JP3386238B2 - Nucleic acid marker for rice blast resistance gene and rice blast resistance gene obtained by this marker - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] This invention relates to rice blast resistance
Gene nucleic acid marker and the nucleic acid marker
Rice blast resistance gene.
It can be labeled with the nucleic acid marker of the present invention.
The rice blast resistance gene was originally developed from rice varieties
A new resistance gene that can be introduced into various plants
It is extremely useful as research material and genetic resources for
You. [0002] 2. Description of the Related Art Resistance of plants to pathogens
Genes are known to exist in combinations of various plants and pathogens
The introduction of the genetic trait is large even in conventional breeding.
Has become a goal. As a result, these resistance genes
Numerous varieties have been produced. Going forward, pesticides
Biological Control Law Utilizing the Innate Function of Plants without Ethanol
To enable low-cost agriculture while harmonizing with health and the environment
Is increasingly important around the world
It is considered something. [0003] Genes resistant to specific pathogens
The first rice blast resistance gene in Japan
And subsequently found that a large number of genes were present.
Resistance, especially from foreign rice
Sex genes are resistant to many blast fungi in Japan
It has high utility as a genetic resource. in
Also,Pi-taandPi-ta ^TwoThe gene is Indian type
And a rice blast resistance gene found in
Gene mapping by D and RFLP and nearby nuclei
Suitable for searching for acid markers. But actually useful
Very few examples have been incorporated into the species. [0004] The reason is that the conventional breeding method is excellent.
Years to introduce resistance genes from different varieties into other varieties
Need for extensive backcrossing
It is necessary to conduct a resistance test to inoculate bacteria.
I can do it. Testing for resistance to foreign pathogens
Is that the import of foreign pathogens is strictly regulated.
Therefore, it is often impossible in Japan. On the other hand, recent advances in plant genetic engineering technology
Therefore, various genes can be identified, isolated, and
They can be introduced into other plants
It has become to. Therefore, for the plant resistance gene,
Even if it can be isolated in the form of nucleic acids,
Easily introduce them into desired varieties by engineering techniques
Time and labor required to grow resistant varieties
Is expected to be able to dramatically reduce power
You. What is the mechanism by which the resistance gene is resistant to plants?
It is also possible to clarify whether
Provides basic information for constructing new resistance genes
You. For this reason, many groups around the world are now resistant
Attempts have been made to isolate the gene, but few have been successful.
This is because the biochemical identity of the resistance gene is unknown,
This is because the clue information is extremely limited. [0006] In recent years, positive methods have been used to isolate genes.
A method called so-called cloning has attracted attention.
You. This method uses markers for nucleic acids in close proximity on a genetic map.
And use it as a label to remove all of the genomic DNA
Markers from the fragmented library
Select the fragment to be identified and place it in the DNA sequence adjacent to the label.
From a target gene. Real
In this case, some of the genes responsible for human genetic diseases
Has been isolated. In plants, this positional cloning
Although there have been few successful cases, rice is
Is considered to be the most suitable plant for this method. Sand
(1) Genome size is the smallest among major crops, (2)
The physical distance to the genetic distance unit is also very small,
(3) It is also possible to introduce and express the isolated gene into cells.
(4) Many breeding efforts have been
Useful gene derived from rice is superior by repeated backcrossing
Since it is incorporated into Japanese rice,
It is easy to narrow down the location on the genome as knowledge
Yes, etc. An important key in this positional cloning
Is whether or not a good proximity marker can be obtained.
You. From rice genome size and genetic map, rice
1 centiMorgan (cM) distance on genetic map is nucleic acid
It is expected to be equivalent to 100-200 kb on a base
On the other hand, live using artificial chromosome (YAC) of yeast
In the rally, quarries with an average of 200 kb or more
A loan has been obtained. Therefore, 100 kb,
A marker marker can be obtained at a distance of 0.5 to 1 cM or less.
For example, a position targeting the rice blast resistance gene
Potential success of null cloning seems to be extremely high
It is. [0009] In addition, the inheritance of rice blast resistance
Nucleic acid markers for offspring can be
Used as a marker for the resistance gene.
Can greatly reduce the labor and time of resistance testing.
It is thought to stand. The present invention has been made in view of the circumstances described above.
The rice blast resistance gene was
New nucleic acid marker to be labeled on the nom and this marker
The rice blast resistance gene obtained by
It is intended to be. [0010] The present invention is to solve the above problems.
In rice chromosome 12 genomic DNA, rice R
The position where the FLP probe XNpb289 hybridizes
The rice RFLP probe XNpb316 hybridizes from the
The region up to the soybean position is the rice blast resistance gene
Pi-ta ^Two Is a genomic region where
Rice blast resistance genePi-ta ^Two Genome region of
Provide an area identification method. Hereinafter, the present invention will be described in more detail.
