JP4437895B2 - Novel genetic markers for non-droplet-free genes and their usage - Google Patents

Description

【図面の簡単な説明】
【図１】オオムギの小穂非脱落性についてのＱＴＬ解析の結果を示す図である。
【図２】本実施の一形態における遺伝マーカーを位置付けたオオムギ３ＨＳ染色体における連鎖地図を示す図である。
【図３】本実施の一形態における遺伝マーカー▲１▼：e05m15-09の塩基配列を示す図である。
【図４】本実施の一形態における他の遺伝マーカー▲２▼：e23m23-07の塩基配列を示す図である。
【図５】本実施の一形態における他の遺伝マーカー▲３▼：e50m21-01の塩基配列を示す図である。
【図６】本実施の一形態における他の遺伝マーカー▲４▼：e09m25-08の塩基配列を示す図である。
【図７】本実施の一形態における他の遺伝マーカー▲５▼：e45m11-11の塩基配列を示す図である。
【図８】本実施の一形態における他の遺伝マーカー▲６▼：e30m07-09の塩基配列を示す図である。
【図９】本実施の一形態における他の遺伝マーカー▲７▼：e40m19-08の塩基配列を示す図である。
【図１０】本実施の一形態における他の遺伝マーカー▲８▼：e39m23-11の塩基配列を示す図である。
【図１１】本実施の一形態における他の遺伝マーカー▲９▼：e04m55-09の塩基配列を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel genetic marker, in particular, a novel genetic marker in the barley 3HS chromosome, and a method for using the same.
[0002]
[Prior art]
Since the late 1980s, genetic markers (eg, DNA markers) and linkage maps (chromosome maps) using them have been rapidly produced in plants. The development of this DNA marker detection technology has greatly advanced the conventional linkage analysis that uses a marker gene involved in morphological traits as a genetic marker. For example, in rice, not only has the efficiency of linkage analysis of traits involving a single gene typified by resistance to insects and insects involved, but also multiple genes that have been difficult to analyze by conventional methods are involved. With respect to traits (quantitative traits), it became possible to analyze the gene loci involved by DNA markers.
[0003]
Here, the “quantitative character” will be briefly described. In contrast to the single dominant gene-dominated trait, many agriculturally important traits typified by heading time and culm length often show continuous variation in hybrid progeny. In general, such traits are often determined by the action of a plurality of genes, and are collectively called quantitative traits. Many traits that are targeted for improvement in crop breeding, such as yield, quality, and taste, are included in this quantitative trait.
[0004]
Individual loci involved in such quantitative traits are called Quantitative Trait Loci (QTL), and before the use of DNA markers, the form of genetics with a clear linkage and biochemical Attempts have been made by linkage analysis with genes that control traits as markers. However, morphological markers, such as pleiotropic expression, often affect the target trait, and it has been virtually impossible to use morphological markers that are distributed throughout the chromosome.
[0005]
Therefore, a method using a new genetic marker was developed. When nuclear DNA is cleaved with a restriction enzyme, the length of the DNA fragment may vary depending on the individual or strain. This is called a DNA polymorphism. Since DNA polymorphism has Mendelian inheritance like the gene that controls normal morphological traits, if these are used as genetic markers and the degree of mutual genetic linkage is estimated and used as an index of distance, the marker A linkage map showing the positions on each chromosome can be created. This marker is called an RFLP (Restriction Fragment Length Polymorphism) marker. In the 1980s, DNA markers represented by RFLP markers were developed, and quantitative trait loci mapping became realistic.
[0006]
By using a detailed linkage map with a DNA marker such as the above RFLP marker, linkage analysis of a locus (QTL) governing quantitative traits has become possible. Furthermore, even if the number of DNA markers on the linkage group increases, if there is no corresponding DNA marker, a gap is formed on the linkage map. However, there is a possibility that the gap is filled by changing the type of the DNA marker, and attempts have been made to compensate for the gap on the linkage map created by the RFLP marker using a microsatellite marker or the like. Currently, as shown in FIG. 1, various markers have been developed and used as DNA markers.
[0007]
Furthermore, the QTL analysis is also used for research on disease resistance that is easily affected by the environment, which has been difficult to analyze until now, and genetics such as culture characteristics (for example, see Non-Patent Documents 1 and 2).
[0008]
Incidentally, in plants such as barley, wheat, and rice, various traits have changed in the process of evolution from wild species to cultivated species. In particular, seeds fall off naturally when mature in wild species (droplet-dropping properties), but seeds do not fall off until cultivated seeds are harvested and threshed by humans (droplet-dropping properties). Have earned.
[0009]
This spikelet loss is one of the most important and important changes in the process of evolution from wild species to cultivated species in plants such as barley. For this reason, many researchers are conducting genetic analysis to investigate this genetic property and to trace the evolutionary process engraved in the genetic information.
[0010]
As a result, it was found that the spikelet loss of barley is controlled by two dominant complementary genes: Btr1 and Btr2, and if one of them is a recessive gene (btr1, btr2), the spikelet loss is acquired. It was. In addition, because these genes are closely linked to each other, the spikelet-free cultivars are either btr1 Btr2 or Btr1 btr2 genotypes and have both recessive genotypes (btr1 btr2) The variety was found to be extremely rare. In addition, there is a geographic distribution of spikelet deciduousness, and it is known that the Btr1 btr2 genotype (E type) is common in the east and the btr1 Btr2 genotype (W type) is common in the west. (See Non-Patent Document 3). Moreover, it is known that the recessive genes of btr1 and btr2 are located in the 3HS chromosome of barley (see Non-Patent Document 4). However, since spikelet detachment is a quantitative trait, its analysis is extremely difficult, and genes btr1 and btr2 involved in spikelet detachment have not been isolated or identified yet. On the other hand, several AFLP (amplified fragment length polymorphism) markers located relatively close to btr1 have been developed by recent research results using QTL analysis or the like (see Non-Patent Document 5).
[0011]
[Non-Patent Document 1]
Mano, Y., Takahashi, H., Sato, K and Takeda, K., "Mapping Genes for Callus Growth and Shoot Regeneration in Barley (Hordeum vulgare L.)", Breeding Science, 46: 137-142, 1996.
[0012]
[Non-Patent Document 2]
Graner, A., Streng, S., Kellermann, A., Schiemann, A., Bauer, E., Waugh, R., pellio, B., Ordon, F, `` Molecular mapping and genetic fine-structure of the rym5 locus encoding resistance To different strains of the Barley Yellow Mosaic Virus Complex. '', Theor Appl Genet 98: 285-290, 1999
[0013]
[Non-Patent Document 3]
Takero Konishi, “Genetic differentiation and geographical distribution of barley”, Genetics, 1987, May, (Vol.41, No.5), p6-10
[0014]
[Non-Patent Document 4]
TAKAHASHI, R et al., "Linkage study of two complementary genes for brittle rachis in barely.", Ber. Ohara lnst. Landw. Biol., Okayama Univ. 12: 99-105.
