114
A . Posch et ill
Anton Posch’ Bartel M. van den Berg’ Wilhelm Postel’ Angelika Gorg’ ‘Lehrstuhl fiir Allgemeine Lebensmitteltechnologie, Technische Universitat Munchen, Freising-Weihenstephan ‘Royal Sluis Holding, Biotech Center, Enkhuizen
Genetic variability of pepper (Capsicum annuurn L.) seed proteins studied by 2-D electrophoresis with immobilized pH gradients Ten pepper (Capsicum annuum L.) inbred lines were successfully differentiated by two-dimensional electrophoresis with immobilized pH gradients. Qualitative polymorphism of water-soluble and urealdetergent-soluble seed proteins, respectively, was investigated by computer analysis and used for establishing a dendrogram derived from maximum-parsimony analysis. The dendrogram calculated from ureddetergent-soluble proteins shows four types of distance indices, whereas water-soluble proteins show two sets of inbred lines with similar intraset distance indices. The validity of the dendrograms with respect to quantitative inherited traits, such as cold tolerance and earliness, will be tested by field trials.
Pepper (Capsicum aiznuuin L.) is a major spice and vegetable crop of great economic importance [l]. Most of the commercially grown peppers are open pollinated varieties, but hybrid breeding is preferred for the following reasons: (i) the crop is more homogeneous concerning plant characteristics (e.g., fruit size, fruit shape, ripening date) and in some cases higher yielding compared to open pollinated varieties. For most vegetables, homogeneity of the crop is the primary reason for hybrid breeding. (ii) Breeder’s material is better protected by growing hybrids, because their selfing gives a highly variable offspring that is of no use to growers and of little use for competitors. An important aspect of hybrid breeding is the efficient selection of inbred lines. Selection, restricted to vegetables, is generally based on the performance of the crop (P.x., fruit characteristics, earliness, yield). The use of morphological markers is difficult because their expression is influenced by environmental conditions. Moreoover, artificial selection has made most agromorphological characteristics unreliable for use as markers of genetic variation [2-41. On the other hand, molecular markers have the advantage that they can be obtained rapidly and in high numbers. They can be utilized to estimate genetic variation and consequently to identify inbred lines that produce hybrids exhibiting increased levels of heterosis. Moreover, molecular markers can b e used for maintaining or increasing genetic variation of the genetic material used in breeding. Extensive knowledge with respect to the association of genetic distance and heterosis has been gathered by hybrid breeding of maize. In several studies, genetic distance between inbred lines of maize has been estimated from isozyme data to predict heterosis. However, genetic distance, as measured by isozymes differences, has not been a good predictor of hybrid perfomance [5,6]. DNA technology, providing a virtually unlimited number of loci and alleles per locus when compared to isozyme electrophoresis, has also been applied to detect molecular genetic polymorCorrespondence: Priv. DOL. Dr. Angelika Gorg, Technische Universitat Miinchen, Lehrstclh 1 fur Allgeineine Lebensmitteltechnologie, DW-8050 Freising-Weihenstephan, Germany Abbreviations: DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; IPG-DALT, two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradient; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate: Tris, Tris (hydroxymethy1)aminomethane; 2-D, two-dimensional
0 VCH Verlags%csell\chafl mbH, D-6940 Weinheim, 1992
phisms in plant species [7].Experimental results of Smith et al. [8], who used a large number of restriction fragment length polymorphism markers, showed much better correlations between the parental distance and the hybrid trait than was the case with isozymes. Leonardi et al. [9] applied two-dimensional (2-D) electrophoresis data in order to predict maize hybrids with high levels of heterosis. They found for maize that distance indices, based on protein amount polymorphisms (PAP), are correlated to hybrid performances, whereas the classical qualitative variability of proteins (spot presentlabsent) failed to be predictive of hybrid values for any of the agromorphological traits investigated. In this study, we characterize 10 inbred pepper lines that show only little morphological variation by qualitative polymorphism of seed proteins using 2-D electrophoresis with immobilized pH gradients (IPG-DALT[lO]).Aquantitative evaluation of the protein polymorphism will follow in order to explore the association between qualitative and quantitative protein polymorphism with quantitative inherited traits, such