I do. The nucleic acid marker used in the method of the present invention is potato
Disease resistance genePi-taOrPi-ta ^Two have
Genomic DNA of several different rice varieties and blast resistance
Genomic DNA of rice varieties without sex genes
In comparison, only varieties with the blast resistance gene
Present and in the vicinity of the blast resistance gene (2.0 cM
Within: about 200-400 kb or less)
Obtained by identifying the DNA sequence. [0012] The comparison of genomic DNA, for example,
An Indian rice cultivar having a resistance gene (donor parent);
Japanese rice varieties without this gene (return parent), and
Quasi-homogeneous material obtained by repeated mating of the return parent to the donor parent
It can be carried out between rice varieties of the gene lineage group. Further
In addition, the nucleic acid marker of the present invention is used for cutting rice genomic DNA.
Hybridize with any of the RFLP probes obtained from the pieces
It can be detected by soybean. That is,
In Ming, the genomic DN of the rice plant to be tested
A is cleaved with any restriction enzyme, and the resulting DNA fragment is
After electrophoresis, hybridize the RFLP probe
Can be used to identify nucleic acid markers.
Wear. [0013] The present invention thus obtained
Is a rice genomic DNA of the Indian rice
Derived regions and regions derived from Japanese rice are indicated by RFLP
Blast resistance genePi-taMa
OrPi-ta ^TwoLocated within 2 cM from
A good marker for the rice blast resistance gene or related genes
Functions as a perception marker. Hereinafter, examples and test examples will be described.
The invention will be described in more detail. [0015] 【Example】 Example 1 Rice blast resistance genePi-ta ^TwoNucleic acid markers
Was identified. (1) Material adjustment (A) DNA extract The following extracts were each prepared by a known method. E. coli plants containing rice libraries
Rasmid DNA   Rice varieties (donor parents) with the blast resistance gene
DNA   Japanese rice varieties without blast resistance gene
(Back parent) DNA   A hybrid obtained by repeatedly mating the returning parent to the donor parent
DNA of Nematode (near isogenic line) (B) RFLP probe Saito et al. (Jpn. J. Breed., Vol 41, 665-67)
0, 1991)
An RFLP probe on the chromosome was used. (2) Hybridization of DNA fragment and RFLP probe
Zation Regarding the DNA extract (a) ~, each genome D
Fragmentation of 2-3 μg of NA with any restriction enzyme (see Table 1)
And 1 × TAE buffer 0.7-1% agarose 3
５5 × 1 mm gel slot, 5 V / cm
Electrophoresis was performed for about 4 hours on a gradient. Then, from the running gel
Transfer the DNA to a nylon membrane by a known method,
Lot. On the other hand, the RFLP probe of (b)
By a known method (for example, the random primer method)³²
Labeled with P or peroxidase, Southern hybrid
DNA immobilized on a membrane by the isomerization method
Specific binding based on the complementarity of
Exposure to DNA detected the bound DNA band. (3) Identification of nucleic acid markers DNA of the above (1)-(a) DNA extract,
For the fragment, the DNA band bound to the RFLP probe
Blast resistance genePi-ta ^TwoHave
DNA and DNAPi-ta ^TwoHave
Mark bands that are not present in the DNA
From among them, F_TwoAnalysis of genesPi-ta ^TwoFrom
Select DNA bands located within a distance of 2.0 cM
And transform those bands into genesPi-ta ^TwoNucleic acid markers
-. Table 1 shows thatPi-ta ^TwoNeighborhood
Probe hybridized to a nucleic acid marker
Type, size and used for cutting genomic DNA
Restriction enzymes were indicated. [0018] [Table 1] These markers are also used in rice genomic libraries.
It is a genePi-ta ^TwoRice products with
Pi-ta when derived from a species^TwoGood mar
It was confirmed that it was used as a car. Next, the characteristics of these nucleic acid markers are examined.
The following shows a test example performed for this purpose. Test example 1 About the nucleic acid marker obtained in Example 1, blast resistance
Genes were introduced from Indian rice by repeated backcrossing
Near isogenic lines (NIL_STest its behavior in
did. Derived from Indian ricePi-ta ^TwoAnd
AndPi-taGenes are known to occupy the same locus
And each of them is subjected to repeated backcrossing as shown in Figure 1.
More than four and three systems have been introduced. Provision of these
In parent, backparent, and the resulting near isogenic lineage
hand,Pi-ta ^TwoExamine nearby RFLPs and find out which chromosome
Revealed whether the part was introduced from the Indian type. DNA was obtained from each of the varieties shown in FIG.
Extract and use the RFLP probe in Table 1 to extract the Southern block.
To determine which nucleic acid marker
Part of each near-isogenic line is an Indian-type donor parent
It was examined whether the chromosome had been inherited (FIG. 2). The result
FruitPi-ta ^TwoThe position on the chromosome map is indicated by the shaded area in FIG.
