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Characterization of Thirteen Date Palm(Phoenix dactylifera L.) Cultivars by Enzyme Electrophoresis using the PhastSystem
J. Plant Physiol. Vol.
145. pp. 62 - 66 (1995)
Characterization of Thirteen Date Palm (Phoenix dactylifera L.) Cultivars by Enzyme Electrophoresis using the PhastSystem I. BOOIJ1,,~, S. MONFORT, and M. FERRy2 G.R.F.P. (French Research Group on date Palm), c/o Conservatoire Botanique National, Hameau Agricole, 83 400 Porquerolles, France 1 Present address: USDAI ARS/BARC-West, PSIIPMBL, Bldg 006, Rm 212,10300 Baltimore Ave., Beltsville, MD 20705-2350, USA 2
Present address: Estaci6n Phoenix, c/Cami. del gat, sin 03203 Elx-Elche, Alicante, Spain
Received February 1, 1994 . Accepted May 10, 1994
Summary
The analysis of date palm (Phoenix dactyli/era L.) enzymatic polymorphism enabled us to establish some reference «identity cards» for the cultivars studied. Thirteen cultivars and ten enzymatic systems were investigated. Isozyme separation was performed with the PhastSystem (Pharmacia, Uppsala), an automated electrophoresis unit, allowing the standardization of migration parameters. Various zymograms have been obtained for five of the ten enzymes studied, showing differences in isozyme number, band intensities, and relative mobility values. We have found six polymorphic loci, expressing thirteen different alleles. These variations are only caused by genetic differences among the cultivars, enabling us to assemble some of them and accurately distinguish nine (69 %) of the thirteen cultivars studied.
Key words: Phoenix dactyli/era L., date palm cultivars, leaf isozymes, varietal identification, enzyme polymorphism, PhastSystem. Abbreviations: BSA = Albumin bovine; EDTA = Ethylenediaminetetraacetic acid; PVP pyrrolidone MW = 40,000; Tris = Tris(Hydroxymethyl) Aminomethane. Introduction
Date palm (Phoenix dactyli/era L.) cultivar identification has been traditionally based on a combination of vegetative and fruiting characteristics. Hence, the different cultivars are sometimes difficult to distinguish from each other. The lack of a practical key for cultivar identification and the long life cycle of date palm promoted the need to establish appropriate and reliable methods of identification. Moreover, it should be interesting to characterize date palm cultivars at an earlier stage of their development. During the past years, isozyme analysis appeared to be a possible alter-
* Corresponding author. © 1995 by Gustav Fischer Verlag, Stuttgart
=
polyvinyl-
native or complementary method for cultivar identification. It has been successfully applied to such diverse species as various fruit trees (Weeden and Lamb, 1985; Menendez and Daley, 1986; Bournival and Korban, 1987; Hauagge et al., 1987), grape (Benin et al., 1988), potatoes (Oliver and MartInez-Zapater, 1985; Contreras and Mansilla, 1989), cereals (Cardy and Kannenberg, 1982; Lallemand and Briand, 1990), forest trees (Bergmann, 1987; Raj ora, 1989), tropical crops Garret and Litz, 1986; Dewald et al., 1988), and palms (Torres and Tisserat, 1980; McClenaghan and Beauchamp, 1986; Baaziz and Saaidi, 1987; Aljibouri et al., 1988). Based on these studies, it appeared feasible that electrophoretic procedures could become a practical means for date palm cultivar identification, depending on the presence of distinc-
Isozymic characterization of date palm cultivars
tive banding patterns of specific isozymes after electropho-
63
Results
reSIS.
This study was conducted to characterize different date palm cultivars by isozyme analysis. It describes a method, which is suitable for the characterization of date palm cultivars phenotypes, using the PhastSystem (Pharmacia, Uppsala, Sweden). This improved method has the advantage of standardization, short separation time, and high resolution.
Materials and Methods Plant material Leaf samples from 13 date palm cultivars were obtained from Saudi Arabia, Algeria, and Morocco. They were Bou Feggous (BFG), Bou Skri (BSK) , Bou Sthammi noire (BST), Khadrawy (KHD), Khalassa (KHL), Mejhoul (MJH), Nabout Seif (NBT), Thoory (THR), Takerbouch (TKR) , Tinakar (TNK), Zahidi (ZHD), Fard # IV (FRD), and Halawy Back-Cross (HLW). The last two are male cultivars.
Leaf extract preparation Leaflets from zero range fronds (fronds from the internal crown, last visible frond) were cut off. One gram of leaflets from each cultivar was chopped into pieces and ground at 4°C in a mortar in 1.7 mL of extraction buffer. It consisted of 0.1 M Tris-HCI pH 7.5 containing 1 mM EDTA, 12 % PVP, 3 % BSA (w/w leaf), and 0.4% {3-mercaptoethanol (v/w leaf) (Torres and Tisserat, 1980, with slight modifications). Because of the very fibrous nature of the palm leaflets, a prior crushing in liquid nitrogen made the extraction easier. The homogenate was centrifuged at 20,000 x g at 4°C for 40 min. Supernatants were collected for immediate electrophoresis analysis or stored in liquid nitrogen.
