CN108085320B

CN108085320B - Dominant early maturing gene Ef-cd of rice and application thereof

Info

Publication number: CN108085320B
Application number: CN201711479841.5A
Authority: CN
Inventors: 方军; 储成才; 邓晓建; 张帆涛
Original assignee: Northeast Institute of Geography and Agroecology of CAS
Current assignee: Northeast Institute of Geography and Agroecology of CAS
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2021-02-12
Anticipated expiration: 2037-12-29
Also published as: CN108085320A

Abstract

A rice dominant early maturing gene Ef-cd and application thereof relate to a rice gene Ef-cd and application thereof. The invention aims to provide a rice dominant precocity gene Ef-cd and application thereof, which are used for promoting the precocity of rice without influencing yield, thereby further improving the yield of hybrid rice and adapting to the short growing period condition in high latitude areas. The nucleotide sequence of the dominant early maturing gene Ef-cd of the rice is shown as SEQ ID NO 1 in a sequence table. Application of dominant early maturing gene Ef-cd of rice in promoting plant early maturing. The Ef-cd mutant can be used for promoting the early maturing of rice without influencing the yield, thereby further improving the yield of hybrid rice, adapting to the short growing period conditions in high latitude areas and simultaneously meeting the early maturing characteristic required by modern light and simple cultivation.

Description

Dominant early maturing gene Ef-cd of rice and application thereof

Technical Field

The invention relates to a rice gene Ef-cd and application thereof.

Background

Rice (Oryza sativa L.) is an important food crop on which humans depend for survival, and more than half of the world's population uses rice as staple food. China is a world rice production major country and a rice history ancient country, is one of the largest rice production bases, and the total rice production is the first world. In recent years, with the increase of the world population, the aggravation of aging, the acceleration of urbanization, the deterioration of the environment caused by the damage of vegetation in partial areas and the like, how to improve the yield and the quality of rice under the condition of limited manpower and environmental resources and ensure the food safety becomes a major problem related to the national civilians.

The grain yield per unit area is improved, and the planting area is enlarged, so that the demand of the continuous population growth on the grains can be effectively solved. Firstly, the most effective method for improving the grain yield per unit area is to carry out more than one year, and the growth period is shortened. Naturally, the desire to increase crop production requires the accumulation of carbohydrates through longer periods of photosynthesis. For example, the first generation varieties IR20, IR24 and IR26 bred by IRRI and the commercial hybrid rice varieties Shanyou 1 and Shanyou 2 bred domestically all require 160 days or more for ripening or harvesting. Although the yield is high, the growth period of the varieties is long, and the varieties can be planted in tropical or subtropical regions only for one season of a year, so that the yield improvement is seriously hindered. On the other hand, a large number of planting areas with sufficient water sources exist in the north of China, but due to low accumulated temperature, the number of varieties which can be planted early after frost is low, and the land utilization is low, so that the breeding period is shortened when the early-maturing and high-yield rice variety is cultivated, and the expansion of the planting area becomes the key point of current breeding research. In rice breeding, high yield and early maturing are long-standing contradictions, namely, high-yield varieties are often late maturing, so that the cultivation of excellent early maturing varieties meets the requirements of various ecological regions and production seasons, and the achievement of high and stable yield is a great challenge to rice production and has very important significance.

Rice breeders have been struggling to develop early maturing varieties for hybrid rice production. In 1976 IRRI issued to IR36 for maturation period of 110 days, IR50 for 105 days and IR58 for 100 days. The test 64 cultivated by the domestic breeder is an early maturing restoring line, and the derived variety and the Shanyou 64 are crops which can be harvested for two seasons in one year. In 1991-2010, the planting area of the dominant variety Minghui 77 and the derived varieties of the other early-maturing restorer line reaches 7,446,700 hectares. The gene for controlling the characteristics of early maturity and high yield of the varieties and the development of markers which can be used for breeding become hot spots for research of rice scientists and breeders.

The earliness of rice is generally considered to be a recessive character controlled by 1-2 pairs of major genes or a quantitative character controlled by multiple genes, and a plurality of Quantitative Trait Loci (QTL) influencing the growth period (heading period) or the maturity are discovered or positioned in the breeding research process. The Japanese rice genome gene (RGP) carries out systematic and intensive research on the positioning of rice heading QTL, the program analyzes Nipponbare/Kasalath derived population to position 14 heading QTLs (Hd1-Hd14), and later researchers position Hd15-17, and the genes Hd1, Hd3a (Hd3 loci are divided into Hd3a and Hd3b), Hd6 and the like are mapped. Researches show that rice flowering is mainly regulated and controlled by homologous genes RFT1 of florigen genes Hd3a and Hd3a, wherein the florigen genes Hd3a are used for controlling rice flowering under a short day condition, and the florigen genes Hd3a and RFT1 are used for promoting expression of Ehd1 and RFT 50 through OsSOC1/OsMADS50 under a long day condition. In addition, 29 heading stage genes and modification genes thereof, and 18 light-sensitive genes and modification genes thereof are reported, wherein the non-light-sensitive genes comprise dominant early genes Ef-1, Ef-x (t), Ef-y (t) and Ef-cd (t), and the recessive late genes comprise Ef-3(t), Ef-4(t) and lf-3 (t); examples of the modifying gene include m-Ef-1, w-Ef-1(t), Su-Ef-ed (t), and the like.

