Cloning method and application of oryza longistaminata interspecific hybrid sterile gene S44

Abstract

The invention discloses a cloning method and application of an interspecific hybrid sterile gene S44 of oryza longistaminata, two candidate genes HUO and SHUI at the site S44 are obtained by a map-based cloning method, the HUO gene in oryza longistaminata is knocked out to obtain a homozygous mutation line with normal self fertility, and the mutation line is hybridized with an Asian cultivated rice parent to obtain the sterile gene S44 of the oryza longistaminata. The S44 locus mediated hybrid sterility effect is eliminated, and mutant hybrids with normal fertility are obtained; or the SHUI gene in the oryza longistaminata is transferred into the hybrid F1 plant, so that the pollen fertility of the transgenic plant is recovered to be normal; the DNA fragment of the oryza longistaminata pollen fertility gene HUO is as shown in SEQ ID NO: 1, and the protein fragment is as shown in SEQ ID NO: 3; the DNA fragment of the Oryza longistaminata pollen fertility gene SHUI is as shown in SEQ ID NO: 2, and the protein fragment is as shown in SEQ ID NO: 4. According to the invention, the gene locus S44 capable of effectively controlling the fertility disorder of interspecific hybrids of oryza longistaminata and Asian cultivated rice is separated, and the gene locus S44 is applied to overcome the fertility disorder of the interspecific hybrids of oryza longistaminata and Asian cultivated rice.

Description

Technical Field

[0001] The invention relates to the technical field of rice gene engineering, and in particular to a method for separating and cloning rice hybrid pollen fertility genes and an application thereof in rice hybrid breeding. Background Art

[0002] Rice is one of the most important food crops in the world and plays a pivotal role in food security. Due to the repeated use of a few core parents by breeders, the gene pool of rice cultivars has become homogenous, and rice yields have stagnated. Wild rice has undergone long-term natural selection and survival competition, and has rich genetic variation. It is a favorable gene pool for genetic improvement of cultivated rice. Longistaminata wild rice has characteristics such as long stigma, large anther, self-incompatibility, cross-pollination and underground stems, as well as many excellent characteristics of resistance to biotic and abiotic stresses. It is one of the excellent gene pools for improving Asian cultivated rice. However, there is serious hybrid sterility between longistaminata wild rice and Asian cultivated rice. The hybrid F1 shows severe embryo abortion, which seriously hinders the utilization of distant hybrid advantages of rice. At present, there is no effective method to overcome the fertility barrier of interspecific hybrids between Asian cultivated rice and longistaminata wild rice.

[0003] So far, the loci that have been cloned and reported to regulate hybrid sterility between rice species or subspecies include S5, Sa, Sc, DPL1 / DPL2, Hsa1, S27 / S28, S1, S7, DGS1 / DGS2, qHMS7, qHMS1, Se / Pf12 / qHMS12, etc. (YongyaoXie, Rongxin Shen, Letian Chen & Yao-Guang Liu (2019) Molecular mechanisms of hybrid sterility in rice, Sci China Life Sci 62; Zhe Zhao, Rongxin Shen, Yao-Guang Liu (2023) Hybrid sterility genes with driving force for speciation in rice, Science Bulletin 68 1845–1848). Previous studies have shown that these genes that control hybrid sterility in rice have no effect on fertility in the homozygous state, but affect the normal development of female or male gametes in the heterozygous state. Zhao Jiying et al. (A new gene controlling hybrid sterility between Oryza sativa and Oryza longistaminata, Euphytica, 2012, DOI 10.1007 / s10681-012-0691-3) located a gene controlling male gamete sterility between Asian cultivated rice and longistaminata wild rice at the end of the long arm of chromosome 6 between RM5814 and RM20695 and named it S44. The researchers found that plants with homozygous longistaminata wild rice genotypes and homozygous Asian cultivated rice RD23 genotypes had normal fertile pollen, while when the site was in a heterozygous state, it showed semi-sterile pollen. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects and shortcomings of the above-mentioned existing technologies, isolate a gene locus S44 that can effectively control the hybrid fertility barrier between longistaminata wild wild rice and Asian cultivated rice, and use it to overcome the problem of hybrid sterility between the two mediated by this site.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A method for cloning the interspecific hybrid sterility gene S44 of Oryza longistaminata, using the map-based cloning method to obtain the candidate genes HUO and SHUI of S44, knocking out the HUO gene in Oryza longistaminata to obtain a homozygous mutant line with normal fertility, using this mutant line to cross with Asian cultivated rice parents to eliminate the hybrid sterility effect mediated by the S44 locus and obtain mutant hybrids with normal fertility; or transferring the SHUI gene in Oryza longistaminata to hybrid F1 plants to restore the pollen fertility of transgenic plants to normal;

[0007] The DNA fragment of the pollen fertility gene HUO of Oryza longistaminata is shown in the sequence listing as SEQ ID NO: 1, and the protein fragment is shown in SEQ ID NO: 3;

[0008] The DNA fragment of the pollen fertility gene SHUI of Oryza longistaminata is shown in the sequence listing as SEQ ID NO:2, and the protein fragment is shown in SEQ ID NO:4.

