JP7328680B2 - DNA marker selection method for extremely early maturing gene on chromosome 7 of rice - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act applies Abstracts of the 134th Lecture Meeting of the Japan Society of Breeding (Research on Breeding, Vol. 20, Separate Volume No. 2), p. September 22nd

Article 30, Paragraph 2 of the Patent Act applies General Incorporated Association Japan Society of Breeding 134th Lecture Date: September 23, 2018

Application of Article 30, Paragraph 2 of the Patent Act Abstracts of the 26th Chubu District Conference of the Japan Society of Breeding, p.

Application of Article 30, Paragraph 2 of the Patent Act The 26th Annual Conference of the Japan Society of Breeding, Chubu Region Date: October 28, 2018

Application of Article 30, Paragraph 2 of the Patent Act Website address https://www2. aeplan. co. jp/mbsj2018/ Publication date: November 9, 2018

Application of Article 30, Paragraph 2 of the Patent Act The 41st Annual Meeting of the Molecular Biology Society of Japan Date: November 28, 2018

Application of Article 30, Paragraph 2 of the Patent Law Summary of the 135th Lecture Meeting of the Japan Society of Breeding (Bedology Research Vol. 21, Separate Volume No. 1), p. March 16, 31

Article 30, Paragraph 2 of the Patent Act applies The 135th lecture of the Japanese Society of Breeding Date: March 17, 2019

The present invention relates to a method for selecting very early rice, which comprises using very early genes e1 and e2 and a DNA marker for determining the presence of the genes.

In recent years, the number of typhoons and landfalls has increased, and there have been a series of large-scale natural disasters such as torrential rains, and the damage to agricultural, forestry and fishery products is increasing. Controlled production in plant factories is considered as one of the measures to avoid such natural disasters. However, in the case of rice, which has a long growing period, the cost of plant factory production is about seven times that of paddy field cultivation, which is not practical.

On the other hand, as an extremely early-maturing variety with a short life cycle, a timeless, extremely early-maturing mutant "Kanto No. 79" induced by γ-ray irradiation of Koshihikari is known. In the previous research of the inventor, genetic analysis of crosses F ₂ and F ₃ between Kanto No. 79 and the original variety Koshihikari revealed a recessive gene that accelerates maturity by 14 days in the genetic background of Koshihikari. Genetic analysis of crosses F ₂ and F ₃ between No. 1 and Tenkoku revealed that they are inherited independently of the short culm gene sd1 (for example, Non-Patent Documents 1 and 2). However, the details of the very early maturing gene have not been elucidated.

Tanisaka et al. (1990) Semi-dwarf genetic analysis of semi-dwarf mutant lines Hokuriku No. 100 and Kanto No. 79 of rice cultivar Koshihikari. Journal of Breeding 40/1:103-117 Tomita, M. (2009) Introgression of Green Revolution sd1 gene into isogenic genome of rice super cultivar Koshihikari to create novel semidwarf cultivar ‘Hikarishinseiki’ (Koshihikari-sd1), Field Crops Research 114/2:173-181

If the life cycle of rice is shortened and year-round production is possible, utilization efficiency will increase, costs will be reduced, and plant factory production will become possible. Therefore, the present invention aims at the development of extremely early-maturing rice, and aims at elucidation of the extremely early-maturing gene.

Under the circumstances as described above, the present inventor first attempted to map the very early maturing gene derived from Kanto No. 79. The gene is referred to as "e1" in this specification. That is, Kanto No. 79 and Nipponbare were crossbred, and e1 homozygous individuals isolated in _F2 were used for mapping with DNA markers. It has been shown to reside in a region of 3 Mb.

Furthermore, the present inventor crossed Koshihikari with an extremely early-maturing mutant ``Inochi-no-Ichi-Wase'' from the large-grain variety ``Inochi-no-Ichi'', and backcrossed the hybrid with the extremely early-maturing property to Koshihikari for genetic analysis. went. As a result, the very early maturing group that matures about 10 days earlier than Koshihikari and the Koshihikari-type mesozoic group separated at a ratio of about 1:3. , named “e2”. Further, mapping with DNA markers revealed that e2 is located in a region of 7.4 to 8.3 Mb from the end of the short arm of chromosome 7 of rice. Thus, the present invention was completed. In the present specification, the allelic loci e1 and e2 relating to extremely early maturation are also referred to as "e1 gene" and "e2 gene" or "extremely early maturing gene e1" and "extremely early maturing gene e2", respectively.