as cold tolerance and earliness in pepper. Equipment for isoelectric focusing (IEF) and horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), (Ultrophor, Multiphor 11, gradient mixer, power supply Macrodrive 5, thermostatic circulator Multitemp II), Immobiline DryPlate (pH 4-7), carrier ampholytes pH 4-6, 2-D Pharmalytes pH 3-10 and GelBond PAG-film were from Pharmacia-LKB (Bromma-Sweden). Electrode wicks (Ultra wicks) for SDS-PAGE were from Bio-Rad (Richmond, CA, USA). Acrylamide (2 X cryst.), N,N’-methylenebisacrylamide (Bis), ammonium persulfate, N,N,N’,N’-tetramethylethylenediamine (TEMED), glycine (Gly), and SDS were from Serva (Heidelberg, Germany). Tris(hydroxymethy1)aminomethane (Tris), Nonidet P-40 (NP-40), dithiothreitol (DTT), and iodoacetamide were from Sigma (St. Louis,MO, USA). Urea, silver nitrate, glycerol, and all other chemicals of analytical grade were from Merck (Darmstadt, Germany). Ten inbred pepper lines (named A-J), based on germplasm from Turkey, were developed by Claude Duranton (Royal Sluis, France), which are homozygous. Twenty to twentyfive pepper seeds were crushed in a liquid nitrogen-cooled mortar and the resulting flour was passed through a 0.5 mm wide sieve. (i) For extraction of water-soluble proteins, 30 mg of the powder was mixed with 250 pLsolubilization buffer of 0.2 O/o w/v carrier ampholytes (pH 4-6), 1 O/o DTT and extracted for 30 min at room temperature with occasional 0173-0838/92/0Y 10-0774 $3.50+.28/0
tlcctl-ophoreii.~1992. 13, 774-777
IPG-DALT or pepper seed proteins
775
the surface of the IPG gel strips, 5 mm from the anode. To facilitate sample entry, voltage was limited to 150 V for the first 30 min and to 300 Vfor the next 60 min. Then IEF was continued with maximum settings of 3500 V, 2 mA and 5 W. After 21000 Vh, constant focusing patterns were obtained. IPG gel strips were either used immediately or stored at -80°C until use. The IPG gel strips were equilibrated for 2 X 15 min in 2 X 10 mL equilibration solution (0.05 M TrisHCI buffer, pH 8.8, containing 6 M urea, 30% w/v glycerol, 2% w/v SDS, and a trace of Bromophenol Blue). DTT (1 O/o w/v) was added to the first equilibration solution and iodoacetamide, 260 mM, to the second [13]. After the equilibration step, the IPG gel strips were blotted on filter paper Horizontal IPG-DALTwas performed according to Gorg et a/. [ 111with minor modifications. All experiments were per- to remove excess equilibration buffer and were then transformed at least in duplicate. For the first dimension, ready- ferred, gel side down, onto the surface of the horizontal SDS pore gradient gel (250 X 190 X 0.5 mm3)cast onto Gelmade IPG gels (IPG DryPlate, 250 X 110 mm), pH 4-7, Bond PAGfilm along the cathodic electrode wick, 1 mm were used. Prior to IEF, the IPG gels were cut into strips (5 mm wide) with the help of a paper cutter. The IPG gel apart. GelBond PAGfilms were washed 6 X 10 min with deionized water prior to use to minimize spot streaking [ 141. strips were rehydrated overnight, in a mold, to the original gel thickness (0.5 mm). Two different rehydration solu- The SDS gels contained a stacking gel which consisted of tions, depending on the sample, were used: (i) water-solu- 6%T and 4%c, and a resolving gel consisting of a linear acrylamide gradient from 12-15YoT. The buffer system was ble proteins - 0.25% w/v Pharmalytes, pH 3-10, 15% w/v 125 mM Tris-HC1,pH 6.8,and 0.1 O/o w/vSDS in the stacking glycerol and 10 mM DTT, and (ii) mealdetergent-soluble proteins-8 M urea,0.50/0~ / ~ N P - 4 0 , 1 0 m ~ D T T a n d 0 . 2 5 %gel, and 375 mM Tris-HCI, pH 8.8, and 0.1 O/o SDS in the resolving gel. The electrode buffer was 25 mM Tris, 192 mM w/v Pharmalytes, pH 3-10. Twenty rehydrated IPG gel glycine and 0.1 O/o SDS [15]. Electrophoresis was performed strips were placed side by side, 2 mm apart, on the flat-bed cooling plate and were cooled at 15°C [12]. The electrode at a maximum voltage of 200 V for about 1 h until the Bromophenol Blue dye had migrated out of the IPG gel strips. strips were soaked with water. A total of 20pL of each sample was applied into silicone rubber frames placed onto Then the IPG gel strips were removed from the surface of
vortexing. Then the suspension was centrifuged (16000 g, 30 min, room temperature). The supernatant was either used immediately for electrophoresis or stored at -80 C until use. (ii) To extract urea/detergent-soluble seed proteins, 15 mg powder was suspended in 500 I.ILlysis solution containing 9 M urea, 1Yo w/vNP-40,1 [Yo w/v DTT and 0.8 O/o w/v Pharmalytes, pH 3-10. The suspension was gently shaken for 30 min at room temperature and then centrifuged (16000 g, 30 min, room temperature). The supernatant was then either used for electrophoresis at once or stored at -80°C until use.
