JP5828451B2 - A method for selecting a DNA marker for a novel short-lived late gene on chromosome 3 of rice - Google Patents

Description

  The present invention relates to a method for selecting a short-spotted late gynecaceous plant using a novel short-spotted late-generation gene d63 (t) and a DNA marker for determining the presence of the gene.

  Gramineae plants are extremely important plants in agriculture including rice, wheat, barley, oats, rye, millet, millet, millet, corn, millet, sorghum and the like. However, Gramineae plants with long vines have been laid down by strong winds, such as typhoons, and there has been a problem that the yield of fruits is greatly reduced. Therefore, breeding of Gramineae plants with short pods was carried out for many years, and as a result, the semi-dwarfing gene sd1 was found. sd1 is a C20 oxidase gene-deficient gene in the gibberellin (GA) biosynthetic system, and is a gene that expresses a trait with a short panicle length but a low overall plant height, called semi-dwarfism.

In recent years, a semi-fertile gene d60 has been newly found (Non-patent Documents 1 and 2). When d60 coexists with the gamete lethal gene gal that is ubiquitous in rice, the male and female gametes are lethal. in cross _F 1 (D60d60Galgal) from the product-line (D60D60galgal), next to pollen fertility and seed 75%, progeny _{F 2} in 6 KaMinorucho稈(4D60D60: 2D60d60GalGal): 2 partially unsaturated Minoricho culm (D60d60Galgal = F1 type): It takes a unique mode of inheritance in a ratio of 1 short rod (d60d60GalGal), and Gal is essential for d60 inheritance. d60 is a valuable short rod gene that could not be obtained in nature without simultaneous artificial mutation of Gal.

The semi-dwarf gene d60 was found to be present on the second chromosome of the grass family plant and was shown to be genetically and functionally independent from sd1 present on the first chromosome. (Patent Document 1). Furthermore, a method for selecting a grass plant having d60 gene and Gal gene by determining the presence of d60 gene and / or the presence of Gal gene essential for inheritance of d60 gene using DNA marker or gene marker Has been developed (Patent Document 1).
However, the d60 gene has not been put into practical use.

Currently, rice culm varieties are cultivated all over the world, such as Carrose 76 in the United States, IR36 in Southeast Asia, and the New Century of Japanese Hikari. However, as a result of gene analysis for these short varieties, it was found that all varieties have the same locus as the semi-fertile gene sd1. That is, the current cultivation of short anthers is controlled by only one specific gene, and there is a problem that only one specific gene is widely used.
Therefore, there is a need for a novel short rod gene that replaces the sd1 gene.

  In addition, due to recent global warming, high temperature ripening failure has occurred, causing problems such as deterioration in rice quality and yield reduction. Therefore, there is a demand for late varieties whose ripening period deviates from the high temperature period of summer.

JP 2008-237138 A

Tomita, M .: The gametic lethal gene gal: activated only in the presence of the semidwarfing gene d60 in rice.In Rice Genetics III (Ed.GS Khush, ISBN 971-22-0087-6), International Rice Research Institute, pp . 396-403 (1996) Tomita, M., Yamagata, H. and Tanisaka, T .: Developmental cytology on gametic abortion caused by induced complementary genes gal and d60 in japonica rice.In Advances in Rice Genetics (Eds.GS Khush, DS Brar and B. Hardy, ISBN 971-22-0199-6), International Rice Research Institute, pp. 178-181 (2003)

  Due to repeated typhoons, a great deal of damage caused by the fall of rice has become a social problem in recent years, and it has become an urgent issue to develop new varieties that are resistant to typhoons by introducing a short cocoon gene. In view of the purpose of breeding to maintain and expand the genetic diversity of varieties, new culm genes should be cultivated and utilized for lodging-resistant breeding without relying solely on the sd1 gene. is there. In recent years, high temperature damage has occurred nationwide due to the global warming phenomenon. Therefore, it is necessary to incubate late so that the ripening period comes around September, and to lower the temperature of the rice ripening period.

