CN114752601B - Gene for regulating and controlling rice stigma exposure and application thereof - Google Patents

Abstract

The invention discloses a gene for regulating and controlling rice stigma exposure, the nucleotide sequence of which 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, thereby improving the fruiting rate and yield of hybrid rice.

Description

Gene for regulating and controlling rice stigma exposure and application thereof

Technical Field

The invention belongs to the field of rice genetic engineering, and in particular relates to newly discovered genes RSE1 and RSE2 for regulating and controlling rice stigma exposure and application thereof in hybridization breeding.

Background

Oryza sativa (Oryza sativa) is produced by domestication of common wild rice (Oryza rufipogon), and is one of the most important food crops for humans. Wild rice is cross-pollinated, while cultivated rice is self-pollinated. The stigma exposure rate is one of key indexes for measuring the level of rice outcrossing. In wild rice, after the full bloom and closure of the palea, a portion or all of the glume remains on the external surface of the glume, a phenomenon known as stigma exposure (Sigma exposure) (Yan, et al 2009). But there is little stigma exposure in cultivated rice or a lower stigma exposure rate. Stigma exposure represents two different reproductive modes, the inbred reproduction of wild rice and the selfed reproduction of cultivated rice. The transformation from the outcrossing reproduction to the selfing reproduction is an important physiological mechanism change in the domestication process of the wild rice to the cultivated rice, the transformation has an important meaning for reducing the filial generation separation of the cultivated rice, and the research on the change of the passage way has an important meaning for the domestication research of the rice (Purugganan and Fuller, 2009).

In the cross breeding process, a breeder selects a series of traits of the parent material that are most interesting in affecting the yield of hybrid seeds, such as the heading and flowering stages of the parent, the number and longevity of pollen, the ear-to-head size and morphology, and the level of outcrossing of the plant, which is one of the most important traits affecting hybrid seed production. Loose spikes, large stigmas and high-level stigmas are exposed to form the developmental basis of the outcrossing behavior of wild rice, and the level of stigmas exposure is one of the core factors determining the outcrossing level of plants. The field observation shows that the exposed stigmas can help plants to obtain more pollen, the exposed stigmas have activity retention time outside longer than the window time for opening and closing the glume flowers in the flowering process, which is favorable for the plants to overcome pollination disorder caused by inconsistent flowering period and increases the probability of forming hybrid seeds by mutual pollination among different rice plants. The field survey shows that the improvement of the stigma exposure level of the hybrid rice female parent can obviously improve the setting rate and the yield of hybrid seeds.

The exposed level of rice stigma plays an extremely important role in the domestication process. Genomics studies have found that the average heterozygosity at the wild rice natural population genome level is nearly 10% whereas the average heterozygosity at the fully domesticated cultivated rice natural population genome is essentially zero (Song, et al 2010). The generation of the difference of the heterozygosity of the genome is the inevitable result that the stigma of the modern cultivated rice is no longer exposed and is fully self-pollinated. Because of exposed stigmas and loose ears, the wild rice individuals can more easily exchange genetic information, and some genome fragments are inevitably brought into other plant genomes, which provides an important mutation source for the wild rice to adapt to different environment selections. The exchange of genetic information also results in more genetic mutations and morphological diversity in the progeny population, which is an important basis for rice acclimatization. In order to meet the increasing grain demands of people, the people select and domesticate wild rice directionally to obtain rice varieties which can stably pass seeds and have high yield. Along with the directional selection of human beings, the pollination mode of the rice is changed from outcrossing to complete selfing, the genetic diversity of the rice is obviously reduced, the genome is changed from high heterozygosity to high purity, and the number of alleles of domesticated traits is also obviously reduced. The genome of cultivated rice has high stability, and the control genes of advantageous traits are stably present and maintained in the progeny. To maintain this stability, stigma exposure was targeted during human acclimation.

At present, research on rice stigma exposure has been advanced to some extent, but the detailed genetic mechanism and development regulation network are still not clear. Genetic studies have found that rice stigma exposure is a complex quantitative trait of typical polygenic control, affected by a variety of external environments, including wind, temperature, humidity, light, etc. (Miyata, et al 2007), which increases the difficulty of the related studies. With the progress of molecular marking technology, genetic research is becoming increasingly available. Over the past decades, a series of segregating populations have been developed for genetic studies of rice stigma exposure, such as F2 populations, recombinant selfing populations, doubled haploid populations, backcross populations, and chromosome fragment replacement populations. Along with the renewal of high throughput genome sequencing technology, GWAS (genome wide association analysis, whole genome association analysis) technology has also been applied to the study of rice stigma exposure (Huang, et al 2012). Using the above-described methods and populations, researchers have cloned at least 38 QTLs (Quantitative Trait Loci ) with exposed control stigma, which are distributed across 12 chromosomes of rice. Most of these QTLs are micro-effective sites, only a small fraction of which are successfully mapped to a smaller extent and can account for more than 10% of the stigma-exposed phenotypic variation.

