CA2206673A1 - Use of molecular markers for genotype determination of the ogura rf gene in brassica napus - Google Patents

Abstract

This invention relates to Brassica plants and methods of breeding Brassica plants. More particularly, the invention relates to the use of molecular markersfor determining the genotype of the Ogura Rf gene in Brassica plants at an early stage in their development. The method involves using a combination of primers derived from RAPD markers in PCR reactions to allow discrimination between homozygous, heterozygous and non-restorer or sterile genotypes.
Also disclosed are combinations of primers which allow such discrimination.

Description

USE OF MOLECULAR MARKERS FOR GENOTYPE DETERMINATION
OF THE OGURA Rf GENE IN BRASSICA NAPUS

Field of the Invention This invention relates to Brassica and Brassica breeding. More particularly, the invention relates to Brassica lines having the Ogura restorer (Rf) gene, and methods to identify these lines using genetic marker analysis.

Background of the Invention Brassica plants, and in particular oilseed from such plants, are increasingly important crops.
As a source of vegetable oil, they presently rank behind only soybeans and palm in 10 commercial importance and they are comparable with sunflowers. The oil is used both as a salad oil and as a cooking oil.

Significant improvement in yield has been demonstrated in Brassica using single cross hybrids (Hutcheson et al. 1981, Sernyk and Stefansson 1983; Grant and Beversdorf 1985).
For commercial hybrid production, a reliable and efficient pollination control system is required. In Brassica, a number of pollination control systems are available: nuclear male sterility, self incompatibility and cytoplasmic male sterility (CMS). There are different types of CMS in Brassica, including, for example, Ogura CMS and Polima CMS.

CMS in plants is characterized by failure to produce normal anthers and functional pollen. It is a m~t~rn~lly inherited trait. The genetic determin~nt~ for CMS reside in the mitochondrial 20 genome as opposed to nuclear genome. A 2.5 Kb NCO1 fragment that gives rise to a 1.4 Kb transcript, has been implicated in Ogura CMS (Bonhomme et al. 1991). This transcript contains two open reading frames, ORF 158 and ORF 138. Subsequent study seems to have elimin~ted the role of ORF 158 and hence reaffirmed the key role of ORF 138 in pollen abortion in Ogura CMS plants (Bonhomme et al. 1992).

To be useful for F, hybrid seed production, a CMS system requires strong fertility restorer and sterility m~ints~iner alleles. What distinguishes the Ogura system from other CMS

systems in Brassica in terms of use for commercial hybrid production is the availability of a workable restorer or Rf gene (in this application, "OGU Rf").

Both Ogura CMS and a good source of fertility restorer gene have been identified in radish Raphanus sativus, (Ogura 1968; Heyn 1976). Ogura CMS has been transferred from radish to B. napus by inter-specific crossing, embryo rescue and backcrossing (Bannerot et al. 1974).
Protoplast fusion with B. napus was required to produce male sterile B. napus cybrids (Pelletier et al. 1983). The restorer gene has also been transferred from radish to B. napus by intergeneric crossing (Heyn 1976), and fully restored Ogura CMS plants were identified carrying a single restorer gene (Pelletier et al. 1987). Unfortunately, decreased female 10 fertility in the restored plant accompanied such a transfer and it was postulated that the radish chromosome segment that carried the restorer gene and that was introgressed from radish also contained radish DNA with genetic information other than the Ogura restorer gene (Pellan-Delourme and Renard 1988). Although retention of radish genetic information around the restorer allele has been recently reduced so as to improve seed production, (Delourme et al.
1991), a linkage study by Delourme and Eber 1992 identified an isozyme locus (marker), PGI
II, linked to the restorer gene and presumably also located on the radish chromosome segment. A biochemical assay is required to determine PGI II expression. PGI II is expressed as a protein and is analyzed by running out the protein on a gel. By analyzing the patterns on the gel it is possible to determine the PGI II genotype of individual samples.

20 Genetic marker analysis has proven useful for detecting the presence of the restorer gene.
Without markers, such as PGI II or genetic markers, breeders need to wait until the plants have flowered to determine whether they carry the restorer gene or not. If the restorer gene is present, the flowers are normal (fertile) and if the restorer gene is absent, they have reduced (abnormal/nonfunctional) anthers and lack pollen.

There are significant advantages to identifying the presence of a gene by marker analysis in a segregating population at an early stage in a plant's development. One advantage is that by detçrminin~ a plant's genotype before it flowers, the breeder saves resources (time and space) associated with growing the plant to flowering and elimin~ting undesired genotypes. In winter napus, the ability to identify the presence of a gene by marker analysis enables the breeder to identify the plants in which the gene is present before vern~li7~tion. This is particularly helpful since it allows breeders to elimin~te any plants, prior to vern~li7~tion, which do not carry the gene, saving valuable space in vern~li7~tion rooms. Winter B. napus requires an eight to twelve week period of vern~li7~tion in order to initiate flowering.

Markers can be used not only to determine the presence of a gene but also its genotype (homozygous vs. heterozygous). Ideally, if the sequence of the actual gene is available, one can use molecular techniques to definitively determine its presence by PCR analysis.
Obtaining a gene sequence is difficult, but identifying loci (markers) that are linked to the gene, though difficult, is less difficult compared to identifying the gene sequence. The 10 "mapping distance" of marker loci to the gene of interest (termed "linkage") will determine the level of confidence in concluding that the gene is present when the marker has been identified as being present. The shorter the "mapping distance" the greater the level of confidence.

The isozyme locus identified by Delourme and Eber (1992) can be used to identify the presence of the restorer gene and this isozyme analysis is often used to determine the genotype of plants cont:~inin~ the OGU Rf gene. However, using this type of marker has certain limitations. The analysis involves assaying a gene product, a protein, which is easily degraded if leaf tissue samples or extracts prepared from leaf tissues are not handled under ideal temperature conditions. In addition, the gene product may be developmentally 20 regulated, i.e. not expressed in certain tissues or at a certain developmental stage. Other PGI
loci (for example, the PGI II isozyme locus) are present in B. napus and, depending on the combination of alleles present in a particular plant, the banding pattern of the PGI-II protein product run out on the gel becomes complex, as has been found in spring napus. Thus, it may not be possible to associate en_yme phenotype with genotype of the restorer gene. Isozyme analysis has been used extensively in winter napus populations, but does not give reliable results in spring napus populations due to the complexity of the banding patterns observed on the gels.

At present, there is no technology or method, other than iso_yme analysis, which uses molecular/biochemical markers to determine the genotype of the OGU Rf gene in Brassica.

PCT Tnt~rn~tional patent application WO 97/02737 of Forman, et al., published on January 30, 1997, claims a method of producing an improved restorer line of Brassica wherein testing the progeny for fertility included the use of AFLP, RFLP and/or RAPD molecular markers, microsatellites, primer and other probes to give genetic fingerprints of Raphanus sativus m~teri~l. In one embodiment, the molecular markers are mapping to a similar region as that of OPC2. Although the molecular markers disclosed indicate the presence of the restorer gene, they do not indicate the absence of the restorer gene, nor determine the genotype of the restorer gene, as do the molecular markers of the present invention.

In the Delourme et al. (1994) study, six random amplified polymorphic DNA (RAPD)10 markers were used in a bulk segregant analysis. However, unlike the present invention of Sequenced Characterized Amplified Region (SCAR) markers, (i) the RAPD markers were generated by the amplification of genomic DNA using only a single primer of albi~
nucleotide sequence to drive the amplification reaction and, (ii) only the presence of the restorer gene (Rf) in plants cont~ining the Ogura CMS system of rapeseed could be determined. The Delourme et al. (1994) study did not teach one skilled in the art how to determine the specific genotype of the variety.

