CN114752601A - Gene for regulating and controlling rice stigma exsertion and application thereof - Google Patents

Abstract

The invention discloses a gene for regulating and controlling the exposure of rice stigma, the nucleotide sequence of the gene is SEQ ID NO. 1 or SEQ ID NO. 2, and the gene can be used for improving the stigma exposure level of cultivated rice so as to improve the maturing rate and the yield of hybrid rice.

Description

Gene for regulating and controlling rice stigma exsertion and application thereof

Technical Field

The invention belongs to the field of rice genetic engineering, and particularly relates to newly discovered genes RSE1 and RSE2 for regulating rice stigma exsertion and application thereof in cross breeding.

Background

Cultivated Asian rice (Oryza sativa) is domesticated from common wild rice (Oryza rufipogon) and is one of the most important food crops for human beings. Wild rice is cross-pollinated, whereas cultivated rice is self-pollinated. The stigma exsertion rate is one of key indexes for measuring the rice outcrossing level. In wild rice, the pistil stigma remains on the outer surface of the glume flowers after some or all of the glume flowers have fully bloomed and the inner and outer palea are closed, a phenomenon known as stigma Exsertion (Yan, et al, 2009). But stigma exsertion rarely occurs in cultivated rice or stigma exsertion rate is low. Stigma exsertion represents two different reproduction modes, i.e., outcrossing reproduction of wild rice and selfing reproduction of cultivated rice. The conversion from cross breeding to self breeding is an important physiological mechanism change in the acclimatization process from wild rice to cultivated rice, the conversion has important significance for reducing the progeny segregation of the cultivated rice, and the research on the change of the passage way has important significance for the acclimatization research of rice (Puruggan and Fuller, 2009).

In the process of hybrid breeding, a breeder selects parent materials most concerned about a series of traits affecting the yield of hybrids, such as the heading and flowering stages of the parents, pollen quantity and pollen longevity, panicle type, stigma size and morphology, and plant outcrossing level, wherein the outcrossing level is one of the most important traits affecting hybrid rice seed production. Loose panicle type, large stigma and high-level stigma exsertion form the developmental foundation of wild rice outcrossing behavior, and the stigma exsertion level is one of the core factors determining the outcrossing level of plants. According to field observation, the exposed stigmas can help plants to obtain more pollen, and the time for the exposed stigmas to retain activity in the outside is obviously longer than the window time for opening and closing glumes in the flowering process, so that pollination obstacles caused by inconsistent flowering periods can be overcome by the plants, and the probability of forming hybrids by mutual pollination among different rice plants is increased. The field survey shows that the rising of the stigma exposure level of the female parent of the hybrid rice can obviously improve the maturing rate and the yield of hybrid seeds.

The exposed level of the rice stigma plays an extremely important role in the domestication process. Genomics studies found that the average heterozygosity rate at the wild rice natural population genome level was close to 10%, while the average heterozygosity rate of the fully acclimated cultivated rice natural population genome was essentially zero (Song, et al.2010). The generation of the difference of the genome heterozygosity degree is the inevitable result of complete self-pollination without exposing the stigma of the modern cultivated rice. Because stigma exsertion and spike type are loose, the wild rice individuals can exchange genetic information more easily, and some genome segments are inevitably brought into the genomes of other plants, so that an important mutation source is provided for adapting to different environmental selections of the wild rice. The exchange of genetic information also leads to the generation of more genetic mutations and morphological diversity in the progeny population, which is an important basis for rice domestication. In order to meet the increasing food demand, human beings domesticate wild rice by directional selection to obtain a rice variety which can be stably propagated and has high yield. Along with the directional selection of human beings, the pollination mode of rice is changed from cross-pollination to complete self-pollination, the genetic diversity of rice is obviously reduced, the genome is changed from high heterozygosity to high purity, and the number of alleles of domesticated characters is also obviously reduced. The genome of cultivated rice has high stability, and the control genes for favorable traits are stably present and maintained in progeny. To maintain this stability, stigma exsertion is directionally selected during human acclimation.

At present, the research of rice stigma exsertion has made some progress, but the detailed genetic mechanism and developmental regulation network are not clear yet. Genetic studies find that rice stigma exsertion is a typical complex quantitative trait controlled by multiple genes and is influenced by various external environments, including wind, temperature, humidity, illumination and the like (Miyata, et al 2007), which increases the difficulty degree of related studies. With the progress of molecular marker technology, means for genetic research are continuously abundant. Over the past decades, a series of segregating populations have been developed for genetic studies of rice stigma exsertion, such as the F2 population, the recombinational inbred population, the doubled haploid population, the backcross population, and the chromosome fragment replacement population. With the advent of high throughput genome sequencing technology, GWAS (genome wide association analysis) technology was also applied to the study of rice stigma exposure (Huang, et al.2012). With the aid of the above-described technical methods and populations, researchers have cloned at least 38 QTLs (Quantitative Trait Loci) that control stigma exsertion, which are distributed on 12 chromosomes of rice. Most of these QTLs are microscopic sites, with only a small fraction of successful map-based clones reaching a smaller range and accounting for over 10% of stigma-revealed phenotypic variation.

Disclosure of Invention

In the rice research, we identify QTL genes controlling stigma exsertion by a map-based cloning method and research the functions of the QTL genes. Firstly, a chromosome single-fragment replacement line GPSL41 which takes indica rice GLA4 as a receptor and ordinary wild rice W1943 as a donor is constructed. The stigma exposure rate of GLA4 is lower than 3%, while the stigma exposure rate of GPSL41 is about 30% -50%, the two are backcrossed to obtain a BC1F1 population, further selfing is carried out to obtain a BC1F2 positioning population, molecular markers are utilized to carry out fine positioning, and the site for controlling stigma exposure is positioned in a 14.1kb interval between the P122 and EC24 molecular markers. Genotype and phenotype alignment analysis confirms that the candidate gene is 2 highly homologous MBD genes, the CDS similarity of the two genes reaches 92.22 percent, the protein sequence similarity reaches 86.98 percent, and the genes are respectively named as RSE1 and RSE 2. Meanwhile, a near isogenic line NIL-RSE is constructed, only a W1943 fragment of about 180kb including RSE1 and RSE2 genes is included, and the stigma exposure rate reaches 30-40%.

The results of transgenic complementation experiments show that the genes RSE1 and RSE2 are respectively complemented with GLA4, and the stigma exposure rate can be improved by about 10%. Therefore, the RSE1 and RSE2 genes are verified to be the genes controlling the stigma exsertion in GPSL 41. Both genes contain an MBD domain and a C2H2 zinc finger domain. Comparison of the parent RSE1 and RSE2 gene sequences revealed variations in the promoter region, UTR region and gene coding region.

The results of microscopic observation of NIL-RSE and GLA4 glume flowers before flowering are shown as follows: the stigma deflection angle of the NIL-RSE is significantly greater than GLA4, and the stigma length of the NIL-RSE is also significantly longer than GLA4, all of which may lead to stigma exsertion. The results of mRNA in situ hybridization detection RSE1 and RSE2 gene expression patterns show that: the RSE1 and RSE2 genes have similar expression patterns in GLA4 and NIL-RSE glume development processes. In GLA 40.8-1 mm glume flowers, RSE1 and RSE2 genes are expressed more uniformly in pistil stigma and each part of the stigma, and in the NIL-RSE glume flowers at the same period, RSE1 and RSE2 genes are strongly expressed in a hairbrush-shaped part at the front end of the pistil stigma, and the expression level is obviously higher than that of a non-hairbrush-shaped part and that of GLA4 glume stigma front end at the same period. We speculate that the specific expression patterns of RSE1 and RSE2 genes on NIL stigma influences the development of the stigma and leads to the stigma exsertion.

RSE1 and RSE2 genes are highly homologous with an Arabidopsis floral development regulatory gene AtMBD8, and MBD genes can enter a nucleus to participate in regulating the dynamic balance of genome methylation. The rice protoplast system is used for confirming that the RSE1 and RSE2 fusion protein can enter the nucleus to perform messenger functions. Comparative analysis of 4-5cm young ear transcriptome found: there were approximately 1176 differentially expressed genes between NIL-RSE and GLA4, of which 582 genes were up-regulated and 594 genes were down-regulated. GO enrichment analysis results show that the differential expression genes relate to a transcription regulation process, hormone regulation pathways such as auxin and cytokinin, related pathways of cell cycle and the like.

The results of nucleic acid diversity analysis and Tajima's D analysis of RSE1 and RSE2 genes using 39 parts of modern cultivar rice material and 14 parts of ordinary wild rice material showed that the RSE1 and RSE2 genes had significantly reduced DNA diversity in the cultivar rice population, significantly deviating from neutral selection. It is indicated that the RSE1 gene and the RSE2 gene may be acclimatized and selected. This finding forms the basis of the present invention. Specifically, the present invention includes the following technical contents.

A gene for regulating and controlling rice stigma exsertion, named RSE in the text, comprises two genes RSE1 or RSE2, and the nucleotide sequence of the gene has homology of more than or equal to 90 percent, more than or equal to 92 percent, more than or equal to 95 percent, preferably more than or equal to 98 percent, and more preferably more than or equal to 99 percent with SEQ ID NO. 1 or SEQ ID NO. 2.

The nucleotide sequence of the above gene is preferably SEQ ID NO:1 (derived from rice W1943) or SEQ ID NO:2 (derived from rice W1943).

In a second aspect, the present invention provides a vector comprising the above gene. The vector is a plasmid for integrating the gene into the genome of rice, particularly cultivated rice.

The backbone plasmid of the above vector may be pCAMBIA series. For example, the backbone plasmid may be pCAMBIA1300, etc., and the constructed RSE1 or RSE2 gene expression vector may be pCAMBIA1300-RSE1 and pCAMBIA1300-RSE2, for example.

In a second aspect of the present invention, there is provided an Agrobacterium transformed with the vector described above. The agrobacterium is used for mediating the introduction of a vector containing the RSE1 or RSE2 gene into rice to complete the transgenic operation. The Agrobacterium is selected from the group consisting of Agrobacterium tumefaciens (Agrobacterium tumefaciens) and Agrobacterium rhizogenes (Agrobacterium rhizogenes).

In a third aspect, the present invention provides the use of the RSE1 or RSE2 gene described above, the vector described above, or the Agrobacterium described above in cross breeding.

In a specific application embodiment, the RSE1 or RSE2 gene can be integrated into the genome of Oryza sativa, especially Oryza sativa (Oryza sativa), by plasmid transformation, homologous recombination technology, or gene editing technology.

