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Seed Surface Architecture and Random Amplified Polymorphic DNA Profiles of Paulownia fortunei, P. tomentosa and their Hybrid
Annals of Botany 83 : 103–107, 1999 Article No. anbo.1998.0780, available online at http:\\www.idealibrary.com on
Seed Surface Architecture and Random Amplified Polymorphic DNA Profiles of Paulownia fortunei, P. tomentosa and their Hybrid P R A K A SH P. K U M AR*, C. D I M P S R A O, G. R A J A S E G E R and A. N. R A O Department of Biological Sciences, National Uniersity of Singapore, 10 Kent Ridge Crescent, Singapore 117600 Received : 25 June 1998
Returned for revision : 24 August 1998
Accepted : 9 September 1998
The surface patterns of winged seeds of Paulownia fortunei, P. tomentosa and P. fortuneiiP. tomentosa were examined by scanning electron microscopy. The pattern of reticulation on the wings and seed coat of P. fortunei and the hybrid are comparable, while that on P. tomentosa is different and more elongated. Also, the wings are more extended at the oblong ends of the seeds in the former when compared to the wings of P. tomentosa. Distinct random amplified polymorphic DNA (RAPD) patterns were obtained for the three taxa and P. kawakamii with five different random oligonucleotide primers, suggesting that the method can yield genetic markers for differentiating the taxa. Also, Southern blot analyses of the RAPD products of the hybrid and the two parent species revealed shared (inherited) genetic polymorphisms. # 1999 Annals of Botany Company Key words : Paulownia species and hybrid, seed surface architecture, reticulated thickening, RAPD markers, Scrophulariaceae.
INTRODUCTION The genus Paulownia (Scrophulariaceae) includes nine species of fast-growing trees, indigenous to China and East Asia (Zhu et al., 1986). These trees were also introduced to North America, Australia, Europe and Japan. The winged seeds of Paulownia are oval, ranging in length from 3 to 7 mm (inclusive of the wing) in the various species (Vujicic, Grubisic and Konjevic, 1993). The wings are laterally extended on the outer edge of the seed and cells of the wing vary in length. Mature walls of these elongated cells have reticulated thickening. The reticulate surface architecture of the wings was examined for eight species of Paulownia (Vujicic et al., 1993), but the earlier work did not include the hybrid taxa of our study. The use of random amplified polymorphic DNA (RAPD) for identification of species and cultivars has been widely recognized (Williams et al., 1990 ; Koller et al., 1993 ; Kumar, Yau and Goh, 1998). In addition, RAPD provides a simple technique for establishing genetic fingerprints for different species, even when no prior genetic information is available. RAPD and RFLP analyses were employed to determine the hybrid origin of P. taiwaniana Shieh & Tsai (Wang et al., 1994). Their data confirmed that P. taiwaniana is a hybrid between P. fortunei Hemsl. and P. kawakamii Ito, and that the maternal parent of the hybrid is P. kawakamii. A putative natural hybrid between P. fortunei and P. tomentosa was obtained from Beijing, China. Morphologically, the hybrid seeds appeared similar to those of P. fortunei but no analytical studies have so far been conducted to determine its hybrid status. The seed surface architecture, including the reticulation on the wings of the hybrid and its * For correspondence. Fax j65-779-2486, e-mail dbskumar! leonis.nus.edu.sg
0305-7364\99\020103j05 $30.00\0
parents, is described in this paper. Total genomic DNA from these taxa were subjected to RAPD analysis. Southern blot analysis was also carried out with five selected RAPD bands in an attempt to ascertain the suitability of the generated DNA markers for identification of Paulownia species and their hybrids. MATERIALS AND METHODS Scanning electron microscopy The seeds of a hybrid between P. fortunei and P. tomentosa (Thumb.) Steud. and those of the parents were obtained from the Chinese Academy of Forestry, Beijing, China. The seed surface architecture of the hybrid was compared with that of the parents using scanning electron microscopy (SEM). Mature dry seeds (without fixation) were glued to aluminum stubs and coated with gold-palladium to a thickness of 40 to 50 nm using a JEOL Finecoat Ion Sputter JFL 1100. Some of the seeds were mounted intact while the others were manually dewinged prior to coating. To facilitate efficient removal of the wings, the seeds were imbibed in water for about 24 h and the wings were dissected out using fine tipped forceps under a stereo dissecting microscope. The dewinged seeds were air-dried and stored (for about 1 week) in a desiccator with silica gel as the desiccant until they were processed for SEM. The specimens were viewed in a JEOL scanning electron microscope (JEOL JSM T220A, Japan) and photographed at different magnifications. For each taxon, at least 15 seeds were randomly selected and studied. Random amplified polymorphic DNA The RAPD patterns of the three taxa along with that of another species, P. kawakamii were examined. Total # 1999 Annals of Botany Company
