J. Res. 110: 187-193, 1997 Journal of Plant Research (~) by The Botanical Society of Japan 1997

Construction of Phylogenetic Tree for Species Based on RAPD Markers

Yung-Luen Yu and Tsai-Yun Lin

Department of Life Science, National Tsing Hua Universiry, No. 101, Sec. 2, Kuang Fu Road, Hsinchu, 30043, Taiwan, R.O.C.

To apply random amplified polymorphlc DNA for ana- sequence. The polymorphisms observed may result from lysis of phylogenetic relationshipe, we used 34 synthetic nucleotide substitutions, insertions, or deletions. The oligonucleotides as primers to examine interspecific and major advantages of this analysis are that (1) the informa- intraspeciflc variations among 18 genotypes, nine species tion of DNA sequence is not required, (2) the protocol is of Nicotlana. The nine species used in this study belong relatively easy to perform, (3) only a small quantity (ng) of to sections Tomentosae and Alatae. In addition, we DNA is needed, (4) a large number of samples can be attempted to clarity the taxonomic position of N. sylves- processed simultaneously in a short period of time, (5) tris. A total of 354 distinct DNA fragments were the technique can be applied to a broad range of species obtained by polymerase chain reaction. Pair-wise com- (Welsh and McClelland 1990, Martin et al. 1991, Reiter et parisons of unique and shared amplification products al. 1992, Torres et al. 1993, Chaparro et al. 1994, Yu and were used to generate Jaccard's similarity coefficients Nguyen 1994), and (6) no radioactive reagent is utilized in and Nei and U's similarity coefficients with the computer the assay. software of numerical and multivariate ana- To apply RAPD for analysis of phylogenetic relation- lysis system. On the basis of the dendrogram construct- ships, nine species from two sections of Nicotiana ed with the similarity coefficients, the 18 Nlcotlana (Tomentosae and Alatae) were examined. Nicotiana genotypes were divided Into two clusters. The classifi- sylvestris is considered a member of Alatae (Goodspeed cation analyzed by RAPD markers is in accordance with 1954). However, Kostoff (1943) observed that F1 hybrids the classification of Goodspeed that N. sylvestris is a of N. sylvestris and any other species in section Alatae member of section Alatae. had lower chromosome associations than F1 hybrids of N. sylvestris and any member of section Tomentosae had. Key words: Nicotlana m Phylogenetic tree m RAPD Hence, he regarded N. sylvestris as a member of section Tomentosae. In this study, we performed RAPD assay to analyze the phylogenetic relationships among 18 The natural distribution of the genus Nicotiana, family genotypes of Nicotiana and determine the taxonomic , is limited to America (75%), Australia and a position of N. sylvestris. few islands of the South Pacific (25%). The estimated 60 species of Nicotiana are classified into 14 sections based Materials and Methods upon distribution and morphological and cytogenetic characteristics (Goodspeed 1954). For example, inflores- Table1 lists the 18 genotypes of nine species of cence expression in section Tomentosae is in thyrse Nicotiana used in this experiment which were provided by panicles except in N. glutinosa. The inflorescence of the US Department of Agriculture (USDA) and the Taiwan section Alatae shows monochasia, often extending di- Tobacco Research Institute (TTRI). were kept in a chasial forks. The traditional taxonomy is established growth chamber under conditions of 22 C day/22 C night primarily on the basis of morphology, distribution and and with light irradiance of 200 ~mol m-2 s-' for 14 hr day -1. cytology. However, factors like the environment, multi- Genomic DNA was extracted and purified according to the genic inheritance, or partial and complete dominance method of Rogers and Bendich (1988) with modifications. often confound the expression of a genetic trait (Tingey Young leaf tissue (1.0 g) was ground in liquid nitrogen and and del Tufo 1993). then mixed with 1.0ml of 2% CTAB extraction Molecular markers such as isozymes and restriction buffer (2% CTAB, 100 mM Tris HCI (pH 8.0), 20 mM fragment length polymorphisms (RFLPs) have been exten- EDTA (pH 8.0), 1.4 M NaCI and 2% /~-mercaptoethanol). sively applied for genetic studies and plant breeding After incubation at 65 C for 5 min with vigorous shaking, (Beckmann and Soller 1983, Tanksley et al. 1989, Yang et the lysate was extracted with an equal volume of chloro- al. 1992). Random amplified polymorphic DNA (RAPD) form/isoamyl alcohol (24 : 1), and centrifuged at 11 • for analysis is based on the amplification of random DNA 10 min. The aqueous phase was transferred to a fresh segments with single primers of the arbitrary nucleotide tube containing 1/10 volume of the 10% CTAB buffer (10% 188 Y.-L. Yu and T.-Y. Lin

