Rice photo-thermo sensitive genic male sterile line breeding method based on whole genome re-sequencing technology

Abstract

The invention provides a rice photo-thermo sensitive genic male sterile line breeding method based on a whole genome re-sequencing technology, and belongs to the technical field of rice breeding. The rice photo-thermo-sensitive genic male sterile line breeding method based on the whole genome re-sequencing technology comprises the following steps: hybridizing sterile line rice red 35S serving as a male parent and photo-thermo-sensitive genic male sterile line rice serving as a female parent to obtain filial generations; carrying out fertility identification on the offspring to obtain a photo-thermo-sensitive genic male sterile offspring; extracting DNA (deoxyribonucleic acid) of the male parent, the female parent and the photo-thermo-sensitive genic male sterile offspring, and performing whole genome re-sequencing; analyzing the sequencing data by using a bioinformatics method to obtain SNP and INDEL information; selecting filial generation planting according to SNP and INDEL information, and performing fertility identification to obtain the rice photo-thermo sensitive genic male sterile line. The method provided by the invention overcomes the defects of long period, low efficiency and high cost of the traditional breeding method, can accurately locate the sterile character related gene, and greatly improves the breeding efficiency and the success rate.

Description

Technical Field

[0001] The invention belongs to the technical field of rice breeding, and in particular relates to a method for breeding rice photothermosensitive genic male sterile lines based on whole genome resequencing technology. Background Art

[0002] Rice is one of the important food crops. With the continuous growth of population and the reduction of cultivated land, increasing rice yield has become a top priority. Hybrid rice has become an important means to increase rice yield due to its significant hybrid vigor. In the process of hybrid rice breeding, the selection of male sterile lines is crucial. Photothermosensitive nuclear male sterile line (PTGMS) is a type of nuclear cytoplasmic male sterile line affected by light and temperature. It has the advantages of convenient fertility conversion and high seed purity, and has been widely used in hybrid rice breeding. However, the traditional PTGMS breeding method has problems such as long cycle, low efficiency and high cost, which can hardly meet the needs of modern breeding.

[0003] In recent years, the rapid development of high-throughput sequencing technology has provided a powerful tool for crop genomics research. Whole genome resequencing technology can comprehensively and quickly obtain genetic variation information of organisms, providing new ideas and methods for crop breeding. However, the application of whole genome resequencing technology to rice PTGMS breeding is still relatively limited.

[0004] Therefore, developing a method for selecting photothermosensitive nuclear male sterile lines of rice based on whole genome resequencing technology is of great significance for improving breeding efficiency, shortening breeding cycle and reducing breeding costs. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for breeding photothermosensitive genic male sterile lines of rice based on whole genome resequencing technology, which has the advantages of high breeding efficiency, short breeding cycle and low breeding cost.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for breeding a photo-thermo-sensitive genic male sterile rice line based on whole genome resequencing technology, comprising the following steps: hybridizing a sterile rice line Hong 35S as a male parent and a photo-thermo-sensitive genic male sterile rice line as a female parent to obtain offspring; performing fertility identification on the offspring to obtain photo-thermo-sensitive genic male sterile offspring; extracting DNA from the male parent, the female parent and the photo-thermo-sensitive genic male sterile offspring, and performing whole genome resequencing; analyzing sequencing data using a bioinformatics method to obtain SNP and INDEL information; selecting offspring for planting according to the SNP and INDEL information, performing fertility identification, and obtaining a photo-thermo-sensitive genic male sterile rice line.

[0008] Preferably, the offspring include the first generation, the second generation and the third generation.

[0009] Preferably, the second generation is obtained by self-pollination of the first generation, and the third generation is obtained by self-pollination of the second generation.

[0010] Preferably, the fertility identification includes at least one of anther identification, pollen viability determination, self-pollination fruit set rate determination and artificial pollination fruit set rate determination.

[0011] Preferably, the anther identification is to observe the color of the anther after determining the pollen fertility conversion period, and if the anther color remains yellow-green and does not change to yellow-white, or the conversion time is significantly delayed, then the anther is identified as sterile.

[0012] Preferably, the pollen viability is determined to be less than 5% fertile pollen, which is then identified as sterile.

[0013] Preferably, if the self-pollination fruit set rate determination and the artificial pollination fruit set rate determination show that no fruit set occurs or the fruit set rate is less than 1%, the plant is identified as sterile.

