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Plant Gene and Trait 2012, Vol.3, No.8, 43
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propagation (seed or cuting) from some long-lived and
holly individuals. The identified long-lived Cyprus
trees showed very different geographical climatical
conditions from humid temprate to semihumid temprate,
semi arid cold and arid warm regions in Iran. The
clustering and PCo revealed that there are only four
group (two big and two very small ones) within popu-
lation so that the seed or cutting origin of individuals
in group 2 were probably Dehbakri 1 (Kerman P.),
Harzevil (Gilan P.) and Abarkooh (Yazd P.) and in
group 3 were Tange kherghe 6 (Fars P.) and Poshtehan
(Gilan P.) because of the oldest trees are attributed to
the group and the hypothesis of artifitial propagation.
More genetic diversity resulted in higher cross polli-
nation and higher effective population size. Further,
the study implies that more variation needs to be
introduced in the existing population for species
persistence. Therefore, the immediate approach and
need of the hour may be to preserve the local geno-
types by collecting seeds from representative ecotypes,
introducing more of micropropagated plants with
variation to increase the actual population and allow
cross breeding.
3 Materials and Methods
3.1 Plant material
The representative plant material was collected from
different locations in 14 provinces of Iran (Table 4).
Needle samples are collected for 63 individuals.
Samples were collected from young branches and
middle of crown lengths. They were immediately
frozen in liquid nitrogen and then stored in
-
80
℃
before DNA isolation.
3.2 DNA extraction and primer selection
Total genomic DNA was extracted using the DNeasy-
TM Plant Mini Kit (QIAGEN) (Valgimigli et al.,
2005). DNA was qualified on Ethidium bromide stained
Agarose gels and spectrophotometer was used to
determine the DNA yield and purity (Doulis et al.,
2000). Eight primers were developed by Sebastiani et
al. (2005) and Valgimigli et al. (2005) applied them
for genetic analysis on
C. sempervirens
(Table 5).
3.3 PCR amplification and product detection
The PCR reaction was performed in a final volume
of 12.5 μL, containing 25 ng of DNA, 1× reaction
buffer, 0.2 mmol/L of each dNTP, 4 mmol/L MgCl
2
and 0.8 μmol/L of each primer and 0.75 U
Taq
polymerase. The following temperature profile was
used for amplification: 94
℃
for 3 min, then 35 cycles
of 94
℃
for 1 min, 60
℃
for 30 s and 72
℃
for 1 min,
ending with 72
℃
for 5 min. 5 μL of each PCR product
were loaded on 1% Agarose gel, run in 1×TAE buffer,
stained with Ethidium bromide and photographed
under UV light to control the success of the
amplifications and to qualify fragments.
Table 5 Characteristics of dinucleotide microsatellite loci developed for
C. sempervirense
(Sebastiani et al., 2005; Valgimigli et al., 2005)
Locus name
Primer sequence 5'
-
3'
Repeated motif
Cyp52
F: CATCCACTGCCAATACTTTT
(GT)21
AY854181
R: AGCATCTTCCCATTACTTGA
Cyp84
F: CATTTCAATTTGCATAAGTTCT
(GT)13(TC)23
AY854182
R: GCAATGGGATGACTACAAAA
Cyp101
F: AGGCCACACTCAAACTTATG
(GT)12
AY854183
R: ATGACAATGGGTGAAGTCAT
Cyp139
F: ACAACTAGAGAGGGAGTGAAAA
(GA)19
AY854184
R: TGGTTGAAACAATAAAGGAGA
Cyp250
F: ATGGATGCAAGAGATTTTGT
(GT)16
AY854186
R: TGGTCCGATAGAAGTACTCG
Cyp257
F: AACTTGCACATTTAGGGATG
(GT)10(TA)4
AY854187
R: TGATGGAATAACATGGACAG
Cyp258
F: AATTTGGGCTCATGAAATTA
(GT)12
AY854188
R: TCTAGACCGATTCTATGGTCA
Cyp293
F: GGCAAGTAATGAAACTCCAC
(GT)14
AY854189
R: TACAAACATGCATGGCTAAC