ILVO - Plant (Applied Genetics and Breeding)http://www.ilvo.vlaanderen.be/plant_uk/

Development of EST markers and evaluation of their use in evergreen azalea analysis

Ellen De Keyser1, Valentina Scariot2 and Jan De Riek1 1Institute for Agricultural and Fisheries Research (ILVO) – Plant Unit, Applied Genetics and Breeding,

Caritasstraat 21, 9090 Melle, Belgium, e-mail: Ellen.DeKeyser@ilvo.vlaanderen.be2Department 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.

Figure 1: Intron-spanning primers were used for amplification on cDNA-containing 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

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.

Conclusions

Source of variation

Degree of

freedom

Sum of squares

Variance components

Percentage of variation

Among hybrid groups 3 33.900 0.623 10.92

Within hybrid groups 40 203.235 5.081 89.08

Total 43 237.136 5.704 AF

LP

Fixation Index Fst: 0.10920

Among hybrid groups 3 11.942 0.219 10.91 Within hybrid groups 40 71.643 1.791 89.09

Total 43 83.585 2.010 ST

MS

Fixation Index Fst: 0.10908

Among hybrid groups 3 10.804 0.226 14.44

Within hybrid groups 40 53.651 1.341 85.56

Total 43 64.455 1.567 ES

T

Fixation Index Fst: 0.14438

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.

Future perspectives

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).

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).

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

References

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.