JP2005278477A - Method for distinguishing strain of strawberry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To establish a method for distinguishing a strain of strawberry by DNA analysis by which high distinguishing accuracy is obtained by a combination of a few primers. <P>SOLUTION: The nearly ten strains of the strawberry can be distinguished by using only one kind of a primer pair by applying an AFLP (amplified fragment length polymorphism) method for detection of polymorphism, and using the primer pair of (EcoRI+AGC) and (MseI+CAA) as a primer for PCR amplification. The accuracy of the distinguishment can be heightened by using several kinds of subservient primer pairs. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for identifying strawberry varieties by DNA analysis.

  With regard to the protection of new plant varieties, a system has been established such as the rights of growers being strengthened by the revision of the Seed and Seed Law. It is necessary to prove that it is the same, that is, the cultivated variety and the suspected illegal use are the same. Therefore, development and use of a DNA analysis technique capable of objective product type identification is desired.

  Identification of plant varieties by DNA analysis is performed by detecting a difference in DNA base sequence (referred to as polymorphism) existing between different varieties. As methods for detecting this polymorphism, methods already developed include, for example, RAPD (Random Amplified Polymorphic DNA) method, RFLP (Restriction Fragment Length Polymorphism) method, CAPS (Cleaved Amplified Polymorphic Sequence) method, AFLP (Amplified Fragment Length Polymorphism) ) There is a method called the law.

  The RAPD method is a method of amplifying DNA with random primers using PCR (Polymerase Chain Reaction). The RFLP method is a method of detecting a specific fragment by cleaving DNA with a restriction enzyme. The CAPS method is a method for identifying DNA. The region is PCR amplified and cleaved with a restriction enzyme. The AFLP method is a method of cleaving DNA with a restriction enzyme and PCR amplification. Each method has advantages and disadvantages in terms of the number of individual base sequences that exhibit polymorphism in a specific region of DNA, reproducibility, the number of specific regions that can be analyzed at one time, the difficulty of analysis, and discrimination ability. It is said that.

Conventionally, strawberry variety identification methods have been carried out by comparing the shape and size of fruits, the shape and size of leaves, the shape of grass, etc., but it is difficult to accurately identify the variety. Therefore, a method for identifying varieties of strawberry by DNA analysis has been studied and disclosed as Patent Document 1. The method for identifying strawberry varieties described in Patent Document 1 uses DNA extracted from a fruit or leaf containing strawberry gourd as a template, and amplifies DNA by a PCR method targeting different base sequences between strawberry varieties. In this method, the varieties of strawberry are identified by distinguishing the polymorphism of the amplified DNA and the polymorphism produced by digestion of the amplified DNA with a restriction enzyme.
JP 2003-339399 A

  The strawberry variety identification method described in Patent Document 1 is an identification method to which the CAPS method is applied. As described above, the polymorphism detection method by the CAPS method is a method in which a specific region of DNA is selected in advance, this specific region is PCR amplified, and cleaved with a restriction enzyme. In the case of strawberries with insufficient DNA analysis compared to the main cereals, in addition to the small number of specific regions of DNA that can be selected for the purpose of variety identification, the number of polymorphisms obtained in one operation is small, In order to identify a large number of varieties, there is a problem that the selected specific region must be treated with a plurality of restriction enzymes having different functions.

  The problem to be solved by the present invention is to identify a variety of strawberry varieties by DNA analysis, particularly in the identification of varieties of an object when there is a suspicion of infringement of a breeder's right by another person for a new strawberry. The purpose is to establish an identification method capable of identifying more varieties in combination.

  The present inventors refer to the identification method applying the CAPS method described in Patent Document 1, which is the prior art of strawberry variety identification, and the identification method applying the AFLP method widely used for plant variety identification. In addition, intensively studying methods for identifying strawberry varieties by DNA analysis, and applying AFLP method to strawberry variety identification, not only can detect many polymorphisms effective for variety identification at once, but also improve the identification accuracy Based on this finding, an optimal primer pair was found as a primer necessary for PCR amplification, and the present invention was completed.

  That is, the present invention is a method for discriminating strawberry varieties by DNA analysis, wherein an adapter is linked to a fragment obtained by cleaving extracted strawberry DNA with restriction enzymes EcoRI and MseI, and preliminarily PCR-amplified to determine the relative number of fragments. As a primer pair for selective PCR amplification using this fragment as a template, a primer “EcoRI + AGC” with 3 bases of AGC added to the EcoRI cleavage side and a primer “3 bases of CAA added to the MseI cleavage side” A method for identifying strawberry varieties using polymorphism detection by the AFLP method, characterized by using “MseI + CAA”. In order to further improve the identification accuracy, “EcoRI + AAC” and “MseI + CAA”, “EcoRI + ACC” and “MseI + CAA”, “EcoRI + ACG” and “MseI + CAA”, “EcoRI + ACT” and “MseI + CTA”, “EcoRI + ACA” and “MseE + CTA”, “MseE + CTA”, ”And“ MseI + CTA ”primer pairs can be used for selective PCR amplification.