The extraction buffer described in the Materials and Methods section provided adequate protection for the leaf extracts so that clear banding patterns were obtained. The 8 - 25 % polyacrylamide gradient gels and the 3 rnA intensity gave the best resolution of the isozymes. Of the ten enzymatic systems investigated, only seven were active under our experimental conditions. They were ACP, ADH, EST, GPI, G6P, PER, and 6PGD. Isozyme banding patterns showed no differences in enzyme forms, relative mobility values (Rf), and intensity of bands among individuals of the same cultivar under the same conditions. However, isozyme patterns revealed obvious differences in enzyme forms and band intensity among the tested cultivars, except for PER zymograms, which presented no significant differences. For this one, only two zones of activity appeared, but no polymorphism was found (data not shown). ACP banding patterns were not reproducible from one migration to another. Thus, we only studied the five following enzymatic systems: ADH, EST, GPI, G6P, 6PGD. G6P was not investigated for TKR and TNK cultivars. We used the same nomenclature as Tisserat and Torres (1979) for the genetic interpretation of our zymograms. The genes are named after the enzymatic system they specify. When two genes code for the same type of enzyme, the locus that specifies the isozyme set that migrates more slowly toward the anode is called 1, the faster 2. The allele that codes for the slower migrating isozyme within a set is called S, the faster F. Representative zymograms of the enzymatic systems studied are illustrated for each cultivar in Fig. 1. The corresponding genotypes are given in Table 1.
Electrophoresis Electrophoretic separation was carried out in an automated Pharmacia gel electrophoresis apparatus, the PhastSystem. It allowed us to accurately and rapidly reproduce the migration parameters. They were 400V, lOrnA, 2.5W, SoC, until 10Vh; then 400 V, 3 rnA, 2.5 W, 4°C for 280 Vh. For each run, two vertical gels SO x 43 x 0.45 mm were used. Each gel accommodated 8 samples. The gels (8-25% polyacrylamide gradient) were made of a 0.112M Tris-Acetate buffer pH 6.4. The running buffer, contained in a 2 % high quality agarose gel, consisted of a 0.25 M Tris and 0.88 M L-Alanine buffer pH 8.8. One ItL (1.5 Itg of protein) of each extract was loaded on the gel. Electrophoresis was carried out for 1 h1 h30, until the bromophenol blue dye had migrated within 0.5 cm of the bottom of the gel.
Enzymatic staining Ten different enzymatic systems were studied, namely acid phosphatase (ACP), alcohol dehydrogenase (ADH), alkaline phosphatase (ALP), esterase (EST), galactose dehydrogenase (GDH), glucose 6-phosphate dehydrogenase (G6P), glucose phosphate isomerase (GPI), 6-phosphogluconate dehydrogenase (6PGD), peroxidase (PER), and phosphoglucose mutase (PGM). All these enzymatic systems were stained according to Vallejos (1983), excepted EST. Staining mixture for EST consisted of 10 mg a-naphtylacetate and 10 mg {3-naphtylacetate in 0.3 mL acetone, 10 mg Fast Blue RR salt, and 10 mL of 0.525 M phosphate buffer pH 6.5.
Alcohol dehydrogenase (ADH) The zymograms produced by ADH consisted of one or three bands depending on the cultivar (Fig. 1). Plant ADH is well known to be a dimeric enzyme. The single-banded pattern suggested a homozygous condition, call it FF, and the three banded pattern suggested the heterozygous condition, call it FS. Only these two genotypes were observed among the cultivars studied. We assume that we are in the presence of one gene, adh-l, with two codominant alleles, F and S.
6-phosphogluconate dehydrogenase (6PGD) The 6PGD zymograms consisted of a four banded pattern. The bands migrating near the origin offer intensity variations (faint or dark) depending on the cultivar (Fig. 1). This observation could be explained if this enzyme is dimeric and if a null or silent allele is hypothesized for 6pgd-1. A missing isozyme is assumed to represent the homozygous null genotype NN, a faint band the heterozygote AN, and the normal dark band the homozygous condition AA. The genotypes AA or AN were represented, but we have never found the homozygous NN.
I. BOOI], S. MONFORT, and M. FERRY
64
PA TIERNS OBTAINED FOR 'THE CULTIVARS STUDIED m BIG BIG KHI BIG IRO Relative MJH fRO HIW BIG an THR NBT KHI BI G NBT m KHI IRO UK m m rnc.bility UK KHI m MJH m values m NBT KHO HIW In ZHO TKR IN K TKR m IRO
(Rf)
ZHO
HIW IHR INK IlR ZHO
IN K fRO ZHO HIW
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m
IH R
NBT KHI MJH IHR ZH0
HIW MJH
m
TN K
m
NBI
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70 60 50 40 30 -
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-
-
10 F5 F5 FS 55
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AA AN NNAA AN NN AA
EST
FF
AA
AN
GPI 6PGD G6P ENZYMA TIC SYSTElvlSANAL'flED
Phosphoglucose isomerase (GP!) The GPI zymograms consisted of enzyme bands in two regions, one close to the origin and the other, nonvariable patterns between Rf 38 and Rf 46 (Fig. 1). There were one or three bands in the region close to the origin, depending on the cultivar, suggesting that these isozymes are dimers specified by one gene, gpi-l, having two alleles F and S. We have found the three genotypes SF, SS and FF.