[0009] Furthermore, a HUO gene knockout vector is transformed into a hybrid F1 plant by an Agrobacterium-mediated genetic transformation method, or a complementary expression vector carrying a SHUI gene is transformed into a hybrid F1 plant by an Agrobacterium-mediated genetic transformation method, both of which can cultivate wide-compatibility varieties.

[0010] Furthermore, the Asian cultivated rice parent is the Thai indica rice variety RD23, the near-isogenic line (NIL-S44) is a material containing the longistaminata wild rice S44 gene fragment in the RD23 background, and the hybrid F1 is an F1 generation plant of the hybrid of RD23 and the near-isogenic line.

[0011] The interspecific hybrid sterility gene S44 of Oryza longistaminata cloned by the method of the present invention is applied to rice breeding.

[0012] The present invention identifies a candidate gene controlling hybrid sterility between Oryza longistaminata and Asian cultivated rice in the localization interval of the interspecific hybrid sterility genetic locus S44 between Oryza longistaminata and Asian cultivated rice through positional cloning, gene prediction and expression analysis, and clarifies the killer-protector role of the closely linked gene HUO-SHUI through gene editing technology and functional complementation experiments. HUO performs the function of a killer in the hybrid sterility system, and SHUI performs the function of a protector in the system. At the same time, a molecular marker for detecting the S44 locus specificity is developed. The cloning of this gene effectively overcomes the problem of hybrid sterility, making it possible to utilize the hybrid advantage between Asian cultivated rice and Oryza longistaminata, and is expected to further increase the yield of hybrid rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram for the construction of near-isogenic lines and the genotypes of the constructed near-isogenic lines;

[0014] Figure 2 The identification and cloning process of S44 is shown;

[0015] Figure 3 It is a protein sequence comparison analysis between Oryza longistaminata HUO and Nipponbare HUO;

[0016] Figure 4 It is a comparison analysis of protein sequences of Oryza longistaminata SHUI and Nipponbare SHUI;

[0017] Figure 5 It is the functional verification status of HUO and SHUI;

[0018] Figure 6 is the expression pattern analysis of HUO and SHUI;

[0019] Figure 7 is RNA in situ hybridization analysis;

[0020] Figure 8 The application of S44 locus-specific molecular marker is shown. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment in the art.

[0022] The present invention relates to the separation and use of genes related to the hybrid fertility gene S44, which controls the pollen fertility of hybrids produced by interspecific hybridization between Oryza longistaminata and Asiatic cultivated rice.

[0023] Example 1

[0024] The method for cloning the interspecific hybrid sterility gene S44 of wild rice is to obtain the candidate genes HUO and SHUI of S44 by using the map-based cloning method, and to knock out the HUO gene in wild rice to obtain a homozygous mutant line with normal fertility. This mutant line is used to cross with Asian rice parents to eliminate the hybrid sterility effect mediated by the S44 locus, and obtain mutant hybrids with normal fertility. The specific method is as follows:

[0025] 1. Construction of near-isogenic lines

[0026] After hybridization with Oryza longistaminata as the donor parent and RD23, an Asian cultivated rice variety from Thailand, as the recurrent parent, the young embryo rescue technique was used to obtain F1 plants, which were then backcrossed with RD23 as the male parent to form a BC1F1 population. Pollen fertility was examined under a microscope, and sterile plants with a pollen fertility of about 50% were selected in each generation and continuously backcrossed with RD23 to BC6F1. The genotype of the BC6F1 sterile plants was evaluated using SSR primers, and the hybrid sterility QTL carried in the population was detected in combination with phenotypic data. Individual plants carrying the sterile loci of Oryza longistaminata and other segments of which were as close to the RD23 background as possible were selected for self-pollination for multiple generations to form BC6F 10 This line was then backcrossed with RD23 to form a BC7F2 population, and the material with only the introgression of the long arm of chromosome 6 with the Oryza longistaminata genome fragment at the end was selected as a near-isogenic line (NIL-S44), see Figure 1 The hybrid F1 used in the present invention is a plant produced by crossing the parents RD23 and NIL-S44. Figure 1 In the figure, a is a schematic diagram of the construction of near-isogenic lines; b is the genotype of the constructed near-isogenic lines, where the gray strips represent the RD23 background and the black strips represent the introgression of the Oryza longistaminata genome.