That is, the present invention
[1] characterized by determining the presence of the e1 gene and/or the e2 gene by detecting a DNA marker present in a region at a distance of 6.6 to 8.3 Mb from the short arm end of chromosome 7 of rice A method for selecting extremely early-maturing rice,
[2] characterized by determining the presence of the e1 gene by detecting a DNA marker present in a region at a distance of 6.8 to 8.3 Mb from the short arm end of chromosome 7 of rice [1] described method,
[3] characterized by determining the presence of the e2 gene by detecting a DNA marker present in a region at a distance of 7.4 to 8.3 Mb from the short arm end of chromosome 7 of rice [1] described method,
[4] The method according to any one of [1] to [3], wherein the DNA marker is selected from the group consisting of RFLP markers, AFLP markers, SSR markers, STS markers, SNP markers, and RAPD markers.
[5] The method of [1], wherein the DNA marker is one or more selected from the group consisting of RM21224, J1753, J1759, and J1763;
[6] The method of [2], wherein the DNA marker is one or more selected from the group consisting of J1753, J1759, and J1763;
[7] The method of [3], wherein the DNA marker is one or more selected from the group consisting of J1759 and J1763;
[8] A method for producing extremely early rice, comprising:
(a) crossing extremely early rice with non-extremely early rice to produce hybrid seeds;
(b) cultivating the hybrid seed to produce a hybrid rice plant;
(c) a method comprising selecting extremely early rice from the hybrid plant by the method according to any one of [1] to [7];
[9] The method according to [8], wherein the non-extremely early-maturing rice is a short-stalked rice or a large-grained rice.
[10] Very early maturing gene e2 located in a region at a distance of 7.4 to 8.3 Mb from the short arm end of chromosome 7 of rice
I will provide a.

According to the present invention, DNA markers linked to the e1 and e2 genes on chromosome 7 of rice are identified. By determining the presence of the e1 gene and/or the e2 gene using the DNA marker, very early-maturing Poaceae plants can be easily selected. According to the present invention, the need for actually cultivating and evaluating test individuals is greatly reduced, and DNA markers linked to target genes can be used even at the stage of seeds and seedlings. can be selected for individuals with Thus, if the life cycle of rice is shortened by making it grow very early, plant factory production will become possible, and damage caused by weather changes such as large typhoons and heavy rains can be avoided. In addition, by making the rice grow very early, it is possible to avoid ripening in August, the hottest month, and to suppress ripening failure.

FIG. 1 shows the linkage status between the very early maturing gene e1 and the SSR and SNP markers on chromosome 7. FIG. FIG. 2 shows the linkage status between the very early maturing gene e2 and the SSR and SNP markers on chromosome 7. FIG.

In the present invention, as rice, Oryza sativa L. can be used. Rice includes both cultivated and wild varieties, preferably cultivated rice, more preferably Asian rice, and particularly preferably japonica rice.

In the present invention, the term "extremely early maturing" means heading about 10 days earlier or 10 days earlier than its original cultivar that does not have the extremely early maturing property or its cross partner that does not have the extremely early maturing property. For example, depending on the cultivation conditions, a grass family plant having a homozygous e1 gene in the genetic background of Koshihikari heads 10 days or more earlier than Koshihikari, for example about 14 days earlier. For example, although depending on the cultivation conditions, a grass family plant having a homozygous e2 gene in the genetic background of Koshihikari heads about 10 days earlier than Koshihikari.

The e1 gene is a gene related to extremely early maturity found in the timeless extremely early maturing mutant "Kanto No. 79" induced by γ-ray irradiation of Koshihikari. In the present invention, in order to clarify the chromosomal position of e1, Nipponbare and Kanto No. 79 were crossed, and e1 homozygous individuals separated in _F2 were used to link with 31 SSRs covering all chromosomes. I did the analysis. As a result, it was found that the e1 gene is located in a region 6.6 to 18.9 Mb from the short arm end of chromosome 7. Furthermore, as a result of detailed linkage analysis using three SNP markers, it was found that the e1 gene is located in a region at a distance of 6.6 to 8.3 Mb from the short arm end of chromosome 7. The strongest linkage with e1 was detected around 6.8 Mb from the end of the short arm of chromosome 7.