104
Kumar et al.—SEM and RAPD Analysis of a Paulownia Hybrid
F. 1. For legend see facing page.
F. 2. For legend see facing page.
genomic DNA was extracted by the method of Honda and Hirai (1990) from leaves of shoots maintained in itro (Rao, Goh and Kumar, 1993, 1996). DNA was treated with RNase (10 µg µl−" ; incubation at 37 mC for 20 min) to remove coprecipitated RNA, followed by phenol-chloroform extraction and ethanol precipitation (Kumar et al., 1998). Five different random primers (Operon Technologies, Alameda, Calif., USA) were used for the analysis : OPA1 : 5h-CAGGCCCTTC ; OPA8 : 5h-GTGACGTAGG ; OPA18 : 5h-AGGTGACCGT ; OPU8 : 5h-GGCGAAGGTT ; OPU9 : 5h-CCACATCGGT. The reaction mixture consisted of 100 ng template DNA, 0n4 m each of the four dNTPs (New England Biolabs, Beverly, MA, USA), 1 µ primer, 3 m MgCl , 5 units of Dynazyme4 II (DNA polymerase), # PCR buffer (both from FinnZymes OY, Finland), and sterile distilled water in a final volume of 50 µl. The reaction mixture was subjected to a cycling program using a PTC100 Programmable Thermal Cycler (MJ Research, MA, USA). The amplification conditions were as follows : 2 min
predenaturing at 95 mC followed by 30 cycles of 1 min denaturation at 95 mC, 3 min primer annealing at 37 mC and 2 min extension at 72 mC with a final extension of 10 min. The RAPD products were fractionated by agarose (1n5 % w\v) gel electrophoresis in the presence of ethidium bromide. The gels were viewed and photographed under UV light. The reactions were carried out in triplicate and a negative control (excluding the DNA template only) was included in all experiments. An unrelated plant, Acacia auriculiformis A. Cunn. ex Benth. was used as an additional control species during RAPD analysis. All the consistently reproducible RAPD bands (between 500 and 2000 bp) in each of the three replicate experiments were scored as discrete variables, using 1 to indicate presence and 0 to indicate absence of a band. Analysis was done using an image capture device with a CCD camera and GelPro Analyzer software Version 3.0 (Media Cybernetics, MD, USA). Genetic similarities between the various genotypes were computed using Nei and Li’s similarity
Kumar et al.—SEM and RAPD Analysis of a Paulownia Hybrid
105
twice at room temperature for 5 min each time, and 0n5iSSC\0n1 % SDS, twice at 60 mC for 15 min each time (Sambrook et al., 1989). Colorimetric detection was performed according to the DIG labelling kit. Some of the blots were probed with α-$#P dCTP-labelled (using Rediprime labelling kit ; Amersham International, UK) probes. The blotted membrane was prehybridized using ExpressHyb hybridization solution (Clontech, USA) for 30 min at 60 mC and hybridized overnight (also at 60 mC). Membranes were washed as described above, exposed to Hyperfilm (Amersham, UK) and developed using a Kodak M-35 X-OMAT film processor. RESULTS AND DISCUSSION
F 1–3. Scanning electron micrographs of Paulownia seeds showing the intact seeds (A), reticulation of the wing cells (B), and the surface of the dewinged seeds (C). Fig. 1. P. fortunei. Fig. 2. P. fortuneiiP. tomentosa. Fig. 3. P. tomentosa. Bars l 500 (A), 10 (B) and 100 (C) µm.