[0027] 2. Fine positioning of S44 and determination of target genes

[0028] The method for obtaining the S44 gene using the map-based cloning method is as follows:

[0029] First, the constructed near-isogenic line of BC7F2 was hybridized with the recurrent parent RD23 to obtain the primary fine-mapping population of BC8F2, from which the heterozygous strains were selected for self-pollination to obtain the secondary fine-mapping population of BC8F3, and a total of 13,298 strains of genetic mapping population were obtained;

[0030] Then, the genome sequences of "Nipponbare" and the indica variety "93-11" were compared to look for differential sequences, and 4 pairs of Indel markers were designed that were polymorphic between the two parents. The genome sequence of Nipponbare was then compared with the genome sequence of Oryza longistaminata, and 18 pairs of OL series InDel markers were designed, of which 9 pairs were polymorphic between the two parents.

[0031] A total of 13 pairs of polymorphic markers were used to perform genotype detection on 13,298 individual plants in the positioning population, and exchange plants were selected according to the genotype for pollen fertility analysis. The pollen detection method is as follows: 5-10 lemmas that are going to bloom on the same day were collected from the branches and stalks in the middle and upper part of the main panicle of a single plant before flowering, and fixed in 70% ethanol. Stained with 1% I2-KI solution, the pollen fertility was divided into three categories under a 160× ordinary optical microscope: empty abortion (typical abortion and round abortion), dyed abortion, and fertile, and the number of pollen of the three categories was recorded respectively. Three fields of view were observed on each piece, and the percentages of the three types of pollen were calculated for each plant;

[0032] Finally, combined with genotypic and phenotypic analysis, S44 was located within the range of 29.6 kb between OL3019-2 and OL3025-1, with 6 and 1 recombinant plants with the markers on both sides, respectively. The Rice Genome Annotation Project (http: / / rice.plantbiology. msu.edu) website was used to compare the genes between OL3019-2 and OL3025-1. The results showed that there were 3 open reading frames in this region of longistaminata wild rice, named MU, HUO, and SHUI, while the RD23 genome had only 1 open reading frame in this region, which was an allele with MU and named JIN, see Figure 2 . Figure 2 In the figure, a is the phenotype of RD23, NIL-S44 and F1, scale bar = 10 cm; b is the iodine-stained image of RD23, NIL-S44 and F1 pollen, red arrows indicate aborted pollen, green arrows indicate fertile pollen, scale bar = 10 μm; c is the quantitative image of pollen fertility and spikelet fertility of RD23, NIL-S44 and F1; d is the analysis of gamete transmission rules of reciprocal crosses between F1 and RD23 and NIL-S44 respectively; e is the positional cloning map of S44 locus; f is the gene structure map of JIN and MU.

[0033] The DNA fragment of the pollen fertility gene HUO of Oryza longistaminata is shown in the sequence listing as SEQ ID NO:1, and the protein fragment is shown in SEQ ID NO:3.

[0034] The DNA fragment of the pollen fertility gene SHUI of Oryza longistaminata is shown in the sequence listing as SEQ ID NO:2, and the protein fragment is shown in SEQ ID NO:4.

[0035] Table 1. Lists the primer sequences used for S44 fine mapping.

[0036] Table 1. Primer sequences used for S44 fine mapping

[0037]

[0038] 3. Comparative analysis of HUO and SHUI sequences

[0039] The amino acid sequences of the two genes were compared and analyzed using DNAMAN software.

[0040] Figure 3 The results of the protein sequence comparison of Oryza longistaminata HUO and Nipponbare HUO are shown. The results of the HUO amino acid sequence comparison showed that the amino acid sequences of Nipponbare and NIL-S44 had 9 amino acid mutations in the exon region. In addition, two bases were inserted into the HUO genome of NIL-S44 at the 975bp position, resulting in a frameshift in the translation of the 3' end protein of HUO. After multiple amino acid mutations occurred in the protein sequence of NIL-S44 HUO, a new Bcr-Abl_Oligo domain was generated, which is involved in the serine / threonine protein kinase activity and the catalytic activity of GTPase.

[0041] Figure 4 The results of the protein sequence comparison between SHUI and Nipponbare are shown. The results of the SHUI amino acid sequence comparison showed that the proteins of Nipponbare and NIL had 4 amino acid mutations.

[0042] 4. Verify the genetic function of HUO

[0043] The HUO gene contained in the S44 locus exists in NIL-S44, but not in RD23. The function of the HUO gene was verified from the fertility phenotype of the transgenic plants by transforming the HUO gene fragment in NIL-S44 into F1 plants or knocking out the HUO gene in the F1 background.

