Method for breeding indica-japonica hybrid semi-glutinous restorer line based on three F1 generations

Abstract

The invention provides a breeding method of an indica-japonica hybrid semi-glutinous restorer line based on three F1 generations, and relates to the technical field of rice genetic breeding. A japonica restorer line containing a semi-glutinous gene Wxmp site and an indica-japonica hybrid rice variety (F1) are hybridized to obtain a composite cross F1 generation; a single plant with excellent comprehensive characters is obtained by combining agronomic character screening and identification of a restoring gene Rf1a, a wide-compatibility gene S5n and a semi-waxy gene Wxmp; the excellent single plant obtained by screening is subjected to test matching with a semi-glutinous japonica sterile line, the comprehensive character of F1 is identified, and the combination with good recovery degree and excellent agronomic character is subjected to anther in-vitro culture to obtain a wide-affinity semi-glutinous indica-japonica hybrid restorer line material with stable inheritance. The indica-japonica hybrid restorer line is rapidly created through three different F1 generations, the restorer gene, the wide-affinity site and the semi-glutinous gene are polymerized through specific molecular marker-assisted selection, the wide-affinity semi-glutinous indica-japonica restorer line variety is obtained, and cultivation and production application of the rice semi-glutinous indica-japonica hybrid variety can be remarkably accelerated.

Description

Technical Field

[0001] This invention relates to the field of rice genetic breeding technology, specifically a breeding method based on three F1 generation indica-japonica semi-glutinous restorer lines. Background Technology

[0002] Currently, my country's indica-japonica hybrid rice technology is developing rapidly, and indica-japonica hybrid rice is becoming the mainstream direction for the breeding of new rice varieties in my country. The breeding goal of indica-japonica restorer lines is to aggregate superior genes to overcome the barriers of interspecific hybridization between indica and japonica subspecies and to cultivate hybrid combinations with strong heterosis.

[0003] Numerous studies have shown that there are significant differences in genetic background among indica and japonica subspecies, and intersubspecies hybrids possess enormous yield potential. Indica-japonica hybrid rice has become a major pathway to further increase rice yield. The F1 generation of intersubspecies indica-japonica hybrids typically exhibits strong heterosis, including enhanced tillering ability, robust stems, large panicles with many grains, high yield potential, and strong resistance to adverse conditions. However, practical breeding applications face multiple obstacles, with hybrid sterility being the most prominent. The F1 generation of indica-japonica hybrids often exhibits low pollen fertility, leading to a significant decrease in seed setting rate and severely limiting the effective expression of heterosis. Furthermore, indica-japonica hybrid combinations often result in overall poor rice quality, particularly in terms of eating taste and appearance. Therefore, breeding broadly compatible indica-japonica hybrid rice with superior rice quality has become a primary goal of rice breeding development.

[0004] In interspecific hybridization breeding of indica and japonica rice, S5 n The introduction of broad-compatibility genes can significantly overcome fertility barriers in the F1 generation of indica-japonica hybrids. Studies have shown that it can increase the seed setting rate of hybrid offspring by about 30% and may restore the seed setting rate to normal levels (over 75%). This can be achieved using S5... n Using molecular markers to assist in selection can quickly and accurately screen for target genotypes, greatly improving breeding efficiency.

[0005] Half-glutinous rice gene (Wx mpThis gene can regulate the amylose content to a moderate level (10-15%), making the cooked rice soft but not mushy, with excellent taste, thus becoming an important target gene for rice quality improvement. The Jiangsu Academy of Agricultural Sciences pioneered genetic breeding of superior-tasting rice, successfully developing a series of excellent-tasting japonica rice varieties suitable for different ecological zones in Jiangsu, such as Nanjing 7718, Nanjing 3908, and Nanjing 5718. Currently, the annual planting area of ​​the Nanjing series varieties in Jiangsu Province accounts for more than one-third of the province's total rice planting area, and has successfully expanded to surrounding areas such as Shanghai, Zhejiang, and Anhui, yielding significant social and economic benefits. The success of these semi-glutinous, high-tasting varieties has opened up new directions for hybrid rice breeding. However, the introduction of the semi-glutinous gene into the restorer line of indica-japonica hybrid rice to cultivate semi-glutinous hybrid rice with even better taste is still in its infancy, with the core bottleneck being the lack of indica-japonica restorer line materials containing the semi-glutinous gene. Using conventional breeding methods to combine semi-glutinous rice genes, male sterility restoration genes, and broad-compatibility genes not only results in a long breeding cycle but also low trait selection efficiency.

