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RAPD analysis of genetic relationships among Alstroemeria L. cultivars
SCIENTIA HORTlCULTulM ELSEVIER
Scientia Horticulhme
68 ( 1997) 18 I- 189
RAPD analysis of genetic relationships among Alstroemeria L. cultivars Joseph G. Dubouzet *, Naho Murata, Koichi Shinoda Hokkaido Nutional Agricultural Experiment Station, Hitsujigaoku- I, Toyohira-ku, Sapporo 062 Japan Accepted
19 August
1996
Abstract A procedure for the rapid extraction of partially purified nucleic acid extracts from Afstroemepolymerase chain reaction (PCR) protocol for the generation of random amplified polymorphic DNA (RAPD) markers were established. Nucleic acid extracts from 23 Afstroemeriu cultivars were amplified with 8 random decamers by PCR. OPCO2,OPCO3, 0PD02, OPDOS, OPDO8, OPDll, 0PD13 and OPDl8 produced 24, 19,21,20, 10, 17,25 and 29 RAPD bands, respectively. The distinctive RAPD patterns generated from these cultivars could be used as genomic ‘fingerprints’ to establish the identity of a given genotype. The ‘Orchid’ and ‘Butterfly’ types were clearly separated in distinct subclusters in a phyllogram obtained by unweighted pair group method analysis (UPGMA) of the genetic distances. The ‘Hybrid’ types were distributed in two major subclusters, reflecting the diversity of the parental species used to generate the population. This phyllogram conformed to expectations based on the available pedigree data. 0 Elsevier Science B.V.
ria cultivars and the corresponding
Keywords: Alstroemeria; RAPD analysis;
genetic distance;
UPGMA
1. Introduction The genus Afstroemeriu is classified under the subclass Monocotyledonae and the family Alstroemeriaceae. The main center of distribution is in the Andes mountains (eastern Chile) with approximately 30 species; a satellite center is located at lower altitudes in central and eastern Brazil with about 23 species (Aker and Healy, 1990; Tombolato et al., 1991). There are four species in Argentina and three in Peru (Bridgen, 1993).
* Corresponding
author.
0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(96)00966-l
182
J.G. Dubouzet et al. / Scientiu Horticulturue 68 11997) 181-189
Modem Alstroemeria cultivars were developed through inter specific hybridization, selection of mutant sports and/or polyploidization (Broertjes and Verboom, 1974; Broertjes and Harten, 1987; Tsuchiya and Hang, 1987). However, the pedigrees of most Alsfroemeria cultivars are secret because they were developed by private entities. Published accounts of cultivar development also claim that their breeding programs were initially based on the seedlings of unknown parentage (Goemans, 1962; Miyake, 1989). Breeders need to know the genetic similarity between potential parents because Alstroemeria are dichogamous species that show a great deal of hybrid vigor upon inter specific hybridization. Breeders unaware of the similarity between their parental species run the risk of inbreeding depression since many of the ‘modem’ Alsfroemeria cultivars were obtained by mere radiation mutation or induced polyploidization (Broertjes and Verboom, 1974; De Jeu et al., 1992). Hence, it is necessary to develop a system of genomic fingerprinting so that the identity of specific cultivars can be rapidly determined. Such a system can be used by geneticists to study the evolution of this genus and it may enable breeders to formulate reasonable breeding plans. A genetic fingerprinting system will also be useful in protecting the rights of the plant patent holders. Previous researchers (Tsuchiya and Hang, 1987; Tsuchiya and Hang, 1989) attempted to trace the ancestry of current Alstroemeria cultivars using cytological techniques but the differences in chromosome morphology, number and Giemsa staining patterns were insufficient to differentiate the various species and cultivars. In recent years, random amplified polymorphic DNA (RAPD) analysis through the polymerase chain reaction (PCR) has become widely used to characterize and trace the phylogeny of diverse plant and animal species. The main advantages of RAPD analysis over other methods (e.g., random fragment length polymorphism analysis) are its low sample DNA requirement and the high frequency of polymorphic bands detected (Williams et al., 1990). This article deals with the establishment of a genomic fingerprinting system for Alsfroemeria, starting with the rapid micro extraction of nucleic acids from 23 cultivars, the generation of RAPD bands by 8 random decamers through PCR estimation of the genetic distances and phylogenetic analysis.
2. Materials and methods Data pertaining to the cultivars included in this study were obtained from various sources (including seed catalogues) and are summarized in Table 1. According to available publications, the cultivars in this study were classified as 1) ‘Orchid’ type (cvs. Canaria, Casablanca, Eleanor, La Paz and Paloma), 2) ‘Butterfly’ type (cvs. Amanda, Ostara and Snow Queen), and 3) ‘Hybrid’ type (rest of the cultivars). 20mg of young (flush) leaf tissues were obtained from 23 Alstroemeria cultivars (Table 1) and minced with scissors. Tissues were homogenized in 200 p,l of the extraction buffer (4M guanidine thiocyanate, 0.1 M EDTA-2Na, 1% cetyltrimethyl-ammonium bromide, 1% polyvinylpyrrolidone in 0.1 M Tris-HCI pH 8) using a plastic homogenizer attached to a battery-powered tooth brush. The homogenate was extracted
ff fff ffff fff
K
s PhytoNova P
Paloma 2. Butterfly Amanda Ostara Snow Queen 3. Hybrid Amor Bianca Carola Cobra Evita Gloria Java Little Sun Nevada Pink Triumph
Orange Red Rose/white Pink
ffff fff ffff ffff
Sunrise Tiara Vienna Wilhelmina
to the number of seasons in a year
Pink
fff
Rosita
’ f corresponds
Yellow White Pink/yellow Red Red Orange Pink Red White Pink
White/ rose Purple/white White
fff fff fff ffff ffff ffff ffff ffff ff ff
s s s K van Zanten (Z) Z K
(P1
Yellow
White/yellow Yellow Yellow
ffff ff
ff
Konst (K) Wulfinghoff K
Yellow
Flower Colors
ff
Casablanca Eleanor La Paz
(W)
in this study.
