Table Of Contentand
Isolation
Characterization
of Micros
Loci
atellite in
the Tree Fern
Alsophila
spinulosa
Yuan
Zhou, Guo-Pei and Wang*
Chen, Ting
Abstract.—
Alsophila
spin
Containing repeats (FIASC
from Guizhou
province, China. The number
of alleles per locus varied
i
observed and
expected
heterozygosity were 0.000-0.750 and
0.218-0.651
the
first microsatellites reported for the Cyatheaceae and will be useful
fi
and
conservation
genetic studies of other remaining
extant populations.
—
Key Words.
Alsophila
spinulosa, genetic
variation, microsatellite
Alsophila
spinulosa (Hook) Try on
(Cyatheaceae) famous
a with
is relict fern
a
tall, erect, arborescent rhizome.
Historically,
distribution has been
its
strongly influenced by
Quaternary
climatic changes
(Tryon, During
1970). the
few
last decades, A. spinulosa has been
experiencing
a drastic decline caused
by
and
habitat loss fragmentation, economic
local and human
exploitation
activities. In China, wild populations and
individuals
of A. spinulosa
are
extremely and
rare are restricted and
to tropical subtropical montane
regions,
occupying warm, humid
and shady
niches low
at latitudes The
(Fu, 1991).
now
species
is listed in the Chinese Red Book
of endangered
species
(Fu,
As
1991). a species with
long
evolutionary
history,
of great
is scientific
it
importance
for investigating pteridophyte
phylogeography,
and
speciation,
adaptive
evolution (Su
et al, 2005).
Information
about the and
level partitioning
of genetic variation within and
among
populations
of threatened
species determine
is critical to appropriate
management
(Dawson
strategies and
Powell,
1999). In a previous
study, the
A
population
genetic and
structure
variation of spinulosa were
inferred using
RAPD
nuclear
markers (Wang
However,
et 2004). observed
al., heterozygosity
and
population
differentiation cannot
be
detected
directly with dominant
makers
(Zhivotovsky, which
prompted
1999),
a search codominant
for
microsatellite
markers.
Microsatellite markers have
already been developed
in a range of plant However,
taxa.
to date there are only few
a reports of
microsatellite markers
in pteridophytes
Pryor
(e.g. et al., 2001; Vitalis et al
Woodhead
2001;
Kang
et 2005;
al., et al, 2006).
E-mail: [email protected]
;
ZHOU
ET MICROSATELLITE LOCI ALSOPHILA SPINULOSA
AL.: IN
43
we
Here and
report the isolation characterization of a of polymorphic
set
genome
from and
microsatellite loci the of A. spinulosa preliminarily assess genetic
diversity using wild individuals from a population in Guizhou province, China.
Materials and Methods
DNA
CTAB
genomic was
Total extracted from following modified
leaf tissue
protocols (Su et al, 1998). Microsatellite markers were isolated following the
FIASCO AFLP
protocol of (Fast Isolation by of Sequences Containing repeats)
A DNA
with minor modifications (Zane et ah, 2002). total of 250 ng genomic
was
completely digested with 3 units of Msel (BioLabs) in a 25 uL volume, and
DNA
then 15 uL of digested was ligated to Msel AFLP adaptor (5'-GACGAT-
DNA
GAGTCCTGAG-375'-TACTCAGGACTCAT-3')
T4
using unit of ligase
1
(BioLabs) in a 30 uL volume at 20 C for 3 h. The digestion-ligation mixture was
diluted and directly amplified using Msel adaptor-specific primers (5'-
(1:10),
mM
uM
GATGAGTCCTGAGTAAN-3')
20 uL with Msel-N, dNTPs,
in 0.9 0.2
mM
DNA
MgCl
Taq polymerase and uL
1.5 1 unit of (Tiangen) 5 diluted
2
,
digestion-ligation DNA. The PCR was performed using a program of 94 C 30
s,
DNA
min
53°C min, 72°C 20 Approximately 1000 ng amplified
1 1 for cycles.
fragments were hybridized with 200 pmol of 5'-biotinylated (AC) probe in a
15
DNA
total volume of 250 uL of SSC 4.2 X and 0.07% SDS, by denaturing for
DNA
min 95°C and incubating 60°C The hybridized was then
5 at at for 2 h.
mixed with 600 uL of Streptavidin MagneSphere Paramagnetic Particles
mM
TEN100
(Promega) which had been treated times with 150 uL of (10
3
mM mM
pH
EDTA,
Tris-HCl, 100 NaCl, allowing a selective binding at
1 7.5),
room temperature 30 min. The beads-probe-DNA complex was separated by
for
DNA
a magnetic After removing nonspecific fragments by nonstringent
field.