Ffgure 1. IPG-DALT [ I l l ofpepper seed proteins. First dimension: IEF with IPG 4-7 (separation distance: 110 mm). Second dimension: SDS-PAGE, 12-15% T. Silver staining according to [16]. (A) Water-soluble proteins of inbred line I. (B) Ureddetergent-soluble proteins of inbred line I.
776
Electrophormis 1992, 1 3 ~174-771
A . Posch ('1 cil
Genetic distances D between two lines were estimated by qualitative protein polymorphism using the statistics of Nei [19]:
the S D S gel and the cathodic electrode wick moved forward so that it overlapped the former application area of the IPG gel strip. Electrophoresis was continued with maxim u m settings of 600 V, 30 mA and 50 W until the Bromophenol Blue front had reached the end of the SDS gel.The gel was then fixed in methanol/acetic/water (4/1/5) for at least 1 h (but usually overnight) and then silver-stained using the method of Merril er al. [16]. 2-D gels were digitized at 80 pm resolution using a Molecular Dynamics 300A laser densitome1.er (Sunnyvale, USA). The resulting 2-D gel images were analyzed using the PDQUEST [17] software package (Protein and DNA Imageware Inc, New York, USA) based on the Quest system of Garrels [18], running on a SPARCstation IPC microcomputer (Sun Microsysterns).
D
= d/(n
+ d)
where n is the number of spots common to both lines and I( the number of spots exclusive to both lines; d was preliminarily determined without any genetic interpretation of the protein pattern. Analyzing homozygous inbred lines, each line displays one and only one polypeptide of an allelic series. Every qualitative difference between two lines was considered as an allelic variation. D is therefore only a rough estimation of the genetic variability between the ten inbred pepper lines examined. The presence or absence of
Figure 2. Simultaneous matching of multiple gels (urea/detergent-soluble proteins). (I) Background reduced image of inbred pepper line A. (11) Enlarged area of (I) marked by window. Comparison of similar areas of2-D gel patterns run in duplicate (mastergel M; inbred lines A, C, D, F, G, I. J).