The above-mentioned gene integration may be Agrobacterium-mediated plasmid transformation.

The gene editing technology can adopt a CRISPR-Cas9 system, a CRISPR-Cpf1 system, a CRISPR-Cas related transposition system INTEGRATE system or a CAST system.

The INTEGRATE system refers to the gene editing tool (Insertion of transposable element for guiding RNA assisted targeting) developed by Sam Sternberg research group; the CAST system is a gene editing tool (CRISPR-associated transposase) developed by the tensor research group.

The INTEGRATE system refers to the gene editing tool (Insertion of transposable element for guiding RNA assisted targeting) developed by Sam Sternberg research group; the CAST system is a gene editing tool (CRISPR-associated transposase) developed by the tensor research group.

The RSE1 or RSE2 gene is used for increasing the stigma exposure level in rice, so that the maturing rate of hybrid rice is increased.

Experiments show that the newly discovered rice RSE1 or RSE2 gene can obviously improve the stigma exposure level of the cultivated rice GLA4, so that the maturing rate and the yield are improved, and the RSE1 or RSE2 gene can be used for improving the existing cultivated rice varieties and has wide development and application prospects.

Drawings

Figure 1 shows a photograph and statistical bar chart comparing stigma exposure levels for GPSL41 and GLA4 in different regions and years. Wherein, A, B: GLA4 and GPSL41 have main ears that flower completely glume flowers, 5mm on a scale; c: stigma exserted phenotype of GLA4 (left) and GPSL41 (right), scale 5 mm; d: the stigma exposure rates of GLA4 and GPSL41 in different regional years are expressed as mean values (SD), and n is 30 (panicle number).

FIG. 2 shows the construction process of the GPSL series chromosome fragment replacement line and the whole genome genotype of GPSL 41. Wherein, A: constructing a GPSL series chromosome fragment replacement line; b: the whole genome genotype of GPSL41, G for homozygous GLA4 genotype, W for homozygous W1943 genotype, and H for W/G heterozygous genotype.

Figure 3 shows photographs of the browning of flowering glumes and stigma. Wherein, A-B: the rice glume flower which is flowering is 5mm in scale; C-H: the process of gradually browning the pistil stigmas of rice in the air.

FIG. 4 shows statistical plots of stigma exsertion rates for different genotype background genetic populations. The Y axis represents the stigma exposure rate, the X axis represents the observation number, nG is 77, nF1 is 133, and nW is 129. G represents GLA4, F1 represents BC1F1, and W represents GPSL 41. Significance of differences between groups was tested using one-way anova with a significance level of 0.01. GLA4 group had an average stigma exposure of 0.053(0.035), F1 group an average stigma exposure of 0.166(0.059), and GPSL41 group an average stigma exposure of 0.286 (0.085). The homogeneity test of variance showed that the F value was 24.342, and p (sig.) was 10^-6The variance among the three groups is irregular. Anova showed a very significant difference in stigma exposure rates between GLA4, F1 and GPSL41 (F308.512, p (sig.) 10)^-6Integrated force testingTest Power is more than or equal to 0.99).

FIG. 5 shows the map-based cloning of the RSE gene. Wherein, A: primary localization of RSE gene; B. c: fine localization of the RSE gene. The values are expressed as mean values (SD) and RSE represents the stigma exposure Rate (Rate/Ratio of stigma exposure). N denotes population size. The numbers in parentheses on the left indicate the recombinants number.

FIG. 6 shows the alignment of the amino acid sequences of the MBD functional domains of the RSE1 gene, the RSE2 gene and the AtMBD8 gene. Where dark colors are labeled as conserved sites.

FIG. 7 shows the alignment of the amino acid sequences of the RSE1 gene and the RSE2 gene.

FIG. 8 shows the structure of a wild rice W1943 BAC fragment comprising the RSE1 gene and the RSE2 gene.

FIG. 9 shows representative photographs and column head exposure rate versus bar graphs of RSE1 complementary transgenic T2. Wherein, A: RSE1 complemented the stigma-exposed phenotype of transgenic T2 plant CP-1. The scale for the whole spike is 1cm, and the scale for the first grade branch is 5 mm. Red arrows are marked as exposed stigma; b: comparing the stigma exsertion rate of parent and CP-1 in the same batch. The values are expressed as mean values (SD), n being 12. The significance of differences between groups was tested using the SNK test and the LSD test,

representing significant differences between groups at a level of 0.05.

FIG. 10 shows representative photographs and a histogram of stigma exsertion ratio of the RSE2 complementary transgenic T2. Wherein, A-C: RSE2 complements stigma exsertion phenotype of transgenic T2 generation plants CP-1, CP-2 and CP-3, the scale of the whole ear is 1cm, the scale of the first-level branch stem is 5mm, and a red arrow mark is an exposed stigma; d: comparing the stigma exposure rate of parent and complementary transgenic strains in the same batch. The values are expressed as mean values (SD), n being 12. The significance of differences between groups was tested using the SNK test and the LSD test,

representing significant differences between groups at a level of 0.05.

Fig. 11 shows a photograph of the morphological structure of an exposed stud and an unexposed stud. Wherein, A: fine structure of rice stigma; b: the form of the unexposed stigma; c: morphological structure of bilateral exposed stigma; d: the morphological structure of the unilateral exposed column head. The scale is 1 mm.

Fig. 12 shows a histogram of statistics of exposed stigma length and AS angle. Wherein, A: comparison of stigma length between GLA4 and NIL-RSE; b: comparison of the column head angle between GLA4 and NIL-RSE. The values are expressed as mean (SD), n being 300. Significance test of differences between groups was performed using t-test of independent samples (levene test value sig ═ 0.082 for equation of variance), bilateral sig ═ 10^-6。

Representing significant differences between groups at a level of 0.05.

FIG. 13 shows the expression level changes of RSE1 gene in different rice panicle development stages. The values are expressed as mean values (SD), n being 3. A one-way anova was used to test the significance of differences between groups. Wherein, Y axis represents relative expression, X axis represents young ear in different development period, and length represents young ear development period.

FIG. 14 shows the expression level changes of RSE2 gene in different development stages of rice panicle. The values are expressed as mean values (SD), with n being 3. A one-way anova was used to test the significance of differences between groups. Wherein, Y axis represents relative expression, X axis represents young ear in different development period, and length represents young ear development period.

FIG. 15 shows photographs of in situ hybridization experiments of RSE1 gene and RSE2 gene. Wherein, A-C: in situ hybridization of the RSE1 gene, A: expression of the RSE1 gene in GLA4 glume flowers; B-C: expression of the RSE1 gene in NIL-RSE glume flowers; D-F: in situ hybridization of RSE2 gene; d: expression of the RSE2 gene in GLA4 glume flowers; E-F: expression of the RSE2 gene in NIL-RSE glume flowers. Scale 20 microns. The black arrow points to the stigma. The material is 4-5cm long young ear.

FIG. 16 is a block diagram of plasmids pCAMBIA1300-RSE1 and pCAMBIA1300-RSE2 constructed according to the present invention. Wherein A is pCAMBIA1300-RSE 1; b is pCAMBIA1300-RSE2 plasmid.

Detailed Description

The invention clones a gene RSE (abbreviation of Stigma exposure Rate of Stigma expression or Ratio of Stigma expression) for regulating and controlling rice Stigma exposure, which comprises two genes RSE1 or RSE2, researches show that the gene RSE can improve the rice Stigma exposure Rate by about 10 percent, and the RSE gene regulates and controls the Stigma exposure Rate by controlling the number and the size of rice Stigma cells. The RSE1 and RSE2 alleles with high stigma exposure rate can obviously improve the population outcrossing rate. The RSE1 and RSE2 loci for controlling the high stigma exposure rate mainly exist in a common wild rice population and are domesticated and selected in an Asia cultivated rice population, so that the common wild rice population can enter most of modern cultivated rice by an artificial selection method, and the genetic diversity of the cultivated rice population and the seed production success rate of hybrid rice are improved.

The RSE1 and RSE2 genes can increase the stigma exposure rate of rice, improve the group outcrossing rate and increase the group genetic diversity and the success rate of hybrid seed production. The common wild rice contains the specific expression genotype of the gene, and the Asian cultivated rice contains the genotype with low stigma exsertion rate and low outcrossing rate, so the gene can be introduced into the Asian cultivated rice through crossbreeding to improve the existing cultivated rice variety.

The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the present invention. Further, it should be understood that various changes and modifications may be made by one skilled in the art after reading the concept of the present invention and those equivalents may also fall within the scope of the invention as defined by the appended claims.

The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.

In the examples herein, if no specific description is made about the reaction temperature or the operation temperature, the temperature is usually referred to as room temperature (15 to 30 ℃).

The whole gene synthesis, primer synthesis and sequencing in this document are all completed by Beijing Optimalaceae Biotechnology Co., Ltd and the national center for gene research of Chinese academy of sciences.

The molecular biology experiments in the examples include plasmid construction, digestion, competent cell preparation, transformation, etc., which were mainly performed with reference to "molecular cloning laboratory Manual" (third edition), J. SammBruker, D.W. Lassel (America), Huang Peitang, et al, science publishers, Beijing, 2002).

Can be operated according to the relevant kit instructions. Specific experimental conditions such as PCR conditions can be determined by simple experiments if necessary.

Examples

1. Materials:

the research mainly uses indica rice GLA4 of indica rice cultivated in Asia with extremely low stigma exsertion level and a chromosome fragment replacement line GPSL41 with high stigma exsertion level. GPSL41 is a replacement line partially substituted on chromosome eight with a fragment of the common wild rice chromosome W1943 against GLA 4.

The genetic population is a recombinant inbred line produced by continuously selfing filial generations after one-generation hybridization of GPSL41 and GLA4 and one-generation backcross. Wherein NIL-RSE is the minimal near isogenic line screened from the inbred progeny population of heterozygous progeny obtained by backcrossing one of the recombinant inbred lines with GLA 4.

2. Paraffin section and in situ hybridization:

materials for histological visualization and in situ hybridization were fixed with 4% paraformaldehyde, dehydrated in a series of ethanol, xylene transparent and embedded in paraffin. The in situ hybridized probe was labeled with digoxin labeling kit from Roche. The labeled in situ sense antisense probe was hybridized on 8- μm paraffin sections.

