This file was created by scanning the printed publication.
Errors identified by the software have been corrected;
however, some errors may remain.
Chapter 15
Differentiation of Poplar Clones Using Random
Amplified Polymorphic DNA Fingerprinting 1
Dolly .c. Lin, Martin Hubbes, and Louis Zsuffa
Introduction
For over a decade, interest in poplars for short rotation
intensive culture plantations for energy, chemicals, and fiber has been developing (Zsuffa et al. 1984). Due to easy
clonal propagation, species and hybrid materials were inadequately defined. Conclusive identification of Populus
species and hybrids is urgently required to: 1) further genetic improvement and characterization; 2) determine the
linkage of important economic traits such as fast growth,
superior wood quality, and ; and 3) enable advanced genetic engineering and breeding.
Recent progress in molecular biology has generated new
analytical tools that are well suited for taxonomic and genetic investigations. Several approaches have been studied
in poplar such as isozyme markers (Rajora 1988, 1989a, 1989b;
Rajora and Zsuffa 1989, 1990, 1991; Rajora et al. 1991), restriction fragment length polymorphism (RFLP) markers
(Bradshaw et al. 1994; Soltes-Rak 1993), STS (sequence tagged
site)/RFLP (Bradshaw et al. 1994), ribosomal DNA (rONA)
polymorphisms (D'Ovidio 1992; D'Ovidio et al. 1990, 1991;
Faivre-Rampant et al. 1992a, 1992b), mitochondrial DNA
(mtDNA) (Barrett et al. 1993), chloroplast DNA (cpDNA)
(Rajora and Dancik 1995a, 1995b, 199Sc; Smith and Sytsma
1990), and random amplified polymorphic DNA (RAPD)
markers (Castiglione et al. 1993; Lin et al. 1994a, 1994b).
Although isozymes have served as genetic markers for
fingerprinting clones, the small number of available alleles
and loci made it impractical to identify all the clones under
investigation (Bournival and Karban 1987). In recent years,
RFLP of rONA, mtDNA, and cpDNA were used intensively
1
Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds.
Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997.
Micropropagation, genetic engineering, and molecular biology
of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO:
U.S. Department of Agriculture, Forest Service, Rocky Mountain
Research Station. 326 p.
116
for inter and intraspecies differentiation in Populus. However, these processes are labor intensive and require a relatively large amount of tissue. Other limitations of RFLP
include low resolution for small size differences between relatively large DNA fragments (Gillet al. 1990) and ambivalent
differences in the migration of DNA fragments across an agarose gel caused by band shifting (Landers 1989).
Amplification of DNA sequences using polymerase chain
reaction (PCR) (Saiki et al. 1988) is currently used with many
organisms for studying populations and systematics, tagging major genes, and constructing genetic maps (Innis et
al. 1990). Advantages of PCR-based DNA markers over the
RFLPs are their rapidity, simplicity, and requirement for only
small amounts of DNA (Castiglione et al. 1993).
RAPD banding patterns were sensitive to slight changes
in reaction conditions, which caused reproducibility problems (Ellsworth et al. 1993; Muralidharan and Wakeland
1993; Penner et al. 1993). However, DNA fingerprints can
be reproduced with standardized conditions such as consistent use of the same thermal cycler, cycling conditions,
and concentrations of the reagents (i.e., template DNA,
buffer, dNTPs, and DNA polymerase). DNA fingerprints
by RAPD markers were chosen in the present study for
species and hybrid differentiation and individual clonal
characterization of Populus.
Materials and Methods
Plant Material
Fifty-five poplar clones representing 8 species and hybrids (table 1) were analyzed. Clones were selected according to frequency of cultivation in Europe and North
America (Castiglione et al. 1993), and for their potential
use in plantations and biomass production (Steenackers
eta1.1990;Zsuffa 1990). Twodormantcuttingsofeachclone
were collected and rooted in the greenhouse at the Faculty of Forestry, University of Toronto.
Differentiation of Poplar Clones Using Random Amplified Polymorphic DNA Fingerprinting
Table l. List of poplar species and hybrids for clonal
characterization.
Number
of clones
Species and hybrids
x balsamifera
xjackii
deltoides
trichocarpa x P. deltoides
deltoides x P. trichocarpa
x euramericana
nigra
nigra x P. maximowiczii
maximowiczii
Total
P.
P.
P.
P.
P.