index and a phenogram was generated using the unweighted pair-group method with arithmetical averages (UPGMA) as described previously (Kumar et al., 1998). Southern blotting The DNA bands from the RAPD gels were transferred to nylon membrane (Hybond N) by the capillary blotting method (Sambrook, Fritsch and Maniatis, 1989). The DNA bands for probe preparation were extracted from duplicate gels using a Qiagen Gel Extraction kit (Qiagen GmbH, Germany). The eluted DNA was labelled using a random primed DIG DNA labelling kit (Boehringer Mannheim, Germany). Membranes were prehybridized (5iSSC, 1 % Blocking reagent, 0n1 % lauroylsarcosine, 0n02 % SDS) for 2 h at 60 mC. Hybridization was carried out overnight at 60 mC. The membranes were washed in 2iSSC\0n1 % SDS
The overall size and seed surface architecture of the hybrid was comparable to that of P. fortunei (Figs 1–3). The seeds of Paulownia tomentosa were about 30 to 40 % smaller than those of P. fortunei and the hybrid. The wings had a reticulate pattern of thickening in all the taxa studied. The reticulations on the wings of P. fortunei and the hybrid were comparable, but differed to those on P. tomentosa (Figs 1–3). Wings were divided into sectors by prominent thickenings at regular intervals radiating from the centre, with several minor compartments and articulations in each major sector (Figs 1–3). Each of the major compartments was an enlarged cell (Vujicic et al., 1993). The minor compartments of reticulation on the wings of P. fortunei and the hybrid within each major sector were arranged along three to five columns in width (Figs 1 B and 2 B), whereas P. tomentosa had mostly one column of rather elongated compartments (Fig. 3 B). The wings were more elongated in the former two taxa compared to P. tomentosa (Figs 1 A, 2 A and 3 A). The reticulation on the surface of the seed coat, after removal of the wings, was also comparable between P. fortunei and the hybrid (Figs 1 C and 2 C). However, repeated attempts at removing the wings of P. tomentosa seeds failed, probably due to their thinness, resulting in portions of the wing still adhering to the inner layer of the seed coat when viewed under the SEM (Fig. 3 C). Cell wall thickenings in hybrid seeds were almost identical to those of P. fortunei, and it is likely that the female parental traits are expressed in the hybrid seed coat. Thus, our observations appear to suggest that the female parent of the hybrid is P. fortunei. RAPD profiles of the two parents with the five random primers tested were distinct from those of the hybrid. One set of representative profiles generated by primer OPA18 is shown in Fig. 4 A. Only bands between 500 and 2000 bp were included in the analysis. Despite polymorphisms, P. fortunei and P. tomentosa exhibited several common (monomorphic) bands, which might explain the success of hybridization between the two species. Also, some distinct parental bands could be observed (common bands between the hybrid and the parents) in the hybrid (Fig. 4, Table 1, see below). This indicates that the RAPD polymorphic bands are useful as genetic markers for the different taxa under study. Five bands generated by OPA18 were chosen for Southern
106
Kumar et al.—SEM and RAPD Analysis of a Paulownia Hybrid A
1
2
3
4
5
6
7
B bp 1900 bp
*
1800
1500
870 800
730
*
550 400
F. 4. Representative RAPD profiles and Southern analysis of selected RAPD bands. A, RAPD profiles of P. fortunei (lane 2), the hybrid (lane 3), P. tomentosa (lane 4), and P. kawakamii (lane 5) generated by primer OPA18. Two of the parental bands in the hybrid are indicated by asterisks between the lanes. Lane 1 is the molecular weight standards (100 bp ladder, Pharmacia Biotech, Uppsala, Sweden), lane 6 is Acacia auriculiformis, and lane 7 is the template DNA-free, negative control. B, Southern blot analysis of five bands from the RAPD profiles generated by OPA18. The approximate molecular weights (Mr) of the bands used as the probe, indicated on the right, also correspond to the Mr of the bands showing the signals. The 550 bp band was probed with $#P-labelled probe, while the rest of the blots were probed with DIG-labelled DNA. The lane numbering corresponds to that in Fig. 4 A.
