Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-17
2001-03-06
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06197518
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the fields of agriculture and disease resistance. More specifically, the present invention relates to markers for Fusarium head blight disease resistance.
BACKGROUND OF THE INVENTION
Fusarium Head Blight (FHB) or scab of wheat is a fungal disease of the genus Fusarium that affects crops such as wheat, oats, barley, rye, corn and some grasses. The disease is of great interest, as it can have significant effects on crop yields. For example, the damage caused by the fungus
Fusarium graminearum
on wheat can be enormous. In particular, during 1993, this fungus caused an estimated three billion dollar loss to farmers in North America alone. The main effects of FHB infection are reduced crop yield and grain quality. Furthermore, FHB also produces mycotoxins which makes contaminated grain unsuitable for human or livestock consumption, for example, causing livestock feeding problems, such as refusal of feed or vomiting.
In order for this disease to occur, a susceptible host must be present and the environmental conditions must be favourable for infection and disease development. Since the environmental conditions cannot be controlled on a large scale, incorporating resistance genes into adapted wheat varieties is the most effective, economic and environmentally safe means of controlling the disease.
Naturally-occurring FHB-resistant cultivars of wheat are known and have been reported as two to three independently segregating genes in some Chinese and Brazilian strains (Van Ginkel et al., 1996,
Plant Disease
80:863). Unfortunately, these resistant cultivars have undesirable agronomic traits, such as small heads and late maturation, and are therefore of little commercial value. However, the resistant cultivars have been used as parents in breeding programs with elite lines (Bai and Shaner, 1994,
Plant Disease
78:760-765), with limited success. This is due to the fact that the inheritance of resistance to FHB is quantitative and controlled by many additive genes. As a consequence, data elucidating both the number and location of these genes has been problematic.
Hexaploid wheat is comprised of three homoeologous genomes (A, B, and D) each having 7 chromosomes. Procunier et al. 1998 , 9
th
lnt. Wheat Gen.Symp., Sask. SK.3:143-147 reported that the D genome ( chromosomes D1 to D7) lacked any significant FHB resistance genes. This placed the resistance genes on the A or B genomes. Two genetic regions ( 3BS and 6BL) for FHB resistance were identified by Anderson et al., 1998, National Scab Forum, St. Paul, Minn. and a single region ( 5A or 6B) is associated with the phenotypic marker awnless and FHB resistance( Ban et al. 1997, Fifth European Fusarium Seminar, Szeged, Hungary). However, Buerstmayr has recently showed that chromosomes 6D, 6B, 5A 4D, and 7A contain the FHB resistance genes ( Buestmayr et al., 1999, Theor. Appl Genet. 98:76-85).
Clearly, identification of the chromosomal locations of the FHB resistance genes followed by molecular mapping of these genes would greatly facilitate breeding FHB resistant strains. Specifically, molecular marker based-assays could be done on seed material, thus eliminating the lengthy growing time for assaying adult plants. Furthermore, seed testing does not require growth cabinet space or a costly nursery. The markers would also allow for gene pyramiding of multiple, independent resistance genes into complex genetic backgrounds. Ideally, the markers should be cost effective, highly reliable and accurate and require only a few hours for testing.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide molecular markers for FHB resistance genes.
According to a first aspect of the invention, there is provided a method of detecting Fusarium head blight resistance in a wheat cell, said method comprising:
providing genomic DNA extracted from the wheat cell;
providing a primer set selected from the group consisting of:
primer set A: a forward primer selected from the group consisting of: Forward Fhb1 primer (SEQ ID No. 1), contiguous primers thereof, noncontiguous primers thereof and homologous primers thereof; and a reverse primer selected from the group consisting of: Reverse Fhb1 primer (SEQ ID No. 2), contiguous primers thereof, noncontiguous primers thereof and homologous primers thereof; or
primer set B: a forward primer selected from the group consisting of: Forward Fhb2 primer (SEQ ID No. 3), contiguous primers thereof, noncontiguous primers thereof and homologous primers thereof; and a reverse primer selected from the group consisting of: Reverse Fhb2 primer (SEQ ID No. 4), contiguous primers hereof, noncontiguous primers thereof and homologous primers thereof; or
primer set C: a forward primer selected from the group consisting of: Forward Fhb3 primer (SEQ ID No. 5), contiguous primers thereof, noncontiguous primers thereof and homologous primers thereof; and a reverse primer selected from the group consisting of: Reverse Fhb3 primer (SEQ ID No. 6), contiguous primers thereof, noncontiguous primers thereof and homologous primers thereof; or
combinations of primer set A, primer set B and primer set C.
combining the genomic DNA and the primer set with reagents suitable for DNA amplification, thereby forming a reaction mixture;
incubating the reaction mixture under conditions wherein the DNA is amplified, thereby producing a DNA fragment of a given molecular weight; and
determining the molecular weight of the DNA fragment, wherein use of primer set A results in synthesis of a DNA molecule of a first molecular weight when Fhb1 is present on the genomic DNA or the synthesis of a DNA molecule of a second molecular weight when Fhb1 is absent on the genomic DNA; use of primer set B results in synthesis of a DNA molecule of a third molecular weight when Fhb2 is present on the genomic DNA or the synthesis of a DNA molecule of a fourth molecular weight when Fhb2 is absent on the genomic DNA; and use of primer set C results in synthesis of a DNA molecule of a fifth molecular weight when Fhb3 is present on the genomic DNA or the synthesis of a DNA molecule of a sixth molecular weight when Fhb3 is absent on the genomic DNA
The molecular weights of the DNA fragments may be determined by loading the reaction mixture onto a suitable support and electrophoresing the reaction mixture; and visualizing the banding pattern of the DNA fragments, wherein the presence of Fhb1 produces a first banding pattern while the absence of Fhb1 produces a second banding pattern, the presence of Fhb2 produces a third banding pattern while the absence of Fhb2 produces a fourth banding pattern, and the presence of Fhb3 produces a fifth banding pattern while the absence of Fhb3 produces a sixth banding pattern.
The wheat cell may be a seed.
The DNA in the reaction mixture may be digested with a restriction enzyme.
According to a second aspect of the invention, there is provided a vector arranged for transformation into wheat cells, said vector including an isolated DNA segment comprising a section of the wheat genome coding for resistance to Fusarium head blight selected from the group consisting of:
a segment of wheat chromosome 3B including Fhb1 Forward primer (SEQ ID NO. 1);
a segment of wheat chromosome 3B including Fhb1 Reverse Primer (SEQ ID NO. 2);
a segment of wheat chromosome 6B including Fhb2 Forward Primer (SEQ ID NO. 3);
a segment of wheat chromosome 6b including Fhb2 Reverse Primer (SEQ ID NO. 4);
a segment of wheat chromosome 5A including Fhb3 Forward Primer (SEQ ID NO. 5); and
a segment of wheat chromosome 5A including Fhb3 Reverse Primer (SEQ ID No. 6).
The vector may include sequences for directing replication of the vector in a wheat cell.
The vector may include sequences for directing integration of the vector into the wheat genome.
The vector may be an artificial chromosome.
According to a third aspect of the invention, there is provided a method of transferring FHB resistance to a wheat cell comprising:
providing a vector containing an isolated DNA segment comprising a sec
Armstrong Ken
Fedak George
Gilbert Jeannie
Procunier James Douglas
Townley-Smith T. Fred
Battison Adrian D.
Her Majesty the Queen in right of Canada as represented by the
Williams Michael
Yucel Remy
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