Development of EST markers and evaluation of their
use in evergreen azalea analysis
Ellen De
1
Keyser ,
Valentina
2
Scariot
and Jan De
1
Riek
1Institute
for Agricultural and Fisheries Research (ILVO) – Plant Unit, Applied Genetics and Breeding, Caritasstraat 21,
9090 Melle, Belgium, e-mail: Ellen.DeKeyser@ilvo.vlaanderen.be
2Department of Agronomy, Forest and Land Management, Università degli Studi di Torino, Via Leonardo Da Vinci 44,
10095 Grugliasco (TO), Italy
EST marker development
Two
hundred
randomly
picked
cDNA
fragments from a cDNA library of the azalea
cultivar ‘Flamenco’ were sequenced. Homology
searches resulted in the assignment of a
putative function to 30% of these sequences.
Primers were developed on 87 of these cDNA
fragments. They were evaluated on the
corresponding cDNA-containing plasmids and
DNA of two distinct rhododendron species (R.
luteum and R. simsii hybrid ‘Flamenco’) for
being intron-spanning. In case no intron was
amplified, new primers were developed once,
else, primers were used on six different species
and cultivars and the presence of polymorphic
bands was evaluated (Fig. 1). This resulted in
32 polymorphic EST markers. Ten of them were
selected for comparison with AFLP and STMS.
0.623
5.081
5.704
10.92
89.08
Among hybrid groups
3
Within hybrid groups
40
Total
43
Fixation Index Fst: 0.10908
11.942
71.643
83.585
0.219
1.791
2.010
10.91
89.09
Among hybrid groups
3
Within hybrid groups
40
Total
43
Fixation Index Fst: 0.14438
10.804
53.651
64.455
0.226
1.341
1.567
14.44
85.56
AFLP
33.900
203.235
237.136
STMS
Sum of
Variance
Percentage
squares components of variation
EST
Degree
Source of variation
of
freedom
Among hybrid groups
3
Within hybrid groups
40
Total
43
Fixation Index Fst: 0.10920
Figure 1: Intron-spanning primers were
used for amplification on cDNAcontaining plasmid DNA (Pl) and DNA
of three Rhododendron simsii hybrids
(‘Lara Rood’ (L), ‘Coelestine’ (C) and
‘Flamenco’ (F)) and three species (R.
luteum (Lu), R. simsii (S) and R.
noriakianum (N)). The left primer pair
resulted in the amplification of
polymorphic bands and was selected as
EST marker, the other primer pair
amplified an intron, but this was not
polymorphic between different cultivars
and species.
Pl Lu F S N L C
Pl Lu F S N L C
Comparison of AFLP, STMS and EST
Forty species and varieties that most concurred in the origin of
the known cultivars and 44 cultivars chosen among
distinguishable horticultural groups were genotyped using 3
AFLP primer combinations, 6 microsatellite loci and 10 ESTs
(Scariot et al.). EST markers revealed a higher genetic distance
detection capacity than AFLPs. Similarity matrices produced for
each marker technique showed weak yet significant
correlations. Performing analysis of molecular variance most of
the genetic diversity was attributable to differences among
cultivars
within
horticultural
groups.
EST
markers
outperformed AFLP and STMS markers concerning Fst values
indicating a low but significant differentiation among
horticultural groups (Table 1).
Table 1: AMOVA (Analysis of Molecular Variance) Significance
tests (1023 permutations) at one level based on AFLP, STMS and
EST data sets (binary Euclidian distance coefficient).
Conclusions
Although ESTs and STMSs appear to be the most appropriate
markers for paternity analysis and assessment of narrow genetic
relationships, AFLP remains the best technique for phylogenetic
studies. EST markers could be particularly useful for QTL mapping
using a candidate gene approach because they target expressed
genes and for comparative mapping studies because they are
derived from gene coding regions which are more likely to be
conserved across populations and species.
Future perspectives
AFLP, STMS and EST markers will be
generated on a number of populations that
are segregating for flower colour and other
plant quality traits. Since azalea has a
rather high chromosome number (2n=26),
integration of the separate population
maps into a consensus genetic map of
azalea will only be successfull when there’s
at least one segregating co-dominant locus
for each chromatide. STMS markers and
EST markers will be used for this purpose.
More functional EST markers are being
developed for genes involved in flower
colour biosynthesis and other plant quality
traits.
References
V. Scariot, E. De Keyser, T. Handa and J. De Riek. Comparative study of the discriminating capacity and effectiveness in assessing genetic relationships of AFLP, STMS and
EST markers in evergreen azalea analysis. Plant Breeding (accepted for publication)
Acknowledgements
The authors wish to thank Veerle Buysens, Veerle Cools and Laurence Desmet for their assistance in DNA preparations, EST development and AFLP, STMS and EST reactions.
The whole staff of the biotech lab of ILVO -Plant Unit was very much appreciated for their support and skilful assistance. This research was funded by the Regione Piemonte,
Settore Servizi di Sviluppo Agricolo and the Ministry of Agriculture of Flanders.
ILVO - Plant (Applied Genetics and Breeding)
http://www.ilvo.vlaanderen.be/plant_uk/