JP4452790B2 - Primer set for detecting DNA marker linked to low molecular weight glutenin gene of wheat and use thereof - Google Patents

Description

  The present invention relates to a primer set for detecting a DNA marker linked to a 42 kDa low molecular weight glutenin gene in wheat that confers strength, a DNA marker linked to the gene, and uses thereof.

In recent years, as a result of searching for seed proteins that make wheat flour more powerful, 42 kDa low molecular weight glutenin enhances the physical properties of flour, and 42 kDa low molecular weight glutenin has an additive effect with 5 + 10 high molecular weight glutenin. It has been found that flour having both of them can be a super strong flour (Non-patent Document 1). Therefore, if there is a technique for simply identifying the presence of 42 kDa low molecular weight glutenin in a wheat individual, that is, the presence or absence of a gene encoding the glutenin, it becomes possible to efficiently produce a powerful wheat line. However, at present, seed proteins are separated and identified by SDS-polyacrylamide gel electrophoresis which takes a long time. In addition, a PCR method has been found to identify the presence or absence of a 5 + 10 high molecular weight glutenin gene (Non-patent Document 2), but a PCR method capable of identifying the presence or absence of a 42 kDa low molecular weight glutenin gene has been established. It has not been.
Takata et al. 2001, Breeding Sci 51: 143-146 M. Ahmad, 2000, Theor. Appl. Genet. 101: 892-896

  Accordingly, an object of the present invention is to provide a primer set for detecting a DNA marker linked to a 42 kDa low molecular weight glutenin gene capable of imparting power to wheat, and a DNA marker linked to the gene.

  Another object of the present invention is to provide a method for efficiently identifying the presence or absence of a 42 kDa low molecular weight glutenin gene in wheat and a method for producing a strong wheat line.

  As a result of intensive studies to solve the above problems, the present inventors have found a primer set capable of detecting a DNA marker linked to the 42 kDa low molecular weight glutenin gene of wheat, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) Detecting a DNA marker linked to a wheat 42 kDa low molecular weight glutenin gene composed of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 Primer set.
(2) A DNA marker linked to the 42 kDa low molecular weight glutenin gene of wheat, which is amplified by performing PCR on DNA extracted from wheat using the primer set described in (1).
(3) Perform PCR on DNA extracted from wheat using the primer set described in (1), and determine whether or not the 42 kDa low molecular weight glutenin gene in wheat depends on the presence of the DNA marker described in (2). A method of identifying presence or absence.
(4) Perform PCR on DNA extracted from wheat using the primer set described in (1), and select a wheat line having a 42 kDa low molecular weight glutenin gene using the DNA marker described in (2) as an index, A method for producing a powerful wheat line, characterized in that a wheat line producing a seed having a 42 kDa low molecular weight glutenin is obtained in a later generation.
(5) A powerful wheat line characterized by having a 42 kDa low molecular weight glutenin obtained by the production method according to (4).
(6) A kit for detecting a 42 kDa low molecular weight glutenin gene in wheat comprising the primer set according to (1).

  According to the present invention, there are provided a primer set for detecting a DNA marker linked to a 42 kDa low molecular weight glutenin gene that imparts power to wheat, and a DNA marker linked to the gene. Moreover, by using these, it is possible to quickly and reliably produce candidate lines of strong wheat varieties.

1. Primer set for detection of DNA marker linked to wheat 42 kDa low molecular weight glutenin gene and kit comprising the same “42 kDa low molecular weight glutenin” as used herein refers to isoelectric focusing and SDS-polyacrylamide gel electrophoresis. This refers to a plurality of glutenins that are separated as spots with a molecular weight of about 42 kDa and an isoelectric point of about 8.0 or more by two-dimensional electrophoresis as a combination, have the N-terminal amino acid sequence SHIPGLERPS, and are dominated by one locus.

  The primer set of the present invention can be obtained, for example, as follows. First, RT-PCR amplifies genes having high homology with known low molecular weight glutenin genes from wheat lines having 42 kDa low molecular weight glutenin and wheat lines not having 42 kDa low molecular weight glutenin. Next, various primer sets were designed with reference to the sequences of these genes, and PCR was performed again on the DNA extracted from the above wheat lines, and primer sets that were confirmed to have clear polymorphisms. Select.