Glucose 6-phosphate dehydrogenase (G6P) G6P occurred in two regions and are apparently specified by two genes. No polymorphism was found within the first set, specified by g6p-1. Isozymes of g6p-2 showed band intensity variations within one band (Fig. 1, band n05). As for 6PGD, we could assume a dimeric enzyme with a null or silent allele for g6p-2, giving the genotypes NN or AN.
Esterase (EST) EST banding pattern appeared to possess the highest number of isozyme molecular forms compared with the other enzyme systems analyzed. EST occurred in four regions (Fig. 1). No differences were found among the bands except in the two regions migrating near the origin, called EST-1
FF
FS
ADH
Fig. 1: Schematic illustration of the zymograms obtained for the thirteen date palm cultivarsa studied, by the analysis of five enzymatic systems b. a: BGF = Bou Feggous, BSK = Bou Skri, BST = Bou Sthammi noire, FRD = Fard # 4, HLW = Halawy back-cross, KHD = Khadrawy, KHL = Khalassa, MJH = Mejhool, NBT = Nabout Seif, TH = Thoory, TKR = Takerbouch, TNK = Tinakar, ZHD = Zahidi. b: EST = esterase, GPI = Phospho glucose isomerase, 6PGD = 6-phosphogluconate dehydrogenase, G6P = Glucose 6-phosphate dehydrogenase, ADH = alcohol dehydrogenase.
Table 1: Isozyme genotypes detected in the thirteen date palm cultivarsa analyzed. ADHb EST-lb EST-2b G6P-2b GPI-l b 6PGD-l b BFG BSK BST FRD HLW KHD KHL MJH NBT THR TKR TNK ZHD
FF FF FF FF FF FF FS FF FF FF FS FF FS
SS FS SS FS FS SS SS FS no bands RF SS SS SS
AA AA AN AN
NN AN AA AA AA NN AA AN AA
AN AN NN NN AN NN AN AN NN AN AN
FS FS FS FS FS FS FF FF SS FS FF FF FF
AA AA AA AN AA AA AN AA AN AN AA AN AA
BFG: Bou Feggous, BSK: Bou Skri, BST: Bou Sthammin noire, FRD: Fard #IV, HLW: Halawy back-cross, KHD: Khadrawy, KHL: Khalassa, MJH: Mejhool, NBT: Nabout Seif, THR: Thoory, TKR: Takerbouch, TNK: Tinakar, ZHD: Zahidi. b ADH: alcohol dehydrogenase; EST: esterase; G6P: 6-phosphoglucose dehydrogenase; GPI: phosphoglucose isomerase; 6PGD: 6-phosphogluconate dehydrogenase.
a
and EST-2. The slowest zone, EST-I, showed one or three bands. Based on the banding patterns obtained for each cultivar, it could be assumed that EST-1 occurs in the date palm
Isozymic characterization of date palm cultivars as a dimeric enzyme and is, apparently, under the control of a single locus with two alleles F and S. Evidence for a third allele of est-I, R (for Retarded), whose product migrated more slowly than that of S, was only found for THR cultivar. The genotypes found were SS, SF, and RF. The EST-2 zone is characterized by the presence of three bands, one of them showing intensity variations (Fig. 1, band n06). As for 6PGD, we could assume a dimeric enzyme with a null or silent allele for est-2, giving both the homozygous (NN or AA) and heterozygous (AN) genotypes.
no bands
Table 2: Enzymatic polymorphism found for the thirteen date palm cultivars studied, by the analysis of five enzymatic systems'. Enzymatic system
Zones observed
studied
2 2 2
slow middle slow fast slow
ADH EST GPI G6P 6PGD
Zones
Loci detected
Enzyme structure
Alleles detected
Dimeric Dimeric
3
Dimeric Dimeric
Dimeric Dimeric
Genotype detected
2 2 2 2 2
2 2 2
a ADH: alcohol dehydrogenase, EST: esterase, GPI: phosphoglucose isomerase, G6P: 6-phosphoglucose dehydrogenase, 6PGD: 6-phosphogluconate dehydrogenase.