3. Real-time quantitative PCR:

fresh plant tissue or frozen tissue at-80 ℃ was extracted with Trizol Reagent. DNA in the total RNA samples was digested with DNase and then SuperScript from Invitrogen^TMII Reverse Transcriptase Reverse transcription into cDNA first strand, further real-time quantitative PCR template. For quantitative PCR, Takara was used

Premix Ex TaqTM kit was performed on an Applied Biosystems 7500real time PCR instrument. Specific gene primers are designed according to the gene sequence, and a rice gene eEF-1 alpha (GenBank access No. AK061464) is selected as an internal reference.

Construction of RSE1 or RSE2 transgenic vectors

The RSE1 gene is subjected to Hind III enzyme digestion BAC and inserted into a plasmid pCAMBIA 1300; the RSE2 gene is subjected to Sal I enzyme digestion BAC and inserted into a plasmid pCAMBIA1300 to obtain RSE gene expression vectors pCAMBIA1300-RSE1 and pCAMBIA1300-RSE2 respectively, as shown in FIG. 16. The plasmid contains RSE gene promoter and terminator from rice besides RSE1 or RSE2 gene, and the screening gene is hygromycin.

Results of the experiment

1. Parent Material and phenotypic Studies

The subject uses cultivated rice Guangdong short No. 4 (GLA4) with the Stigma Exsertion Rate of less than 3 percent and a chromosome fragment replacement line GPSL41 with the Stigma Exsertion Rate of 30 percent to 50 percent to carry out map-based cloning of RSE (Rate of Stigma Exsertion, Stigma Exsertion Rate) locus, and is shown as A, B, C in figure 1. The stigma exposure rates of GLA4 and GPSL41 vary with the environment, but phenotypic differences between parents persist, as shown at D in figure 1.

GPSL41 is a chromosome fragment replacement line (GPSL series, fig. 2) constructed in the laboratory with ordinary wild rice W1943 as the donor parent, GLA4 as the acceptor parent, and a large range of W1943 fragment replacements on chromosome 8, which contains one stigma-exposed QTL locus RSE (Huang, et al.2012) located by natural population whole genome association analysis in the laboratory, which is strongly domesticated and selected simultaneously in indica rice populations and japonica rice populations (Π W/Π c >4, where the value represents genetic diversity, W represents wild rice, and c represents cultivated rice).

2. Construction of genetic populations and establishment of phenotypic Studies

2.1 construction of the genetic population

The main construction process of the replacement series material GPSL41 used in the study (see A in FIG. 2) is as follows: 1. hybridizing ordinary wild rice W1943 and indica rice GLA4 to obtain F1 generation; 2. backcrossing F1 with GLA4 to obtain BC1F 1; 3. carrying out backcross on BC1F1 and GLA4 to obtain BC2F1, and repeating the backcross to BC5F 1; 4. BC5F1 is selfed to obtain a BC5F2 population, and a fragment replacement line with different chromosome position replacements is obtained through molecular assisted screening.

The mapping populations used in this study were obtained by backcrossing GPSL41 and GLA4 as follows: 1. hybridizing GPSL41 and GLA4 to obtain an F1 population; 2. selfing the F1 population to obtain an F2 population, and performing primary positioning by using the population; 3. performing molecular assisted screening on an F2 population, selfing an individual subjected to key recombination to obtain an F3 population, and performing fine positioning by using the population; 4. f4 population obtained by F3 selfing and subsequent selfing population are used for fine positioning and phenotype verification, and near isogenic lines are constructed by the population.

2.2 establishment of phenotypic examination method

Although the literature reports that several grain type genes (GS3, GW2, GW5 and the like) can influence the stigma exsertion of rice (Zhou, et al.2017), no gene for controlling the stigma exsertion of rice has been cloned by a population genetics method so far, and the most important reason is the complexity of the stigma exsertion phenotype investigation. The stigma exsertion is a complex character which is controlled by multiple genes and influenced by environmental factors, and the exposure level of the stigma is influenced by the environmental temperature, the photoperiod, the air humidity and the like. Meanwhile, the stigma exsertion phenotype presents different forms including unilateral (left and right) stigma exsertion, bilateral stigma exsertion and the like, and the complexity of character investigation is increased. To accurately assess stigma exsertion phenotype, finding a suitable test method, we performed a careful study of a large sample of the two parents and the F1 heterozygous phenotype in the shanghai of 2016 and the hainan of 2017, respectively, and found the following factors affecting phenotype test:

(1) examine the time window in which the stigma is exposed. The stigma exserts from flowering, after flowering, the stigma is exposed in the peripalea space at 1/3 below the glume, the naked stigma gradually browns and leaves a significant brown mark on the glume (see fig. 3), and counting the browned mark can expand the time window of phenotype investigation.

(2) The flowering time between rice tillers on GLA4 background is not uniform, and later tillers and early tillers are affected by different photoperiods and temperatures, possibly interfering with stigma exposure levels.

(3) In GLA4 and GPSL41 parental populations, small fluctuations in the stigma exposure rate of individuals in the same genetic background can occur, possibly leading to phenotypic errors (see figure 4).

(4) The stigma exsertion rate is a typical semi-dominant quantitative trait, and the F1 phenotype and two parental phenotypes present continuous distribution, so that the heterozygote phenotype identification is difficult, and phenotype misjudgment is easy to occur (figure 4).

Therefore, in order to eliminate environmental interference and human errors to the maximum extent and accurately measure the stigma exposure level, a set of new rules for phenotype investigation is established, and the new rules are as follows:

(1) the stigma exposure Rate (Ratio/Rate of Sigma expression, RSE), i.e., the Ratio of the number of glumes exposed on one spike stigma to the total number of glumes, was examined, and the exposure pattern (unilateral or bilateral exposure) was not distinguished.

(2) And (3) inspecting the average stigma exposure rate of the small population with the specific genotype, planting 24 small populations according to the heading characteristics of GLA4, randomly selecting 5 single plants, inspecting the stigma exposure rate (5 plants with 3 ears) of each main ear, and calculating the average value of the stigma exposure rate to be used as phenotype data.

(3) Only a small population of recombinants homozygous for the genotype in the targeted population was examined for average stigma exsertion rate, and the M2 single plant phenotype was rechecked using the M3 small population. A W1943 phenotype was defined as an average stigma exposure rate of greater than 20%, while a GLA4 phenotype was defined as an average stigma exposure rate of less than 3%.

Map-based cloning of RSE and construction of near-isogenic lines

3.1 map-based cloning of RSE sites

We obtained hybrid F1 populations of GPSL41 and GLA4 in 2015 south hainan, and selfed F2 populations in 2016 shanghai. The F2 population was screened to obtain about 1000 individuals with a W1943 fragment replacement on chromosome 8 alone, followed by genotyping and phenotyping, targeting the target fragment on the long arm of chromosome 8 using indel molecular markers P2 and P90, and narrowing the target region to an interval of about 2Mb using molecular markers EC22 and P100, consistent with previous GWAS results (A in FIG. 5).

The F3 population was further expanded by about 15000 strains, and about 3000 useful recombinants were obtained by screening. Meanwhile, finer indels and SNP molecular markers are designed between molecular markers EC22 and P100, 70 key recombinants are obtained by screening with molecular markers P79 and P48, and the target region is reduced to a region of about 180kb by further using molecular markers P42 and P70 (B in FIG. 5).

In the Shanghai of 2018, the localization interval was further narrowed by about 10000 strains of the F4 population. A series of new SNP molecular markers were designed every 10kb (every 1kb for partial fragments) between P42 and P70 and 15 recombinants were screened. In the course of further mapping, we obtained 3 key recombinants R1, R3 and R4 (C in fig. 5). Using the molecular markers P122 and P107, the target region was narrowed to 14.1kb according to the phenotypic differences of recombinants R3 and R4, which includes an unknown functional gene RSE1 and an mango gene EPF (C in FIG. 5).

Interestingly, recombinants R1 and R7 contained larger substitutions of the W1943 fragment than the recombinant R3, and the average stigma exposure rate of the recombinants was significantly higher than that of R3 in different regions and years (C in fig. 5). It is illustrated that R1 and R7 contain another site for controlling stigma exsertion, located between P107-P68, and designated RSE2 (C in FIG. 5).

3.2 construction of NIL-RSE near-isogenic line

During the mapping process, we simultaneously screened a near isogenic line with genotype at about 180Kb between molecular markers P42 and P70 as W1943, and the remaining region genotype as GLA4, and named the near isogenic line NIL-RSE.

3.3 candidate genes for stigma exsertion

We analyzed the last location region for candidate genes using RAP-DB (https:// rapdb. dna. affrc. go. jp/index. html). Two complete genes were included between molecular markers P123 and EC24, one of which was a known functional gene, EPF, and one MBD gene, which we speculated might be a candidate gene for RSE 1. Also included between markers P107 and P68 was an MBD gene adjacent to the candidate RSE1 gene, which was 92.22% similar to RSE1 at the coding DNA level and 86.98% similar to the two protein sequences (see FIGS. 6 and 7), presumably the RSE2 candidate gene.

4. Genetic transformation verification of genes RSE1 and RSE2

In order to verify whether the RSE1 gene and the RSE2 gene are actually rice stigma exsertion control genes, transgenic experiments are carried out. The wild rice BAC (see FIG. 8) containing the RSE1 gene (containing the EPF gene) and the RSE2 gene was digested separately and introduced into the pCAMBIA1300 vector to construct a complementary vector of the RSE1 gene and the RSE2 gene (see FIG. 16). Since GLA4 transformed by the complementary vector is indica rice which is difficult to genetically transform, the complementary vector is selected to be handed to a company for transformation, and finally, complementary transgenic plants of the RSE1 gene and the RSE2 gene are obtained in batches.

The complementary transgenes of T0 generations of RSE1 genes have 16 strains, hygromycin is identified to be all positive, gene specific molecular markers are identified to be all positive, 4 transgenic plants obtained in the first batch are accidentally killed in the field planting process, phenotype investigation is conducted on the T2 generations of the remaining 1 transgenic plants, the stigma exposure rate of the T2 generations is found to be 8% and is significantly higher than 0.4% of the negative parent GLA4 in the same batch (see figure 9), the same trend is observed in phenotype investigation of 2 transgenic T1 plants in the second batch (see B, C in figure 10), the remaining 9 transgenic T1 generations and the transgenic T2 generations in the third batch are just sown in Hainan, and subsequent phenotype investigation is waited.

