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Varieties with closed flowers - a useful strategy for restricting pollen dispersal in oilseed rape?

(2008 – 2011) Julius-Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants (JKI), Institute for Biosafety of Genetically Modified Plants, Quedlingburg

Topic

Plants that pollinate and fertilize themselves inside closed flowers are called cleistogamic. As part of a breeding programme French scientists have been able to produce cleistogamy in oilseed rape by means of mutations.

Recently, research has been carried out into cleistogamy to examine whether it could be used for biological confinement in transgenic oilseed rape. This could restrict pollen-mediated gene transfer to neighbouring plants.

However, the trait is also of interest for conventional varieties when it comes to traceability and separating production of crops of different origins and qualities (identity preservation).

This project examined the following questions:

  • What outcrossing behaviour is demonstrated by cleistogamic oilseed rape lines in realistic growing conditions?

The project is part of the joint project on “Developing and testing confinement strategies for oilseed rape”. The network partners - the University of Hohenheim, the University of Göttingen and the Julius-Kühn-Institut (JKI) - are collaborating on laboratory analyses, the use of trial fields and data exchange for modelling.

Summary

The investigated gene sequences from the cleistogamic and conventional rape variety differed only slightly, so it was not possible to develop suitable molecular markers or a PCR method for differentiating between the oilseed rape lines.

Because of this, it was not possible to analyse the samples obtained from the field or to determine to what extent the use of cleistogamic rape plants can reduce outcrossing.

Experiment description

Conventional oilseed rape - open flowers

Oilseed rape with closed flowers (Cleistogamic oilseed rape)

Cleistogamic oilseed rape

Plot trial with cleistogamic and conventional oilseed rape

Plot trial with cleistogamic and conventional oilseed rape

Photos: Dr. Alexandra Hüsken, JKI

Developing a method for quantifying outcrossing in cleistogamic varieties

The project aims to develop a suitable PCR method for quantifying outcrossing from cleistogamic varieties to conventional non-cleistogamic varieties. Since only a few genetic details are available about the cleistogamy gene in the oilseed rape line under investigation, molecular markers will have to be identified that can be used to detect and quantify incrossing from a cleistogamic oilseed rape plot to a recipient plot.

Outcrossing of cleistogamic varieties in the field

Ring tests at several locations were conducted to determine the extent of incrossing of cleistogamic varieties into recipient oilseed rape varieties. Research will focus on the separation distance that needs to be observed between a cleistogamic and a non-cleistogamic oilseed rape plot in order to avoid exceeding a particular outcrossing threshold. To this end, a 0.5 hectare field will be planted with cleistogamic oilseed rape as a pollen source (donor). A 0.5 hectare plot will be planted downwind with non-cleistogamic oilseed rape as a pollen recipient. Oilseed rape plants at varying distances from the donor plot will be scored during the flowering period and samples will be collected at harvest time.

Secondary dormancy in seeds of cleistogamic varieties

Laboratory and field experiments will investigate whether there are differences between the conventional high-yield varieties and the cleistogamic line with regards to the expression of the secondary dormancy trait. The research (laboratory and field experiments) will be conducted on freshly harvested rape seeds.

Results

Diagram of the trial field with cleistogamic oilseed rape as the pollen donor and the Marcant variety as the recipient. Oilseed rape plants that had finished flowering were harvested at various distances (dotted lines) from the donor plot (site 1 from 0 to 50 metres, site 2 from 0 to 36 metres)

Developing a method for quantifying outcrossing in cleistogamic varieties

Identifying molecular markers: Gene sequences that are responsible for the erucic acid content appeared promising as potential markers. Apart from the cleistogamy trait, the cleistogamic rape and the Marcant line differ in terms of the erucic acid levels in their seeds. The cleistogamic line is a low erucic acid rapeseed (LEAR), while the Marcant line has a high erucic acid level (HEAR).

The low erucic acid level in the cleistogamic rapeseed is due to a mutation in the fae1 (= fatty acid elongation 1) gene. Oilseed rape lines with different erucic acid levels therefore have different fae1 gene sequences, so it should be possible to use these to develop molecular markers for molecular biological analysis. However, despite a great deal of research, it was not possible to develop any suitable primers.

The cleistogamy gene is another potential marker gene. This is a point mutation that leads to an amino acid being exchanged in the protein sequence. The DNA sequence of the mutated Clg gene from the cleistogamic oilseed rape was used to develop primers with which it was possible to amplify segments of the DNA sequence from the Marcant Clg gene. However, it was not possible to develop a sufficiently sensitive method for this marker either.

Outcrossing of cleistogamic varieties in the field

In the summer of 2009 and 2010, the trial fields were harvested. The main stems were harvested from plants of the pollen recipient (Marcant variety) that had clearly finished flowering to obtain seeds for the molecular biological investigations. The individual plants were harvested at eight different distances from the donor field (cleistogamic rape). Between 112 and 128 individual plants were harvested from the sites in question.

Since the PCR methods developed were not sensitive enough for a quantitative differentiation of the rape lines, it has not yet been possible to analyse the samples obtained from the field. If technical progress makes sensitive quantitative differentiation possible, the analyses will be carried out at a later date.