[0014] Preferably, the bioinformatics method is to use the bioinformatics analysis platform BMKCloud for processing and analysis.

[0015] Preferably, the processing and analysis include data quality control, sequence alignment, variant detection and variant annotation.

[0016] Beneficial effects of the present invention:

[0017] The method for selecting photothermosensitive nuclear sterile rice lines based on whole genome resequencing technology provided by the present invention overcomes the shortcomings of traditional breeding methods such as long cycle, low efficiency and high cost, can accurately locate genes related to sterility traits, and greatly improve breeding efficiency and success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the changes in pollen vitality of rice in different generations. A is the pollen of F1 generation rice, B is the pollen of F2 generation rice, C is the pollen of F3 generation rice, D is the pollen of female rice, and E is the pollen of male rice.

[0019] Figure 2 This is the distribution diagram of SNP mutation types in the whole genome;

[0020] Figure 3 This is a distribution diagram of the length of insertions and deletions (InDels) detected in the whole genome (Genome) and coding region (CDS). The vertical axis represents the length of InDel, ranging from -10 to +10 base pairs (bp), where positive values ​​represent insertions and negative values ​​represent deletions; the horizontal axis represents the number of InDels of corresponding lengths; if the number of samples exceeds 50, the figure will only display data for 50 samples by default. DETAILED DESCRIPTION

[0021] The present invention provides a method for breeding a photo-thermo-sensitive genic male sterile rice line based on whole genome resequencing technology, comprising the following steps: hybridizing a sterile rice line Hong 35S as a male parent and a photo-thermo-sensitive genic male sterile rice line as a female parent to obtain offspring; performing fertility identification on the offspring to obtain photo-thermo-sensitive genic male sterile offspring; extracting DNA from the male parent, the female parent and the photo-thermo-sensitive genic male sterile offspring, and performing whole genome resequencing; analyzing sequencing data using a bioinformatics method to obtain SNP and INDEL information; selecting offspring for planting according to the SNP and INDEL information, performing fertility identification, and obtaining a photo-thermo-sensitive genic male sterile rice line.

[0022] In the present invention, the photothermosensitive genic male sterile rice is preferably the photothermosensitive genic male sterile rice line 19XS. In the present invention, the progeny preferably include the first generation, the second generation and the third generation. The second generation is preferably obtained by self-pollination of the first generation, and the third generation is preferably obtained by self-pollination of the second generation. In the present invention, when the progeny are subjected to fertility identification, at least one of anther identification, pollen viability determination, self-pollination fruit set rate determination and artificial pollination fruit set rate determination is preferably included. The anther identification is preferably to observe the anther color after determining the pollen fertility conversion period. If the anther color remains yellow-green and does not turn into yellow-white, or the conversion time is significantly delayed, it is identified as sterile. The method for determining the pollen fertility conversion period is preferably: at the heading stage, observe the anther color changes of the mother parent and its hybrid offspring every day, record the date and duration when the anther color changes from yellow-green to yellow-white, and compare the differences between different genotypes at two locations to determine the pollen fertility conversion period. In the present invention, if the pollen viability is determined to be less than 5% of fertile pollen, the plant is identified as sterile; if the self-pollination fruit set rate and artificial pollination fruit set rate are determined to be unable to set fruit or the fruit set rate is less than 1%, the plant is identified as sterile.

[0023] In the present invention, the method for extracting DNA from the male parent, the female parent and the photothermosensitive nuclear sterile progeny is preferably the CTAB method. In order to ensure the quality of DNA, the operation process must be strictly controlled during the extraction process to avoid DNA degradation and contamination. NanoDrop 2000 (Thermo Fisher Scientific) is used to measure the DNA concentration and purity to ensure that it meets the library sequencing requirements. In the present invention, the bioinformatics method is preferably processed and analyzed using the bioinformatics analysis platform BMKCloud. The processing and analysis preferably include data quality control, sequence alignment, variation detection and variation annotation.

[0024] The present invention can help optimize phenotypic screening by analyzing the data of the above-mentioned whole genome resequencing: improve the fertility identification method based on the key trait characteristics discovered in the study. For example, if a specific anther characteristic is highly correlated with sterility, it can be used as the focus of early screening. It can also improve the accuracy of selection: based on the research results, more precise selection criteria can be formulated, which may include specific quantitative requirements for indicators such as pollen fertility, anther morphology, and self-fertility rate.