The Delourme et al (1994) study mentioned the possibility of SCAR markers from the Randomly Amplified Polymorphic DNA (RAPD) fragments which were found to be linked to the restorer locus and postulated that these markers could then be used to facilitate 20 breeding programs involving this gene. Delourme et al. (1994) did not teach one skilled in the art (i) how to use markers to determine the genotype of Brassica plants for Ogura CMS, (ii) how to develop SCAR markers from the 'rf' associated RAPD markers, (iii) how to develop and use a combination of SCAR markers in a single, multiplex amplification reaction to determine the specific genotype of a plant variety (rather than simply determining whether a plant variety has a fertility restorer locus or a fertility m~int~iner locus), or (iv) how to develop and use SCAR markers linked to the absence of the restorer locus, where the markers are derived from OPY5. Without SCAR markers from the 'rf' associated RAPD markers, one would be unable to differentiate reliably between a plant that is homozygous or heterozygous for the Rf gene.

, CA 02206673 1997-06-10 The we~knesses of current technology and methods for determining the genotype of the OGU
Rf restorer gene in Brassica are that:

i) isozyme analysis is ineffective, especially in spring Brassica napus, for determining OGU
Rf genotype because the analysis involves assaying a protein which may degrade or which may be developmentally regulated. Depending on the combination of alleles present in a particular plant, the banding pattern of the protein product may be too complex to interpret (as is the case for spring Brassica napus).

ii) RAPD markers are unreliable since reactions are performed at low annealing temperatures.
Slight variations in reaction conditions or components, for example, DNA quality, or concentration of primer or DNA, often results in total or partial failure of RAPD products to be amplified.

iii) RFLP markers are cumbersome in breeding programs because a large amount of DNA is required, a large number of samples is required for throughput, and a slow turn-around time is associated with RFLP analysis.

iv) lltili7ing SCAR markers linked to the restorer gene (Rf) allows one to distinguish varieties carrying the restorer gene from varieties not carrying the restorer gene. The markers will show up in individuals carrying the restorer gene but will be absent in individuals not carrying the restorer gene. However, this system:

. does not dirrelellLiate between homozygous 'RfRf and heterozygous 'Rfrf' restorer types. The marker will show up in both of these genotypes since they both contain the restorer gene; and ~ can provides false negatives. An individual in which the marker does not show up would score as 'rfrf~ indicating that it does not carry the restorer gene. In fact, the PCR reaction may have simply failed (for any one of a number of reasons) - this is a fairly common phenomenon in PCR based systems.

It is an object of the present invention to provide a simple, efficient and reliable method for identifying the genotype of Brassica germplasm for the Ogura restorer gene.

, CA 02206673 1997-06-10 It is another object of the present invention to identify markers that can be used in combination to determine the genotypic state of the gene in segregating populations, including both winter and spring Brassica napus populations, as well as in other Brassica species.

It is another object of the present invention to distinguish between homozygous and heterozygous restorer types by using a combination of standard PCR primers to amplify markers for the restorer gene 'Rf' and for the "absence of restorer gene" 'rf'.

It is a further object to provide a method to rapidly screen large numbers of samples of Brassica germplasm to determine the genotype for the OGU restorer gene, without having to 10 grow the plant to maturity (i.e. before the plant flowers).

Summary of the Invention This invention relates to a method for deterrnining the genotype of Brassica germplasm for the Ogura (Rf) restorer gene in a Brassica breeding program, comprising the steps of: (1) amplifying the Brassica germplasm for the Ogura (rf) restorer gene using at least one primer;
and (2) determining the genotype using at least two nucleic acid markers, wherein one marker indicates the presence of the OGU Rf gene (Rf) and the other marker indicates the absence of the OGU Rf gene (rf), and using a dot blot assay to detect the genotype. The Brassica germplasm may be a winter or spring Brassica napus, Brassica rapa or Brassica juncea The markers may be SCAR markers, RAPD markers or AFLP markers. The SCAR marker 20 indicating the absence of the OGU Rf gene (rf) may be Y5, a marker having partial homology to the Y5 sequence, or any other marker selected from a sequence of the RAPD band from which that marker is derived. The SCAR marker indicating the presence of the OGU Rf gene (rf) may be C2, N20, F10, a marker having partial homology to one of these sequences, or any other marker selected from a sequence of the RAPD band from which these markers are derived. The RAPD marker indicating the absence of the OGU Rf gene (rf) may be OPY5, OPG8, a marker having partial homology to one of these sequences, or any other marker selected from a sequence of the band i~rom which these markers are derived. The RAPD
marker indicating the presence of the OGU Rf gene (rf) may be OPC2, OPN20, OPH3, , CA 02206673 1997-06-10 OPF10, OPH15, a marker having partial homology to one of these sequences, or any other marker selected from a sequence of the band from which these markers are derived. The AFLP marker indicating the presence of the OGU Rf gene (rf) may be E36XM48AIII, E35XM62AV, E33XM47AI, E38XM60AI, a marker having partial homology to one of these sequences, or any other marker selected from a sequence of the band from which these markers are derived.

The amplification step in the method to which this invention relates may comprise at least one polymerase chain reaction (PCR) and primers for PCR, which may be primers described in Table 2 below or primers having partial homology to those primers. The method involving this amplification step may also comprise a multiplex polymerase chain reaction (PCR) using all of the primers in a single reaction.

The amplification step may also comprise using primer sets C2 and Y5 in combination in a multiplex polymerase chain reaction (PCR), and the step of determining the genotype may comprise:
(A) the steps of (1) dotting PCR reaction onto two identical membranes, (2) probing the membranes with probes, wherein one membrane is probed with a probe which hybridizes to a marker indicating the presence of Rf (which may be a marker of 677 base pairs) and the second membrane is probed with a probe which hybridizes to a marker indicating the absence of rf (which may be a marker of about 774 base pairs), and (3) comparing the two membranes to cletermine the genotype of Brassica germplasm for the Ogura restorer; or (B) the steps of (1) running out the products of PCR on an electrophoresis gel, wherein the reaction products may be (a) one band (about 677 bp), indicating a genotype of RfRf, (b) two bands (about 677 bp and about 774 bp), indicating a genotype of Rfrf, or (c) one band (about 774 bp), indicating a genotype of rfrf, and (3) reading the gel to determine the genotype of Brassica germplasm for the Ogura restorer.

The invention also includes a homozygous locus associated with the presence of the Ogura (RfRf) restorer gene (said locus mapping to at least one of the markers C2, N20, F10, a marker having partial homology to one of these sequences, or any other marker selected from a sequence of the RAPD band from which these markers are derived) or a homozygous locus , CA 02206673 1997-06-10 associated with the absence of the Ogura (rfrf) restorer gene (said locus mapping to one of the markers Y5, a marker having partial homology to that sequence or any other marker selected from a sequence of the RAPD band from which that marker is derived). A heterozygous locus associated with the presence of the Ogura (Rfrf) restorer gene and that maps to one of the aforementioned markers is also part of this invention.

A combination of markers for det~rmining the genotype of Brassica germplasm for the Ogura (Ri[~ restorer gene is part of this invention, said combination comprising at least one marker that indicates the presence of the Ogura (Rf) restorer gene and at least one marker that indicates the absence of the Ogura (rf) restorer gene. The combination of markers may also comprise a first set of nucleic acid markers, which may be C2, N20, F10, a marker having partial homology to one of these sequences, or any other marker selected from a sequence of the RAPD band from which these markers are derived, and a second set of nucleic acid markers, which may be Y5, a marker having partial homology to the Y5 sequences, or any other marker selected from a sequence of the RAPD band from which that marker is derived.

The invention also relates to SCAR markers: (1) for determining the presence of the Ogura (Rf) restorer gene in the genotype of Brassica germplasm, which may be the nucleic acid marker C2, N20, F10, a marker having partial homology to one of these sequences, or any other marker selected from a sequence of the RAPD band from which these markers are derived; or (2) for detçrmining the absence of the Ogura (Rf) restorer gene in the genotype of 20 Brassica germplasm, which may be the nucleic acid marker Y5, a marker having partial homology to that sequences, or any other marker selected from a sequence of the RAPD band from which that marker is derived.