SF
A
FF
SF
~ ~ ~
FF
SF
FF
Discussion
Six polymorphic loci have been found for the five enzymatic systems studied, with thirteen different alleles: 3 for EST-1locus and two for each ADH, EST-2, GPI, 6PGD and G6P locus (Table 1, Table 2). These results partly confirmed those obtained by Torres and Tisserat (1980) and Baaziz (1987). Variations found may be caused by the nature of vegetal material tested (older palms used by the above authors) or the conditions of the experiments (10 % polyacrylamide gels or starch gels used). Regarding the zymograms obtained for each enzymatic system studied, we had established a classification key by hierarchically sorting the enzymes showing the greatest number of genotypes (Bennaceur, 1991). The cultivars showing the same allozymic patterns were grouped together. The enzymatic systems showing a null or silent allele were latter observed, because the hypothesis for their genotypic interpretation should be verified. For instance, variations observed could only be the result of experimental conditions. However, zymograms obtained were exactly reproducible, and the isozymes of the other loci had showed the same intensity banding patterns. On the other hand, intensity variations observed on the litigious locus (gene with a silent or null allele) corresponded well with different proportions of its alleles. Nevertheless, these differences could also be caused by the presence in the extracts of an exogen factor limiting the isozyme revelation (Pasteur et al., 1987). Eleven groups of «standard» patterns have been established within the thirteen cultivars analyzed (Fig. 2). We can accurately distinguish nine of them (69 %): BFG, BSK, FRD, HLW, KHL, MJH, NBT, THR, TNK. Three ofthese cult ivars showed an unique genotype for EST: NBT (no isozy-
RF
BT
65
SF
NK 'KJ{L
zJi'J)
Fig. 2: Classification of thirteen date palm cultivars" based on their enzymatic genotypes. The enzymaticb genotypes obtained with an accurate interpretation (EST-I, ADH and GPI-I) have been differentiated from those based on the hypothesis of a null or silent allele (6PGD-I, G6P and EST-2). aBGF: Bou Feggous, BSK: Bou Skri, BST: Bou Sthammi noire, FRD: Fard # IV, HLW: Halawy backcross, KHD: Khadrawy, KHL: Khalassa, MJH: Mejhool, NBT: Nabout Seif, TH: Thoory, TKR: Takerbouch, TNK: Tinakar, ZHD: Zahidi. bEST: esterase, GPI: phosphoglucose isomerase, ADH: alcohol dehydrogenase, 6PGD: 6-phosphogluconate dehydrogenase.
mes in the EST-1 zone), THR (RF genotype for the EST-1 zone), and HLW (FS genotype for the EST-1 zone and NN genotype for the EST-2 zone). The last four cultivars were classified into two groups: BST-KHD, and TKR-ZHD (Fig. 2). The analysis of other key enzymes or a correlation between the isozyme identification and conventional identification (at fruiting stages) should allow us to distinguish between them and complete our classification. The separation of different cultivars with the PhastSystem gave us good results, with a high analysis capacity per day. This method, more than standardization advantages, required short preparation and separation times and gave a high resolution. Even if it should be necessary to be cautious in interpreting the zymograms, enzyme electrophoresis with the PhastSystem appeared to be such an efficient and performing tool that we could rapidly and accurately analyze numerous samples at early stages of development. Date palm cultivar identification will enable us to verify the homogeneity of in vitro shoots and to distinguish between the different cultivars. We should find and eliminate any variant produced, as well as the processes generating them, without waiting for the first fructification (usually four to five years). It is an appreciable gain of time. This technique could be used to control the vitroplant production.
66
I. BoorJ, S. MONFORT, and M. FERRY
Acknowledgements
We gratefully acknowledge F. Kjellberg and R. Lumaret for their help in the interpretation of zymograms and for providing useful commentaries on a preliminary draft of this manuscript.
References ALJIBOURI, A. A. M., K. M. ADHAM, and K. N. JASSAM: Identification of date palm cultivars by isozyme analysis. Symposium IrakoFrans;ais sur la culture de tissus du palmier dattier, 26-29 Sept. 1988, p. 50-64 (1988). BAAZIZ, M. and M. SAAIDI: Preliminary identification of date palm cultivars by esterase isoenzymes and peroxidase activities. Can. J. Bot. 66, 89-93 (1987). BENIN, M., J. GASQUEZ, A. MAHFOUDI, and R. BESSIS: Caracterisation biochimique des cepages de Vitis vini/era L. par electrophon!se d'isozymes foliaires: Essai de classification des varietes. Vitis 27, 157 -172 (1988). BERGMANN, F.: Characterization of multiclonal aspen cultivars using isozyme electrophoresis. Forest Ecology and Management 22, 167 -172 (1987). BoURNIVAL, B. L. and S. S. KORBAN: Electrophoretic analysis of genetic variability in the apple. Scientia Horticulturae 31, 233 -243 (1987). CARDY, B. J. and L. W. KANNENBERG: Allozymic variability among maize inbred lines and hybrids. Applications for cultivar identification. Crop Science 22, 1016-1020 (1982). CONTRERAS, A. and R. MANSILLA: Electroforesis de protefnas y esterasas como metodo qufmico de identificati6n en papas. Turrialba 39(2),193-198 (1989). DEWALD, M. G., G. A. MOORE, and W. B. SHERMAN: Identification of pineapple cultivars by isozyme genotypes. J. Am. Soc. Hort. Science 113 (6), 935-938 (1988).