[0025] The bioinformatics analysis method provided by the present invention utilizes the developed analysis process to perform comparison, variation detection and other analyses on whole genome resequencing data, identify SNPs and InDels related to the traits of photothermosensitive nuclear male sterile rice lines, and can be helpful for the development of molecular markers. For example, the development of molecular markers based on the detected variations will be more helpful for the rapid breeding of photothermosensitive nuclear male sterile rice lines.

[0026] In the present invention, when selecting offspring for planting according to SNP information, selection is made based on the number of SNPs, the ratio of conversion and transversion type SNPs, and the ratio of heterozygous type SNPs. Preferably, offspring whose number of SNPs, the ratio of conversion and transversion type SNPs, and the ratio of heterozygous type SNPs are close to those of the mother are selected for planting; when selecting offspring for planting according to InDel information, selection is made based on the InDel statistical data, the number of insertions, the number of deletions, and the number of homozygous InDels in the coding region. Preferably, offspring whose InDel statistical data, the number of insertions, the number of deletions, and the number of homozygous InDels in the coding region are close to those of the mother are selected for planting.

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] Example 1

[0031] The selected thermosensitive genic male sterile rice line 19XS was used as the female parent. This sterile line showed male sterility in Wuhan (representing the Yangtze River Basin environment) but was fertile in Hainan (representing the South China environment). Another sterile line, Hong 35S, was used as the male parent for artificial hybridization to construct segregating pools for subsequent analysis.

[0032] 1.1 Field trials

[0033] To explore the thermosensitive nuclear male sterility characteristics of 19XS and the fertility segregation of its hybrid progeny with Red 35S, field experiments were conducted in Wuhan City, Hubei Province and Lingshui County, Hainan Province, representing different light and temperature environments.

[0034] Wuhan test site: The sowing time is May 10, the transplanting time is early June, and the planting materials include: 19XS, Red 35S, hybrid first generation (F1), hybrid second generation (F2) and hybrid third generation (F3) plants.

[0035] Hainan test site: The sowing time is late November, the heading time is early March of the following year, and the planting materials include 19XS, Red 35S, F1, F2 and F3 generation plants.

[0036] Each location was set up with 3-5 replicates, and each plot was 10-20 square meters in size. A randomized block design was used to reduce field test errors. Field management measures (such as water and fertilizer management, pest and disease control, etc.) were carried out in accordance with local conventional rice cultivation techniques.

[0037] 1.2 Fertility identification

[0038] In view of the thermosensitive nuclear sterility of 19XS and the fact that both parents are sterile lines, fertility identification is particularly important. The following methods are used for detailed fertility identification. If it is identified as a sterile line by one of the following four methods, it means that the strain is a sterile line:

[0039] (1) Anther observation: During the heading period, observe the anther color changes of 19XS and its hybrid offspring every day. Record the date and duration when the anther color changes from yellow-green to yellow-white, and compare the differences between different genotypes at two locations to determine the period of pollen fertility conversion. If the anther color remains yellow-green and does not change to yellow-white, or the conversion time is significantly delayed, it is judged as a sterile line.

[0040] (2) Pollen vitality determination: Collect fresh anthers from 9:00 to 10:00 a.m. every day during the flowering period and stain them with 1% I2-KI solution. Observe the morphology and staining of pollen grains under an optical microscope, count the number of pollen grains with normal staining and full morphology, and calculate the percentage of fertile pollen to evaluate the fertility level of pollen. If most pollen grains are irregular in shape or abnormally stained, and the percentage of fertile pollen is extremely low, less than 5%, then it is judged as a sterile line. The results are as follows: Figure 1 As shown, dark (black or dark brown) pollen grains indicate viability, while light or blank pollen grains indicate inviability. Figure 1It can be seen that the pollen vitality changes from parents to offspring in different generations. The female parent (D) has almost no viable pollen, which is consistent with the characteristics of the sterile line; the male parent (E) presents a large number of incompletely mature potentially viable pollen; the F1 generation (A) begins to have some viable pollen; the number of viable pollen in the F2 generation (B) and the F3 generation (C) increases significantly and tends to be stable. This series of changes intuitively reflects the inheritance law of photothermosensitive nuclear sterility between generations, and shows the process of gradually restoring fertility from the sterile female parent to the offspring, providing an important phenotypic basis for understanding and utilizing the photothermosensitive nuclear sterile line in rice hybrid breeding.