Disclosure of Invention

In rice studies, we identified QTL genes controlling stigma exposure by means of map-based cloning and studied their function. First, a chromosome single-piece substitution line GPSL41 was constructed using indica GLA4 as an acceptor and ordinary wild rice W1943 as a donor. Wherein the stigma exposure rate of GLA4 is lower than 3%, and the stigma exposure rate of GPSL41 is about 30% -50%, the two are backcrossed to obtain BC1F1 population, the BC1F2 positioning population is obtained by further selfing, the molecular marker is utilized for fine positioning, and the site for controlling the stigma exposure is positioned in a 14.1kb interval between the P122 and EC24 molecular markers. Genotype and phenotype comparison analysis determined that the candidate genes were 2 highly homologous MBD genes, with CDS similarity reaching 92.22% and protein sequence similarity reaching 86.98%, designated RSE1 and RSE2 genes, respectively. Meanwhile, a near isogenic line NIL-RSE is constructed, and the column head exposure rate reaches 30% -40% only by 180kb W1943 fragments containing RSE1 and RSE2 genes.

The transgenic complementation experiment result shows that the RSE1 and RSE2 genes respectively complement GLA4, and the stigma exposure rate can be improved by about 10 percent. Thus we verify that the RSE1 and RSE2 genes are exposed genes in the control stigma in GPSL41. Both genes contained one MBD domain and a C2H2 zinc finger domain. Comparing the gene sequences of the parent RSE1 and RSE2, it was found that there was variation in the promoter region, UTR region and gene coding region.

Microscopic observations of pre-flowering NIL-RSE and GLA4 glume flowers revealed that: the NIL-RSE has a significantly greater stigma deflection angle than GLA4 and the NIL-RSE has a significantly longer stigma length than GLA4, all of which may result in stigma exposure. The results of the mRNA in situ hybridization detection of the expression patterns of the RSE1 and RSE2 genes show that: the expression patterns of RSE1 and RSE2 genes during GLA4 and NIL-RSE glume development are similar. In GLA 4.8-1 mm glume flowers, the expression of RSE1 and RSE2 genes in the parts of the pistil stigma and the style of the glume flowers is relatively uniform, and in the same-time NIL-RSE glume flowers, the RSE1 and RSE2 genes are strongly expressed in the hairbrush-shaped part at the front end of the pistil stigma, and the expression level is obviously higher than that of the hairbrush-shaped part at the front end of the non-hairbrush-shaped part and the synchronous GLA4 glume flower stigma. We speculate that the specific expression pattern of RSE1 and RSE2 genes on NIL glume-like flower posts influences the development of the post head and leads to the exposure of the post head.

RSE1 and RSE2 genes are highly homologous to the Arabidopsis thaliana flower development regulatory gene AtMBD8, and the MBD genes can enter the nucleus to participate in regulating genome methylation homeostasis. The rice protoplast system was used to demonstrate that the fusion proteins RSE1 and RSE2 can enter the nucleus for functional purposes. Comparative analysis of 4-5cm young ear transcriptome found: there are approximately 1176 differentially expressed genes between NIL-RSE and GLA4, of which 582 genes were up-regulated and 594 genes were down-regulated. The GO enrichment analysis result shows that the differential expression gene relates to the transcription regulation process, hormone regulation and control paths such as auxin, cytokinin and the like, cell cycle related paths and the like.

The results of nucleic acid diversity analysis and Tajima's D analysis of the RSE1 and RSE2 genes using 39 parts of modern cultivated rice material and 14 parts of ordinary wild rice material showed a significant decrease in DNA diversity of the RSE1 and RSE2 genes in the cultivated rice population, significantly deviating from neutral selection. It was demonstrated that RSE1 gene and RSE2 gene may be selected by acclimatization. This finding forms the basis of the present invention. Specifically, the invention comprises the following technical contents.

A gene regulating the exposure of rice stigma, designated herein as RSE, comprises two genes RSE1 or RSE2, the nucleotide sequence of which has more than or equal to 90%, more than or equal to 92%, more than or equal to 95%, preferably more than or equal to 98%, more preferably more than or equal to 99% homology 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 invention provides a vector comprising the gene as described above. 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 of the pCAMBIA series. For example, the backbone plasmid may be pCAMBIA1300 or the like, and the constructed RSE1 or RSE2 gene expression vector may be pCAMBIA1300-RSE1 or pCAMBIA1300-RSE2, for example.

In a second aspect the invention provides an agrobacterium transformed with the vector described above. The agrobacterium is used for mediating the vector containing the RSE1 or RSE2 gene to be introduced into rice to complete the transgenic operation. The agrobacterium is selected from agrobacterium tumefaciens (Agrobacterium tumefaciens) and agrobacterium rhizogenes (Agrobacterium rhizogenes).

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

In particular embodiments, the RSE1 or RSE2 genes described above may be integrated into the genome of Oryza sativa, particularly Oryza sativa (Oryza sativa), using plasmid transformation, homologous recombination techniques, or gene editing techniques.

