Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-05-06
1999-09-07
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 911, 536 221, 536 243, 536 253, C12P 1934, C07H 2104, C07H 2100
Patent
active
059486503
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method of identifying hop varieties, and in particular, to an identifying method which utilizes genetic engineering technology.
BACKGROUND OF THE INVENTION
Hops gives beer its unique aroma and bitterness, and also helps to sediment out excess protein and suppress bacterial proliferation. However, as the extent to which hops act in this way varies mainly according to the variety, it is necessary to identify hop varieties in order to manufacture beer of consistent quality and develop new beers.
Conventionally, the identification of hop varieties was based on their constituent components, e.g. bitter components (.alpha. acid/.beta. acid cohumulone/humulone, colupulone/lupulone) and essential oil components (farnesene, caryophyllene).
However, in addition to the fact that this identifying method based on bitterness and oils requires a great amount of effort for measuring all the components, it was also unable to determine the varieties precisely because, even for the same variety, these components vary widely depending on the harvesting location and year. Due to recent progress in genetic engineering, various attempts have been made to elucidate sequences of chromosomes depending on the plant species, and based on the base sequences found, to identify strains from differences in genetic information between different plants or varieties of the same plant, i.e. from the polymorphism of DNA sequences.
An identification of plant variety based on this kind of polymorphism is reliable because the DNA sequence itself is not easily affected by environmental influences, and is therefore invariant.
In this regard, the present invention provides a reliable and simple method of identifying hop varieties using the aforesaid genetic technique.
DISCLOSURE OF THE INVENTION
The method of identifying hop varieties according to this invention detects this polymorphism genetically based on differences in DNA sequences between varieties. Specifically, portions of the DNA sequence of hops which show polymorphism are amplified by a polymerase chain reaction (referred to hereafter as PCR) using an identifying primer, and the varieties are then identified by analyzing this amplified DNA.
To use the above method, it is necessary to understand the polymorphism of DNA between hop varieties.
The term polymorphism specifically covers differences in the genetic sequence due to insertions, deletions or substitutions. The length of the sequence involved may be 1 bp, or several tens of bp or more, but is preferably in a range from several bp to several tens of bp.
This sequential polymorphism between varieties may be detected by determining sequences of parts corresponding to chromosome DNA for each variety, and then performing a comparative analysis on the sequences. A simpler method however is to use the RAPD (Random Amplified Polymorphic DNA) technique developed by Williams et al (Nucleic Acids Research, Vol. 18, p. 6531, 1990).
This RAPD technique detects polymorphism between unknown DNA sequences using PCR. Specifically, primers of low specificity comprising a relatively short synthetic oligonucleotide of about 10 bases are mixed with a plurality of DNA types, and PCR is performed.
When the DNA sequence of the hop comprises a sequence which is fully or partially complementary to the primers, the primers anneal with the full or partial complementary sequence, and the portion enclosed between primers is synthesized and is amplified. When there are differences in the sequence such as insertions or deletions in the DNA within the portion enclosed between primers, PCR amplified fragments of different size are obtained depending on the variety. Further, when one variety comprises a sequence with which a primer anneals and another variety does not, PCR amplified fragments are obtained only for the first variety. The polymorphism may then be detected by fractionation, e.g. by electrophoresis.
The RAPD method may be used not only to detect polymorphism according to this invention as describ
REFERENCES:
Pillay et al. Chloroplast DNA between cultivated hop, Humulus Iupulus and the related species H. japonuicus Theor. Appl. Genet vol. 89, pp. 372-378), 1994.
Pillay et al. Random amplified polymorphic DNA (RAPD) markers in hop, Humulus lupulus: level of genetic variability and segregation F1 progeny, Theor. Appl. Genet. vol. 92, pp. 334-339, 1996.
Williams et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Research vol. 18(22), pp. 6531-6535, 1990.
Plant Breeding, vol. 113 (1994) G. Mosges et al., "Genetics Fingerprinting of Sunflower Lines and F1 Hybrids using Isozymers, Simple and Repetitive Sequences as Hybridization Probes, and Random Primers for PCR", pp. 114-124.
Methods in Molecular and Cellular Biology, vol. 5 (1994) Perry B. Gregan, et al., "Microsatellite Fingerprinting and Mapping of Soybean" pp. 49-61.
Theor. Appl.Genet., vol. 85 (1992) S. He et al., "Detection of DNA Sequence Polymorphisms Among Wheat Varieties" pp. 573-578.
Euphytica, vol. 80 (1994), H.L. Ko et al. "Random Amplified Polymorphic DNA Analysis of Australian Rice (Qryza Sativa L.) Varieties" pp. 179-189.
J. American Society Hort. Sci., vol. 119(4) (1994) J. F. Hancock et al. "Randomly Amplified Polymorphic DNAs in the Cultivated Strawberry FragariaXananassa" pp. 862-864.
Araki Shigeki
Tsuchiya Yohichi
Marschel Ardin H.
Sapporo Breweries Ltd.
Tung Joyce
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