[0044] The construction and genetic transformation method of the complementary vector are as follows: the total DNA of NIL-S44 is extracted by the CTAB method, and the primers F:ccagctgcagAAGGTTGGTTGGTGAGTTTGAGAAG and R:TCAATCAGACTTCAAAAGGCA AACA are used to amplify the HUO gene from NIL-S44 by PCR technology. The PCR reaction of this embodiment uses the high-fidelity enzyme KOD fx enzyme (Toyobo Co., Ltd.) and amplifies 30 cycles (94°C 30s, 62°C 30s, 68°C 5min). After digestion with PstI and BstEII, it is connected to pCAMBIA1305. The positive plasmid is transferred into the Agrobacterium competent cell EHA105, and F1 is transformed by the Agrobacterium tumefaciens-mediated method (Hier et al., Plant J.6:271-282).

[0045] The present invention obtained a total of 31 independent transgenic T0 rice plants, all of which were positive. Pollen was taken from the positive transgenic plants at heading and stained with 1% I2-KI for observation. All strains showed high sterility (pollen fertility <20%). Analysis of three independent T1 transgenic families showed that the ratio of RD23 genotype offspring was significantly lower than that of the control, and the offspring segregation was more serious, which indicated that the gene could affect the fertility of hybrid pollen.

[0046] With "ATCGGATGGGGTGGATCCACCGG" as the target, the CRISPR-Cas9 gene knockout vector of HUO was constructed and transformed into F1 plants. The genetic transformation method was the same as above. After PCR amplification and sequencing identification (F: GCATATCCGTCCGATCAGT; R: GCCACGTTCTC CATAAGTTG CGG), two homozygous lines had a deletion of 1 "G" and an insertion of 1 "T" at 88bp in the coding region, respectively. Pollen from the two homozygous knockout lines was taken for I2-KI staining, and it was found that the pollen fertility was restored to normal. Three T1 generation groups of 114, 239 and 304 plants were selected from them, and it was found that the gamete segregation of the three lines was restored to 1:2:1, see Figure 5 .

[0047] The above verification results indicate that the HUO gene in the present invention is involved in the development of male gametes in the interspecific hybrid offspring of Asian cultivated rice and longistaminata wild rice. Knocking out the HUO gene in NIL-S44 using gene editing technology can artificially create a wide-compatibility line, which can then be hybridized with Asian cultivated rice to effectively break the interspecific hybrid sterility.

[0048] 5. Verify the functionality of SHUI

[0049] The SHUI gene contained in the S44 site exists in NIL-S44, but not in RD23. The present invention adopts the method of transforming the SHUI gene fragment in NIL-S44 into F1 plants, or knocking out the SHUI gene in the NIL background, and verifies the function of the SHUI gene from the fertility phenotype of the transgenic plants.

[0050] The construction of complementary vector and genetic transformation method are: utilize the method for CTAB to extract the total DNA of NIL-S44, use primer F:ccagctgcagAAGGTTGGTTGGTGAGTTTGAGAAG and R:ccctgtcgacTCTCGCCTCACACTCTCACTCTCCC, use PCR technology to increase SHUI gene from NIL-S44.The present embodiment PCR reaction uses high-fidelity enzyme KOD fx enzyme (Toyobo Co., Ltd.), and increases 30 cycles (94 ℃ 30s, 62 ℃ 30s, 68 ℃ 7min).After cutting with SalI and BstEII, be connected among the pCAMNIA1300.Positive plasmid is transferred among the Agrobacterium competent cell EHA105, and transforms F1 by Agrobacterium tumefaciens mediated method (Hier etc., Plant J.6:271-282).

[0051] A total of 3 independent transgenic lines were analyzed, and the pollen fertility of the T0 generation transgenic lines was restored to 72.63%, and the pollen fertility of the T1 generation transgenic homozygous plants was restored to normal. Analysis of three T1 generation populations of 89, 196 and 208 strains found that the segregation ratio of RD23 type, heterozygous type and NIL-S44 type gametes met the ratio of 1:2:1. This shows that the SHUI gene in the present invention can restore hybrid pollen fertility.

[0052] The method of knocking out the SHUI gene is: first, using "agtcgtcgctgagcacacgatgg" and "gacctccaagtcctcaagactgg" as targets, respectively, and using gene editing methods to knock out the SHUI gene in the NIL-S44 background, and sequencing the obtained transgenic plants (F:CAAACTACTTCTGCGTAATAACAATA / R:CTAGAACAGCCCCTAAAATCCA;F:TTCTATAAAGCCACACTCGG / R:ATTTGAACAGCTAAGTGAGC), it was found that the pollen fertility phenotype of the two knockout strains in the heterozygous state was completely sterile. Subsequently, the HUO and SHUI genes were knocked out at the same time in the F1 background, and the test found that the sports fertility of the huoshui double mutant was restored to normal, and the segregation ratio of the three double mutant offspring also conformed to the Mendel segregation ratio, see Figure 5 .