P.
P.
P.
P.
3
3
5
3
2
25
6
5
3
55
DNA Extraction
Young leaves were collected from rooted cuttings. After washing with distilled water and blotting dry, leaf tissue (0.5 g) was ground to powder in liquid nitrogen, then
transferred to 10 ml of preheated 2X CTAB isolation buffer
(100 mM Tris-HCI, pH 8.0, 1.4 M NaCI, 20 mM EDTA, 2
percent hexadecyltrimethylammonium bromide 'CTAB,'
0.2 percent 2-mercaptoethanol). After thoroughly stirring,
the mixture was incubated at 60 oc for 30 min, extracted
twice with chloroform-isoamyl alcohol (24:1), and precipitated with 2/3 volume of cold isopropanol (Doyle and
Doyle 1987). DNA concentration was estimated by com-
paring serial dilutions of DNA with those in agarose gels
stained with ethidium bromide.
Primers
Sequences of 17 primers used for the PCR reaction are
in table 2.
Amplification of DNA by PCR
The amplification reaction was performed in 15 JJI of
buffer containing 50 mM Tris-HCl (pH 8.3), 5 mM
MgCI 2, 200 11M each of dATP, dCTP, dGTP and dTTP,
0.5 units of Taq DNA polymerase (Boehringer
Mannheim), 15 pmol primer and 10 ng of DNA. The
amplification reaction was performed in the PerkinElmer DNA Thermal Cycler (Model TC-1). After aninitial denaturation at 94 oc for 2 min, the reaction program
continued for 40 cycles consisting of 1 min denaturing
at 94 oc, 1 min annealing at 40 oc, and 2 min extension
at 72 °C. This program was followed by an extension
phase at 72 oc for 10 min. DNA from all clones was subjected to 3 independent amplifications, and only fragments that were observed in all assays were scored for
species and clonal characterization.
Electrophoresis in Agarose Gel
Amplification products (15 ill) were analyzed by electrophoresis in 1.5 percent agarose gel (15 x 15 em) at 100 V
until the tracking dye (tartrazine) migrated toward the end
Table 2. Sequences and sources of primers used in the RAPD analyses.
Name
Sequence
References
Chl-1
Chl-2
Chl-4
Chl-10
Deca-2
Deca-4
Deca-5
Deca-7
Deca-9
Deca-10
Deca-11
Deca-12
Deca-13
5'-GAGGCCTACGCCCCATAGAA-3'
5'-AATGCGTTGAGGCGCAGCAG-3'
5'-TTCCCGTGTCTTCCGGCTTAC-3'
5'-TTCTTCTCCTACCAGTATCG-3'
5'-GCGATCCGGC-3'
5'-CGTTGGCCCG-3'
5'-CCAAGGGGGC-3'
5'-CCGCCCGGAT-3'
5'-TGGCCCCGGT-3'
5'-AGCCGGCCTT-3'
5'-ATCGGCTGGG-3'
5'-CTTGCCCACG-3'
5'-GTGGCAAGCC-3'
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
Castiglione et
2114
2115
2116
2117
5'-GACTGCCTCT-3'
5'-GAAACGGGTC-3'
5'-GTGACCGAGT-3'
5'-CAGAAGCGGA-3'
Operon
Operon
Operon
Operon
1
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
al.
1993
1993
1993
1993
1993
1993
1993
1993
1993
1993
1993
1993
1993
Technologies 1
Technologies
Technologies
Technologies
1000-T Atlantic Ave. Suite 108, Alameda, CA 94501-1147, USA
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
117
Section Ill Molecular Biology
of the gel. Gels were stained in 0.5 11g/ml ethidium bromide solution and photographed in UV-light.
Results and Discussion
The clone and origin of poplar materials subjected to RAPD
analyses are listed in table 3. Of 17 primers tested, only 4 (DecatO, Deca-2, Chl-1, and Deca-7) (figures 1 to 4) were needed to
distinguish all 55 poplar clones into species and hybrids (table
4). The molecular sizes of RAPD markers used for species and
hybrid differentiation are in table 4. Individual clones could be
characterized by primer Chl-1 (figure 3) and/ or Deca-7 (figure
4). DTAC clones from different parental origin (i.e., P. deltoides x
P. trichocarpa and P. trichocarpa x P. deltoides) could be distinguished
by primer Chl-1 (figure 3).