According to breeding practice and the genetic basis of heterosis, namely the theory that the heterosis effect and dominant effect of single gene control dominate, breeding early-maturing hybrid rice requires that the sterile line is dominant early-maturing or the restoring line is recessive late-maturing to achieve better effect. The discovery and utilization of the early maturing gene of the rice are beneficial to solving the contradiction that the early maturing and the high yield are difficult to be considered simultaneously, and are also beneficial to overcoming the obstacle of super-relativity and late maturing between indica subspecies. Therefore, the discovery and identification of the rice heading stage genes comprise the research on heading stage gene positioning, cloning, backcross transformation and the like, particularly the combination of molecular marker assistance and agronomic trait selection is utilized, the breeding utilization of the heading stage genes is deeply researched on the basis of traditional genetic breeding, and the method has important theoretical significance and application value.

Disclosure of Invention

The invention aims to provide a rice dominant precocity gene Ef-cd and application thereof, which are used for promoting the precocity of rice without influencing yield, thereby further improving the yield of hybrid rice and adapting to the short growing period condition in high latitude areas.

The dominant early maturing gene Ef-cd of the rice is from an indica rice sterile line 6442S-7, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1 in a sequence table.

The accession number of the Nipponbare allele corresponding to the Ef-cd is Os03g0122500, and the nucleotide sequence is shown as SEQ ID NO. 2 in the sequence table.

The cloned rice dominant precocity gene Ef-cd mutant is long noncoding RNA (lncRNA) and comes from an antisense strand of an OsSOC1 gene, and the precocity of rice is controlled by positively regulating and controlling the expression of the OsSOC1 gene. The mutant sequence comprises 4 deletions, 1 insertion and 22 SNPs in the Os03g0122500 promoter region, and 1 insertion and one SNP mutation respectively positioned in a first intron and a second intron in the gene. The presence of the variant Ef-cd can obviously promote the early maturing of rice. It was also found by genotyping different cultivars that a total of 72 cultivars of 1479 cultivars contained this dominant early-maturing genetic variation, of which 29 were from the International Rice Institute (IRRI); in 1,439 Chinese excellent hybrid rice produced in field, 299 hybrid genotype and 16 homozygous genotype both promote rice heading obviously.

The Ef-cd mutant can be used for promoting the early maturing of rice without influencing the yield, thereby further improving the yield of hybrid rice, adapting to the short growing period conditions in high latitude areas and simultaneously meeting the early maturing characteristic required by modern light and simple cultivation.

Drawings

FIG. 1 shows the phenotype of the Ef-cd near isogenic line D248 and the recurrent parent Shuhui 881(SH 881);

FIG. 2 is an Ef-cd fine positioning interval;

FIG. 3 is the Os03g0122500 gene sequence aligned D248 with Shuhui 881(SH 881);

FIG. 4 shows the phenotype of the T-DNA insertion mutants ef-cd-1 and ef-cd-2 of the Os03g0122500 gene;

FIG. 5 shows the RNAi mutant phenotype of Os03g0122500 gene;

FIG. 6 shows the expression identification of the gene of the RNAi mutant line Os03g 0122500;

FIG. 7 is an Ef-cd-Cas9 knockout mutant phenotype;

FIG. 8 is a schematic structural diagram of Os03g0122500 gene; the knockout site is an Os03g0122500 promoter region;

figure 9 compares the luciferase activity regulated by the Os03g0122500 promoter in D248 and its recurrent parent SH881 in protoplasts with significance p < 0.01;

FIG. 10 is the relative expression level of Ef-cd in D248 and SH881 15-60 days after sowing;

FIG. 11 is the relative expression level of OsSOC1 in D248 and SH881 15-60 days after sowing;

FIG. 12 is the relative expression level of Ef-cd and OsSOC1 in wild type and Ef-cd-2 mutant (PFG _2A-10486.R) at 45 days after sowing;

FIG. 13 shows the relative expression levels of Ef-cd and OsSOC1 in wild type and Ossoc1 mutant (PFG-3A-05349. L) at 45 days after sowing;

FIG. 14 is the relative expression levels of Hd3a and RFT1 in D248 and SH881 after 30 days of short-day sun treatment after sowing;

FIG. 15 is the relative expression levels of Hd3a and RFT1 in D248 and SH881 after 40 days of short-day sun treatment after sowing;

FIG. 16 shows the relative expression levels of OsSOC1 genes in early-maturing and late-maturing varieties of different genotypes,^*represents p<0.05；^**Represents p<0.01; NS is not significant;

FIG. 17 shows the relative expression levels of the Ef-cd genes in early-maturing and late-maturing varieties of different genotypes.^*Represents p<0.05；^**Represents p<0.01; NS is not significant;

FIG. 18 shows a comparison of heading date of Beijing (39 ℃ 54' N) recurrent parent;

FIG. 19 is a comparison of the heading date of the Sichuan adults (30 ℃ 42' N) recurrent parent;

FIG. 20 is a comparison of heading date of the Hainan Ling Water (18 ℃ 22' N) recurrent parent;

FIG. 21 is a comparison of the heading maturity of the Ef-cd isogenic line hybrid rice ZS97A/D330 with the control hybrid rice ZS97A/MH 63;

FIG. 22 is a comparison of the heading maturity of the Ef-cd isogenic line hybrid rice E-II-32A/FH838 with that of the control hybrid rice II-32A/FH 838;

FIG. 23 is a graph showing the comparison of yield data of hybrid rice SY63, E-SY63, II-You838 and E-II-You838 in Beijing;

FIG. 24 is a graph showing the comparison of yield data of Jiaxing hybrid rice SY63, E-SY63, II-You838 and E-II-You 838;

FIG. 25 is a graph comparing yield data for oryza sativa SY63, E-SY63, II-You838, and E-II-You 838;

FIG. 26 is a graph showing the comparison of yield data for hybrid rice SY63, E-SY63, II-You838 and E-II-You838 in Fuzhou province;

FIG. 27 is pedigree information for D248;

FIG. 28 is a graph showing the analysis of the prevalence of the Ef-cd gene in rice cultivars using the 122500indel-1 marker (adjacent to the 36bp insertion/deletion gene site);

FIG. 29 is a graph showing the use of the 122500indel-1 marker to analyze the prevalence of the Ef-cd gene in rice cultivars;

FIG. 30 is a phenotypic and genotypic interaction analysis of the trilogy rice variety Ef-cd;

FIG. 31 is a phenotypic and genotypic interaction analysis of Hangzhou rice variety Ef-cd.