  A primer set suitable for the present invention is composed of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2.

  The primer set of the present invention can also be provided as a 42 kDa low molecular weight glutenin gene detection kit. The kit of the present invention is sufficient if it contains at least the above primer set, and if necessary, PCR extraction reagents (excluding primers) such as DNA extraction reagents, PCR buffer solutions and DNA polymerases, staining agents, and electricity. A detection reagent such as a gel for electrophoresis, instructions and the like may be included.

2. DNA marker linked to 42 kDa low molecular weight glutenin gene The DNA marker of the present invention refers to a DNA band that is linked to wheat 42 kDa low molecular weight glutenin gene and appears when 42 kDa low molecular weight glutenin is expressed. Specifically, PCR product obtained by performing PCR on DNA extracted from wheat using the above primer set and exhibiting a band pattern at about 700 bp by agarose gel electrophoresis. The DNA marker of the present invention can be used for detection of the presence or absence of a 42 kDa low molecular weight glutenin gene, discrimination of wheat having a 42 kDa low molecular weight glutenin gene, and production of a strong wheat line.

3. A method for discriminating the presence or absence of a 42 kDa low molecular weight glutenin gene and a method for producing a strong wheat line The primer set or kit and DNA marker of the present invention can be used in the following manner.
For example, according to the present invention: 1. PCR is performed on DNA extracted from wheat using the primer set described in 1. A method for discriminating the presence or absence of the 42 kDa low molecular weight glutenin gene in wheat is provided depending on the presence or absence of the DNA marker described in 1.

  According to the present invention, 1. 1. PCR is performed on DNA extracted from wheat using the primer set described in 2 above. A method for producing a strong wheat line, characterized in that a wheat line having a 42 kDa low molecular weight glutenin gene is selected using the DNA marker described in the above as an index to obtain a wheat line that produces seeds having a 42 kDa low molecular weight glutenin in its progeny Is provided.

  According to the present invention, “strong wheat line” refers to a candidate line of wheat varieties that serve as raw materials for strong flour, and “strong flour” has the same physical properties as foreign strong flour, such as bread and Chinese noodles The flour that can be used for A “strong wheat line” that can be produced by the method of the present invention is characterized by having a low molecular weight glutenin of 42 kDa.

The wheat line to be subjected to PCR in the above method may be any wheat line as long as it is a wheat cultivar or line having a 42 kDa low molecular weight glutenin and having one cross as a parent. Examples of wheat varieties or lines having a low molecular weight glutenin of 42 kDa include “Glenlea”, Kansas State University breeding line “KS831957”, Hokkaido Agricultural Experiment Station breeding lines “Katsukei 33”, “Katsukei 34”, and the like. The other mating parent may be any wheat cultivar / line, preferably a domestic wheat cultivar or line. The wheat line obtained by this crossing and subjected to PCR is typically a F ₂ generation line, but may be a line advanced from F _{3 to} F _{10 in} order to confirm traits other than strength. .

  Wheat seeds are collected from the wheat line obtained above, grown, and DNA is extracted from the leaves. DNA extraction can be performed by a conventional method for plant materials, such as a cesium chloride density gradient method or a CTAB method, and these methods can be modified as necessary. Further, a commercially available DNA extraction kit may be used.

  PCR is performed using the DNA extracted above as a template. The primer set is used under appropriate PCR conditions. Examples of PCR conditions include, for example, one cycle of 94 ° C. for 30 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 1 minute, for a total of 50 cycles, but is not limited thereto.

  The PCR product is subjected to agarose gel electrophoresis, and a wheat line having a band at about 700 bp is selected. A candidate line of a strong wheat variety can be obtained in the progeny by self-breeding this wheat line or crossing with another wheat variety or line. Here, other wheat varieties or strains used for crossing are not particularly limited, but imparting desired traits other than strength (super strength, cold resistance, disease resistance) to modify wheat What can do is preferable.