In the process of constructing RSE1 gene complementary transgene, the wild rice BAC enzyme cutting fragment containing RSE1 gene additionally contains an EPF gene (RAE2/EPFL1/GAD1), the gene is positioned at about 4kb upstream of the initiation codon of RSE1 gene and encodes a 125AA short peptide for controlling the growth of miscanthus, and the RSE1 transgenic plant can form miscanthus. The EPF gene belongs to a plant epidermal cell growth factor, and mainly regulates and controls the development of leaf epidermal stomatocace cells in arabidopsis thaliana. The research on the TaEPFL1 gene of wheat shows that the stamen development of the TaEPFL1 mutant is obviously abnormal, but the pistil development is not influenced. The OsEPFL1 gene of the rice is mainly specifically expressed in mango primordium, the expression of the OsEPFL1 gene is strongest in the later period of SP7 and the early period of SP8, wild type complementation of Nipponbare, NA93-11 and Koshihikari can cause transgenic plants to form long awns, but the exposed stigmas of long awn grains are not found. The microdissection observation shows that the development of the rice stigma exsertion is based on the length and angle change of the pistil stigma, which is inconsistent with the tissue expression pattern of the EPF but completely consistent with the expression pattern of the RSE1, so that the EPF gene is considered not to influence the stigma exsertion. At present, we have sent the company transformation GLA4 with a complementary vector containing only the RSE1 gene in order to compensate for the experimental errors.

The complementary transgenes of T0 generation of RSE2 gene have 10 lines, hygromycin is identified to be all positive, but gene specific molecular marker identification is only 7 positive, 1 transgenic death of 4 strains in the first batch is carried out, phenotype investigation is carried out on T2 generation of the rest 3 transgenic plants, the stigma exposure rate is respectively 12%, 9% and 10% and is obviously higher than 0.3% of that of the negative parent GLA4 in the same batch (see figure 10), and the same trend is observed on phenotype investigation of 3 transgenic T1 generation plants in the second batch.

The above results indicate that the complementary RSE1 gene and RSE2 gene can indeed significantly increase the stigma exposure rate of GLA 4.

NIL-RSE stigma exserted phenotype dissection

To study the developmental mechanisms of stigma exsertion, we performed anatomical analysis of the florets of GLA4 and NIL-RSE, selected the glumes of pre-heading ears from the GLA4 and NIL-RSE populations, dissected and observed comparing pistil structures. An intact carpel is divided into three parts, namely an ovary, a style and a Stigma, and the Stigma is further divided into a brush-shaped part (SBP) and a non-brush-shaped part (SNBP). Defining an included Angle between vertical central axes of concentric skins of a stigma hairbrush-shaped area (SBP) AS an AS Angle (Angle of SBP, AS); while the Angle of the concentric vertical neutral axes of the non-brush shaped region (SNBP) is defined as the ASN Angle of SNBP, ASN) (see A in FIG. 11).

Anatomical observation shows that pistils without exposed headings in GLA4 are bilaterally symmetrical in the overall structure, and the lengths, ASNs and AS angles of the two stigma are basically consistent. The pistil with the unilateral stigma exposed in the NIL-RSE loses the bilateral symmetry structure; the length, ASN and AS angles of the unexposed stigma are not obviously different from those of the unexposed stigma in GLA4, and compared with the unexposed stigma of GLA4, the length of the exposed stigma is obviously increased, the ASN and AS angles are simultaneously and obviously increased, or the AS angle is obviously increased, the SBP area of part of the exposed stigma is greatly twisted, and the AS can reach more than 90 degrees. There was also a small amount of bilateral stigma exsertion in the NIL-RSE population, whose pistils did not necessarily lose bilateral symmetry, but bilateral stigma lengths, ASN and AS angles were simultaneously significantly increased, AS shown in figures 11 and 12.

The above results illustrate that the mast head length and AS angle are the structural basis for determining whether the mast head can be exposed.

Spatio-temporal expression Pattern analysis of RSE genes

6.1 analysis of expression level

We first searched the tissue expression patterns of RSE1 gene and RSE2 gene in the RiceXPro database (https:// RiceXPro. dna. affrc. go. jp /), and the data showed that RSE1 gene and RSE2 gene both expressed at a lower level in different rice tissues, indicating that RSE1 and RSE2 are widely expressed genes. The expression levels of the RSE1 gene and the RSE2 gene in different stages of the GLA4 and the NIL-RSE spike development are detected, and the results show that the expression levels of the RSE1 gene and the RSE2 gene have no significant difference between the different stages of the spike development. Also, there was no significant change in the expression levels of RSE1 gene and RSE2 gene when comparing GLA4 and NIL-RSE during the same panicle development period (as shown in FIGS. 13 and 14).

The above results indicate that there was no significant difference in the expression levels of the RSE1 gene and the RSE2 gene in the young panicles.

6.2 expression Pattern analysis

In order to research the expression patterns of the RSE1 gene and the RSE2 gene, digoxin-labeled RNA probes are constructed by taking sequences specific to the RSE1 gene and the RSE2 gene as templates respectively, and RNA in situ hybridization is carried out by using the probes to detect the spatiotemporal expression patterns of the RSE1 gene and the RSE2 gene in the development process of young ears. In situ hybridization results showed that the expression patterns of RSE1 gene and RSE2 gene were substantially identical whether in GLA4 or in NIL-RSE floret development (see FIG. 15). In 4-5cm young ear florets, both in GLA4 and in NIL-RSE, the RSE1 gene and the RSE2 gene are expressed at a low level in the palea-palea, and in the ovary, pistil and stamen. Especially in ovule, anther, filament, and stigma. We note that: in GLA4, the RSE1 gene and the RSE2 gene are relatively uniformly expressed on the stigma and style of pistil of floret with 4-5cm ear, while in NIL-RSE floret at the same period, the RSE1 gene and the RSE2 gene are strongly expressed in the brush-growing region (SBP) of the head front segment of pistil, and the expression level is obviously higher than that of the non-brush region (SNBP) of the head and that of the brush-growing region (SBP) of the head front segment of pistil in corresponding developmental stage GLA 4. In the NIL-RSE near isogenic line, the strong expression of the RSE1 gene and the RSE2 gene through the anterior-segment brush-colonizing region (SBP) of the stigma may affect the growth direction or growth rate of the stigma, resulting in an stigma-exposed phenotype.

By combining the previous glume dissection experiment results, the statistics results of the stigma length and angle and the in situ hybridization experiment results, the specific expression mode of the RSE gene on the stigma is considered to finally influence the development of the stigma and cause the stigma to be exposed.

Acclimatization analysis of RSE Gene

During the map-based cloning of the RSE1 gene and the RSE2 gene, the allele from the normal wild rice W1943 contributed to the stigma exsertion phenotype, whereas the allele from GLA4 did not cause, or caused only a very low level of, stigma exsertion. To study RSE1^WAnd RSE2^WEvolved to RSE1^GAnd RSE2^GWhether the process of (1) is artificially selected, 39 deeply sequenced modern cultivated rice materials and 14 common wild rice materials are selected for the alignment analysis of the full-length genome sequences of the RSE1 gene and the RSE2 gene. Multiple alignments were performed using cluster-W and the results were modified using MEGA alignment followed by Tajima's D neutral selection using DnaSP-v6, the results are shown in Table 1.

Tajima's D testing of the RSE1 Gene and the RSE2 Gene in Table 1

Wherein: n, sequence number; l, sequence length (bp); s, number of polymorphic (separation) sites; Π, nucleic acid diversity.

The results showed that the Tajima's D value of the RSE1 gene in the cultivated rice population was-2.6596, indicating that the number of haploids was less than the number of polymorphic sites, rare alleles were present at high frequency, while the population was less heterozygous on average and selective clearance occurred; the p-value <0.001 negative neutral selection hypothesis indicates that the RSE1 gene is subject to targeted selection in a cultivated rice population. The Tajima's D value of the RSE1 gene in the wild rice population is-0.6336, which indicates that the RSE1 gene is also selectively cleared in the population; p-value >0.1 accepted the neutral selection hypothesis, suggesting that allelic variation of the RSE1 gene in a wild rice population is due to random mutations and is subject to balanced selection. Like the RSE1 gene, the results of the Tajima's D test showed that the RSE2 gene was subject to balanced selection in the wild rice population and to targeted selection in the cultivated rice population.

To verify the results of the Tajima's D assay, we examined the nucleic acid diversity of RSE1 gene and RSE2 gene in the cultivated rice population and the wild rice population, respectively, using MEGA, and the results are shown in Table 2.

TABLE 2 nucleic acid diversity analysis of RSE1 Gene and RSE2 Gene

Wherein: m, sequence number; s, number of polymorphic (separation) sites; ps, S/n, n is sequence length; Π, nucleic acid diversity.

II in Table 2^WNucleic acid diversity, Π, representing wild Rice population^CRepresenting the nucleic acid diversity of the cultivated rice population. The calculation results showed that II of RSE1 gene^W/Π^C7.765, and II of RSE2 gene^W/Π^C9.5348, which is significantly higher than pi commonly used^W/Π^CThreshold 3 or 3.5. The above results indicate that the nucleic acids of the RSE1 gene and the RSE2 gene range from the wild rice population to the cultivated rice populationThe diversity is remarkably reduced and is strongly domesticated.

As described above, both RSE1 gene and RSE2 gene were strongly selected by human and were domesticated genes.

The experimental results show that the RSE1 or RSE2 gene improves the stigma exposure level of the cultivated rice GLA4, and the suggestion that the genes can be used for improving the genetic diversity of cultivated rice groups and the seed production success rate of hybrid rice, so that the maturing rate and the yield of the hybrid rice are improved, the existing cultivated rice varieties are improved, and the application prospect is wide.

Reference documents

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Huang,X.,et al.Natural variation at the DEP1 locus enhances grain yield in rice[J].Nat Genet,2009a,41(4):494-7.

Huang,X.Y.,et al.A previously unknown zinc finger protein,DST,regulates drought and salt tolerance in rice via stomatal aperture control[J].Genes Dev,2009b,23(15):1805-17.

Miyata,M.,et al.Marker-assisted selection and evaluation of the QTL for stigma exsertion under japonica rice genetic background[J].Theor Appl Genet,2007,114(3):539-48.

Purugganan,M.D.,and D.Q.Fuller.The nature of selection during plant domestication[J].Nature,2009,457(7231):843-8.

Song,G.S.,et al.Comparative transcriptional profiling and preliminary study on heterosis mechanism of super-hybrid rice[J].Mol Plant,2010,3(6):1012-25.

Song,Xian-Jun,et al.A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase[J].Nature Genetics,2007,39(5):623-630.

Wing,Rod A.,Michael D.Purugganan,and Qifa Zhang.The rice genome revolution:from an ancient grain to Green Super Rice[J].Nature Reviews Genetics,2018,19(8):505-517.