A RAPD marker for deterrnining the presence of the Ogura (Rf) restorer gene in the genotype of Brassica germplasm, which may be the nucleic acid marker OPN20, OPH3, OPF10, OPH15, a marker having partial homology to one of these sequences, or any other marker selected from a sequence of the band from which these markers are derived, is also part of this invention.

The invention also includes a combination of primers for use in a polymerase chain reaction (PCR) to determine the genotype of Brassica germplasm for the Ogura (Rf) restorer gene, , CA 02206673 1997-06-10 which primers may be those described in Table 2 below, primers having at least partial homology to those primers, primers which anneal to a sequence from which those primers are derived, or primers derived from any portion of the RAPD fragment or amplified RAPD
fragment from which those primers are derived. A primer kit comprising the aforementioned primers is also part of this invention.

In addition, this invention includes a system for detçrminin~ the genotype of Brassica germplasm for the Ogura (Rf) restorer gene, comprising the steps of:
(a) amplifying the genotype in a single multiplex polymerase chain reaction (PCR), using primers described in Table 2 below, primers having at least partial homology to those primers, primers which anneal to a sequence from which those primers are derived, or primers derived from any portion of the RAPD fragment or amplified RAPD fragmentfrom which those primers are derived; and (b) determining the genotype using a combination of nucleic acid markers, wherein the markers may be a C2 marker indicating the presence of the OGU Rf gene (Rf), or markers having at least partial homology with a C2 marker, and a Y5 marker indicating the absence of the OGU Rf gene (rf), or markers having at least partial homology with a Y5 marker.

Brief Description of the Drawin~s In drawings which illustrate embodiments of this invention, FIG. 1 illustrates by way of exemplification the markers OPH15 and OPF10 linked to the Rf gene, but distal to other RAPD markers.

These and other objects and advantages of the invention will be al)par~illl to those skilled in the art from the following description and appended claims.

~ CA 02206673 1997-06-10 Detailed Description of the Invention This invention enables a breeder to distinguish between homozygous and heterozygous Ogura restorer types by using a combination of two SCAR markers, one linked to the restorer gene 'Rf' and one linked to 'rf', i.e., absence of restorer gene.

In one embodiment, two PCR reactions for each DNA sample are used, one with primers for the 'Rf' marker and one with primers for the 'rf' marker. By comparing the results of these two reactions it is possible to determine the genotype of individual plants, either 'Rf~, 'Rfrf', or 'rfrf'. Homozygous restorer types 'RfR~ would contain only the marker linked to 'Rf'. Heterozygous plants would contain the markers linked to both 'Rf' and 'rf'.
10 Homozygous non-restorer types would contain only the marker linked to 'rf'.

In another embodiment, a single PCR reaction for each DNA sample is used, by multiplexing both sets of primers in the single PCR reaction. The two sets of primers, when used together in a multiplex PCR reaction, allow for genotypic determination of the OGU Rf gene in individual plants of segregating OGU Rf populations.

These two sets of standard PCR primers were derived via bulked segregant analysis of a segregating OGU Rf population using RAPD PCR (Example 1). RAPD markers (bands) linked to the OGU Rf gene (Rf) were discovered. Other RAPD markers were found to be present only in the absence of OGU Rf (rf). The markers (bands) of interest were cloned and sequenced and the standard (SCAR) PCR primers were designed from these sequenced20 fragments.

One standard PCR primer pair, derived from a RAPD band generated using the Operon primer OPC2, gives a band which is linked to the OGU Rf gene (Rf). The second primer pair, derived from a RAPD band generated using the Operon primer OPY5, gives a band which is only present in the absence of the restorer gene, i.e., is linked to rf. The two sets of primer pairs dec~i~n~ted as C2 and Y5, derived from RAPD markers using OPC2-linked to Rf and using OPY5-linked to rf are described below in Table 2.

, CA 02206673 1997-06-10 When used in standard PCR reactions, primer pair C2 gives a band of 677 bp (base pairs) indicating the presence of the OGU Rf gene (Rf). Primer pair Y5 gives a band of 774 bp indicating the absence of the OGU Rf gene (rf). When used together in multiplex PCR
reactions the two sets of primer pairs allow for discrimin~tion between RfRf (homozygous restorer), Rfrf (heterozygous restorer) and rfrf (non-restorer/sterile) genotypes.

Bands observed in multiplex PCR using primer sets C2 and Y5 in combination are:

~ RfRf- one band (677 bp) is seen ~ Rfrf - two bands (677 bp and 774 bp) are seen ~ rfrf- one band (774 bp) is seen 10 A dot blot assay detection system based on the same two primer sets has also been developed, which works as follows:

~ set up multiplex PCR using primer sets C2 and Y5 in combination;

make two identical membranes by dotting PCR reaction onto membranes, ~ probe the membranes with radioactive or non-radioactive probes. One membrane is probed with the 677 bp fragment linked to Rf, the second membrane is probed with the 774 bp fragment associated with rf; and ~ compare the two membranes to determine OGU Rf genotype.

The system which is being utilized ~ ellLly to genotype OGU populations is a combination of two SCAR markers, developed from RAPD bands. One marker C2 is linked to the restorer 20 gene 'Rf' (developed using RAPD primer OPC2) and the other marker Y5 is linked to the absence of the restorer gene 'rf' (developed using RAPD primer OPY5). The combination of these two markers allows for the determination of OGU Rf genotype in individual plants, either 'RfRf', 'Rfrf', or 'rfrf'.

For each of the two SCAR markers there is a set of two standard PCR primers, one forward and one reverse, which were developed to amplify only the bands of interest (Table 2). One set of primers (C2) amplifies a band associated with the restorer gene, 'Rf', while the second set of primers (Y5) amplifies a band associated with the absence of the restorer gene 'rf.
These two sets of primers were developed so that they can both be utilized in the same PCR
reaction. By using the two sets of primers in the same PCR reaction the problem of false negatives is elimin~ted, i.e. a failed PCR reaction will be obvious because no detectable result will be obtained by electrophoresis or by probing, either with radioactive or non-radioactive probes.

By using a multiplex format of PCR reactions, in this case two primer pairs in one reaction, at least one product should be formed. Absence of both products would signify failure of the 10 PCR reaction. If the 'Rf' associated band is the only band amplified the individual plant is homozygous for the restorer gene (RfRf), if both the 'Rf' and 'rf' associated bands are amplified then the individual is heterozygous for the restorer gene (Rfrf). If only the 'rf' associated band is amplified then the individual is a homozygous non-restorer type (rfrf). If there are no bands amplified then the PCR reaction has failed for that sample and the reaction can be repeated. This procedure elimin~tes false negatives.

There are two main methods which can be utilized to visualize the results of the PCR
amplifications. One method is to run the reaction products on an agarose gel and to stain with ethidium bromide to detect bands. While this method is adequate for small numbers of samples it is not conducive to large scale analysis of hundreds of samples. The pouring, 20 loading, running of gels and staining/detection is very time consuming, labour intensive and costly.

This invention provides a dot blot system which allows for the rapid analysis of a large number of samples. In combination with a high throughput DNA extraction system, the dot blot system allows for the simultaneous testing of over 400 samples. With automation, the number of samples tested could be increased tremendously.

The dot blot assay involves "dotting" PCR products on duplicate blots and probing with the respective cloned RAPD band (discussed in more detail below).

, CA 02206673 1997-06-10 The following Examples are presented as specific illustrations of the present invention. It should be understood, however, that the invention is not limited to the specific details set forth in the Examples.

Example 1 - Screenin~ of Population 94CWN2133 to Identify RAPD Markers Linked to the Restorer Gene and~ to Determine Which of These Markers Most Accurately Reflected the Genotype of the Plants From seeds of the F2 population 94CWN2133, 369 plants were grown in greenhouse.
Isozyme analysis was conducted on each plant using leaf tissue. In addition, plants were scored for flowering phenotype, either fertile or sterile. Of these 369 plants, 175 were selected to be utilized in the linkage analysis to develop markers for the OGU restorer gene (Rf). Other plants were excluded from the study due to inconclusive isozyme score or because plants did not flower or were too late flowering. It was from these 175 plants that the two sets of bulk DNA were formed.