Table 1. The genotypes in Nicotiana used for RAPD analysis fications were performed with a programmable thermal controller (MJ Research Inc) programmed for 2 cycles of 2 Chromo- Acc. min at 94 C, 1 min at 36 C and 2 min at 72 C, followed by Section Species no. no.some (2n) Source 43 cycles of 1 min at 94 C, 1 min at 36 C and 2 min at 72 C. After all cycles were completed, the reactions were Alatae held at 72 C for 10 min and slowly cooled to 4 C. The N. Iongiflora (NIo) 30 20 USDA DNA fragments amplified were analyzed by electropho- 30A 20 USDA resis in 1.5% NuSieve (FMC) agarose gels running in 1X 30B 20 USDA TBE buffer (89 mM Tris, 89 mM boric acid and 2 mM 30C 20 USDA EDTA) at 150 V for 3.5 hr. The gels were then stained N. plumbaginifolia (Np) 4,3A 20 USDA with ethidium bromide and photographed under UV light. 43B 20 USDA All the amplifications were tested at least twice for re- 43s 20 USDA producibility. N. bonariensis (Nb) 18 "l-rRI Photographs from ethidium bromide stained agarose N. a/ata (Na) 3 18 USDA gels were used for RAPD analysis. Bands on gels were N. /angsdorffii (Nla) 28A 18 USDA scored as present (1) or absent (0) for all the taxa studi- 28B 18 USDA ed. Pair-wise comparisons of these taxa were analyzed N. sylves~ris (Ns) 56A 24 USDA with the computer software of the numerical taxonomy Tomentosae and multivariate analysis system (NTSYS-pc, version 1.80; N. otophora (No) 38 24 USDA Rohlf 1993) and Jaccard's similarity coefficient and Nei and Li's similarity coefficient. The similarity coefficients 38A 24 USDA obtained were then used to construct a dendrogram using 38B 24 USDA the unweighted pair-group method with arithmetical aver- 30(3 24 USDA ages (UPGMA) employing the sequential, agglomerative, N. tomentosiformis (Nt) 24 "I-FRI hierarchical and nested clustering (SAHN) routine in 24 "H'RI N. glutinosa (Ng) NTSYS programs.