Flowering Time a
included
Van Staaveren (S)
Source
of Alsfroemeria cultivars
1. Orchid Canaria
Hybrid Type/ Cultivar
Table 1 Characteristics
180-200 180-220
110
120-160 140
150-180
140-160 180-220
180 165
150-160
150
200-220
200-210 160-180 200-220
150-200
Flower Stem Length (cm)
(Hachinohe,
1994)
‘Butterfly’ ‘Butterfly’
type X ‘Orchid’ type (Ohkawa, 1994) type X A. aurea lutea (Ohkawa, 1994)
Two homologous and one different genome (Tsuchiya and Hang, 1987) Mutant of cv. Regina (A. pelegrina X A. uureu ) (Broertjes and Verboom, 1974)
Sport of cv. Wilhelmina
Mutant of cv. Orchid (A. pauperculu X A. aurea ) (Van Scheepen, 1991) cv. Orchid X cv. Paloma (Ohkawa, 1994) ? X A. aurecr lutea (Ohkawa, 1994) Radiation mutant of cv. Rio (? X A. urea luteu ) (Ohkawa, 1994) A. aurea X A. aureu (Ohkawa, 1994)
Notes
184
J.G. Dubouzet et al./kientia
Horticulturae 68 (1997) 181-189
with 200 pl 1-bromo-3-chloropropane and the phases were allowed to separate naturally. The nucleic acids were precipitated from the 150 cl.1aqueous supemate by adding 450 p,l ethanol and centrifuging at 15 OOOrpm for 1 min. The precipitate was resuspended in 450$70% ethanol and then centrifuged at 15 000 t-pm for 5 min. The washed precipitate was air dried and then suspended in 200 pl TE at pH 7.5. Replicate extracts were prepared using tissues from a different leaf of the same shoot. UV absorbance of the nucleic acid extracts was measured using a Beckman DU 65 spectrophotometer. The extracts had an average optical density at 260nm (A,,) = 14.5 k 7.7 and an average A,,,/A,s, = 1.71 f 0.1. These extracts were adjusted to long nucleic acid per ~1 and used for PCR. The 10 (~1PCR reaction mix consisted of 5 pl DNA extract, 1.6 pl 25 mM MgCl,, 1.35 pl de-ionized water, 1.Opl 10X Buffer II, 0.8 p.1 1OmM dNTP mix, 0.2 ~1 10 pM random decamer (Operon Industries, USA) and 0.05l~l AmpliTaq DNA polymerase (Perkin Elmer, USA). The random decamers were 0PC02 (5’-GTGAGGCGTC-3’), OPC03 (5’~GGGGGTCTIT-3’1, OPD 02 (5’-GGACCCAACC-3’1, 0PD05 (5’TGAGCGGACA-3’), OPDOS (5’-GTGTGCCCCA-3’), OPDl 1 (5’-AGCGCCA’ITG-3’), 0PD13 (5’~GGGGTGACGA-3’) and OPD18 (5’-GAGAGCCAAC-3’). The replicate DNA extracts were simultaneously amplified. Thermal cycling was performed using the GeneAmp PCR System 9600 from Perkin Elmer. The thermal profile consisted of an initial denaturation step at 94°C for 1.5 min followed by 45 cycles of 94°C for 15 s, 36 “C for 15 s and 72 “C for 2 min and a final exposure to 72 “C for 5 min. The 10 ~1 amplified products and 0.5 pg Hin dIII-Eco RI double digest molecular marker were electrophoresed in a minigel consisting of 1.5% Agarose S (Nippon Gene, Japan) and 1 kgml -’ ethidium bromide in 40mM Tris-acetate and 1 mM EDTA (TAE) buffer at 1OOV. The gels were photographed using Polaroid 667 film. The prints were enlarged 350% using an A935 Fuji Xerox (Japan) photocopier to facilitate RAPD band comparisons. The RAPD bands were scored as either present (1) or absent (0). The relationship between each pair of entries was estimated using a genetic distance formula derived from the similarity index recommended by Nei and Li (1979): =I,2
GeneticDistance = 1 - G,
+G2
where G, and G, are the total number of RAPD bands of genotypes 1 and 2, respectively, and G,,2 is the number of bands common to both genotypes. The genetic distance values were analyzed by unweighted pair group analysis (UPGMA) using J. Felsenstein’s package of phylogenetic programs (Phylip v.3.572). The analytical results were illustrated using R. Page’s TreeView v. 1. Both programs are available through anonymous file transfer protocol from the Internet.
