Bot. Bull. Acad. Sin. (1998) 39: 153_160

Yang et al. — Phylogenetic position of Raphanus in relation to Brassica species

Phylogenetic position of Raphanus in relation to Brassica

species based on 5S rRNA spacer sequence data

Yau-Wen Yang^1,3, Pey-Feng Tseng¹, Pon-Yean Tai¹ and Cheng-Ju Chang²

¹Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China

²Institute of Biomedical Science, Academia Sinica, Taiwan, Republic of China

(Received August 23, 1997; Accepted March 4, 1998)

Abstract. Based on RFLP analysis, two evolutionary lineages for Brassica diploid species have been proposed. These are (I) the "nigra" lineage and (II) the "rapa/oleracea" lineage. The phylogenetic relationship of Raphanus species to these two lineages is still unclear because chloroplast and mitochondrial DNA genomic restriction site variation suggests that Raphanus is more closely related to the "rapa/oleracea" lineage, whereas nuclear RFLPs and other lines of evidence suggest that Raphanus belongs to the "nigra" lineage. Here, we present evidence of the intergenic spacer of nuclear 5S rRNA to support that Raphanus is more closely related to the "nigra" lineage than to the "rapa/oleracea" lineage. Genetic polymorphism within species is also discussed.

Keywords: Brassica; Phylogeny; Raphanus; 5S rRNA spacer.

Introduction

Brassica is one of the most important plant groups, containing species widely used in our daily life. Brassica rapa (n=10), B. nigra (n=8), and B. oleracea (n=9) are the three basic groups with three amphidiploid species, B. napus (n=19), B. juncea (n=18) and B. carinata (n=17) derived from interspecific hybridization between pairs of these diploid species, B. rapa × B. oleracea, B. rapa × B. nigra and B. nigra × B. oleracea, respectively (U, 1935). Based on chloroplast DNA (Warwick and Black, 1991), mitochondrial DNA (Palmer and Herbon, 1988), and nuclear DNA variation (Song et al., 1988; 1990) the phylogenetic relationships in Brassica and its related genera have been proposed. Brassica species can be divided into two evolutionary pathways: the "nigra" lineage and the "rapa/oleracea" lineage. Raphanus is thought to be closely related to the Brassica species. However, its relation to either lineage remains unsolved. Based on chloroplast and mitochondrial DNA restriction site variation (Warwick and Black, 1991; Palmer and Herbon, 1988), Raphanus was proposed to be more closely related to the "rapa/oleracea" lineage, but nuclear RFLPs and other RAPD data have suggested that Raphanus is more closely related to the "nigra" lineage (Song et al., 1990; Thormann et al., 1994).

5S rRNA genes are arranged in tandom arrays in the nuclear genome with highly conserved regions, separated by the non-transcribed intergenic spacer (IGS) that may vary in length or sequence between and even within spe

cies (Gerlach and Dyer, 1980; Long and Dawid, 1980). IGS evolves rapidly and is informative at the level of genus and species, so it has been chosen to study the phylogenetic relationships between and within genera (Appels et al., 1989, 1992; Baum and Appels, 1992; McIntyre et al., 1992; Moran et al., 1992; Playford et al., 1992; Reddy and Appels, 1989; Udovicic et al., 1995). Bhatia et al. (1993) demonstrated great polymorphism in IGS within B. rapa and related species. However, the phylogenetic relationship of Raphanus to these two lineages has not been studied using IGS sequences. Here we present data from the IGS of nuclear 5S rRNA to support the hypothesis that Raphanus is more closely related to the "nigra" lineage.

Materials and Methods

Plant Material

A total of twelve accessions of plant materials, which include seven accessions of B. rapa, one accession of B. oleracea, two accessions of R. sativus, one accession of Rorippa indica and one accession of Lepidium virginicum were used for PCR amplification of 5S rRNA sequences (Table 1). Among them, four accessions were provided by the Asian Vegetable Research and Development Center (AVRDC), and rest of them were collected by the authors. Also, another six 5S rRNA sequences from Genbank, which include sequences from B. rapa (X60723), Eruca sativa (X63524), B. nigra (X65710, X65711), Sinapis alba (X56866) and Arabidopsis athaliana (M65137) were used in this study (Bhatia et al., 1993; Campell et al., 1992; Capesius, 1991; 1993; Singh et al., 1994).