[0053] The above verification results indicate that the exogenous SHUI gene transformed in the present invention can overcome the hybrid fertility barrier between Asian cultivated rice and Oryza longistaminata at this site.

[0054] Figure 5In the figure, a is the restoration of pollen fertility by knocking out HUO gene in T0 generation plants, scale bar = 100 μm; b is the quantitative analysis of pollen of HUO knockout gene; c is the segregation ratio of the three genotypes in the offspring of HUO knockout gene plants; d is the introduction of exogenous HUO gene into F1 plants resulting in high pollen sterility; e is the quantitative analysis of pollen fertility of F1 plants after the introduction of exogenous HUO gene; f is the exogenous HUO gene causing aggravated skewed segregation of offspring; g is the phenotype of pollen fertility restoration by introducing exogenous SHUI gene into F1 plants, scale bar = 100 μm; h is the quantitative analysis of pollen fertility of F1 plants after the introduction of exogenous SHUI gene; i is the restoration of normal Mendelian segregation ratio of offspring by exogenous SHUI gene; j is the mutant plant of double knockout HUO / SHUI stained with I2-KI, scale bar = 100 μm; k is the pollen fertility of the huoshui double mutant; l is the phenotype of gamete segregation distortion in hybrid offspring that can be restored by simultaneous knockout of HUO / SHUI.

[0055] 6. Analysis of S44 expression pattern

[0056] Real-time quantitative PCR and RNA in situ hybridization were used to detect the expression of HUO and SHUI genes in various tissues. The primer sequences used for S44 expression pattern analysis are shown in Table 2. In the NIL-S44 background, HUO and SHUI were not expressed during the vegetative growth period, but only during reproductive growth; however, in the F1 background, both genes were expressed in various tissues, as shown in Table 2. Figure 6 . Figure 6 In the figure, a is the expression pattern analysis of HUO; b is the expression pattern analysis of SHUI; c is the expression of HUO and SHUI in anther, scale bar = 200 μm; d is the expression of HUO and SHUI in pollen, scale bar = 10 μm.

[0057] Real-time quantitative PCR was performed according to the experimental method of Biorad (pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, 40 cycles; melting curve program, within the range of 65-95°C, increasing by 0.5°C every 2-5 s), see Figure 6 .

[0058] Table 2 Primer sequences used for expression pattern analysis

[0059]

[0060] Figure 7 RNA in situ hybridization analysis is shown. Figure 7In the figure, a is the expression pattern analysis of HUO in S6, S8 and S10, scale bar = 50 μm; b is the expression pattern analysis of SHUI in S6, S8 and S10, scale bar = 50 μm. S6: pollen mother cell stage; S8: meiosis stage; S10, dinucleate stage.

[0061] RNA in situ hybridization results showed that JIN / MU, HUO, and SHUI were highly expressed in the tapetum and microspores.

[0062] The RNA in situ hybridization method is as follows:

[0063] (1) Fixation and embedding of materials

[0064] 1) Fixation: Fix the florets of the parent RD23 at various developmental stages in FAA solution, vacuum the solution to make the material sink below the liquid surface, then replace with new FAA solution and place at 4°C overnight;

[0065] 2) Dehydration: The fixed material was dehydrated in ethanol gradient, 1 h per step;

[0066] 3) Transparency: The dehydrated material was transparentized by passing through a gradient solution of xylene / ethanol with a volume ratio of 1:3, 1:1 and 3:1, each step for 1 h; then, transparentized with pure xylene 3 times, each time for 1 h;

[0067] 4) Embedding: slowly pour the paraffin (Sigma) melted at 65°C into the xylene solution containing the material, so that the volume ratio of paraffin and xylene is 1:1, and place it at 42°C for 1 day; continue to add pure paraffin, so that the volume of paraffin and xylene is 2:1, and place it at 42°C for 3-4 h; then transfer the material to a 60°C incubator, replace it with pure paraffin, and place it at 60°C for 2 days, during which time the pure paraffin is replaced 4 times; pour the infiltrated material and paraffin into a small paper box, arrange the material with tweezers, and after the paraffin cools to room temperature, store it in a 4°C refrigerator for later use.