3. Results The eight random primers generated a total of 165 RAPD bands: OPCO2,OPCO3 and OPDO2,OPDO5, OPDOS, OPDll, 0PD13 and 0PD18 produced 24, 19, 21,20, 10, 17,
J.G. Dubouzet et al./Scientia kb
Horticulturae 68 (1997) 181-189
185
M
1.38
-I
8.53
+
Fig. 1. RAPD bands generated by OPDS from 11 Ahtroemeria cvs. Lane M: Eco R I/ Hin dII1 double digest size marker; lane 1 - cv. Canaria; lane 2 - cv. La Paz; lane 3 - cv. Eleanor; lane 4 - cv. Paloma; lane 5- cv. Amanda; lane 6 - cv. Ostara; lane 7 - cv. Snow Queen; lane 8 - cv. Amor; lane 9 - cv. Bianca; lane 10 - cv. Java and lane 11
- cv. Wilhelmina.
25 and 29 polymorphic bands, respectively. Fig. 1 shows the RAPD pattern generated by 0PD05 from genomic DNA of four ‘Orchid’ types (cvs. Canaria, La Paz, Eleanor and Paloma), three ‘Butterfly’ types (cvs. Amanda, Ostara and Snow Queen) and four ‘Hybrid’ types (cvs. Amor, Bianca, Java and Wilhelmina). RAPD analysis was based on the amplification patterns of replicate extracts.
flmanda
-p
L
0.1 Branch
I
OSt al-a snow ttueen
length
’
Fig. 2. Phyllogram obtained by unweighted pair group method analysis of 165 RAPD markers generated by 8 random primers from 23 Alstroemeria cultivars. Numbers indicate specific subclusters discussed in the text.
186
J.G. Dubouzet et al./Scientia
Horticulturae 68 (1997) 181-189
The genetic distance values obtained using the formula derived from Nei and Li’s similarity index are available from the authors upon request. Fig. 2 shows the relationships among cultivars according to UPGM Analysis of the genetic distances.
4. Discussion The cv. Orchid (syn. cv. Walter Fleming) is a product of A. pauperculu Philippi (syn. A. oiohcea Philippi) X A. aureu Graham (Van Scheepen, 1991). The original diploid hybrid between A. uureu and A. puupercufu was sterile but a tetraploid sport was subsequently found to be fertile. This sport was crossed with other diploid species to produce the triploid ‘Orchid’ types (De Jeu et al., 1992). Tsuchiya et al. (1987) reported that cv. Orchid had some chromosome types similar to those found in A. area. The ‘Orchid’ types included in this study are descendants of A. uureu (Table 1). ‘Orchid’ types flower for 3-5 months in the spring with little or no flower production in the rest of the year (Bridgen, 1993). The cv. Eleanor (Table 1) seems to be a major exception to this generalization. ‘Butterfly’ types generally have both Brazilian and Chilean genes in their family tree (Bridgen, 1993). The ‘Butterfly’ hybrids consist of hybrids between A. pefegrinu L. or A. puuperculu and A. psittucinu Lehm. (syn. A. pulchellu) or A. inodoru. The genomes of these sterile diploids were artificially doubled to produce fertile allotetraploids (De Jeu et al., 1992). Induced tetraploids in Alstroemeriu have large, thick, dark green leaves, large, wide and thick flower petals, and longer vase lives (Tsuchiya et al. 1987). ‘Butterfly’ types have short growth habits and they have larger and more open flowers. Cool temperatures will induce flowering and long photo periods will maintain flowering of the ‘Butterfly’ cultivars for 9-12 months in a year (Bridgen, 1993). ‘Hybrid’ types were produced in Europe by crossing different Alstroemeriu species from Chile, Brazil and Peru (De Jeu et al., 1992). Some ‘Hybrid’ types were obtained by crossing ‘Butterfly’ and ‘Orchid’ types, e.g., cv. Vienna (Ohkawa, 1994). The flower colors, flower stem lengths and flowering times of ‘Hybrid’ types (as shown in Table 1) reflect the diversity of the ancestries of these cultivars. A. uureu, A. puuperculu, A. pelegrinu, A. ligtu L., and A. ligtu ssp simsii (Spreng.) Bayer have been suggested as contributory progenitors to these modem European cultivars (Aker and Healy, 1990; Broertjes and Verboom, 1974; Langton, 1987). However, Rustanius et al. (1991) claimed that A. ligtu hybrids may not have been used in the production of modem European and North American Alstroemeriu cultivars. The genetic basis for the classification of modem Alstroemeriu cultivars is not clear since pedigree data are either lacking or ambiguous. The following discussion describes a possible classification of 23 Alstroemeriu cultivars based on RAPD and phylogenetic analyses. The eight random decamers generated distinctive amplification products from the entries. The representative RAPD patterns shown in Fig. 1 were generated by 0PD05. The RAPD patterns obtained from the ‘Orchid’ types (lanes l-4) are relatively more
J.G. Dubouzet et al. / Scientia Horticulturae 68 (1997) 181 -I 89
187
similar compared with those generated from the ‘Butterfly’ types (lanes 5-7). This can be ascribed to the difference in parentage of these two types. The ‘Hybrid’ types (lanes 8-10) showed correspondingly more variable RAPD patterns. The RAPD pattern generated from cv. Java (lane 10) was almost identical to that obtained from cv. Wilhelmina (lane 11); this is because cv. Java is just a mutant sport of cv. Wilhelmina. These distinctive RAPD patterns can be used as genomic ‘fingerprints’ to establish the identity of a given genotype. The genetic distance value provides a useful estimate of the relationship between a specific pair or a small number of genotypes. Phylogenetic analysis, however, is more appropriate for the interpretation of all possible relationships among a large group of genotypes. The various relationships among these cultivars are shown in the phyllogram (Fig. 2) generated by UPGM