[0041] (3) Self-pollination fruit set rate determination: 19XS and its hybrid offspring were bagged and self-pollinated to prevent interference from foreign pollen. Bagging was carried out in Wuhan and Hainan respectively, and the fruit set rate of rice ears after self-pollination was counted to evaluate its self-pollination fruit set ability. If the rice ear fruit set rate after bagging and self-pollination is less than 1%, it is judged as a sterile line.

[0042] (4) Artificial pollination: Use 19XS and Hong 35S as female parents and pollen from another variety as male parent for artificial pollination, and observe the fruit setting to determine the sterility type. If no fruit is set or the fruit setting rate is less than 1%, it is determined to be a sterile line.

[0043] 1.3 DNA extraction and sequencing

[0044] In order to understand the genetic mechanism of thermosensitive genic male sterility of 19XS and the fertility segregation rules of hybrid progenies with Red 35S, genome resequencing was performed on 19XS, Red 35S, F1, F2 and F3 generations.

[0045] (1) Sample collection: Young leaves of the 19XS, Hong 35S, F1, F2, and F3 sterile lines determined in step 1.2 were collected, with 3-5 biological replicates for each genotype. The leaves were immediately frozen in liquid nitrogen and then stored in a -80°C freezer until DNA extraction.

[0046] (2) DNA extraction: The genomic DNA of rice leaves was extracted using the modified CTAB method. The DNA concentration and purity were measured using NanoDrop 2000 (Thermo Fisher Scientific). The DNA concentration was between 20 and 100 ng / μL, the 260 / 280 ratio was between 1.8 and 2.0, and the 260 / 230 ratio was between 2.0 and 2.2 to ensure that it met the requirements for library construction and sequencing.

[0047] (3) Library construction and sequencing: Use an ultrasonic DNA shearer to fragment the DNA to about 350 bp and construct a sequencing library. According to the requirements of the Illumina / BGI sequencing platform, the library is quality tested and paired-end sequencing is performed after passing the test. The sequencing read length is 150 bp. The sequencing depth must meet the requirements of subsequent analysis, and the sequencing depth of each genome must not be less than 10×.

[0048] 1.4 Data Analysis

[0049] The raw data obtained by sequencing were processed and analyzed using the bioinformatics analysis platform BMKCloud (www.biocloud.net). The specific steps are as follows:

[0050] 1.4.1 Data Quality Control

[0051] (1) Use fastp software to perform quality control on the raw data in Fastq format, remove low-quality reads generated during sequencing, as well as adapter sequences and sequences containing ploy-N, to improve data quality.

[0052] (2) Calculate indicators such as Q20, Q30, GC content and sequence repetition level to evaluate sequencing quality. Q20 and Q30 refer to the proportion of bases with base quality values ​​greater than or equal to 20 and 30 in the sequencing results. The higher these two indicators are, the better the sequencing quality. GC content refers to the proportion of guanine (G) and cytosine (C) in the DNA sequence, which can be used to evaluate the complexity of the DNA sequence. Sequence repetition level refers to the proportion of repeated sequences in the sequencing data, which can be used to evaluate the quality of library construction and sequencing depth.

[0053] (3) All downstream analyses were performed based on high-quality Cleandata.

[0054] 1.4.2 Sequence Alignment

[0055] (1) Use bwa-mem2 software to align the clean data to the rice reference genome sequence (MSU Rice Genome Annotation Project Release 7 or higher) to obtain the position information of each sample on the reference genome. During the alignment process, appropriate parameters need to be set to ensure the accuracy and efficiency of the alignment.

[0056] (2) After the alignment is completed, use samtools software to sort and index the bam files to facilitate subsequent analysis.

[0057] 1.4.3 Mutation Detection

[0058] (1) Samtools software was used to sort the alignment results and remove the redundant information generated during the PCR process to obtain high-quality alignment results.

[0059] (2) Use GATK software to perform variation detection and identify variation types such as SNP (Single Nucleotide Polymorphism) and INDEL (Insertion and Deletion).

[0060] (3) In order to ensure the accuracy of variation detection, it is necessary to set appropriate filtering parameters according to the actual situation. The present invention uses the following parameters for hard filtering: QD<2.0||MQ<40.0||FS>60.0||QUAL<30.0||MQrankSum<-12.5||ReadPosRankSum<-8.0-clusterSize 2-clusterWindowSize5. After filtering, high-quality SNP and INDEL data are obtained for subsequent analysis.