The above-described gene integration may be agrobacterium-mediated plasmid transformation.

The gene editing technique described above may employ a CRISPR-Cas9 system, a CRISPR-Cpf1 system, a CRISPR-Cas related transposition system INTEGRATE system, or a CAST system.

The INTEGRATE system described above refers to the gene editing tool developed by Sam Sternberg research group (Insertion of transposable elements by guide RNA-assisted targeting, guiding insertion of RNA-assisted targeted transposable elements); the CAST system refers to the gene editing tool developed by the Zhang Feng research group (CRISPR-associated transposase ).

The INTEGRATE system described above refers to the gene editing tool developed by Sam Sternberg research group (Insertion of transposable elements by guide RNA-assisted targeting, guiding insertion of RNA-assisted targeted transposable elements); the CAST system refers to the gene editing tool developed by the Zhang Feng research group (CRISPR-associated transposase ).

The RSE1 or RSE2 gene is used in raising the stigma exposing level in rice to raise the setting rate of hybrid rice.

Experiments show that the newly discovered rice RSE1 or RSE2 gene can obviously improve the stigma exposure level of cultivated rice GLA4, thereby improving the fruiting rate and yield, and indicating that the RSE1 or RSE2 gene can be used for improving the existing cultivated rice variety, and has wide development and application prospects.

Drawings

Fig. 1 shows comparative photographs and statistical bar graphs of the column head exposure levels of GPSL41 and GLA4 in different regions and years. Wherein A, B: GLA4 and GPSL41 main ears bloom completely glume flowers, scale 5mm; c: the stigma-exposed phenotype of GLA4 (left) and GPSL41 (right), scale 5mm; d: the stigma exposure rates of GLA4 and GPSL41 in different regions were expressed as average (SD), n=30 (spike number).

FIG. 2 shows the construction process of GPSL series chromosome fragment substitution lines and GPSL41 whole genome genotypes. Wherein A: the construction process of GPSL series chromosome fragment substitution system; b: the whole genome genotype of GPSL41, G represents the homozygous GLA4 genotype, W represents the homozygous W1943 genotype, and H represents the W/G heterozygous genotype.

Fig. 3 shows a photograph of a brown flower of glume and stigma that is flowering. Wherein, A-B: the rice glume flowers which are flowering are 5mm on a scale; C-H: the rice pistil stigma is gradually brown in the air.

Figure 4 shows statistical plots of stigma exposure for background genetic populations of different genotypes. Wherein, the Y-axis represents the column head exposure rate, the X-axis represents the observation number, ng=77, nf1=133, nw=129. G represents GLA4, F1 represents BC1F1, and W represents GPSL41. Inter-group difference significance detection was performed using one-way analysis of variance with a significance level of 0.01.GLA4 group column cap exposure averaged 0.053 (0.035), F1 group average column cap exposure averaged 0.166 (0.059), GPSL41 group average column cap exposure averaged 0.286 (0.085). The variance alignment test shows F value=24.342, p (sig.) =10 ^-6 The variance between the three groups is irregular. Analysis of variance showed a very significant difference in stigma exposure rate between GLA4, F1 and GPSL41 (f=308.512, p (sig.) =10 ^-6 The unified force test Power is greater than or equal to 0.99).

FIG. 5 shows the map-based cloning of the RSE gene. Wherein A: initial localization of RSE gene; B. c: fine localization of RSE genes. The values are expressed as average (SD) and RSE is the Rate of column head exposure (Rate/Ratio of stigma exsertion). N represents population size. The numbers in left Bian Kuohao represent the number of recombinants.

FIG. 6 shows amino acid sequence alignment of MBD domains of RSE1 gene, RSE2 gene and AtMBD8 gene. Wherein the dark color is marked as a conserved site.

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

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

FIG. 9 shows a photograph of a representation of RSE1 complementation transgene T2 and a comparison of stigma-up exposure versus bar graph. Wherein A: RSE1 complementation transgene T2 generation plant CP-1 column head outerThe dew type. The whole spike scale is 1cm, and the primary branch scale is 5mm. Red arrows are marked as exposed stigmas; b: comparison of the stigma Exposure Rate of the same batch of parent and CP-1. The values are expressed in the form of an average (SD), n=12. The SNK test and LSD test were used for the inter-group difference significance test,representing significant differences between groups at the 0.05 level.

FIG. 10 shows a photograph of a representation of RSE2 complementation transgene T2 and a comparison of stigma-up exposure versus bar graph. Wherein, A-C: the post heads of the plants CP-1, CP-2 and CP-3 of the T2 generation of the RSE2 complementation transgene are exposed, the whole spike scale is 1cm, the primary branch scale is 5mm, and the red arrow mark is the exposed post head; d: and comparing the stigma exposure rate of the parent and complementary transgenic lines in the same batch. The values are expressed in the form of an average (SD), n=12. The SNK test and LSD test were used for the inter-group difference significance test,representing significant differences between groups at the 0.05 level.