³Corresponding author.

Botanical Bulletin of Academia Sinica, Vol. 39, 1998

Table 1. Different accessions of Brassica and its related species used in phylogenetic analysis of 5S rRNA sequence.

Abbr. Species Subspecies Cultivar or accession Sources

Brc1 B. rapa chinensis 20 days Pai-Tai Sc^a

Brc2 B. rapa chinensis Chin-Chiang Pai-Tai Sc^a

Brc3 B. rapa chinensis Peng-Hop Pai-tai B00049^b

Brp1 B. rapa pekinensis Chinese cabbage Sc^a

Brp2 B. rapa pekinensis Chinese head cabbage Sc^a

Brp3 B. rapa pekinensis Dwarf leaf Chinese cabbage B00026^b

Brr1 B. rapa rapifera Goseki B00475^b

Brf B. rapa Field mustard X60723^c

Boc B. oleracea capitata Sc^a

Rs1 R. sativus Mei Hwa radish Sc^a

Rs2 R. sativus Meei-Nong radish Sc^a

Es E. sativa X63524^c

Bn1 B. nigra X65711^c

Bn2 B. nigra X65710^c

Sa S. alba X56866^c

Ri R. indica Sc^a

At A. thalina M65137^c

Lv L. virginicum Sc^a

^aSc = Collected by the authors.

^bAccession no. from AVRDC.

^cAccession no. for 5S rRNA sequence from Genbank.

DNA Isolation and Amplification

Genomic DNA was isolated from 2 g fresh leaves taken from 2_3 plants for each accession grown in the growth chamber according to the method described by Junghans and Metzlaff (1990). This DNA was then used as template for PCR amplification of 5S rRNA repeat based on two primers from Sinapis alba (Capesius, 1991). For PCR reaction, DNA was first denatured at 94°C for 4 min prior to the start of each PCR cycle. The complete PCR mixtures in 100 ul contained 100 ng DNA containing 10 mM Tris HCl, pH 8.3, 50 mM KCl, 0.1 mg/ml gelatin, 1.5 mM MgCl₂, 0.1 mM dNTP, 0.2 µM primer, and 0.5 unit taq polymerase. Amplification was performed using DNA Thermal Cycler (Perkin Elmer Cetus, Model 2400). DNAs were amplified for 35 cycles of 1 min at 94°C, 45 sec at 55°C, 1 min at 72°C and one final cycle of 5 min at 72°C. The PCR products were then run on 1% agarose gels. Amplified bands representing monomer and dimer of the 5S rRNA genes were cut and purified with JETpure kit (Genomed Inc, NC, USA). These fragments were then ligated with pBluescript II, Sk^_ (Stratagene, CA, USA) and transformed into E. coli XL-1 cells. The clones were then sequenced with ABI 373 automated sequencer (Applied Biosystem, NJ, USA).

Data Analysis

The IGS sequences from different accessions were aligned using the "MEGALIGN" program of Lasergene system (DNASTAR Inc. Madison, WI, USA) and manual adjustment. The distance between each pair of sequences (OTUs) was then determined by Kimura's two-parameter method (1980) using the "Mega" program (Kumar et al., 1993). Bootstrap values were also obtained with the same program.

Results and Discussion

The 5S rRNA gene in Brassica and related genera is 119 bp long. (Bhatia et al., 1993; Campell et al., 1992; Capesius, 1991; 1993; Singh et al., 1994). A forward primer, 5'-GGATGGGTGACCTCCCGGGAAGTCC-3' (positions from 81 to 105 of 5S rRNA) and a reverse primer, 5'-CGCTTAACTGCGGAGTTCTGATGGG-3' (positions from 58 to 34 of 5S rRNA gene) (Capesius, 1991) were used for amplification of 5S rRNA genes. The positive clones containing the regions from position 81 to 119 of 5S rRNA, IGS, and the regions from position 1 to 58 of 5S rRNA were sequenced and aligned with other published DNA sequences in the IGS region (Figure 1). Immediately following the 3'end of 5S RNA, there is a T-rich region of about 20 bp (positions 1 to 31) thought to be required for termination of the 5S rRNA (Hemleben and Werts, 1988). A GC motif with a consensus sequence, (G/T)GGGCGG(G/A)(G/A)(C/T), which may be involved in the regulation of transcription (Bhatia et al., 1993; Hart and Folk, 1982; Sorensen and Frederiksen, 1991) was located in the positions from 159 to 174. A putative TATA box, ATATATA, critical in the initiation of transcription of 5S rRNA genes (Korn, 1982; Selker et al., 1986), is found 5' to the 5S rRNA from the position 394 to 400. The IGS in the Brassica and its related genera ranges from 367 to 399 nucleotides, and the most divergent region is from position 206 to 387. In the polymorphic region, Rs1 and Rs2 of R. sativus were found to share the same nucleotide with "Bn1" and "Bn2" of B. nigra and "Sa" of S. alba in many locations (positions 36_38, 41, 127_128, 168, 170, 172, 215, 235, 249, 251, 258, 275_276, 289_290, 294, 360_363, 365_366, 370), while these two accessions of R. sativus were found to share same nucleotides with accessions of B. rapa or B. oleracea in fewer locations