[0061] 1.4.4 Variant Annotation

[0062] (1) SNPs and INDELs were annotated using snpEff software, and SNPs were classified into intergenic regions, upstream or downstream regions, and exons or introns according to their location information on the genome.

[0063] (2) SNPs located in the coding region are further classified as synonymous SNPs or non-synonymous SNPs. Synonymous SNPs refer to SNPs that do not change the amino acid sequence, while non-synonymous SNPs refer to SNPs that change the amino acid sequence.

[0064] (3) For INDELs, determine whether they cause frameshift mutations and then analyze their effects on gene function. Frameshift mutations refer to mutations in which INDELs cause changes in the gene coding frame, thereby affecting the protein translation process.

[0065] The obtained SNP results are shown in Table 1 and Figure 2 As shown. Table 1 clearly shows the dynamic changes in the number of SNPs and heterozygosity in different generations. The F1 generation samples (such as F101 and F102) showed the highest genetic diversity, with a total number of SNPs reaching about 1-1.1 million and a heterozygosity of 70-80%. This reflects the significant genetic differences between the parents. As the generations progressed, the number of SNPs in the F2 and F3 generation samples dropped to about 600,000-650,000, and the heterozygosity dropped significantly to about 20%, reflecting the process of genetic separation and homozygosity. Figure 2The distribution patterns of six SNP mutation types are intuitively presented, with the horizontal axis representing the SNP mutation type (T:A>C:G, T:A>G:C, T:A>A:T, C:G>A:T, C:G>G:C, C:G>T:A), and the vertical axis representing the number of SNPs of each type. Each mutation type contains two complementary changes, for example, T:A>C:G includes T>C and A>G. This classification method takes into account the complementarity of DNA double strands and the possibility that sequencing data may be aligned to the positive or negative strand of the reference genome, thus providing a comprehensive and accurate overview of the distribution of SNP mutations. Among them, transition mutations T:A>C:G and C:G>T:A dominate in all samples, while the frequency of transversion mutations (T:A>A:T, T:A>G:C, C:G>A:T, C:G>G:C) is relatively low. This distribution pattern remained relatively consistent among different samples, which is consistent with the data shown in Table 1 that the ratio of transition to transversion type SNPs in most samples was between 2.34 and 2.47.

[0066] In addition, Table 1 also shows the differences in SNP characteristics of the parents. The number of SNPs in the father (PF01, PF02) is lower (about 550,000-560,000), while the number of SNPs in the mother (PM01, PM02) is higher (about 1 million-1.1 million). This difference may be closely related to the genetic basis of the photothermosensitive genic sterility trait.

[0067] The detailed data in Table 1 and Figure 2 The intuitive display of the SNP variation patterns of the photothermosensitive genic male sterile rice lines in different generations can be clearly seen. These findings not only reveal the genetic diversity and segregation rules of this trait, but also provide important clues for a deeper understanding of its molecular mechanism, laying a solid genomic foundation for subsequent functional research and molecular marker development.

[0068] Table 1 Statistics of SNPs detected

[0069]

[0070]

[0071] The obtained InDel results are shown in Table 2 and Figure 3 As shown. As can be seen from Table 2, the total number of InDels in the F1 generation samples (such as F101 and F102) is the highest, about 210,000 to 240,000. This is consistent with the high number of SNPs in the F1 generation observed in Table 1, which once again reflects the significant genetic differences between the parents. As the generations advance to F2 and F3, the number of InDels mostly drops to about 140,000 to 160,000, indicating that genetic variation is gradually fixed between generations. In terms of the type of InDel, Table 2 shows that in most samples, there are slightly more deletions than insertions, with a ratio of about 52:48. This pattern is Figure 3This was also verified in Table 2. Whether it is the whole genome or the coding region, the peak of deletion is slightly higher than that of insertion. It is worth noting that Table 2 reveals that the F1 generation samples (such as F101 and F102) show extremely high heterozygosity, while the proportion of homozygous InDel in the F2 and F3 generations increases to about 80%. This trend is similar to the separation pattern of SNPs and conforms to the classic Mendelian inheritance law.

[0072] Figure 3 The distribution characteristics of InDel length are clearly shown. Short InDels of 1-5 bp dominate the whole genome, among which 1 bp InDels are the most common. As the length increases, the frequency of InDels decreases rapidly. This distribution pattern is also basically consistent in the coding region, but the frequency is generally low, reflecting the low tolerance of the coding region to InDels. Figure 3 The distribution characteristics of InDels in the coding region are also worth noting. InDels with multiples of 3 bp are relatively more common in the coding region, which may reflect the selection pressure to maintain the protein reading frame. However, InDels with multiples of non-3 bp still exist in the coding region, suggesting potential functional changes or regulatory mechanisms.