  In the strawberry variety identification by DNA analysis, the strawberry variety identification with less DNA information can be efficiently performed by applying the AFLP method to polymorphism detection. In addition, by using “EcoRI + AGC” and “MseI + CAA” primer pairs as primers necessary for PCR amplification, it is possible to identify about 10 varieties of strawberry by using only one primer pair. Become. In addition to this, by using a plurality of types of auxiliary primer pairs, it is possible to increase the identification accuracy of about 10 types to 99% or more.

In strawberry variety identification by DNA analysis according to the present invention, polymorphism detection is performed by the AFLP method. The AFLP method itself is a known method, and is performed according to a known AFLP method except for DNA extraction method and primer selection specific to strawberries. Strawberries are rich in polysaccharides, and the degree of removal greatly affects the success or failure of subsequent DNA analysis. Therefore, DNA extraction is preferably performed according to the following procedure.
(1) Collect a small amount of any one or more of fresh strawberry leaves and gourd pieces and crush them as finely as possible at extremely low temperatures.
(2) After crushing, a buffer solution containing sorbitol and polyethylene glycol is injected and stirred.
(3) After stirring and centrifuging to remove the supernatant as much as possible, the DNA is purified, separated and extracted by a DNA extractant effective for removing polysaccharides.

  The selection of primers necessary for PCR amplification is the most important matter for the present invention. In the AFLP method, an adapter is ligated to a fragment obtained by cleaving extracted strawberry DNA with restriction enzymes EcoRI and MseI, preliminarily PCR-amplified to change the relative number of fragments, and selective PCR amplification is performed using this fragment as a template. The primers used for the second PCR amplification include a primer added with 3 bases on the restriction enzyme EcoRI cleavage side and a primer added with 3 bases on the restriction enzyme MseI cleavage side.

  As the sequence of 3 bases added to the EcoRI cleavage side, there are 4 × 4 × 4 = 64 types including AAA, AAT, AAG and AAC, and there are also 64 types of 3 base sequences added to the MseI cleavage side. Therefore, there are 64 × 64 = 4,096 combinations of pairs of EcoRI cleavage side primers and MseI cleavage side primers. If selective PCR amplification was performed using all of these 4,096 combinations of primer pairs, enormous time was required for selective PCR amplification and subsequent analysis. In order to efficiently identify the varieties, it is necessary to select a primer pair that can obtain the maximum discrimination ability with the minimum number of primer pairs.

  In the present invention, as a primer pair used for selective PCR amplification, a primer “EcoRI + AGC” with 3 bases of AGC added to the EcoRI cleavage side and a primer “MseI + CAA” with 3 bases of CAA added to the MseI cleavage side are used. Only by using this pair of primers, about 10 varieties of strawberries can be identified. In order to further improve the identification accuracy, primer pairs “EcoRI + AAC” and “MseI + CAA”, “EcoRI + ACC” and “MseI + CAA”, “EcoRI + ACG” and “MseI + CAA”, “EcoRI + ACT” and “MseI + CTA”, “EcoRI + ACA” and “EcoRI + ACA” PCR amplification is performed using each primer pair of “MseI + CTA”, “EcoRI + ACC” and “MseI + CTA”. As a result, it is possible to finally identify the product with a probability of 99% or more. Although these primers can be produced by themselves, a commercially available reagent kit (for example, a reagent kit manufactured by Applied Biosystem Japan Co., Ltd.) can also be used.

  Hereinafter, an example in which varieties are identified by DNA analysis for 10 varieties including new varieties will be described. The names of the strawberry varieties to be identified are “Fukuoka S6”, “Toyonoka”, “Sachinoka”, “Nyomine”, “Tochiotome”, “Ascalbee”, “Red Pearl”, “Sahonoka” ”,“ Akihime ”,“ Iberry ”, and“ Fukuoka S6 ”is a new variety.

  0.05 g of undeveloped leaves or gourd pieces of the above-mentioned varieties were each dispensed into 2.0 ml Eppendorf tubes containing zirconia balls and stored at −80 ° C. After these tubes were submerged in liquid nitrogen, they were set in a Waring blender 300MM (manufactured by Retch) and crushed for 30 seconds. After crushing, 1 ml each of 50 mM Tris-HCl buffer solution (containing 5 mM EDTA, 350 mM Sorbitol, 10% PEG) supplemented with 0.1% thioglycolic acid was added and stirred. Thereafter, the mixture was centrifuged for 5 minutes at a temperature of 4 ° C. and 6000 rpm, the supernatant was removed as much as possible, and then total DNA was extracted using a DNA extraction kit Nucleon PHYTO Pure (manufactured by Amersham Pharmacia Biotech). The extracted DNA was dissolved in 100 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), quantified with an ultraviolet absorption measuring device DU640 (manufactured by BECKMAN), and the concentration was adjusted based on the value and used as a test. did.