  Traditionally, to obtain a grass plant that is short or late, genetically cultivated a large number of individuals, measure the culm length after each individual heads, and record the ripening period Thus, the traits of the individual had to be confirmed, and a vast field, great effort and considerable years were required.

  Therefore, the present invention develops a method for discriminating plants having the above-mentioned desired traits, that is, plants that are short and late, by using DNA markers in the laboratory without actually cultivating them. For the purpose. Furthermore, an object of the present invention is to develop a Gramineae plant having a combination of two traits that exhibit a short culm trait based on a novel short culm gene and that it is late life.

  Under the circumstances as described above, the present inventor conducted genetic analysis by paying attention to the traits that are shorter and later than Koshihikari in the rice cultivar Konpaku. As a result, a novel short anther late QTL (quantitative trait locus) d63 (t) was found and mapped onto the third chromosome of a grass family plant that has not been reported to have a short anther gene. In addition, a DNA marker linked to d63 (t) was identified. Thus, the present invention has been completed. In the present specification, the novel short moth late life QTL d63 (t) found in the present invention is referred to as “d63 (t) gene” or “short moth late life gene d63 (t)”.

That is, the present invention
[1] To determine the presence of a d63 (t) gene by detecting a DNA marker present on the third chromosome of a grass family and linked to the d63 (t) gene. A selection method for short-growth late plantaceae,
[2] The method according to [1] above, wherein the DNA marker is linked to the d63 (t) gene within a recombination value of 15%,
[3] The method according to [1] or [2] above, wherein the DNA marker is at a distance of 34 to 37 Mb from the short arm end of chromosome 3.
[4] The method according to any one of [1] to [3] above, wherein the DNA marker is selected from the group consisting of an RFLP marker, AFLP marker, SSR marker, STS marker, SNP marker, and RAPD marker,
[5] The method according to [4] above, wherein the DNA marker is one or more selected from the group consisting of RM1038, RM1373 and RM2187,
[6] The short culm late gene d63 (t) and [7] The short culm late gene d63 (t) present at a position of 34 to 37 Mb from the short arm end of the chromosome 3 of the grass family plant
I will provide a.

  According to the present invention, by using a DNA marker on the third chromosome of a gramineous plant to determine the presence of the d63 (t) gene, it is possible to easily select a short and late-grown gramineous plant. . In addition, by selecting for a new short culm late gene d63 (t), it has a short culm and late phenotype that is completely different from the short culm plant that relied solely on the conventional sd1 gene. New grasses can be bred.

FIG. 1 shows the chromosomal location of the SSR marker used for the mapping of the dwarf late gene d63 (t). FIG. 2 is an electrophoretogram showing the linkage status between the short culm late gene d63 (t) and the SSR markers RM1373, RM5916 and RM8068. FIG. 3 is a diagram showing mapping of the short cocoon late gene d63 (t) to the end of the third chromosome, and the relationship between the short cocoon late gene d63 (t) and the SSR markers RM1038, RM1373 and RM2187 at the end of the third chromosome. It is an electrophoretic diagram which shows a chain | linkage condition. FIG. 4 shows the SSR markers used for DNA diagnosis of the short anther gene d60 and the late anther late gene d63 (t). FIG. 5 shows the results of identification by DNA diagnosis of the short anther gene d60 and the late anther gene d63 (t).

In the present invention, a novel short culm late gene d63 (t) was found by the following method. 530 individuals of F ₃ 54 line arbitrarily selected from the cross F ₂ of Koshihikari and Kogane Haru were cultivated, and the culm length, the number of ears reached and the grass type were investigated for each individual, and the genotype was determined by the progeny test. As a result, the progeny of the individuals who were Koshihikari-type Nagatoro early seedlings in F ₂ were 12 lines of Nagatoro early seedlings and 31 lines from which the short-spotted late seedlings and Nagatoro early seedlings were isolated. Progeny 11 strains of individuals was Koganebare type short稈晩production in F ₂ were all fixed at short稈晩production. In other words, the separation ratio of F ₂ is Koshihikari type (Nagato early-mature homozygous) 12 lines: Heterotype 31 lines: Golden brilliant (short-spotted homozygous type) 11 lines, which is compatible with a 1: 2: 1 separation ratio. (Χ ² = 1.26, 0.50 <P <0.75). From this, it became clear that the short culm traits and the late larvae are dominated by one leading gene. This gene was named “d63 (t)”.