From bulk segregant analysis (see Materials and Methods, RAPD analysis) of the 500 RAPD
primers screened, five [OPC2, OPH3, OPN20, OPH15, and OPF10] revealed amplification products that were present in fertile bulks and parent NW3002 (Rfl~f) and absent in sterile bulks, while four primers [OPG8, OPF6, OPY5, and OPG2] revealed amplification products that were present in the sterile bulks and parental sample, Bristol (rfrf), but were absent in the 20 fertile bulks (Table 1).

, CA 02206673 1997-06-10 Table 1. RAPD markers identified as bein~ linked to OGU Rf (restored plants) or absence of OGU Rf (sterile plants) RAPD presence in presence in RAPD SCAR marker markers restored sterile plants Band size developed?
plants (bp) OPC2 + - 1139 yes OPN20 + - 1782 yes OPY5 - + 839 yes OPH15 + - 1345 yes OPF10 + - 883 yes OPH3 + - 613 yes OPG2 - + 1350* no OPG8 - + 749 yes OPF6 - + 1240* no * bands were not sequenced - size estimated from gels Individual F2 plants from population 94CWN2133, including those used to form bulks, were screened using the 9 RAPD markers which had been identified by bulked segregant analysis.
The five markers associated with the fertile bulks co-segregated almost perfectly in the 175 individuals, i.e., in any individual either all five bands were present or all were absent. The same was observed for the four primers that were associated with the absence of the restorer 10 gene. This indicated that recombination between these markers and the restorer gene is relatively rare.

Linkage analysis performed by using Mapmaker 3.0 (Whitehead Institute for Biomedical Research) indicated that OPC2, OPN20, OPH3 (markers associated with the Rf gene) and OPY5, OPG8, OPG2 and OPF6 (markers associated with the absence of the Rf gene) mapped the same locus, 1.4 cM from the Rf gene. The PGI II locus was mapped 1.5 cM on the opposite side of the restorer gene from the RAPD markers. Two other markers, OPH15 and OPF10, were also linked to the Rf gene but were distal to the other RAPD markers, 0.6 cM
and 1.7 cM, respectively (Fig. 1).

RAPD markers linked to the absence of the restorer gene were further tested on a set of ten regular canola varieties, i.e., normal cytoplasm and lacking the OGU Rf gene. Five of these 10 varieties were winter B. napus and five were spring B. napus. Since none of the ten varieties contained the restorer gene it was expected that the four markers associated with the absence of the restorer gene should be present in all of these varieties. Two of the markers, OPG2 and OPY5, were found to be present in all ten varieties while the other two markers, OPF6 and OPG8, were inconsistent (and thus elimin~ted from further testing). It was concluded that OPY5 and OPG2 were more accurate for det~rmining the absence of the restorer gene.

Example 2 - Conversion of RAPD Marker Bands to SCAR Markers and use of SCAR
Markers to Determine Genotype RAPD markers are somewhat unreliable and are not easily adapted to systems involving routine genotypic det~rmin~tions. For this reason several SCAR markers, and primers for 20 these markers, were developed from the RAPD markers. The selective use of SCAR primers in combination was used to determine plant genotype (RfRf, Rfrf, or rfrf) through detection of PCR products by gel electrophoresis or dot blot analysis.

Two SCAR markers, C2 and Y5, were found to work well together in combination.

In order to utilize the SCAR markers DNA was first extracted from the samples to be tested.
Any DNA extraction system could be used, as is known to those skilled in the art. After DNA
extraction, PCR reactions were set up using two sets of primer pairs in the same reaction.
One set of primer pairs, set C2, amplified a 677 bp band associated with the restorer gene 'Rf'; the second set of primer pairs, set Y5, amplified a 774 bp band associated with the absence of the restorer gene 'rf' .

Table 2 shows the nucleotide sequence of the primer pairs involved. The PCR reaction mixture and techniques used for DNA amplification and visl~li7~tion of the results were those described below in the Materials and Methods section.

Table 2 Nucleotide sequence of primers used to determine OGU Rf genotype Primer Sequence forward reverse forward reverse Example 3 - Vis~ tion of PCR Results by Gel Electrophoresis on a 1.4% Agarose Gel PCR products were loaded onto 1.4% agarose gels, either lX TAE (0.04M Tris-acetate;
0.001M EDTA, pH 8.0) or 0.5X TBE (0.045M Tris-borate; 0.001M EDTA), cont~inin~ 0.4 ~g/ml ethidium bromide. Gels were run in the corresponding buffer for 4 hours at 80V and were then viewed under UV light. Photographs were taken to document the gels and sample genotypes were determined from these photos. For homozygous restorer individuals one band of approximately 677 bp was seen. For heterozygous individuals two bands were seen, one of about 677 bp and a second of about 774 bp. For individuals which did not contain any copies of the restorer gene one band of approximately 774 bp was seen.

Example 4 - Development of Hi~h Throu~hput Dot Blot Detection System A high throughput dot blot detection system was developed. For blot analysis, the procedure described below in the Materials and Methods section was followed.

Example 5 - Determination of OGU Rf Genotype Usin~ the Dot Blot Detection System By comparing the autorads formed by the sets of identical membranes it was possible to correctly determine the OGU Rf genotype of the samples. For example, for a given sample on the membrane, if it showed a dot when probed by C2 but the duplicate sample on the sister membrane did not show a dot when probed with Y5 then the sample was homozygous restorer (RfRf). If one membrane showed a dot when probed with C2 and its sister membrane 10 also showed a dot when probed with Y5 then the sample was heterozygous for the restorer gene (Rfrf). If one membrane showed a dot when probed with Y5 but its sister membrane did not show a dot when probed with C2 then the sample was homozygous non-restorer (rfrf). If neither membrane showed a dot then the PCR reaction had failed.

Example 6 - Testinp of SCAR Markers Developed From RAPD Markers In order to test the validity of the SCAR markers in terms of linkage to Rf gene, as well as to the PGI II locus, a second sample of 137 F2 plants developed from population 94CWN2133 were screened with the SCAR primer sets C2 and Y5. Results are summarized in Table 3:

Table 3. Numbers of plants in which SCAR markers were present (+) or absent (-) in restored (RR or Rr) and unrestored (rr) plants.

SCAR marker Fertile, restored plants Sterile, unrestored plants (106 plants) (36 plants) + +

20 a - data was unavailable for five plants These markers were further tested using a number of segregating populations from the spring and winter napus breeding programs. Table below illustrates the agreement of marker scores with PGI II and/or flowering scores (hence presence of Rf gene). For the purposes of illustration data for 10 plants per population are presented in the table, populations consisted of many more individuals and additional populations, other than those presented in the table, have also been tested. Test crosses were made on individuals from several populations in order to determine the accuracy of the SCAR markers. In almost all cases the expected segregation ratios were observed.

Table 4. Confirm~tion of markers Line number Plant Score Score OGU Rf PGI II Flowering number using C2 using Y5 Genotype isozyme score (1~ (rf~ data primer primer set set 96SN1049 1 + + heterozygous *Spring fertile napus *sterile plants 4 + + heterozygous PGI II fertile had been culled + + heterozygous scores not fertile - tested using available 6 + + heterozygous fertile C2 and Y5 primers 8 + + heterozygous fertile separately, run 9 + + heterozygous fertile on gels + + heterozygous fertile 11 + - homozygous fertile 13 + + heterozygous fertile 14 + - homozygous fertile 96FNW1348-1 1 - + sterile rr flowering score - tested using 2 + + heterozygous Rr unavailable dot blot protocol 3 + + heterozygous Rr 4 - + sterile rr + - homozygous RR

6 + + heteroz,vgous Rr 7 - + sterile rr 8 + + heterozygous Rr 9 - + sterile rr + + heterozygous Rr 94CWN2133 1 - + sterile rr sterile *second half of 2 + + heterozygous Rr fertile population 3 - + sterile rr sterile - tested using 4 + + heterozygous Rr fertile dot blot protocol + + heterozygous Rr fertile 6 + + heterozygous Rr fertile 7 + + heterozygous Rr fertile 8 - + sterile rr sterile 9 + + heterozygous Rr fertile + + heterozygous Rr fertile Example 7 - Identification of AFLP Markers Experiments to identify AFLP markers linked both to the OGU Rf restorer gene and to the absence of the restorer gene used bulked DNA samples derived from the second section of population 94CWN2133. Four bulks were used, two consisted of ten individuals each of homozygous restored plants while the other two bulks consisted of ten sterile individuals each. Screening of the population 94CWN2133 to identify AFLP markers linked to the restorer gene is done using techniques known to those skilled in the art. To date seventeen putative AFLP markers linked to the restorer gene have been identified along with eighteen possible markers linked to the absence of the restorer gene. Four of the AFLP markers that have been developed are E336XM48AIII, E35XM62AV, E33XM47AI and E38XM60AI.
SCAR markers, and primers for these markers, are developed from these AFLP markers using techniques known to those skilled in the art.