Results CTAB and 0.7 M NaCI) and extracted with an equal volume of chloroform/isoamyl alcohol (24:1). DNA was The size of the amplified fragments ranged from 0.2 to precipitated with the addition of an equal volume of CTAB 1.5 kb. The profile of the amplified products obtained precipitation buffer (1% CTAB, 50 mM Tris ~ HCI (pH 8.0) with primer 48 is shown in Figure 1. Of the 354 amplified and 10 mM EDTA (pH 8.0)) and recovered by centrifuga- products, 339 (95.8%) were polymorphic and 15 (4.2%) tion for 15 min at 3,500Xg after incubation at --70 C for were monomorphic (Table 2). For each primer evaluated, 15 min. The pellet was dried and redissolved in high-salt the number of amplified products ranged from 6 to 14. In TE buffer (10 mM Tris ~ HCI (pH 8.0), 1 mM EDTA (pH 8.0) terms of the Nicotiana species, the products obtained with and 1 M NaCI), and then precipitated by ethanol. The the 34 primers ranged from 108 (N. glutinosa) to 158 (N. pellet was washed with 80% ethanol, dried under vacuum Iongiflora accession 30 C) and had an average of 4.1 and resuspended in sterile deionized water. The DNA amplified fragments per primer (Table3). Amplified samples were aliquoted and stored at -20 C prior to use. polymorphic DNA fragments were scored for computer To produce distinct DNA bands, genomic DNA from N. analysis based on Jaccard's similarity coefficients and Iongiflora accession 30 C and N. plumbaginifolia acces- Nei and Li's similarity coefficients and ranged from 0.099 sion 43C was used to screen 400 random oligonu- to 0.980 and 0.181 to 0.990, respectively (Table 4). The cleotides (decamers) with G-I-C contents of 50-80%, result of pair-wise comparisons indicated that Ficus which were obtained from Dr. J.B. Hobbs at the Oligonu- awkeotsang Makino was the most distantly related to all cleotide Synthesis Laboratory, Nucleic Acid-Protein Ser- genotypes in Nicotiana. vice Unit, University of British Columbia (UBC-1 to 100, 201 All of the genotypes in Nicotiana had similarity indices to 300, 401 to 500 and 601 to 700). Thirty-four primers ranging from 0.142 to 0.980 based on Jaccard's similarity (Table 2) were selected for amplification of DNA frag- coefficients and from 0.248 to 0.990 based on Nei and Li's ments with genomic DNA of 18 Nicotiana genotypes and of similarity coefficients (Figure2). Interestingly, the 18 Ficus awkeotsang Makino (jelly fig) as a control. genotypes analyzed were obviously divided into two Genomic DNA amplifications were performed according to clusters, A and B, on the basis of both Jaccard's and Nei the method of Williams et al. (1993) with some modifica- and Li's similarity coefficients. Cluster A comprised 12 tions. Each reaction was prepared on ice with a volume genotypes with a Jaccard's similarity index of 0.331 and a of 25/.el containing 10 mM Tris ~ HCI (pH 8.8), 50 mM KCI, Nei and Li's similarity index of 0.496 and was subdivided 1.5 mM MgCI2, 0.1% Triton X-100, 200~M dNTPs (Phar- into two subclusters (a and b). Cluster B comprised 6 macia), 0.2/~M primer, 25 ng genomic DNA and 2 units of genotypes with a Jaccard's similarity index of 0.320 and a PrimeZyme DNA polymerase (Biometra, Ver. 2.0). Ampli- Nei and Li's similarity index of 0.485. In cluster A, Phylogenetic Tree for Nicotiana 189

Table 2. Syrdh~c oligonucleotides used as primers for RAPE) analysis in N/cot/aria

Primer Nucleotide No. of No. of % polyrnorphism sequence amplified polym~phic (b/ax 1(30) number (5' to 3") fragments (a) fragments Co)

9 CCTGCGCTTA 6 5 88.33 21 ACCGGG i I tC 9 9 100.00 35 CCGGGG'I-FAA 9 9 100.00 36 CCCCCCTTAG 8 8 1(30.00 44 "]-rA CCC_,CGC-,-,-,-~ 11 11 100.00 47 "r'TcCCCAAGC 12 12 100.00 48 I-I'AACGGGGA 12 12 100.00 53 CTCCCTGAGC 8 7 87.50 201 CTGGGGA'ITI" 10 10 100.00 214 CATGTGC'I-I'G 6 3 50.00 221 CCCGTCAATA 14 14 100.00 224 TCTCCGGTAT 11 11 100.00 2"34 TCCACX3CmlkCG 7 7 100.00 247 TACCGACGGA 10 10 100.00 288 CCTC_,C'FFGAC 12 11 91.67 4O2 CCCGC_,CG'I-FG 10 10 100.00 4O4 TCTCTACGAC 13 12 92.31 CCGTCTC'I-I'T 13 12 92.31 427 GTAATCGACG 12 12 1(30.00 429 AAACCTGGAC 8 7 87.50 435 CTAGTAGGGG 12 11 91.67 446 GCCAGCG'n'c 10 10 100.00 475 CCAGCGTATr 12 12 100.00 483 GCACTAAGAC 8 8 100.00 497 GCATAGTGCG 10 9 90.00 605 CCGATCATTC 10 10 100.00 611 CCATCGTACC 7 6 85.71 630 CACTCTCTGG 14 14 100.00 645 TACAGCG'I-FG 12 12 100.00 653 CATGCAAGAC 7 7 100.00 657 GTCC'I-I'TAGC 14 12 85.71 661 CCTGC'I-rAC,,G 14 14 100.00 662 GGCTACGTCT 9 8 88.89 684 CCACACGTAG 14 14 100.00 Total 354 339 95.76