T 1. RAPD markers generated by the primer OPA18 (5h-AGGTGACCGT) Molecular weight (bp)
Pf
PH
Pt
Pk
1900 1800 1700 1500 1400 1080 1020 950 870 730 690 600 590 570 550 500
0 1 0 1 0 1 0 1 1 1 1 1 1 1 1 1
1 1 0 1 1 0 0 0 1 1 1 0 1 0 1 1
1 1 1 1 1 1 0 1 1 0 1 0 1 0 1 0
0 1 1 1 1 1 1 1 1 0 1 0 1 0 1 0
T 2. The total number of RAPD bands generated for the arious Paulownia taxa by the fie random primers used Number of bands
The bands are represented as present (1) or absent (0) in the four taxa examined : P. fortunei (Pf), the hybrid (PH), P. tomentosa (Pt), and P. kawakamii (Pk).
blotting analysis. These data clearly indicated that the monomorphic bands (e.g. 1800 bp, 870 bp and 550 bp bands, Fig. 4) were genetically related, because they gave clear positive signals in all four Paulownia species tested. Furthermore, the band at 1900 bp was specific for the
Primer
Pf
PH
Pt
Pk
Total
OPA1 OPA8 OPA18 OPU8 OPU9 Total
11 10 12 10 02 45
13 11 10 10 06 50
15 12 11 12 07 57
12 10 11 10 07 50
51 43 44 42 22 202
P. fortunei (Pf), the hybrid (PH), P. tomentosa (Pt) and P. kawakamii (Pk).
hybrid and P. tomentosa only (Fig. 4). Similarly, the band visible in the ethidium bromide stained gel at 730 bp was confirmed to be specific for P. fortunei and the hybrid. These bands did not hybridize with any other bands on the gel, including the Acacia DNA, indicating that cross hybridization due to the shared primer sequence in the various RAPD bands was not a problem at the stringency of hybridization used (Fig. 4). A total of 202 RAPD bands were scored from the five random primers for the Paulownia species studied (Table 2). When a phenogram was constructed using the pooled data, P. fortunei and the hybrid were clearly grouped under one node, while P. tomentosa and another species, P. kawakamii, grouped under a distinct node (Fig. 5). This is in agreement
Kumar et al.—SEM and RAPD Analysis of a Paulownia Hybrid
107
P. fortunei P. f. × P. t. hybrid
P. tomentosa
P. kawakamii
Level 0.64
0.72
0.80
0.88
F. 5. Phenogram generated with all the RAPD bands scored from five different random primers.
with our observation that the seed morphology of the hybrid resembles that of P. fortunei rather than that of P. tomentosa. The third species tested, P. kawakamii, exhibited distinct RAPD profiles with all the primers tested when compared to P. fortunei and P. tomentosa. The fact that P. tomentosa appears to be genetically closer to P. kawakamii than to P. fortunei (Fig. 5), suggests that hybridization between these two species could be possible. As mentioned earlier, evidence for the hybrid nature of P. taiwaniana was obtained using RAPD and RFLP analyses (Wang et al., 1994). It should be pointed out that besides the work of Wang et al. (1994), there is no other genetic information available for Paulownia species. In the absence of such data, establishing ‘ genetic ’ or DNA-based markers for species identification, e.g. using RAPDs as in the present study, is of significance. Our observations confirm the hybrid status of the taxon under study and suggest that P. fortunei is likely to be the female parent of this natural hybrid. Based on our observations from RAPD analysis we can also conclude that RAPD markers are suitable for identification of the economically important taxa studied. A C K N O W L E D G E M E N TS We thank Prof. Z. H. Zhu, Chinese Academy of Forestry, Beijing, China for providing the Paulownia seeds, Mr Pavan Prakash for help with the Southern blots and Mr T. K. Ong for assistance with photography.