Fig. 11 shows photographs of morphological structures of an exposed column head and an unexposed column head. Wherein A: fine structure of rice stigma; b: a form of non-exposed column head; c: morphology of the bilateral exposed column heads; d: morphology of unilateral exposed column head. Scale 1mm.

Figure 12 shows a bar chart of the length of the exposed column head and AS angle statistics. Wherein A: comparison of stigma length between GLA4 and NIL-RSE; b: comparison of the stigma angle between GLA4 and NIL-RSE. The values are expressed in the form of an average (SD), n=300. Inter-group difference significance test (level test value sig=0.082 of variance equation), double-sided sig=10, using t-test of independent samples ^-6 。Representing significant differences between groups at the 0.05 level.

FIG. 13 shows the variation of the expression level of RSE1 gene in different spike development stages of rice. The values are expressed in the form of an average (SD), n=3. The significance of the differences between groups was checked using one-way anova. Wherein, the Y axis represents relative expression quantity, the X axis represents young spike in different development periods, and the length represents young spike development period.

FIG. 14 shows the variation of the expression level of RSE2 gene in different rice spike development stages. The values are expressed in the form of an average (SD), n=3. The significance of the differences between groups was checked using one-way anova. Wherein, the Y axis represents relative expression quantity, the X axis represents young spike in different development periods, and the length represents young spike development period.

FIG. 15 shows photographs of in situ hybridization experiments of RSE1 gene and RSE2 gene. Wherein, A-C: in situ hybridization of RSE1 gene, a: expression of the RSE1 gene in GLA4 glume flowers; B-C: expression of the RSE1 gene in NIL-RSE glume; 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. And a scale 20 micro. The black arrows point to the column heads. The material is young spike with length of 4-5 cm.

FIG. 16 is a frame diagram of plasmids pCAMBIA1300-RSE1 and pCAMBIA1300-RSE2 constructed in accordance with the present invention. Wherein A is pCAMBIA1300-RSE1; b is the pCAMBIA1300-RSE2 plasmid.

Detailed Description

The gene RSE (abbreviation of the stigma exposure rate Rate of Stigma Exsertion or Ratio of Stigma Exsertion) for regulating and controlling the stigma exposure of the rice is cloned and obtained, and comprises two genes RSE1 or RSE2, which are found to improve the stigma exposure rate of the rice by about 10 percent through research, and the RSE gene regulates and controls the stigma exposure rate by controlling the number and the cell size of the glume-like flower stigma of the rice. The RSE1 and RSE2 alleles with high stigma exposure rate can obviously improve the population outcrossing rate. RSE1 and RSE2 sites for controlling the high stigma exposure rate are mainly present in common wild rice groups and are domesticated and selected in Asian cultivated rice groups, so that the RSE1 and RSE2 sites enter most modern cultivated rice groups through a manual selection method, and the genetic diversity of the cultivated rice groups and the seed production success rate of hybrid rice are improved.

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 hybrid seed production success rate. The common wild rice contains the specific expression genotype of the gene, and the Asian cultivated rice contains the genotype with low stigma exposure rate and low outcrossing rate, so that the gene can be introduced into the Asian cultivated rice by cross breeding to improve the existing cultivated rice variety.

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

The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.

In the examples herein, if no specific explanation is made for the reaction temperature or the operating temperature, this temperature is usually referred to as room temperature (15-30 ℃).

The whole gene synthesis, primer synthesis and sequencing are all completed by Beijing qingke biotechnology limited company and national gene research center of China academy of sciences.

The molecular biology experiments in the examples include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., and are mainly performed by referring to "molecular cloning experiment guidelines (third edition), J.Sam Broker, D.W. Lassel, huang Peitang, et al, science Press, beijing, 2002).

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

Examples

1. Materials:

the present study used mainly the low stigma exposure level of indica oryza sativa GLA4 and the high stigma exposure level of the chromosomal fragment replacement line GPSL41.GPSL41 is a substitution line from the part on chromosome eight in the context of GLA4 to the chromosomal fragment of wild-type rice W1943.

The genetic population is a recombinant inbred line generated by continuously selfing filial generations of GPSL41 and GLA4 after backcrossing the filial generations. Wherein the NIL-RSE is the smallest near isogenic line selected from the population of the inbred offspring of one of the recombinant inbred lines and GLA4 backcross.

2. Paraffin section and in situ hybridization:

materials for histological observation and in situ hybridization were fixed with 4% paraformaldehyde, dehydrated with ethanol series, and xylene transparent and embedded in paraffin. Probes hybridized in situ were labeled with the rogowski digoxigenin-labeling kit. The labeled in situ sense antisense probe was hybridized on 8- μm paraffin sections.