Yang et al. — Phylogenetic position of Raphanus in relation to Brassica species

Botanical Bulletin of Academia Sinica, Vol. 39, 1998

Yang et al. — Phylogenetic position of Raphanus in relation to Brassica species

Figure 1. Alignment of IGS sequences begins at the position 1 and ends at the position 422. Different OTUs are indicated in Table 1.

Botanical Bulletin of Academia Sinica, Vol. 39, 1998

(positions 45_46, 67, 122_123, 157, 227, 328, 332_333, 337, 353, 375). For example, in location 38, all accessions of B. rapa and B. oleracea have nucleotide "C," while all accessions of B. nigra, S. alba, and R. sativus have nucleotide "T."

The distances between different OTUs (sequences) were determined at the level of nucleotide substitution using Kimura's two-parameter method (Table 2). The neighbor joining method (Saito and Nei, 1987) was then used to

construct a phylogenetic tree among these OTUs and the bootstrap test was performed to determine the bootstrap confidence level (BCL) for each node of the constructed tree (Figure 2). Great polymorphism was observed within B. rapa; however, the distances between OTUs within species are always smaller than those between species (Table 2 and Figure 2). Two accessions (Brp2 and Brp3) of ssp. pekinensis are clustered with two accessions of ssp. chinensis (Brc1 and Brc3) before clustering with Bcf; then

Figure 2. A NJ tree is constructed using the sequence divergences estimated with Kimura's two-parameter method. BCL values are labeled to indicate the percentage of trees that support the node.

Table 2. Number of substitutions per nucleotide site in the intergenic spacer of 5S rRNA genes calculated with Kimura's two-parameter method (Kimura, 1980).

OTUs Brc1 Brc2 Brc3 Brp1 Brp2 Brp3 Brr1 Brf Boc Rs1 Rs2 Es Bn1 Bn2 Sa Ri At Lv

Brc1

Brc2 0.095

Brc3 0.046 0.083

Brp1 0.058 0.089 0.055

Brp2 0.046 0.095 0.038 0.072

Brp3 0.043 0.088 0.027 0.063 0.032

Brr1 0.052 0.068 0.027 0.043 0.043 0.038

Brf 0.052 0.083 0.032 0.055 0.044 0.035 0.032

Boc 0.202 0.199 0.181 0.181 0.191 0.177 0.167 0.176

Rs1 0.409 0.396 0.398 0.390 0.403 0.417 0.403 0.401 0.387

Rs2 0.391 0.395 0.385 0.381 0.385 0.398 0.380 0.378 0.382 0.103

Es 0.402 0.379 0.368 0.403 0.359 0.382 0.368 0.386 0.463 0.430 0.364

Bn1 0.384 0.392 0.369 0.364 0.369 0.387 0.369 0.388 0.389 0.269 0.266 0.418

Bn2 0.383 0.392 0.368 0.363 0.377 0.386 0.368 0.373 0.385 0.274 0.272 0.415 0.011

Sa 0.407 0.402 0.378 0.383 0.401 0.396 0.387 0.396 0.403 0.274 0.246 0.428 0.165 0.165

Ri 0.806 0.728 0.737 0.744 0.759 0.750 0.730 0.768 0.752 0.760 0.731 0.803 0.667 0.683 0.649

At 0.812 0.796 0.833 0.790 0.804 0.781 0.798 0.794 0.877 0.819 0.798 0.884 0.834 0.841 0.909 0.544

