A genomic library was constructed from DNA of two azalea genotypes: a Belgian pot azalea R. simsii hybrid Mevr. Van Belle and a Chinese R. simsii from Daoxian. An enrichment of microsatellite containing sequences was performed as in Van de Wiel et al. (1999). Fragments were sequenced and primers were designed that allow the amplification of the microsatellite repeat. About 220 microsatellite containing clones were selected from the enrichment procedure. Mainly dinucleotide repeats and some trinucleotide repeats were found. The selected primers were tested in a small set of reference varieties to check their value (specificity and polymorphic rate) and to set up the PCR-conditions. Five primer pairs have been tested, two of them gave a specific and polymorphic pattern. They were further screened by radioactive PCR on a selection of 5 plants from the azalea breeders gene pool which included the two genotypes used library construction. These 2 STMS markers uniquely identified the 5 plants

Arens, P.

Bockstaele, E., van

Dendauw, J.

Loose, M., de

Riek, J., de

Vosman, B.

English

Name not available

Development of Sequence Tagged Microsatellite Site (STMS) markers in Azalea

Wageningen Yield 

Proc. Int. Symp. on Molecular Markers 
Eds. Doré, Dosba & Baril 
Acta Hort. 546, ISHS 2001 
 
 
193 
DEVELOPMENT OF SEQUENCED TAGGED MICROSATELLITE SITE 
(STMS) MARKERS IN AZALEA 
UTILISATION DES MARQUEURS MICROSATELLITES A SEQUENCES 
ETIQUETEES CHEZ LES AZALEES 
 
 
J. Dendauw1, J. De Riek1, P. Arens2, E. Van Bockstaele1, B. Vosman2 and M. De Loose1 
1Dept. of Plant Genetics and Breeding, CLO-Gent, Caritasstraat 21, B-9090 Melle, 
Belgium 
2Centre for Plant Breeding and Reproduction Research, Postbox 16, 6700 AA 
Wageningen, The Netherlands 
 
 
Keywords: Rhododendron species, Belgian pot azalea, microsatellite enrichment, 
dinucleotide and trinucleotide repeats 
 
Abstract 
 
A genomic library was constructed from DNA of two azalea genotypes: a Belgian pot 
azalea R. simsii hybrid Mevr. Van Belle and a Chinese R. simsii from Daoxian. An 
enrichment of microsatellite containing sequences was performed as in Van de Wiel et al. 
(1999). Fragments were sequenced and primers were designed that allow the 
amplification of the microsatellite repeat. About 220 microsatellite containing clones 
were selected from the enrichment procedure. Mainly dinucleotide repeats and some 
trinucleotide repeats were found. The selected primers were tested in a small set of 
reference varieties to check their value (specificity and polymorphic rate) and to set up 
the PCR-conditions. Five primer pairs have been tested, two of them gave a specific and 
polymorphic pattern. They were further screened by radioactive PCR on a selection of 5 
plants from the azalea breeders gene pool which included the two genotypes used library 
construction. These 2 STMS markers uniquely identified the 5 plants.  
 
1. Introduction  
 
Azaleas belong to the genus Rhododendron of the family of Ericaceae. The 
azaleas were first classified by Linneaus in Species plantarum (1753) as Azalea indica. 
Azaleas can be divided in two groups, 1°) deciduous azaleas which belong to the 
subgenus Pentanthera and 2°) evergreen azaleas which belong to the subgenus Tsutsusi 
(Chamberlain and Rae, 1990). In Belgium Rhododendron simsii hybrids also known as 
Belgian pot azaleas are an important export product. Identification based on 
morphological and physiological traits is not always clear and is labour intensive. In 
Rhododendron spp. till now some DNA-marker analysis is done. Feasibility of 
Rhododendron DNA for RAPD–analysis has been evaluated (Iqbal et al., 1994). 
Phylogenetic relationships of Rhododendroideae are measured using rbcL and matK 
sequencing data (Kron, 1997). Introgression of R. kaempferi into R. kiusianum is studied 
by (Kobayashi et al., 1998) using PCR on cp-DNA.  
DNA markers, especially AFLP (De Riek et al., 1999) and STMS, can give 
complementary data to the morphological and physiological data. 
 