[0068] (2) Sectioning and spreading

[0069] First, the large wax block containing the material was divided, and then the divided small wax blocks were glued to the rootstock in the horizontal or vertical direction as needed; the small wax block was trimmed into a trapezoid with a blade, and the excess paraffin around the material was removed; the material was cut into 10 μm thick slices, and the slices were placed on paper in order with a brush and a dissecting needle; for microscopic examination, appropriate slices were selected, placed on a poly-lysine coated slide (Sigma), a few drops of RNase-free ddH2O were added, and the slides were placed on a 42°C slide table for 5-10 minutes; when the slices became transparent, the water on the slides was aspirated, and the slides were placed in a 42°C incubator for overnight adhesion.

[0070] (3) Preparation of RNA probe

[0071] 1) Preparation of RNA probe in vitro transcription template: amplify the specific sequence in the target gene cDNA with specific primers, clone into the pGEM-T easy (Promaga) vector, screen the clones with forward and reverse insertions by colony PCR, and sequence them;

[0072] 2) Prepare sense and antisense probes using DIG RNA Labeling Kit (Roche);

[0073] 3) RNA probe semi-quantification:

[0074] Dig labeling RNA control (initial concentration 100 ng / μl) was diluted to 10 ng / μl, 1 ng / μl, 100 pg / μl, 10 pg / μl, 3 pg / μl, 1 pg / μl with RNase-free ddH2O; in vitro transcribed labeled RNA probes were diluted in the following proportions: 1 / 10, 1 / 100, 1 / 1000, 1 / 10000, 1 / 100000. 1 μl of each of the diluted solutions was spotted on a Hybond-N+ nylon membrane (Amersham Biosciences) and baked at 80 °C for 2 h to crosslink RNA. The details are as follows:

[0075] a) Immerse the membrane in 10 ml Washing Buffer (0.3% Tween-20, 0.1 M Maleic acid, 0.15 M NaCl, pH 7.5) and shake gently at room temperature for 2 min;

[0076] b) Discard the Washing buffer, add 10 ml 1× Blocking Solution (1% Block Reagent, 0.1 M Maleic acid, 0.15 M NaCl, pH 7.5), and shake gently at room temperature for 30 min;

[0077] c) Discard 1×Blocking Solution, add 10 ml Antibody Solution (dilute Anti-DIG-AP antibody with 1×Blocking Solution at a ratio of 1:10000), and shake gently at room temperature for 30 min;

[0078] d) Discard Antibody Solution, add Washing Buffer and wash the membrane twice, 15 min each time;

[0079] e) Discard the Washing Buffer, add 10 ml Detection Buffer (0.1 M Tris-Cl, 0.1 M NaCl, pH 9.5), and equilibrate at room temperature for 2-5 min;

[0080] f) Discard the Detection Buffer, add the color developing solution NBT / BCIP (Ameresco), and develop the color in the dark;

[0081] g) Estimate the probe concentration based on RNA control, aliquot into 10 μl / tube, and store at -80 ℃ for future use;

[0082] (4) Pretreatment. All glassware used were baked at 180℃ for 5-6 h, and the solutions were prepared with RNase-free ddH2O. The details are as follows:

[0083] 1) Dewaxing and rehydration: Place the slides with the attached materials into a staining jar and soak them in 100% xylene twice, 20 min each time; then, immerse the slides in xylene / ethanol (3:1), xylene / ethanol (1:3), 100% ethanol, 100% ethanol, 90% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, 10% ethanol, RNase-free ddH2O, and RNase-free ddH2O in sequence, 2 min each step;

[0084] 2) Proteinase K digestion: Preheat proteinase K buffer (100 mM Tris-Cl, 50 mM EDTA, pH 7.5) at 37°C, add proteinase K stock solution (20 mg / ml) to a final concentration of 3 μg / ml; treat the sections at 37°C for 20 min; soak the sections in 37°C RNase-free ddH2O for 5 min, then transfer to room temperature and soak in RNase-free ddH2O for 5 min;

[0085] 3) Acetylation: Add 40 ml RNase-free ddH2O, 536 μl triethanolamine, 160 μl concentrated hydrochloric acid and 100 μl acetic anhydride to the staining jar, stir quickly and put the slide in, keep at room temperature for 10 min; wash twice with 2×SSC, 5 min each time;

[0086] 4) Dehydration: Immerse the slide in RNase-free ddH2O, RNase-free ddH2O, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol, 100% ethanol, and 100% ethanol in sequence, 2 min per step; take out the slide, place it in an empty staining jar, and dry it at room temperature for 0.5~1 h before using it for hybridization.

[0087] (5) Hybridization

[0088] Drop 100-150 μl of hybridization solution (RNA probe concentration is 0.5-5 ng / μl) on each slide, cover with a parafilm of appropriate size, gently cover from one side, and flatten the hybridization solution without creating bubbles; put the slide into a wet box (lined with absorbent paper saturated with 0.3 M NaCl + 50% formamide), seal it, and hybridize overnight at 42°C.