Furthermore, the d63 (t) gene was mapped onto the third chromosome using gene linkage. Specifically, with respect to short稈晩producing homozygous individuals in crosses F ₂ of Koshihikari and Koganebare, applying an appropriate number of DNA markers selected to cover the entire chromosome of rice, d63 (t) gene It was found to be linked to a DNA marker on chromosome 3.

  Therefore, the present invention discriminates the presence of the d63 (t) gene by detecting a DNA marker that is present on the third chromosome of the grass family and is linked to the d63 (t) gene. The present invention provides a method for selecting short-growth late grasses, characterized by:

The “DNA marker present on the third chromosome of the grass family and linked to the d63 (t) gene” used in the present invention can be found, for example, by the following method. .
d63 (t) to expand the cross _{F 2} with other strains having no strain and d63 (t) gene with a gene, to genomic DNA from d63 (t) homozygote _{F 2} individuals, specific pattern Find DNA markers on chromosome 3 of gramineous plants showing As a strain having the d63 (t) gene, for example, Koganei is used. Although it does not limit as another strain | stump | stock which does not have d63 (t) gene, For example, Koshihikari can be used.

A gene or chromosomal site linked to the d63 (t) gene thus found is used as a DNA marker in the present invention.
“DNA marker” means a DNA polymorphism that can be used as a marker (marker) for genetic analysis and has a difference in nucleotide sequence depending on individuals or strains. For example, RFLP (restriction enzyme DNA fragment length) Polymorphism), AFLP (amplified fragment length polymorphism), SSR (simple repetitive sequence), STS (sequence tag site), SNP (single nucleotide polymorphism), RAPD (random amplified fragment polymorphism) and the like.

In the present invention, the d63 (t) gene and recombination value are within 15%, preferably within 10%, more preferably within 5%, compared to the recombination value (0%) when completely linked. Furthermore, it is more preferable to use a gene or chromosomal site linked within 1% as a DNA marker from the viewpoint of improving the accuracy of discrimination.
The recombination value is obtained by the following formula.
Recombination value (%) = (Number of recombinant gametes / total number of gametes) × 100

  In the present invention, a preferred DNA marker is present on the long arm of chromosome 3 of the grass family, more preferably near the end of the long arm. Further preferred DNA markers are located at a distance of about 30 Mb to long arm end, preferably about 32 to 37 Mb, more preferably about 33 to 37 Mb, more preferably about 34 to 37 Mb from the short arm end of chromosome 3 of the grass family. It is a marker.

  In the present invention, as a DNA marker, any marker of RFLP marker, AFLP marker, SSR marker, STS marker, SNP marker, or RAPD marker may be used.

  Examples of RFLP markers used in the present invention include AU090467 (C5), C98296 (C1468), D46782 (S11669), AU029959 (E50341S), C19151 (E10030S), D23971 (R689), AU031900 (R2462), D15409 (C595) ), AU029545 (E31057S), D13530 (G249), AU032992 (S770), C98285 (C1401), AU031775 (R1618), C26945 (C50518S), D40140 (S1912), C91691 (E31254), D28318U (R2311U) ), D23280 (C2540), AU031889 (R2404), C98850 ( 3180S), D25346 (G1015), AU033071 (S2516), AU068822 (E11382SA), C20258 (E11757S), C97959 (C217), AU031895 (R2443), C98248 (C1329), C23589 (S13428, 106646A, D46168U, D46168S, 9046) C831), AU032999 (S851), D24593 (R2224), AU068874 (C50771S), C98026 (C393A), AU063818 (E765S), AU031709 (R707), C98289 (C1442), AU031816 (R1927), 983 R3020), AU082662 (C1484), D135 8 (G164), AU069869 (E10579S), AU031654 (R273), AU032469 (S10594), AU032056 (R3385), C98026 (C3938), C98222 (C1219), C98214 (C1164), D47644 (S13262), D40113 (S13186) AU086018 (S11301), D39845 (S1469), AU031815 (R1925), C19276 (E10195S), C98265 (C1354), D25378 (G1318), AU032841 (S15179S), and AU032661 (S13122).