analysis of the genetic distances. The ‘Butterfly’ types (cvs. Amanda, Ostara and Snow Queen; subcluster 4) are grouped in a distinctly separate subcluster in this phyllogram. The ‘Butterfly’ types are well separated from the ‘Orchid’ types (cvs. Canaria, Eleanor, La Paz and Paloma; subcluster 7). Thus, this phyllogram supports previous reports that claimed these two types were derived from different sets of parental species. The phyllogram also indicates that the modem hybrids are more closely related to the ‘Orchid’ types than to the ‘Butterfly’ types. This implies that A. aureu, which is the main parent identified in the pedigrees of these “Orchid” types (Table l), may be a common parent to these ‘Hybrid’ types. This hypothesis needs to be verified in future studies. Although located on a different branch, the cv. Casablanca is clearly associated with the ‘Orchid’ types, as shown by its location in this phyllogram. The ‘Orchid’ types (cvs. Canaria, Eleanor and La Paz; subcluster 8) were closely related. This is also indicated by the great similarity in the flower structure and color patterns of these three cultivars. The close similarity between cvs. Eleanor and La Paz may be because they have the same male parent (A. uurea lutea). Paloma and Casablanca, which are also classified as ‘Orchid’ types, show a slightly different flower structure and color pattern from the former three cultivars; this difference is reflected in a greater separation from cvs. Canaria, Eleanor and La Paz. The ‘Hybrid’ types are separated in two subclusters (5 and 6) between the ‘Butterfly’ and ‘Orchid’ types. Subcluster 5, which is closer to the ‘Orchid’ types, includes the cvs. Amor, Bianca, Cobra, Gloria and Little Sun. Except for cv. Little Sun, these cultivars are similar to the ‘Orchid’ types in floral morphology. The flowering characteristic of cv. Bianca is quite similar to the ‘Orchid’ types. The rest of the ‘Hybrid’ types, which are more genetically different from the ‘Orchid’ types, are grouped in subcluster 6. The cv. Rosita is a mutant of a product of A. pelegrinu X A. area. As mentioned above, A. pelegrinu was putatively used in the production of some of the ‘Butterfly’ types; it has never been reported as a parent of the ‘Orchid’ types. The cvs. Java, Wilhelmina and Vienna were obtained from crosses using ‘Butterfly’ types as maternal parents. The distribution of the ‘Hybrid’ types into different subclusters may be an indication of the diversity of the parental species used to generate this population. The cvs. Java and Wilhelmina were the most closely related among the ‘Hybrid’ types; this close similarity is expected since cv. Java is a mutant sport of cv. Wilhelmina (Hachinohe,
188
J.G. Dubouzet et al./ Scientia Horticulturae 68 (I 997) 181-189
1994). These two cultivars also have a partial resemblance in gross flower structure and coloration. The phyllogram can be used in the formulation of breeding plans. For example, if crosses involving the most distantly related cultivars are desired, then crosses between the ‘Orchid’ and the ‘Butterfly’ types can be recommended. Crosses between closely related genotypes like cvs. Eleanor and La Paz or Java and Wilhelmina are less likely to produce heterosis.
5. Conclusion
The rapid nucleic acid extraction procedure and the corresponding PCR protocol for the generation of RAPD markers have proved useful in the fingerprinting and classification of Alstroemeria cultivars. A phyllogram generated by unweighted pair group method analysis of the genetic distances conformed to the expectations based on the available pedigree data. Hence, RAPD analysis should be a primary tool in future research into the evolution of this genus.
Acknowledgements
The authors are indebted to Ms. S. Tachikawa of the Southern Hokkaido Agricultural Experiment Station for providing the leaf materials from the various Alstroemeria cultivars used in this study. This study was supported in part by a post doctoral fellowship awarded to J.G. Dubouzet by the Research Development Corporation of Japan.
References Aker, S. and W. Healy. 1990. The phytogeography of the genus Alstroemeria. Herbertia, 46(2): 76-87. Bridgen, M.P. 1993. Alstroemeria. In: A.A. de Hertogh and M. Le Nard @ditors), The Physiology of Flower Bulbs. Elsevier Science Publ, Netherlands, pp. 201-209. Broertjes, C. and A.M.V. Harten. 1987. Application of mutation breeding methods. In: Abbot, A.J. and R.J. Atkin (Editors), Improving vegetatively propagated crops. Academic Press, London, pp. 335-348. Broertjes, C. and H. Verboom. 1974. Mutation breeding of Alstroemeria. Euphytica, 23: 39-44. De Jeu, M.J., H. Sasbrink, F. Ganiga Caldere and J. Piket. 1992. Sexual reproduction biology of Alstroemeria. Acta Hortic., 325: 571-575. C&mans, J.A.M. 1962. Breeding of Alstroemerias. J. Royal Hort. Sot., 87: 282-284. Hachinohe, A. 1994. Arusutoromeria no shin hinshu. Noukou to engei, 49(5): 139 (In Japanese). Langton, F. A. 1987. Breeding for improved ornamental plants. In: Abbot, A.J. and R.J. Atkm (Editors), Improving vegetatively propagated crops. Academic Press, London, pp. 159- 180. Miyake, I. 1989. Breeding spotless Alstroemeria in Japan. Herbertia, 45(1.2): 40-45. Nei, M. and Li, W.H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. (USA)., 76: 5269-5273. Ohkawa, K. 1994. Hbshu kairyou no rekishi. In: Ohkawa, K. (Editor). Arusutoromeria. Seibundou Shinkousha, Tokyo, pp. !26-35 (In Japanese).