The e2 gene is a novel gene related to extremely early maturation found in the extremely early maturing mutant 'Inochi no Ichi Wase' of 'Inochi no Ichi'. Specifically, Koshihikari and Inochi no Ichi early maturing were crossed, and the earliest maturing (hereinafter referred to as extremely early maturing) individual that flowered 10 days earlier than Koshihikari isolated in cross _F2 was used as the first parent, and Koshihikari was used as the recurrent parent. Backcrossing was performed twice, and an isogenic line was bred by transferring the extremely early maturing trait to Koshihikari. As a result, BC ₁ F ₂ and BC ₂ F ₂ were separated into very early type: Koshihikari type = 1:3. Therefore, it was found that this very early maturation is controlled by one recessive very early maturation gene. This gene was named "e2".

In order to examine the allele between the e1 gene and the e2 gene, the ``Koshihikari e1sd1 strain'' (extremely early maturity/short culm isogenic strain produced by crossing Kanto No. 79 with Tokkoku) and the ``Koshihikari e2 strain'' (Koshihikari and life (1) Early crossing ( _F2 ) was backcrossed once to Koshihikari (e2 homozygous individual), and test crossing was performed to obtain _F2 individuals. In the test cross _F2 , the Koshihikari e1sd1 line and the Koshihikari e2 line, which are the parents, have similar number of days to heading (47.8 days and 49.0 days, respectively), whereas the number of days to heading is much later than that of the parents. Individuals with days ranging from 43 to 60 days corresponding to Koshihikari appeared. That is, it turned out that e1 and e2 are non-opposed.

In order to clarify the chromosomal location of e2, Nipponbare or Koshihikari was crossed with Inochi no Ichiwase, and the e2 homozygous individual isolated in _F2 was used to identify 53 SSR and SNP markers covering the entire chromosome. Linkage analysis was performed. As a result, the e2 gene was found to be located in a region 7.4 to 8.3 Mb from the short arm end of chromosome 7, and the strongest linkage with e2 was detected around 8.3 Mb.

Therefore, the present invention determines the presence of the e1 gene and/or the e2 gene by detecting a DNA marker present in a region at a distance of 6.6 to 8.3 Mb from the short arm end of chromosome 7 of rice. A method for selecting extremely early-maturing rice (hereinafter also referred to as "the method for selecting extremely early-maturing rice of the present invention") characterized by the following is provided.

"DNA marker" means a DNA polymorphism that can be used as a mark (marker) for genetic analysis and shows a difference in base sequence depending on individuals or strains. polymorphism), AFLP (amplified fragment length polymorphism), SSR (simple repeat sequence), STS (sequence tag site), SNP (single nucleotide polymorphism), RAPD (randomly amplified fragment polymorphism), and the like. In the present invention, one or more DNA markers selected from the group consisting of RFLP markers, AFLP markers, SSR markers, STS markers, SNP markers, and RAPD markers may be used. DNA markers can be retrieved, for example, from public databases. For example, databases of SNP markers include the Q-TARO (QTL Annotation Rice Online) database (http://qtaro.abr.affrc.go.jp/).

The "DNA marker present in the region at a distance of 6.6 to 8.3 Mb from the short arm end of chromosome 7 of rice" used in the present invention can be found by, for example, the following method. A cross F2 between a line having the e1 gene or the e2 gene and a line not having the _e1 gene or the e2 gene is developed, and a specific pattern is generated for the genomic DNA obtained from the e1e1 homozygous or e2e2 homozygous F2 _individual . A DNA marker present in the region 6.6 to 8.3 Mb from the short arm end on chromosome 7 of rice shown is found. Furthermore, for the DNA markers showing the specific pattern, a recombination titer is determined, and a low recombination titer, for example, a recombination titer of 30%, preferably 20%, more preferably within 10% of the e1 gene or e2 gene. Those that are linked may be used as DNA markers. The recombination value is obtained by the following formula.
Recombination value (%) = (number of recombinant gametes/total number of gametes) x 100

Further, the genome replacement rate may be determined by backcrossing the DNA markers showing the specific pattern, and those with a low replacement rate, for example, 50% or less, may be used as the DNA marker. For example, a gene or chromosomal site with a genome replacement rate of 50% or less to strains lacking the e1 or e2 gene is used as a DNA marker.