3. Real-time quantitative PCR:

fresh plant tissue or tissue frozen at-80℃was extracted with Trizol Reagent. DNA in the total RNA sample was digested with DNase and then SuperScript from Invitrogen ^TM II Reverse Transcriptase reverse transcription into cDNA first strand, further templates for real-time quantitative PCR. Quantitative PCR was performed by Takara CorpThe Premix Ex taq kit was performed on a Applied Biosystems 7500real time PCR instrument. Specific gene primers are designed according to the gene sequence, and rice gene eEF-1 alpha (GenBank accession 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 the plasmid pCAMBIA 1300; the RSE2 gene is subjected to Sal I digestion BAC and inserted into the 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 in addition to RSE1 or RSE2 gene, and the screening gene is hygromycin.

Experimental results

1. Parental material and phenotypic investigation

The subject uses cultivated rice Guangland dwarf No. 4 (GLA 4) with a stigma exposure rate lower than 3% and a chromosome fragment substitution line GPSL41 with a stigma exposure rate reaching 30% -50% to perform map cloning of RSE (Rate of Stigma Exsertion, stigma exposure rate) sites, see A, B, C in FIG. 1. The stigma exposure rates of GLA4 and GPSL41 varied with the environment, but the phenotypic differences between the parents were stable, as shown in fig. 1D.

GPSL41 is a chromosome fragment substitution line (GPSL series, figure 2) constructed in the laboratory and using common wild rice W1943 as a donor parent, GLA4 as an acceptor parent and a large-scale W1943 fragment substitution on chromosome 8, wherein the fragment comprises a stigma-exposed QTL locus RSE (Huang, et al 2012) which is positioned in the laboratory by using natural population whole genome association analysis, the QTL locus is subjected to strong domestication selection in indica rice population and non-glutinous rice population simultaneously (pi W/pi c >4, pi value represents genetic diversity, W represents wild rice and c represents cultivated rice).

2. Construction of genetic population and construction of phenotype investigation method

2.1 construction of genetic populations

The main construction process of the replacement material GPSL41 (see FIG. 2A) used in the study is as follows: 1. hybridizing common wild rice W1943 and indica rice GLA4 to obtain F1 generation; 2. backcrossing F1 and GLA4 to obtain BC1F1;3. continuously backcrossing the BC1F1 and the GLA4 to obtain BC2F1, and repeatedly backcrossing to BC5F1;4. the BC5F1 is selfed to obtain a BC5F2 population, and fragment substitution lines with different chromosome position substitutions are obtained through molecular assisted screening.

The locating population used in this study was obtained from GPSL41 and GLA4 backcross, as follows: 1. hybridizing GPSL41 with GLA4 to obtain an F1 population; 2. selfing the F1 group to obtain an F2 group, and performing initial positioning by using the group; 3. molecular auxiliary screening of F2 population, selfing individuals with key recombination to obtain F3 population, and fine positioning by using the population; 4. f4 colony is obtained by F3 selfing, fine localization and phenotype verification are carried out on subsequent selfing colony, and meanwhile, the colony is utilized to construct an near isogenic line.

2.2 establishment of phenotypic investigation method

Although the literature reports that several grain genes (GS 3, GW2, GW5, etc.) can affect rice stigma exposure (Zhou, et al 2017), there has not been cloning to date by methods of population genetics to genes controlling rice stigma exposure, most importantly due to the complexity of stigma exposure phenotype investigation. The stigma exposure is a complex character which is regulated by multiple genes and is influenced by environmental factors, and the stigma exposure level can be influenced by environmental temperature, photoperiod, air humidity and the like. Meanwhile, the stigma exposure phenotype shows different forms, including unilateral (left and right) stigma exposure, bilateral stigma exposure and the like, so that the complexity of property investigation is increased. In order to accurately evaluate the stigma-exposed phenotype and find a suitable investigation method, we have conducted a careful investigation of a large sample of the two parents and the F1 heterozygous phenotype in the 2016 s Shanghai and 2017 s Hainan respectively, and found the following factors affecting the phenotype investigation:

(1) And (5) examining a time window for exposing the column head. Stigma exposure begins with flowering, after which stigma is exposed 1/3 of the palea space under the glume, the exposed stigma is gradually brown and a marked brown mark is left on glume (see figure 3), and statistical brown marks can expand the time window of phenotype investigation.

(2) Flowering time among rice tillers of GLA4 background is not uniform, and later tillers and early tillers can be affected by different photoperiod and temperature, so that the stigma exposure level can be interfered.

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

(4) The stigma exposure rate is a quantitative trait typical of semi-dominant, the F1 phenotype and the two parental phenotypes are continuously distributed, which results in difficult heterozygous subtyping and susceptibility to phenotype misinterpretation (FIG. 4).

Therefore, in order to eliminate environmental interference and human error to the greatest extent and accurately measure the stigma exposure level, a new set of phenotype investigation rules is established, and the new rule is specifically as follows:

(1) Examining the stigma exposure rate (Ratio/Rate of Sigma Exsertion, RSE), i.e. the Ratio of the number of glumes exposed by a spike to the total number of glumes, and distinguishing the exposure form (single-side or double-side exposure).