[0015]
[Non-Patent Document 5]
KOMATUDA, T., and Y. MANO, co-authored, `` Molecular mapping of the intermedium spike-c (int-c) and non-brittle rachis 1 (btr1) loci in barley (Hordeum vulgare L.). '', Theor Appl Genet 105: 85-90
[0016]
[Problems to be solved by the invention]
Here, a precondition for the effectiveness of the QTL analysis is that a linkage map with genetic markers (for example, DNA markers) having an accurate order is created. In general, the higher the density between the markers, the higher the accuracy of the position of the newly mapped QTL, and the estimation accuracy of the QTL map by interval mapping tends to be higher as the recombination value between the markers is smaller. . If a higher-density linkage map is used, QTL, that is, gene isolation / identification becomes possible.
[0017]
However, in the conventional barley 3HS chromosome as described above, only the AFLP marker near btr1 does not reveal the exact positions of the two recessive genes btr1 and btr2, and the relationship between the positions of btr1 and btr2 is unknown. . Therefore, there is a problem that a detailed linkage map for isolating and identifying these two recessive genes btr1 and btr2 cannot be prepared.
[0018]
In addition, if the spikelet non-dropping genes btr1 and btr2 can be isolated and identified, the use of these genes may control the drop-off time of cereal seeds. It seems to be a useful gene with high utility value.
[0019]
Therefore, in order to produce a reliable linkage map, which is an effective means for isolating and identifying the genes btr1 and btr2 involved in barley spikelet loss, btr1, in the 3HS chromosome of barley, A genetic marker located near btr2 has been strongly sought.
[0020]
The present invention has been made in view of the above-mentioned problems, and the object thereof is a novel genetic marker, in particular, a novel genetic marker located in the vicinity of the non- spikelet deciduous genes btr1, btr2 in the barley 3HS chromosome, and The purpose is to provide usage.
[0021]
[Means for Solving the Problems]
As a result of intensive investigations in view of the above problems, the present inventors have found that 84 genetic markers are present in the vicinity of btr1 and btr2 and are closely linked to btr1 and btr2 in the 3HS chromosome of barley. The present invention has been completed by mapping the genetic marker, creating a linkage map, and further identifying the base sequence of 9 genetic markers out of 84 genetic markers.
[0022]
That is, the genetic marker according to the present invention is a genetic marker obtained by treating barley genomic DNA with AFLP, the same position as the position of the barley spikelet-free genes btr1, btr2 on the chromosome, and the above From the positions of btr1 and btr2 on the chromosome 0.5cM, 1.0cM and 2.0cM on the telomere side, and from the positions of btr1 and btr2 on the chromosome 2.7cM, 3.2cM and 3.7cM, It is characterized by being seated at any position selected from 4.2 cM and 4.7 cM centromere side positions.
[0023]
The above genetic marker is located near the position of the barley spikelet non-dropping genes btr1 and btr2 on the chromosome, and is closely linked to btr1 and btr2. For this reason, it is considered that the barley spikelet non-dropping genes btr1 and btr2 can be cloned by using the above genetic marker.
[0024]
The “position on the chromosome of barley spikeless genes btr1 and btr2” is considered to be a position of 50 cM from the telomere side and 20 cM from the centromere side in the 3HS chromosome of barley.
[0025]
The genetic marker according to the present invention is characterized by comprising a polynucleotide having the base sequence shown in the following (a) or (b) or a part thereof. (A) The base sequence shown in any one of SEQ ID NOs: 1 to 9. (B) a base sequence in which one or more bases are substituted, deleted, inserted, and / or added in the base sequence shown in any of SEQ ID NOs: 1 to 9, and the spikelet of the plant is not lost A nucleotide sequence linked to a gene or QTL involved in sex.
[0026]
These genetic markers are linked to the btr1 and btr2 genes involved in barley spikelet defoliation, enabling marker assisted selection in crop breeding as well as linkage mapping. It can be used as a base for map-based cloning using map information. That is, according to the above genetic marker, a linkage map useful for QTL analysis or the like for isolating and identifying a gene involved in barley spikelet defoliation can be prepared. Furthermore, it becomes possible to selectively breed a spikelet non-dropping plant using the genetic marker as an index.
[0027]
In addition, it is preferable that the said plant is a gramineous plant. In particular, the genetic marker according to the present invention is preferably derived from barley. Further, it is preferably a genetic marker in the barley 3HS chromosome. Furthermore, the genetic marker is preferably an AFLP marker.
[0028]
The genetic marker group according to the present invention includes at least one of the above genetic markers, and is characterized in that it is used to create a linkage map of barley chromosomes. Furthermore, the genetic marker group according to the present invention preferably includes at least one marker selected from restriction enzyme fragment length polymorphism (RFLP) marker, AFLP marker, STS marker and microsatellite marker.
[0029]
By using the above genetic marker group, a more accurate linkage map can be created.
[0030]
In addition, the linkage map according to the present invention is characterized by being created using the genetic marker group described above. According to the linkage map, since many genetic markers linked to the spikelet non-dropping gene or QTL are used, the position of the spikelet non-dropping gene can be identified more accurately.
[0031]
In addition, the method for isolating a spikelet deciduous gene according to the present invention uses at least the genetic marker, genetic marker group, or linkage map described above, and the locus of a gene involved in spikelet deciduousness from a plant chromosome. It includes a step of specifying the riding position. According to the above method for isolating spikelet-free genes, spikelet-free genes can be efficiently isolated.
[0032]
Moreover, in the method for isolating the spikelet non-dropping gene according to the present invention, it is preferable to use the genetic marker as a primer or a probe. Moreover, it is preferable that the process of specifying the locus position of the said gene includes the process of performing the QTL analysis about the gene involved in the spikelet non-dropping property of barley.
[0033]
In addition, the method for selecting plant individuals according to the present invention is characterized by selecting plantlets that are not deciduous into spikelets using the genetic marker, genetic marker group, or linkage map described above.
[0034]
According to the above configuration, it is possible to easily and efficiently select a plant individual that does not fall off spikelets using the genetic marker, genetic marker group, or linkage map as an index.
[0035]
DETAILED DESCRIPTION OF THE INVENTION
In the process of evolution from wild to cultivated barley, barley has acquired the property that it does not drop seeds until it is maturated by humans and does not drop seeds. It is known that the genes btr1 and btr2 (non-brittle rachis 1 (btr1) and 2 (btr2)) involved in barley spikelet loss are located on the barley 3HS chromosome. However, spikelet detachment is a quantitative trait and simple isolation and identification of btr1 and btr2 is difficult, and it is considered that analysis through QTL analysis is the most efficient. However, the positions of btr1 and btr2 cannot be accurately specified only with DNA markers near btr1 and btr2, which have been known so far.