Detailed Description

The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.

The first embodiment is as follows: the dominant early maturing gene Ef-cd of the rice of the embodiment is derived from an indica rice sterile line 6442S-7, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1 in a sequence table.

The second embodiment is as follows: the accession number of the Nipponbare allele corresponding to the Ef-cd of the present embodiment is Os03g0122500, and the nucleotide sequence is shown as SEQ ID NO. 2 in the sequence Listing.

The third concrete implementation mode: the application of the embodiment in promoting the early maturing of the plant.

The fourth concrete implementation mode: the third difference between the present embodiment and the specific embodiment is that: the plant is rice. The rest is the same as the third embodiment.

The fifth concrete implementation mode: the third difference between the present embodiment and the specific embodiment is that: the plant precocity promotion is specifically promoting rice heading. The rest is the same as the third embodiment.

The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.

Example 1: fine localization of rice Ef-cd gene

The Ef-cd is the first site of dominant precocity, found in male sterile line 6442S-7 of indica rice and located on chromosome 3 of rice. In order to finely locate the Ef-cd gene, serial backcross is carried out between Shuhui 881(SH881) as recurrent parent and sterile line 6442S-7, then selfing selection is carried out to construct BC₅F₁₁Near isogenic line D248, BC₅F₁₁The near isogenic line D248 is hybridized with recurrent parent Shuhui 881(SH881) to obtain F₁Seed, F₁Selfing to obtain F₂. From F₂The DNA extracted from 4,800 late flowering individuals is screened and identified in the generation group, and the published SSR markers of rice and the gene sequences flanking the Ef-cd in SH881 and 6442S-7 are utilized to develop Indel markers (Table 1) to finely locate the Ef-cd genes.

DNA extracted by CTAB method is used as PCR reaction template, the total volume of PCR reaction system is 10 μ l, the DNA template of rice genome is 1 μ l (about 200ng), 2 × Master Mix is 5 μ l, 10 μ M primers are 0.5 μ l each, add ddH₂O to 10. mu.l. Reaction procedure: denaturation at 94 deg.C for 5 min; 30s at 94 ℃; 30s at 58 ℃; 20s at 72 ℃ for 35 cycles; extension at 72 ℃ for 10 min.

TABLE 1 map-based molecular marker sequence information

The results are shown in fig. 1 and fig. 2, the heading date of the near isogenic line D248 is significantly earlier than that of the recurrent parent holhou 881 (fig. 1); using F₂Population selection identified multiple Indel markers, mapping the Ef-cd to a 12.9Kb intervalThe selected region contained the flowering activator Os03g0122600(OsSOC1/OsMADS50/DTH3) and Os03g0122500 genes (FIG. 2). The heading stage refers to the time from sowing to heading when the scab is extracted.

In order to shorten the localization interval and more finely localize the mutant gene, total RNA of SH881 and D248 leaves was extracted by TRIzol (Invitrogen, USA), 1. mu.g of total RNA was reverse transcribed by ReverTra Ace qPCR RT Master Mix (Toyobo, Japan) to synthesize cDNA, the full-length cDNA sequences of the OsSOC1 and Os03g0122500 genes of SH881 and D248 were amplified by PCR, respectively, and PCR products were recovered for cloning and sequencing. The total volume of the PCR reaction system was 50. mu.l, 1. mu.l (about 200ng) of the rice genomic DNA template, 25. mu.l of 2XPCR buffer for KOD Fx, 10. mu.l of 2mM dNTP, 1. mu.l of KOD Fx (1U/. mu.l), 1.5. mu.l of each 10. mu.M primer, and ddH2O to 50. mu.l. Reaction procedure: denaturation at 98 deg.C for 2 min; 10s at 98 ℃; 90s at 60 ℃; 2min at 68 ℃ and 30 cycles; extension at 78 ℃ for 10 min. The PCR product was recovered and the clones were subjected to Sanger sequencing analysis.

The sequencing results were compared, and it was found that the cDNA sequence of OsSOC1 gene derived from SH881 and D248 did not differ by SNP, while Os03g0122500 transcribed from the antisense strand of OsSOC1 belongs to IncRNA, and the accession number of database (https:// shigen. nig. ac. jp/rice/oryzae) is AK242050, and that differences existed in both promoter region and intron region. Compared with SH881, D248 has deletion of 4 large fragments (including a large deletion mutation of 36bp and 267 bp), 1 single-base insertion and 22 SNPs in the Os03g0122500 promoter region; and one insertion and one SNP in the first and second introns, respectively, within the gene (figure 3).

To verify the function of the candidate gene, two mutants of the Os03g0122500 gene obtained from a pool of rice T-DNA insertion mutants (http:// signal. salk. edu/cgi-bin/Rice) were designated ef-cd-1(PFG-1D-05526.R) and ef-cd-2(PFG-2A-10486. R). The T-DNA insertion sites for ef-cd-1 and ef-cd-2 were located in the first intron and promoter region of the Os03g0122500 gene, respectively (the mutation regions were identical to D248), and both exhibited a delayed heading date phenotype (FIG. 4). The results indicate that the variation of the promoter region and the intron region of the Os03g0122500 influences the growth period of rice, and the Os03g0122500 may be a candidate gene of the Ef-cd.