  The restriction enzyme treatment was performed as follows using DNA extracted from each strawberry variety, restriction enzymes EcoRI (15 u / μl, Takara), and MseI (10 u / μl, BioLabs). Using 0.5 μg of DNA, 10 u of enzyme and 10-fold concentration buffer, the solution was adjusted to 20 μl with sterilized distilled water and treated at a temperature of 37 ° C. for 2 hours. In carrying out the AFLP method, an AFLP startup module (for regular use) manufactured by PE Applied Biosystems and PRISM310 Genetic Analyzer were used, and analysis was performed using the analysis software GeneScan.

  First of all, for the purpose of identifying the new variety “Fukuoka S6”, the polymorphism of the three varieties by the AFLP method was added between “Toyonoka” and “Sachinoka” used for breeding this variety and the parental cross. Detection was performed. The result is shown in FIG. In the figure, the symbol ◎ indicates “Fukuoka S6”, the symbol ● indicates “Toyonoka”, and the symbol ○ indicates “Sachinoka” a combination of primers each having a single polymorphism. The symbol-indicates a combination of primers for which no single polymorphism was obtained.

  As shown in FIG. 1, polymorphism was detected in 53 primer pairs out of 64 primer pairs in which 8 kinds of EcoRI cleavage side primers and 8 kinds of MseI cleavage side primers were combined. Of these, a single polymorphism was observed in many of the three varieties tested with a combination using a primer with 3 bases of CAA or CTA added to the MseI cleavage side. From this, it was considered that a combination using a primer having 3 bases of CAA or CTA added to the MseI cleavage side should be used for detecting polymorphisms of all varieties tested.

  Therefore, for all 10 varieties tested, polymorphism by AFLP method was used using 16 primer pairs in which 8 types of EcoRI cleavage side primers were combined with a primer obtained by adding 3 bases of CAA or CTA to MseI cleavage. Detection was performed. As a result, polymorphisms were detected in all combinations. FIG. 2 is a diagram showing a part of the results. In the figure, the symbol + indicates that a DNA fragment corresponding to the marker is present, and the symbol-indicates that it does not exist. As shown in the figure, among many combinations, in the combination of the primer “EcoRI + AGC” added with 3 bases of AGC on the EcoRI cleavage side and the primer “MseI + CAA” added with 3 bases of CAA on the MseI cleavage side, Eight polymorphisms were recognized, and it was confirmed that the same or different between the new variety “Fukuoka S6” and the other nine varieties could be identified only by using this primer pair.

  In order to further improve the identification accuracy, "EcoRI + AAC" and "MseI + CAA", "EcoRI + ACC" and "MseI + CAA", "EcoRI + ACG" and "MseI + CAA", "EcoRI + ACT" and "MseI + CTA", "EcoRI + ACA" and "MseE + CTA" ”And“ MseI + CTA ”using the respective primer pairs, and adding these results, for example, for“ Fukuoka S6 ”, the polymorphism using the“ EcoRI + AGC ”and“ MseI + CAA ”primer pairs. In the detection, the discrimination probability is about 96.6%, but by using the auxiliary primer pair, the discrimination probability is sequentially increased, and finally the discrimination is possible with a probability of 99.9999%.

  In the above embodiment, the strawberry varieties “Fukuoka S6”, “Toyonoka”, “Sachinoka”, “Nyomine”, “Tochiotome”, “Ascalbee”, “Red Pearl”, “Sagahonoka” Ten types of “Akihime” and “Iberry” were tested, but it was confirmed by experiments that other types can be identified by the same method.

  By adopting the method for identifying strawberry varieties according to the present invention, it becomes possible to identify varieties objectively and with high accuracy, so that it becomes easy to prove the fact of infringement of the breeder's rights by others, Can contribute to protection.

It is a figure which shows the result of the polymorphism detection performed preliminary about DNA of three kinds of strawberries. It is a figure which shows the result of having discriminate | determined the seed | species by DNA analysis about 10 kinds of strawberries by the method of this invention.

Claims (2)

  Primer when selective PCR amplification is carried out using the extracted strawberry DNA fragmented with restriction enzymes EcoRI and MseI, preliminarily PCR-amplified to change the relative number of fragments, and using this fragment as a template Uses polymorphism detection by AFLP method, using primer “EcoRI + AGC” with 3 bases of AGC added to EcoRI cleavage side and primer “MseI + CAA” with 3 bases of CAA added to MseI cleavage side Strawberry variety identification method.   In addition to the selective PCR amplification using the “EcoRI + AGC” and “MseI + CAA” primer pairs, “EcoRI + AAC” and “MseI + CAA”, “EcoRI + ACC” and “MseI + CAA”, “EcoRI + ACG” and “MseI + CAA”, “EcoRI + ACT” The strawberry variety identification method according to claim 1, wherein selective PCR amplification is performed using each of the primer pairs of "MseI + CTA", "EcoRI + ACA" and "MseI + CTA", "EcoRI + ACC" and "MseI + CTA".

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