LITERATURE CITED Honda H, Hirai A. 1990. A simple and efficient method for identification of hybrids using non-radioactive ribosomal DNA as probe. Japanese Journal of Breeding 40 : 339–349. Koller B, Lehman A, McDermott JM, Gessler C. 1993. Identification of apple cultivars using RAPD markers. Theoretical and Applied Genetics 85 : 901–904. Kumar PP, Yau CKJ, Goh CJ. 1998. Genetic analyses of Heliconia species and cultivars with randomly amplified polymorphic DNA (RAPD) markers. Journal of American Society for Horticultural Science 123 : 91–97. Rao CD, Goh CJ, Kumar PP. 1993. High frequency plant regeneration from excised leaves of Paulownia fortunei. In Vitro Cellular and Deelopmental Biology–Plant 29P : 72–76. Rao CD, Goh CJ, Kumar PP. 1996. High frequency adventitious shoot regeneration from excised leaves of Paulownia spp. cultured in itro. Plant Cell Reports 16 : 204–209. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning : A laboratory manual. 2nd edn. New York : Cold Spring Harbor Laboratory Press. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18 : 6531–6535. Vujicic R, Grubisic D, Konjevic R. 1993. Scanning electron microscopy of the seed coat in the genus Paulownia (Scrophulariaceae). Botanical Journal of the Linnean Society 111 : 505–511. Wang WY, Pai RC, Lai CC, Lin TP. 1994. Molecular evidence for the hybrid origin of Paulownia taiwaniana based on RAPD markers and RFLP of chloroplast DNA. Theoretical and Applied Genetics 89 : 271–275. Zhu ZH, Chao CJ, Lu XY, Xiong YG. 1986. Paulownia in China : Cultiation and utilization. Singapore : Asian Network Biological Sciences and International Development Research Center of Canada.
Seed Surface Architecture and Random Amplified Polymorphic DNA Profiles of Paulownia fortunei, P. tomentosa and their Hybrid P R A K A SH P. K U M AR*, C. D I M P S R A O, G. R A J A S E G E R and A. N. R A O Department of Biological Sciences, National Uniersity of Singapore, 10 Kent Ridge Crescent, Singapore 117600 Received : 25 June 1998
Returned for revision : 24 August 1998
Accepted : 9 September 1998
The surface patterns of winged seeds of Paulownia fortunei, P. tomentosa and P. fortuneiiP. tomentosa were examined by scanning electron microscopy. The pattern of reticulation on the wings and seed coat of P. fortunei and the hybrid are comparable, while that on P. tomentosa is different and more elongated. Also, the wings are more extended at the oblong ends of the seeds in the former when compared to the wings of P. tomentosa. Distinct random amplified polymorphic DNA (RAPD) patterns were obtained for the three taxa and P. kawakamii with five different random oligonucleotide primers, suggesting that the method can yield genetic markers for differentiating the taxa. Also, Southern blot analyses of the RAPD products of the hybrid and the two parent species revealed shared (inherited) genetic polymorphisms. # 1999 Annals of Botany Company Key words : Paulownia species and hybrid, seed surface architecture, reticulated thickening, RAPD markers, Scrophulariaceae.
INTRODUCTION The genus Paulownia (Scrophulariaceae) includes nine species of fast-growing trees, indigenous to China and East Asia (Zhu et al., 1986). These trees were also introduced to North America, Australia, Europe and Japan. The winged seeds of Paulownia are oval, ranging in length from 3 to 7 mm (inclusive of the wing) in the various species (Vujicic, Grubisic and Konjevic, 1993). The wings are laterally extended on the outer edge of the seed and cells of the wing vary in length. Mature walls of these elongated cells have reticulated thickening. The reticulate surface architecture of the wings was examined for eight species of Paulownia (Vujicic et al., 1993), but the earlier work did not include the hybrid taxa of our study. The use of random amplified polymorphic DNA (RAPD) for identification of species and cultivars has been widely recognized (Williams et al., 1990 ; Koller et al., 1993 ; Kumar, Yau and Goh, 1998). In addition, RAPD provides a simple technique for establishing genetic fingerprints for different species, even when no prior genetic information is available. RAPD and RFLP analyses were employed to determine the hybrid origin of P. taiwaniana Shieh & Tsai (Wang et al., 1994). Their data confirmed that P. taiwaniana is a hybrid between P. fortunei Hemsl. and P. kawakamii Ito, and that the maternal parent of the hybrid is P. kawakamii. A putative natural hybrid between P. fortunei and P. tomentosa was obtained from Beijing, China. Morphologically, the hybrid seeds appeared similar to those of P. fortunei but no analytical studies have so far been conducted to determine its hybrid status. The seed surface architecture, including the reticulation on the wings of the hybrid and its * For correspondence. Fax j65-779-2486, e-mail dbskumar! leonis.nus.edu.sg