J.G. Dubouzet et al./Scientia
Horticulturae 68 (1997) 181-189
189
Rustanius, P., A. Hang, H.G. Hughes and T. Tsuchiya. 1991. Chromosome analysis of Alstroemeria ligtu hybrids. HortScience, 26: 902-904. Tomboiato, A.F.C., R.B. Tones and C. Azevedo. 1991. Programa de melhoramento genetic0 de madressilva ( Alstroemeria spp. Alstroemeriaceae). Agronomic0 (Campinas). 43: 13-24 (In Portuguese). Tsuchiya, T. and A. Hang. 1987. Chromosome studies in the genus Alstroemeria. Acta Hortic.. 20.5:281-287. Tsuchiya, T. and A. Hang. 1989. Cytogenetics in the genus Alstroemeria. Herbertia, 45( 1.2): 163- 170. Tsuchiya, T., A. Hang, W.E. Healy, Jr. and H. Hughes. 1987. Chromosome studies in the genus Alstroemeria I. Chromosome numbers in 10 cultivars. Bot. Gaz., 148: 519-524. Van Scheepen, J. (Editor) 1991. International Checklist for Hyacinths and miscellaneous bulbs. Royal General Bulbgrowers’ Association, Hillegom, Netherlands, pp. 15-47. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and SC. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: 6531-6535.
Scientia Horticulhme
68 ( 1997) 18 I- 189
RAPD analysis of genetic relationships among Alstroemeria L. cultivars Joseph G. Dubouzet *, Naho Murata, Koichi Shinoda Hokkaido Nutional Agricultural Experiment Station, Hitsujigaoku- I, Toyohira-ku, Sapporo 062 Japan Accepted
19 August
1996
Abstract A procedure for the rapid extraction of partially purified nucleic acid extracts from Afstroemepolymerase chain reaction (PCR) protocol for the generation of random amplified polymorphic DNA (RAPD) markers were established. Nucleic acid extracts from 23 Afstroemeriu cultivars were amplified with 8 random decamers by PCR. OPCO2,OPCO3, 0PD02, OPDOS, OPDO8, OPDll, 0PD13 and OPDl8 produced 24, 19,21,20, 10, 17,25 and 29 RAPD bands, respectively. The distinctive RAPD patterns generated from these cultivars could be used as genomic ‘fingerprints’ to establish the identity of a given genotype. The ‘Orchid’ and ‘Butterfly’ types were clearly separated in distinct subclusters in a phyllogram obtained by unweighted pair group method analysis (UPGMA) of the genetic distances. The ‘Hybrid’ types were distributed in two major subclusters, reflecting the diversity of the parental species used to generate the population. This phyllogram conformed to expectations based on the available pedigree data. 0 Elsevier Science B.V.
ria cultivars and the corresponding
Keywords: Alstroemeria; RAPD analysis;
genetic distance;
UPGMA
1. Introduction The genus Afstroemeriu is classified under the subclass Monocotyledonae and the family Alstroemeriaceae. The main center of distribution is in the Andes mountains (eastern Chile) with approximately 30 species; a satellite center is located at lower altitudes in central and eastern Brazil with about 23 species (Aker and Healy, 1990; Tombolato et al., 1991). There are four species in Argentina and three in Peru (Bridgen, 1993).
* Corresponding
author.
0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(96)00966-l
182
J.G. Dubouzet et al. / Scientiu Horticulturue 68 11997) 181-189
Modem Alstroemeria cultivars were developed through inter specific hybridization, selection of mutant sports and/or polyploidization (Broertjes and Verboom, 1974; Broertjes and Harten, 1987; Tsuchiya and Hang, 1987). However, the pedigrees of most Alsfroemeria cultivars are secret because they were developed by private entities. Published accounts of cultivar development also claim that their breeding programs were initially based on the seedlings of unknown parentage (Goemans, 1962; Miyake, 1989). Breeders need to know the genetic similarity between potential parents because Alstroemeria are dichogamous species that show a great deal of hybrid vigor upon inter specific hybridization. Breeders unaware of the similarity between their parental species run the risk of inbreeding depression since many of the ‘modem’ Alsfroemeria cultivars were obtained by mere radiation mutation or induced polyploidization (Broertjes and Verboom, 1974; De Jeu et al., 1992). Hence, it is necessary to develop a system of genomic fingerprinting so that the identity of specific cultivars can be rapidly determined. Such a system can be used by geneticists to study the evolution of this genus and it may enable breeders to formulate reasonable breeding plans. A genetic fingerprinting system will also be useful in protecting the rights of the plant patent holders. Previous researchers (Tsuchiya and Hang, 1987; Tsuchiya and Hang, 1989) attempted to trace the ancestry of current Alstroemeria cultivars using cytological techniques but the differences in chromosome morphology, number and Giemsa staining patterns were insufficient to differentiate the various species and cultivars. In recent years, random amplified polymorphic DNA (RAPD) analysis through the polymerase chain reaction (PCR) has become widely used to characterize and trace the phylogeny of diverse plant and animal species. The main advantages of RAPD analysis over other methods (e.g., random fragment length polymorphism analysis) are its low sample DNA requirement and the high frequency of polymorphic bands detected (Williams et al., 1990). This article deals with the establishment of a genomic fingerprinting system for Alsfroemeria, starting with the rapid micro extraction of nucleic acids from 23 cultivars, the generation of RAPD bands by 8 random decamers through PCR estimation of the genetic distances and phylogenetic analysis.