Recently, several specific PCR primers were developed for
DNA fingerprinting. These primers were determined from
known DNA sequences, which include the M13 universal
primer (Chong et al. 1995), simple sequence repeats (SSRs)
(Gupta et al. 1994), and microsatellite repeats (Morgante and
Olivieri 1993). DNA fingerprinting with these specific prim-
Table 3. Poplar (Populus spp.) clones and their origin for RAPD analyses (note figures 1 - 4).
Clone no.
Upper Gel
TAC8
TAC21
TAC51
JAC4
JAC7
JAC28
035
039
0196
0391
OTAC7
DTAC8
DTAC26
DTAC9
OTAC10
ON5
ON14
DN16
ON17
Lower Gel
DN93
145/51
1214
1455
DN21
ON30
ON173
DN177
N75
N84
N91
N100
NM1
NM2
NM8
M900
M901
M908
118
Species and hybrid
Origin
P. balsamifera
P. balsamifera
P. balsamifera
P.xjackii
P.xjackii
P.xjackii
P. deltoides (W-1-2) Larsson
P. deltoides
P. x deltoides cv. 'Northwest'
P. deltoides (Mixoploid) (Brockville #C136)
P. trichocarpa x P. deltoides cv. 'Unal'
P. trichocarpa x P. deltoides cv. 'Beaupre'
P. trichocarpa x P. deltoides cv. 'Boelare'
P. deltoides x P. trichocarpa cv. 'Donk'
P. deltoides x P. trichocarpa cv. 'Barn'
P. x euramericana cv. 'Gel rica'
P. x euramericana cv. 'Hartt'
P. x euramericana cv. 'Batarde d'Hauterive'
P. x euramericana cv. 'Robusta'
Ontario
Ontario
Manitoba
Ontario
Ontario
Manitoba
Ontario
Ontario
Saskatchewan
Ontario
Geraardsbergen, Belgium
Geraardsbergen, Belgium
Geraardsbergen, Belgium
Wageningen, Holland
Wageningen, Holland
W. Germany
W. Germany
France
France
P. x euramericana cv. 'Triplo'; cl. '137/61'
P. x euramericana
P. x euramericana
P. x euramericana
P. x euramericana cl. '178B' (LW42)
P. x euramericana cv. 'Canada Blanc'
P. x euramericana (cv. 'Oorskamp')
(Koster 925)
P. x euramericana (cv. 'Spijk') (Koster 2195)
P. nigra (#113)
P. nigra var. italica (#555/50)
P. nigra cv. 'Purkersdorf' (#44/62(1 0))
P. nigra cv. 'Kunovice' (312/65(002/66)
P. nigra x P. maximowiczii
P. nigra x P. maximowiczii (cl. 'Max-1 ')
P. nigra x P. maximowiczii (#62-7)
P. maximowiczii
P. maximowiczii
P. maximowiczii
Casale Monferrato,
Casale Monferrato,
Casale Monferrato,
Casale Monferrato,
Italy
Spain
Holland
Italy
Italy
Italy
Italy
Holland
Hungary
W. Germany
Austria
Czechoslavakia
Japan
W. Germany
Korea
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Differentiation of Poplar Clones Using Random Amplified Polymorphic DNA Fingerprinting
T
u
1
J
2 3
4
TO
0
56
7 8
DT _ 0=-=--=N-
M
910111213141516171819
Figure 1. DNA polymorphism of poplar clones
amplified with primer Deca-1 0.
T = P. balsamifera
ON = P. dettoides x P. nigra
J
P. xjackii
EU
=
=
OT =
NM =
TO
0
N
M
L
EU
1 2 3
ON
4 5 6 7
N
NM
Ma
Ma
M
P. x euramericana
P. deltoides
P. nigra
P. de/toides x P. trichocarpa
P. nigra x P. maximowiczii
P. trichocarpa x P. deltoides
P. maximowiczii
Molecular Markers VI from Boehringer Mannheim
Corporation
species differences
8 9 101112131415161718
~
Table 4. Identification of poplar species and
hybrids by primers and molecular marker.
Primers
Marker size Species and hybrids'
Deca- 10 922
ers was also performed in our study (Lin unpublished data) to generate
highly reproducible Dt A-ba nding patterns. However, our results from
these experiments (1993 to 1995) indica te tha t either random or specific
primers can be used for Dt A fingerprinting without major problems.