[0036]
Accordingly, the present invention provides a novel genetic marker and a method for using the same in order to more accurately identify the positions of btr1 and btr2 in the 3HS chromosome. The final objective is to isolate and identify btr1 and btr2, which are spikelet non-dropping genes, using these inventions. Hereinafter, an embodiment of the genetic marker of the present invention and a method for using the same will be described in order based on FIGS. Note that the present invention is not limited to this. Further, in the present invention, the spikelet non-dropping genes btr1 and btr2 are sometimes referred to as genes involved in spikelet non-dropping property, spikelet non-dropping genes, or simply btr1 and btr2.
[0037]
(1) Genetic marker according to the present invention
As described above, the genetic marker according to the present invention is a genetic marker obtained by subjecting barley genomic DNA to AFLP treatment, and is the same position on the chromosome of the barley spikelet-free genes btr1 and btr2. , And btr1 and btr2 from the position on the chromosome 0.5cM, 1.0cM and 2.0cM respectively on the telomere side, and btr1 and btr2 from the position on the chromosome 2.7cM, 3.2cM, 3 respectively Any genetic marker may be used as long as it is seated at any position selected from positions on the centromere side of .7 cM, 4.2 cM, and 4.7 cM.
[0038]
“AFLP treatment” as used in the present invention refers to an adhesive which is prepared by treating a DNA sample prepared from a test plant (barley) with two types of restriction enzymes and then cleaving with the restriction enzymes, as shown in the examples described later. A predetermined AFLP adapter that selectively binds to the ends is connected, PCR is performed using a primer complementary to the adapter, a primary amplification product of the restriction enzyme fragment is obtained, and this primary amplification product is obtained. PCR is carried out selectively using a primer that is complementary to the above-mentioned AFLP adapter as a template and a selected nucleotide obtained by extending several bases (three bases in this example) in a restriction enzyme fragment. Any known AFLP treatment (method) for obtaining a genetic marker may be used.
[0039]
For example, EcoRI and MseI can be used as the two types of restriction enzymes, but are not particularly limited. The “primer obtained by adding a selection nucleotide extended several bases into a restriction enzyme fragment” may be any primer that can selectively amplify a genetic marker by PCR using the primary amplification product as a template. The phrase “extended several bases” includes, for example, a primer extended by 3 bases, but is not particularly limited, and may be extended to about 1 base to about 10 bases. Further, other AFLP processing conditions such as PCR can be appropriately set based on conventionally known AFLP processing, and are not particularly limited.
[0040]
Examples of the genetic marker include 84 genetic markers positioned on the linkage map shown in FIG. 2 (in FIG. 2, the upward direction is the telomere side and the downward direction is the centromere side). That is, as genetic markers that sit (position) on “the same positions on the chromosomes of the barley spikelet loss genes btr1 and btr2”, for example, e09m-25-08, e24m42-08, e26m58- There are 10 genetic markers, e30m07-09, e45m11-11, e50m21-01, e57m10-07, e58m61-09-1, e58m61-09-2, and e59m33-13. In addition, genetic markers that sit at the "position of 0.5 cM telomere from the position on the chromosome of the barley spikelet-free deciduous genes btr1, btr2" include e01m12-09, e23m23-07, e40m19-08, for example. 4 genetic markers of e62m32-08. Examples of genetic markers that sit at the “position on the chromosome side of the barley spikelet-free genes btr1 and btr2 on the 1.0 cM telomere side” include e05m15-09 and e51m34-13. Examples of genetic markers that sit on the "barley spikeless genes btr1 and btr2 positions on the 2.0 cM telomere side from the chromosome position" include e07m31-09, e22m24-13-1, and e22m24. 11 genetic markers included in the same frame in the figure as -13-2 etc. In addition, as a genetic marker that sits at the “position of 2.7 cM centromere from the position on the chromosome of btr1, btr2 of barley spikelets”, for example, e37-m44-13 can be mentioned. In addition, genetic markers that sit at the “position on the chromosome side of the barley spikelet non-dropping genes btr1, btr2 from the position of 3.2 cM centromere” include e23m12-13-1, e23m12-13- 2, 4 genetic markers, e23m44-13 and e48m02-08. In addition, as genetic markers seated at “positions on the chromosome side of the barley spikelet non-dropping genes btr1 and btr2 on the 3.7 cM centromere side”, for example, e04m55-09, e06m49-08, etc. Among them, 14 genetic markers included in the same frame can be listed. In addition, examples of genetic markers that sit at the “position of 4.2 cM centromere from the position on the chromosome of barley spikelet-free btr1 and btr2” include e34m11-10, e39m08-08, and the like. Among them, 6 genetic markers included in the same frame can be listed. In addition, genetic markers that sit at the “position of 4.7 cM centromere from the position on the chromosome of the barley spikelet-free deciduous genes btr1 and btr2” include e03m36-13-1 and e03m36-13-, for example. 32 genetic markers included in the same frame in the figure as 2 nd.
[0041]
Among these genetic markers, in particular, 10 genetic markers that sit at the same chromosome position as the spikelet non-dropping genes btr1 and btr2, and the chromosome position of the spikelet non-dropping genes btr1 and btr2 , A position on both sides of the btr1 / btr2 chromosome position “position of 0.5 cM telomere from the position of the barley spikelet non-dropping gene btr1, btr2 on the chromosome” The genetic marker that sits at the position of 2.7 cM centromere from the position of the barley spikelet-free genes btr1 and btr2 on the chromosome is for cloning the spikelet-free genes btr1 and btr2. Very useful to.
[0042]
Moreover, the genetic marker which concerns on this invention should just be a genetic marker which has a base sequence shown by the following (a) or (b) as mentioned above. (A) The base sequence shown in any one of SEQ ID NOs: 1 to 9. (B) A base sequence in which one or more bases are substituted, deleted, inserted and / or added in the base sequence shown in any one of SEQ ID NOs: 1 to 9, and the spikelet of the plant is not lost A nucleotide sequence linked to a gene or QTL involved in sex.
[0043]
As used herein, “QTL (Quantitative Trait Loci)” refers to a quantitative trait locus. “Linkage” refers to a phenomenon in which two or more genes or genetic traits located on the same chromosome are inherited in pairs.
