E-mail: brudolp@gwdg.de

ABSTRACT

Genomic DNA libraries of Brassica napus ‘Mansholt’s Hamburger Raps’ and ‘Samourai’ were screened for the presence of (GA)_n and (GT)_n motifs and 185 positive clones were isolated. Of these clones 123 were fully sequenced and 101 microsatellites could be identified. Primer pairs for PCR were designed for 72 microsatellites. Of 41 microsatellites that have been tested for polymorphisms in the varieties ‘Mansholt’s Hamburger Raps’ and ‘Samourai’ – the parents of a double haploid mapping population 15 microsatellites showed polymorphism and could be mapped on 7 linkage groups of an RFLP map of rapeseed (Uzunova et al. 1995). In addition 7 of these microsatellite markers were analysed in 34 varieties of Brassica napus and their number of alleles ranged from 2 to 7. No relationship between degree of polymorphism and repeat length was observed.

INTRODUCTION

Eukaryotic genomes have been shown to be densely interspersed with a class of repetitive elements, termed microsatellites or simple sequence repeats (SSRs), that consist of short in-tandem-arranged repeat motifs of one to a few nucleotides in length (Hamada et al. 1982; Tautz and Rentz 1984). In mammalian genomes, where microsatellites were first analysed, the most frequently occurring repeat sequence is (CA/TG)_n (Hamada et al. 1982). In plants, DNA sequence database searches indicated microsatellites to be less abundant compared to mammals with (A)_n and (AT)_n the most prevalent repeat motif, followed by (GA/TC)n (Lagercrantz et al. 1993; Morgante and Olivieri 1993; Wang et al. 1994).

At many microsatellite loci, a high frequency of variation in the number of repeat units has been observed (Tautz et al. 1986). These length variants can easily be analysed by polymerase chain reaction using primer pairs specific to unique sequences flanking individual loci (Weber and May 1989; Litt and Luty 1989), making microsatellites an abundant source of highly polymorphic and codominant genetic markers. SSR markers have been developed in a number of plant species and have been shown to be useful in genetic mapping (Liu et al. 1996), variety identification (Rongwen et al. 1995) or the analysis of genetic variation (Plaschke et al. 1995).

Objective of this work is the development and characterization of a set of microsatellite markers for rapeseed. Where possible, the microsatellite markers should be integrated into an existing genetic map of the rapeseed genome (Uzunova et al. 1995).

MATERIALS AND METHODS

Plant material

For genetic mapping of the microsatellite loci a double haploid segregating population of 151 lines from a cross between the winter rapeseed varieties ‘Mansholt’s Hamburger Raps’ and ‘Samourai’ was available. This population had previously been used for the development a genetic map of the rapeseed genome (Uzunova et al. 1995). Furthermore, a test array of 34 Brassica napus genotypes was selected for characterisation of the oilseed rape microsatellite markers.

Isolation of microsatellites

Microsatellites were isolated from two genomic DNA libraries of small (200 – 1.200 bp) inserts from the cultivars ‘Mansholt’s Hamburger Raps’ and ‘Samourai’. Oilseed rape genomic DNA was digested with Sau3AI and cloned into the BamHI site of the lambda phage vector ZAP Express (Stratagene). The library was screened for the presence of dinucleotide repeats by hybridization with poly GA/CT and poly GT/CA. Random primed radiolabelling and hybridisation to the filters from the plated phage library were performed according to Uzunova et al. (1995). After plaque hybridisation, purified single plaques were converted into plasmids via the in vivo excision system and the plasmids were sequenced on an automatic laser fluorescence DNA sequencer (ALFexpress, Amersham Pharmacia Biotech) according to standard procedures.

PCR analysis

Primer pairs complementary to the flanking regions of the microsatellite sequences were designed using the computer program Oligo 6.0. PCR reactions were carried out in a volume of 10 µl containing 25 ng of DNA template, 0.5 µM of each primer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 1x reaction buffer and 1 unit of Taq DNA polymerase (Amersham Pharmacia Biotech or PeqLeb). After a denaturing step of 2 min at 94 °C, a “touch down” amplification profile was used (Kresovich et al. 1995), including a denaturing step of 60 s at 94 °C, an extension step of 45 s at 72 °C and an annealing step of 30 s. The initial temperature was 65 °C for two cycles and was subsequently dropped by 1 °C every two cycles until a final temperature of 55 °C was reached. The annealing temperature of 55 °C was employed for the last 20 cycles of the amplification. PCR products were detected using 6% or 8% polyacrylamide gels and the automatic laser fluorescence DNA sequencer. The fragment length analyses were conducted using the computer program AlleleLinks (Amersham Pharmacia Biotech).