2. Materials and methods Data pertaining to the cultivars included in this study were obtained from various sources (including seed catalogues) and are summarized in Table 1. According to available publications, the cultivars in this study were classified as 1) ‘Orchid’ type (cvs. Canaria, Casablanca, Eleanor, La Paz and Paloma), 2) ‘Butterfly’ type (cvs. Amanda, Ostara and Snow Queen), and 3) ‘Hybrid’ type (rest of the cultivars). 20mg of young (flush) leaf tissues were obtained from 23 Alstroemeria cultivars (Table 1) and minced with scissors. Tissues were homogenized in 200 p,l of the extraction buffer (4M guanidine thiocyanate, 0.1 M EDTA-2Na, 1% cetyltrimethyl-ammonium bromide, 1% polyvinylpyrrolidone in 0.1 M Tris-HCI pH 8) using a plastic homogenizer attached to a battery-powered tooth brush. The homogenate was extracted
ff fff ffff fff
K
s PhytoNova P
Paloma 2. Butterfly Amanda Ostara Snow Queen 3. Hybrid Amor Bianca Carola Cobra Evita Gloria Java Little Sun Nevada Pink Triumph
Orange Red Rose/white Pink
ffff fff ffff ffff
Sunrise Tiara Vienna Wilhelmina
to the number of seasons in a year
Pink
fff
Rosita
’ f corresponds
Yellow White Pink/yellow Red Red Orange Pink Red White Pink
White/ rose Purple/white White
fff fff fff ffff ffff ffff ffff ffff ff ff
s s s K van Zanten (Z) Z K
(P1
Yellow
White/yellow Yellow Yellow
ffff ff
ff
Konst (K) Wulfinghoff K
Yellow
Flower Colors
ff
Casablanca Eleanor La Paz
(W)
in this study.
Flowering Time a
included
Van Staaveren (S)
Source
of Alsfroemeria cultivars
1. Orchid Canaria
Hybrid Type/ Cultivar
Table 1 Characteristics
180-200 180-220
110
120-160 140
150-180
140-160 180-220
180 165
150-160
150
200-220
200-210 160-180 200-220
150-200
Flower Stem Length (cm)
(Hachinohe,
1994)
‘Butterfly’ ‘Butterfly’
type X ‘Orchid’ type (Ohkawa, 1994) type X A. aurea lutea (Ohkawa, 1994)
Two homologous and one different genome (Tsuchiya and Hang, 1987) Mutant of cv. Regina (A. pelegrina X A. uureu ) (Broertjes and Verboom, 1974)
Sport of cv. Wilhelmina
Mutant of cv. Orchid (A. pauperculu X A. aurea ) (Van Scheepen, 1991) cv. Orchid X cv. Paloma (Ohkawa, 1994) ? X A. aurecr lutea (Ohkawa, 1994) Radiation mutant of cv. Rio (? X A. urea luteu ) (Ohkawa, 1994) A. aurea X A. aureu (Ohkawa, 1994)
Notes
184
J.G. Dubouzet et al./kientia
Horticulturae 68 (1997) 181-189
with 200 pl 1-bromo-3-chloropropane and the phases were allowed to separate naturally. The nucleic acids were precipitated from the 150 cl.1aqueous supemate by adding 450 p,l ethanol and centrifuging at 15 OOOrpm for 1 min. The precipitate was resuspended in 450$70% ethanol and then centrifuged at 15 000 t-pm for 5 min. The washed precipitate was air dried and then suspended in 200 pl TE at pH 7.5. Replicate extracts were prepared using tissues from a different leaf of the same shoot. UV absorbance of the nucleic acid extracts was measured using a Beckman DU 65 spectrophotometer. The extracts had an average optical density at 260nm (A,,) = 14.5 k 7.7 and an average A,,,/A,s, = 1.71 f 0.1. These extracts were adjusted to long nucleic acid per ~1 and used for PCR. The 10 (~1PCR reaction mix consisted of 5 pl DNA extract, 1.6 pl 25 mM MgCl,, 1.35 pl de-ionized water, 1.Opl 10X Buffer II, 0.8 p.1 1OmM dNTP mix, 0.2 ~1 10 pM random decamer (Operon Industries, USA) and 0.05l~l AmpliTaq DNA polymerase (Perkin Elmer, USA). The random decamers were 0PC02 (5’-GTGAGGCGTC-3’), OPC03 (5’~GGGGGTCTIT-3’1, OPD 02 (5’-GGACCCAACC-3’1, 0PD05 (5’TGAGCGGACA-3’), OPDOS (5’-GTGTGCCCCA-3’), OPDl 1 (5’-AGCGCCA’ITG-3’), 0PD13 (5’~GGGGTGACGA-3’) and OPD18 (5’-GAGAGCCAAC-3’). The replicate DNA extracts were simultaneously amplified. Thermal cycling was performed using the GeneAmp PCR System 9600 from Perkin Elmer. The thermal profile consisted of an initial denaturation step at 94°C for 1.5 min followed by 45 cycles of 94°C for 15 s, 36 “C for 15 s and 72 “C for 2 min and a final exposure to 72 “C for 5 min. The 10 ~1 amplified products and 0.5 pg Hin dIII-Eco RI double digest molecular marker were electrophoresed in a minigel consisting of 1.5% Agarose S (Nippon Gene, Japan) and 1 kgml -’ ethidium bromide in 40mM Tris-acetate and 1 mM EDTA (TAE) buffer at 1OOV. The gels were photographed using Polaroid 667 film. The prints were enlarged 350% using an A935 Fuji Xerox (Japan) photocopier to facilitate RAPD band comparisons. The RAPD bands were scored as either present (1) or absent (0). The relationship between each pair of entries was estimated using a genetic distance formula derived from the similarity index recommended by Nei and Li (1979): =I,2
GeneticDistance = 1 - G,
+G2
where G, and G, are the total number of RAPD bands of genotypes 1 and 2, respectively, and G,,2 is the number of bands common to both genotypes. The genetic distance values were analyzed by unweighted pair group analysis (UPGMA) using J. Felsenstein’s package of phylogenetic programs (Phylip v.3.572). The analytical results were illustrated using R. Page’s TreeView v. 1. Both programs are available through anonymous file transfer protocol from the Internet.
