Agriculture Reference
In-Depth Information
then proceeds to have DNA's information
translated.
Just like human beings, all grapevines have
distinct DNA profi les. DNA testing was intro-
duced by Jeffreys, Wilson, and Thein in 1985
and has been successfully used in the fi eld of
criminal law—it was first applied to vine
research by Thomas and Scott in 1993. DNA
testing represents the current state of the art for
grape variety identifi cation, as DNA is relatively
easy to obtain with a little experience, testing
can be carried out at any time of the year, and
tests are not infl uenced by the environment.
Initially, the molecular techniques used
included restriction fragment length polymor-
phism (RFLP), amplifi ed fragment length poly-
morphism (AFLP), and random amplifi ed poly-
morphic DNA (RAPD), but these have been
largely replaced by the use of microsatellite
technology, which is characterized by high
reproducibility and standardization. Microsatel-
lite markers are the most commonly used DNA
test for identifi cation of grapevine cultivars
today. There are two types of microsatellites
most used at present: nuclear microsatellites (or
nuclear simple sequence repeats, nSSR), which
are the best known and most used to date, and
chloroplast microsatellites (CpSSR), which may
be more useful in parentage studies.
To genetically identify a vine, specifi c, small
segments of DNA called alleles are targeted and
the polymerase chain reaction (PCR) technique
is used to make millions of copies of selected
marker segments. The alleles in one marker
site of a given variety are compared with those
present in the same marker site of another vari-
ety. Though I am simplifying greatly, two vines
are considered to belong to the same variety
if they possess identical sequences of DNA
in a suffi cient number of these marker sites,
called microsatellites and also known as single
sequence repeats, or SSR. Microsatellites,
found in all vegetal and animal species, includ-
ing grapes, are simple sequence repetitions
scattered randomly throughout an organism's
DNA that were long believed not to be tran-
scribed and translated. In other words, not to
code for proteins. (However, as we shall see
later in this chapter, it is becoming apparent
that this view is increasingly obsolete.) As
microsatellites are constant within each variety
and differ from those of other varieties, they act
as molecular fi ngerprints for varieties. The
identifi cation of unknown vines is done by com-
paring the SSR profi les obtained from the sam-
ple with reference SSR profi les of cultivars
stored in internationally available databases,
much as with fi ngerprints in criminology. A
variety is considered “new” if the microsatellites
do not match any in the database. Depending
on the number of sites studied, the probability
of different varieties having the same SSR pat-
tern by chance is roughly one in eight billion.
While several sets of SSR markers have
been proposed for grape research, the best
known are the set of six suggested by the grape
Genetic Resources (GENRES) projects. This
project established the European Vitis Database
(EVDB), which now includes data for more
than 28,200 accessions, representing the
Vitis
collections of eighteen European contributors.
The research group also selected six microsatel-
lite loci allowing for the identifi cation of grape-
vine cultivars (VVS2, VVMD5, VVMD7,
VVMD27, VrZag62, and VrZag79). Though
most researchers believe that six loci are
enough to determine genetic identity between
most species, at least nine are indicated for vari-
eties that are close genetically. Statistically, the
probability identity value increases by six pow-
ers of ten when nine, rather than six, SSR loci
are studied. Analyzing more than ten loci of the
grapevine genome further increases the accu-
racy of cultivar identifi cation: the ten microsat-
ellite loci internationally accepted are VVS2,
VVMD5, VVMD7, VVMD25, VVM27,
VVMD28, VVMD31, VVMD32, VrZAG62, and
VrZAG79. Recently however, researchers have
begun to think that even more loci might be
necessary to increase the discrimination power
of investigations performed.
Recently, another molecular DNA marker
has come into use: single nucleotide polymor-
phisms, or SNPs (pronounced “snips”). Future