As a line having the e1 gene or the e2 gene, for example, a hybrid plant of Kanto No. 79, Inochi no Ichiwase, Kanto No. 79 or Inochi no Ichiwase, or a plant having Kanto No. 79 or Inochi no Ichiwase as an ancestor can be used. The above line without the e1 gene or the e2 gene is not limited, but for example, Koshihikari, Nipponbare, etc. can be used.

The DNA marker used in the present invention is a marker located in a region about 6.6 to 8.3 Mb from the end of the short arm of rice chromosome 7. For example, the distance from the short arm end of chromosome 7 is about 6.6 to 8.3 Mb, preferably about 6.8 to 8.3 Mb DNA marker can be used to determine the presence of the e1 gene. good. For example, DNA markers in a region approximately 7.4-8.3 Mb from the short arm end of chromosome 7 may be used to determine the presence of the e2 gene.

Examples of DNA markers for use in the present invention include, but are not limited to, RM21224, J1753, J1759, and J1763. RM21224 is an SSR marker. J1753, J1759, and J1763 are SNP markers at distances of 6.8 Mb, 7.4 Mb, and 8.3 Mb from the short arm end of rice chromosome 7, respectively. In the present invention, preferably one or more DNA markers selected from the group consisting of RM21224, J1753, J1759 and J1763 are used.

In the present invention, "detection" may be performed using a method commonly used in the art for detecting the above DNA marker, and a person skilled in the art can appropriately select according to the type of DNA marker used. can be done. Examples thereof include the PCR method, Southern blotting method, AFLP method and the like.

In the case of SSR markers, for example, using primers specific to each SSR, PCR is performed using DNA extracted from the sample as a template, and the length of the amplified product is detected to determine whether the sample has the target gene. can be judged.

In the case of SNP markers, for example, a probe that specifically hybridizes to each SNP allele is designed, the end of the probe is modified with a different fluorescent dye and quencher for each SNP allele, and a primer designed outside the probe can be detected by the PCR method using The probe is degraded by the extension reaction of the primer, and by measuring the fluorescence emitted when the fluorescent dye and the quencher are separated, an allele-specific fluorescence amplification curve is obtained to determine which SNP allele the sample has. can judge. Examples of fluorescent dyes include, but are not limited to, FAM, HEX, and the like. Allele-specific probes and primers can be appropriately designed by those skilled in the art based on the genomic information of the sample.

The method for selecting extremely early-maturing rice plants of the present invention is used, for example, in breeding methods for producing new extremely early-maturing rice plants. Such breeding methods include general methods known in the art, such as methods characterized by crossing a cultivar having the e1 gene or the e2 gene with another cultivar. Accordingly, the present invention further provides a method for producing extremely early rice, comprising:
(a) crossing a rice plant having the very early maturing trait with a rice plant not having the very early maturing trait to produce hybrid seeds;
(b) cultivating the hybrid seed to prepare a hybrid rice plant; and (c) selecting an extremely early rice plant from the hybrid plant by the method for selecting an extremely early rice plant of the present invention;
(hereinafter also referred to as "method for producing extremely early rice of the present invention").

In the method for producing extremely early rice plants of the present invention, rice plants having extremely early maturing traits include, for example, Kanto No. 79, Inochi no Ichiwase, Kanto No. 79 or Inochi no Ichiwase hybrid plants, or Kanto No. 79 or Inochi It is possible to use plants that have early maturity as ancestors. As the rice that does not have the extremely early maturity trait, for example, a cultivar that does not have the e1 gene and the e2 gene is used. Varieties lacking the e1 gene and the e2 gene include, but are not limited to, Koshihikari, Nipponbare, and the like. In addition, the cultivar lacking the e1 gene and the e2 gene may be short-culm rice having short-culm genes such as sd1 and d60, or large-grain rice having large-grain genes such as Gg.