HAUAGGE, R., D. E. KESTER, S. ARULSEKAR, D. E. PARFITT, and L. LIU: Isozyme variation among California almond cultivars - II. Cultivar characterization and origins. J. Amer. Soc. Hort. Science 112 (4), 693 - 698 (1987). JARRET, R. L. and R. E. LITz: Enzyme polymorphism in Musa acu· minata ColI. J. Heredity 77, 183 -188 (1986). LALLEMAND, J. and F. BRIAND: Identification varietale des orges par electrophorese - Description de 280 varietes. Agronomie 6, 447 -450 (1990). MCCLENAGHAN, L. R. and A. C. BEAUCHAMP: Low genic differentiation among isolated populations of California Fan palm (Wash· ingtonia fili/era). Evolution 40 (2),315- 322 (1986). MENENDEZ, R. A. and L. S. DALEY: Characterization of Pyrus species and cultivars using gradient polyacrylamide gel electrophoresis. J. Environ. Horticulture 4 (2),56-60 (1986). OLIVER, J. L. and J. M. MARTINEZ-ZAPATER: A genetic classification of potato cultivars based on allozyme patterns. Theorical and Applied Genetics 69, 305 - 311 (1985). PASTEUR, N., G. PASTEUR, F. BONHOMME, J. CATALAN, and J. BRITTON-DAVIDIAN: Manuel technique de genetique par electrophorese de proteines. Lavoisier (eds.), Paris (1987). RAJORA, O. P.: Characterization of 43 Populus nigra L. clones representing selections, cultivars and botanical varieties based on their multilocus allozyme genotypes. Euphytica 43, 197 - 206 (1989). TISSERAT, B. and A. M. TORRES: Isozymes as genetic indicators in date palm. Date Grower's Institute Reports 54, 24-28 (1979). TORRES, A. M. and B. TISSERAT: Leaf isozymes as genetic markers in date palm. Am. J. Bot. 67 (2), 162 -167 (1980). VALLEJos, C. E.: Enzyme activity staining. In: Isozymes in plants genetics and breeding. Tanksley and Orton (eds.). Part A, pp. 469 (1983). WEEDEN, N. F. and R. C. LAMB: Identification of apple isozyme phenotypes. J. Am. Soc. Hort. Science 110 (4),509-515 (1985).
145. pp. 62 - 66 (1995)
Characterization of Thirteen Date Palm (Phoenix dactylifera L.) Cultivars by Enzyme Electrophoresis using the PhastSystem I. BOOIJ1,,~, S. MONFORT, and M. FERRy2 G.R.F.P. (French Research Group on date Palm), c/o Conservatoire Botanique National, Hameau Agricole, 83 400 Porquerolles, France 1 Present address: USDAI ARS/BARC-West, PSIIPMBL, Bldg 006, Rm 212,10300 Baltimore Ave., Beltsville, MD 20705-2350, USA 2
Present address: Estaci6n Phoenix, c/Cami. del gat, sin 03203 Elx-Elche, Alicante, Spain
Received February 1, 1994 . Accepted May 10, 1994
Summary
The analysis of date palm (Phoenix dactyli/era L.) enzymatic polymorphism enabled us to establish some reference «identity cards» for the cultivars studied. Thirteen cultivars and ten enzymatic systems were investigated. Isozyme separation was performed with the PhastSystem (Pharmacia, Uppsala), an automated electrophoresis unit, allowing the standardization of migration parameters. Various zymograms have been obtained for five of the ten enzymes studied, showing differences in isozyme number, band intensities, and relative mobility values. We have found six polymorphic loci, expressing thirteen different alleles. These variations are only caused by genetic differences among the cultivars, enabling us to assemble some of them and accurately distinguish nine (69 %) of the thirteen cultivars studied.
Key words: Phoenix dactyli/era L., date palm cultivars, leaf isozymes, varietal identification, enzyme polymorphism, PhastSystem. Abbreviations: BSA = Albumin bovine; EDTA = Ethylenediaminetetraacetic acid; PVP pyrrolidone MW = 40,000; Tris = Tris(Hydroxymethyl) Aminomethane. Introduction
Date palm (Phoenix dactyli/era L.) cultivar identification has been traditionally based on a combination of vegetative and fruiting characteristics. Hence, the different cultivars are sometimes difficult to distinguish from each other. The lack of a practical key for cultivar identification and the long life cycle of date palm promoted the need to establish appropriate and reliable methods of identification. Moreover, it should be interesting to characterize date palm cultivars at an earlier stage of their development. During the past years, isozyme analysis appeared to be a possible alter-
* Corresponding author. © 1995 by Gustav Fischer Verlag, Stuttgart
=
polyvinyl-
native or complementary method for cultivar identification. It has been successfully applied to such diverse species as various fruit trees (Weeden and Lamb, 1985; Menendez and Daley, 1986; Bournival and Korban, 1987; Hauagge et al., 1987), grape (Benin et al., 1988), potatoes (Oliver and MartInez-Zapater, 1985; Contreras and Mansilla, 1989), cereals (Cardy and Kannenberg, 1982; Lallemand and Briand, 1990), forest trees (Bergmann, 1987; Raj ora, 1989), tropical crops Garret and Litz, 1986; Dewald et al., 1988), and palms (Torres and Tisserat, 1980; McClenaghan and Beauchamp, 1986; Baaziz and Saaidi, 1987; Aljibouri et al., 1988). Based on these studies, it appeared feasible that electrophoretic procedures could become a practical means for date palm cultivar identification, depending on the presence of distinc-
Isozymic characterization of date palm cultivars
tive banding patterns of specific isozymes after electropho-
63
Results
reSIS.