0305-7364\99\020103j05 $30.00\0
parents, is described in this paper. Total genomic DNA from these taxa were subjected to RAPD analysis. Southern blot analysis was also carried out with five selected RAPD bands in an attempt to ascertain the suitability of the generated DNA markers for identification of Paulownia species and their hybrids. MATERIALS AND METHODS Scanning electron microscopy The seeds of a hybrid between P. fortunei and P. tomentosa (Thumb.) Steud. and those of the parents were obtained from the Chinese Academy of Forestry, Beijing, China. The seed surface architecture of the hybrid was compared with that of the parents using scanning electron microscopy (SEM). Mature dry seeds (without fixation) were glued to aluminum stubs and coated with gold-palladium to a thickness of 40 to 50 nm using a JEOL Finecoat Ion Sputter JFL 1100. Some of the seeds were mounted intact while the others were manually dewinged prior to coating. To facilitate efficient removal of the wings, the seeds were imbibed in water for about 24 h and the wings were dissected out using fine tipped forceps under a stereo dissecting microscope. The dewinged seeds were air-dried and stored (for about 1 week) in a desiccator with silica gel as the desiccant until they were processed for SEM. The specimens were viewed in a JEOL scanning electron microscope (JEOL JSM T220A, Japan) and photographed at different magnifications. For each taxon, at least 15 seeds were randomly selected and studied. Random amplified polymorphic DNA The RAPD patterns of the three taxa along with that of another species, P. kawakamii were examined. Total # 1999 Annals of Botany Company
104
Kumar et al.—SEM and RAPD Analysis of a Paulownia Hybrid
F. 1. For legend see facing page.
F. 2. For legend see facing page.
genomic DNA was extracted by the method of Honda and Hirai (1990) from leaves of shoots maintained in itro (Rao, Goh and Kumar, 1993, 1996). DNA was treated with RNase (10 µg µl−" ; incubation at 37 mC for 20 min) to remove coprecipitated RNA, followed by phenol-chloroform extraction and ethanol precipitation (Kumar et al., 1998). Five different random primers (Operon Technologies, Alameda, Calif., USA) were used for the analysis : OPA1 : 5h-CAGGCCCTTC ; OPA8 : 5h-GTGACGTAGG ; OPA18 : 5h-AGGTGACCGT ; OPU8 : 5h-GGCGAAGGTT ; OPU9 : 5h-CCACATCGGT. The reaction mixture consisted of 100 ng template DNA, 0n4 m each of the four dNTPs (New England Biolabs, Beverly, MA, USA), 1 µ primer, 3 m MgCl , 5 units of Dynazyme4 II (DNA polymerase), # PCR buffer (both from FinnZymes OY, Finland), and sterile distilled water in a final volume of 50 µl. The reaction mixture was subjected to a cycling program using a PTC100 Programmable Thermal Cycler (MJ Research, MA, USA). The amplification conditions were as follows : 2 min
predenaturing at 95 mC followed by 30 cycles of 1 min denaturation at 95 mC, 3 min primer annealing at 37 mC and 2 min extension at 72 mC with a final extension of 10 min. The RAPD products were fractionated by agarose (1n5 % w\v) gel electrophoresis in the presence of ethidium bromide. The gels were viewed and photographed under UV light. The reactions were carried out in triplicate and a negative control (excluding the DNA template only) was included in all experiments. An unrelated plant, Acacia auriculiformis A. Cunn. ex Benth. was used as an additional control species during RAPD analysis. All the consistently reproducible RAPD bands (between 500 and 2000 bp) in each of the three replicate experiments were scored as discrete variables, using 1 to indicate presence and 0 to indicate absence of a band. Analysis was done using an image capture device with a CCD camera and GelPro Analyzer software Version 3.0 (Media Cybernetics, MD, USA). Genetic similarities between the various genotypes were computed using Nei and Li’s similarity
Kumar et al.—SEM and RAPD Analysis of a Paulownia Hybrid
105
twice at room temperature for 5 min each time, and 0n5iSSC\0n1 % SDS, twice at 60 mC for 15 min each time (Sambrook et al., 1989). Colorimetric detection was performed according to the DIG labelling kit. Some of the blots were probed with α-$#P dCTP-labelled (using Rediprime labelling kit ; Amersham International, UK) probes. The blotted membrane was prehybridized using ExpressHyb hybridization solution (Clontech, USA) for 30 min at 60 mC and hybridized overnight (also at 60 mC). Membranes were washed as described above, exposed to Hyperfilm (Amersham, UK) and developed using a Kodak M-35 X-OMAT film processor. RESULTS AND DISCUSSION
F 1–3. Scanning electron micrographs of Paulownia seeds showing the intact seeds (A), reticulation of the wing cells (B), and the surface of the dewinged seeds (C). Fig. 1. P. fortunei. Fig. 2. P. fortuneiiP. tomentosa. Fig. 3. P. tomentosa. Bars l 500 (A), 10 (B) and 100 (C) µm.