Example 2: analysis of promoter function

(1) RNAi vector construction

Amplifying 168bp of the 5 ' region of the Os03g0122500 gene by using the Ef-cd full-length gene of D248 as a template, wherein the sequence of an amplification primer is RNAiP forward primer: 5'-GTCGACTCAAAGCGATCTGTCACCTGAA-3'; RNAiP reverse primer: 5'-GGATCCTACTTGTACTCCTGCTTG-3' was constructed into a pUCriRNA vector using a XhoI/BglII and SalI/BamHI double ligation. Finally, the stem loop was cloned into pCAMBIA2301 vector. The rice Nipponbare is transformed by an agrobacterium-mediated method. The expression level of the Ef-cd gene was detected by a semiquantitative method.

(2) CRISP/Cas9 vector construction

The Ef-cd-Cas9 mutant was constructed by Biogle (Hangzhou, China). The construction process is as follows: two different sgRNA targets, sg1213: 5'-GTCGATTCGGTGCGTGATAA-3' and sg1214: 5'-GTCATATACAACAGGGGTAC-3', were designed for the Os03g0122500 promoter region. The two sgRNAs are constructed on a BGK032-DSG vector containing Cas9 protein, agrobacterium tumefaciens EHA105 is transformed, and then rice Nipponbare is genetically transformed. Knocking out and detecting transgenic plants: extracting the genome DNA of the transgenic plant, and performing PCR amplification and sequencing analysis on the knock-out fragment by using a primer Efcd-cas9 near a target point.

Efcd-cas9 forward primer:5’-TCAGACTCCAGAGTTGGAAAA-3’

Efcd-cas9 reverse primer:5’-TGCGAAATGCTAGACGCATA-3’。

As shown in FIGS. 5-9, the RNA interference technique was applied to the promoter region sequence of Os03g0122500, which resulted in the phenomenon of delayed heading date after the expression level of Os03g0122500 gene was decreased (FIGS. 5 and 6). An Ef-cd-Cas9 mutant of an Os03g0122500 gene knocked out by using a CRISP/Cas9 technology is sequenced, 158bp knocking-out exists in a promoter region of the Os03g0122500, and the mutant presents a late-flowering phenotype (figure 7 and figure 8). These results also indicate that the Ef-cd (Os03g0122500) gene promoter region variation affects the rice growth period, resulting in a late-maturing phenotype.

(3) LUC assays luciferase Activity assays

There are many nucleotide site variations in the Os03g0122500 promoter region, and it is therefore presumed that these variations may affect the activity of the promoter of the gene.

The 2050 base upstream promoter sequence of the gene was cloned from D248 and its recurrent parent SH881, respectively, and inserted into the target vector pGWB535 by the In-Fusion kit from Clontech and fused with the firefly luciferase coding sequence. The luciferase activity of the reporter gene was measured using a dual-luciferase kit from Promega.

As a result, as shown in FIG. 9, the luciferase activity of pD248:: LUC was 7.34 times higher than that of pSH881: LUC, indicating that the promoter of Ef-cd had a stronger activity in D248.

Example 3: QPCR comparative analysis of the changes in the expression of Ef-cd and OsSOC1

(1) Expression changes of Ef-cd and OsSOC1 in D248 and SH881

Total RNA was extracted from rice leaves 15 to 60 days after sowing using TRIzol (Invitrogen, USA). cDNA was synthesized by reverse transcription of 1. mu.g total RNA using ReverTra Ace qPCR RT Master Mix (Toyobo, Japan), and qPCR primers were designed:

Ef-cdQ forward primer:5’-ACTCCTCCTTTCGTCTTCCT-3’，

Ef-cdQ reverse primer:5’-GCTTGATTGTGGCTTGGTATTT-3’，

OsSOC1Q forward primer:5’-CTGCGTTGAGAAAGGAGATGAT-3’，

OsSOC1Q reverse primer:5’-CATGGACGACAATACGGGTAAG。

the qPCR reaction system was mixed according to SYBR Green Real-Time PCR Master Mix reagent (Toyobo, Japan), the conditions of the qPCR reaction: 2min at 95 ℃; 15s at 95 ℃; 30s at 60 ℃; goto step 240 cycles at 95 ℃ for 1min, 55 ℃ for 1min, 95 ℃ for 30 s. The primer sequences are shown as follows by taking rice OsActin as an internal reference:

OsActin1Q forward primer:5’-ACCATTGGTGCTGAGCGTTT-3’，

OsActin1Q reverse primer:5’-CGCAGCTTCCATTCCTATGAA-3’。

QPCR adopts comparative CT method (^ΔΔCT) is adopted to calculate the gene expression quantity, the rice OsActin gene is taken as an internal reference gene, and an untreated sample is taken as a reference. Target Gene TableThe difference in arrival is expressed by the multiple of the treated samples relative to the untreated samples at each time point. Each sample included 3 biological replicates and 3 technical replicates, and the data were averaged over 3 biological replicates, and two data were averaged if there was a large deviation in one value. The raw data is normalized. And (5) carrying out difference significance analysis on the data after the normalization treatment by a T test. The relative expression amount calculation method comprises the following steps: 2^-ΔΔCT＝2^{- (Delta CT treatment-Delta CT control)}＝2^{- [ (CT treatment target Gene-CT treatment reference Gene) - (CT control target Gene-CT control reference Gene)]}。