As a specific example of the method for producing extremely early rice of the present invention,
(a′) crossing a rice plant having the very early maturing trait with a rice plant not having the very early maturing trait to produce _F1 ;
(b') crossing _F1 with a rice plant that does not have the very early maturity trait;
(c') selecting an extremely early rice plant from the hybrid plant by the method for selecting an extremely early rice plant of the present invention; A method comprising crossing with rice that does not have Steps (c') and (d') above may be repeated.

Another specific example of the method for producing extremely early-maturing rice of the present invention is as follows.
(a'') crossing a rice plant having the very early maturing trait with a rice plant not having the very early maturing trait to create _F1 ;
(b'') inbreeding _F1 to create _F2 , and (c'') selecting an extremely early rice plant from the hybrid plant by the method for selecting an extremely early rice plant of the present invention. A method comprising: The above steps (b'') and (c'') may be repeated.

In the method for producing extremely early rice plants of the present invention, a DNA marker linked to the e1 gene or the e2 gene is used, so desired plants can be easily selected with high probability. In addition, breeding using such DNA markers is efficient because only a relatively small number of individuals can be cultivated and selection can be made at an early stage such as seeds and young seedlings.

The present invention further provides a novel very early maturing gene e2. The use of the e1 gene or e2 gene not only enables the selection and breeding of extremely early rice, but also allows direct introduction into rice to develop new extremely early varieties.

Example 1: Mapping of extremely early maturing gene e1 Kanto No. 79 and Nipponbare were crossed, and in 313 crossed F ₂ individuals, a very early maturing group whose heading date is distributed around the average value of the heading date of Kanto No. 79 and a heading date of A later mesozoic group appeared and separated into 65 very early (e1 homozygous): 248 mesozoic (E1 homozygous + E1e1 type) ≈ 1:3 (χ ² =2.991, 0.05<P <0.10). Thirty-nine individuals of e1 homozygous type Kanto No. 79 type segregated in _F2 were tested, and selected so that polymorphism was shown between Kanto No. 79/Nihonbare and all chromosomes of rice were covered. Linkage analysis with e1 was performed using 2 SSR markers.

Genomic DNA was extracted from leaves of 39 e1 homozygous individuals of Kanto No. 79 at the heading stage isolated in _F2 according to a conventional method. Briefly, leaves stored at −80° C. were placed in a 2 ml crushing tube (CK28) containing ceramic beads (YTZ beads) and immersed in liquid nitrogen for 1 minute to freeze. After being spun at 5000 rpm for 20 seconds with a bead-type high-speed cell disruptor (Precellys 24, bertin), the leaves were taken out, immersed in liquid nitrogen again, frozen, and spun again at 5000 rpm for 20 seconds to pulverize the leaves. 500 μL of 2×CTAB was injected into the pulverized leaves and incubated in a constant temperature bath at 55° C. for 30 minutes. 500 μL of chloroform/isoamyl alcohol (24:1) was injected, shaken for 15 minutes, centrifuged at 5000 rpm and 4° C. for 10 minutes, and the supernatant was dispensed into autoclaved sterilized 1.5 ml tubes. To this, 50 μL of 3M sodium acetate (pH 7.8) and 500 μL of isopropanol were added to aggregate the DNA. This was centrifuged at 5000 rpm and 4° C. for 10 minutes to precipitate, the supernatant was discarded, 300 μL of 70% ethanol was added, and the mixture was centrifuged at 5000 rpm and 4° C. for 10 minutes. After centrifugation, the supernatant was discarded, air-dried, and the DNA was dissolved in 100 μL of sterilized Milli-Q water to extract the DNA.

Using the extracted DNA sample derived from each _F2 plant as a template, a PCR reaction was performed with the composition shown in Table 1. The PCR program consisted of heat denaturation at 95°C for 2 minutes, followed by 35 cycles of heat denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and extension reaction at 72°C for 30 seconds. PCR products were electrophoresed on a 2% agarose gel at 100 V for 45 minutes.

As a result, linkage with the SSR marker on chromosome 7 was observed, and e1 was found to be located on chromosome 7. Therefore, furthermore, in the 6.6 to 18.9 Mb region from the short arm end of chromosome 7 where significant linkage with e1 was detected, three SNP markers showing polymorphism between Kanto No. 79 and Nipponbare were used. detailed linkage analysis was performed.