index and a phenogram was generated using the unweighted pair-group method with arithmetical averages (UPGMA) as described previously (Kumar et al., 1998). Southern blotting The DNA bands from the RAPD gels were transferred to nylon membrane (Hybond N) by the capillary blotting method (Sambrook, Fritsch and Maniatis, 1989). The DNA bands for probe preparation were extracted from duplicate gels using a Qiagen Gel Extraction kit (Qiagen GmbH, Germany). The eluted DNA was labelled using a random primed DIG DNA labelling kit (Boehringer Mannheim, Germany). Membranes were prehybridized (5iSSC, 1 % Blocking reagent, 0n1 % lauroylsarcosine, 0n02 % SDS) for 2 h at 60 mC. Hybridization was carried out overnight at 60 mC. The membranes were washed in 2iSSC\0n1 % SDS
The overall size and seed surface architecture of the hybrid was comparable to that of P. fortunei (Figs 1–3). The seeds of Paulownia tomentosa were about 30 to 40 % smaller than those of P. fortunei and the hybrid. The wings had a reticulate pattern of thickening in all the taxa studied. The reticulations on the wings of P. fortunei and the hybrid were comparable, but differed to those on P. tomentosa (Figs 1–3). Wings were divided into sectors by prominent thickenings at regular intervals radiating from the centre, with several minor compartments and articulations in each major sector (Figs 1–3). Each of the major compartments was an enlarged cell (Vujicic et al., 1993). The minor compartments of reticulation on the wings of P. fortunei and the hybrid within each major sector were arranged along three to five columns in width (Figs 1 B and 2 B), whereas P. tomentosa had mostly one column of rather elongated compartments (Fig. 3 B). The wings were more elongated in the former two taxa compared to P. tomentosa (Figs 1 A, 2 A and 3 A). The reticulation on the surface of the seed coat, after removal of the wings, was also comparable between P. fortunei and the hybrid (Figs 1 C and 2 C). However, repeated attempts at removing the wings of P. tomentosa seeds failed, probably due to their thinness, resulting in portions of the wing still adhering to the inner layer of the seed coat when viewed under the SEM (Fig. 3 C). Cell wall thickenings in hybrid seeds were almost identical to those of P. fortunei, and it is likely that the female parental traits are expressed in the hybrid seed coat. Thus, our observations appear to suggest that the female parent of the hybrid is P. fortunei. RAPD profiles of the two parents with the five random primers tested were distinct from those of the hybrid. One set of representative profiles generated by primer OPA18 is shown in Fig. 4 A. Only bands between 500 and 2000 bp were included in the analysis. Despite polymorphisms, P. fortunei and P. tomentosa exhibited several common (monomorphic) bands, which might explain the success of hybridization between the two species. Also, some distinct parental bands could be observed (common bands between the hybrid and the parents) in the hybrid (Fig. 4, Table 1, see below). This indicates that the RAPD polymorphic bands are useful as genetic markers for the different taxa under study. Five bands generated by OPA18 were chosen for Southern
106
Kumar et al.—SEM and RAPD Analysis of a Paulownia Hybrid A
1
2
3
4
5
6
7
B bp 1900 bp
*
1800
1500
870 800
730
*
550 400
F. 4. Representative RAPD profiles and Southern analysis of selected RAPD bands. A, RAPD profiles of P. fortunei (lane 2), the hybrid (lane 3), P. tomentosa (lane 4), and P. kawakamii (lane 5) generated by primer OPA18. Two of the parental bands in the hybrid are indicated by asterisks between the lanes. Lane 1 is the molecular weight standards (100 bp ladder, Pharmacia Biotech, Uppsala, Sweden), lane 6 is Acacia auriculiformis, and lane 7 is the template DNA-free, negative control. B, Southern blot analysis of five bands from the RAPD profiles generated by OPA18. The approximate molecular weights (Mr) of the bands used as the probe, indicated on the right, also correspond to the Mr of the bands showing the signals. The 550 bp band was probed with $#P-labelled probe, while the rest of the blots were probed with DIG-labelled DNA. The lane numbering corresponds to that in Fig. 4 A.