  Examples of the SSR marker used in the present invention include RM3829, RM8269, RM8203, RM6970, RM6806, RM6876, RM3684, RM6987, RM3815, RM6736, RM2593, Os03ssr024241900, Os03sr0242100, Os03ssr0242100, Os03ssr0242100, , RM15883, RM15884, RM15888, Os03ssr02433000, RM15886, RM15887, RM15888, RM15889, RM15890, RM15891, RM15892, RM15893, RM15889, RM15895, RM15 98, RM15899, RM15900, RM15902, RM15903, RM15904, RM15906, RM15907, RM15909, RM15914, RM15915, RM15918, RM15919, RM15992, RM15992, RM15925, RM15925, RM15925 RM1230, 94O03-4, RM16088, RM1373, RM16092, RM1038, RM2187, and RM232, preferably RM1038 (SEQ ID NO: 1 and SEQ ID NO: 2), RM1373 (SEQ ID NO: 3 and SEQ ID NO: 4) and RM2187 (sequence) From No. 5 and SEQ ID No. 6) It is used one or more markers selected from that group.

  In the present invention, “detection” may be carried out using a conventional method in the art for detecting the above DNA marker, and is appropriately selected by those skilled in the art depending on the type of DNA marker used. Can do. For example, PCR method, Southern blotting method, AFLP method and the like can be mentioned.

As a method for detecting a DNA marker using PCR, for example, it can be carried out as follows.
[DNA extraction]
Plants (or parts thereof) are freeze-ground with liquid nitrogen, DNA extract is added and incubated with shaking. The solution is spooled out of the polymer DNA by extraction and polymerization precipitation. The spooled-out DNA is purified to produce a DNA solution.
[PCR]
Using the genomic DNA extracted from each Gramineae plant body as a template, a reaction solution containing a primer specific for the DNA marker, DNA polymerase and the like is prepared. Using a thermal cycler, etc., a series of reactions of denaturation, annealing, and extension are repeated. The resulting PCR product is then electrophoresed. The reaction conditions and primers for PCR can be referred to, for example, Theor Appl Genet (2000) 100: 697-712 (this document is incorporated herein by reference).

  Furthermore, the present invention provides a novel short culm late gene d63 (t). By using the d63 (t) gene, it is possible not only to select and breed rice plants having two traits of short culm and late bryophyte, but also to introduce new short culm late cultivars by directly introducing them into gramineous plants. Development is also possible.

Detection of short-spotted late QTL Fostered K 2 and Kosei Hara's cross F ₂ and cultivated 530 individuals of F ₃ 54 strains arbitrarily selected from the F ₂ strain, and investigated the culm length, the number of ears reached and the plant type for all individuals. .
As a result, among the 43 strains had been Koshihikari type length稈早production in F _2, 12 lines are long稈早production in F _3, 31 strains were separated into short稈晩production and long稈早production in F _3. Progeny 11 strains of individuals was Koganebare type short稈晩production in F ₂ were all fixed at short稈晩production. That is, the separation ratio of _{F 2} is Koshihikari type length稈早raw homozygous (D63 (t) D63 (t )) 12 System: heterozygous (D63 (t) d63 (t )) 31 System: Koganebare type short稈晩raw Homozygous (d63 (t) d63 (t)) 11 lines were obtained, which matched a separation ratio of 1: 2: 1 (χ ² = 1.26, 0.50 <P <0.75). From this, it became clear that the short culm traits and the late larvae are dominated by one leading gene. This gene was named “d63 (t)”.