SNP markers were detected by the TaqMan probe method by designing FAM- and HEX-labeled TaqMan probes for each allele. Three SNPs [J1753 (6.8 Mb), J1759 ( 7.4 Mb), J1763 (8.3 Mb)], probes that specifically hybridize to SNP alleles differing by only one base were designed and labeled with different fluorescent dyes FAM and HEX, respectively (Table 3-1). . The TaqMan probe thus prepared is cleaved by the extension reaction of the outer designed primer (Table 3-2), and emits light when the fluorescent dye and quencher (BHQ-1) are separated. It was determined which SNP allele was possessed by this allele-specific fluorescence amplification curve.

In the table, single nucleotide polymorphisms are underlined.

SNP analysis was performed using the DNA sample extracted above. SNP analysis contains 10 μL SSo Advanced Universal Probes Supermix (BIO-RAD, USA), 1 μL forward primer, 1 μL reverse primer, 0.4 μL probe FAM, 0.4 μL probe HEX, 2 μL DNA sample (100 ng), 5.2 μL sterile water. After heat denaturation with the reaction solution at 95°C for 3 minutes, PCR reaction was performed at 95°C for 15 seconds and 64.2°C for 30 seconds for 40 cycles to detect fluorescence.

When the recombination value between each DNA marker on chromosome 7 and the e1 gene was calculated, it was 23.0% for RM8010 at the 4.1 Mb point from the short arm end, 6.4% for RM21224 at the 6.6 Mb point, and 6.4%. 3.8% at J1753 at 8 Mb, 5.1% at J1759 at 7.4 Mb, 5.1% at J1763 at 8.3 Mb, and 27.2% at RM3300 at 18.9 Mb ( Figure 1). Therefore, the e1 gene could be mapped onto chromosome 7, and the e1 gene was found to be linked to DNA markers in the region 6.6 Mb to 8.3 Mb from the short arm end of chromosome 7. In particular, the strongest linkage with the e1 gene was detected at 6.8 Mb from the short arm end of chromosome 7.

Example 2: Detection of extremely early maturing gene e2 (1) Genetic analysis of Ichi no Ichi early maturity of life Koshihikari and Ichi no Ichi early maturity of life were crossed, and an individual that matured 10 days earlier than Koshihikari separated by cross _F2 was used as a parent once, Backcrossing was performed with Koshihikari as the recurrent parent. As a result, in BC ₁ F ₂ and BC ₂ F ₂ , the extremely early maturing group that matured 10 days earlier than Koshihikari and the Koshihikari type intermediate group were 18 extremely early: 79 intermediate ≒ 1: 3 and 16 extremely early. Type: 56 mesomorphs≅1:3 separated bimodally. Therefore, it was found that the very early maturity of the 1 early maturity of life is controlled by one very early maturity gene. This gene was named e2.

(2) Allele test between e2 and e1 In order to examine the allele between the extremely early maturing gene e1 that is derived from Kanto No. 79 and causes early maturity by 14 days and the extremely early maturing gene e2 of Inochi no Ichi early maturing, Koshihikari rice with e1 was tested. The isogenic line "Koshihikari e1sd1" and the e2 homozygous type isolated by backcrossing BC ₁ F ₂ with Koshihikari as the recurrent parent and the extremely early maturing type individual isolated in the cross F ₂ of Koshihikari x Inochi no Ichi Wase as a single parent. A cross _F2 with an individual "Koshihikari e2 strain" was tested. Koshihikari e1sd1 was grown as described in Non-Patent Document 2.

Briefly, in the process of cultivating a Koshihikari isogenic line (Koshihikari sd1) having a short culm gene sd1, a next-generation _F3 line for each individual was nurtured from 100 Kanto No. 79 x Tenkoku cross _F2 individuals, We obtained a line in which the heading date was fixed to the Kanto No. 79 type, which was extremely early, and the Koshihikari type, which was middle maturing, and the culm length was fixed to the sd1 homozygous type derived from Tenkoku. Of these, in the backcross BC ₁ F ₂ with the e1sd1 homozygous individual fixed at F3 of Koshihikari sd1 × Kanto No. ₇₉ as a single parent and Koshihikari as a recurrent parent, very early maturing type (e1 homotype): mid-life type (E1 homozygote + E1e1 type) ≈ 1:3, and short-culm type (sd1 homozygous type): long-culm type (Sd1 homozygous + Sd1sd1 type) ≈ 1:3 in the extremely early and intermediate groups. As described above, a very early maturing/short culm isogenic line 'Koshihikari e1sd1' was bred from Koshihikari by 10 days early and with a short culm of 30 cm.