The AFLP method of amplification was conducted in accordance with the technique described in the Materials and Methods section of Vos, et al (1995).

Materials and Methods RAPD Markers Plant material: Plant material used was the winter B. napus F2 OGU restorer population 94CWN2133, and its parents NW3002 and Bristol, which were obtained from the winter canola breeding group at Pioneer Hi-Bred. Plants had been grown to the four leaf stage, 20 vernalized for 12 weeks at 4~C, and then grown in the greenhouse. Plants from this population were screened for the presence of the restorer gene using the Pgi-2 isozyme marker (Delourme and Eber, 1992). Isozyme analysis was done at Pioneer's Johnston, Iowa, electrophoresis laboratory. Based on isozyme pattern plants from this population were classified according to genotype, either homozygous restorer (E~, heterozygous restorer (Rfrf), or unrestored (rfrf). Individual plants were also scored as male fertile or sterile based on flower phenotype, i.e., whether pollenlanther were normal or non-functional.

DNA Extraction: DNA was extracted from lyophilized ground leaf tissue using a modified CTAB extraction protocol (Doyle and Doyle, 1990). For each sample, 0.3g of ground leaf tissue was placed into tubes co~ il-g 10ml of CTAB buffer (0.lM Tris, 0.7M NaCl, 10mM
EDTA, 27mM CTAB, 1% B-mercapotethanol). Tubes were incubated at 60~C for 1 hour.Chloroform:isoamyl alcohol, 24:1, (5 ml) was added and the blended suspensions were centrifuged at 6,000 rpm for 5 min in a Beckman J2-HS centrifuge. Supern~t~nt~ were recovered and a second chloroform/isoamyl extraction was carried out. Supern:~t~nt~ were then transferred to new tubes and 8ml of isopropanol was added to precipitate the DNA.
After centrifuging for S min. at 5000 rpm the supern~t~nt~ were poured off and 4 mls of 76%
EtOH, 0.2M NaOAc was added. This solution was left for 20 min. before being poured off.
The pellets were washed in 76% EtOH, 1 OmM NH40Ac and then resuspended in 9 mls of TE
10 buffer (pH 8.0). Once DNA was resuspended an additional two chloroform/isoamyl extractions were carried out as needed. After the last chloroform/isoamyl extraction 7.0 mls of 20% PEG, 2.5M NaCl was added to the supern~t~nt~, the two solutions were mixed and tubes were placed on ice for 1 hour. After 1 hour tubes were centrifuged at 8,000 rpm for 10 min. at 4~C. Supern~t~nt~ were discarded and pellets were washed in 70% chilled ethanol prior to being resuspended in 0.5 ml TE cont~ining 50 ug/ml RNAse A. DNA concentrations were determined by fluorescence in the presence of bisbenzimide (Hoescht dye 33258) using a TKO 100 fluorometer (Hoefer).

RAPD analysis: The Bulk Segregant Analysis (BSA) method (Michelmore et al. 1991) was used for the identification of putative markers linked to the Ogura Rf gene.

20 DNA aliquots of 2 ug each were collected from homozygous fertile individuals and from homozygous sterile individuals. These aliquots were combined to form four separate DNA
pools, two pools of homozygous fertile individuals (Rfl~f) and two pools of homozygous sterile (rfrf) individuals. The number of individuals in each bulk was either 17 or 18.

The DNA "bulks" along with DNA from parental lines, NW3002 (homozygous restorer) and Bristol (homozygous non-restorer), were diluted to 25 ng/~ll. RAPD markers were tested for their ability to detect polymorphism between the two types of pooled DNA "bulks", as well as, between the two parents. Primer kits A through Y from Operon Technologies, consisting of a total of 500 oligonucleotides, were used as primers. One ~l of the diluted DNA was used as template in a final reaction volume of 15,u1. The PCR reaction solution contained lX PCR

Buffer II (Perkin Elmer), 2.5 mM MgCl2, 200 ~lM each of dATP, dCTP, dGTP, and dTTP, 0.4 IlM Operon primer, 0.375 Units - of Taq polymerase (Perkin Elmer). DNA amplification was performed in a Perkin Elmer Geneamp PCR System 9600 thermocycler programmed as follows: 2 min. at 94~C for 1 cycle, 5 sec. at 94~C, 30 sec. at 36~C, and 1 min. at 72~C for 35 cycles; 5 min. at 72~C for 1 cycle; hold at 4~C. Amplification products were separated by gel electrophoresis on 1.4% agarose gels and visualized by ethidium bromide st:~ining. Primers detecting polymorphisms were then tested on all individuals from population 94CWN2133 to determine which primers gave polymorphic bands linked either to the presence or absence of the restorer gene. Gels for this analysis were either 1.4% agarose or 2% agarose (1%
10 Metaphore and 1% NuSieve 3: 1; FMC BioProducts).

Conversion to SCAR Primers: Bands which showed linkage to either the restorer gene or the absence of the restorer gene were cloned using the TA Cloning Kit (Invitrogen) and were sequenced. Based on the sequence of the cloned bands oligonucleotides were designed such that the fragment of interest could be amplified by PCR, using more reproducible reaction conditions. PCR products or marker bands developed this way are referred to as Sequence Characterized Amplification Regions or SCARS. The primers were designed with similar annealing temperatures so that they might be used together in multiplex PCR reactions.
These SCAR primers were tested against individuals from the 94CWN2133 segregating population to determine if they accurately reflected the genotype of the plants as 20 characterized by the RAPD primers.

The PCR reaction mixture (20.0 ~l) for testing the SCAR marker primers contained 25 ng genomic DNA, lX PCR Buffer II (Perkin Elmer), 1.5 mM MgCl2 250 ~M each of dATP, dCTP, dGTP, and dTTP, 22.5 pmoles each of forward and reverse primers, 0.5 U of Taq polymerase (Perkin Elmer). DNA amplification was performed in a Perkin Elmer Geneamp PCR System 9600 thermocycler programmed as follows: 2 min. at 94~C for 1 cycle; 1 min.
at 94~C, 1 min. at 60~C, and 1 min. at 72~C for 35 cycles; 5 min. at 72~C for 1 cycle; hold at 4~C. Amplification products were visualized on 1.4% agarose gels.

From a total of nine SCAR markers developed, one linked to the restorer gene (marker C2) and a second linked to the absence of the restorer gene (marker Y5), gave very reproducible and clear bands when tested together in multiplex PCR reactions. SCAR markers H3 and H 15 (linked to the restorer gene) and G8 (linked to the absence of the restorer gene) did not give reproducible, clear bands when used in multiplex PCR reactions. The PCR reaction mixture (20.0 ul) which was found to give the best results for multiplexing these two primer sets was:
10-25 ng genomic DNA, lX PCR Buffer II (Perkin Elmer), 1.5 mM MgCl2 250 ,uM each of dATP, dCTP, dGTP, and dTTP, 19.5 pmoles C2 primers (forward and reverse in equal mix), 25.5 pmoles Y5 primers (forward and reverse in equal mix), 0.5 U of Taq polymerase (Perkin Elmer). DNA amplification was performed in a Perkin Elmer Geneamp PCR System 9600 thermocycler programmed as follows: 2 min. at 94~C for 1 cycle; 1 min. at 94~C, 1 min.
at 60~C, and 1 min. at 72~C for 35 cycles, 5 min. at 72~C for 1 cycle, hold at 4~C. After PCR, results were visualized by gel electrophoresis on a 1.4% agarose gel or, results were visualized by means of a dot blot assay (as described earlier).