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Sequence listing

<110> prominent innovation center of molecular plant science of Chinese academy of sciences

<120> gene for regulating and controlling rice stigma exsertion and application thereof

<130> SHPI2010673

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9033

<212> DNA

<213> Oryza rufipogon W1943

<400> 1

cgcgctcgcg ctctcgctgt gaattacggt gtgtgtggcc gcgtcgcgcg cgcgccgctg 60

cccgtgcccg gcagcacatg gcgctgcact gctgctgatg ctggtggtgg tggaatcgtt 120

gtcgtcgcgt cgggctgaga gggattgttg atagattccg tgtaatatgc caggacaaaa 180

ttttgtcacc gctgctgctg cccatgcagc tggatcggct cggctactct caccacctat 240

actgtatctt tcactggcat ctgctcgccg ttttggaatc tctgcggtgt ggggttgcct 300

cttgcatgta cacatgtttt tcatgtatcg atccatgcct cctccatgga atctaatggg 360

aatccatcat cgttcatgct ggatggatgg atggatgtag tgaatggtag tttttcttat 420

ttttgttgga gatggatatt ttttacttta catctaatcg gatatatgtt gccttttaaa 480

ttgagaattt agctactcaa acaatccatt ctgaaattcg ttcaaacgaa gatttgaact 540

tagtatctta ggctgcgttc ggcagaccag gctcccaact cctccttatt ttccgcgcgc 600

acgcttttca aactattaaa cggtgcgttt ttgcaaaaag tttctatacg aaagttgttt 660

aaaaaattaa attaatctat ttttgaaaaa aatagctaat atttaattaa atcacgcgct 720

aattgctact ccgttttgcg tgccggggag taaggattcc ccgaacacag actagagtgt 780

tattcagatc gctgcaatct acatgccctt tcgcaaagtg aacggtatag ttctagtggt 840

aaattggtaa ttggtccctc tctgcatctg ctatgcatgc atgcatttgc ttgatagtat 900

gtttttgtgc gttgatgtgc ataggtcagt tttgttcatt tgaggtttca tgccagggaa 960

aatcgatcat gcactacatc ctttattatt gccgtaagta cgattgatta tgtaatcgac 1020

atattcatca atcctcccat gatccaaaaa aacgttccat gccggtgtga tcgtggtgat 1080

ttggttgatt tgaaaggaac gtaatgattg aatttttttt atgggaaggc caacttcggt 1140

agaacacttg tgtgcagcca tcaaagacga gtggaaactt gcaatgcggc attcctctta 1200

accgattgag gtcagttgca gcccatcgcg cagtggtgta atcagcagtg ttttaaatag 1260

cgggtttaaa acgtttagtt ggttttctca aacagttata gcgggctaaa tagagctata 1320

acggctaaaa ttgtataaga gagtaaatat ctataccctt ctcagacagt tatagtcaaa 1380

gatagtggca gctatagccg gaaatttaaa acctggtaac cagtaacggc tcgacactag 1440

caccgttctt acgaggtctg gttttgagtt ggcggcgacc cctccatttc ccaattgcta 1500

aataatctgt ttttccttgc tcaaatcttc gataacatat tacttcgccc aatgttttcc 1560

catgaggcca cgatatccct ctaattcaca tctcaccatt tttttccaga gcaaatgttc 1620

atgtttttag aaaaaaaata gggataaatc cacatcgcag attgcacccc ttaattgttg 1680

gaattgccta aaaactcctc tcaactaggg gtgtaagtgg ctaacccgcg aaacccactt 1740

atagactaaa aaataagcca cgaacccgct tattttgacc tataagttgg ttcccggctg 1800

acccacgtac acccctactc tcaactatgg ccctctttgg ccaccattgt tgctacagtg 1860

taccaaaact gataagtaca atcacagcta gcacaaatgc acaatcgatc acctcatgta 1920

aaaccggata taggctgcgt tcttttcagg ctattactcc tcgttttccg ttagcacgct 1980

tttcaaacta ctaaacggtg catttttttg taaaaagttt ctatatgaaa gttacttaaa 2040

aaaatcatat tagtccattt ttttttaaaa aaatagcaga tacttaatta atcatgtgct 2100

aatggatcgc tccgttttcc gtgagcactg ttgggggtgg gaaagaacgc agccatagtg 2160

taatatagaa ttctccaaga aggatagcta gggtcttggg aacatcgtac catccggtag 2220

tttatgattc tcgcaataga gatttgtttt cccattcatc gtagtgggcc gtcgcgatcc 2280

ctggttgtgc atcgtaaatt cgtaatgggc cctccaaatc ctacaccaat acaccatctt 2340

ccacgcatgt tgcatgctca aggttaatgg gccgtgtact ttcttcactc ttatcggttg 2400

gccatatgtt tatggtccat gagatatcgg cccacgtagc atttgggctt gtagccccca 2460

cctcgctccc atccataacc gcccaacctg tgcggctcct tttgtcgttg cgcgcgacgc 2520

acctcattgc ctcggtcccc cacctctctc ctcatcggac ggcgccacgc cacccaagtc 2580

cagatccaac gatcagaacg acagccaata gtagggatgg cagtattgcc cgtgggttcg 2640

ggtatccgcg gatacctggc ccgataggca tgggcgtggc accttttttt aacccgtggg 2700

catgtttgat ccgatgcccg agaacttagt gggtatacat gggtttatat tttgctcgtc 2760

gataacctat gcttgactaa aaaatttttt agcccatgta tatctttcat gccttcaata 2820

gttaacctct cctcaaaacc caatagccca tttagcccat ttgtatatag caatatatgt 2880

gttcactcaa cgaaacccta gccttatcct tttccccacc accacctctc aatcatctat 2940

tcctctacgc ggcggcggcg cacgagaaag aagcaggagg tggctggtgg cgcgacgcaa 3000

cggcgcggcg cgagctagcg acacgacggc gatggctagc gcacggctgg cggtgcacaa 3060

gcgtgggcgg cagcaggctc gtggccggcg gcacatgagc gtggagcgcg acgggcaact 3120

tagcggtgag ggcgacaacc ggtgaccggt gatgcggttg cgcacaagcc gcgactggcg 3180

gcgcggcggg cggcggcaca tggcaggtaa gcccagtggg tacccgtcaa gcatacccgt 3240

gcccgacagg catgggcata ggcatgagat tttgcccgat aggttttgcg ggcgtgggtc 3300

aggcacgagc acgtggatct cgggcgggac aaggttttgc tatgcacatg cccgacccga 3360

cccgttgccc tccctaccct ataggctata gctcacacca aacccgccct ctctctctct 3420

ctcttcccgt ggagccccaa ctacgccgcc agcccgccat ctcatccccc ggcgagcgac 3480

acggtcgcat cccacctgag ctccgaccgc cgcagcggcg gtcgcctcgc cggagcagaa 3540

gccgcgcgtg ggggggaagg cggaggaggg gaggggttca atggagacgg aggtgttccc 3600

cgtggttgac ctccgcgtgc tcacgcagtc cgatctggac gagctcgccg ccgcctccgc 3660

ccacgccgtg gacccgcgga gcagctgccc cgaccgcgcc gtcttcaacg agagcgccgg 3720

ctcgcgtaag cagaccttct cccgcgtccg cttcgtcccc gccgccgccg ccgccgctgc 3780

cgccgcctcc gcctccgccg ccgccgccgc caagctgccc aggggcaacg acaaggagga 3840

cagcttcatc gcctaccacc tccgccgcct cttcgcgccc gacgacccct ccctcaccga 3900

gaacccctcc tttccccaaa cccaaaccct agctcggtcg ccgtctcccg accccgacca 3960

gttgaccacc aactccaggg gggtctccgt tgatctggtg agcctctcac ggcttgccga 4020

cccgtacgac gcggagctcg ggaagcggac tgccaggatg accacggagg aggagctcat 4080

gggctttatc tctagcctcg cgggtcagtg ggtgaaccag aggatgcgga ggaagttagt 4140

tgatgcgtcc ttcttcgggg atcacctccc cagcgggtgg aggctgcagc ttgggatcga 4200

gcggaaggac cgcaaggcct gggtgaactg cttcagttat gtgaggtgag cctcatcatc 4260

ctttccttgc aaatgctgca atgctatggg atccgttgtt agtaactgga atgggggtta 4320

taacttgcaa taagctagag ggctggatgc ttgctatgat gtttagagat gcggtaatgt 4380

gtgacttata gatgcaatgg atgtgtggtg gatttggttt gtgaagattg ttttatactc 4440

ttttagtgta gtcaactgtt gctaatcaat catgcacaag tttatcagat cagttcttgg 4500

tagttttgaa tgattgattt ttttttcaac atggccttca tgctgtcatt ctgtggattg 4560

cctattttcc atggaagaat aaatcttatt tgttaacaat attattatgc aaacatacaa 4620

taactgaata cccccaggac gagctgtttc atgacgaggg ggatgctatg gctggacatt 4680

atggtatagt atatatagta gtggtgtcgg tatgacaagg cctcaggatt agcatgtgct 4740

gcagcaattg gagaatcata gtagacaaat agatattcta ctccttattt ctccattaga 4800

gtttcctagt tttgtcattg gagtcctagt agtattagat actagtttgt ttcctttgcc 4860

ttatctagag gcacctcaat atacaaaggt agcctagtac tggattatgc cttgagcaat 4920

aagaaatatc taaaggggat tgacccaaaa gcccagtcat ttcaatgaca tactagttaa 4980

atgataatgc tgaataatct ggggggcaaa gacaagactt ttgcctacca tagtataaga 5040

gcagcgtgga gcggtattgc tttaagataa tgagttttgt tttattcctt tgatatttgc 5100

ttttccgcac ttcatttgat ctgtagtttt aaggcatgca aatgcgattg cagcagcatt 5160

atttagcaat ggcttgattg atgcttactg ctacctccgt ttcaggttat aagactttct 5220

aaaatctaga aagtcttata atatgaaaca gaggaagtat atgataggtt aaacataaag 5280

atagatttgt atggcgcata tcatatctca agtatataac ataacacatg tatgtgtaaa 5340

tttgcttctc ctgccgagtt ctcactgata cacaggtcct aatccaacct gtcaatgaga 5400

accagtggat aaagtgaatt aaggacatgg catggtgtct aaaatgtcta aaataaacag 5460

taattttcat ttgctttatt taactgcaat gcctatttga agtagtgcct agagcacatg 5520

agcatttcaa tcattatctc gaatgttcct cagaactcag gtgttccacc atatatgctc 5580