similarity coefficients ranged from 0.980 (Jaccard's coeffi- coefficient of 0.4A.4. cient) and 0.990 (Nei and Li's coefficient) for the most closely related genotypes, N. plumbaginifolia accession Discussion 43A and N. plumbaginifolia accession 43 C, to 0.265 (Jac- card's coefficient) and 0.419 (Nei and Li's coefficient) for Wheat cultivars generally have a low level of polymor- the most distantly related genotypes, N. Iongiflora acces- phism on the basis of restriction patterns so that RAPD sion 30A and N. sylvestris. In cluster B, the greatest analysis has been used to generate more polymorphic similarity was between N. otophora accession 38A and N. markers (Joshi and Nguyen 1993). Similarly, RAPD assay otophora accession 38B with a Jaccard's similarity coeffi- was employed to analyze phylogenetic relationships cient of 0.834 and a Nei and Li's similarity coefficient of among species of Nicotiana in this study. In Brassica, a 0.910, whilst the least similarity was observed between N. minimum of ten primers with a total of 100 RAPD bands otophora accession 38A and N. glutinosa with a Jaccard's was needed to demonstrate the genetic relationships similarity coefficient of 0.285 and a Nei and Li's similarity (Demeke et a/. 1992). In our study, a minimum of 19 ]90 Y.-L. Yu and T.-Y. Lin

Fig. 1. Genomic DNA from 18 genotypes of Nicotiana and Ficus awkeotsang (jelly fig) amplified with primer 46 (5'-TFAACGGGGA-3'). LaneM: DNA size marker (Haelll cut-&x174 RF DNA). Lane Fa: Ficus awkeotsang. Lanes 1-18: 1=30, 2=30A, 3=30B, 4=30C, 5=43A, 6=43B, 7=43(3, 8=Nb, 9=Na, 10=28A, 11=28B, 12=58A, 13=38, 14=38A, 15=38B, 16=35C, 17=Nt and 18=Ng. The accession numbers and codes for this figure can be found in Table 1.

Table 3. Datailed list of RAPD fragments produced with 34 primers output file from our data. Moreover, the same topology was obtained using a hundred data files with randomized No.of Average No. of Genotypesa scored fragments polymorphic input orders. fragments per primer fi'agments In accordance with the classification of Goodspeed (1954), the 18 genotypes of 30 158 4.6 143 Nicotiana were divided into two clusters (A and B) based on the phylogenetic tree con- 30A 151 4.4 136 structed (Figure 2), cluster A belonging to section Alatae 30B 155 4.8 140 and cluster B belonging to section Tomentosae. Our 30(3 151 4.4 136 results could also suggest that the dendrogram divides 43A 149 4.4 134 these species into three clusters B, b and a. In that case 43B 139 4.1 124 section Alatae is divided into two clusters with N. Iongi- 4,3(3 150 4.4 135 flora and N. plumbaginifolia, the two species of 10-paired Nb 124 3.8 109 chromosome, belonging to cluster a and the other four Na 127 3.7 112 species in Alatae belonging to cluster b. Nicotiana Iongi- 28A 150 4.4 135 flora and N. plumbaginifolia are almost identical in exter- 28B 142 4.2 127 nal morphology, chromosome number and karyotype 56A 121 3.6 106 (Goodspeed 1954). The species of 9-paired chromo- 35 135 4.0 120 some, N. alata and N. langsdorffii are located in subcluster 35A 149 4.4 134 b of cluster A. In cluster B the similarity coefficient 38B 139 4.1 124 between N. otophora and N. tomentosiformis is higher 35C 146 4.3 131 (0.497 to 0.511 for Jaccard's similarity coefficients and 0. 664 to 0.677 for Nei and Li's similarity coefficients) than Nt 134 3.9 119 that between N. otophora and N. glutinosa (0.323 to 0,335 Ng 108 3.2 93 for Jaccard's similarity coefficients and 0.488 to 0.502 for Average 140___T~ 4.1 ___0.2b 125_T~ Nei and Li's similarity coefficients). Nicotiana otophora a The abbreviationfor the genotypes of Nicotiana are indicated in and N. tomentosiformis are two of the three core species Table 1. in section Tomentosa and exhibit affinity in external b Values are mean___95%confidence interval. morphology. However, N. glutinosa is more or less mar- ginal species in section Tomentosa. , which is a member of section Alatae according to the primers with a total of 200 RAPD bands resulted in a classification of Goodspeed (1954), was regarded as a phylogenetic tree similar to that in Figure2. Multiple member of section Tomentosae according to cytogenetic UPGMA may occur due to ties resulted from input order studies (Kostoff 1943). N. sylvestris has 2n=24 chromo- effects (Backeljau et al. 1996). To cope with ties in somes as in species of section Tomentosa. This data UPGMA trees, the FIND option was specified for up to 25 suggest that N. sylvestris is at least basal to the clade of alternative trees. Only a single tree was written in the section Alatae. Our results from the RAPD analysis Phylogenetic Tree for Nicotiana 191