The application of RFLP of rON A, mtDNA, and cpDNA in differentiation of Populus is limited to the inte r and intraspecies level. Compared to RAPD markers, the recently d eveloped technique of amplified
restriction fragm ent polymorp hism (AFLP) (Vas et a!. 1995) may provide an ad vantage in reliability for DNA fingerprinting. In practice,
however, DNA fingerprinting by RAPD markers remains an easier and
less expensive technique for individua l clonal characteriza tion.
807
600
583
Deca-2
Chl-1
11 02
M
953
T
884
1710
DN, EU, N, NM
N, NM , M
TD
DT
DN,EU
NM,M
1035
77
Deca-7
J, D, TD, DT, DN, EU,
N, NM , M
M
ON, EU, N, NM
J, D, TD, DT
1649, 1474
272
T
ON
J
Acknowledgments
This study was supported by the Genetic Improvement Activity, Task
Vill, Bioenergy Agreement, International Energy Agency, and the Natu ra l
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
P. balsamifera
P. deltoides x P. nigra
P. xjackii
P. x euramericana
EU
P. deltoides
D
N
P. nigra
P. deltoides x P. trichocarpa
DT
NM = P. nigra x P. maximowiczii
P. trichocarpa x P. deltoides
TO
M = P. maximowiczii
11 9
Section Ill Molecular Biology
u
1
T
J
2 3 4
5 6 7 8
D
TO --=D::....:T'--- --=D::...::..N-=-- M
9 10 1112 13 14 151617 18 19
Figure 2. DNA polymorphism of poplar
clones amplified with primer
Deca-2.
T
= P. balsamifera
ON = P. deltoides x P. nigra
J
P. xjackii
EU = P. x euramericana
0
= P. defloides
=
=
=
P. nigra
DT = P. deltoides x P. trichocarpa
NM
P. nigra x P. maximowiczii
TO
P. trichocarpa x P. defloides
Ma = P. maximowiczii
M
Molecular Markers VI from Boehringer Mannheim
Corporation
-+ = species differences
N
M
L
Sciences and Engineering Research Counci l of Canada
(1 SERC) operating grant to L. Zsuffa. We gratefully acknowledge the technical support of Mr. K. Vitols in the PCR work.
Literature Cited
Barrett, J.W.; Rajora, O.P.; Yeh, F.C.H.; Dancik, B.P. 1993.
Mitochondrial D A variation and genetic rela tionships
of Populus species. Genome. 36: 87-93.
Boumival, B.L.; Korban, S.S. 1987. Electrophoretic analysis
of genetic variability in the apple. Sci. Hortic. 31: 233-243.
Bradshaw, H.D., Jr.; Villar, M.; Watson, B.D.; Otto, K.G.;
Stewart, S.; Stettler, R.F. 1994. Molecular genetics of
120
ON
EU
1 2 3
4 5
6 7
N
NM
Ma
8 9 101112131415161718
grow th and development in Populus. 3. A genetic linkage map of a hybrid poplar composed of RFLP, STS,
and RAPD markers. Theor. Appl. Genet. 89: 167-178.
Castiglione, S.; Wang, G.; Damiani, G.; Bandi, C.; Bisoffi,
S.; Sa la, F. 1993. Identification of elite poplar (Populus
spp.) clones using RAPD fingerprints. Theor. Appl.
Genet. 87: 54-59.
Chong, K.X.; Yeh, F.; Zsuffa, L.; Aravanopoulos, F.A. 1995.
Dt A fingerprinting of willows (Salix L.) using polymerase chain reaction with M13 universal prime r. Scan.
J. For. Res. 10: 27-31.
D'Ovidio, R. 1992. Nucleotide sequence of a 5.8S rONA
gene and of the inte rnal transcribed spacer from Populus
rleltoirles. Plant Mol. Bioi. 19: 1069-1072.
D'Ovidio, R.; Mugnozza, G.S.; Tanzarella, O.A. 1990. Ribosoma l DNA genes s tructure in some Popu l us spp.
(Salicaceae) and their hybrids. Pl. Syst. Evol. 173: 187-196.
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
DiHerentiation of Poplar Clones Using Random Amplified Polymorphic DNA Fingerprinting
J
T
u
1
2 3
4
5
D
6
7 8
TD
DT ----=D
: ..N
.:.-=----- M
9 10 1.1 12 1314 1516 17 18 19
Figure 3. DNA polymorphism of poplar clones
amplified with primer Chl-1 .