The results are shown in FIGS. 10-13: the Ef-cd began to rise at day 30 of D248 seedling development and reached a maximum at day 60, while there was little change in expression during SH881 seedling development (FIG. 10, where Curve 1 represents SH881 and Curve 2 represents D248); OsSOC1 shows increased expression level in 15-60 days of D248 and SH881 seedling development, while the expression level in D248 is higher, and particularly the expression level is increased more remarkably in 30-60 days (FIG. 11, wherein curve 1 represents SH881 and curve 2 represents D248). The expression level of OsSOC1 in the ef-cd mutant (ef-cd-2) is obviously reduced (figure 12); whereas the expression level of Ef-cd was not significantly reduced in the Ossoc1 mutant (PFG-3A-05349. L) (FIG. 13). The above results indicate that the expression level of D248 is higher in the Ef-cd and OsSOC1, and particularly that the Ef-cd regulates the expression of OsSOC1 30-60 days after sowing.

(2) Relative expression levels of anthocyanin genes Hd3a and RFT1 in D248 and SH881

The OsSOC1 influences the growth period of rice by regulating the expression of downstream florigen genes Hd3a and RFT1, so whether the variation of the Ef-cd influences the relative expression of Hd3a and RFT1 genes is analyzed by a qPCR method.

Extracting total RNA of D248 and SH881 under the conditions of 30 days short day and 40 days long day after sowing, carrying out reverse transcription and qPCR analysis on the relative expression quantity of Hd3a and RFT1 genes on seedling leaves, and carrying out the following analysis steps as in example 3(1) and primer sequences of Hd3a and RFT 1:

Hd3aQ forward primer:5’-TCGAGGTCGGCGGCAATGAC-3’

Hd3aQ reverse primer:5’-CCTCGCGCTGGCAGTTGAAGTAGAC-3’；

RFT1Q forward primer:5’-CCATTCGTCCGGATCACTAACCTCA-3’，

RFT1Q reverse primer:5’-CGCGCTGGCAGTTGAAGTAGAC-3’。

the study is shown in figures 14 and 15: under the short-day and long-day conditions of 30 days and 40 days after sowing, the expression of Hd3a and RFT1 in D248 of seedlings is obviously higher than that of SH881, which shows that the expression of Hd3a and RFT1 genes is promoted by the Ef-cd variation under the long-day or short-day conditions.

(3) Expression changes of Ef-cd and OsSOC1 in different genotype heading stage varieties

dth3 and Hd9 are heading stage genes mapped to chromosome 3, and are derived from African rice and Japanese variety Kasalath, respectively, and are different from the genotypes of the IRRI source and its derivative line varieties. The nearly isogenic systems dth3-NIL (the African Rice Pyrex Roche parent Dianjingyou1, DJY1), Hd9-NIL (the Kasalath Pyrex Roche parent Nipponbare, Nip) were all late-maturing phenotypes.

Total RNA of the following different precocious and late materials was extracted, and qPCR analysis was performed after reverse transcription into cDNA to analyze the relative expression amounts of the Ef-cd and OsSOC1 genes, in the same manner as in example 3 (1).

TABLE 2 different breeding genotype materials and their control materials

Precocity	Late maturing
		DJY1	dth3-NIL (Pyrolu Africa parent DJY1)
Nip	Hd9-NIL (Kasalath gamma gyrus parent Nip)
		Zaohui89(IR50 gamma gyrus parent IR24)	IR24
D330	Minghui 63(MH63)

The qPCR analysis results are shown in FIGS. 16 and 17, and the relative expression amounts of the Ef-cd and OsSOC1 genes in the early-maturing variety are obviously higher than those of the late-maturing variety; sequence analysis shows that the Ef-cd sequence has 30 SNPs and 3 large fragment insertions in dth3-NIL, and Hd9-NIL has 11Kb large fragment deletion. And the genotype of the early recovery 89 and the D330 are the same as that of the D248; the genotypes of IR24 and Minghui 63 are the same as SH 881. The above results indicate that the characteristics of precocity in varieties of different genotypes are positively correlated with the expression levels of Ef-cd and OsSOC 1.

Example 4 close linkage of the Ef-cd genotype to the Rice early maturing trait

According to the family data, the D248 Ef-cd genotype was deduced to be from the international rice research institute-developed variety IR9761-19, and was detected in the progeny of the breeding variety (FIG. 27).

The Ef-cd genotype (a 36 base deletion in the promoter region of the Ef-cd compared to the late-maturing genotype) was examined using the 122500indel-1 marker. Research shows that early-maturing varieties (IR30, IR36, IR50, IR58, IR64 and IR74) bred in later period of international rice plant all contain an Ef-cd locus like detection 64 (as shown in FIG. 28, early-maturing varieties Ce64, IR30, IR36, IR50, IR58, IR64 and IR74 all contain an Ef-cd gene locus, but late-maturing varieties IR24 and IR29 do not contain the Ef-cd gene locus); meanwhile, the major early-maturing restorer lines in China all contain the Ef-cd early-maturing genes, such as test 64-7 (80 th 20), Minghui 77 (90 th 20), R402 (beginning of 21 st) and To463 (modern), but do not exist in the late-maturing restorer lines such as Minghui 63, 9311, Shuhui 881 and Fuhui 838 (as shown in FIG. 29, the Ef-cd gene loci exist in the major early-maturing restorer line varieties in China, such as Ce64-7, Minghui 77(MH77), R402 and To463, but do not exist in the major late-maturing restorer varieties in China, such as Minghui 63(MH63),9311, Shuhui 881(SH881) and Fuhui 838(FH 838)).