It was estimated to be in the range indicated by. Test example 2 Blast resistance genePi-ta ^TwoOn the RELP map
F to determine the position of_TwoAnalysis was performed. Blast resistance genePi-ta ^Twohave
Variety PiNo. No. 4 and No. 22 which do not have this
Mating between many hybrid second generations (F_Two) Got. This
F _TwoRice blast resistance genePi-ta ^TwoAnd nucleic acid mer
Determine the recombination rate between the car and the genetic distance between the two
The required experiment was performed. F_TwoInoculate about 400 seeds of
Conidia of rice blast strain Kita 1 at about 5 leaf stage
1 × 10^Five/ Ml of the suspension by spray inoculation of the leaves,
After 24 hours at 25 ° C and 100% relative humidity, return to greenhouse
7 days after inoculation, the resistance and susceptibility of each individual in the presence or absence of lesions
The sex was determined. Here, 84 individuals determined to be susceptible were
And extract DNA from each of them.
Hybridization using RFLP probe
Test to determine if each probe is Indian or Japanese
Was done. As a result, XNpb088 and 316
No recombination was found with the illibridized nucleic acid marker
And the genetic distance between the gene and the resistance gene is 0.6c
M or less. This distance is
It corresponds to a distance of about 60 to 120 kb or less, and artificially dyes yeast.
In the chromosome (YAC), a 200-300 kb genomic fragment
Considering that it can be obtained easily,
Position cloning is possible
You. Also hybridized to another RFLP probe
Nucleic acid markers also show that their RFLP is 2.0
Because it is located within a distance of cM,Pi-ta ^TwoHeredity
Suggested to function as a good marker for offspring
(FIG. 3). Note that F_TwoAnalyze only sensitive individuals
Spray inoculation was sufficient for individuals with no lesions
Susceptible individuals that are apparently resistant
On the contrary, a large number of resistant individuals
The potential for lesions is extremely small. Therefore,
This improves the accuracy of the test. Also the resistance
Because sexual genes are dominant, sensitive individualsPi-ta ^Twoand
Pi-taBecomes a Japanese recessive homozygous at the locus
I have. Electrophoretic band pattern of the marker in the vicinity
Are expected to be Japanese type, but due to recombination
One of the two homologous chromosomes is indian
However, it is easy to detect this. Therefore, the population
Can detect recombination on the chromosome twice as high
Thus, the efficiency of the measurement of the recombination rate is doubled. These two
The advantage is finding the distance between two genes with very low recombination rates.
Is very important for From the results of Test Examples 1 and 2 above, Example 1
The nucleic acid marker obtained inPi-t
aandPi-ta ^TwoFrom 0 to 2.0c on the genetic map
Located at a distance of MPi-taandPi-ta
^TwoHas been confirmed to be a good marker for
Was. [0026] As described in detail above, according to the present invention,
What is the rice blast resistance genePi-ta ^TwoOrP
i-taA good nucleic acid marker is provided. This
Blast resistance from various rice plants
Easy isolation of sex genes and related genes
And the development and breeding of excellent varieties are promoted. Ma
In addition, it is possible to easily perform a resistance test on the rice.
It opens up new ways to construct new resistance genes. [0027]

[Brief description of the drawings] [0028] Fig. 1 Blast resistance genePi-taandPi-
ta ^TwoIs a genealogy diagram showing the introduction of Japanese to rice. under
Lines indicate varieties with each resistance gene. [0029] FIG. 2 Pi-ta^TwoNear-isogenic lines with genes
FIG. 1 is a schematic diagram showing rice chromosome 12. Black or gray
The shaded area indicates an area derived from Indian rice. [0030] FIG. 3 Blast resistance genePi-ta ^TwoIndicates the position of
FIG. 4 is a partially enlarged schematic diagram of the rice chromosome 12 obtained.
(A) shows PiNo. 4 and Norin 22
F _TwoAnalysis (b) shows Kasalath (Indian rice) and Koshi
F by the combination of Hikari (Japanese rice)_TwoTable analysis
(C) is expected from these results and the result of FIG.
Pi-ta ^TwoIndicates the range of positions. The small key isPi-
taWhenPi-ta ^TwoIf is strictly a double allele,
A good key indicates the range otherwise.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Naoki Katsura 1144-209 Yatabe, Tsukuba, Ibaraki (72) Inventor Seiya Sato 3-7-2-106, Azuma, Tsukuba, Ibaraki (72) Inventor Ikuo Ando Ibaraki 1-11481-1- 402-1003, Azuma, Tsukuba-shi, Prefecture (72) Inventor Akira Saito 4-11-1-416- 303, Matsushiro, Tsukuba-shi, Ibaraki (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/09 C12Q 1/68 C12N 15/00

Claims (1)

(57) [Claim 1] In rice chromosome 12 genomic DNA, rice RFLP probe XNpb316 is located from the position where rice RFLP probe XNpb289 hybridizes.
Is a genomic region where the rice blast resistance gene Pi-ta ² is present, wherein the rice blast resistance gene Pi-ta ^{2 is located.}
Genomic region identification method.