Table 1. Genetic distance often inbred pepperlines (A-.I) using data from 2-D electrophoresis ofwater-soluble proteins (bottom diagonal) and urea/detergent-soluble proteins (top diagonal) A
A B C
D E F G
H 1 J
0.020 0.020 0.007 0.014 0.324 0.317 0.328 0.3 1 I 0.298
B
C
D
E
F
G
H
I
J
0.012 -
0.015 0.002
0.000 0.014 0.020 0.317 0.309 (18.320 0.304 0.291
-
0.010 0.002 0.005
0.014 0.020 0.317 0.309 0.320 0.304 0.291
-
0.019 0.012 0.010 0.010
0.007 0.318 0.311 0.322 0.306 0.292
0.017 0.010 0.012 0.007 0.012
0.324 0.317 0.328 0.3 11 0.298
-
0.050 0.043 0.046 0.041 0.050 0.048
0.019 0.063 0.049 0.067
-
0.019 0.017 0.019 0.015 0.024 0.022 0.036
0.080 0.031 0.049
-
0.029 0.022 0.024 0.020 0.024 0.012 0.060 0.034
0.098 0.104
0.017 0.010 0.012 0.007 0.017 0.01s 0.038 0.007 0.027
0.019
~
Elrcirophuresi~1992. 13, 774-777
IPG-DALT of pepper seed proteins
proteins was then transformed into a 1 (present) or 0 (absent) matrix. The character matrix was analyzed using the Wagner method contained in the PAUP computer package. This algorithm derives minimum-phylogenetic-length trees based upon the principle of parsimony [20]. By using horizontal IPG-DALT, highly reproducible protein patterns were obtained. Figure 1 illustrates typical high resolution 2-D gels of both kinds of protein samples. The silver stained gels were digitized and subjected to computer analysis (Fig. 2). After background subtraction, editing to remove streaks and spot detection, only the most distinct spots were chosen for estimation of genetic distance. These were 184 from about 230 water-soluble and 419 from about 550 urea/detergent-soluble proteins. Of the water-soluble and urea/detergent-soluble proteins, 37% and 8 %, respectively, were variable in presence/absence. Genetic distance indices based on 2-D electrophoresis between pairs of the ten inbred lines are presented in Table 1. The same data from 2-D electrophoresis were transferred into a 1 (pres-
A.
777
ent) or 0 (dbsent) matrix over all varieties. Unrooted minimum length trees (shown in Fig. 3) were constructed using the Wagner method of the PAUP computer program 1201. The dendrogram calculated from the variability of the water-soluble proteins shows 2 sets of inbred lines (Line A, B,C,D,E;LineF,G,H,I,J) with similarintraset distanceindices. The dendrogram revealed after analyzing the variability of the urea/detergent-soluble proteins shows more groups. There are four types of distance indices when c o v paring all inbred lines to each other. These studies have indicated that genetic distances can easily be estimated using 2-D electrophoresis in a reliable way as to number of mutations. A genetic distance analysis needs at least 20 protein differences.Now this can easily be done by 2-D electrophoresis in contrast to other techniques, such as isozyme electrophoresis which is very timeconsuming, because one has to use several gel systems, different staining and extraction techniques, and different tissues (from roots, leaves, etc.). However, whether all visible differences are different mutations and are meaningful for distance analysis will be the subject of further studies (e.g. genetic linkage and secondary modifications of proteins). Received August 10, 1992
C
References
- D E F
-
I - J
-H
B.
c: F I
H
Figure 3. (A) Dendrogram derived from maximum-parsimony analysis of the data from 2-D electrophorsis using the presence/absence variability of 34 urealdetergent-soluble proteins. Branch and internodal distances are proportional to the number of changes required. The total length was 37. and the consistency index (a measure of homoplasy) was 0.019. (B) Dendrogram derived from maximum-parsimony analysis of the data from 2-D electrophoresis using the presence/absence variability of 68 water-soluble pepper seed proteins. The total length was 76, and the consistency index was 0.895.