In the test cross _F2 of the Koshihikari e1sd1 line and the Koshihikari e2 line, the average number of days to head of the parents was 47.8 days (Koshihikari e1sd1 line) and 49.0 days (Koshihikari e2 line), respectively. On the other hand, from the 43rd day, an individual that lived later than the parents and reached the 60th day appeared. Therefore, it turned out that e1 and e2 are non-conflicting. That is, since the genotypes of the parents are e1e1E2E2 and E1E1e2e2, the genotype with dominant alleles at both loci in _F2 grows late and grows bivalently 13 early (1e1e1 (1E2E2:2E2e2:1e2e2): (1E1E1 :2E1e1) 1e2e2: (1E1E1:2E1e1)2E2e2): 3 late maturing (1E1E1E2E2:2E1e1E2E2).

(3) Mapping of extremely early maturing gene e2 In 159 F ₂ individuals crossed between Nipponbare and Inochi no Ichi early maturing, segregation was made into a bimodality of 42 early maturing types: 117 middle maturing types ≈ 1:3. The Nipponbare x Inochi no Ichi early maturing F ₂ , Koshihikari x Inochi no Ichi early maturing F ₂ , and Koshihikari/(Koshihikari/Inochi no Ichi early maturing F ₂ ) BC ₁ F ₂ homozygous extremely early maturing individuals were tested. Then, linkage analysis was performed between 51 SSRs and SNP markers covering the entire chromosome of rice and e2. SSR markers and SNP markers were detected in the same manner as in Example 1. TaqMan probes and primers used for detection of SNP markers are also shown in Tables 3-1 and 3-2 of Example 1.

As a result, linkage with a DNA marker on chromosome 7 was observed, and e2 was found to be located on chromosome 7. When the recombination value between each DNA marker on chromosome 7 and the e2 gene was calculated, RM8010 at the 4.1 Mb point from the short arm end was 15.0%, J1753 at the 6.8 Mb point was 8.3%, 7 5.0% for J1759 at 4 Mb, 5.0% for J1763 at 8.3 Mb and 32.8% for RM3300 at 18.9 Mb (Fig. 2). Therefore, it was found that e2 is located in a region of 7.4 Mb to 8.3 Mb from the short arm end of chromosome 7.

INDUSTRIAL APPLICABILITY According to the present invention, DNA markers closely linked to very early genes e1 and e2 and having high selection efficiency are developed, and a method for developing very early rice by marker selection for e1 gene and/or e2 gene is provided. Therefore, the present invention can be used in the field of agriculture, particularly in the field of breeding of grasses.

Claims (5)

The presence of the e1 gene and/or the e2 gene is determined by detecting a DNA marker present in a region at a distance of 6.6 to 8.3 Mb from the short arm end of the chromosome 7 of rice, A method for selecting very early rice, wherein the DNA marker is one or more selected from the group consisting of RM21224, J1753, J1759, and J1763. The presence of the e1 gene is determined by detecting a DNA marker present in a region at a distance of 6.8 to 8.3 Mb from the short arm end of chromosome 7 of rice , wherein the DNA marker is J1753, 2. The method of claim 1, wherein one or more selected from the group consisting of J1759 and J1763 . The presence of the e2 gene is determined by detecting a DNA marker present in a region at a distance of 7.4 to 8.3 Mb from the short arm end of chromosome 7 of rice , wherein the DNA marker is J1759 and 2. The method of claim 1, which is one or more selected from the group consisting of J1763 . A method for producing extremely early rice, comprising:
(a) crossing extremely early rice with non-extremely early rice to produce hybrid seeds;
(b) cultivating the hybrid seed to produce a hybrid rice plant;
(c) a method comprising selecting very early rice from the hybrid plants by the method according to any one of claims 1 to 3 ; 5. The method according to claim 4 , wherein the non-extremely early-maturing rice plant is a short-culm rice plant or a large-grain rice plant.