[0044]
The plant is preferably a gramineous plant. The “Poaceae plant” here is not particularly limited as long as it is a plant of the genus Hordeum, the genus Wheat, or the rice. The genetic marker is preferably derived from barley, and may be derived from barley genomic DNA or cDNA. It goes without saying that the properties of barley genus are not particularly limited in terms of properties such as two- and six-rows, nakedness, skininess, parallelism and vorticity. Furthermore, the genetic marker is preferably a genetic marker in the 3HS chromosome of barley.
[0045]
The genetic marker is preferably an AFLP marker. As used herein, “AFLP marker” refers to a sample DNA that is cleaved with two types of restriction enzymes, an adapter is ligated to their sticky ends, specific to the adapter, and a few nucleotides in the restriction enzyme fragment. A genetic marker obtained by amplifying the restriction enzyme fragment using an extended primer.
[0046]
The “polynucleotide” referred to in the present invention includes RNA and DNA. RNA includes mRNA, and DNA includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof. The DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding DNA serving as a sense strand or an anti-coding strand serving as an antisense strand. Furthermore, the “genetic marker” of the present invention includes other base sequences other than the base sequence of (a) or (b), for example, untranslated region (UTR) sequences and vector sequences (including expression vector sequences). It may contain a base sequence such as
[0047]
The genetic marker according to the present invention may be a polynucleotide itself having the base sequence represented by SEQ ID NOs: 1 to 9, or may be a polynucleotide comprising a part of the base sequence. The “part of the base sequence” used herein indicates a base sequence having a length that can be used as a genetic marker, and the length is not particularly limited.
[0048]
In the present invention, “one or more bases are substituted, deleted, inserted and / or added” can be replaced, deleted, inserted and / or added by a known artificial method. It means a mutation in which a certain number of bases are substituted, deleted, inserted, and / or added. Further, the mutation does not have to be an artificially introduced mutation. For example, a polynucleotide having a similar mutation may be isolated from a naturally occurring plant genome and used as a genetic marker according to the present invention.
[0049]
As the genetic marker, in the present embodiment, for example, (1) genomic DNA having the nucleotide sequence represented by SEQ ID NO: 1 (marker name: e05m15-09), (2) the nucleotide sequence represented by SEQ ID NO: 2 is used. Genomic DNA (marker name: e23m23-07), (3) genomic DNA having the base sequence shown in SEQ ID NO: 3 (marker name: e50m21-01), (4) genome having the base sequence shown in SEQ ID NO: 4 DNA (marker name: e09m25-08), (5) genomic DNA having the base sequence shown in SEQ ID NO: 5 (marker name: e45m11-11), (6) genomic DNA having the base sequence shown in SEQ ID NO: 6 ( Marker name: e30m07-09), (7) genomic DNA having the base sequence shown in SEQ ID NO: 7 (marker name: e40m19-08), (8) genomic DNA having the base sequence shown in SEQ ID NO: 8 (marker name) : E 39m23-11), (9) In addition to genomic DNA having the base sequence shown in SEQ ID NO: 9 (marker name: e04m55-09), mRNA having a base sequence corresponding to the base sequence of the genomic DNA is exemplified. .
[0050]
Hereinafter, the genetic markers (DNA markers) (1) to (9) will be described in detail. Note that the genetic markers (1) to (9) are all STS markers. Moreover, the sequence | arrangement shown by the underline part in FIGS. 3-11 shows the base sequence of a primer. This is very useful because it can be used as a primer for directly amplifying the DNA marker shown below (not AFLP treatment) using DNA extracted from a plant as a template.
[0051]
(1) DNA marker: e05m15-09
e05m15-09 is a DNA marker having a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1, which is located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0052]
Specifically, as shown in FIG. 3, e05m15-09 is a DNA marker consisting of 166 base pairs and is an AFLP marker amplified in the barley variety “Azumamugi”, but in the barley variety “Kanto Nakate Gold” Is not amplified.
[0053]
(2) DNA marker: e23m23-07
e23m23-07 is a DNA marker having a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, and is located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0054]
Specifically, as shown in FIG. 4, e23m23-07 is a DNA marker consisting of 280 base pairs and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”, but in the barley variety “Azumamugi” Is not amplified.
[0055]
(3) DNA marker: e50m21-01
e50m21-01 is a DNA marker having a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 3, which is located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0056]
Specifically, as shown in FIG. 5, e50m21-01 is a DNA marker consisting of 1209 base pairs and an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. Furthermore, the position of the base “A (adenine)” indicated by a double underline in the figure is a position having a single nucleotide polymorphism (also referred to as SNPs (Single Nucleotide Polymorphisms), snips), and the single nucleotide polymorphism is a restriction enzyme. Can be detected by Ava II.
[0057]
(4) DNA marker: e09m25-08
e09m25-08 is a DNA marker having a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4, which is located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0058]
Specifically, as shown in FIG. 6, e09m25-08 is a DNA marker consisting of 207 base pairs, and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”, but in the barley variety “Azumamugi” Is not amplified.
[0059]
(5) DNA marker: e45m11-11
e45m11-11 is a DNA marker having a polynucleotide of the base sequence shown in SEQ ID NO: 5, located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0060]
Specifically, as shown in FIG. 7, e45m11-11 is a DNA marker consisting of 105 base pairs and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”, but in the barley variety “Azumamugi” Is not amplified.
[0061]
(6) DNA marker: e30m07-09
e30m07-09 is a DNA marker having a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 6, which is located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0062]
Specifically, as shown in FIG. 8, e30m07-09 is a DNA marker consisting of 166 base pairs and an AFLP marker amplified in the barley variety “Azumamugi”, but in the barley variety “Kanto Nakate Gold” Is not amplified.
[0063]
(7) DNA marker: e40m19-08
e40m19-08 is a DNA marker having a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 7, which is located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0064]
Specifically, e40m19-08 is a DNA marker composed of 247 base pairs as shown in FIG. 9, and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. Furthermore, the position of the base “A (adenine)” indicated by a double underline in the figure is a position having a single nucleotide polymorphism, and the single nucleotide polymorphism can be detected by the restriction enzyme DraI.
[0065]
(8) DNA marker: e39m23-11
e39m23-11 is a DNA marker having a polynucleotide of the base sequence shown in SEQ ID NO: 8, located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0066]
Specifically, as shown in FIG. 10, e39m23-11 is a DNA marker consisting of 95 base pairs and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”, but in the barley variety “Azumamugi” Is not amplified.
[0067]
(9) DNA marker: e04m55-09
e04m55-09 is a DNA marker having a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 9, which is located in the vicinity of the spikelet non-dropping genes btr1 and btr2, and linked to the spikelet non-dropping gene or QTL. It is a DNA marker.
[0068]
Specifically, e04m55-09 is a DNA marker consisting of 164 base pairs as shown in FIG. 11, and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. Furthermore, the position of the base “C (cytosine)” indicated by the double underline in the figure is a position having a single nucleotide polymorphism, and the single nucleotide polymorphism can be detected by the restriction enzyme Msl I.