Hereinafter, the present invention will be described more specifically with reference to examples. However, these examples do not limit the present invention.
Example 1 Design of Primer Set and Detection of 42 kDa Low Molecular Weight Glutenin Gene Using This Primer Spring Spring Cultivar “Spring no Akebono” has a 48 kDa low molecular weight glutenin controlled by an allele of the 42 kDa low molecular weight glutenin gene. The physical properties of “Spring Akebono” flour are weaker than “Glenlea”, which possesses 42kDa low molecular weight glutenin. First, primers were searched for polymorphisms for “Spring Akebono” and “Glenlea”. When RT-PCR was performed on "Spring Akebono" and "Glenlea" with a certain combination of primers, genes of different sizes were obtained, both of which have a repeat sequence specific to the low molecular weight glutenin gene. Therefore, it was considered to be a low molecular weight glutenin gene. Among them, the base sequence of the gene obtained from “Haru no Akebono” showed 99% homology with the previously reported low molecular weight glutenin gene (EMBL Data library accession no. Y17845). 20 pairs of primers were created with reference to the base sequence of the low molecular weight glutenin gene derived from this "Spring Spring" and genomic PCR was performed on the DNA of "Spring Spring" and "Glenlea". A polymorphism was observed in the combination of primer HaruF1: 5′-CCATCCAACAACAACCACACCA-3 ′ (SEQ ID NO: 1) and reverse primer HaruR0: 5′-CCCGAGTTGCTGTTGTGACTGT-3 ′. However, in this combination, only a few products were amplified from “Glenlea”, so the 3 ′ base (T) of HaruR0 was replaced with “C” corresponding to the gene obtained from RT by using “Glenlea”. When genomic PCR was performed again using the primer HaruR1: 5′-CCCGAGTTGCTGTTGTGACTGC-3 ′ (SEQ ID NO: 2), the amplification product in “Glenlea” increased. Thus, PCR was performed on 65 individuals of the B ₅ F ₂ generation obtained by crossing “Spring Akebono” and “Glenlea” using a combination of HaruF1 and HaruR1. First, glutenin protein was extracted from half seed grains, and the presence or absence of 42 kDa low molecular weight glutenin and 48 kDa low molecular weight glutenin was confirmed by SDS-polyacrylamide gel electrophoresis. DNA was extracted and PCR was performed using this as a template. For DNA extraction, about 8 cm of young leaves placed in a 1.5 ml tube is pulverized using liquid nitrogen and glass beads, boiled for 30 seconds with 80 μl of 0.25 N sodium hydroxide, then 80 μl of 0.25 N hydrochloric acid, 40 μl of buffer (0.5 M Tris-HCl, 0.25% IGEPAL CA-630, pH 8.0) was added and boiled for 2 minutes. PCR is 2 μl 10 × PCR buffer, 0.8 μl 2.5 mM dNTP, 1 μl 1% IGEPAL, 0.4 μl 5 μμ each primer, 1 μl DNA extract, 0.08 μl (0.4 U) AmpliTaq Gold (Applied Biosystems) Was carried out in a 20 μl reaction system to which was added. The PCR cycle was 50 cycles, with one cycle at 94 ° C. for 30 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 1 minute. This reaction was performed using GeneAmp PCR System 2400 (manufactured by Perkin-Elmer). The result is an approximately 700 bp DNA fragment for all individuals with 42 kDa low molecular weight glutenin, and an approximately 800 bp DNA fragment for all individuals with 48 kDa low molecular weight glutenin, both 42 kDa and 48 kDa low molecular weight Both about 800 bp and about 700 bp DNA fragments were obtained for all heterozygous individuals containing both glutenins (FIG. 1). From this result, it was shown that a DNA fragment of about 700 bp was linked to a gene encoding 42 kDa low molecular weight glutenin, and it was shown that a wheat individual containing 42 kDa low molecular weight glutenin can be easily distinguished by PCR. The base sequence of the DNA fragment of about 700 bp (692 bp) is shown in SEQ ID NO: 3.