Development of a Dot Blot System: A dot blot detection system for vis-l~li7ing the results of multiplex PCR reactions using the two SCAR primer sets C2 and Y5 was developed. This same system could be utilized using any other combination of primers which lend themselves to multiplex PCR.

For dot blot analysis, 5 ~l of PCR product from the multiplex PCR reactions were dotted onto each of two HybondTM N+ nylon membranes. The membranes were then removed and placed DNA side up on blotting pad saturated with 0.6M NaCl, 0.4M NaOH for 2 min, then transferred to blotting pad saturated with 0.5M Tris (pH 7.5), 1.5M NaCl for 10 min.
Samples were bound to the membranes by baking for 30 min. to 1 hour at 80~C. Of the two identical membranes, one was probed with the 'Rf' associated probe (C2) and the other probed with the 'rf' associated probe (Y5).

Prehybridization was done in a HybaidTM oven at 65~C for 2 hours to overnight.
Prehybridization/hybridization solution consisted of 1% SDS, 10% dextran sulphate sodium salt 500K, 5X SSPE (0.9M NaCl, 50mM Na2HPO4.7H2O, 5mM EDTA, pH 7.7), 10X
Denhardts (0.2% BSA fraction V, 0.2% ficoll 400K, 0.2% polyvinylpyrolidone 360K). DNA
probes used for hybridization were fragments amplified from the plasmid clones obtained from cloning of original RAPD marker bands, using the same primer sets (C2 and Y5).

Amplified probes were gel purified and isolated using a GENECLEAN kit (BIO 101, Inc.).
During prehybridization, probes were labeled with 32p using the Amersham multiprime DNA
labeling system (RPN.1600Z) and purified using the protocols for NICK~ columns (Pharmacia Biotech). After prehybridization, labeled probes were added to the prehybridization solution and membranes were hybridized for 1 hour to overnight. From the sets of two identical membranes, one membrane was probed with the C2 probe (Rf associated) while its sister membrane was probed with the Y5 probe which is associated with the absence of the restorer gene. After hybridization membranes were washed once at 60~C
in 2 X SSC, 0.1% SDS for 20 min followed by two washes in 0.1 X SSC, 0.1% SDS for 30 10 min. at 60~C. Dot blots were exposed to Amersham HyperfilmTM-MP for periods varying from 4 hours to overnight.

The OGU Rf genotype of the samples was determined by comparing the autorads formed by the sets of identical membranes. For example, for a given sample on the membrane, if it showed a dot when probed by C2 but the duplicate sample on the sister membrane did not show a dot when probed with Y5 then the sample is homozygous restorer (Rfl~f). If one membrane showed a dot when probed with C2 and its sister membrane also showed a dot when probed with Y5 then the sample is heterozygous for the restorer gene (Rfrf~. If one membrane showed a dot when probed with Y5 but its sister membrane did not show a dot when probed with C2 then the sample is homozygous non-restorer (rfrf). If neither 20 membrane showed a dot then the PCR reaction had failed.

AFLP Markers Plant material: Plant material used for AFLP analysis was a winter B. napus population which was derived from the winter B. napus OGU population, 94CWN2133, used for RAPD
analysis. The population used for AFLP analysis was derived by crossing homozygous restorer plants from population 94CWN2133 with sterile plants from the same population.
The resulting heterozygous offspring were then selfed resulting in a population segregating for the restorer gene. This population is referred to as section 2 of population 94CWN2133.
Plants from section 2 of population 94CWN2133 were vern~ .l, screened with the Pgi-2 isozyme marker, and scored for flowering phenotype following the same protocols used for the original 94CWN2133 population. The second section of population 94CWN2133 consisted of 151 individuals. Several of these individuals were elimin~ted due to lack of flowering or PGI II scores, making the final population 137 individuals.

DNA Extraction: DNA was extracted following the same protocol used for the original 94CWN2133 population, a modified CTAB extraction protocol (Doyle and Doyle, 1990).
For section two of population 94CWN2133 0.1g of ground, lyophilized leaf tissue was used for extraction. Following the final chloroform/isoamyl extraction DNA was precipitated with 1/10 volume of 3M NaOAc and 2/3 volume propanol instead of with 20%PEG, 2.5M NaCl .
Tubes were inverted to mix and then centrifuged at 8,000 rpm for 10min. at 4~C prior to washing with 70% ethanol and resuspending in TE.

AFLP analysis: As with the RAPD analysis, Bulk Segregant Analysis (Michelmore et al.
1991) was used for the identification of putative AFLP markers linked to the Ogura Rf gene.
To form bulked DNA samples with concentrations of 50 ng/ul, DNA aliquots of 5 ng each were collected from homozygous fertile individuals and from homozygous sterile individuals.
The 5 ng aliquots were combined to form two homozygous fertile (RfRf) pools and two homozygous sterile (rfrf) pools. The number of individuals in each pool/bulk was 10.

20 DNA bulks and parental samples, NW3002 and Bristol, were screened using the AFLP
protocol (Vos et al. 1995). DNA was digested using EcoRI and MseI enzymes and first step amplification was performed using AFLP primers having a single selective nucleotide. For first step amplification 50ul PCR reactions were set up cont~inin~ 75 ng of both AFLP
primers, 5ul of DNA template (1:10 dilution of digestion/ligation mix), lU Taq polymerase (Perkin Elmer), lX PCR buffer (Perkin Elmer) and 1.25 mM of all four dNTPs. After PCR, first step amplification reaction mixes were diluted 20-fold before being used as templates for the second amplification reaction. Reactions for labeling of EcoRI primers for the second amplification were performed in lX One-Phor-All buffer PLUS (Pharmacia Biotech).

30 After the second amplification an equal volume of formamide dye (98% formamide, 10mM
EDTA pH 8.0, and bromophenol blue and xylene cyanol as kacking dyes) was added and the resulting mixes were then heated for 3min. at 90~C and cooled on ice. Each sample (3-5ul depending on combs used) was loaded on a 4.5% denaturing polyacrylamide gel. Gels were prepared using a solution cont:~ining 4.5% acrylamide/Bis Solution 19:1 (Bio-RadLaboratories), 7.5 M urea, 50mM Tris, 50mM Boric acid, lmM EDTA. A volume of 120 ml of gel solution was degassed in a desiccator attached to a vacuum source for 20 min. To this solution 100 ul TEMED and 500 ul 10% APS were added and gels were cast using a Sequi-Gen~GT Nucleic Acid Electrophoresis Cell (Bio-Rad Laboratories). The bottom buffer tray contained 1.25 M sodium acetate, lOOmM Tris, lOOmM Boric acid, 2mM EDTA and, lOOmM Tris, lOOmM Boric acid, 2mM EDTA was used as a running buffer. Electrophoresis was performed at 120W, 45~C for approximately 2 hours. After electrophoresis gels were dried for 1 hr at 80~C in a dual temperature slab gel dryer (Bio-Rad Laboratories) and were exposed to film (HyperfilmTM-MP, Amersham) for 4-7 days.

Polymorphic bands were identified as being putative markers for the Ogu restorer gene.
These putative markers are then screened against the individuals from section 2 of population 94CWN2133 to determine how closely linked the markers are to the restorer gene.