[0069]
The genetic markers of (1) to (9) above are fragments that are close to the positions of the spikelet non-dropping genes btr1 and btr2 on the chromosome among the 84 genetic markers shown in the examples and FIG. The nucleotide sequence is specified for the reason that it is not too short (about 100 bp) and the fragment size is not too small.
[0070]
As described above, the genetic markers (1) to (9) are considered to be closely linked to the spikelet non-dropping genes btr1 and btr2, and can be used as genetic markers for btr1 and btr2. Therefore, the genetic markers of (1) to (9) above can be used not only for marker selection in crop breeding but also for the preparation of linkage maps in the barley 3HS chromosome, and btr1 using the map information, It can also be used to isolate btr2.
[0071]
Moreover, a partial sequence of the genetic marker according to the present invention can be used as a probe or primer. As such a probe, for example, a so-called DNA chip (including a microarray) in which a partial sequence of the genetic marker of the present invention is fixed on a carrier such as a chip or silicon is used. Examples include cases used for inspection and diagnosis.
[0072]
Further, if the sequence of the probe is selected from the nucleotide sequence of the genetic marker according to the present invention and a genomic DNA (or cDNA) library of wheat, rice, or other plants is screened, the same as the genetic markers described above. It is highly possible to isolate a genetic marker encoding a homologous molecule or a similar molecule having the above functions.
[0073]
Therefore, by using the above genetic marker, it is possible to isolate and identify not only barley but also other plants such as wheat, rice and other genes involved in spikelet defoliation by QTL analysis. It is considered to be.
[0074]
The genetic marker production method according to the present invention is produced by subjecting DNA extracted from barley leaves to AFLP treatment, as will be described in detail in the above method and the examples described later.
[0075]
Specifically, a DNA sample prepared from a test plant is first treated with two types of restriction enzymes (in this example, EcoRI and MseI), and then a predetermined AFLP adapter is attached to the sticky ends cleaved by the restriction enzymes. Then, PCR is performed using a primer complementary to the adapter, and an amplification product of the restriction enzyme fragment is obtained. Using this amplified product as a template, PCR was performed using a primer obtained by adding a selected nucleotide obtained by extending several bases (three bases in this example) in the restriction enzyme fragment to the above-mentioned primer to obtain a genetic marker. Yes. The obtained amplification product can be analyzed by electrophoresis, and the band pattern can be compared with the parent cultivar to determine which parental trait it has.
[0076]
Of course, the method for producing the genetic marker according to the present invention is not limited to this, and it may be produced using other known methods. That is, in the present invention, any method may be adopted as long as it can produce the above-described genetic marker (SNP marker) or a group of genetic markers including the genetic marker.
[0077]
(2) Use of genetic marker according to the present invention (usefulness)
As a use of the genetic marker according to the present invention, it can be widely used as a marker for tracking the location of a target gene in experiments such as gene cloning, cell fusion, and mating. In particular, among the genetic markers according to the present invention, those containing single nucleotide polymorphisms (SNPs) in the nucleotide sequence have the advantage of being easy to detect.
[0078]
In addition, the organism to be a target using the genetic marker according to the present invention is not particularly limited, but since these genetic markers are derived from barley, particularly preferred targets include barley and wheat series to which the barley belongs, Menaceae can be mentioned. Of course, depending on the application, plants other than Gramineae plants and animals in some cases can be targeted.
[0079]
The genetic markers according to the present invention can be used alone for the various uses described above, but in particular, in order to use them for preparing a linkage map of barley chromosomes, it is preferable to use a plurality of them as a genetic marker group. The “linkage map” in the present invention is a linkage map in a broad sense including what is called a genetic map, a genetic map, or a chromosome map. Therefore, hereinafter, the linkage map may be referred to as a genetic map, a genetic map, or a chromosome map.
[0080]
The linkage map obtained by mapping the genetic marker group according to the present invention can be suitably used for the purpose of specifying the target gene from the chromosome. In the present invention, the spikelet non-dropping gene btr1 is particularly preferred. , Btr2 can be suitably used for specifying the seating position.
[0081]
As mentioned above, the spikelet non-dropping genes btr1 and btr2 may be able to control the drop-off timing of grain seeds, and are considered to be useful genes that are extremely useful in agriculture. It is. Thus, the spikelet non-dropping gene is useful both academically and industrially, and if the genetic marker or genetic marker group according to the present invention is used, at least the spikelet non-dropping gene is effectively obtained from barley. It becomes possible to isolate and identify.
[0082]
The genetic marker group according to the present invention only needs to include the above-described genetic markers composed of oligonucleotides, but may further include other genetic markers. The other genetic marker is not particularly limited as long as it can be used as a genetic marker on the chromosome, and specifically, for example, a restriction fragment length polymorphism (RFLP) marker. And conventionally known genetic markers such as microsatellite markers (SSR (Simple Sequenced Repeat) markers), AFLP markers, and STS (Sequence Tagged Site) markers.
[0083]
As will be described in the examples described later, in the present invention, genetic markers converted to STS are prepared from the barley genome by AFLP. Therefore, since the genetic marker according to the present invention includes a specific base sequence that exists only on the genome, it is more useful than the RFLP marker or the SSR marker. However, if the genetic marker according to the present invention and other RFLP markers, SSR markers, AFLP markers, and STS markers are used in combination as a genetic marker group, a more detailed linkage map can be created. Is further improved.
[0084]
For example, in the present invention, as shown in FIG. 2, a genetic map according to the present invention and other AFLP markers can be combined to create a barley linkage map. In this linkage map, genetic markers are located on the 3HS chromosome of barley as follows. In FIG. 2, the upper direction is the telomere side and the lower direction is the kinematic body side.
[0085]
That is, as shown in FIG. 2, 86 genetic markers are located in the barley 3H chromosome. Among them, the nine genetic markers according to the present invention shown in (1) to (9) above are all located in the vicinity of the spikelet non-dropping genes btr1 and btr2 (shaded in the figure). marker). Specifically, conventionally known genetic markers: e14m-27-04-01 and e15m19-07 (see non-patent document 5, marker shown in bold in FIG. 2) are shown in FIG. A distance of about 2.0 cM on the telomere side and 4.7 cM on the centromere side from the region containing the non-dropping genes btr1 and btr2, respectively. On the other hand, the genetic markers (3) to (6) were found to be contained in the same region as that containing btr1 and btr2. The genetic marker (1) is about 1.0 cM away from the region containing btr1 and btr2 to the telomere side, and the genetic markers (2) and (7) are telomere from the region containing btr1 and btr2. The genetic markers (8) and (9) are separated by about 3.7 to 4.7 cM from the region containing btr1 and btr2 to the centromere side. In addition, the full length of 3H chromosome shown in FIG. 2 is 9.9 cM.