(Example 2) Detection of 42 kDa low molecular weight glutenin gene The autumn sowing wheat line "Katsukei 32" has a 48 kDa low molecular weight glutenin controlled by alleles of 5 + 10 high molecular weight glutenin and 42 kDa low molecular weight glutenin gene, “Katsukei 34” has a 2 + 12 high molecular weight glutenin and a 42 kDa low molecular weight glutenin that are dominated by alleles of the 5 + 10 high molecular weight glutenin gene. The physical properties of “Katsukei 32” and “Katsukei 34” are comparable. PCR was performed using primers HaruF1, HaruR1 for the "winning system 32 No." and the "winning system No. 34" and F ₆ generation 79 obtained by mating the recombinant inbred line (RIL). First, glutenin protein was extracted from a plurality of seeds, and the presence or absence of 42 kDa low molecular weight glutenin and 48 kDa low molecular weight glutenin was confirmed by SDS-polyacrylamide gel electrophoresis. DNA was extracted from leaves developed from different seeds by the same method as in Example 1, and PCR was performed using this as a template. PCR is 2 μl of 10 × PCR buffer, 2 μl of 10 × PCR enhancer, 0.4 μl of 50 mM magnesium sulfate, 2 μl of 2.5 μm dNTP, 1 μl of 5 μM each primer, 1 μl of DNA extract, 0.5 μl (1.25 U) Platinum Pfx The reaction was performed in a 20 μl reaction system to which DNA polymerase (manufactured by Invitrogen) was added. A total of 45 cycles of PCR were performed, with one cycle being 94 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 1 minute. This reaction was performed using GeneAmp PCR System 2400 (Perkin-Elmer). As a result, a DNA fragment of about 700 bp was obtained for all of the RIL containing 42 kDa low molecular weight glutenin, and an 800 bp DNA fragment was obtained for all of the RIL containing 48 kDa low molecular weight glutenin (FIG. 2). This result also showed that the DNA fragment of about 700 bp was linked to a gene encoding 42 kDa low molecular weight glutenin, and it was shown that wheat individuals containing 42 kDa low molecular weight glutenin can be easily distinguished by PCR.

FIG. 1A shows an electropherogram of the glutenin protein of a B ₅ F ₂ generation individual derived from the cross between “Spring Akebono” and “Glenlea”. FIG. 1B shows an electrophoretogram of the DNA fragment obtained by PCR (lane 1: “Spring Akebono”, lane 2: near-isogenic line with 42 kDa low molecular weight glutenin, lane 3: “Glenlea”, lanes 4-11. : 42 kDa, 42 kDa and 48 kDa, 42 kDa and 48 kDa, 42 kDa and 48 kDa, 48 kDa, 42 kDa and 48 kDa, 48 kDa, 42 kDa, low molecular weight glutenin individuals of the B ₅ F ₂ generation, right arrow: 42 kDa and 48 kDa low molecular weight glutenin, And their corresponding DNA fragments, arrows on the left side of the figure: positions of protein and DNA size markers). FIG. 2A shows an electrophoretogram of glutenin proteins of “Katsukei 32” and “Katsukei 34” recombinant inbred lines (RIL). FIG. 2B shows an electrophoretogram of the DNA fragment obtained by PCR (lane 1: “Katsukei 32”, lane 2: “Katsukei 34”, lanes 3-8: 42 kDa, 42 kDa, 48 kDa in this order. RIL with 42 kDa, 48 kDa, 48 kDa low molecular weight glutenin, right arrow: 42 kDa and 48 kDa low molecular weight glutenin, and corresponding DNA fragments, left arrow: position of protein and DNA size markers).

Claims (5)

Primer set capable of detecting a DNA marker linked to a wheat 42 kDa low molecular weight glutenin gene, comprising an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 .   A DNA marker linked to a 42 kDa low molecular weight glutenin gene of wheat, which is amplified by performing PCR on DNA extracted from wheat using the primer set according to claim 1. PCR is performed on DNA extracted from wheat using the primer set according to claim 1,
A method for discriminating the presence or absence of a 42 kDa low molecular weight glutenin gene in wheat based on whether or not the DNA marker according to claim 2 is present. PCR is performed on DNA extracted from wheat using the primer set according to claim 1,
A wheat line having a 42 kDa low molecular weight glutenin gene selected using the DNA marker according to claim 2 as an index, and obtaining a wheat line that produces seeds having a 42 kDa low molecular weight glutenin in its progeny, How to make A kit for detecting a 42 kDa low molecular weight glutenin gene in wheat comprising the primer set according to claim 1.

Images