[l] Panda, R.C., Theor. Appl. Genet. 1986, 72, 665-670. [2] Shifriss, C. and Sacks, J.M., Theor. Appl. Genet. 1980, j8,253-256. [3] Smith, J.S.C. and Smith, 0,s.in: Proc. U P O V Workshop, Versuilles, 1989. [4] Ghaderi, A., Adams, M.W. and Nassib, A.M., Crop Science 1984,24, 37-42. [5] Price, S.C., Kahler,A.L.,Hallauer, A.R., Charmley, P. and Giegel, P., J. Hered. 1986, 77, 341-344. [6] Lamkey, K.R., Hallauer, A.R. and Kahler, A.L., J. Hered 1987, 78, 231-234. [7] Lee, M. Godshalk, E.B., Lamkey, K.R. and Woodman, W.W., Crop Science 1989,2Y, 1067-1071. [8] Smith,O.S.,Smith,J.S.C.,Bowen,S.L.,Tenborg,R.A.andWall,S.J. Theor. Appl. Genet. 1990, 80, 833-840. (91 Leonardi, A., Damerval, C., Herbert, Y., Gallais, A. and de Vienne, D., Theor. Appl. Genet. 1991, 82, 552-560. [lo] Gorg, A. Nuture 1991, 349, 545-546. [ll] Gorg, A,, Postel, W. and Gunther, S., Electrophoresis 1988, 9, 531546. [12] Gorg, A,, Postel, W., Friedrich, C., Kuick, R., Strahler, J.R. and Hanash, S.M., Electrophoresis 1991, 12, 653-658. [13] Gorg, A., Postel, W., Weser, J., Gunther, S., Strahler, J.R., Hanash, S.M. and Somerlot, L., Electrphoresis 1987, 8, 122-124. [14] Gorg,A.,Postel,W., Gunter, S. and Weser,J.,Electrophoresis 1985,6, 599-604. [I51 Laemmli, U.K., Nuture 1970, 227, 680-685. [16] Merril, C.R.,Goldman, D., Sedman, S.A. andEbert. H.,Science 1981, 211, 1437-1438. [17] Krauss, M.R., Collins, P.J. and Blose, S.H., BioTechniques 1990, 8, 218-223. [18] Garrels, J.I., J. Biol. Chem. 1989, 264, 5269-5282. [I91 Nei, M.. Arnrr. Nutu,: 1972, 106, 283-292. [20] Swofford, D.L., PAUP (Phylogenetic Analysis Using Parsimony), Documentation of program, Illinois Natural History Survey, Champaign 1985.
A . Posch et ill
Anton Posch’ Bartel M. van den Berg’ Wilhelm Postel’ Angelika Gorg’ ‘Lehrstuhl fiir Allgemeine Lebensmitteltechnologie, Technische Universitat Munchen, Freising-Weihenstephan ‘Royal Sluis Holding, Biotech Center, Enkhuizen
Genetic variability of pepper (Capsicum annuurn L.) seed proteins studied by 2-D electrophoresis with immobilized pH gradients Ten pepper (Capsicum annuum L.) inbred lines were successfully differentiated by two-dimensional electrophoresis with immobilized pH gradients. Qualitative polymorphism of water-soluble and urealdetergent-soluble seed proteins, respectively, was investigated by computer analysis and used for establishing a dendrogram derived from maximum-parsimony analysis. The dendrogram calculated from ureddetergent-soluble proteins shows four types of distance indices, whereas water-soluble proteins show two sets of inbred lines with similar intraset distance indices. The validity of the dendrograms with respect to quantitative inherited traits, such as cold tolerance and earliness, will be tested by field trials.
Pepper (Capsicum aiznuuin L.) is a major spice and vegetable crop of great economic importance [l]. Most of the commercially grown peppers are open pollinated varieties, but hybrid breeding is preferred for the following reasons: (i) the crop is more homogeneous concerning plant characteristics (e.g., fruit size, fruit shape, ripening date) and in some cases higher yielding compared to open pollinated varieties. For most vegetables, homogeneity of the crop is the primary reason for hybrid breeding. (ii) Breeder’s material is better protected by growing hybrids, because their selfing gives a highly variable offspring that is of no use to growers and of little use for competitors. An important aspect of hybrid breeding is the efficient selection of inbred lines. Selection, restricted to vegetables, is generally based on the performance of the crop (P.x., fruit characteristics, earliness, yield). The use of morphological markers is difficult because their expression is influenced by environmental conditions. Moreoover, artificial selection has made most agromorphological characteristics unreliable for use as markers of genetic variation [2-41. On the other hand, molecular markers have the advantage that they can be obtained rapidly and in high numbers. They can be utilized to estimate genetic variation and consequently to identify inbred lines that produce hybrids exhibiting increased levels of heterosis. Moreover, molecular markers can b e used for maintaining or increasing genetic variation of the genetic material used in breeding. Extensive knowledge with respect to the association of genetic distance and heterosis has been gathered by hybrid breeding of maize. In several studies, genetic distance between inbred lines of maize has been estimated from isozyme data to predict heterosis. However, genetic distance, as measured by isozymes differences, has not been a good predictor of hybrid perfomance [5,6]. DNA technology, providing a virtually unlimited number of loci and alleles per locus when compared to isozyme electrophoresis, has also been applied to detect molecular genetic polymorCorrespondence: Priv. DOL. Dr. Angelika Gorg, Technische Universitat Miinchen, Lehrstclh 1 fur Allgeineine Lebensmitteltechnologie, DW-8050 Freising-Weihenstephan, Germany Abbreviations: DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; IPG-DALT, two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradient; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate: Tris, Tris (hydroxymethy1)aminomethane; 2-D, two-dimensional