[0086]
That is, all the genetic markers according to the present invention are located closer to the spikelet non-dropping genes btr1 and btr2 than the previously published genetic markers: e14m-27-04-01 and e15m19-07. I found out to ride. That is, since the genetic marker according to the present invention is more closely linked to the spikelet non-dropping genes btr1 and btr2, the usefulness of the present invention is also apparent from this point.
[0087]
As described above, the linkage map according to the present invention is not particularly limited as long as it is a linkage map in the barley chromosome prepared using the above genetic marker group.
[0088]
As described above, in the linkage map, the higher the density between the markers, the higher the accuracy of the position of the gene. As described above, the linkage map according to the present invention is a high-density linkage map. It may be possible to isolate and identify a non-drop-off gene.
[0089]
In addition, the conventionally well-known method can be utilized for the production method of the linkage map concerning this invention, It does not specifically limit. For example, the usual method using calculation software can be mentioned (LANDER, E., P. GREEN, J. ABRAHAMSON, A. BARLOW, M. DALY et al., 1987 Mapmaker: an interactive computer package for constructing primary genetic linkage Genomics 1: 174-181. or LINCOLN, S., M. DALY and E. LANDER, 1993 Constructing genetic linkage maps with Mapmaker / Exp 3.0.Whitehead Institute for Biomedical Research Technical Report. 3rd ed , Cambridge, MA, USA.
[0090]
In addition, the genetic marker group according to the present invention preferably includes a genetic marker located on the 3HS chromosome of barley for use in identifying a spikelet non-dropping gene. As described above, it has been reported that the spikelet non-dropping gene in barley sits on the 3HS chromosome. Therefore, in order to prepare a genetic map used for isolating and identifying the gene, it is highly preferable that it contains many genetic markers located on the 3HS chromosome.
[0091]
As described above, by using the genetic marker or genetic marker group according to the present invention, a genetic map for identifying a spikelet non-dropping gene can be created. For this reason, the present invention uses a genetic marker or genetic marker group, or the linkage map, to identify a spikelet-dropping gene comprising a step of identifying the locus position of a spikelet-dropping gene from a plant chromosome. Isolation methods (cloning methods) are included.
[0092]
The method of cloning the spikelet non-dropping gene is not particularly limited, and a known method can be used. For example, the spikelet non-dropping gene may be isolated and identified by using the genetic marker or genetic marker group according to the present invention as a primer or a probe.
[0093]
In addition, based on the above-mentioned genetic marker, or a linkage map in which the genetic marker group is located, the position of the spikelet non-dropping gene is identified and cloned in more detail, so-called map-based cloning is performed, It is also possible to isolate and identify spikelet non-dropping genes. One specific method for that purpose is a method using so-called QTL analysis.
[0094]
The QTL analysis method uses any one of the above genetic markers, genetic marker groups, or linkage maps (hereinafter sometimes referred to as genetic markers) and the genes btr1 and btr2 involved in barley spikelet loss Any other method, method, etc. can be used as long as it is a method for performing QTL analysis with respect to the above, and a conventionally known QTL analysis method can be used, and is not particularly limited.
[0095]
The principle of QTL analysis begins by classifying each individual of the hybrid population into a genotype group of markers and comparing their average values. If the marker and one of the QTLs involved in the analysis target trait are linked, a bias occurs between the average values of the genotype groups. If they are not chained, there is no bias between them. An analysis in which this is developed and associated between markers without markers is a QTL analysis by an interval mapping method. When this analysis is repeated using markers distributed throughout the chromosomes of the species to be analyzed, the chromosomal region involved in the analysis target trait is revealed.
[0096]
Among important items in the QTL analysis, it is important to select a large difference regarding the trait to be analyzed as a mating parent, that is, selection of genetically different varieties. Thereby, it can be expected that the main one in QTL involved in the trait is detected.
[0097]
That is, “QTL analysis” may be QTL mapping. Further, specific methods for isolating the mapped QTL include the following methods. First, two varieties of parent individuals are mated to produce a large number of lines as experimental materials. Next, the genome of this lineage group is analyzed using the target trait as an index, and genetic markers (DNA markers) considered to be present in the vicinity of the target gene are statistically analyzed and found. Using this genetic marker, a linkage map is prepared, and backcrossing is repeated with one parent using each genetic marker as an index to produce an individual in which only the periphery of the target gene is derived from the other parent. The individual obtained here is called a quasi-isogenic line, and using this quasi-isogenic line, the target QTL, that is, the gene is isolated by narrowing down the position of the target gene on the chromosome. .
[0098]
According to the QTL analysis method of the present invention, since the above genetic marker or the above linkage map is used, as a result of performing QTL analysis on the genes involved in barley spikelet loss, the results of btr1 and btr2 The accuracy and reliability of the position can be improved. Therefore, it is considered that the positions of genes btr1 and btr2 involved in barley spikelet detachment can be more accurately identified by the QTL analysis of the present invention, and the genes can be isolated and identified.
[0099]
Furthermore, for example, a plant (individual) having a spikelet non-dropping property can be selected using any of the above genetic markers, genetic marker groups, or linkage map as an index. The plant (individual) selection method is not particularly limited as long as the above genetic marker or the like is used as an index, and the other steps, conditions, materials, and the like are not particularly limited.
[0100]
As a specific method of the plant selection method, a conventionally known method in plant breeding can be used and is not particularly limited. Specifically, for example, there is a method in which genomic DNA of a plant produced by crossing or the like is extracted, and the plant is selected using as an index whether or not it has the genetic marker or genetic marker group according to the present invention. In addition, as a means for detecting the genetic marker or genetic marker group, for example, a method using a DNA chip on which a partial sequence of the genetic marker or the like is immobilized, or a partial sequence of the genetic marker or the like is used as a primer A conventionally known method such as a method of amplifying the genetic marker by PCR as described above can be used and is not particularly limited.
[0101]
In addition, for example, there is a possibility that a plant having spikelet detachability can be produced using any one of the above genetic markers as an index. The said plant production method should just use the said genetic marker as a parameter | index, Other processes, conditions, materials, etc. are not specifically limited.
[0102]
As a specific method of the plant production method according to the present invention, a conventionally known technique or genetic engineering technique in plant breeding can be used and is not particularly limited. Specifically, for example, a method of producing a plant individual having the genetic marker by mating a plant having the genetic marker as a parent individual, or a gene introduction using a DNA region containing the genetic marker using a vector, etc. A method for producing a plant having the above-described genetic marker by a genetic engineering technique is mentioned. Whether the produced plant individual has the genetic marker can be easily examined using the above-described DNA chip or PCR method.