Through genome analysis of 1495 high-quality hybrid rice varieties, the major QTL related to the change of the panicle stage in Sansui and Hangzhou is the gene locus of Ef-cd-OsSOC 1. Therefore, the heading date of 1439 high-quality hybrid rice varieties (indica-indica hybrids) in san and Hangzhou was analyzed by using 122500indel-1 marker. As shown in FIGS. 30 and 31, in the third generation (a) and the Hangzhou (b), the early D248 homozygous genotype (AA) and heterozygous genotype (AT) hybrid varieties identified by the SNP marker vf0301285586 generally heading earlier than the wild SH881 genotype (TT) variety. Significance p <0.01, NS means not significant. All hybrids with homozygous or heterozygous Ef-cd genotypes shed early. These results also indicate that the Ef-cd gene is closely linked to the early maturity of rice.

Example 5: growth period effect and yield effect test application of Ef-cd gene

(1) The ear sprouting period of the Ef-cd near isogenic line is obviously shortened

The method is characterized in that the method is planted in rice planting areas in 2015 and planted in China academy of sciences (39 degrees 54 ' N, Beijing), Jiaxing agriculture academy of sciences (30 degrees 75 ' N, Jiaxing Zhejiang), Sichuan agriculture university (30 degrees 42 ' N, Sichuan Chengdu), Fujian agriculture academy of sciences (26 degrees 08 ' N, Fujian, Fuzhou) and Hainan Ling water (18 degrees 22 ' N) in a random block mode. The row spacing of rice planting is 26cm, and the spacing is 17 cm. The field management follows the following normal agricultural fertilization experience (per hectare): 50kg of nitrogen, 60kg of phosphorus and 95kg of potassium as base fertilizers; 90kg of tillering nitrogen; 30kg of nitrogen in heading stage. All rice material was grown under the same culture conditions. Block experiments were repeated 3 times. Heading date is the number of days from sowing to emergence of the first inflorescence above the sword-like leaves, and heading date is recorded every day.

The results are shown in FIGS. 18, 19 and 20, where the near isogenic lines of the higher generation (Table 3) are phenotypically similar to the recurrent parent, but with an early heading stage. When the rice is planted in 3 rice planting areas with different photoperiods, such as Hainan Lingshui (18 degrees 22 ' N), Sichuan Chengdu (30 degrees 42 ' N), Beijing (39 degrees 54 ' N) and the like, the heading period of the near isogenic line is shortened by 8.9-19.7 days compared with that of the wild type.

TABLE 3

Recurrent parents	NILs	Combination	Generation
				Shuhui881	D248
	6442S-7/SH881	BC₅F₁₁
			Minghui63	D330	6442S-7/MH63	BC₉F₁₂
Shuhui527	D488	6442S-7/SH527					BC₉F₁₂
			Mianhui725	D439	6442S-7/MH725	BC₁₃F₁₀
Yihui577	D374	6442S-7/YH1577					BC₁₀F₉
			II-32B	B3115		6442S-7/II-32B		BC₆F₁₄

(2) The Ef-cd mutant genotype is premature without reduced yield

Rice was planted in 2015 at the institute of genetics and development of the chinese academy of sciences (39 ° 54 'N, beijing), the jiaxing agricultural academy (30 ° 75' N, jiaxing, zhejiang), the sichuan agricultural university (30 ° 42 'N, changchun), the fujian agricultural academy (26 ° 08' N, fujian, fuzhou), in the same manner as in (1) of example 5.

The planting material is early-maturing near isogenic lines D248 and D330 and corresponding late-maturing parents SH881 and MH 63; southern hybrid rice Shanyou 63(ZS97AXMH63), II-You838(II-32AXFH 838); the use of a near-isogenic line for introducing the Ef-cd into the above hybrid rice: ZS97A/D330, wherein D330 is near isogenic line of MH63 introduced into Ef-cd gene, E-II-You838(E-II-32A/FH838), wherein E-II-32A is near isogenic line of II-32A introduced into Ef-cd gene.

Comparison of the yield-related traits of the early-maturing near-isogenic lines (D248, D330) and the corresponding late-maturing parents (SH881, MH63) showed no significant differences including panicle number, grain number, seed set rate and thousand kernel weight, indicating that the Ef-cd gene did not affect yield.

In order to further detect whether the Ef-cd can be introduced into the hybrid rice to shorten the heading stage without influencing the yield, the investigation finds that the heading and the mature stages of all the hybrid rice containing the Ef-cd near isogenic lines of the four sites are 6.9-15.9 days earlier than those of the corresponding hybrid rice (figures 21 and 22), and the spike number, the plant height, the grain number, the maturing rate and the thousand grain weight show no significant difference. Comparing the yields of the hybrid rice with the Ef-cd genotypes with the corresponding control hybrid rice, it was found that the yields of hybrid rice with both the Ef-cd genotypes in Beijing were higher than the control hybrid rice (FIG. 23), that the yields of E-II-You838(E-II-32A/FH838) in hybrid rice with only one Ef-cd genotype in Jiaxing were higher than the control (FIG. 24), and that the yields were the same elsewhere (FIGS. 25 and 26). These results strongly suggest that the Ef-cd-OsSOC1 locus can be applied to the sterile line or the restorer line of hybrid rice to promote the early maturity of the hybrid rice without influencing the yield. (in FIGS. 23-26, p < 0.01; NS is not significant).