0 VCH Verlags%csell\chafl mbH, D-6940 Weinheim, 1992
phisms in plant species [7].Experimental results of Smith et al. [8], who used a large number of restriction fragment length polymorphism markers, showed much better correlations between the parental distance and the hybrid trait than was the case with isozymes. Leonardi et al. [9] applied two-dimensional (2-D) electrophoresis data in order to predict maize hybrids with high levels of heterosis. They found for maize that distance indices, based on protein amount polymorphisms (PAP), are correlated to hybrid performances, whereas the classical qualitative variability of proteins (spot presentlabsent) failed to be predictive of hybrid values for any of the agromorphological traits investigated. In this study, we characterize 10 inbred pepper lines that show only little morphological variation by qualitative polymorphism of seed proteins using 2-D electrophoresis with immobilized pH gradients (IPG-DALT[lO]).Aquantitative evaluation of the protein polymorphism will follow in order to explore the association between qualitative and quantitative protein polymorphism with quantitative inherited traits, such as cold tolerance and earliness in pepper. Equipment for isoelectric focusing (IEF) and horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), (Ultrophor, Multiphor 11, gradient mixer, power supply Macrodrive 5, thermostatic circulator Multitemp II), Immobiline DryPlate (pH 4-7), carrier ampholytes pH 4-6, 2-D Pharmalytes pH 3-10 and GelBond PAG-film were from Pharmacia-LKB (Bromma-Sweden). Electrode wicks (Ultra wicks) for SDS-PAGE were from Bio-Rad (Richmond, CA, USA). Acrylamide (2 X cryst.), N,N’-methylenebisacrylamide (Bis), ammonium persulfate, N,N,N’,N’-tetramethylethylenediamine (TEMED), glycine (Gly), and SDS were from Serva (Heidelberg, Germany). Tris(hydroxymethy1)aminomethane (Tris), Nonidet P-40 (NP-40), dithiothreitol (DTT), and iodoacetamide were from Sigma (St. Louis,MO, USA). Urea, silver nitrate, glycerol, and all other chemicals of analytical grade were from Merck (Darmstadt, Germany). Ten inbred pepper lines (named A-J), based on germplasm from Turkey, were developed by Claude Duranton (Royal Sluis, France), which are homozygous. Twenty to twentyfive pepper seeds were crushed in a liquid nitrogen-cooled mortar and the resulting flour was passed through a 0.5 mm wide sieve. (i) For extraction of water-soluble proteins, 30 mg of the powder was mixed with 250 pLsolubilization buffer of 0.2 O/o w/v carrier ampholytes (pH 4-6), 1 O/o DTT and extracted for 30 min at room temperature with occasional 0173-0838/92/0Y 10-0774 $3.50+.28/0
tlcctl-ophoreii.~1992. 13, 774-777
IPG-DALT or pepper seed proteins
775
the surface of the IPG gel strips, 5 mm from the anode. To facilitate sample entry, voltage was limited to 150 V for the first 30 min and to 300 Vfor the next 60 min. Then IEF was continued with maximum settings of 3500 V, 2 mA and 5 W. After 21000 Vh, constant focusing patterns were obtained. IPG gel strips were either used immediately or stored at -80°C until use. The IPG gel strips were equilibrated for 2 X 15 min in 2 X 10 mL equilibration solution (0.05 M TrisHCI buffer, pH 8.8, containing 6 M urea, 30% w/v glycerol, 2% w/v SDS, and a trace of Bromophenol Blue). DTT (1 O/o w/v) was added to the first equilibration solution and iodoacetamide, 260 mM, to the second [13]. After the equilibration step, the IPG gel strips were blotted on filter paper Horizontal IPG-DALTwas performed according to Gorg et a/. [ 111with minor modifications. All experiments were per- to remove excess equilibration buffer and were then transformed at least in duplicate. For the first dimension, ready- ferred, gel side down, onto the surface