3. Results The eight random primers generated a total of 165 RAPD bands: OPCO2,OPCO3 and OPDO2,OPDO5, OPDOS, OPDll, 0PD13 and 0PD18 produced 24, 19, 21,20, 10, 17,
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M
1.38
-I
8.53
+
Fig. 1. RAPD bands generated by OPDS from 11 Ahtroemeria cvs. Lane M: Eco R I/ Hin dII1 double digest size marker; lane 1 - cv. Canaria; lane 2 - cv. La Paz; lane 3 - cv. Eleanor; lane 4 - cv. Paloma; lane 5- cv. Amanda; lane 6 - cv. Ostara; lane 7 - cv. Snow Queen; lane 8 - cv. Amor; lane 9 - cv. Bianca; lane 10 - cv. Java and lane 11
- cv. Wilhelmina.
25 and 29 polymorphic bands, respectively. Fig. 1 shows the RAPD pattern generated by 0PD05 from genomic DNA of four ‘Orchid’ types (cvs. Canaria, La Paz, Eleanor and Paloma), three ‘Butterfly’ types (cvs. Amanda, Ostara and Snow Queen) and four ‘Hybrid’ types (cvs. Amor, Bianca, Java and Wilhelmina). RAPD analysis was based on the amplification patterns of replicate extracts.
flmanda
-p
L
0.1 Branch
I
OSt al-a snow ttueen
length
’
Fig. 2. Phyllogram obtained by unweighted pair group method analysis of 165 RAPD markers generated by 8 random primers from 23 Alstroemeria cultivars. Numbers indicate specific subclusters discussed in the text.
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The genetic distance values obtained using the formula derived from Nei and Li’s similarity index are available from the authors upon request. Fig. 2 shows the relationships among cultivars according to UPGM Analysis of the genetic distances.
4. Discussion The cv. Orchid (syn. cv. Walter Fleming) is a product of A. pauperculu Philippi (syn. A. oiohcea Philippi) X A. aureu Graham (Van Scheepen, 1991). The original diploid hybrid between A. uureu and A. puupercufu was sterile but a tetraploid sport was subsequently found to be fertile. This sport was crossed with other diploid species to produce the triploid ‘Orchid’ types (De Jeu et al., 1992). Tsuchiya et al. (1987) reported that cv. Orchid had some chromosome types similar to those found in A. area. The ‘Orchid’ types included in this study are descendants of A. uureu (Table 1). ‘Orchid’ types flower for 3-5 months in the spring with little or no flower production in the rest of the year (Bridgen, 1993). The cv. Eleanor (Table 1) seems to be a major exception to this generalization. ‘Butterfly’ types generally have both Brazilian and Chilean genes in their family tree (Bridgen, 1993). The ‘Butterfly’ hybrids consist of hybrids between A. pefegrinu L. or A. puuperculu and A. psittucinu Lehm. (syn. A. pulchellu) or A. inodoru. The genomes of these sterile diploids were artificially doubled to produce fertile allotetraploids (De Jeu et al., 1992). Induced tetraploids in Alstroemeriu have large, thick, dark green leaves, large, wide and thick flower petals, and longer vase lives (Tsuchiya et al. 1987). ‘Butterfly’ types have short growth habits and they have larger and more open flowers. Cool temperatures will induce flowering and long photo periods will maintain flowering of the ‘Butterfly’ cultivars for 9-12 months in a year (Bridgen, 1993). ‘Hybrid’ types were produced in Europe by crossing different Alstroemeriu species from Chile, Brazil and Peru (De Jeu et al., 1992). Some ‘Hybrid’ types were obtained by crossing ‘Butterfly’ and ‘Orchid’ types, e.g., cv. Vienna (Ohkawa, 1994). The flower colors, flower stem lengths and flowering times of ‘Hybrid’ types (as shown in Table 1) reflect the diversity of the ancestries of these cultivars. A. uureu, A. puuperculu, A. pelegrinu, A. ligtu L., and A. ligtu ssp simsii (Spreng.) Bayer have been suggested as contributory progenitors to these modem European cultivars (Aker and Healy, 1990; Broertjes and Verboom, 1974; Langton, 1987). However, Rustanius et al. (1991) claimed that A. ligtu hybrids may not have been used in the production of modem European and North American Alstroemeriu cultivars. The genetic basis for the classification of modem Alstroemeriu cultivars is not clear since pedigree data are either lacking or ambiguous. The following discussion describes a possible classification of 23 Alstroemeriu cultivars based on RAPD and phylogenetic analyses. The eight random decamers generated distinctive amplification products from the entries. The representative RAPD patterns shown in Fig. 1 were generated by 0PD05. The RAPD patterns obtained from the ‘Orchid’ types (lanes l-4) are relatively more