2. Materials and methods  
 
2.1. DNA extraction  
 
The DNA of 2 plants (R. simsii hybrid Mevr. Van Belle and R.simsii Daoxian) was 
used for constructing the genomic library. For testing the developed microsatellites the 
DNA of those 2 plants plus the DNA of 4 other plants (R. simsii “Knut Erwen”, 
Proc. Int. Symp. on Molecular Markers 
Eds. Doré, Dosba & Baril 
Acta Hort. 546, ISHS 2001 
 
 
194 
“Ambrosiana”, the Hirado hybrid “Heiwa-no-hikari”, and R. simsii Daoxian) was used. 
For DNA extraction, young leaf material was harvested in the greenhouse and 
immediately frozen in liquid nitrogen. The leaves were either directly ground in liquid 
nitrogen or stored at -80 °C until lyophilisation. After 48 h lyophilisation the material was 
ground in a mechanical mill (Culatti). DNA-extraction was done using a two-step 
protocol, modification of the protocol described by Greenwood et al., (1989). The second 
extraction buffer was changed to improve yield for small amounts of leaf material (De 
Riek et al., 1999). 
 
2.2. Isolation and of the microsatellites 
 
A genomic library, prepared from a Alu I digest of R. simsii hybrid Mevr. Van 
Belle was enriched for GA/GT, TGT/GTG/GAG/GCT/TGTT/GATA and 
TCT/CGT/AGT/TGA/GTAT containing DNA-fragments, according to Van de Wiel et al. 
(1999). The enriched fragments were cloned into a pBluescript SK+ vector and 
transformed into E. coli DH5α. Glycerol stocks were made for longterm storage. Plasmid 
DNA extraction was performed with the GFX™ Micro Plasmid Prep Kit (Amhersham 
Pharmacia Biotech). 
Sequencing analysis was done using universal T3 and T7-pimers with the Big 
Dyes Terminator Kit (PE Biosystems) on a ABI Prism™ 377 DNA XL upgrade 
Sequencer.  
 
2.3. PCR reactions and detection of the amplified fragments 
 
Forward and reverse primers were developed using Primer 0.5 (Whitehead 
Institute for Biomedical Research). Amplifications were performed in a total volume of 
20 µl which contained 10 ng genomic DNA, 0.1µM of each primer, 100 µM 
deoxyribonucleotides, 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 0.125% 
(w/v) BSA and 1.25 U AmpliTaq (PE Biosystems). The unlabelled and radioactive PCR 
reactions were performed in tubes using a Perkin Elmer 9600 thermocycler with cycling 
conditions as follows: 1 cycle of 94°C for 5 min and 35 cycles of 55°C for 1 min, 72°C 
for 2 min, and 94°C for 1 min. After the final cycle, one cycle of 55°C for 1 min and 
72°C for 7 min was added. PCR products were electrophoresed using 2,5 % agarose gels 
and ethidium bromide staining for a preliminary evaluation. To obtain higher resolution 
polyacrylamide gel electrophoresis (PAGE) on vertical gels (6% polyacryamide, 7.5 M 
urea, Tris-borate-EDTA buffer) with a Sequi-Gen GT Sequencing cell (BioRad) was 
performed. Here the DNA bands were labelled, by PCR using a γ-ATP33 kinated forward 
primer. The sizes of the PCR-products were determined by loading a Sequa Mark ladder 
(Research Genetics) near the samples on the gel. 
 
3. Results  
 
Two hundred and twenty microsatellite containing clones derived from the 
genomic bank of the R. simsii hybrid Mevr. Van Belle were sequenced. Of these 125 
clones contained microsatellites (Table 1). The other 95 clones contained no repeat or 
resulted in bad sequencing reactions. 
From those 125 clones, 70 (52 dinucleotide and 18 trinucleotide repeats) were 
useful for primer development. 
So far, 5 primers have been developed using dincleotide repeat containing 
sequences as template. The developed primer pairs were tested on a selection of 5 plants, 
including the cultivars used to construct the genomic library.  
The PCR products were first detected on agarose gels. From the five primer pairs 
used, two resulted in a good pattern, without smears or non-specific bands. 
Those two primer pairs (AZA-002 flanking an (AC)8(TC)8(AC)17-repeat and AZA-
003 flanking a (TG)15-repeat ) were further used for radioactive detection on 
Proc. Int. Symp. on Molecular Markers 
Eds. Doré, Dosba & Baril 
Acta Hort. 546, ISHS 2001 
 
 
195 
polyacrylamide gel electrophoresis (PAGE). The higher resolution resulted in a better 
separation of the different alleles. With the 2 primer pairs it was possible to distinguish 
the 5 plants from each other (Figure 1). 
 