To mapping Koshihikari and _{F 2} fixation was confirmed by Koganebare crosses _{F 3} short稈晩raw homozygote (d63 (t) d63 (t )) individual short稈晩raw gene d63 (t), covering the rice genome Thus, 3-5 (each 10 in the third chromosome) for each 12 chromosomes were applied, and SSR markers showing 57 DNA polymorphisms were applied to examine the linkage with each SSR marker. . In addition, all the SSR markers on the 3rd chromosome showed DNA polymorphism between the parental varieties (FIG. 1).

From each individual of F _2, genomic DNA was extracted as follows. The leaves were taken from the plants and stored frozen at -80 ° C. The leaves were frozen with liquid nitrogen and ground. To this was added a DNA extract (2% CTAB, 100 mM Tris-HCl, 20 mM EDTA.2Na, 1.4 M NaCl, pH 8.0) equivalent to the weight of the leaf powder, and incubated for 90 minutes with shaking at 55 ° C. did. This solution was extracted with chloroform / isoamyl alcohol (24: 1). After adding 1/10 amount of 3M sodium acetate pH 5.2 to the supernatant, an equal amount of 99.5% isopropanol at −20 ° C. was added to spool out the polymerized DNA. The spooled-out DNA is dissolved in 1.5 ml of High-Salt TE (1M NaCl, 10 mM Tris · HCl, 1 mM EDTA · 2Na, pH 8.0), and after ethanol precipitation, 500 μl of TE (10 mM Tris-HCl, 1 mM EDTA) -Dissolved in 2Na, pH 8.0). 1/100 amount of RNase solution (1 mg / ml) was added thereto and incubated at 37 ° C. overnight. Further, phenol-chloroform extraction and chloroform extraction were performed once, followed by ethanol precipitation and dissolution in 500 μl of TE (10 mM Tris-HCl, 1 mM EDTA · 2Na, pH 8.0).

Using 200 ng of genomic DNA derived from each F ₂ plant extracted as described above as a template, 200 nM forward primer, reverse primer, 400 μM dNTPs, 2.5 mM MgCl ₂ , 1 × PCR Buffer II (TAKARA), 0.5 U LA A total volume of 25 μl of a reaction solution containing Taq DNA polymerase (TAKARA) was prepared. Using a thermal cycler, a series of reactions of denaturation 94 ° C. for 30 seconds, annealing 58 ° C. for 30 seconds, and extension at 72 ° C. for 1 minute were performed 35 times. The first denaturation 94 ° C. and the final synthesis 72 ° C. were performed for 5 minutes each. The PCR product was electrophoresed on a 2% agarose gel at 100 V for 45 minutes (FIGS. 2 and 3).

The primers used in PCR are as follows.
For marker RM1038, forward primer: TGGTTCGATTCGGATTTC (SEQ ID NO: 1) and reverse primer: AAGCTATTCACAAGCAGCTC (SEQ ID NO: 2).
For marker RM1373, forward primer: TGCTATACCCAAATGTCCAAGC (SEQ ID NO: 3) and reverse primer: ATCTTCTGAGTGCTGCCAAGC (SEQ ID NO: 4).
For marker RM2187, forward primer: GTCATTTGAAGTAAATCCGT (SEQ ID NO: 5) and reverse primer: GGTCTACTTGCGAAATAAGT (SEQ ID NO: 6).
For marker RM5916, forward primer: GCTATAAGAATCGTATTAAG (SEQ ID NO: 7) and reverse primer: TACTGCTATTAAAGTCAGAA (SEQ ID NO: 8).
For marker RM8068, forward primer: AAACCTCTCGCTGTAATTAG (SEQ ID NO: 9) and reverse primer: TGAACATTTATTGATATGGTAAA (SEQ ID NO: 10).