T
ON
= P. balsamifera
= P. delloides x P. nigra
J
P. x jackii
EU = P. x euramericana
0 = P. deltoides
N = P. nigra
OT = P. deltoides x P. trichocarpa
NM = P. nigra x P. maximowiczii
TO = P. trichocarpa x P. deltoides
Ma = P. maximowiczii
M
Molecular Markers VI from Boehringer Mannheim
Corporation
4
= species differences
M
L
EU
1 2 3 4 5
DN
6 7
N
NM
Ma
8 9 1011 12 13 14 1516 17 18
D'Ovidio, R.; Mugnozza, G.S.; Tanza rella, O.A. 1991. rONA
cloning and rapid hybrid identification in Populus spp.
(Salicaceae). Pl. Syst. Evol. 177: 165-174.
Doyle, J.J.; Doyle, J.L. 1987. A rapid DNA isolation p rocedure for sma ll quantities of fres h leaf tissue. Phytochem.
Bull. 19: 11015.
Ellsworth, D.L.; Rittenhouse, K.D.; Honeycutt, R.L. 1993.
Artifactural va ria tion in randomly amplified polymorphic D A banding patterns. BioTechniques. 14:214-216.
Faivre-Ra mpant, P.; Bodergat, R.; Servi lle, A. 1992a. Une
me thode molecu laire d eclasse men t des clones de
Peuplie rs (Populrts) dans les section s Taca mah aca,
Aigeiros, Le uce et Leucoi.des par d es fragments de restriction des unites ribosomiques. C.R. Acad . Sci. Paris,
315(Ill): 133-138.
Faivre-Rampant, P.; Jeandroz, S.; Le fevre, F.; Le moine, M.;
Villar, M.; Be rville, A. 1992b. Ribosomal 0\!A stu d ies
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
in poplars: Populus deltoides, P. 11igm, P. triclrocarpa, P.
maximowiczii, and P. alba. Genome. 35: 733-740.
Gill, P.L.; Sulli va n, K.; We rre tt, D.J. 1990. The ana lysis of
hypervariable DNA profiles: p roblems associated w ith
the objective determination of the probabi lity of a match.
Hum. Genet. 85: 75-79 .
Gup ta, M.; C hy i, Y,S.; Romero-Severson, J.; Owen, J.L.
1994. Amplification of DNA markers fro m evolutionarily d iverse genomes using sing le primers of simp leseque nce repeats. Theor. Ap pl. Genet. 89: 998-1006.
Innis, M.A.; Gelfand, G.l-1.; Sninsky, J.J.; White, T.J. 1990.
PCR protocols: A g u ide to me thod s and a pp lications.
San Diego, CA, U.S.A.: Academic Press. 482 p.
Landers, E.S. 1989. DNA fingerprinting on trial. Na ture.
339: 501-505.
Lin, D.; 1-Iubbes, M.; Zsu ffa, L. 1994a. Differentiation of
popla r and w illow clones using RAPD fingerprints . Tree
121
Section Ill Molecular Biology
u
T
1
4
TD
D
J
2 3
5
6
7 8
DT ---=0::...=.-N~ M
9 10 1112 1314 15 16 17 18 19
Figure 4. DNA polymorphism of poplar clones
amplified with primer Deca-7.
T
ON
= P. balsamifera
=
P. deltoides x P. nigra
P. xjackii
P. x euramericana
P. delloides
P. nigra
P. del/oides x P. trichocarpa
NM
P. nigra x P. maximowiczii
TO = P. trichocarpa x P. del/oides
Ma = P. maximowiczii
J
EU =
0 =
N =
OT =
=
M
-+
Molecular Markers VI from Boehringer Mannheim
Corporation
= species differences
M
EU
DN
4 5 6 7
Physiol. 14: 1097-1105.
Lin, 0.; Hubbes, M.; Zsuffa, L. 1994b. Differentiatio n of
poplar a nd willow clones u sing RAPD fingerprints.
Norwegian J. of Ag ric. Sci., Suppl. No. 18: 67-81
Morgante, M.; Olivieri, A.M. 1993. PCR-amplificed microsatellites as markers in plant genetics. Plant J. 3: 175-182.
Muralidharan, K.; Wakeland, E.K. 1993. Concentration of
primer and template qu alitatively affects products in
random-amp li fied po ly morphic ON A PCR .