T 1. RAPD markers generated by the primer OPA18 (5h-AGGTGACCGT) Molecular weight (bp)
Pf
PH
Pt
Pk
1900 1800 1700 1500 1400 1080 1020 950 870 730 690 600 590 570 550 500
0 1 0 1 0 1 0 1 1 1 1 1 1 1 1 1
1 1 0 1 1 0 0 0 1 1 1 0 1 0 1 1
1 1 1 1 1 1 0 1 1 0 1 0 1 0 1 0
0 1 1 1 1 1 1 1 1 0 1 0 1 0 1 0
T 2. The total number of RAPD bands generated for the arious Paulownia taxa by the fie random primers used Number of bands
The bands are represented as present (1) or absent (0) in the four taxa examined : P. fortunei (Pf), the hybrid (PH), P. tomentosa (Pt), and P. kawakamii (Pk).
blotting analysis. These data clearly indicated that the monomorphic bands (e.g. 1800 bp, 870 bp and 550 bp bands, Fig. 4) were genetically related, because they gave clear positive signals in all four Paulownia species tested. Furthermore, the band at 1900 bp was specific for the
Primer
Pf
PH
Pt
Pk
Total
OPA1 OPA8 OPA18 OPU8 OPU9 Total
11 10 12 10 02 45
13 11 10 10 06 50
15 12 11 12 07 57
12 10 11 10 07 50
51 43 44 42 22 202
P. fortunei (Pf), the hybrid (PH), P. tomentosa (Pt) and P. kawakamii (Pk).
hybrid and P. tomentosa only (Fig. 4). Similarly, the band visible in the ethidium bromide stained gel at 730 bp was confirmed to be specific for P. fortunei and the hybrid. These bands did not hybridize with any other bands on the gel, including the Acacia DNA, indicating that cross hybridization due to the shared primer sequence in the various RAPD bands was not a problem at the stringency of hybridization used (Fig. 4). A total of 202 RAPD bands were scored from the five random primers for the Paulownia species studied (Table 2). When a phenogram was constructed using the pooled data, P. fortunei and the hybrid were clearly grouped under one node, while P. tomentosa and another species, P. kawakamii, grouped under a distinct node (Fig. 5). This is in agreement
Kumar et al.—SEM and RAPD Analysis of a Paulownia Hybrid
107
P. fortunei P. f. × P. t. hybrid
P. tomentosa
P. kawakamii
Level 0.64
0.72
0.80
0.88
F. 5. Phenogram generated with all the RAPD bands scored from five different random primers.
with our observation that the seed morphology of the hybrid resembles that of P. fortunei rather than that of P. tomentosa. The third species tested, P. kawakamii, exhibited distinct RAPD profiles with all the primers tested when compared to P. fortunei and P. tomentosa. The fact that P. tomentosa appears to be genetically closer to P. kawakamii than to P. fortunei (Fig. 5), suggests that hybridization between these two species could be possible. As mentioned earlier, evidence for the hybrid nature of P. taiwaniana was obtained using RAPD and RFLP analyses (Wang et al., 1994). It should be pointed out that besides the work of Wang et al. (1994), there is no other genetic information available for Paulownia species. In the absence of such data, establishing ‘ genetic ’ or DNA-based markers for species identification, e.g. using RAPDs as in the present study, is of significance. Our observations confirm the hybrid status of the taxon under study and suggest that P. fortunei is likely to be the female parent of this natural hybrid. Based on our observations from RAPD analysis we can also conclude that RAPD markers are suitable for identification of the economically important taxa studied. A C K N O W L E D G E M E N TS We thank Prof. Z. H. Zhu, Chinese Academy of Forestry, Beijing, China for providing the Paulownia seeds, Mr Pavan Prakash for help with the Southern blots and Mr T. K. Ong for assistance with photography.
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