agatttatca tctgtcgtag atgaataatt ttgttgtaat aacagtaaag atcacttctc 5640

atcactataa gttcgaaagt atctgttaag ataacaaatt cctatttatc tgcttcacct 5700

ttatctatca tgtgtgctac tcatcagcga acatatgttg tggatggctg catcatttca 5760

gcgcaataat tttgttcgtc acatttttcc cctgtactaa atgggcatgt tgtactttcc 5820

agccccaagg gacagagttt tgctacttgc caagaggttt ctgcatacct catgtcactt 5880

cttgggtatc cggagttcaa aacggataat attgagtatg gtagcacaca acaacatggc 5940

ttgtgtgctg acgatggtgt taatgtaagt tgaaagtgta tgtcacttta tcttgtgaac 6000

ttgagtttgt ctagtttaat ttttgggttt gtggcaggtt ttaggtgttc aacaccaaat 6060

tggtacaagt atggacagtc aaagtaattt gccagttgct tctgctactt tttatagtca 6120

ttcaagagat caagacgaaa cggttgcaga tgatataaat tcttatgaat gtcaacaatg 6180

caatttaact tttcatggtc agagtgccta tgcgcatcac ctgatcactt ttcacaaagt 6240

gagttctaag aggcgtaaga gtaacaaggt tagcaaattt ggtgagccag tgataggcaa 6300

agatgggaaa tttgaatgcc cagtatgtaa caagacgttt gaggaacagt cacggtactt 6360

tggtcacatt ggatcccatg caaagtatca ggggctgact cctgaagcat tcctacaaac 6420

cttttcagga aaggttggta acaattcttt tgcaggtttg tcatctagcc ttcaagtatt 6480

ggtcggatca ccacaactga atgagaagac tactgcttgt gaagcacgat cacagcatca 6540

cgattgttca actaaacatg gaggcaatag tacaagaggt atagaccttt ttaactcaaa 6600

tcatcctgct aacttcaatg ggcataatca atcttggtgt agatctgatg aaattcctcc 6660

caccacagaa gctcaaagca catggactta tagaaataat gagatgaact gtgctgatag 6720

aactgttccg agaacagtac ctcagcccaa tgatcacgag gattgcaggg ttagtggctt 6780

tgctgaagca actaatttta atgatcaagc aggaagacac caaggtttta gacctttctc 6840

ctttggaact accaaccatt gtcaaggcca gataattgat catgcagtag ctgcttccaa 6900

gcatgctgag gttaataata gtatgaaatc aagagatgtc aacctcaatt cacgcctgaa 6960

cacaatatcc tttcctattg caactgcaaa caatgaaaca tcgactgccc ttaatgacgt 7020

gaatcggtca tgcattactg gaaaaggttt tagtggaagt ttcagtaaca atgatggtgc 7080

tgcatctatc gtgttgccca gttctggatt aaacaataaa atttctagct ccctcggtgt 7140

agctgacaga tcatctattg ctgccagatc cttcaatgct ggttatgctt atgaaaatgg 7200

cgcttctgaa gctaacaata ttggcaacaa aaacaatacc atggtgtatc aaacaagttt 7260

ggccatgcgt ccactctctc ctgtaagtaa catggattgc ttcacgtttt gttcagtgca 7320

tttaaaaata gcaactgaca tctattgatg cttataataa taacatggat agcatagaaa 7380

acaaaacaag taactaagct agattttctg ttgatgctta taataatatc ttgactggca 7440

atgtcactga aaggagtcta cccagtgtaa ttgcaactcc agtcacttag ggcctaatat 7500

ttcaagccat aattcacttc ctaaatcaag cacactagcg accagtaact taagcaaggg 7560

gagtgatgtc aactggaaag ttgttttaaa aatcatatta atccatttgt taaaaaaata 7620

actaatactc cctcgtttaa ggttataaga cgttttgact ttggttgaaa tcaaactact 7680

ctaagttttt ctaagtttga ctaactctat agacaaaagt agtagtaata tttacaacac 7740

tagcatagtt tcattaaacc tataattgaa taaattttca taatatattt atgttgggtt 7800

aaatatatta ctactttttt ctacaaaatt agtcaaactt aagagtagtt tgactttgac 7860

caaagtcaaa aatgtcttac tacctgaaac ggagggagta cttaattaat cttgcgctag 7920

ccatcgcttc gttttgtgcg cactcccgac ctcagccctg tagcggctcg acgctagcat 7980

tgttcatatg aggtaaataa tagggaaaat tccatctata ccacaaagat tttagcagta 8040

ccaaataata ccaccagatt ttttctcctt gttgtttaaa aaaaaaggat ttttcctcct 8100

tccaaaaata ccacaaactt cacggagaac cgacagagta agtaagtaga tgaaactgcc 8160

ctcacatctc tcgttctctc atctctttaa gtgatagggt tggcaatctc tgctcagcga 8220

tagggattga atagtttcat taaacatata ataattgaat aatctctgca tcgcgtctgg 8280

gacgaaggag atcgccgctg gtccggcccg cgtggaggca cttttctgct tatgccgccg 8340

cggtgttgtg tccgggcagg agaccccact ggtccagcac tccggctccg gcggtttggc 8400

cccgtccggc cgtcccccgt acggaggcac ttttatgctc cgtcgctggt gtttgcgctt 8460

cctccgattt tctaacccat tgccgtgcga agcagggcaa acaaagcagc ggcgttgctt 8520

cgagaaggac agccgctgtg gtgtccgagc ggcatggagg ccaagttgtg agacacccac 8580

ggactgcttc atcagtttac atgcctgagc aaggtcctgg atggttgtga gctccaccag 8640

ctaatcacca aaggcagctt atgcatcatg cagctggatt ggagaactcc taaagctgaa 8700

ggccacgatg ggattggaga actcctaaat cttgttcaca cagggtacct cagtactctt 8760

cttttcaaac acaggtaacg gagacaaatt agtttgtatg agattgggta acgtctgttg 8820

aaatatgtgg tattcttaaa agaaacgttg ttgcactgtg gtattgagtg gagatgaaag 8880

aatcttgtgg tattatttgg ttggtcaaat ctttgtggta tagttggaat tttcatataa 8940

taatatattt ttccttggtc aaatcttcga taacatttta ctttgcccga tgttttccca 9000

tgatatccct cttagtcaca tcccaccatt tct 9033

<210> 2

<211> 13149

<212> DNA

<213> Oryza rufipogon W1943

<400> 2

gtcgacgagg cttccgtgga gccattcgtg tgcagcggcg atgcgcatat ggtactacct 60

gcaccggtgg ttgagctgac caagtattct caaccaaatc tcatcatgaa tcccaggagt 120

tctgaaccgg aggcaagcaa caaagagacc acaacgattc acaaggtgag ctagtttatt 180

ttctgcaaca aggaaaccac atggtttctg atttttgtgt gttgattcga atgaggagtg 240

gtggtgtact atctcatttt ttttcttaga cactgtgctt tcagatctga gacaagggtg 300

atttgtggaa tgaacaaagt catatgtctt tcttagacac catgcttttc agatctgagc 360

aagggtcatt tttgtggaat gaacaaagcg atatgtcttc ttagacattt tgtttgcaga 420

tgtaagcaag ggtgatttgg catgaacaac cgtgtgcact tttctgattt tgtgatgtca 480

gtttaacact tagattttcc agttttcaga aatgcctact gttttgtgcc tgttgtggaa 540

tttgtggaaa tatgcagtca tggtttgttt gctagttgag ttaggatttg tctgaagaat 600

actgtacaac aacataactg cagaagcgaa atatgtatgc caaattttaa tttcaggcac 660

caccagacaa tttgtatgca ctctgggctt atctgaatgt tgtaggggtt tatcttaatg 720

ttgtagattt tttttcacac aatggtctga atactgtaga taagcagaac tagtagtagc 780

agaatgcttg aatcgtatgt aaatatgagg cgcatctggg gctactacta ttgttactca 840

gacttaaaat ggatgtatgg aaacaagttc aagcctgcat ttgtctattc aacttgttac 900

aaaatgatca tgtcagtagt tttagtgtga aatttgaaat ttgaaattca tgttgctgaa 960

tttttctgaa ccctacttat gtaattctga actttgctgt caaaggaatg catggcgtgc 1020

tgtggcagtt ttggtccctt ttttttttgt ttgttctaga ttttccagtt tttgtaaatt 1080

ttgtcaaaag aattgtaatt tattatctct ctgtgtttgt ccaacctgaa acacaatttg 1140

ttagttttga aataacaaaa acttttacaa ctatattttg taattttgtc agttttgaaa 1200

attttgtcaa aaaaatgtaa tttattatct ctatgctttg tccaacctaa aacacgattt 1260

gttagttttg taattacaaa aacttttaca actatatttt gtaaatttgt cagttttgta 1320

aattttgtca aaaaaatgta atttattatc tctatccttt gtccaacctg aaacacaatt 1380

tgttattttc tttaacttct caaaataagt tatttcttca tatttgtatt ttgtttttgt 1440

cagttttttt taaaacttct caaaataagt ggttttgtca tatttgtatt tttgtctttt 1500

gtttttgtca gttttttaac ttctcaaaat aagtggtttt gtcatatttg tattttgttt 1560

ttgtcagttt tttaaacttc tcaaaataag tggttttgtt atttttgtat cttgtttttg 1620

tcagtttttt aaattaatgc atgtgttcct caggtctgtt gtagatctcc tccaccgttc 1680

atccttagcc cgacgccgat gccactgcca cctgccattc cctcctcacc aaggtaaaag 1740

cctggctgac ttccatatag ctctgagcta gtaacagtac ttccaaaagg gccataaaat 1800

caagctacaa tgtttcagac ttccaactgc tattaacagt acttacttcc aaagtgccat 1860

tacagtcttc cagcagctac catgtttcaa aataaagagc tcagtacaaa ttcatctgac 1920

ttttttttta actactatac tgtgtcaggt tactgtccgt tgtaaaaaat tgtaatttat 1980

tatctctctg ctttgtccaa cccgaaacat aatttgtatg tttggtttta tcaattttgt 2040

tagttttgta aaccagctgt agaaaagatt gatagaaata ttgcatgctt cataccatgt 2100

ttcatatttg tcagaaatag tttatgaatg atcgtgctct ttgttatctc ctacttttgt 2160

cagttttgta aaagaatttt gtaatttatt atctctctgt gtttgttcaa cctgaaacac 2220

aatttatatg tttagttttg tcagttttgt aaagttttta gttttataaa ccagctgtag 2280

aaaaaaatac aacaaaaaga ttacatgctt cacaccaggc aaaatgtaac cacaactata 2340

tctagttttt cttagggcct catcgtttgt ttttttttct aataagccaa aacggcttat 2400