Sequence listing

<110> institute of geography and agroecology of northeast China academy of sciences

<120> dominant early maturing gene Ef-cd of rice and application thereof

<160> 18

<210> 1

<211>2327

<212> DNA

<213> Rice (Oryza sativa)

<220>

<223> dominant early maturing gene Ef-cd of rice

<400> 1

attggttgtg aagagaatgt tagtggagaa gttgttatat tttagaacag agggagtaat 60

aattaaaata ctcaacttta tgaaatatcg atacagttat tcttttcttt gtatacttcc 120

aatgcattta tatcctaatt tcacaaactt cgatataata attgtttgag aaagaactta 180

ttttagaaca agtcgtataa gggttaaaaa caaacttatt taagatggag gtatttaggt 240

ggtgtttggt tggagggact aaagtggggc taaactttag tccctctcac aaaaacataa 300

gtctctagtt atatttttgt gagagggact aaatttagtc cctagttacc aaacaccccc 360

ttaagatgga gctagtaccc tgcatgacgg ccatgtgcta gtttatctct cgcctcggga 420

gtttgtgggc catatgctac gtagatgttg ggccatcttc gtgggctcag gctccaccag 480

cacgtccgct cacggcgcat gggatgcacg cggcgccatt tgttccccac actcccgtgg 540

gccgcatact acccaggacg gtggcgcgtt gggccgtatc gtccccaagc ccatcacggc 600

ttttgcccta catcgcctcg atcgcgtttg ggcccgctag tccatgctag tatacgcgac 660

agcgacgtgt acgtacgtct ccaacgcgaa tacaacacct ccacgtggcg tacttgcatg 720

tgaggcagtc ttcaacgtat gtttccctgt caaaattcta ccgattcctg cggtttcctg 780

catccttttt gcctctaatt gcgtgtgacc actagctttt cgcctatgtt cccttccgtg 840

cctccgcgtg acacgagctg tttgagcagc agtgtatatc aactgaacat cgaataaata 900

ctacttcatc cgtttcataa tataagttat tctagcattt ttcacattca tattgattat 960

atatatatat atatatatat atgagaaacg gagggagtaa tgatttatta ctaccatgta 1020

tactcgctgt aaagagaaat atactactac ttcagactcc agagttggaa aaatcacatc 1080

gcggctttgc aagcaaaaac cgttatcacg caccgaatcg agggatacta ctagaagcga 1140

tggggattag ggtccagaaa tctcctcggc ttccactgcc caatcgacac gtcgaccaca 1200

ggtagcattc aagagatcga aacgtcgacc aataccagca tagcgcgtca tatacaacag 1260

gggtacaggt caaagcgatc tgtcacctga atgtactccc tccctcttta aaactcctcc 1320

tttcgtcttc ctcccgttcc ggccttgtgc agagaatctc aacagagata tgttcagcat 1380

atgtttgctc caacatcaca tcaaatacca agccacaata aagcaggagt acaagtacgc 1440

acttgagatc tctcgattta gatttagatg ggtagtggag tctgccgatc gaagggtttc 1500

tgcctggtgg tgctgaatgg gtgggtgcat gcgtctagca tttcgcatcc atggacgaca 1560

atacgggtaa gtaaaatata cagcgtggtc tgtttttagg atggtttggt gtcattgcat 1620

gcctagctat catctccttt ctcaacgcag agatccagct tattcctggc ctgttactta 1680

agaatggggc atcgcttggc tatcttctgc agcaccgccg ctggagcgac ttctgccagg 1740

cagccctatg aatagctcag tttcgacatc catgttgtcg ttggtggtgt tgatgttacg 1800

gtccgggttc tcatcttcag cccggacagt caaaggagca gacaagggag gctgattctt 1860

acactgcaca aagttcatga tcaagggaag caaatcaggt gagttgatct ttcagtgtcg 1920

cagaaaattc tcggcattta tattggtgtg cacataagat atcgcacgaa ttagctcaaa 1980

taatcatatg ctaggctggg cgatcgactt aagctggctg ctggtgtctt ctcggcagta 2040

aaagtttcac tcgtcttacc ttttcgcgta actcttcatt gtccttgcgc agcttcatct 2100

cctggtcgat gttaatgcaa aaaagagaca aacacgacat taggaacaga tgattggtcc 2160

tgcgtttcag gttatatata tatttattta tttatagtaa gtacgaagtt ggtgaggtac 2220

cttctctctc agtttggcaa cctgctcctc aagcagcttt gtctagtatg cagagaattg 2280

gttgtaagtt tgtgcacatc atatatatag ataaattaat tgctgat 2327

<210> 2

<211>2633

<212> DNA

<213> Rice (Oryza sativa)