This study was conducted to characterize different date palm cultivars by isozyme analysis. It describes a method, which is suitable for the characterization of date palm cultivars phenotypes, using the PhastSystem (Pharmacia, Uppsala, Sweden). This improved method has the advantage of standardization, short separation time, and high resolution.
Materials and Methods Plant material Leaf samples from 13 date palm cultivars were obtained from Saudi Arabia, Algeria, and Morocco. They were Bou Feggous (BFG), Bou Skri (BSK) , Bou Sthammi noire (BST), Khadrawy (KHD), Khalassa (KHL), Mejhoul (MJH), Nabout Seif (NBT), Thoory (THR), Takerbouch (TKR) , Tinakar (TNK), Zahidi (ZHD), Fard # IV (FRD), and Halawy Back-Cross (HLW). The last two are male cultivars.
Leaf extract preparation Leaflets from zero range fronds (fronds from the internal crown, last visible frond) were cut off. One gram of leaflets from each cultivar was chopped into pieces and ground at 4°C in a mortar in 1.7 mL of extraction buffer. It consisted of 0.1 M Tris-HCI pH 7.5 containing 1 mM EDTA, 12 % PVP, 3 % BSA (w/w leaf), and 0.4% {3-mercaptoethanol (v/w leaf) (Torres and Tisserat, 1980, with slight modifications). Because of the very fibrous nature of the palm leaflets, a prior crushing in liquid nitrogen made the extraction easier. The homogenate was centrifuged at 20,000 x g at 4°C for 40 min. Supernatants were collected for immediate electrophoresis analysis or stored in liquid nitrogen.
The extraction buffer described in the Materials and Methods section provided adequate protection for the leaf extracts so that clear banding patterns were obtained. The 8 - 25 % polyacrylamide gradient gels and the 3 rnA intensity gave the best resolution of the isozymes. Of the ten enzymatic systems investigated, only seven were active under our experimental conditions. They were ACP, ADH, EST, GPI, G6P, PER, and 6PGD. Isozyme banding patterns showed no differences in enzyme forms, relative mobility values (Rf), and intensity of bands among individuals of the same cultivar under the same conditions. However, isozyme patterns revealed obvious differences in enzyme forms and band intensity among the tested cultivars, except for PER zymograms, which presented no significant differences. For this one, only two zones of activity appeared, but no polymorphism was found (data not shown). ACP banding patterns were not reproducible from one migration to another. Thus, we only studied the five following enzymatic systems: ADH, EST, GPI, G6P, 6PGD. G6P was not investigated for TKR and TNK cultivars. We used the same nomenclature as Tisserat and Torres (1979) for the genetic interpretation of our zymograms. The genes are named after the enzymatic system they specify. When two genes code for the same type of enzyme, the locus that specifies the isozyme set that migrates more slowly toward the anode is called 1, the faster 2. The allele that codes for the slower migrating isozyme within a set is called S, the faster F. Representative zymograms of the enzymatic systems studied are illustrated for each cultivar in Fig. 1. The corresponding genotypes are given in Table 1.
Electrophoresis Electrophoretic separation was carried out in an automated Pharmacia gel electrophoresis apparatus, the PhastSystem. It allowed us to accurately and rapidly reproduce the migration parameters. They were 400V, lOrnA, 2.5W, SoC, until 10Vh; then 400 V, 3 rnA, 2.5 W, 4°C for 280 Vh. For each run, two vertical gels SO x 43 x 0.45 mm were used. Each gel accommodated 8 samples. The gels (8-25% polyacrylamide gradient) were made of a 0.112M Tris-Acetate buffer pH 6.4. The running buffer, contained in a 2 % high quality agarose gel, consisted of a 0.25 M Tris and 0.88 M L-Alanine buffer pH 8.8. One ItL (1.5 Itg of protein) of each extract was loaded on the gel. Electrophoresis was carried out for 1 h1 h30, until the bromophenol blue dye had migrated within 0.5 cm of the bottom of the gel.
Enzymatic staining Ten different enzymatic systems were studied, namely acid phosphatase (ACP), alcohol dehydrogenase (ADH), alkaline phosphatase (ALP), esterase (EST), galactose dehydrogenase (GDH), glucose 6-phosphate dehydrogenase (G6P), glucose phosphate isomerase (GPI), 6-phosphogluconate dehydrogenase (6PGD), peroxidase (PER), and phosphoglucose mutase (PGM). All these enzymatic systems were stained according to Vallejos (1983), excepted EST. Staining mixture for EST consisted of 10 mg a-naphtylacetate and 10 mg {3-naphtylacetate in 0.3 mL acetone, 10 mg Fast Blue RR salt, and 10 mL of 0.525 M phosphate buffer pH 6.5.
Alcohol dehydrogenase (ADH) The zymograms produced by ADH consisted of one or three bands depending on the cultivar (Fig. 1). Plant ADH is well known to be a dimeric enzyme. The single-banded pattern suggested a homozygous condition, call it FF, and the three banded pattern suggested the heterozygous condition, call it FS. Only these two genotypes were observed among the cultivars studied. We assume that we are in the presence of one gene, adh-l, with two codominant alleles, F and S.