Lv 0.852 0.771 0.774 0.794 0.793 0.788 0.754 0.773 0.832 0.793 0.746 0.876 0.773 0.767 0.766 0.814 0.742

Yang et al. — Phylogenetic position of Raphanus in relation to Brassica species

they were clustered with Brr1 of ssp. rapifera before they met Brp1 of ssp. pekinensis. Brc2 of ssp. chinensis is located outside these 7 OTUs. Since most values of BCL are low for these accessions within B. rapa, the phylogenetic relationship among subspecies is hard to determine on the basis of this DNA sequence. The result may be due to intensive cultivation and occasional outcrossing between different subspecies in B. rapa. Nevertheless, 8 OTUs from B. rapa were grouped into one composite OTU that is significantly different from the other OTUs in this study. As shown by RFLP data (Song et al., 1988; 1990; Thormann et al., 1994), B. oleracea (Boc) is more closely related to B. rapa than to B. nigra. Two accessions (Bn1 and Bn2) of B. nigra were clustered with "Sa" of Sinapis alba before they met two accessions (Rs1 and Rs2) of Raphanus sativus (Figure 2). The close relation between S. alba and B. nigra is also reported based on RFLP data (Warwick and Black, 1991). By using Arabidopsis athaliana, Rorippa indica, and Lepidium virginicum as outgroups, it was clearly shown that Raphanus sativus is more closely related to the B. nigra lineage than to the B. rapa/oleracea lineage. In Table 2, the average distance between R. sativus and B. rapa is also significantly larger than that between R. sativus and B. nigra (0.396 ± 0.010 vs. 0.270 ± 0.004). In addition, similar phylogenetic relationships among these OTUs were also observed in the maximum parsimony tree based on the branch and bound search method using the MEGA program (Kumar et al., 1993) (results not shown). Also, data analysis based on the nucleotide sequences of the 18S - 25S spacer region showed a similar result (unpublished data). The discrepancy between nuclear DNA sequence data and chloroplast (or mitochondrial) RFLP data suggests that R. sativus may have been derived from hybridization between species belonging to different lineages, as Song et al. (1990) proposed. If this hypothesis is true, two distinct types of IGS must exist in the Raphanus with one close to B. nigra and the other close to B. oleracea/rapa since nuclear DNA sequences are biparentally inherited. However, no such nuclear DNA sequences in Raphanus have been found to date.

In conclusion, the IGS of the 5S rRNA gene has been used succesfully to illustrate the phylogenetic relationships among Brasssica rapa and its related species. The phylogenetic position of R. sativus to the "nigra" lineage was demostrated. However, the origin of R. sativus may need to be further studied by using more data from chloroplast DNA sequences and other nuclear markers to test the views presented by Palmer and Herbon (1988) and Song et al. (1990). In addition, due to the intensive cultivation or hybridization between cultivars or subspecies, it is hard to determine the phylogenetic relationship among different subspecies within B. rapa based on this DNA sequence.

Acknowledgements. We thank Dr. H.H. Ho, Mr. L.-C. Chang, Y.-K. Huang, Dr. H.F. Yen and Dr. C.H. Tsou for giving us valuable advice, and Miss Y.-J. Peng for technical assistance in DNA sequencing in this research. This work is supported in part by National Science Council and Academia Sinca, Taiwan.

Literature Cited

Appels, R., B.R. Baum, and B.C. Clarke. 1992. The 5S DNA units of bread wheat (Triticum aestivum). Plant Syst. Evol. 183: 183_194.

Appels, R., P. Reddy, C.L. McIntyre, L.B. Moran, O.H. Frankel, and B.C. Clarke. 1989. The molecular-cytogenetic analysis of grasses and its applications to studying relationships among species of the Triticeae. Genome 31: 122_133.

Baum, B.R. and R. Appels. 1992. Evolutionary change at the 5S Dna loci of species in the Triticeae. Plant Syst. Evol. 183: 195_208.

Bhatia, S., K. Singh, V. Jagannathan, and M. Lakshmikumaran. 1993. Organization and sequence analysis of the 5S rRNA genes in Brassica campestris. Plant Sci. 92: 47_55.

Campell, B.R., Y. Song, T.E. Posch, C.A. Cullis, and C.D. Town. 1992. Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana. Gene 112: 225_228.

Capesius, I. 1991. Sequence of the 5S rRNA gene from Sinapis alba. Plant Mol. Biol. 17: 169_170.

