The microsatellite technique (SSR) is well described as a highly polymorphic, codominant marker system for animals and plants. To use these advantages for the analyses of rapeseed several activities had been started in the last year to speed up the development of microsatellites for B. napus. More than 60 well-working SSRs have been selected and their suitability for different applications and in addition for automation had been checked.
KEYWORD SSR, genetic distance, essential derived varieties, hybridity testing, homogenity testing
Microsatellites as genetic markers detect a much higher level of polymorphism per locus than any other marker system. Based on this advantages SSR markers have been already efficiently used for studies of genetic diversity (Plaschke et al., 1995), mapping (Warland et al., 1998), and variety identification (Bredemeijer et al., 1998) in different crops. Until now marker techniques like RAPD and RFLP have been used for the estimation of genetic distance in Brassica napus (Knaak, 1995; Förster et al., 1995). In contrast to this techniques SSRs offer a much higher degree of automation which is needed to deal with the great number of individuels generally handled in rapeseed breeding.
Material and methods
A 96 well leaf crusher (CrushExpress, available from SU Resistenzlabor) enable us to improve the automation and the handling of large number of samples while saving cost and time for genomic DNA isolation of winter and spring oilseed rape varieties and different breeding lines. Up to now we selected 60 well-working SSRs with mainly one single defined fragment and no unspecific amplification background. To accuratly determine the allelic patterns of the analysed rapeseed varietes automated DNA sequencing mashines (ALF) were used for fragment analyses. The resulting allele sizes for each marker and variety had been stored in a database for further analysis.
First mapping work was carried out on a subset of SSRs by analysing 156 dihaploid lines of the mapping population Mansholt’s x Samourai (Uzunova et al., 1995). The results of 26 mapped loci (table 1) indicated a good distribution across the rapeseed genome underlining the suspected good suitability of SSRs for estimating genetic distances.
Table 1: Distribution of mapped SSRs in the mapping population Mansholt’s x Samourai
Number of SSRs
each 1 SSR
each 2 SSRs
each 3 SSRs
Estimation of genetic distance
The good suitability of SSRs for estimating the genetic distances in rapeseed (Plieske et al., 1998) can be confirmed with our first results Until now 48 SSRs had been applied to a set of 33 winter- and 34 spring rapeseed varieties. The clusteranalysis (figure 1) and the direct comparison of the genetic distance values indicated the overall agreement with well established data from RFLP analyses (Knaak, 1995). For future calculations and comparisons the scored SSR data points were collected in a database. This will allow an unequivocal identification or distinguishing of known and unknown varieties and breeding lines as well as giving hints for essential derived varieties (see figure 1, genotypes Falcon and MSL 004c).
Figure 1: Dendrogram of spring and winter rapeseed varieties clustered by UPGMA based on genetic distance estimates of 45 SSRs. Groups already identified with RFLPs are marked with symbols and colours.
Testing homogenity and hybridity
In order to get plant breeders right a variety must have sufficient homogenity which needs a lot of selfing during breeeding. In this background SSRs might perhaps become a useful tool to simplify the testing of homogenity and hybridity. Our results from single plants of different seed lots clearly pointed out the applicability of SSRs to identify homozygous or heterozygous plants (figure 2A).
In addition for registration the high number of already listed varieties in Europe more and more lead to problems in dinstinguishing. By support of SSRs this problem probably could be solved. For instance the analyses of two new hybrids that could not distinguished by their phenotype could be differentiated with SSR markers. In our tests SSRs generate different allelic patterns for the two male lines visualizing the differences between the two hybrids (figure 2B).
Figure 2: A) Identification of homozygous and heterozygous single plants with one SSR. B) Two hybrids (hybrid 11, hybrid12) with the same female but different male lines (male1, male2) could not be distinguished by their phenotype but with SSRs.
The results slead to the conclusion that the SSR technique is an accurate and reliable, easy handling and cost-effective tool for the main applications of markers in breeding programs. In regard to the increasing number of SSR markers we can assume that in future also mapping approaches, the identification of closely linked markers via bulk segregant analysis and the testing of essential derived varieties are possible applications.
We thank the Deutsche Saatveredelung Lippstadt-Bremen and the Norddeutsche Pflanzenzucht Hans Georg Lembke KG, Germany for having made available the SSRs and Dr. habil. W. Ecke, University of Göttingen, Germany for providing the DNA of the mapping population and the calculation of the mapping data.
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