6-phosphogluconate dehydrogenase (6PGD) The 6PGD zymograms consisted of a four banded pattern. The bands migrating near the origin offer intensity variations (faint or dark) depending on the cultivar (Fig. 1). This observation could be explained if this enzyme is dimeric and if a null or silent allele is hypothesized for 6pgd-1. A missing isozyme is assumed to represent the homozygous null genotype NN, a faint band the heterozygote AN, and the normal dark band the homozygous condition AA. The genotypes AA or AN were represented, but we have never found the homozygous NN.
I. BOOI], S. MONFORT, and M. FERRY
64
PA TIERNS OBTAINED FOR 'THE CULTIVARS STUDIED m BIG BIG KHI BIG IRO Relative MJH fRO HIW BIG an THR NBT KHI BI G NBT m KHI IRO UK m m rnc.bility UK KHI m MJH m values m NBT KHO HIW In ZHO TKR IN K TKR m IRO
(Rf)
ZHO
HIW IHR INK IlR ZHO
IN K fRO ZHO HIW
~HlH
m
IH R
NBT KHI MJH IHR ZH0
HIW MJH
m
TN K
m
NBI
+100 90 80 -
--- --- --- --- --- ---11 __________________ 1D --___
- - ------ -
,-
- - - - - -8-
_ _ _ _ _ _1 _
70 60 50 40 30 -
...
... ,
----- -
••• ••• •••••
5
4
]
.2
-,
-5
_4
_1 -]
2
1
20 -
- ---
-
2
_1
-
-
F5
~
-
_4 _ -]
-, _1
5 _4
---
---
-2
-
NN
AN
-]
-1
]
-2 1
-
-
10 F5 F5 FS 55
~
RF -
AA AN NNAA AN NN AA
EST
FF
AA
AN
GPI 6PGD G6P ENZYMA TIC SYSTElvlSANAL'flED
Phosphoglucose isomerase (GP!) The GPI zymograms consisted of enzyme bands in two regions, one close to the origin and the other, nonvariable patterns between Rf 38 and Rf 46 (Fig. 1). There were one or three bands in the region close to the origin, depending on the cultivar, suggesting that these isozymes are dimers specified by one gene, gpi-l, having two alleles F and S. We have found the three genotypes SF, SS and FF.
Glucose 6-phosphate dehydrogenase (G6P) G6P occurred in two regions and are apparently specified by two genes. No polymorphism was found within the first set, specified by g6p-1. Isozymes of g6p-2 showed band intensity variations within one band (Fig. 1, band n05). As for 6PGD, we could assume a dimeric enzyme with a null or silent allele for g6p-2, giving the genotypes NN or AN.
Esterase (EST) EST banding pattern appeared to possess the highest number of isozyme molecular forms compared with the other enzyme systems analyzed. EST occurred in four regions (Fig. 1). No differences were found among the bands except in the two regions migrating near the origin, called EST-1
FF
FS
ADH
Fig. 1: Schematic illustration of the zymograms obtained for the thirteen date palm cultivarsa studied, by the analysis of five enzymatic systems b. a: BGF = Bou Feggous, BSK = Bou Skri, BST = Bou Sthammi noire, FRD = Fard # 4, HLW = Halawy back-cross, KHD = Khadrawy, KHL = Khalassa, MJH = Mejhool, NBT = Nabout Seif, TH = Thoory, TKR = Takerbouch, TNK = Tinakar, ZHD = Zahidi. b: EST = esterase, GPI = Phospho glucose isomerase, 6PGD = 6-phosphogluconate dehydrogenase, G6P = Glucose 6-phosphate dehydrogenase, ADH = alcohol dehydrogenase.
Table 1: Isozyme genotypes detected in the thirteen date palm cultivarsa analyzed. ADHb EST-lb EST-2b G6P-2b GPI-l b 6PGD-l b BFG BSK BST FRD HLW KHD KHL MJH NBT THR TKR TNK ZHD
FF FF FF FF FF FF FS FF FF FF FS FF FS
SS FS SS FS FS SS SS FS no bands RF SS SS SS
AA AA AN AN
NN AN AA AA AA NN AA AN AA
AN AN NN NN AN NN AN AN NN AN AN
FS FS FS FS FS FS FF FF SS FS FF FF FF
AA AA AA AN AA AA AN AA AN AN AA AN AA
BFG: Bou Feggous, BSK: Bou Skri, BST: Bou Sthammin noire, FRD: Fard #IV, HLW: Halawy back-cross, KHD: Khadrawy, KHL: Khalassa, MJH: Mejhool, NBT: Nabout Seif, THR: Thoory, TKR: Takerbouch, TNK: Tinakar, ZHD: Zahidi. b ADH: alcohol dehydrogenase; EST: esterase; G6P: 6-phosphoglucose dehydrogenase; GPI: phosphoglucose isomerase; 6PGD: 6-phosphogluconate dehydrogenase.