RESULTS AND DISCUSSION

The objectives of this study are to produce a set of mapped microsatellite markers that are interspersed in the oilseed rape genome. For this two size fractionated phage libraries of oilseed rape containing 60.000 and 45.000 pfu respectively, was screened for the presence of GA/TC- and CA/TG-motifs and 185 positive clones were found. 123 of these clones have been sequenced and 101 microsatellites could be identified, showing all three types of microsatellite structures: 61 microsatellites are perfect in their repeats, while 37 show an imperfect repeat and three were compound repeats. In addition to GA- and CA-repeats other motifs were found: two T/A-, five AT-repeats and one CTT/AAG-repeat. Nevertheless, GA-SSRs are four-fold more abundant than CA-SSRs. This is in agreement with the results of Lagercrantz et al. (1993) and other authors, who reported that the GT/AC- motif, being the most abundant dinucleotide repeat in mammals, was found to be considerably less frequent in plants.

So far, for 71 of the 101 identified microsatellites primer pairs were designed and 41 of them were tested in PCR with template DNA from ‘Mansholt’s Hamburger Raps’, ‘Samourai’ and the F₁ of these two winter rapeseed varieties. A total of 15 microsatellite markers showed polymorphism in the screening and could be mapped on the previously established genetic map of rapeseed (Tab. 1). The mapped markers were distributed across 7 linkage groups with 1 to 5 microsatellite markers per group (Fig. 1). No clustering was observed on linkage groups with more than one mapped microsatellite, indicating an even distribution of microsatellite markers in the rapeseed genome.

Tab. 1: Characteristics of the mapped Oilseed Rape Microsatellite Markers

*) Range of PCR product length between ‘Mansholt’s’ and ‘Samourai’

On the other hand, several sequenced clones contained two to four microsatellites with different repeat motifs. This might indicate a small scale clustering of microsatellites. For two clones primer pairs were designed for each of the clustered microsatellites allowing independent analysis. In both cases, only one of the two microsatellites proved to be polymorphic in the mapping population. The corresponding markers, MD 2.2 and MR 133.2, were located on linkage groups 13 and 3.

The variability of seven of the mapped microsatellite loci was investigated in an array of 34 Brassica napus varieties and significantly more variation was detected with the microsatellite markers compared to RFLP and RAPD markers. The number of alleles ranged from 2 to 7, with an average of 3.9 different alleles per microsatellite locus (Tab. 1).

This work will continue to include all microsatellites identified in the sequenced clones. As the current results have shown, it will not be possible to map all the microsatellites in the mapping population. Nevertheless, many of the non mapped microsatellites may still be useful because they will show polymorphisms between other rapeseed varieties. In addition, new mapping populations are currently being developed that will allow to increase the number of mapped microsatellites.

Fig. 1: Map positions of microsatellite markers on the oilseed rape genetic map. Distances between markers (on the left-hand side of the linkage group) are given in cM, calculated from recombination frequencies according to the Kosambi mapping function.

CONCLUSION

In this project 15 microsatellites were mapped so far and 7 have been characterized using a set of 34 rapeseed varieties. The degree of polymorphisms found with microsatellite markers was significantly higher than with RFLP markers. In addition, the mapped microsatellite markers were distributed across 7 linkage groups with no apparent clustering within linkage groups. These results indicate that microsatellites may be evenly distributed across the rapeseed genome. Their genomic distribution, the high degree of polymorphism and their large potential for automated analysis will make microsatellites in rapeseed a near ideal marker type especially for identifying varieties and hybrid components or distinguishing between new varieties and essentially derived varieties.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the company Norddeutsche Pflanzenzucht (NPZ), Holtsee, Germany and the Bundesministerium für Bildung und Forschung (BMBF) for their financial support.

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