of the horizontal SDS pore gradient gel (250 X 190 X 0.5 mm3)cast onto Gelmade IPG gels (IPG DryPlate, 250 X 110 mm), pH 4-7, Bond PAGfilm along the cathodic electrode wick, 1 mm were used. Prior to IEF, the IPG gels were cut into strips (5 mm wide) with the help of a paper cutter. The IPG gel apart. GelBond PAGfilms were washed 6 X 10 min with deionized water prior to use to minimize spot streaking [ 141. strips were rehydrated overnight, in a mold, to the original gel thickness (0.5 mm). Two different rehydration solu- The SDS gels contained a stacking gel which consisted of tions, depending on the sample, were used: (i) water-solu- 6%T and 4%c, and a resolving gel consisting of a linear acrylamide gradient from 12-15YoT. The buffer system was ble proteins - 0.25% w/v Pharmalytes, pH 3-10, 15% w/v 125 mM Tris-HC1,pH 6.8,and 0.1 O/o w/vSDS in the stacking glycerol and 10 mM DTT, and (ii) mealdetergent-soluble proteins-8 M urea,0.50/0~ / ~ N P - 4 0 , 1 0 m ~ D T T a n d 0 . 2 5 %gel, and 375 mM Tris-HCI, pH 8.8, and 0.1 O/o SDS in the resolving gel. The electrode buffer was 25 mM Tris, 192 mM w/v Pharmalytes, pH 3-10. Twenty rehydrated IPG gel glycine and 0.1 O/o SDS [15]. Electrophoresis was performed strips were placed side by side, 2 mm apart, on the flat-bed cooling plate and were cooled at 15°C [12]. The electrode at a maximum voltage of 200 V for about 1 h until the Bromophenol Blue dye had migrated out of the IPG gel strips. strips were soaked with water. A total of 20pL of each sample was applied into silicone rubber frames placed onto Then the IPG gel strips were removed from the surface of
vortexing. Then the suspension was centrifuged (16000 g, 30 min, room temperature). The supernatant was either used immediately for electrophoresis or stored at -80 C until use. (ii) To extract urea/detergent-soluble seed proteins, 15 mg powder was suspended in 500 I.ILlysis solution containing 9 M urea, 1Yo w/vNP-40,1 [Yo w/v DTT and 0.8 O/o w/v Pharmalytes, pH 3-10. The suspension was gently shaken for 30 min at room temperature and then centrifuged (16000 g, 30 min, room temperature). The supernatant was then either used for electrophoresis at once or stored at -80°C until use.
Ffgure 1. IPG-DALT [ I l l ofpepper seed proteins. First dimension: IEF with IPG 4-7 (separation distance: 110 mm). Second dimension: SDS-PAGE, 12-15% T. Silver staining according to [16]. (A) Water-soluble proteins of inbred line I. (B) Ureddetergent-soluble proteins of inbred line I.
776
Electrophormis 1992, 1 3 ~174-771
A . Posch ('1 cil
Genetic distances D between two lines were estimated by qualitative protein polymorphism using the statistics of Nei [19]:
the S D S gel and the cathodic electrode wick moved forward so that it overlapped the former application area of the IPG gel strip. Electrophoresis was continued with maxim u m settings of 600 V, 30 mA and 50 W until the Bromophenol Blue front had reached the end of the SDS gel.The gel was then fixed in methanol/acetic/water (4/1/5) for at least 1 h (but usually overnight) and then silver-stained using the method of Merril er al. [16]. 2-D gels were digitized at 80 pm resolution using a Molecular Dynamics 300A laser densitome1.er (Sunnyvale, USA). The resulting 2-D gel images were analyzed using the PDQUEST [17] software package (Protein and DNA Imageware Inc, New York, USA) based on the Quest system of Garrels [18], running on a SPARCstation IPC microcomputer (Sun Microsysterns).
D
= d/(n
+ d)
where n is the number of spots common to both lines and I( the number of spots exclusive to both lines; d was preliminarily determined without any genetic interpretation of the protein pattern. Analyzing homozygous inbred lines, each line displays one and only one polypeptide of an allelic series. Every qualitative difference between two lines was considered as an allelic variation. D is therefore only a rough estimation of the genetic variability between the ten inbred pepper lines examined. The presence or absence of
Figure 2. Simultaneous matching of multiple gels (urea/detergent-soluble proteins). (I) Background reduced image of inbred pepper line A. (11) Enlarged area of (I) marked by window. Comparison of similar areas of2-D gel patterns run in duplicate (mastergel M; inbred lines A, C, D, F, G, I. J).
