Protocols for Micropropagation of Woody Trees and Fruits

3. In addition to testing various buffers, selection of tissues with lower or no
phenolic compounds may enable unbiased estimations (Suda, 2004). If no
tissue/buffer provides acceptable results, changing the type of buffer additives
(in WPB metabisulfite acts as an antioxidant, and PVP-10 as a tannincomplexing
agent) or their concentration may reduce the negative effect of
cytosolic compounds, and is therefore recommended.
However, the absence of major ploidy changes does not exclude the possible existence
of genetic differences such as DNA polymorphism. Molecular methods like
microsatellite marker analysis can be the ideal tool to assess DNA polymorphism,
and a protocol developed for Q. suber by Lopes et al. (2006) is presented thereafter.
2.2. Microsatellite Markers
PLANT GENETIC FIDELITY 75
Genotyping (PCR analysis) of SSR fragments can be achieved in various ways: radioactive
detection (incorporation of labelled nucleotides and end-labelling of one
of the PCR primers), non-radioactive detection (high resolution gel electrophoresis,
silver staining, blotting and hybridization and fluorescent dyes on automated sequ-
encers).
The protocol given here is for genotyping using fluoro-labelled primers. The
dyes: 6-FAM, JOE, HEX and TET) to allow detection of the polymerase chain
reaction (PCR) products (see Table 2). About 0.01 µmol of each primer are ordered,
described in this protocol. Primers can generally be purified by standard liquid chromato-
graphy (e.g. the HPSF
tography (HPLC). For this individual application, we recommend the use of standard
markers and was adapted from Lopes et al. (2006). Protocols for isolation of microsatellite
markers in Quercus species can be found elsewhere (Isagi & Suhandono,
1997; Steinkellner et al., 1997a; Kampfer et al., 1998). From the available nuclear
microsatellites (nSSRs) developed in the Quercus genus that have been transferred
with success to Q. suber, eight were chosen for study according to their degree of
polymorphism (heterozygosity and number of alleles) and the quality of the PCR
product. Briefly, of the eight nSSRs assayed, QM58TGT and QM50–3M were first
described by Isagi & Suhandono (1997) in Q. myrsinifolia Blume, and QpZAG9,
QpZAG15, QpZAG36 and QpZAG110 were first described in Q. petraea (Matts.)
previously been reported (Gomez et al., 2001; Hornero et al., 2001b). The other two
nSSRs, QrZAG7 and QrZAG11 were first described in Q. robur (Kampfer et al.,
1998) and their transferability to Q. suber was reported by Hornero et al. (2001b).
® forward primers are synthesized with a fluorescent label attached to the 5′ end (ABI
which is sufficient to perform at least 2,500 PCR runs under the standard conditions
method, MWG Biotech) or by high performance liquid chroma-
liquid chromatography for unlabelled primers and the use of HPLC purification for
labelled primers, as it is more sensitive. We resuspend the primers in sterile deionized
water or sterile buffer (i.e. TE; 10 mM Tris pH 8, 1 mM EDTA). The standard concentra-
tion for stock solution of PCR primers is 100 µM. To obtain a concentration of 100 µM
the synthesis report of the vendor should provide you with the appropriate
diluent volume.
The protocol described below refers only to genotyping with previously known
Liebl. (Steinkellner et al., 1997a,b). The transferability of these loci to Q. suber has