tagagaataa aaataaattc gtaggtaaaa cttttatata tgtgttttcg gtgacttaaa 2460

agccaatgct gaaaaagaaa ctacgttgaa aatatctcaa aatcaatgtc aaaattaagt 2520

ttaaagattt aaattttagc tttttcttta gttgaatagg ccatccgaat ggagcttcga 2580

ataccttgag taatatcaac tccttcagcc ttccttttgc caagatcaca cacctgcgcc 2640

tctcctgaaa atttaattaa gtacaacaga ttatattttt gtcatctaat agccattcat 2700

agttcaacat attttgttca taagccccct tatctgaaca cctagagcta gtatttaaag 2760

tgggacaaac aagcagtagc tttgtgtttt gtaatttgtt agcaattttc agttgttttc 2820

agtgagaact gaacatctta gaggcaatgt tgttaagata aatcgtcgtg tattttttct 2880

tatatttttc ggtgggctgt gagagacaaa cctaagccca taaaaaagga gtgggatttg 2940

tttctcaaag cgctacgcgc tctgttattt tattgcacgc agggggaggg ggagggatgg 3000

acgacgcgtg tatcaatcgc acggcgcaaa aaacggctga aagcccaaaa tttttcagcc 3060

gtttgtcaca tagagtccct ttttttaagt gtccagagca attgttcatt tttttaaaaa 3120

aaatataggg ataaatccac attgcagatt gcaccctctt aattgttgga tttgcctaaa 3180

aactcctctc aactatggcc ctctttgggt accattgttg ctacaatgta ccaaactaat 3240

aggtacaatc atagctagca caatcgatca cctcctgtaa aaccggatat agtgtaatat 3300

agaattatcc aagaaggatg gctagggaac atcataccat ccattagttt acgatcctcg 3360

caagcgaaaa gtacaccgaa ggtcggtcct acaacttata atcgagttac aaaatcgttc 3420

ttaaaccgta aaaccggata aaatgcatcc ctcaacttgc aaaaccagtg caaactaggt 3480

ctctcggcgg ttttgactcc ggttttgtct gatgtggcag tggattcaat gcgggaccca 3540

cgtgggcctc acacgtcagc ctcttcttcc cctcccctct ccacctctct tcctctctcc 3600

tttcctctcc acggcggcga ccggcaggct gggcgcagta ggttggtgcg gcgggcgtag 3660

ccagtggcta gcggggaagg aactgtgcgc cagcgacaca gagctcgact ggcgtcgtag 3720

agagaggggt cgccgccgtg tgaagagatg cagtcgccgg catcgctctt cccctccact 3780

ctcctcgctc tcttccctga gttcgatgcg gagcgtcgtt tccgtccagg tcgccaccta 3840

ggtgccacga gcccgacgtg tagtgccgca tccatcaaca tgtcgtcgtc gtcgtcgccg 3900

ctgcatccat ctctttctct ctcgtcacca tcgtcctcgt cactgctgcg ggacaaagcc 3960

gctcatcggc caccgactgc acccgactca ctctccggaa tcacactata catggcgttg 4020

gtgaggcgtc gccgcctcac tacctacggg cctccttcgc tcctgccgct cctacctctc 4080

ttcgttggca atcgcggtcg ccggtgagct cacaggagct tggggcatct ggcggtggtg 4140

gcgacattgg cgtggggagg gccatgaagc ggcaagactc gcggttcttc caccgactag 4200

agttgcagct ggtgtggggt gtccccttct cccccgtcgc cgccgccaaa ccttctcctc 4260

tgccgcctcg ccccaccgac ctccttccca tccgtcggcc tgccgcgctc agcttttccc 4320

tcgccggtca ccgctcgtgg agaggggagg agagagagag aggaagagag atgaagaggg 4380

gaggggaaga agaggctgac gcgtggggcc cacgtgggtc ccacaccgac tcagctgcca 4440

cataagacaa aactggagtc aaaaccacca aaggacctaa agtgaatggt tttgtaagtt 4500

aagggatgtc atatatctgg ttttacagtt aggggatgat tttgtaactc gatgacaagc 4560

tgagggtttc gatatacttt ttccttctcg caatagcaat ttttcccacg cgtaagtggg 4620

ccgtctcgat ccccttgtcg tgcgtcaaag gaaaaagtac accgaaggtc cctcaacttg 4680

tcattgagtt acaaaatcgt ccctgaaccg caaaaccaga cttatgatat cccttaacaa 4740

aaccatttac tttaggtcct tcggtggttt taaccccagt tttgtccgac gtggcggctg 4800

agttagcgtg ggacccatgt gggccccaca tgtcaggatg ccacctcatc tcttccctct 4860

tatttcccct tttctgcctt tctctctctc acttttctca ggccggacag gcagcgctgt 4920

ggggaggagg ccgccggggg gaggggagag gaggaggtct ggcagccggt gtcgcgccgg 4980

cggaggcaac cccgccgtcc gcatccttgt tgccgcgccg tggcacagcg ccgcctgtcg 5040

ccagccgctt tgccactgct gactccgccg cctccacata ctcgccgcgc tctgccgagg 5100

acatgcggag agagaggtcg gagccgtgct ggccggagga gcacgacggc tgccatggtg 5160

actcaccgga ggagcacgac ggctgccatg gtgcgcacct gacctaccca tcccgaactc 5220

ttgctcttac gatggcggcg acaacgcgct ccctcacgtc gctctcgtcc gcctccgcat 5280

ctgtcggcct cgtgcgcacc tccatcagcg gtggcggtgt ggcgcttggt gagggagcct 5340

aggacggtga ggcgggtgaa gagcttgagg aggcggcggc gcggcgcaca gagcatgtgg 5400

cggcgcctgg tggagaccgg ctctgcgcca ccgcgctcgc ccgctgacgc cgcctcgccc 5460

tgccgatgcc gcctctccgt caagctcgcc tgcgccctag ccacggccaa gaaaagtgag 5520

agagagagag gatgaaggga gaagagggaa aataagagag aggtgatgag gtggcatcct 5580

aacatgtggg gcccacgtgg gtcccacgct gacttagccg ccaagtaaga caaaaccggg 5640

atcaaatccg ccgagggact tattgtgacc ggttttgatt agttaaggga cgcaggatat 5700

ctggttttgc ggtttgagga caattttgta actcgatgac aagttgaggg accttcggtg 5760

tactttttcc gcgtcgtaat gggccctact gatcccaagc atcacaatgg ggcaatgggc 5820

cctcctaatc ctacaccatc ttcccctcat gtcggatgcg caaggttaat gggccgagta 5880

ctttcttcac tctgtttatt ggtcagccca tatgtttatg gtccatgaga tatcggccca 5940

gtagcattta cgcttgtagc ccccaccccg ctcccatcaa taaccgccca agtcaagctt 6000

gggcggctcc attttgtcgt tgcgcgcgac gcgcctcact ccccccggtc cccccacctc 6060

tctcctcatc ggacggcgcc gcgccaccca agcccagatc caacggtcag aacgacagcc 6120

tatagctcac accaaaccac tctctctctc tctctctctc tctcctcgtg gagccaaccc 6180

aactacgccg ccgccacctc atccccccgg cgagcgacac ggtcgcatcc caccggagct 6240

ccgaccgccg cagcggcggt cgcctcgccg gagaagaagc cgcgtggggg ggaaggcgga 6300

ggaggggggg gggtgggggt gcaatggaga cggaggtgtt ccccgtggtt gacctccgcg 6360

tgctctcgca gtccgacctg gacgcgctcg ccgccgcctc agcccacgcg gtggccccgg 6420

ggggcagctg ccccgacgcc gaccaactcc cgccgctgaa gatcgaccgc gccgtcttca 6480

acgagagcgc cggctcgcgc aagcagacct tctcccgcgt ccgcttcggc gccgccgccg 6540

ccgtcgccgc ctccccttcc tccccctccc cctccgccgc cgccaagctg cccaggggca 6600

acgacaagga ggacagcttc atcgcctacc acctccgccg cctcttcgcg cccgacgacc 6660

cctcctctcc ccaaacccaa accctagctc tacccgcgcc gccgtctccc gaccccgacc 6720

agttgaccac caactccaag ggggtctccg ttgatctggt gagcctctca cggcttgccg 6780

acccgtacga cgcggagctc gggaagcgga ctgcggggat gaccacggag gaggagctga 6840

tgggcttcat ctctagcctc gcgggtcagt gggtgagcca gaggatgcgg aggaagttag 6900

ttgatgcgtc cttcttcggg gatcacctcc ccagcgggtg gaggctgcag cttgggatca 6960

agcggaagga ccgcaaggcc tgggtgaact gcttcagtta tgtgaggtga gcctcatcat 7020

cctttactga caaatgctgc aatgttaggg gatctgttgt tagtaactgg aatgggggtt 7080

atatattgca ataagctagc tgcctggatg cttgctatga tgtttagaga tgcggttatg 7140

tgtgactata gatgcaaact tgcaaaggat gtgtggtgga tttggtttgt gaagattgtt 7200

ttatactctt ctagcgtagt caactgatgc taatcaatcc gtgcacaagt ttatcagatc 7260

agttcttgac agttttgaat gattgatttt ttttttcaac atggccttct tgctgtcatt 7320

ctgtggattg cctactttct aaggaagaat aaatcttatt ggttaacaat aactgaatac 7380

cccctaggat gagctgtttc atgacgaggg ggaaatgcta tggctggcct caggattagc 7440

atgtgctgca gcaattggag aatcatagta gacaaataga tattctactc cttatttctc 7500

cattagagtt tcctagtttt gtcattggag tcctagtagt attagatact agtttgtttc 7560

ctttgcctta tctagaggca cctcaatata caaaggtagc ctagtactgg attatgcctt 7620

aagcaataag aaatatctaa aggggattga cccaaaagcc cagtcgtttc aatgacatac 7680

tagttaaatg ataattctga atattctggg gggcaaagac atgacttttg ccttccatag 7740

tataagagca gcgtggagtg ttattgcttt aagataatga gttttgtttt attcctttga 7800

tatttgtttt tcggcacttc atttgatctg tgcttttaag gcatgcatgt tatgcgattg 7860

cagcagcatt atctagcaat ggcttgattg atgcttacta tatcataggt taaacattaa 7920

gatagattcg tatggcacat atgatatctc aagtacataa cataacacat gtatgtgtaa 7980

atttgcttct cctgccgagt tctcactgat acacaggtcc taatccaact tgtcaatgag 8040

aaccagtgga taaagtgaat taaggccatg gcatggtgtc taaaacaata attaattttc 8100

atttgcttta tttaactgca atgcctattt gaagtagtgc ctagagcaca tgagcatttc 8160

aatcattatc ttgaatgttc cacagaactc aggtgttcca ccatatatgc tcagatttat 8220

catctgtcgt tgatgaataa ttttgttgta ataatagtaa agatcacttc tcatcactat 8280