<220>

<223> nitrile japonica alleles corresponding to Ef-cd

<400> 2

attggttgtg aagagaatgt tagtggagaa gttgttatat tttaggacaa atcctgagag 60

ttaaaagttg ttatatttta gaacagaggg agtaataatt aaaatactca actttatgaa 120

attttgatac agttattctt ttctttgtat acttccaatg catttatatc ctaatttcac 180

aaacttcgat ataataattg tttgagaaag aacttatttt agaacaaatc gtaaaagggt 240

taaaaacaaa cttatttaag atggaggtat ttaggtggtg tttggttcga gggactaaag 300

tggggctaaa ctttagtccc tctcataaaa acataaatcc ctagttatat ttttgtgaga 360

gagactaaat ttagtcccta gttaccaaac acccccttaa gatggagcta gtactctgca 420

tgacggccat gtgctagttt atctctcgcc acgggagttt gtgggccata tgctacgtag 480

atgttgggcc atcttcgtgg gctcaggctc caccagcacg tccgctcacg gcgcatggga 540

tgcacgcggc gccgtttgtt cccccacact cccgtggccg catactaccc aggacggtgg 600

cgcgttgggc cgtatcgtcc ccaagcccat cacggctttt gccctacatc gcctcgatcg 660

cgtttgggcc cgctagtcca tgctagtata cgcgacagcg acgtgtacgt acatctccaa 720

cgcgaataca acacctccac gtggcgtact tgcatgtgag gcagtcttca acgtatgttt 780

ccctgtcaaa attctaccga ttcctgcggt ttcctgcatc ctttttgcct ctaattgcgt 840

gtgaccacta gcttttcgcc tatgttccct tccgtgcctc cgcgtgacac gagctgtttg 900

agcagcagtg tgtatcaact gaacatcgaa taaatactac ttcatccatt tcataatata 960

agttattcta acatttttca cattcatatt gatcatatat atatatatat atgtatatgt 1020

atatatgtat gtatatatgt atgtatgtat atatgtatat atgtatatat atgtatatat 1080

atgtatatat gtatatatat gtatatatat gtatatgtat atatatatat atatgtatat 1140

atatatatat atatgtatat atatatatat gtatatatat atatgtatat atatatatat 1200

gtatgtatat atatatatgt atatatatat atatatatat atatatatat atatatatat 1260

atatatatat atatatatat atatatatga aacggagtga gtaatgattt attactacca 1320

tatatactcg ctgtaaagag aaatatactg ctactacttc agactccaga gttggaaaaa 1380

tcacatcgcg gctttgcaag caaaaaccgt tatcacgcac cgaatcgagg gatactacta 1440

gaagcgatgg ggattagggt ccagaagtct cctcggcttc cactgcccaa tcgacacgtc 1500

gaccacaggt agcattcaag agatcgaaac gtcgaccaat accagcatag cgcgccatat 1560

acaacagggg tacaggtcaa agcgatctgt cacctgaatg tactccctcc ctctttaaaa 1620

ctcctccttt cgtcttcctc ccgttccggc cttgtgcaga gaatctcaac agagatatgt 1680

tcagcatatg tttgctccaa catcacatca aataccaagc cacaataaag caggagtaca 1740

agtacgcact tgagatctct cgatttagat ttagatgggt agtggagtct gccgatcgaa 1800

gggtttctgc ctggtggtgc tgaatgggtg ggtgcatgcg tctagcattt cgcatccatg 1860

gacgacaata cgggtaagta aaatatacag cgtggtctgt ttttaggatg gtttggtgtc 1920

attgcatgcc tagcatcatc tcctttctca acgcagagat ccagcttatt cctggcctgt 1980

tacttaagaa tggggcatcg cttggctatc ttctgcagca ccgccgctgg agcgacttct 2040

gccaggcagc cctatgaata gctcagtttc gacatccatg ttgtcgttgg tggtgttgat 2100

gttacggtcc gggttctcat cttcagcccg gacagtcaaa ggagcagaca agggaggctg 2160

attcttacac tgcacaaagt tcatgatcaa gggaagcaaa tcaggtgagt tgatctttca 2220

gtgtcgcaga aaattctcgg catttatatt ggtgtgcaca taagatatcg cacgaattag 2280

ctcaaataat catatgctag gctgggcgat cgacttaagc tggctgctgg tgtcttctcg 2340

gcagtaaaag tttcactcat cttacctttt cgcgtaactc ttcattgtcc ttgcgcagct 2400

tcatctcctg gtcgatgtta atgcaaaaaa gagacaaaca cgacattagg aacagatgat 2460

tggtcctgcg tttcaggtta tatatatatt tatttattta tagtaagtac gaagttggtg 2520

aggtaccttc tctctcagtt tggcaacctg ctcctcaagc agctttgtct agtatgcaga 2580

gaattggttg taagtttgtg cacatcatat atatagataa attaattgct gat 2633

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> RNAiP forward primer

<400> 3

gtcgactcaaagcgatctgtcacctgaa 28

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> RNAiP reverse primer

<400> 4

ggatcctacttgtactcctgcttg 24

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sg1213

<400> 5

gtcgattcggtgcgtgataa 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sg1214

<400> 6

gtcatatacaacaggggtac 20

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Efcd-cas9 forward primer

<400> 7

tcagactccagagttggaaaa 21

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Efcd-cas9 reverse primer

<400> 8

tgcgaaatgctagacgcata 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Ef-cdQ forward primer

<400> 9

actcctcctttcgtcttcct 20

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Ef-cdQ reverse primer

<400> 10

gcttgattgtggcttggtattt 22

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> OsSOC1Q forward primer

<400> 11

ctgcgttgagaaaggagatgat 22

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> OsSOC1Q reverse primer

<400> 12

catggacgacaatacgggtaag 22

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> OsActin1Q forward primer

<400> 13

accattggtgctgagcgttt 20

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> OsActin1Q reverse primer

<400> 14

cgcagcttccattcctatgaa 21

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Hd3aQ forward primer

<400> 15

tcgaggtcggcggcaatgac 20

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Hd3aQ reverse primer

<400> 16

cctcgcgctggcagttgaagtagac 25

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> RFT1Q forward primer

<400> 17

ccattcgtccggatcactaacctca 25

<210> 18

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> RFT1Q reverse primer

<400> 18

cgcgctggcagttgaagtagac 22

Claims

1. Dominant early maturing gene of riceEf-cdCharacterized in that the rice dominant early maturing geneEf-cdThe gene is derived from indica rice sterile line 6442S-7, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1 in a sequence table.

2. The dominant early-maturing gene of rice of claim 1Ef-cdThe nucleotide sequence of the corresponding Nipponbare allele is shown as SEQ ID NO. 2 in the sequence table.

3. The dominant early-maturing gene of rice of claim 1Ef-cdApplication in promoting early maturing of rice.

4. Use according to claim 3, characterized in that: the promotion of the early maturing of the rice is specifically the promotion of the heading of the rice.