a
and EST-2. The slowest zone, EST-I, showed one or three bands. Based on the banding patterns obtained for each cultivar, it could be assumed that EST-1 occurs in the date palm
Isozymic characterization of date palm cultivars as a dimeric enzyme and is, apparently, under the control of a single locus with two alleles F and S. Evidence for a third allele of est-I, R (for Retarded), whose product migrated more slowly than that of S, was only found for THR cultivar. The genotypes found were SS, SF, and RF. The EST-2 zone is characterized by the presence of three bands, one of them showing intensity variations (Fig. 1, band n06). As for 6PGD, we could assume a dimeric enzyme with a null or silent allele for est-2, giving both the homozygous (NN or AA) and heterozygous (AN) genotypes.
no bands
Table 2: Enzymatic polymorphism found for the thirteen date palm cultivars studied, by the analysis of five enzymatic systems'. Enzymatic system
Zones observed
studied
2 2 2
slow middle slow fast slow
ADH EST GPI G6P 6PGD
Zones
Loci detected
Enzyme structure
Alleles detected
Dimeric Dimeric
3
Dimeric Dimeric
Dimeric Dimeric
Genotype detected
2 2 2 2 2
2 2 2
a ADH: alcohol dehydrogenase, EST: esterase, GPI: phosphoglucose isomerase, G6P: 6-phosphoglucose dehydrogenase, 6PGD: 6-phosphogluconate dehydrogenase.
SF
A
FF
SF
~ ~ ~
FF
SF
FF
Discussion
Six polymorphic loci have been found for the five enzymatic systems studied, with thirteen different alleles: 3 for EST-1locus and two for each ADH, EST-2, GPI, 6PGD and G6P locus (Table 1, Table 2). These results partly confirmed those obtained by Torres and Tisserat (1980) and Baaziz (1987). Variations found may be caused by the nature of vegetal material tested (older palms used by the above authors) or the conditions of the experiments (10 % polyacrylamide gels or starch gels used). Regarding the zymograms obtained for each enzymatic system studied, we had established a classification key by hierarchically sorting the enzymes showing the greatest number of genotypes (Bennaceur, 1991). The cultivars showing the same allozymic patterns were grouped together. The enzymatic systems showing a null or silent allele were latter observed, because the hypothesis for their genotypic interpretation should be verified. For instance, variations observed could only be the result of experimental conditions. However, zymograms obtained were exactly reproducible, and the isozymes of the other loci had showed the same intensity banding patterns. On the other hand, intensity variations observed on the litigious locus (gene with a silent or null allele) corresponded well with different proportions of its alleles. Nevertheless, these differences could also be caused by the presence in the extracts of an exogen factor limiting the isozyme revelation (Pasteur et al., 1987). Eleven groups of «standard» patterns have been established within the thirteen cultivars analyzed (Fig. 2). We can accurately distinguish nine of them (69 %): BFG, BSK, FRD, HLW, KHL, MJH, NBT, THR, TNK. Three ofthese cult ivars showed an unique genotype for EST: NBT (no isozy-
RF
BT
65
SF
NK 'KJ{L
zJi'J)
Fig. 2: Classification of thirteen date palm cultivars" based on their enzymatic genotypes. The enzymaticb genotypes obtained with an accurate interpretation (EST-I, ADH and GPI-I) have been differentiated from those based on the hypothesis of a null or silent allele (6PGD-I, G6P and EST-2). aBGF: Bou Feggous, BSK: Bou Skri, BST: Bou Sthammi noire, FRD: Fard # IV, HLW: Halawy backcross, KHD: Khadrawy, KHL: Khalassa, MJH: Mejhool, NBT: Nabout Seif, TH: Thoory, TKR: Takerbouch, TNK: Tinakar, ZHD: Zahidi. bEST: esterase, GPI: phosphoglucose isomerase, ADH: alcohol dehydrogenase, 6PGD: 6-phosphogluconate dehydrogenase.
mes in the EST-1 zone), THR (RF genotype for the EST-1 zone), and HLW (FS genotype for the EST-1 zone and NN genotype for the EST-2 zone). The last four cultivars were classified into two groups: BST-KHD, and TKR-ZHD (Fig. 2). The analysis of other key enzymes or a correlation between the isozyme identification and conventional identification (at fruiting stages) should allow us to distinguish between them and complete our classification. The separation of different cultivars with the PhastSystem gave us good results, with a high analysis capacity per day. This method, more than standardization advantages, required short preparation and separation times and gave a high resolution. Even if it should be necessary to be cautious in interpreting the zymograms, enzyme electrophoresis with the PhastSystem appeared to be such an efficient and performing tool that we could rapidly and accurately analyze numerous samples at early stages of development. Date palm cultivar identification will enable us to verify the homogeneity of in vitro shoots and to distinguish between the different cultivars. We should find and eliminate any variant produced, as well as the processes generating them, without waiting for the first fructification (usually four to five years). It is an appreciable gain of time. This technique could be used to control the vitroplant production.
66
I. BoorJ, S. MONFORT, and M. FERRY
Acknowledgements
We gratefully acknowledge F. Kjellberg and R. Lumaret for their help in the interpretation of zymograms and for providing useful commentaries on a preliminary draft of this manuscript.
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