Table 1. Genetic distance often inbred pepperlines (A-.I) using data from 2-D electrophoresis ofwater-soluble proteins (bottom diagonal) and urea/detergent-soluble proteins (top diagonal) A
A B C
D E F G
H 1 J
0.020 0.020 0.007 0.014 0.324 0.317 0.328 0.3 1 I 0.298
B
C
D
E
F
G
H
I
J
0.012 -
0.015 0.002
0.000 0.014 0.020 0.317 0.309 (18.320 0.304 0.291
-
0.010 0.002 0.005
0.014 0.020 0.317 0.309 0.320 0.304 0.291
-
0.019 0.012 0.010 0.010
0.007 0.318 0.311 0.322 0.306 0.292
0.017 0.010 0.012 0.007 0.012
0.324 0.317 0.328 0.3 11 0.298
-
0.050 0.043 0.046 0.041 0.050 0.048
0.019 0.063 0.049 0.067
-
0.019 0.017 0.019 0.015 0.024 0.022 0.036
0.080 0.031 0.049
-
0.029 0.022 0.024 0.020 0.024 0.012 0.060 0.034
0.098 0.104
0.017 0.010 0.012 0.007 0.017 0.01s 0.038 0.007 0.027
0.019
~
Elrcirophuresi~1992. 13, 774-777
IPG-DALT of pepper seed proteins
proteins was then transformed into a 1 (present) or 0 (absent) matrix. The character matrix was analyzed using the Wagner method contained in the PAUP computer package. This algorithm derives minimum-phylogenetic-length trees based upon the principle of parsimony [20]. By using horizontal IPG-DALT, highly reproducible protein patterns were obtained. Figure 1 illustrates typical high resolution 2-D gels of both kinds of protein samples. The silver stained gels were digitized and subjected to computer analysis (Fig. 2). After background subtraction, editing to remove streaks and spot detection, only the most distinct spots were chosen for estimation of genetic distance. These were 184 from about 230 water-soluble and 419 from about 550 urea/detergent-soluble proteins. Of the water-soluble and urea/detergent-soluble proteins, 37% and 8 %, respectively, were variable in presence/absence. Genetic distance indices based on 2-D electrophoresis between pairs of the ten inbred lines are presented in Table 1. The same data from 2-D electrophoresis were transferred into a 1 (pres-
A.
777
ent) or 0 (dbsent) matrix over all varieties. Unrooted minimum length trees (shown in Fig. 3) were constructed using the Wagner method of the PAUP computer program 1201. The dendrogram calculated from the variability of the water-soluble proteins shows 2 sets of inbred lines (Line A, B,C,D,E;LineF,G,H,I,J) with similarintraset distanceindices. The dendrogram revealed after analyzing the variability of the urea/detergent-soluble proteins shows more groups. There are four types of distance indices when c o v paring all inbred lines to each other. These studies have indicated that genetic distances can easily be estimated using 2-D electrophoresis in a reliable way as to number of mutations. A genetic distance analysis needs at least 20 protein differences.Now this can easily be done by 2-D electrophoresis in contrast to other techniques, such as isozyme electrophoresis which is very timeconsuming, because one has to use several gel systems, different staining and extraction techniques, and different tissues (from roots, leaves, etc.). However, whether all visible differences are different mutations and are meaningful for distance analysis will be the subject of further studies (e.g. genetic linkage and secondary modifications of proteins). Received August 10, 1992
C
References
- D E F
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I - J
-H
B.
c: F I
H
Figure 3. (A) Dendrogram derived from maximum-parsimony analysis of the data from 2-D electrophorsis using the presence/absence variability of 34 urealdetergent-soluble proteins. Branch and internodal distances are proportional to the number of changes required. The total length was 37. and the consistency index (a measure of homoplasy) was 0.019. (B) Dendrogram derived from maximum-parsimony analysis of the data from 2-D electrophoresis using the presence/absence variability of 68 water-soluble pepper seed proteins. The total length was 76, and the consistency index was 0.895.
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