Table 4. Similarity matrix of 18 genotypes in Nicotiana and Ficus awkeotsang using Jaccard's coeffients (I) and Nei and Li's coefficients (11) : range of value from 0 to 1. with values closer to 1 indicating increasing similarity. I: Jaccard's similarity coeffients

Genotypea Fab 30 30A 30B 30C 43A 438 4,3C Nb Na 28A 28B 56A 38 38A 38B 38(3 Nt Ng

Fa 1.000 30 0.113 1.000 30A 0.099 0.577 1.000 30B 0.115 0.573 0.962 1.000 30C 0.117 0.896 0.549 0.553 1.000 43A 0.118 0.574 0.579 0.583 0.538 1.000 43B 0.112 0.580 0.551 0.556 0.576 0.756 1.000 43C 0.118 0.571 0.584 0.589 0.5,36 0.980 0.752 1.000 Nb 0.102 0.343 0.329 0.335 0.335 0.365 0.349 0.377 1.000 Na 0.134 0.301 0.343 0.343 0.293 0.387 0.330 0.392 0.521 1.000 28A 0.112 0.322 0.344 0.356 0.326 0.359 0.320 0.364 0.531 0.530 1.000 28B 0.135 0.297 0.324 0.330 0.301 0.358 0.330 0.363 0.500 0.508 0.776 1.000 56A 0.154 0.292 0.265 0.266 0.289 0.311 0.319 0.316 0.361 0.348 0.369 0.397 1.000 38 0.183 0.172 0.149 0.142 0.167 0.183 0.176 0.153 0.239 0.242 0.239 0.258 0.255 1.000 38A 0.182 0.195 0.154 0.152 0.195 0.196 0.166 0.168 0.219 0.232 0.230 0.248 0.233 0.711 1.000 38B 0.179 0.193 0.150 0.153 0.193 0.180 0.173 0.180 0.212 0.237 0.235 0.237 0.221 0.734 0.834 1.000 38C 0.152 0.202 0.165 0.162 0.128 0.190 0.183 0.189 0.227 0.252 0.254 0.240 0.242 0.683 0.788 0.792 1.000 Nt 0.177 0.2"32 0.203 0.199 0.218 0.225 0.203 0.224 0.217 0.273 0.251 0.237 0.232 0.511 0.466 0.484 0.497 1.000 Ng 0.150 0.209 0.205 0.206 0.199 0.236 0.223 0.240 0.234 0.250 0.248 0.265 0.272 0.335 0.285 0.321 0.323 0.337 1.000

II. Nei and U's similarity coefficients

Genotyp~ Fab 30 30A 3013 30(3 48A 43B 4,3(3 Nb Na 28A 28B 68A 38 35A 35B 38(3 Nt Ng

