436 430 456 420 415 408 456 450 476 440 435 428 The presence of two somewhat distinct classes of 58 spacer also correlates well with the observation of two 58 arrays in different species of Populus as revealed by in situ hybridization (Prado et a!. 1 996). Indeed, based on the pres­ ence of these two discrete classes of sequence, we predict that class 1 and 2 58 repeats of Populus correspond to two cytologically characterized 58 loci. This finding mirrors similar observations for 58 sequences from tetraploid spe­ cies of cotton (Cronn et a!. 1 996). Fluorescent in situ hybrid­ ization reveals that diploid cottons possess one 58 locus, whereas the allotetraploid species of cotton possess two 58 loci. Extensive sampling of the 58 clones from allotetraploid cottons also revealed two discrete classes of sequence, one from each of the two genomes, which were present in ap­ proximately equal proportions. If our prediction that class 1 and 2 sequences each repre­ sent a 58 locus in the P. deltoides genome is correct, then these two arrays exhibit very different properties. For exam pie, one of the two major loci shows a high degree of se­ quence conservation and is characterized by the presence of a (potentially highly) variable GAA microsatellite, whereas the other locus shows greater nucleotide diversity and lacks microsatellite repeats. The genesis of this marked difference in intra-array polymorphism (class 2 repeats) is unknown. Processes like sequential array contraction-expansion events are known to effectively reduce heterogeneity within arrays © 2002 NRC Canada Genome Vol. 45, 2002 1186 Table 1. Distance matrix for P. deltoides 5S NTS sequences. G3-5 G3-5 G3-l G48-19 G3-8 G48-17 G48-20 0.0164 O.ol08 0.1333 0.1466 0.1082 G3-1 G48-19 G3-8 G48-17 G48-20 0.0166 0.0109 0.0279 0.1468 0.163 0.14 0.1631 0.1799 0.1534 0.1392 0.1168 0.1339 0.1038 0.1469 0.1534 0.0274 0.1465 0.1599 0.1226 0.1277 0.1387 0.0969 0.1271 0.1334 0.1388 Note: Uncorrected (p) distances are shown below diagonal, and Jukes-Cantor distances. are shown above the diagonal. Fig. 4. Distance dendrogram of 5S rDNA spacer sequences from Populus deltoides constructed using Jukes-Cantor distances and the minimum evolution criterion of PAUP* version 4.0. Terminal branches and internodes with distances greater than 0.01 are in­ dicated; terminal branches leading to clones G3-5 and G48-19 are 0.000 and 0.005 units in length, respectively. G3-1 Acknowledgements G48-19 Class 1 Spacers ----------· Class 2 Spacers }--o-.o -55-3 - G3-8 microsatellite in one of the major arrays suggests that repeat length may be quite variable in this tree species. Because the number of repeats in a microsatellite can change quickly over evolutionary time, concerted evolution may act to spread and fix length variants within reproductive popula­ tions rapidly. Such a process could account for the larger ( 650 bp) 5S repeat described from another P. deltoides ac­ cession (Gottlob-McHugh et a!. 1 990). G48-20 The authors thank Dr. D.K. Khurana of Yashwant Singh Parmar University of Horticulture and Forestry, Solan, India, for providing P. deltoides germplasm and for useful discus­ sions. W are indebted to Dr. Yateendra Joshi for critical ed­ iting of the manuscript. This work was funded by a grant from the Department of Biotechnology, Goverment of India, New Delhi, to M. Lakshmikumaran. We are grateful to the Director General, TERl, for his kind support. References 0.0630 0.05 substitutions/site G48-17 (Dover 1 994), and such events may have occurred in the re­ cent history of P. de!toides. Alternatively, the class 2 se­ quences we isolated may actually be localized on two or more arrays. Although this scenario seems unlikely, because P. deltoides only shows two major 5S arrays (Prado et a!. 1 996), it is possible that we sampled 5S units that are local­ ized in dispersed, minor arrays that may be distributed throughout the genome. 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