(2) And (3) examining the average stigma exposure rate of small groups of specific genotypes, planting 24 small groups according to the characteristics of GLA4 heading, randomly selecting 5 single plants, examining the stigma exposure rate (5 plants by 3 spikes) of 3 main ears of each plant, and calculating the average value of the stigma exposure rate as phenotype data.

(3) Only the average stigma exposure rate of the genotype homozygous recombinants offspring small populations in the mapped population was examined, and the M3 small populations were used to review the M2 individual phenotype. A W1943 phenotype was defined as having an average stigma exposure of greater than 20% and a GLA4 phenotype was defined as having an average stigma exposure 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 a cross F1 population of GPSL41 and GLA4 in Hainan 2015 and an inbred F2 population in 2016. Screening of the F2 population resulted in about 1000 individuals with W1943 fragment substitution on chromosome 8 alone, followed by genotyping and phenotyping, targeting of the target fragment to chromosome 8 long arm using indel molecular markers P2 and P90, and narrowing of the target region to a region of about 2Mb using molecular markers EC22 and P100, consistent with previous GWAS results (fig. 5 a).

Further expanding the F3 population by about 15000 strains, screening to obtain about 3000 available recombinants. Meanwhile, finer indel and SNP molecular markers are designed between the molecular markers EC22 and P100, 70 key recombinants are obtained by screening through the molecular markers P79 and P48, and the target region is further reduced to a region of about 180kb by using the molecular markers P42 and P70 (B in FIG. 5).

In the Shanghai 2018, the F4 group was used to further narrow the localization zone by about 10000 plants. A series of new SNP molecular markers were designed every 10kb (every 1kb for a partial fragment) between P42 and P70 and 15 recombinants were selected. During further localization we obtained 3 key recombinants R1, R3 and R4 (C in fig. 5). By using the molecular markers P122 and P107, the target region can be narrowed to within 14.1kb, which contains an unknown functional gene RSE1 and a mango gene EPF (C in FIG. 5), based on the phenotype differences of recombinants R3 and R4.

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

3.2 Construction of NIL-RSE near isogenic line

In the localization process, we screened a near isogenic line of which the genotype of about 180Kb region between the molecular markers P42 and P70 is W1943 type, and the genotype of the other region is GLA4 type, and named NIL-RSE.

3.3 stigma-exposed candidate Gene

Using the rice gene annotation database RAP-DB (https:// rapdb. Dna. Affrc. Go. Jp/index. Html), we analyzed the last located interval for candidate genes. Between the molecular markers P123 and EC24, two complete genes are included, one of which is known as functional gene EPF and one as MBD gene, which we speculate to be a candidate gene for RSE 1. An MBD gene adjacent to the candidate RSE1 gene was also included between molecular markers P107 and P68, and was 92.22% similar to RSE1 at the coding region DNA level, 86.98% similar to the two protein sequences (see fig. 6, 7), presumably the RSE2 candidate gene.

4. Genetic transformation verification of genes RSE1 and RSE2

To verify whether the RSE1 gene and RSE2 gene are indeed rice stigma exposure control genes, we performed transgenic experiments. The enzyme-cut fragments of wild rice BAC (see FIG. 8) containing RSE1 gene (containing EPF gene) and RSE2 gene were introduced into pCAMBIA1300 vector, respectively, to construct the complementary vector of RSE1 gene and RSE2 gene (see FIG. 16). Because GLA4 transformed by the complementary vector is indica rice with great genetic transformation difficulty, the complementary vector is selected to be delivered to a company for transformation, and finally, complementary transgenic plants of RSE1 genes and RSE2 genes are obtained in batches.

The T0 generation complementary transgenes of RSE1 genes are totally 16 strains, hygromycin identification is totally positive, gene specific molecular markers are totally positive, 4 transgenic plants of 5 strains are obtained in the first batch and accidentally die in the field planting process, the phenotype investigation of T2 generation of the residual 1 transgenic plants is found to be 8 percent higher than 0.4 percent of negative parent GLA4 in the same batch (see figure 9), the phenotype investigation of T1 generation plants of 2 strains in the second batch is also observed to be the same trend (see B, C in figure 10), and the T1 generation of the residual 9 transgenic plants and the T2 generation of the transgenes are just sown in Hainan and wait for the subsequent phenotype investigation.