Conversion of AFLP Markers to SCAR Markers: AFLP bands showing linkage to the restorer gene are cloned and sequenced. Based on the sequence information SCAR primers 20 are developed to amplify the bands of interest. These SCAR primers are tested on section two of population 94CNW2133 to determine if they accurately reflect the genotype of the individuals. The protocol used for testing of SCAR primers is the same as that used to test SCAR primers developed from RAPD bands. These primers are designed with similar annealing temperatures to those which were developed from RAPD bands so that the primers might be used together in multiplex PCR reactions.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was 30 specifically and individually indicated to be incorporated by reference in its entirety.

The present invention has been described in detail and with particular reference to the p~efelled embodiments, however, it will be understood by one having ordinary skill in the art that changes can be made thereto without departing from the spirit and scope thereof.

References Bannerot, et al. (1974) Cytoplasmic male sterility transfer from Raphanus to Brassica, Proc.
EUCAR-PIA Meet. Crop Sect. Cruciferae, vol. 25, pp. 52-54.

Bonhomme, et al. (1991) A 2.5 kb NcoI fragment of Ogura radish mitochondrial DNA is correlated with cytoplasmic male-sterility in Brassica hybrids, Curr. Genet., vol. 19, pp. 121-127.

Bonhomme, et al. (1992) Sequence and transcript analysis of the Nco2.5 Ogura-specific fragment correlated with cytoplasmic male sterility in Brassica hybrids, Mol. Gen. Genef., vol. 235, pp. 340-348.

10 Delourme and Eber (1992) Linkage between an isozyme marker and a restorer gene in radish cytoplasmic male sterility of rapeseed (Brassica napus L.), Theor. Appl. Genet., vol. 85, pp.
222-228.

Delourme, et al. (1991) Radish cytoplasmic male sterility in rapeseed: breeding restorer lines with a good female fertility, Proc. 8th Int. Rapeseed Conf., vol. 5, pp. 1056.

Delourme, et al. (1994) Identification of RAPD markers linked to a fertility restorer gene for the Ogura radish cytoplasmic male sterility of rapeseed ( Brassica napus L.), Theor. Appl.
Genet., vol. 88, pp. 741-748.

Doyle and Doyle (1990) Isolation of plant DNA from fresh tissue, Focus, vol. 12, pp. 13-15.

Grant and Beversdorf (1985) Heterosis and combining ability estimates in spring-planted 20 oilseed rape (Brassica napus L.) Can. J: of Genet. and Cyt., vol. 26, pp. 472-478.

Heyn (1976) Transfer of restorer genes from Raphanus to cytoplasmic male-sterile Brassica napus, Cruciferae Newslett, vol. 1, pp. 15-16.

Hutcheson, et al. (1981) Performance of a naturally occurring subspecies hybrid in B.
campestris L. var oleifera Metzg, Can. J: Plant Sci., vol. 61, pp. 895-900.

Michelmore, et al. (1991) Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genome regions by using segregating populations, Proc. Natl. Acad. Sci. USA, vol. 88, pp. 9828-9832.

Ogura (1968) Studies on the new male sterility in Japanese radish, with special references on the utilization of this sterility towards the practical raising of hybrid seeds. Mem. Fac.. Agri.
Kagoshima Univ., vol. 6, pp. 39-78.

Pellan-Delourme and Renard (1988) Cytoplasmic male sterility in rapeseed (Brassica napus L.): Female fertility of restored rapeseed with "Ogura" and cybrids cytoplasms. Genome, vol.
30, pp. 234-238.

10 Pelletier, et al. (1983) Intergeneric cytoplasmic hybridization in Cruciferae by protoplast fusion. Mol. Gen. Genet, vol. 191, pp. 244-250.

Pelletier et al. (1987) Molecular, phenotypic and genetic characterization of mitochondrial recombinants in rapeseed. Proc. 7th Int. Rapeseed ConJ:, Pozan, Poland, pp. 113-118.

Sernyk and Stefansson (1983) Heterosis in summer rape (Brassica napus L.) Can. ~ Plant Sci., vol. 63, pp. 407-413.

Vos, et al. (1995) AFLP: a new technique for DNA fing~ inLillg, Nucleic Acids Res., vol.
23, No. 21, pp. 4407-4414.

SEQUENCE LISTING

(1) GENERAL INFORMATION:
ti) APPLICANT: Pioneer Hi-Bred International, Inc.
(ii) TITLE OF INVENTION: USE OF MOLECULAR MARKERS FOR GENOTYPE
DETERMINATION OF THE OGURA Rf GENE IN BRASSICA NAPUS
(iii) NUMBER OF SEQUENCES: 15 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Deeth Williams Wall (B) STREET: 150 York Street, Suite 400 (C) CITY: Toronto (D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP: M5H 3S5 (V) COM~U'1'~ READABLE FORM:
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McMahon, Eileen M.
(C) REFERENCE/DOCKET NUMBER: 4245 0037 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 416-941-9440 (B) TELEFAX: 416-941-9443 (2) INFORMATION FOR SEQ ID NO:1:
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(A) NAME/KEY: -(B) LOCATION: 1..1782 (D) OTHER INFORMATION: /note= "OPN20"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

ACAACGGTAC TTATGAATTT CTATTTACTA GGATAATGTA CCTTGTCGTT TT~ ll 480 ATGGTCCTGC CCAGGCTCCA AGTTTCAGAC ATGGAACAGG TTTGATATAT ~ ATGG 840 CCTTAAAGCA A~'l"l"l"l"l'CCG ATGGTGGTTA TGATAAAAAC TCAAGAGGGG ATGACTGCAA 1080 TTGTCTCTAG TTTTAGTTCT ACAGTATTTC GCTTATTAAC TGGTCTTTCT G~l"l"l"l"l"lCTT 1740 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 613 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: -(B) MAP POSITION: -(C) UNITS: -(ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1..613 (D) OTHER INFORMATION: /note= "OPH3"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

ATATATCGAT CAATTTCAGA T~ll~lllGTC CCTTGTGTAT TCCAACATTT TGTATATGGT 600 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 883 base pairs (B) TYPE ! nu~lei~ a~id (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: -(B) MAP POSITION: -(C) UNITS: -(ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1..883 (D) OTHER INFORMATION: /note= "OPF10"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GTTTGGTTTT GTTTAGAAGG A~lllllAAT CCTTTGGGGT AAAGAGAAAT TAGGGTAATT 720 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1345 base pairs (B) TYPE- nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: -(B) MAP POSITION: -(C) UNITS: -(ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1..1345 (D) OTHER INFORMATION: /note= "OPH15"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

TAAA~'l"l"l"l"l' TTTATAATGT TATAATATAT TTGCAGGTTA TCAGTTTGAC TATGTATTTG 540 ~ AGA GGCATATGCT AGAAGAAATG GCTCAGGAAG CGGTGCGGTT CAAGCTGATA 960 TGAGTCATCT TAA'l"l"l"l"l"l"l' CTTTATCAAA ATCTTGTCAT AAATGCTCTT GAAATATTAC 1080 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: -(B) MAP POSITION: -(C) UNITS: -(ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1..419 (D) OTHER INFORMATION: /note= "E36XM48AIII"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 259 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: -(B) MAP POSITION: -(C) UNITS: -(ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1..259 (D) OTHER INFORMATION: /note= "E35XM62AV"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 143 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: -(B) MAP POSITION: -(C) UNITS: -(ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1..143 (D) OTHER INFORMATION: /note= "E33XM47A1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

GTATATGAAA ACGCACGTAT GTTTTATA~A TAAAATCCCT TACTTTATAA CAAAACCTGT 120 (2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: -(B) MAP POSITION: -(C) UNITS: -(ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1..130 (D) OTHER INFORMATION: /note= "E38XM6OAl"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Claims (27)

1. A method for determining the genotype of Brassica germplasm for the Ogura (Rf) restorer gene in a Brassica breeding program, comprising the steps of:
~ amplifying the Brassica germplasm for the Ogura (rf) restorer gene using at least one primer, and ~ determining the genotype using at least two nucleic acid markers, wherein one marker indicates the presence of the OGU Rf gene (Rf) and the other marker indicates the absence of the OGU Rf gene (rf).

2. The method of claim 1, wherein the markers are selected from a group consisting of SCAR markers, RAPD markers and AFLP markers.