tagtttgaaa gtacgtgtta agatacatgc ctatttatct gctcagtgaa catatgttgt 8340

gaatggctgc atcatttcag cgcaataatt ttgtttgtcg cattttttcc cctgtactaa 8400

atgggcatat tgtactttcc agccccaagg gacagagttt tgctacttgc caagaggttt 8460

ctgcatacct catgtcactt cttgggtatc cggagttcaa aacggataat attgagtatg 8520

gtagcacaca acaacatggc ttgtgtgctg acgatggtgt taatgtaagt tgaaagtgta 8580

tgtcacttta tcttgtgaac ttgagtttgt ctagtttaat ttttgggttt gtggcaggtt 8640

ttaggtgttc aacaccaaat tggtacaggt atggacagtc aaagtatttt gccagttgct 8700

tctattacct tttctagtca ttcaagagat caagacgaaa cagatgcaga tgatataaat 8760

tcttatgaat gtcaacaatg caatttaact tttcatggtc agagtgccta tgcgcatcac 8820

ctgatcactt ttcacaaaat gggttctaaa aggcgtaaga ttaacaaggt tggcaaattt 8880

ggtgagccag tgataggcaa agatgggaaa tttgaatgcc cagtatgtaa taagacgttt 8940

gaggaacagt cacggtactt tggtcacgtt ggatcccatg caaagtatca cgggctgact 9000

cctgaagcat tcctacaaac cttgtcagga aaggttggta acgattcttt tgcaggtttg 9060

tcatgtagcc ttcaagattt ggtcggatca ccacaactga atgagaagac tactgctagt 9120

gaagcacgat cacagcatca caattgttca actaaacatg gaggcaatag tacaagaggt 9180

atagaccttt ttaactcaaa ttgtccagct aacttcaatg ggcataatca aacttggtgt 9240

agacctgatg aaattcctcc caccacagat gctccaagca catggactta tagaaataat 9300

gtgacgaact gtgctgatag aactgttccg agaacagcac ctcagcccaa tgatcacatg 9360

gattgcaggg ttagtggctt tgctgaagca actaatttta acgatcaagc aggaagacac 9420

caaggtttta gaccttcctc ctttggaact accaaccatt gtcaaggcca gataattgat 9480

catgcagtag ctgcttccaa gcatgctgag gttaataata gtatgaaatc aagagatgtc 9540

aacctcaatt cacgcctgaa cacaatatcc tttcctattg caactgcgaa caatgaaaca 9600

tcgactgccc ttaatgacgt gaatcggtca tgcattactg gaaaaggttt tagtggaagt 9660

ttcagtaaca atgatggtgc tgcatctatc gtgttgccca gttctggatt aaacaataaa 9720

attcctagct ccctcggtgt agctgacaga tcatctattg ctgcaagatc cttcaatgct 9780

ggttatgtta atgaaaatgg tgcttctgaa gctaacaata ttggcaacaa aaacaatacc 9840

atggtgtatc agacaagttt ggccatgcgc ccagtctctc ctgtaagtaa catggattgc 9900

ttcatttcct gttcagtgca tttaaaaata gcaacagaca tctgtatgat acaaatgaat 9960

gaatggatag catacaaaac aaaactagta actaagctag attttctgtt gatgcttata 10020

ataataaaat attgactggc agtgtcactg aaaggagtct acccagtgta atagaaactc 10080

cagtcacgta ggacctaata tttcaagcca cgattcatca cttcctaaat caagcacact 10140

agcagccagt aacttaagca aggggagtga tgtcaactgg aaggtttctt ttgttaatag 10200

aagcaattcc aactgcataa tgggctcttt tgttacccac tagtttctgc ttgaggtttt 10260

gggaaggact agtagtgtta tgcaaaatcg atacaatgat tgtgccactg cttgtaactt 10320

gctcgcttca gcaagtacta gccagaatgc caacaatctt atgcctatgt aggacaattt 10380

tggtcatgtg tagtttacgc tctggtttgt tctgttggtg atgttccaat cagcagcaca 10440

accagagatc aggtagactg catcaaacat tctttttagt ctgatcactc ctgtgatgtt 10500

taaaaatgct ttcgatattt tatttttgta aatctagtgg aacccagtaa tataatgcct 10560

ctttaggtca gttcatttga cataaatcaa ccagtataat tagactatgc aataggaaag 10620

gaggaaatga acattgtgtt tctcaagact attgcttata agttgttttg cttttgtcat 10680

tgttaccagt tttgttaaag tgtgcataga gtgaatggtt ttttcctctc atgaacgact 10740

atgacatttc atgctgagtg tcctacatgc tgatttgatc ttatttttcc tacacagtgt 10800

gatctgcaac ttggatttag tggtcagaag cagcagatat tgcctggtta tggagaactt 10860

agaccagctg cgtctgggtc ccctcagctt gggggcatgg cagcaaacag ttcaattccc 10920

accagaccct ctcagccgca gtttgggagt atggccagaa ccgatgcttt gcctactgga 10980

ccctctcaac cagggagctt ggccagacct aattttgtgc ccacaggatt ttctcagttt 11040

gcaagcaggc cacctacttc tgtaccacca gccgattcct ctcagtttgc agggggcatg 11100

gccaggcaaa acattccgac catgtctgaa ccaactctag tattgggcta tactcctcag 11160

atggtcaatg gccctccagc ccagctagga tgggatctat ctttgtcaag gatggtcagt 11220

gaaggcatgc tcccagtgtt atgtatatgg tgcaacagcc aattccacca ttttggcccc 11280

attgatgcac agcaatctgg ttcgtttggt ttcatttgcc cagcttgcaa ggagaagatg 11340

tcaggcaatc ctaatgcgcc caataacggt ccatggcaac catgataact gttgtggctg 11400

gtctacaatc atttctgttg ctggatcttt ggtctaaggt tagctgcggt gtgcctgcac 11460

cccagagttt tagtagggta tctaggaacg atgtctgtat tttgtggact ctcttctgta 11520

cgtacttctg ttgaaatgtg aagaaaaccc caagagaaaa acccactgaa tgttgtactt 11580

ttggcaggat ggatctccag taagattcaa gggttgtacg cagccatcat ccaaaaagag 11640

tttatgagat agattttgat gtagtattat tgtttggcaa caaaggtaca agttgatcgc 11700

acgctgatcg cgtgatagac ctggtactgg tagaatccat acgacaacga tttggacgaa 11760

tcttctttca tgacgaacat tcactatcat catctgatac aaaagttctc tttttttttt 11820

tcagccttac agccttagaa cctttggtgt ttctactaat tatatattcc aggcttccag 11880

cagataaaaa tacatcaata catgcatgct gaacattaat tcactaaacc attgtgggaa 11940

cttcttttat ttaacgatga cataaccgct aggataaaca gaacccaaca ttattatatt 12000

cctttctttt atttaacgat gacataaccg ctagggttaa cagaacccaa cattattaca 12060

ttcctcttat atttttcttc caggaacatt attgcgttat attactactg aaaatctgaa 12120

atatctatcc ttcttgggtt cttgcctcga cgtggcaaat gtgatgacta taataataat 12180

tatatggtct actccctccg tttcacaatg taagtcattc tagtattttc cacatttata 12240

ttgatgttaa tgaatctaga tacattgatg ttaatgtgga aaatactaga atgacttaca 12300

ttgtgaaatg gaggaagtag ctccgaaact ctacggacat cccctgaaag aaaaagagga 12360

gatgaaatgc accacattca tgtggaaaat ctcaaacaca atatattttt ttttatatta 12420

agatagcttg aaaaaaagct accacatcca ctatagaggc caactggtca agtaattaac 12480

acactaatta aagaaaagga aaaatatctg caacgtttga ttatggtggg ttcagattat 12540

aacaatatag attgattgat ataattgtat acaacgtaaa aataaactta gagtgaattt 12600

tattctggac cattttttat taccaatgtt ttcctttgga tcatatttta accaatgttt 12660

ttactttgga ccgggtattc aatatttttt attggatcgg acaattttac ctttataaca 12720

ctcaaatcac tcttttgctt ctccgttgtt tttattttcc taaagggcca tgtaagactc 12780

tttttctcct aatatatccg acaaatctcc tgccgtttaa tgtttaaaaa aaatactgta 12840

gaccactctc cactctttat ccatctacat ttaacttcat attaaaaaga tgaccttaca 12900

tatagccgtg agagtttatt aacgagtaga agcgaaaatt cagttagaaa cattttcttc 12960

cagaaatcga aagcgaaaat gcaggaaata attatatcga gtgtggccag agccgggggc 13020

gtactatacg tacgttgggt tgtaggtgat cctgatgacg ccggcgtcga ggttggcgat 13080

cttggcgaag gcctccctgg agaggtcgat ggtgctcgtg cacccgtcgc tgctggcgca 13140

gttgtcgac 13149

Claims (10)

1. The gene for regulating rice stigma exsertion has the nucleotide sequence with homology of 90% or more, 92% or more, 95% or more, preferably 98% or more, and more preferably 99% or more with SEQ ID NO 1 or SEQ ID NO 2.

2. The gene of claim 1, wherein the nucleotide sequence is SEQ ID NO 1 or SEQ ID NO 2.

3. A vector comprising the gene of claim 1 or 2.

4. The vector of claim 3, wherein the backbone plasmid is of the pCAMBIA series.

5. The vector of claim 4, wherein the backbone plasmid is pCAMBIA 1300.

6. An agrobacterium transformed with the vector of any one of claims 3-5.

7. Use of the gene according to claim 1 or 2, the vector according to any one of claims 3 to 5, or the agrobacterium according to claim 6 in cross breeding.

8. Use according to claim 7, wherein the gene according to claim 1 or 2 is integrated into the genome of oryza sativa using plasmid transformation, homologous recombination techniques or gene editing techniques.

9. Use according to claim 7, for increasing the level of stigma exsertion.

10. Use according to claim 9 for increasing the set percentage of hybrid rice.

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