In the construction of the RSE1 gene complementation transgene, the wild rice BAC enzyme fragment containing the RSE1 gene additionally contains an EPF gene (RAE 2/EPFL1/GAD 1) located about 4kb upstream of the RSE1 gene start codon, encoding a 125AA short peptide controlling mango development, which can lead to the formation of a long mango in the RSE1 transgenic plant. EPF gene belongs to a kind of plant epidermal cell growth factor, and is mainly used for regulating and controlling the development of leaf epidermal stomata cells in Arabidopsis thaliana. Studies on the wheat TaEPFL1 gene found that the TaEPFL1 mutant stamen developed significantly abnormally, but pistil development was not affected. Rice OsEPFL1 gene is mainly specifically expressed in mango primordium, the expression in SP7 later stage and SP8 early stage is strongest, and wild type complementation of Japanese sunny, NA93-11 and Koshihikari can lead to transgenic plants to form long mango, but no long mango seed is found to be accompanied by exposed stigmas. Microscopic anatomical observations revealed that the development basis of rice stigma exposure is changes in the length and angle of pistil stigma, which are inconsistent with the tissue expression pattern of EPF but completely consistent with the expression pattern of RSE1, so we believe that the EPF gene does not affect stigma exposure. Currently, complementary vectors comprising only the RSE1 gene have been sent to the company to transform GLA4 in an attempt to compensate for the experimental errors described above.

The T0 generation complementary transgenes of RSE2 genes are totally positive in 10 lines, hygromycin identification is positive in all, but the identification of the gene specific molecular markers is positive in 7 lines, the first 4 transgenic lines die 1, the other 3 transgenic lines are obviously higher than 0.3 percent of negative parent GLA4 in the same batch in 12 percent, 9 percent and 10 percent respectively through phenotype examination on T2 generation of the rest 3 transgenic plants (see figure 10), and the same trend is observed through phenotype examination on the second 3 transgenic T1 generation plants.

The above results demonstrate that complementing the RSE1 gene and the RSE2 gene does significantly increase the stigma-disclosure rate of GLA 4.

NIL-RSE stigma-exposed phenotypic dissection

To study the developmental mechanisms of stigma exposure, we performed dissection analyses on flowers of GLA4 and NIL-RSE, dissections of glume flowers of pre-heading spike selected from GLA4 and NIL-RSE populations and observations comparing pistil structure. A complete carpel is divided into three parts, ovary, flower column and Stigma, and the Stigma is further divided into brush-shaped part (SBP) and non-brush-shaped part (SNBP). We define the Angle between the central axis of the concentric skin of the column cap brush-like region (SBP) AS Angle of SBP, AS; whereas the Angle of the non-brush region (SNBP) concentric skin perpendicular to the central axis is defined as ASN Angle of SNBP, ASN (see FIG. 11A).

Anatomical observations show that the pistil of GLA4 with the column head not exposed is bilaterally symmetrical on the whole structure, and the lengths of the two column heads, ASN and AS angles are basically consistent. The pistil exposed from the unilateral column head in the NIL-RSE loses the bilateral symmetry structure; the length, ASN and AS angles of the non-exposed column cap are not obviously different from those of the non-exposed column cap in GLA4, and compared with the non-exposed column cap of GLA4, the length of the non-exposed column cap is obviously increased, the ASN and AS angles are obviously increased at the same time, or the AS angle is obviously increased, the SBP area of part of the non-exposed column cap is greatly twisted, and the AS can reach more than 90 degrees. There is also a small amount of bilateral stigma exposure in the NIL-RSE population, whose pistil does not necessarily lose bilateral symmetry, but the bilateral stigma length, ASN and AS angle are significantly increased simultaneously, AS shown in fig. 11 and 12.

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

Space-time expression pattern analysis of RSE Gene

6.1 analysis of expression level

We first searched the tissue expression patterns of the RSE1 gene and the RSE2 gene in the RiceXPro database (https:// RiceXPro. Dna. Affrc. Go. Jp /), and the data show that both the RSE1 gene and the RSE2 gene are expressed at lower levels in different rice tissues, indicating that RSE1 and RSE2 are widely expressed genes. Subsequently, the expression levels of the RSE1 gene and the RSE2 gene in different periods of GLA4 and NIL-RSE spike development were detected, and the results show that the expression levels of the RSE1 and the RSE2 genes have no significant difference between the different periods of spike development. Furthermore, there was no significant change in the expression levels of RSE1 gene and RSE2 gene in GLA4 and NIL-RSE compared during the same spike development period (as shown in FIGS. 13 and 14).

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

6.2 expression Pattern analysis

In order to study the expression patterns of the RSE1 gene and the RSE2 gene, digoxin-marked RNA probes are respectively constructed by taking specific sequences of the RSE1 gene and the RSE2 gene as templates, and the probes are used for carrying out RNA in situ hybridization to detect the space-time expression patterns of the RSE1 gene and the RSE2 gene in the young ear development process. In situ hybridization results showed that the expression patterns of the RSE1 gene and the RSE2 gene were substantially identical during either GLA4 or NIL-RSE floret development (see FIG. 15). In young spike flowers of 4-5cm, both GLA4 and NIL-RSE, the RSE1 gene and RSE2 gene are expressed at very low levels in the palea and palea, but in ovary, pistil and stamen. In particular, the expression is relatively strong in ovules, anthers, filaments and stigma. We note that: in GLA4, the RSE1 gene and the RSE2 gene were expressed relatively uniformly on the stigma and style of the pistil of the 4-5cm ear, whereas in NIL-RSE flowers of the same period, the RSE1 gene and the RSE2 gene were strongly expressed in the brush-promoting region (SBP) of the anterior segment of the pistil stigma at a significantly higher level than in the brush-not-stigma region (SNBP) and also significantly higher than in the brush-promoting region (SBP) of the anterior segment of the stigma in GLA4 at the corresponding developmental stage. In NIL-RSE near isogenic lines, strong expression of the RSE1 gene and RSE2 gene through the brush-growing region (SBP) of the anterior column head may affect the growth direction or growth rate of the column head resulting in the stigma-exposed phenotype.

