Distinguishing Among Magnolia Cultivars Using Fluorescent Amplified Fragment Length Polymorphism AFLP Analysis
Distinguishing Among Magnolia Cultivars Using Fluorescent Amplified Fragment Length Polymorphism AFLP Analysis
Distinguishing Among Magnolia Cultivars Using Fluorescent Amplified Fragment Length Polymorphism AFLP Analysis
To cite this article: Anthony D. Mitchell , Roy A. Edwards & Chris M. Frampton (2001)
Distinguishing among Magnolia cultivars using fluorescent Amplified Fragment Length
Polymorphism (AFLP) analysis, New Zealand Journal of Crop and Horticultural Science, 29:2,
77-83, DOI: 10.1080/01140671.2001.9514165
Article views: 85
per reaction than other fingerprinting techniques, the (10 mMTris-HCl, pH 8.0; 0.1 mMEDTA) buffer.
large number of putative loci sampled providing a DNA concentrations were estimated and standard-
measure of variation across a wide portion of the ised against known concentrations of Lambda DNA
genome (Sharma et al. 1996). on 1% agarose gels.
In the present investigation, we used fluorescent Initially 10 \i\ of the total DNA extract was di-
AFLP analysis to study genetic variation in M. gested with 6 jLtl digestion buffer (50 mAfTris-HCl,
denudata Desr., M. liliiflora Desr., and cultivars of pH 7.5; 50 mM (CH3COO)2Mg.4H2O; 250 mM
their hybrid cross. M. campbellii Hook. & Thorns, CH3COOK), 5 nl dH2O, 1 \i\ of each restriction
and M. stellata (Sieb. & Zucc.) Maxim, were in- enzyme; EcoRl (10 U/jj.1, Roche Molecular Bio-
cluded for reference because these species are esti- chemicals) and Tru91 (an isoschizomer forAfeel) (10
mated (Qui et al. 1995) to be among the closest U/(xl, Roche Molecular Biochemicals), for 2 h at
relatives of M. denudata and M. liliiflora. The main 37°C followed by 15 min at 70°C. A 5 \l\ aliquot was
question we addressed was: can Magnolia cultivars run on a 1% agarose gel to check digestion was
be distinguished using AFLP markers? complete and the remaining DNA was then ligated
by adding 1 \i\ of each of the EcoRl (5 pmol/^l)
and Msel (50 pmol/|xl) adapters (Life Technologies),
MATERIALS AND METHODS 2 (il of ligation buffer (100 mMTris-HCl, pH 8.3;
15 mMMgCl2; 500 mMKCl), 1 \ll T4 DNA ligase
Plant materials and DNA samples (1 U/Hl, Gibco), and 5 \il dH2O. The mixture of
Samples from 12 mature Magnolia specimens were ligation cocktail plus digested DNA was incubated
used for the present study (Table 1). A single cultivar overnight at 4°C.
of unknown origin was included for investigation,
plus samples of M. campbellii and M. stellata. Two Pre-and selective amplifications
leaves were used for separate extractions from M. x The total pre-amplification reaction volume was 40
soulangeana and carried through the complete AFLP (0.1. This reaction consisted of 2 (il ligated DNA (di-
process as a control. All selective amplifications luted 1:5), 4 (al of 10 x PCR reaction buffer (100 mM
were repeated for the first primer combination and Tris-HCl, pH 8.3; 15 mMMgCl 2 ; 500 mMKCl),
subsequently carried through as a test of reproduc- 0.8 jil 10 mMdNTP, 31.04 Hi dH2O, 0.16 jal Taq
ibility. Polymerase (5 U/HI, Roche Molecular Biochem-
icals), and 1 Hi of each pre-amplification primer,
DNA extraction, digestion, and ligation each with one selective nucleotide; EcoR 1+1 primer
Total DNA was extracted using a modification of the 5'-GACTGCGTACCAATTCA-3' andMsel+l 5'-
CTAB method (Doyle & Doyle 1987). DNA was GATGAGTCCTGAGTAC-3' (50 ng/\i\, Life Tech-
precipitated in ethanol and resuspended in 25 jj.1 TE nologies). Pre-amplification was carried out in a
1
# M . /////flora 'Nigra'
M. ff/H/tora
-0.56 —I—i—i r#-i—i—r I —I
-0.27 -0.27 0.14 0.35 0.56
Principal coordinate 1
M. x soulangeana cultivars, 64%; and (3) between in concordance, both showing that: (1) M.
species, 55%. campbellii and M. stellata were separated from
A total of 1401 AFLP fragments were recorded other OTUs; (2) M. x soulangeana cultivars
for 13 OTUs for the four pooled AFLP primer grouped more closely with M. denudata than with
combinations, with an average polymorphism of M. liliiflora; (3) samples originating from separate
81.2% (Table 2). The Mantel test showed a highly leaves of M. x soulangeana grouped closely to-
significant (P = <0.001) correlation between all gether; and (4) the sample of unknown hybrid ori-
combinations of primer matrices. The matrix cor- gin appeared among the OTUs representing M.
relation statistics for each primer combination were: denudata and M. x soulangeana cultivars. No ties
r = 0.62 for primer 1 versus 2, r = 0.69 for primer were encountered during UPGMA analysis.
1 versus 3, /• = 0.69 for primer 1 versus 4, r = 0.85 Magnolia x soulangeana cultivars contained
for primer 2 versus 3, r = 0.86 for primer 2 versus greater proportions of fragments inherited from M.
4, r = 0.86 for primer 3 versus 4. Good separation denudata compared with M. liliiflora (Fig. 3). Num-
was achieved using principal coordinates analysis, bers of fragments shared between cultivars and par-
with the first coordinate explaining 64.2% of the ents ranged from M. x soulangeana 'Alba' (165
variation. The second and third coordinates ex- fragments shared with M. denudata and 73 frag-
plained only 5.1 and 4.3% of the total variation re- ments shared withM liliiflora), to M x soulangeana
spectively. Principal coordinate axes 1 and 2 (Fig. 'Lennei' (139 fragments shared with M. denudata
1) and UPGMA clustering (Fig. 2) were generally and 99 fragments shared with M. liliiflora).
Mitchell et al.—Distinguishing among Magnolia cultivars 81
40 -
0-
^ A° </
Cultivars
(Fig. 1 and 2) and compared with the other cultivars, profiling as a tool for registration purposes. Accu-
contained a similar number of fragments inherited rate identification of cultivars would also require a
fromM denudata andM liliiflora (Fig. 3). Unpub- database containing DNA fingerprints for all recog-
lished flowering records of magnolias at Lincoln for nised cultivars for comparison.
3 years from 1988 showed that the unknown mag-
nolia was more similar to M. x soulangeana 'San
Jose' thanM. x soulangeana 'Lennei' in terms of the
spring flowering period and accumulated thermal
time. But M. "unknown" was unlike both M. x ACKNOWLEDGMENTS
soulangeana 'San Jose' and M. x soulangeana We are grateful to the Brian Mason Scientific and Tech-
'Lennei' in that only these two cultivars of M. x nical Trust for funding. Thanks to Jacqui Kent for her
soulangeana have a second and significant flower- assistance with the ABI. Thanks to R. R. Scott and I.
ing period in late summer. M. x soulangeana 'Ruby' Schonberger and three anonymous referees for critical
has also shown some tendency to flower in late sum- reading of this manuscript and to the staff and students
mer, but to a lesser extent. A significant period of of the Ecology and Entomology Group for their support.
flowering in late summer was also recorded for M.
liliiflora and M. liliiflora 'Nigra' for each of the 3
years.
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