As a result, linkage with the SSR marker on the third chromosome was observed, and no linkage was observed except for the third chromosome (see FIGS. 2 and 3). The third marker on chromosome RM1038, RM1373, and RM2187, while indicating DNA polymorphisms among parents (Koshihikari and Koganebare), about 85% to 95% of the short culm d63 (t) homozygous individuals in crosses _{F 2} golden It showed the same DNA pattern as that of Hare (ie, short cocoon type) (see FIGS. 2 and 3). On the other hand, the marker RM8068 markers RM5916 and on chromosome 1 on chromosome 2 shows a DNA polymorphisms among parents, in the short culm d63 (t) homozygous individuals in crosses _{F 2,} short稈型, Koshihikari type and Heterogeneous DNA pattern was exhibited and inherited independently. Therefore, the d63 (t) gene could be mapped onto the third chromosome.

  Furthermore, the recombination value with marker RM1373 near the end of the long arm of chromosome 3 is 4.3%, the recombination value with RM1038 is 8.6%, and the recombination value with RM2187 is 13.4%. It was found that d63 (t) is near the end of the long arm of chromosome 3 (FIG. 3). Therefore, a DNA marker located at a distance of about 30 Mb to the long arm end from the short arm end of the third chromosome can be used in the selection of short crocodile late grasses.

To foster cross _F 2 144 individual identification Koshihikari d60 system and golden sunny by DNA diagnosis of short culm gene d60 and short稈晩raw gene d63 (t), heading date for all individuals, were investigated culm length, the grass type. As a result, in F ₂ , the long rice individuals that exceeded the parent rice varieties (Koshihikari d60 line and Kogane Haru) were separated from the extremely short rice individuals that were lower.

Next, DNA diagnosis was performed on all 144 F ₂ individuals using SSR markers linked to each of d63 (t) and d60. Chromosome 3 RM1038 was used as a marker for d63 (t), and Chromosome 2 RM6375 (SEQ ID NO: 9) was used as a marker for d60 (FIG. 4). RM6375 is known to be useful as a d60 marker.

The DNA diagnosis method was performed as follows.
According to the method described in Example 2, the genomic DNA was extracted from each individual in F _2, the DNA as a template was PCR amplified using primers specific for the marker. Primers for RM1038 are as described in Example 2. Primers for RM6375 were forward primer: CGAATGGAACGACAAGAGATGC (SEQ ID NO: 11) and reverse primer: ATAGATCGACAACGTAGATCCACAG (SEQ ID NO: 12).

As a result, there were 30 d63 (t) gene homozygous individuals, 13 d60 gene homozygous individuals, and 3 extremely short individuals with both d63 (t) and d60 genes homozygous. was detected. This agreed well with the theoretical frequency of 1/4: 1/9: 1/36 when d63 (t) and d60 are inherited independently (χ ² = 0.08, 0.950 <P <0.975). . Here, since the short rod gene d60 becomes sterile when it coexists with the gamete lethal gene gal, Gal is indispensable for the inheritance of d60, and the theoretical value of the F ₂ segregation ratio of d60 homozygous short rod individuals is 1 / 9
Therefore, with these two SSR markers, d63 (t) gene and d60 gene can be clearly distinguished from each other in the variety breeding process, and DNA diagnosis can be made. Short duck rice having d63 (t) gene and d63 (t) gene and d60 gene It is possible to quickly select short rice that has both.

  According to the present invention, in order to expand the genetic diversity of short varieties and counter unforeseen weather fluctuations, the short varieties are closely linked to a new short culm gene d63 (t) genetically different from sd1. A DNA marker having high selection efficiency is developed, and a method for developing a short rice field rice by d63 (t) marker selection is provided. By applying this method, it is possible to isolate the d63 (t) main body, and it is also possible to adjust the plant type by elucidating the function of d63 (t). Expanding the potential of short culm breeding with a bias on GA by selecting markers for the short culm gene d63 (t) will help to develop varieties in public testing and research institutions related to agricultural test sites and plant factories across the country. Therefore, the present invention can be used in the agricultural field, particularly in the field of breeding of gramineous plants.

Claims (1)

A method for selecting a short-grown late plantaceae, wherein the presence of a d63 (t) gene is determined by detecting one or more DNA markers selected from the group consisting of RM1038, RM1373 and RM2187 .