Combining the previous glume dissection experiment result, the statistical result of the stigma length and angle and the in-situ hybridization experiment result, the specific expression mode of the RSE gene on the stigma is considered to finally influence the development of the stigma and lead to the exposure of the stigma.

Domestication analysis of RSE Gene

During the cloning of the RSE1 and RSE2 genes at the map position, the allele from the common wild rice W1943 contributed to the stigma exposure phenotype, whereas the allele from GLA4 did not cause, or caused only very low levels of stigma exposure. To study RSE1 ^W And RSE2 ^W Evolved into RSE1 ^G And RSE2 ^G If it is manually selected, we selected 39 parts of modern cultivated rice material and 14 parts of ordinary wild rice material which are deeply sequenced to perform comparison analysis of the full-length genome sequences of RSE1 gene and RSE2 gene. Multiple alignments were performed using cluster-W and the results were modified with MEGA alignment, followed by Tajima's D neutral selection of the modified alignment using DnasP-v6, results are shown in Table 1.

Tajima's D assay of Table 1, RSE1 Gene and RSE2 Gene

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

The results show that the Tajima's D value of the RSE1 gene in the cultivated rice population is-2.6596, which shows that the number of haplotypes is less than the number of polymorphic sites, rare alleles exist at high frequency, and the average heterozygosity of the population is lower and selective elimination occurs; negative neutral selection hypothesis of p-value <0.001 suggests that the RSE1 gene is targeted for selection in the 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 selectively cleared in the population; the p-value >0.1 receives a neutral selection hypothesis, indicating that allelic variation of the RSE1 gene in the wild rice population is caused by random mutation and is subject to balanced selection. As with the RSE1 gene, the Tajima's D assay showed that the RSE2 gene was selected in balance in the wild rice population and targeted in the cultivated rice population.

To verify the Tajima's D detection results, we used MEGA to detect the nucleic acid diversity of RSE1 and RSE2 genes in the oryza sativa populations and the wild oryza sativa populations, respectively, the results are shown in table 2.

Table 2, analysis of nucleic acid diversity of RSE1 Gene and RSE2 Gene

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Wherein: m, the number of sequences; s, number of polymorphic (isolated) sites; ps, S/n, n is the sequence length; pi, nucleic acid diversity.

Pi in table 2 ^W Nucleic acid diversity representing wild rice populations, pi ^C Nucleic acid diversity representing a cultivated rice population. Calculation results show that pi of RSE1 gene ^W /Π ^C N of RSE2 Gene = 7.765 ^W /Π ^C = 9.5348, all significantly higher than the commonly used pi ^W /Π ^C Threshold 3 or 3.5. The above results demonstrate that the nucleic acid diversity of both RSE1 gene and RSE2 gene is significantly reduced from the wild rice population to the cultivated rice population, and that they are strongly domesticated and selected.

In summary, both the RSE1 gene and the RSE2 gene are strongly selected artificially and are domesticated genes.

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

Reference to the literature

Huang,X.,et al.A map of rice genome variation reveals the origin of cultivated rice[J].Nature,2012,490(7421):497-501.

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.

Yan,W[J].G.,et al.Association mapping of stigma and spikelet characteristics in rice(Oryza sativa L.)[J].Mol Breed,2009,24(3):277-292.

Sequence listing

<110> molecular plant science Excellent innovation center of China academy of sciences

<120> gene for regulating and controlling rice stigma exposure 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 (6)

1. A gene for regulating and controlling rice stigma exposure is characterized in that the nucleotide sequence of the gene is SEQ ID NO. 1 or SEQ ID NO. 2.

2. A vector comprising the gene of claim 1.

3. The vector of claim 2, wherein the backbone plasmid of the vector is the pCAMBIA series.

4. The vector of claim 3, wherein the backbone plasmid of the vector is pCAMBIA1300.

5. An agrobacterium, wherein the agrobacterium is transformed with a vector according to any of claims 2-4.

6. Use of the gene according to claim 1, the vector according to any one of claims 2 to 4, or the agrobacterium according to claim 5 in rice cross breeding, wherein the gene according to claim 1 is integrated into the genome of cultivated rice using plasmid transformation, homologous recombination techniques or gene editing techniques; the rice crossbreeding is used for improving the stigma exposure level.