[0089] (6) Film development

[0090] 1) Wash off the hybridization solution: wash 4 times with 4×SSC at 55°C, 10 min each time;

[0091] 2) RNase A treatment to remove unbound probes: Preheat RNase buffer (500 mM NaCl, 10 mM Tris-Cl, 1 mM EDTA, pH 7.5) at 37°C, add RNase A to a final concentration of 30 μg / ml; place the slide in it and treat at 37°C for 30 min, then wash the slide twice with RNase buffer at 37°C, 15 min each time;

[0092] 3) Wash away non-specifically bound probes: wash the slides at 55°C with 2×SSC, 2×SSC, 0.5×SSC, and 0.5×SSC, for 30 min each time.

[0093] (7) Antibody binding and color development

[0094] 1) Blocking of antibody nonspecific sites: Place the slide in Buffer I (150 mM NaCl, 100 mM Tris-Cl, pH 7.5) at room temperature for 2 times, 5 min each time; then, immerse the slide in Buffer I containing 0.5% Blocking Reagent at room temperature for 30 min; then immerse the slide in Buffer II (0.3% Tween-20, 150 mM NaCl, 100 mM Tris-Cl, pH 7.5) containing 1% BSA (Takara) at room temperature for 30 min;

[0095] 2) Antibody binding: Add 150 μl of antibody solution to each slide, cover with parafilm to avoid bubbles, place in a wet box (lined with absorbent paper saturated with Buffer II), and incubate at room temperature for 3 h.

[0096] 3) Wash away unbound antibodies: Wash three times with Buffer I at room temperature, 10 min each time; then, immerse the slide in Buffer III (100 mM NaCl, 50 mM MgCl;

[0097] 4) Color development: Immerse the slide in NBT / BCIP solution and leave it at room temperature in the dark overnight or longer;

[0098] 5) Microscopic examination and photography: Wash the developed slides three times with ddH2O at room temperature, 5 min each time; remove the slides, remove excess water from the surface, add 50 μl 20% glycerol to each slide, cover with a coverslip, and directly examine and photograph under a microscope, or place in a 4°C refrigerator and complete microscopic examination and photography within one week.

[0099] Example 2. Application of S44 marker in molecular marker-assisted breeding

[0100] Table 3 lists the S44 locus-specific molecular markers.

[0101] Table 3 S44 site-specific molecular markers

[0102]

[0103] The present invention developed two pairs of HUO gene-specific markers and two pairs of SHUI gene-specific markers for detecting whether rice materials contain HUO genes and SHUI genes. The presence of these two genes was detected in indica rice (3 / 13), japonica rice (3 / 14), longistaminata wild rice (3 / 3), short-tongued wild rice (5 / 5), common wild rice (2 / 6), Nivara wild rice (2 / 3), and southern wild rice (1 / 1). Figure 8 The results of detecting 27 Asian cultivated rice accessions and 17 wild rice accessions using S44 locus-specific molecular markers are shown. Figure 8 Among them, 920-1 and 920-2 are HUO gene-specific markers; 930-1 and 930-2 are SHUI gene-specific markers.

[0104] The hybrid fertility gene provided by the present invention has important application value. One of the applications of the present invention is to use the isolated hybrid fertility gene sequence to cultivate hybrid fertility compatible lines. The present invention provides a method of knocking out the HUO gene by genetic engineering technology such as using gene knockout technology or other effective technologies, and the mutant can be used as a parent to cross with other rice varieties or strains to cultivate hybrid compatible lines. By genetic engineering technology such as transgenic technology or other effective technologies, the SHUI gene is introduced, and the transgenic material is used as a parent and then cross-bred with other materials to obtain hybrid materials with normal fertility.

[0105] According to the present invention, molecular markers can be designed based on the differences in the two alleles HUO and SHUI. The molecular markers can amplify target bands for materials containing HUO and SHUI genes. However, materials without the two genes have no target bands. The molecular markers can be used to detect parents, hybrid offspring and transgenic materials, and can also be used for molecular marker-assisted breeding.

[0106] Another application of the hybrid fertility gene provided by the present invention is to generate specific molecular markers according to the gene sequence information, including SNP and Indel markers, etc. These markers can be used to identify whether wild rice or cultivated rice contains functional S44 sites, providing guidance for parent selection.

[0107] Using these specific molecular markers, we can breed compatible lines of cultivated rice with wild rice or wild rice with cultivated rice. These compatible lines can be hybridized with other wild rice or cultivated rice to produce hybrids with normal pollen fertility, which is conducive to the breeding of distant hybrid varieties with strong advantages.

[0108] Sequence Listing SEQ ID No: 1 shows the DNA fragment sequence of the HUO gene coding region isolated and cloned in the present invention.