mM mM mM
pH
EDTA, and
washes (10 Tris-HCl, 1 NaCl, 7.5) stringent
1
DNA
washes (SSC 0.2 X and 0.1% SDS) for 3 times each, the target was released
mM, EDTA mM, pH
TE
from the bead-probes with 50 uL (Tris-HCl 10 1.0 8.0)
95 and soon
°C min, transferred as as possible.
at for 5
DNA
containing repeats were amplified for 30 cycles with Msel-N primers
and same program mentioned above was used. Fragments ranging from 400
the
to 1000 bp were isolated and purified (Omega Biotek). They were then ligated
pMDl9-T plasmid vector (TaKaRa) and were transformed into
into the
competent Escherichia DH-5a. Positive clones were identified by
coli cells
M13
and
blue/white then were amplified using universal primers
selection,
with
visualized by agarose gel electrophoresis. Eighty clones different insert
85% which contained simple sequence
fragments were sequenced, of repeats.
were developed from simple sequence
Subsequently, 33 primer pairs repeats
containing ten or more repeats with suitable flanking sequences.
All of the 33 pairs of primers were tested using 74 A. spinulosa individuals
PCR
Guizhou
sampled from population located in province, southern China.
a
were performed 10 uL volume containing approximately 20 ng
reactions in a
mM mM
uM
DNA, MgCl dNTPs, 0.25 each primer, IX Taq buffer and
0.2
1.5
2
,
PCR
The
0.5 unit Taq polymerase (Tiangen). profiles included an initial
AMERICAN FERN VOLUME NUMBER
JOURNAL:
98
(2008)
1
GenBank
e
mn
Repeat
T„
e
g
N
H
(bp)
A
t
Asl EU036670 F:GCACGGGTAGCCCAGATG
(GA) (Ac), 215-219
54 3 0.000 0.493
5
R:ATGGGTTCGCCCCTTCTT
(AG),,, (AG),
3
As2 EU036671 F:TCAAACATTCTACCAC-
(GA) 225-228
2
19
GAAGC
R:TCATTTCATACCATTCC
TCCC
W
TATTTGGTGGAAAGT-
As3 EU036672 (GT) (GA)
F: 0.750 0.651
U) 7
...
.
GAA
ATCTTGGTTTGCGTCTAA
(TG) (AG)
R:
H
(i
C
denaturation at 94 for 5 min, followed by 35 cycles of 50 94 50
C,
s at s at
C
annealing temperature, 90 s at 72 and then 10 min at 72 C. Amplified products
6%
were
electrophoresized in denaturation polyacrylamide and
gel visualized
A DNA
by
silver staining. 25 bp ladder (Promega) was used
to identify
alleles.
Results and
Discussion
DNA
Fourteen of the 33 primer
pairs successfully amplified fragments, but only
three yielded polymorphic
Preliminary population were
loci. genetics analyses
GENEPOP
performed using (Raymond and number
Rousset, 1995].The
of
alleles,
the observed and expected heterozygosities, Hardy-Weinberg equilibrium and
linkage disequilibrium were examined. The markers low
reveal relatively level of
variation within the population. The number of per locus ranged from
alleles 2 to
H
H
ranged from and
4, to 0.750 from 0.218 to 0.651, respectively (Table
E
1).
All three polymorphic deviated from Hardy-Weinberg
loci significantly
equilib-
rium and showed <
notable deficits of heterozygotes (P 0.001). This deviation
from Hardy-Weinberg
suggests high levels of inbreeding within population.
this
No
was among
significant linkage disequilibrium observed
the three pairs of
loci.
The
polymorphic
three loci presented here provide the first set of codominant
markers
for population genetic study of A. spinulosa. Although these markers
revealed very low
genetic diversity, it is not surprising for a potential self-
We
fertilization fern. believe that these makers will be useful for the ongoing
population and
conservation
genetic studies of other remaining
extant
populations
of A. spinulosa. Moreover, since limited population
genetic studies
on
exist pteridophytes using SSRs, may
further analyses of A. spinulosa provide
more
data of general
relevance.
was
ject supported by the "100 Talent Projecf'of the Chinese Academy
of Sciences
0729281F02) and Young
the "Outstanding
Scientist Project" of the Natural Science
:
Hubei
l of Province, China (Grant No.: O631061H01).
LOCI ALSOPHILA SPINULOSA
IN
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