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Bioscience Methods
BM 2010, Vol.1, No.1
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and Roose, 1997) had been tagged by molecular
markers using BSA. Ling et al. (2000) used the
second bulks of increased individuals to confirm the
validity of markers that was selected by the first
bulk screening.
In this work, bulks of different number of
individuals between 6 and 12 were tried to select
markers and the results were almost the same. A few
of the markers selected by BSA, such as 34d12,
63F7R, not matching up with the known phenotypes
of the individuals among ‘9145 family’, although
they were much well fitted to the phenotypes of the
bulked segregating samples and even their parents,
were taken out in the earlier stage. Some markers
such as 3p21S, 45b9T and 4L17L, after screening the
phenotype-known rootstocks, were thought to be not
specific and unsuitable for application in MAS of
citrus rootstock breeding. So, BSA is just the first and
initial steps for marker development, but not the last.
Much works of selection and evaluation needs to done
before extended application might be expected in
genetic identification of MAS and MBC in breeding.
2.2 The RGC gene clustering in citrus and the
genetic distance between Ctv and
Tyr1
By this study, it is obvious that the gene
Ctv
for
resistance to CTV and the major gene
Tyr1
for
resistance to citrus nematode are closely linked on
the chromosome of CN family (9145). Both Pt8a
and Pt9a markers are co-segregated with
Ctv
locus
not only in R family (Deng et al., 2000), but also
their derived markers co-segregated with
Tyr1
in
9145 family. This can also be confirmed by the
previously mapped many
Ctv
-linked markers such
as SCAD08, SCAm02, SCT08, SCO07 and a Ctv
candidate sequence-derived marker Y65R+F that
are also associated with resistance and mapped with
Tyr1
in the 9145 family. The
Ctv-Tyr1
region surely
contains a major cluster of resistance genes. In
addition to previously cloned RGC sequences, three
NBS-LRR class gene sequences were obtained by
BAC clone primer walking (GenBank accession
number: AY336943). This phenomenon has also
been found in other plants resistance genes research
(Meyers et al., 1999; Shen et al., 1998).
The incorporation of markers from linkage with
Tyr1
to the
Ctv
locus region of‘9401 family’ was the
first try to get an initial picture of major clustering
of resistance genes in
Ctv-Tyr1
genomic region. The
results showed that the two resistance genes loci
were not so far away from each other. The structural
rearrange ment in the
Ctv-Tyr1
chromosomal region
was approved by the different markers' order
in‘9145’ and ‘9401’ family linkage map (Figure 3A;
Figure 3B), although the eight markers' linkage in
9401 family was rather roughly constructed. A
mutation or recombination event occurred in the
Ctv
region was also found in at least one group of
P.
trifoliata
(Fang et al., 1998). The
R
gene cluster
between
Ctv-Tyr1
regions might have arisen from
the kinds of recombination and rearrangement
events.
2.3 Other factors affecting the efficiency of the
linkage map
Once the linkage mapping of the genes of interest
with high resolution is complete, the genes may
become targets for map-based cloning. As
demonstrated in some model plants, molecular
marker technologies have been providing powerful
tools for tagging genes of interest rapidly for
subsequent marker-assisted selection (MAS) and
map-based cloning (MBC). The degree of
correctness and accurateness of the genetic map is
presumably based on its density of the existing
markers. Well-saturated markers are proved to be
decisive to localize the gene of interest to a short
genomic region and facilitate the cloning the target
sequence. The success will depend on the following
two factors: (1) the size of the population; (2) the
development of new markers and more specific
markers.
One of the essential requirements for MAS in
breeding program is the specificity and precision of
markers with low error rate in scoring, that is as
close to the gene as possible for its utility across all
population, which is relied on the size of population
used for mapping. If the linked marker used for
selection is at a distance away from the gene of
interest, leading to cross-overs between the marker
and
the
gene,
a
high
percentage
of