3. The method of claim 1, wherein the marker indicating the absence of the OGU Rf gene (rf) is a SCAR marker selected from a group consisting of ~ Y5, represented by SEQ ID NO: 1, ~ a marker having partial homology to that sequence, and ~ any other marker selected from a sequence of the RAPD band from which that marker is derived.

4. The method of claim 1, wherein the marker indicating the presence of the OGU Rf gene (rf) is a SCAR marker selected from a group consisting of ~ C2, represented by SEQ ID NO: 2, ~ N20, represented by SEQ ID NO: 3, ~ F10, represented by SEQ ID NO: 4, ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the RAPD band from which these markers are derived.

5. The method of claim 1, wherein the markers are SCAR markers and the marker indicating the presence of the OGU Rf gene (Rf) is C2 having the sequence set out in SEQ ID NO 2, or a marker having partial homology to C2, and the marker indicating the absence of the OGU Rf gene (rf) is Y5 having the sequence set out in SEQ ID NO
1, or a marker having partial homology to Y5.

6. The method of claim 1, wherein the marker indicating the absence of the OGU Rf gene (rf) is a RAPD marker selected from a group consisting of ~ OPY5, represented by SEQ ID NO: 5, ~ OPG8, represented by SEQ ID NO: 6, ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the band from which these markers are derived.

7. The method of claim 1, wherein the marker indicating the presence of the OGU Rf gene (rf) is a RAPD marker selected from a group consisting of ~ OPC2, represented by SEQ ID NO: 7, ~ OPN20, represented by SEQ ID NO: 8, ~ OPH3, represented by SEQ ID NO: 9, ~ OPF10, represented by SEQ ID NO: 10, ~ OPH15, represented by SEQ ID NO: 11, ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the band from which these markers are derived.

8. The method of claim 1, wherein the marker indicating the presence of the OGU Rf gene (rf) is a AFLP marker selected from a group consisting of ~ E36XM48AIII, represented by SEQ ID NO: 12, ~ E35XM62AV, represented by SEQ ID NO: 13, ~ E33XM47AI, represented by SEQ ID NO: 14, ~ E38XM60AI, represented by SEQ ID NO: 15, ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the band from which these markers are derived.

9. The method of claim 1, wherein the amplification step comprises at least one polymerase chain reaction (PCR) and primers for PCR are selected from the following group of primers, or from primers having partial homology to the following group of primers:

forward reverse forward reverse

10. The method of claim 9, wherein the method comprises a multiplex polymerase chain reaction (PCR) using all of the primers in a single reaction.

11. The method of claim 1, wherein the step of determining the genotype comprises the use of a dot blot assay to detect the genotype.

12. The method of claim 1, wherein the amplification step comprises using primer sets C2 and Y5 in combination in a multiplex polymerase chain reaction (PCR), and the step of determining the genotype comprises ~ dotting PCR reaction onto two identical membranes, ~ probing the membranes with probes, wherein one membrane is probed with a probe which hybridizes to a marker indicating the presence of Rf and the second membrane is probed with a probe which hybridizes to a marker indicating the absence of rf, and ~ comparing the two membranes to determine the genotype of Brassica germplasm for the Ogura restorer.

13. The method of claim 12, wherein the marker indicating the presence of Rf is about 677 base pairs and the marker indicating the absence of rf is about 774 base pairs.

14. The method of claim 1, wherein the amplification step comprises using primer sets C2 and Y5 in combination in a multiplex polymerase chain reaction (PCR), and the step of determining the genotype comprises ~ running out the products of PCR on an electrophoresis gel, wherein the reaction products are from a group consisting of:

~ one band (about 677 bp), indicating a genotype of RfRf, ~ two bands (about 677 bp and about 774 bp), indicating a genotype of Rfrf, and one band (about 774 bp), indicating a genotype of rfrf, and ~ reading the gel to determine the genotype of Brassica germplasm for the Ogura restorer.

15. The method of claim 1, wherein the Brassica germplasm is selected from a group consisting of winter and spring Brassica napus, Brassica rapa and Brassica juncea.

16. A homozygous locus associated with the presence of the Ogura (RfRf) restorer gene, said locus mapping to at least one of the following markers:
C2, represented by SEQ ID NO: 2, ~ N20, represented by SEQ ID NO: 3, ~ F10, represented by SEQ ID NO: 4, a marker having partial homology to one of those sequences, and any other marker selected from a sequence of the RAPD band from which these markers are derived.

17. A homozygous locus associated with the absence of the Ogura (rfrf) restorer gene, said locus mapping to one of the following markers:
~ Y5, represented by SEQ ID NO: 1, a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the RAPD band from which these markers are derived.

18. A heterozygous locus associated with the presence of the Ogura (Rfrf) restorer gene, said locus mapping to one of the markers of claim 16 and one of the markers of claim 17.

19. A combination of markers for determining the genotype of Brassica germplasm for the Ogura (Rf) restorer gene, wherein at least one marker indicates the presence of the Ogura (Rf) restorer gene and at least one marker indicates the absence of the Ogura (rf) restorer gene.

20. A combination of markers for determining the genotype of Brassica germplasm for the Ogura (Rf) restorer gene, comprising a first set of nucleic acid markers selected from the group consisting of ~ C2, represented by SEQ ID NO:2, N20, represented by SEQ ID NO:3, ~ F10, represented by SEQ ID NO:4, ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the RAPD band from which these markers are derived, and markers comprising a second set of nucleic acid markers selected from a group consisting of Y5, represented by SEQ ID NO: 1 ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the RAPD band from which these markers are derived.

21. A SCAR marker for determining the presence of the Ogura (Rf) restorer gene in the genotype of Brassica germplasm, comprising a nucleic acid marker selected from the group consisting of C2, represented by SEQ ID NO: 2, ~ N20, represented by SEQ ID NO: 3, ~ F10, represented by SEQ ID NO: 4, ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the RAPD band from which these markers are derived.

22. A SCAR marker for determining the absence of the Ogura (Rf) restorer gene in the genotype of Brassica germplasm, comprising a nucleic acid marker selected from the group consisting of ~ Y5, represented by SEQ ID NO: 1, ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the RAPD band from which these markers are derived.

23. A RAPD marker for determining the presence of the Ogura (Rf) restorer gene in the genotype of Brassica germplasm, comprising a nucleic acid marker selected from the group consisting of ~ OPN20, represented by SEQ ID NO: 8, ~ OPH3, represented by SEQ ID NO: 9, ~ OPF10, represented by SEQ ID NO: 10, ~ OPH15, represented by SEQ ID NO: 11, ~ a marker having partial homology to one of those sequences, and ~ any other marker selected from a sequence of the band from which these markersare derived.
~ a sequence of the band from which these markers are derived.

24. A combination of primers for use in a polymerase chain reaction (PCR) to determine the genotype of Brassica germplasm for the Ogura (Rf) restorer gene, comprising primers selected from the following group of primers, from primers having at least partial homology to the following group of primers, from primers which anneal to a sequence from which these primers are derived, or from primers derived from any portion of the RAPD fragment or amplified RAPD fragment from which the following primers are derived:

forward reverse forward reverse

25. A primer kit, comprising the primers of claim 25.

26. A system for determining the genotype of Brassica germplasm for the Ogura (Rf) restorer gene, comprising the steps of:

27. amplifying the genotype in a single multiplex polymerase chain reaction (PCR), using primers selected from the following group of primers, from primers having at least partial homology to the following group of primers, from primers which are complementary to these primers, from primers which anneal to a sequence from which these primers are derived, or from primers derived from any portion of the RAPD
fragment or amplified RAPD fragment from which the following primers are derived:

forward reverse forward reverse ~ determining the genotype using a combination of nucleic acid markers, wherein the markers are selected from a group consisting of: a C2 marker indicating the presence of the OGU Rf gene (Rf), or markers having at least partial homology with the C2 marker, and a Y5 marker indicating the absence of the OGU Rf gene (rf), or markers having at least partial homology with the Y5 marker.