[0109] >HUO CDS

[0110]

[0111] Sequence Listing SEQ ID No: 2 shows the DNA fragment sequence of the SHUI gene coding region isolated and cloned in the present invention.

[0112] >SHUI CDS

[0113]

[0114] Sequence Listing SEQ ID No: 3 shows the HUO protein sequence isolated and cloned in the present invention.

[0115] >HUO AA

[0116] MDPPQRIFGAASSPPIGIPPTSIAAPVDPPHPIFGAASSSPIAHRSLLPTPSVATAVAVQVDPPPPHSESELVQALSDKYRLPVRFLLSSIPPTYLSAWELSDGKAQGLVWSHSDALDITHHYQSLGLHEIRFLYSEFISLRCVQDMEYMIARTPFSMRVLNPDVAKEKSRML SKLNGYCCSLREALRQVQAGGNLVEMFNLRSKFAQEHELCTCRTELLRQMKVDALNRVLVLGLGEGASSAHSADMIWILKHHRPDEYAVAVNDDGTVSWPIIHGLVQNIRNMMGNKLTSNDTLANSRTSLLGVIGRFQFEHKVQEVLEKTRSHSTVHDRSYFKIPGCHGYQVSA

[0117] Sequence Listing SEQ ID No: 4 shows the SHUI protein sequence isolated and cloned in the present invention.

[0118] >SHUI AA

[0119] MENTMAAKRSWMEHHRVLSDDSSFHMEDLPEQQPQLMENVAVAKRSRLEQHRTLSDNSFRVEDLPEEIQSLLLSLLSLKEAASTSIVSRNWRKLWTRYPNLCFDGSKDGPADMDSVKIERMKFIDTVNSIIQQHSGIGLNKFSIRCNLLKDDSDILDRWIRFATASKAKIIDMNLCTNRNNKGPTKHLYDFPLEAFGDQDIPFIQCLFLNNVSIKPHSDIGFTKLRSLHLHCVQIIGDLSGLLFNCSSLEDLEVFACLGVTALNIPHQLNKLRHLLICNMRIQMLEFHVPGLSHFEYKGTMIPIMLHGCSKLQKATLNFHQTWLEEDNNKVLGHVFHGIPSVSAVEVLNLLVDICTKQSVWSSQVHTLTARPTIMFMNLKHLTYEILIFTKDPNSHSGVLQLAQYLAFAPQLETLELHMLYHSTHCRCWHEGAGVSYGHMPHHHLKMVYMSGFRCYRAQVELLFAILEMGDELEHVTIDPMTRVPYSPDLMNLGIPEDEICQWANRTSQRFGKAISVVKPP

Claims

1. The interspecific hybrid sterility gene of wild rice S44 The cloning method is characterized in that The positional cloning method was used to obtain S44 Candidate genes HUO and SHUI , the wild rice HUO Gene knockout was performed to obtain a homozygous mutant line with normal fertility. This mutant line was hybridized with the Asian cultivated rice parent to eliminate S44 locus-mediated hybrid sterility effect to obtain mutant hybrids with normal fertility; or SHUI The gene was transferred into hybrid F1 plants, restoring the pollen fertility of the transgenic plants to normal; The pollen fertility gene of Oryza longistaminata HUO The DNA fragment is shown in the sequence listing as SEQ ID NO: 1, and the protein fragment is shown in SEQ ID NO: 3; The pollen fertility gene of Oryza longistaminata SHUI The DNA fragment is shown in SEQ ID NO:2, and the protein fragment is shown in SEQ ID NO:

4.

2. The interspecific hybrid sterility gene of Oryza longistaminata as claimed in claim 1 S44 The cloning method is characterized in that Will carry the HUO The gene knockout vector is transformed into hybrid F1 plants through Agrobacterium-mediated genetic transformation, or the gene knockout vector is transformed into hybrid F1 plants through Agrobacterium-mediated genetic transformation. SHUI The complementary expression vector of the gene is transformed into hybrid F1 plants through Agrobacterium-mediated genetic transformation, and wide-compatibility varieties of the site can be cultivated.

3. The interspecific hybrid sterility gene of Oryza longistaminata according to claim 1 or 2 S44 The cloning method is characterized in that The Asian cultivated rice parent is a Thai indica rice variety RD23, a near isogenic line (NIL- S44 ) is a wild rice containing Oryza longistaminata in the RD23 background S44 The material of gene fragments, hybrid F1 is the F1 generation plant of hybridization between RD23 and near isogenic line.

4. The interspecific hybrid sterility gene of Oryza longistaminata cloned by the method of claim 1, 2 or 3 S44 Application in rice breeding.

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