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similar compared with those generated from the ‘Butterfly’ types (lanes 5-7). This can be ascribed to the difference in parentage of these two types. The ‘Hybrid’ types (lanes 8-10) showed correspondingly more variable RAPD patterns. The RAPD pattern generated from cv. Java (lane 10) was almost identical to that obtained from cv. Wilhelmina (lane 11); this is because cv. Java is just a mutant sport of cv. Wilhelmina. These distinctive RAPD patterns can be used as genomic ‘fingerprints’ to establish the identity of a given genotype. The genetic distance value provides a useful estimate of the relationship between a specific pair or a small number of genotypes. Phylogenetic analysis, however, is more appropriate for the interpretation of all possible relationships among a large group of genotypes. The various relationships among these cultivars are shown in the phyllogram (Fig. 2) generated by UPGM analysis of the genetic distances. The ‘Butterfly’ types (cvs. Amanda, Ostara and Snow Queen; subcluster 4) are grouped in a distinctly separate subcluster in this phyllogram. The ‘Butterfly’ types are well separated from the ‘Orchid’ types (cvs. Canaria, Eleanor, La Paz and Paloma; subcluster 7). Thus, this phyllogram supports previous reports that claimed these two types were derived from different sets of parental species. The phyllogram also indicates that the modem hybrids are more closely related to the ‘Orchid’ types than to the ‘Butterfly’ types. This implies that A. aureu, which is the main parent identified in the pedigrees of these “Orchid” types (Table l), may be a common parent to these ‘Hybrid’ types. This hypothesis needs to be verified in future studies. Although located on a different branch, the cv. Casablanca is clearly associated with the ‘Orchid’ types, as shown by its location in this phyllogram. The ‘Orchid’ types (cvs. Canaria, Eleanor and La Paz; subcluster 8) were closely related. This is also indicated by the great similarity in the flower structure and color patterns of these three cultivars. The close similarity between cvs. Eleanor and La Paz may be because they have the same male parent (A. uurea lutea). Paloma and Casablanca, which are also classified as ‘Orchid’ types, show a slightly different flower structure and color pattern from the former three cultivars; this difference is reflected in a greater separation from cvs. Canaria, Eleanor and La Paz. The ‘Hybrid’ types are separated in two subclusters (5 and 6) between the ‘Butterfly’ and ‘Orchid’ types. Subcluster 5, which is closer to the ‘Orchid’ types, includes the cvs. Amor, Bianca, Cobra, Gloria and Little Sun. Except for cv. Little Sun, these cultivars are similar to the ‘Orchid’ types in floral morphology. The flowering characteristic of cv. Bianca is quite similar to the ‘Orchid’ types. The rest of the ‘Hybrid’ types, which are more genetically different from the ‘Orchid’ types, are grouped in subcluster 6. The cv. Rosita is a mutant of a product of A. pelegrinu X A. area. As mentioned above, A. pelegrinu was putatively used in the production of some of the ‘Butterfly’ types; it has never been reported as a parent of the ‘Orchid’ types. The cvs. Java, Wilhelmina and Vienna were obtained from crosses using ‘Butterfly’ types as maternal parents. The distribution of the ‘Hybrid’ types into different subclusters may be an indication of the diversity of the parental species used to generate this population. The cvs. Java and Wilhelmina were the most closely related among the ‘Hybrid’ types; this close similarity is expected since cv. Java is a mutant sport of cv. Wilhelmina (Hachinohe,
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1994). These two cultivars also have a partial resemblance in gross flower structure and coloration. The phyllogram can be used in the formulation of breeding plans. For example, if crosses involving the most distantly related cultivars are desired, then crosses between the ‘Orchid’ and the ‘Butterfly’ types can be recommended. Crosses between closely related genotypes like cvs. Eleanor and La Paz or Java and Wilhelmina are less likely to produce heterosis.
5. Conclusion
The rapid nucleic acid extraction procedure and the corresponding PCR protocol for the generation of RAPD markers have proved useful in the fingerprinting and classification of Alstroemeria cultivars. A phyllogram generated by unweighted pair group method analysis of the genetic distances conformed to the expectations based on the available pedigree data. Hence, RAPD analysis should be a primary tool in future research into the evolution of this genus.
Acknowledgements
The authors are indebted to Ms. S. Tachikawa of the Southern Hokkaido Agricultural Experiment Station for providing the leaf materials from the various Alstroemeria cultivars used in this study. This study was supported in part by a post doctoral fellowship awarded to J.G. Dubouzet by the Research Development Corporation of Japan.
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