[0103]
The “plant” referred to here is preferably a gramineous plant such as barley, wheat, and rice.
[0104]
As described above, according to the plant selection method, it is possible to select a plant having a spikelet non-dropping property with a very simple and high probability. Moreover, according to the said plant production method, the plant which has a spikelet non-dropping property can be produced very easily and with high probability.
[0105]
Embodiments will be described below in more detail with reference to the accompanying drawings. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.
[0106]
【Example】
Genetic markers were prepared for the purpose of isolating and identifying the genes btr1 and btr2 of the barley spikelet-free genes btr1 and btr2 in detail. Specifically, it was performed as follows.
[0107]
[1] Experimental materials and experimental methods
[1-1] Plant used
In this example, the following barley varieties were used. Hordeum vulgare ssp. Vulgare cvs. Azumamugi, Kanto Nakate Gold (Barley Breeding Laboratory, National Institute of Crop Science, Tsukuba).
[0108]
Genomic DNA was extracted from the above barley cultivar and used to prepare the following AFLP markers.
[0109]
[1-2] Preparation of AFLP marker
The AFLP marker was basically produced according to the method of MANO et al. (MANO, Y., et al., “Construction of a genetic map of barley (Hordeum vulgare L.) cross' Azumamugi 'x' Kanto Nakate Gold 'using a simple and efficient amplified fragment-length polymorphism system. "Genome 44: 284-292). Specifically, an AFLP adapter that specifically binds to the restriction enzyme fragments after binding a DNA sample prepared from a plant of the barley variety with restriction enzymes EcoRI and MseI. Connected. As the AFLP adapter, an E-side AFLP adapter that binds to the sticky end by the restriction enzyme EcoRI and an M-side AFLP adapter that binds to the sticky end by the restriction enzyme MseI were used. These AFLP adapters are double-stranded DNAs. Specifically, E-adapter1 having the base sequence shown in SEQ ID NO: 10 and E-adapter having the base sequence shown in SEQ ID NO: 11 are used as the E-side AFLP adapter. adapter2 was used as a mixture, and M-adapter1 having the base sequence shown in SEQ ID NO: 12 and M-adapter2 having the base sequence shown in SEQ ID NO: 13 were used as an M-side AFLP adapter.
[0110]
Next, PCR is performed using a primer complementary to the AFLP adapter to obtain a primary amplification product. Specifically, E-000 having the nucleotide sequence shown in SEQ ID NO: 14 is used as a primer complementary to the E-side AFLP adapter, and SEQ ID NO: 15 is used as a primer complementary to the M-side AFLP adapter. M-000 having the base sequence shown was used.
[0111]
Subsequently, PCR was performed using the primary amplification product as a template and a primer complementary to the AFLP adapter, which was a primer obtained by adding three selected nucleotides to E-000 or M-000. As the primer obtained by adding three selected nucleotides to E-000, 64 types of primers e01 to e64 having a base sequence represented by any of SEQ ID NOs: 16 to 79 were used, and the M-000 was used. 64 primers m01 to m64 having a base sequence represented by any of SEQ ID NOs: 80 to 143 were used as primers in which 3 selected nucleotides were added. As a result of the PCR, the obtained amplification product was used as an AFLP marker, and the nucleotide sequence was determined. In addition, detailed conditions, such as PCR, followed the said literature.
[0112]
[1-3] QTL analysis
QTL analysis of Composite interval mapping (CIM) is based on the computer program QTL Cartographer Version 1.14 (Basten et al., “A reference manual and tutorial for QTL mapping.”, Department of Statictics, North Carolina State University, Raleigh, NC, USA) The brittleness (%) of the data was arcsin√p transformed.
[0113]
100 markers were used for the base map. CIM was performed with model 6 (5 background markers and 10 cM window size) prepared by default. Background marker inclusions can be accurately analyzed to enable efficient QTLs mapping. A region including a virtual position associated with a trait is used as a threshold value of 3.0 for a log-likelihood (LOD) score.
[0114]
[1-4] AFLP marker mapping
The method for preparing a linkage map in which the AFLP marker obtained in [1-2] above was prepared was basically according to the method of MANO et al. (MANO, Y., et al., “Construction of a genetic map of barley (Hordeum vulgare L.) cross 'Azumamugi' x 'Kanto Nakate Gold' using a simple and efficient amplified fragment-length polymorphism system. ", Genome 44: 284-292). Specifically, it was performed by the usual method using calculation software (LANDER, E., P. GREEN, J. ABRAHAMSON, A. BARLOW, M. DALY et al., 1987 Mapmaker: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations.Genomics 1: 174-181., or LINCOLN, S., M. DALY and E. LANDER, 1993 Constructing genetic linkage maps with Mapmaker / Exp 3.0.Whitehead Institute for Biomedical Research Technical Report 3rd ed, Cambridge, MA, USA.).
[0115]
[2] Results
First, QTL analysis was performed by the method described in [1-3] above in order to identify the positions of the genes btr1 and btr2 involved in the absence of spikelets in barley. As a result, as shown in FIG. 1, a large peak was detected between the conventionally known AFLP markers: e14m27-4-1 and e15m19-7 in the 3H chromosome of barley. From this, it was considered that the spikelet non-dropping genes btr1 and btr2 sit between AFLP markers: e14m27-4-1 and e15m19-7.
[0116]
Then, in order to construct a genetic marker that sits between AFLP markers: e14m27-4-1 and e15m19-7 and is located closer to the spikelet non-dropping genes btr1 and btr2, [1-2 In this way, 84 AFLP markers were obtained. Next, a linkage map in which these 84 AFLP markers were positioned was prepared according to the method [1-4]. FIG. 2 shows this linkage map. In the figure, markers shown in bold are AFLP markers conventionally known: e14m27-4-1 and e15m19-7. Others are AFLP markers created this time. In FIG. 2, the upper direction is the telomere side and the lower direction is the kinematic body side.
[0117]
As shown in the figure, it was found that all the 84 AFLP markers sit on the chromosome positions of the spikelet-free deciduous genes btr1 and btr2.
[0118]
Then, among the 84 AFLP markers, the base sequences of nine AFLP markers were determined according to the criteria such as closer to the spikelet non-dropping genes btr1 and btr2 and the fragment not too short (about 100 bp). These nine markers are shaded markers in the linkage map of FIG.