[0073] Comprehensive Table 2 and Figure 3 Based on the information from the previous study, we can draw the following conclusions: InDel variation plays an important role in the inheritance of photothermosensitive genic male sterile lines in rice. Their number, type, and distribution pattern not only reflect genetic diversity, but also reveal potential functional impacts. In particular, the characteristics of InDel in the coding region provide important clues for understanding the molecular mechanism of photothermosensitive genic male sterile traits. These findings lay the foundation for the subsequent development of molecular markers and precision breeding, and also point the way for in-depth exploration of the impact of InDel on gene function.

[0074] Table 2 InDel statistics for the whole genome and coding regions

[0075]

[0076]

[0077] Note: CDS: InDel statistics in coding regions; Genome: InDel statistics across the entire genome; Insertion: The number of insertions detected; Deletion: The number of deletions detected; Het: The number of heterozygous InDels; Homo: The number of homozygous InDels; Total: The total number of InDels detected (excluding duplicates).

[0078] 1.5 Based on the SNP and INDEL information, select progeny with potential photothermosensitive nuclear sterility characteristics for planting, wherein the progeny with potential photothermosensitive nuclear sterility characteristics are progeny whose SNP number, conversion and transversion type SNP ratio, and heterozygous type SNP ratio are close to those of the mother parent, and the InDel statistical data, insertion number, deletion number, and homozygous InDel number in the coding region are also close to those of the mother parent; perform fertility identification on the selected progeny (fertility identification method is the same as 1.2), conduct multi-point and multi-year tests under different light and temperature conditions to verify the stability of its photothermosensitive characteristics; evaluate agronomic traits and combination capabilities, and finally screen and obtain a rice photothermosensitive nuclear sterile line that maintains stability.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for breeding rice photothermosensitive genic male sterile lines based on whole genome resequencing technology, characterized in that: The method comprises the following steps: hybridizing the sterile rice line Hong 35S as the male parent and the photothermosensitive nuclear sterile rice line as the female parent to obtain offspring; conducting fertility identification on the offspring to obtain photothermosensitive nuclear sterile offspring; extracting DNA from the male parent, the female parent and the photothermosensitive nuclear sterile offspring to perform whole genome resequencing; analyzing the sequencing data by using a bioinformatics method to obtain SNP and INDEL information; selecting offspring for planting according to the SNP and INDEL information, conducting fertility identification, and obtaining a photothermosensitive nuclear sterile rice line.

2. The method for breeding photothermosensitive genic male sterile rice lines according to claim 1, characterized in that: The offspring include the first generation, the second generation and the third generation.

3. The method for breeding photothermosensitive genic male sterile rice lines according to claim 2, characterized in that: The second generation is obtained by self-pollination of the first generation, and the third generation is obtained by self-pollination of the second generation.

4. The method for breeding photothermosensitive genic male sterile rice lines according to claim 1, characterized in that: The fertility identification includes at least one of anther identification, pollen viability determination, self-pollination fruit set rate determination and artificial pollination fruit set rate determination.

5. The method for breeding photothermosensitive genic male sterile rice lines according to claim 4, characterized in that: The anther identification is to observe the color of the anther after determining the pollen fertility conversion period. If the anther color remains yellow-green and does not change to yellow-white, or the conversion time is significantly delayed, it is identified as sterile.

6. The method for breeding photothermosensitive genic male sterile rice lines according to claim 4, characterized in that: The pollen viability test shows that the fertile pollen is less than 5%, which is considered sterile.

7. The method for breeding photothermosensitive genic male sterile rice lines according to claim 4, characterized in that: If the self-pollination fruit set rate determination and the artificial pollination fruit set rate determination show that no fruit set occurs or the fruit set rate is lower than 1%, the plant is identified as sterile.

8. The method for breeding photothermosensitive genic male sterile rice lines according to claim 1, characterized in that: The bioinformatics method is to use the bioinformatics analysis platform BMKCloud for processing and analysis.

9. The method for breeding photothermosensitive genic male sterile rice lines according to claim 8, characterized in that: The processing and analysis include data quality control, sequence alignment, variant detection and variant annotation.

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