Fa 1.000 30 0.203 1.000 30A 0.181 0.731 1.000 30B 0.206 0.728 0.960 1.000 30C 0.210 0.945 0.709 0.712 1.000 43A 0.212 0.730 0.733 0.737 0.700 1.000 43B 0.202 0.734 0.710 0.714 0.731 0.861 1.000 4,3C 0.211 0.727 0.738 0.741 0.696 0.990 0.858 1.000 Nb 0.186 0.511 0.495 0.502 0.502 0.535 0.517 0.547 1.000 Na 0.237 0.463 0.511 0.511 0.453 O.558 O.4,96 O.563 0.685 1.000 28A 0.201 0.487 0.512 0.525 0.492 0.528 0.484 0.533 0.693 0.693 1.000 28B 0.238 0.458 0.490 0.497 0.463 0.527 0.496 0.532 0.667 0.674 0.874 1.000 56A 0.267 0.452 0.419 0.420 0.449 0.474 0.485 0.480 0.531 0.516 0.5,39 0.568 1.000 38 0.309 0.294 0.259 0.248 0.287 0.310 0.299 0.309 0.386 0.289 0.386 0.410 0.406 1.000 38A 0.308 0.326 0.267 0.263 0.327 0.289 0.285 0.288 0.359 0.377 0.375 0.390 0.378 0.831 1.000 38B 0.303 0.323 0.276 0.265 0.324 0.306 0.295 0.304 0.350 0.383 0.381 0.383 0.362 0.847 0.910 1.000 38C 0.263 0.336 0.283 0.279 0.330 0.319 0.309 0.318 0.370 0.403 0.405 0.388 0.390 0.811 0.881 0.884 1.000 Nt 0.301 0.377 0.337 0.332 0.358 0.367 0.337 0.366 0.357 0.429 0.401 0.383 0.376 0.677 0.636 0.652 0.664 1.000 Ng 0.275 0.346 0.340 0.342 0.332 0.381 0.364 0.388 0.379 0.400 0.395 0.406 0.428 0.502 0.444 0.486 0.488 0.504 1.000

a The abbreviations for the genotypes of Nicotiana are indicated in Table 1. b Fa re~'esents Ficus awkeo~ang. 192 Y.-L. Yu and T.-Y. Lin

I. Jaccard's Similarity Indices

Similarity 0.000 0.200 0.400 0.600 0.800 1.000 I I I I I I Fa F. awkeotsang [---- 8o N. Iongiflora 3~ N. Iongiflora E 30A N. iongiflora 30B N. longiflora 43A N. plumbaginifolia 43C IV. plumbaglnifol/a 43B N. plumbaginifolia Nb N. bonariensis Na N. alata 28A N. langsdorffii 28B N. langsdorffll 56A N. sylvestris 38 N. otophora 38A N. otophora 38B N. otophora 38C N. otophora Nt N. tomentosiformis Ng N. glutinosa