[0119]
The base sequences of these nine AFLP markers are shown in SEQ ID NOs: 1 to 9, respectively. Specifically, (1) SEQ ID NO: 1 is the base sequence of e05m15-09, (2) SEQ ID NO: 2 is the base sequence of e23m23-07, (3) SEQ ID NO: 3 is the base sequence of e50m21-01, (4) SEQ ID NO: 4 is the base sequence of e09m25-08, (5) SEQ ID NO: 5 is the base sequence of e45m11-11, (6) SEQ ID NO: 6 is the base sequence of e30m07-09, and (7) SEQ ID NO: 7 shows the base sequence of e40m19-08, (8) SEQ ID NO: 8 shows the base sequence of e39m23-11, and (9) SEQ ID NO: 9 shows the base sequence of e04m55-09.
[0120]
(1) As shown in FIG. 3, e05m15-09 is a DNA marker consisting of 166 base pairs and is an AFLP marker amplified in the barley variety “Azumamugi”, but in the barley variety “Kanto Nakate Gold” Not amplified.
[0121]
(2) As shown in FIG. 4, e23m23-07 is a DNA marker consisting of 280 base pairs and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. In the barley variety “Azumamugi”, Not amplified.
[0122]
(3) As shown in FIG. 5, e50m21-01 is a DNA marker consisting of 1209 base pairs and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. Furthermore, the position of the base “A (adenine)” indicated by a double underline in the figure is a position having a single nucleotide polymorphism (also referred to as SNPs (Single Nucleotide Polymorphisms), snips), and the single nucleotide polymorphism is a restriction enzyme. Can be detected by Ava II.
[0123]
(4) As shown in FIG. 6, e09m25-08 is a DNA marker consisting of 207 base pairs and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. In the barley variety “Azumamugi”, Not amplified.
[0124]
(5) As shown in FIG. 7, e45m11-11 is a DNA marker consisting of 105 base pairs, and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. In the barley variety “Azumamugi”, Not amplified.
[0125]
(6) As shown in FIG. 8, e30m07-09 is a DNA marker consisting of 166 base pairs and is an AFLP marker amplified in the barley variety “Azumamugi”, but in the barley variety “Kanto Nakate Gold” Not amplified.
[0126]
(7) e40m19-08 is a DNA marker consisting of 247 base pairs, as shown in FIG. 9, and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. Furthermore, the position of the base “A (adenine)” indicated by a double underline in the figure is a position having a single nucleotide polymorphism, and the single nucleotide polymorphism can be detected by the restriction enzyme DraI.
[0127]
(8) As shown in FIG. 10, e39m23-11 is a DNA marker consisting of 95 base pairs and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. In the barley variety “Azumamugi”, Not amplified.
[0128]
{Circle around (9)} e04m55-09 is a DNA marker consisting of 164 base pairs as shown in FIG. 11, and is an AFLP marker amplified in the barley variety “Kanto Nakate Gold”. Furthermore, the position of the base “C (cytosine)” indicated by the double underline in the figure is a position having a single nucleotide polymorphism, and the single nucleotide polymorphism can be detected by the restriction enzyme Msl I.
[0129]
These nine AFLP markers are closely linked to the spikelet non-dropping genes btr1 and btr2 because they are located in the vicinity of the spikelet dropping-off genes btr1 and btr2, as shown in the linkage map of FIG. it is conceivable that.
[0130]
In addition, since the linkage map shown in FIG. 2 is a linkage map in which high-density AFLP markers are located, the locus positions of the spikelet non-dropping genes btr1 and btr2 can be accurately identified. The non-dropout genes btr1 and btr2 can be isolated and identified.
[0131]
【The invention's effect】
As described above, according to the genetic marker of the present invention, not only can a highly reliable linkage map be prepared, but also a more accurate QTL analysis can be performed for the genes btr1 and btr2 involved in spikelet loss. There is an effect that it can be performed. Therefore, there is an effect that it becomes a very useful means for isolating and identifying the genes btr1 and btr2 involved in spikelet detachment.
[0132]
[Sequence Listing]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the results of a QTL analysis of barley spikelet detachment.
FIG. 2 is a diagram showing a linkage map in the barley 3HS chromosome in which genetic markers in one embodiment are positioned.
FIG. 3 is a diagram showing the base sequence of genetic marker {circle around (1)}: e05m15-09 in one embodiment of the present invention.
FIG. 4 is a diagram showing the base sequence of another genetic marker {circle around (2)}: e23m23-07 in one embodiment of the present invention.
FIG. 5 is a diagram showing the base sequence of another genetic marker {circle around (3)}: e50m21-01 in one embodiment of the present invention.
FIG. 6 is a diagram showing the base sequence of another genetic marker (4): e09m25-08 in one embodiment of the present invention.
FIG. 7 is a drawing showing the base sequence of another genetic marker {circle around (5)}: e45m11-11 in one embodiment of the present invention.
FIG. 8 is a view showing the base sequence of another genetic marker {circle around (6)}: e30m07-09 in one embodiment of the present invention.
FIG. 9 is a diagram showing the base sequence of another genetic marker (7): e40m19-08 in one embodiment of the present invention.
FIG. 10 is a drawing showing the base sequence of another genetic marker {circle around (8)}: e39m23-11 in one embodiment of the present invention.
FIG. 11 is a drawing showing the base sequence of another genetic marker {circle around (9)}: e04m55-09 in one embodiment of the present invention.

Claims (12)

A genetic marker comprising a polynucleotide having a base sequence represented by any one of SEQ ID NOs: 1 to 9 .   The base sequence is linked to a gene or QTL involved in plant spikelet non-dropping property,
  The genetic marker according to claim 1, wherein the plant is a gramineous plant.   The genetic marker according to claim 1 or 2, wherein the genetic marker is derived from barley.   The genetic marker according to any one of claims 1 to 3, wherein the genetic marker is a genetic marker in barley 3HS chromosome.   The genetic marker according to any one of claims 1 to 4, wherein the genetic marker is an AFLP marker.   A genetic marker group comprising at least the genetic marker according to any one of claims 1 to 5 and used for preparing a linkage map of a barley chromosome.   The genetic marker group according to claim 6, further comprising at least one marker selected from a restriction enzyme fragment length polymorphism marker, an AFLP marker, an STS marker, and a microsatellite marker.   A linkage map of barley chromosomes produced using the genetic marker group according to claim 6 or 7.   Using at least the genetic marker, genetic marker group, or linkage map according to any one of claims 1 to 8 to identify the locus position of a gene involved in spikelet detachment from a plant chromosome; A method for isolating a spikelet non-dropping gene, comprising:   The method for isolating a spikelet non-dropping gene according to claim 9, wherein the genetic marker is used as a primer or a probe.   11. The spikelet detachment property according to claim 9 or 10, wherein the step of specifying the locus position of the gene comprises a step of performing QTL analysis on a gene involved in barley spikelet detachment property. Methods for gene isolation.   A method for selecting a spikelet non-dropping plant individual using the genetic marker, genetic marker group, or linkage map according to any one of claims 1 to 8.

Images