Capesius, I. 1993. Sequence of the 5S rRNA gene from Brassica nigra and its relation to other 5S rRNA genes from Brassicaceae. J. Plant Physiol. 142: 112_114.

Gerlach, W.L. and T.A. Dyer. 1980. Sequence organization of the repeating units in the nucleus of wheat which contain 5S rRNA genes. Nucleic Acids Res. 8: 4851_4865.

Hart, R.P. and W.R. Folk. 1982. Structure and organization of a mamalian 5S gene cluster. J. Biol. Chem. 257: 11706_11711.

Hemleben, V. and D. Werts. 1988. Sequence organization and putative regulatory elements in the 5S rRNA genes of two higher plants (Vigna radiata and Matthiola incana). Gene 62: 165_169.

Junghans, H. and M. Metzlaff. 1990. A simple and rapid method for the preparation of total plant DNA. Biotechniques 8: 176.

Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111_120.

Korn, L.J. 1982. Transcription of Xenopus 5S ribosomal RNA genes. Nature 295: 101_105.

Kumar, S., K. Tamura, and M. Nei. 1993. MEGA-Molecular Evolutionary Genetics Analysis. Version 1.01. Penn. State Uni. University Park, PA, USA.

Long, E.O. and I.B. Dawid. 1980. Repeated genes in eukaryotes. Annu. Rev. Biochem. 49: 727_764.

McIntyre, C.L., B. Winberg, K. Houchins, R. Appels, and B.R. Baum. 1992. Relationships between Oryza species (Poaceae) based on 5S DNA sequences. Plant Syst. Evol. 183: 249_264.

Moran, G.F., D. Smith, J.C. Bell, and R. Appels. 1992. The 5S RNA genes in Pinus radiata and the spacer region as a probe for relationships between Pinus species. Plant Syst. Evol. 183: 209_221.

Palmer, J.D. and L.A. Herbon. 1988. Plant mitochondrial DNA evolves rapidly in structure, but slowly in sequence. J. Mol. Evol. 28: 87_97.

Playford, J., R. Appels, and B.R. Baum. 1992. The 5S DNA units of Acacia species (Mimosaceae). Plant Syst. Evol. 183:

Botanical Bulletin of Academia Sinica, Vol. 39, 1998

235_247.

Reddy, P. and R. Appels. 1989. A second locus for the 5S multigene family in Secale L: Sequence divergence in two lineages of the family. Genome 32: 456_467.

Saito, N. and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406_425.

Selker, E.U., E. Morzycka-Wroblewska, J.N. Stevens, and R.L. Metzenberg. 1986. An upstream signal is required for in vitro transcription of Neurospora 5S RNA genes. Mol. Gen. Genet. 205: 189_192.

Singh, K., S. Bhatia, and M. Lakshmikumaran. 1994. Novel variants of the 5S rRNA genes in Eruca sativa. Genome 37: 121_128.

Song, K.M., T.C. Osborn, and P.H. Williams. 1988. Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs) 1. Genome evolution of diploid and amphidiploid species. Theor. Appl. Genet. 75: 784_794.

Song, K.M., T.C. Osborn, and P.H. Williams. 1990. Brassica taxonomy based on nuclear restriction fragment length poly

morphisms (RFLPs) 3. Genome relationships in Brassica and related genera and the origin of B. oleracea and B. rapa (syn. campestris). Theor. Appl. Genet. 76: 497_506.

Sorensen, P.D. and S. Frederiksen. 1991. Characterization of human 5S rRNA genes. Nucleic Acids Res. 19: 4147_4151.

Thormann, C.E., M.E. Ferreira, L.E.A. Camargo, J.G. Tivang, and T.C. Osborn. 1994. Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species. Theor. Appl. Genet. 88: 973_980.

U, N. 1935. Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Jpn. J. Bot. 7: 389_452.

Udovicic, F., G.I. McFadden, and P.Y. Ladiges. 1995. Phylogeny of Eucalyptus and Angophora based on 5S rDNA spacer sequence data. Mol. Phylo. and Evol. 4: 247_256.

Warwick, S.I. and L.D. Black. 1991. Molecular systematics of Brassica and allied genera (Subtribe Brassicinae, Brassiceae)- Chloroplast genome and cytodeme congruence. Theor. Appl. Genet. 82: 81_92.