I1. Nei and Li's similarity indices

Similarity 0.000 0.200 0.400 0.600 0.800 1,000 I I I I I I Fa F. awkeotsang 30 N. Iongiflora 30C N. Iongiflora 30A N. Iongiflora C 30B N. Iongiflora 43A N. plumbaginifolia 43C N. plumbaginifolia 43B N. plumbaginifolia Nb N. bonariensls Na N. alata 28A N. langsdorffii 28B N. langsdorffii 56A N. sylvestris 38 N. otophora 38A N. otophora 38B N. otophora 38C N. otophora Nt N. tomentosiformis Ng N. glutinosa Fig. 2. Dendrogramconstructed using the UPGMA based on Jacoard's similarity coefficients (I) and Nei and Li's similarity coefficients (11) illustrating the genetic relationships among 18 genotypes of Nicotiana and Ficus awkeotsang. Relative lengths indicate similarity indices. The abbreviations of samples are the same as those indicated in Table 1 and Figure1. Phylogenetic Tree for Nicotiana 193 conform to the classification of Goodspeed (1954) and (RAPD): a case study in Brassica. Theor. Appl. coincide with a previous study of physical maps of some Genet. 84: 990-994. Nicotiana chloroplast DNA (Yang et al. 1992). The size Goodspeed, T.H. 1954. The Genus Nicotiana. Waltham, and basic structure of the chloroplast DNA from N. sylves- Massachusetts, Chronica, Botanica. tris were found to be almost identical to that from N. Joshi, C.P. and Nguyen, H.T. 1993. RAPD (random am- otophora and N. plumbaginifolia; however, the wealth of plified polymorphic DNA) analysis based intervarietal genetic relationships among hexaploid wheats. restriction site variation in N. otophora chloroplast DNA Plant Sci. 93: 95-103. compared to N. sylvestris and N. plumbaginifolia suggest- Kostoff, D. 1943. Cytogenetics of the Genus Nicotiana. ed that N. sylvestris and N. plumbaginifolia were distantly Karyosystematics, Genetics, Cytology, Cytogenetics related to N. otophora. and Phylesis of Tobaccos. Sofia, State Printing PCR that is used to produce informative amplification House. products often produces artifactual products as well. To Lamboy, W.F. 1994. Computing genetic similarity coeffi- eliminate RAPD artifacts, we carried out at least two cients from RAPD data: the effects of PCR artifacts. replicates and analyzed only those bands that were repro- PCR Methods Applic. 4: 31-37. ducible. However, discarding faint or inconsistant bands Martin, G.B., Williams, J.G.K. and Tanksley, S.D. 1991. could introduce false negatives into the data. It is also Rapid identification of markers linked to a possible Li's coefficient is recommanded as it displays Pseudomonas resistance gene in tomato by using less percent bias than Jaccard's coefficient (Lamboy random primers and near-isogenic lines. Proc. Natl. 1994). Based on our analyses, the values of Nei and Li's Acad. Sci. USA 88: 2336-2340. coefficients are always higher than that of Jaccard's Reiter, R.S., Williams, J.G.K., Feldmann, K.A., Rafalski, J.A., Tingey, S.V. and Scolnik, P.A. 1992. Global and coefficients for each pair of data. The difference in the local genome mapping in Arabidopsis thaliana by values might be resulted from the bias included in Jac- using recombinant inbred lines and random amplified card's coefficient. Our results showed the advantage of polymorphic DNAs. Proc. Natl. Acad. Sci. USA 89: Nei and Li's coefficient for computing genetic similarity 1477-1481. coefficients for closely related species. Rogers, S.O. and Bendich, A.J. 1988. Extraction of DNA On the basis of the dendrogram constructed with the from plant tissues. In S.B. Gelvin, R.A. Schilperoot, similarity coefficients generated from RAPD markers, the and D.P.S. Verma, ads., Plant Molecular Biology 18 Nicotiana genotypes were divided into two sections, Manual, Dordrecht, Kluwer Academic Publishers, pp. Tomentosae and Alatae, in accordance with the classifi- A6: 1-10. cation of Goodspeed (1954). This study demonstrated Rohlf, F.J. 1993. NTSYS-pc Numerical Taxonomy and that RAPD assay is a rapid and sensitive technique for Multivariate Analysis System, Version 1.80. New identifying phylogenetic relationships at the interspecific York, Applied Biostatistics. and the intraspecific levels in Nicotiana. Tanksley, S.D., Young, N.D., Paterson, A.H. and Bonierale, M.W. 1989. RFLP mapping in plant breeding: new We thank Drs. LC. Chao, C.C. Chen, J. Katchen and P. tools for an old science. Biotechnology 7: 257-264. Keng for critical review of the manuscript. We also thank Tingey, S.V. and del Tufo, J.P. 1993. Genetic analysis with random amplified polymorphic DNA markers. the USDA and the TTRI for providing seeds of Nicotiana. Plant Physiol. 101: 349-352. This work was supported by Grant No. NSC84-2321-B007- Torres, A.M., Weeden, N.F. and Martin, A. 1993. Linkage 004-B05 from the National Science Council, Republic of among isozyme, RFLP and RAPD markers in Vicia China. faba. Theor. Appl. Genet. 85: 937-945. 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