4. Discussion 
 
From the microsatellite containing clones mainly dinucleotide and some 
trinucleotide repeats were found, using the enrichment procedure like described above. 
One of the problems encountered for dinucleotide repeats is stuttering of the Taq DNA-
polymerase, which results in shadow bands. With the 2 primer pairs AZA-002 and AZA-
003 some stuttering was observed. This can be reduced by the choice of the repeat used 
for primer development, optimising the PCR-program or using a hot start enzyme. In the 
near future hot start enzymes will be tested. Also detection by fluorescent PCR and 
automatic analysis with genotyping software will be evaluated to obtain higher troughput. 
In a next step the whole genepool of Belgian pot azaleas will be screened using 
microsatellites.  
DNA-markers become more and more a helpful tool for several crops. In the case 
of azalea, DNA-markers can be useful for identification purposes. Because in Belgium 
the pot azaleas are mainly an export product, an identification system on the juvenile, 
non-flowering, plant stage is of interest. Also, for the protection of commercial azalea 
cultivars and for granting Plant Breeders Rights DNA-markers can be unambiguous tool. 
To introduce DNA-markers for these purposes, a quick and inexpensive detection system 
is required. Once developed, a set of microsatellite markers can satisfy those applications.  
 
Acknowledgements 
 
This work is a co-operation between DvP-CLO (B) and CPRO-IGD (Nl). It was 
supported by a grant of the Ministry of Small Enterprises, Traders and Agriculture - DG 6 
Research and Development (Project D1/2 - 5781 A - section 1). 
 
References  
 
Chamberlain D.F., and Rae S.J., 1990. A revision of Rhododendron IV Subgenus 
Tsutsusi. Edinburgh J. Bot., 47:89-200. 
De Riek, J., Dendauw J., Mertens M., De Loose M., Heursel J., and Van Bockstaele E. 
1999. Validation of criteria for the selection of AFLP markers to assess the genetic 
variation of a breeders’ collection of evergreen azaleas. Theor. appl. Genet., 99:1155-
1165. 
Kobayashi N., Kawashima T., and Arisumi K., 1998. Introgression in Japanese evergreen 
azaleas (Rhododendron kiusianum and R. kaempferi). Proc. Third. Symp. On New 
Floricultural Crops. Acta Hortic., 454, ISHS 1998. 
Kron K.A. ,1997. Phylogenetic relationships of Rhododendroideae (Ericaceae). Am. J. of 
Bot., 84,7: 973-980. 
Greenwood M.S., Hopper C.A., and Hutchison K.W., 1989. Maturation in Larch. I. Effect 
of age on shoot growth, foliar characteristics and DNA methylation. Plant physiol., 
90:406-412. 
Iqbal M.J., Gray L.E., Paden D.W., and Rayburn A.L., 1994. Feasibility of Rhododendron 
DNA profiling by RAPD analysis. Plant Varieties and Seeds, 7, 59-63. 
Van de Wiel C., Arens P., and Vosman B., 1999. Microsatellites retrieval in lettuce 
(Lactuca sativa L.). Genome, 42: 139-149. 
Proc. Int. Symp. on Molecular Markers 
Eds. Doré, Dosba & Baril 
Acta Hort. 546, ISHS 2001 
 
 
196 
Tables 
 
 
 
1. Types of di- and trinucleotide repeats isolated from azalea. 
 
REPEAT TYPE NUMBER OF CLONES % 
Dinucleotide    
(AC) 30 57.7 
(TG) 10 19.2 
(CT) 2 3.8 
(AC) (TC) 4 7.7 
(AG) (TG) 4 7.7 
Rest 2 3.8 
Sub Total 52 100 
Trinucleotide    
(CCA) 42 57.5 
(TGC) 12 16.4 
(CAG) 6 8.2 
(CAA) 1 1.3 
(GAC) 1 1.3 
(TAG) (TCG) 5 7 
(CTG) (TTG) 3 4.2 
(TAC) (GAC) 2 2.8 
(CAG) (CAA) 1 1.3 
 SubTotal 73 100 
 Grand Total   125 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Proc. Int. Symp. on Molecular Markers 
Eds. Doré, Dosba & Baril 
Acta Hort. 546, ISHS 2001 
 
 
197 
Figures 
 
1. Fraction of STMS-pattern of five plants with two microsatellite marker using PAGE. 
 
 
 
 
  
1    2    3    4    5    
1 = R. simsii Knut Erwen 
2 = R. simsii Ambrosiana 
3 = Hirado Heiwa-No-Hikari   
4 = R. simsii Mevr. Van Belle 
5 = R. simsii Daoxian 
Primerpair AZA-003   
(TG)15 
+ 170 bp 
       Primerpair AZA-002  
           (AC)8(TC)8(AC)17 
+ 260 bp 
    3       4       5  


A revision of Rhododendron IV Subgenus Tsutsusi.

Feasibility of Rhododendron DNA profiling by RAPD analysis.

Maturation in Larch. I. Effect of age on shoot growth, foliar characteristics and DNA methylation. Plant physiol.,

Phylogenetic relationships of Rhododendroideae (Ericaceae).

Validation of criteria for the selection of AFLP markers to assess the genetic variation of a breeders’ collection of evergreen azaleas.

Development of Sequence Tagged Microsatellite Site (STMS) markers in Azalea

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