BioTechniques. 14: 362-364.
Penner, G. A.; Bush, A.; Wise, R.; Kim, W.; Domier, L.; Kasha,
K.; Laroche, A.; Scoles, G.; Molnar, S.J.; Fedak, G. 1993.
Reproducibility of random amplified polymorphic DNA
(RAPD) analysis among laboratories. PCR Methods
Applic. 2: 341-345.
Rajora, O.P. 1988. Allozy mes as aids for identification and
differentiation of some Populus maximowiczii clonal va ria-
122
N
NM
Ma
8 9 101112131415161718
tion. Biochemical Systematics and Ecology. 16: 635-640.
Rajora, O.P. 1989a. Genetic structure and identification of
Populus deltoides clones based on allozyme. Genome. 32:
440-448.
Rajora, O.P. 1989b. Characterization of 43 Populus nigra L.
clones representing selection, cultivars and botanical varieties based on their a llozyme genotypes. Euphytica.
43: 197-206.
Rajora, O.P.; Dancik, B.P. 1995a. Chloroplas t DNA va riation in Populus. I. Intraspecific restriction fragment diversi ty w ithin Populus deltoides, P. nigra and P.
maximowiczii. Theor. Appl. Genet. 90: 317-323.
Rajora, O.P.; Dancik, B. P. 1995b. Chloroplast D NA va ri ation in Populus. II. Interspecific restriction fragment
poly morphisms and genetic re lationships among
Populus deltoides, P. nigra, P. maximowiczii, and P. x
canadensis. Theor. Appl. Genet. 90: 324-330.
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Differentiation of Poplar Clones Using Random Amplified Polymorphic DNA Fingerprinting
Rajora~
O.P.; Dancik~ B.P. 1995c. Chloroplast DNA variation in Populus. III. Novel chloroplast DNA variants in
natural Populus x canadensis hybrids. Theor. Appl. Genet.
90: 331-334.
Rajora~ O.P.; Zsuffa, L. 1989. Multilocus genetic structures,
characterization and relationships of some Populus x
canadensis cultivars. Genome. 32: 99-108.
Rajora, O.P.; Zsuffa~ L. 1990. Allozyme divergence and
evolutionary relationships among P. deltoides, P. nigra,
and P. maximowiczii. Genome. 33:44-49.
Rajora O.P.; Zsuffa L. 1991. Screening Populus deltoides
Marsh. clones to assure and correct identity. Scan. J.
For. Res. 6:471-478.
Rajora O.P.; Zsuffa, L.; Dancik, B.P. 1991. Allozyme and
leaf morphological variation of eastern cottonwood at
the northern limits of its range in Ontario. For. Sci. 37:
688-702.
Saiki, R.K.; Gelfand, D.H; Stoffel, S.; Scharf, S.T.; Higuchi,
R.; Hom, G.T.; Mullis, K.B.; Erlich, H.A. 1988. Primerdetected enzymatic amplification of DNA with athermostable DNA polymerase. Science. 239: 487-491.
1
1
1
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Smith, R.L.; Sytsma, K.J. 1990. Evolution of Populus nigra
(sect. Aigeiros): introgressive hybridization and the
chloroplast contribution of Populus alba (sect. Populus).
Am. J. Bot. 77: 1176-1187.
Soltes-Rak, E. 1993. Clonal characterization of poplar and
willow using DNA and protein markers. IEA/BA Task
VIII Genetic Improvement Activity Newsletter No.2.
Steenackers, V.; Strobl, S.; Steenackers, M. 1990. Collection and distribution of poplar species, hybrids and
clones. Biomass. 22: 1-20.
Vos, P.; Hogers, R.; Bleeker~ M.; Reijans, M.; Vandelee, T.;
Hornes, M.; Frijters, A.; Pot, J.; Peleman, J.; Kuiper, M.;
Zabeau, M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research. 23:4407-4414.
Zsuffa, L. 1990. Genetic improvement of willows for energy plantations. Biomass. 22: 35-47.
Zsuffa, L.; Mosseler, A.; Raj, Y. 1984. Prospects for interspecific hybridization in willows for biomass production. In: Perttu, K., ed. Ecology and management of
forest biomass production systems. Dept. Ecol. and
Environ. Res., Swed. Univ. Agric. Rep. 15: 261-281.
123