[0006] Problems and shortcomings of existing technologies: Existing technologies for breeding broad-compatibility, semi-glutinous indica-japonica restorer lines generally suffer from long breeding cycles and low efficiency in selecting target traits. Placing this quality objective within the more complex context of inter-subspecies hybridization of indica and japonica, and breeding an indica-japonica restorer line possessing both "broad compatibility" and "semi-glutinous" characteristics, presents even greater technical challenges. The core deficiency lies in the difficulty of traditional breeding methods in rapidly and synergistically integrating multiple key traits such as broad-compatibility genes, semi-glutinous genes, and strong restorer genes, leading to a slow and uncertain breeding process.

[0007] The technical challenges of solving this problem are immense: using traditional breeding methods such as conventional hybridization and backcrossing to address these issues is extremely difficult. Breeders must invest a very long timeframe and expend significant resources on genotyping of offspring materials and assessing the appearance and eating quality of rice. Particularly challenging is that for key traits such as indica-japonica compatibility, amylose content (semi-glutinous) in rice, and fertility recovery ability, reliable phenotypic identification can only be conducted after several generations of genetically stable materials. This severely limits breeding efficiency and accuracy. Summary of the Invention

[0008] In response to the above-mentioned existing technologies, this invention proposes a breeding method based on three F1 generation indica-japonica hybrid semi-glutinous restorer lines. Its core innovation lies in the design of three consecutive F1 generations to achieve rapid aggregation and trait optimization of key genes, so as to achieve efficient aggregation of restorer genes, wide-compatibility genes and semi-glutinous genes, and to accelerate the homozygous stabilization process of materials by using anther culture technology.

[0009] This invention systematically overcomes the technical bottlenecks faced by traditional breeding methods in creating wide-compatibility semi-glutinous indica-japonica restorer lines. Unlike conventional methods that rely on phenotypic selection and multiple backcrosses, this invention proposes using three different F1 generations to rapidly aggregate the target gene and simultaneously employing molecular marker-assisted selection technology to target the wide-compatibility gene (S5). n ), semi-glutinous gene (Wx mp Parallel and precise genotyping was conducted using the fertility restorer gene (Rf1a). This multi-gene aggregation strategy has multiple advantages: First, by aggregating broad-compatibility genes and restorer genes, both indica-japonica hybrid sterility and cytoplasmic male sterility can be overcome simultaneously, ensuring normal seed setting in hybrids; second, the introduction of the semi-glutinous gene can improve the eating quality of indica-japonica hybrids, achieving synergistic improvement in yield and quality; and third, through marker-assisted selection and anther culture, the trait stabilization process can be accelerated, the breeding cycle shortened, and the selection speed of superior combinations increased.

[0010] This invention successfully transforms the breeding concept of "widely compatible semi-glutinous indica-japonica cross-linking restorer line" into an efficient and feasible technical system, providing key germplasm resources and methodological support for achieving synergistic improvement of hybrid vigor and excellent eating quality among indica-japonica subspecies, and laying a solid material and methodological foundation for cultivating new indica-japonica hybrid rice varieties with high yield, high quality and excellent eating taste.

[0011] The present invention provides a breeding method based on three F1 generation indica-japonica semi-glutinous restorer lines, comprising the following steps:

[0012] S01: Contains the heterozygous Rf1arf1a, which contains the male sterility restoration gene, and the broad-affinity S5. n The heterozygous indica-japonica hybrid rice variety Yongyou 2640 (F1) was used as the donor parent, and was coupled with the homozygous Rf1aRf1a allele containing the male sterility restorer gene and the Wx allele containing the low amylose content in the endosperm. mp Wx mp The semi-glutinous japonica restorer line Ninghui 346 was used as the recipient parent to obtain the F1 generation population;

[0013] S02: Based on plant type, ear type, and other major agronomic traits, the F1 generation population was initially screened to obtain individual plants with excellent comprehensive traits. Using marker-assisted selection technology, the F1 generation plants obtained from phenotypic screening were further genotype-screened to determine if the male sterility restorer gene Rfla was homozygous. S5 n and Wx mp Plants with excellent combined traits due to heterozygous genes;

[0014] S03: The target plants obtained from S02 screening were mated with the japonica semi-glutinous male-sterile line Jia 58A to generate new F1 hybrids. Molecular marker detection was performed on the F1 hybrids generated by the mating groups, and those containing S5 were selected. n The combination of genes was examined, and related agronomic traits were investigated to screen out F1 combinations with good resilience, high combining ability, and excellent overall variety traits.

[0015] S04: The superior combinations obtained from S03 were subjected to anther culture, and chromosome doubling was performed to obtain doubled haploid (DH) plants, resulting in genetically stable lines. Molecular markers were used to differentiate Rfla and S5. n and Wx mp The gene underwent genotyping and obtained the genotype Wx. mp Wx mp S5 n S5 n RflaRfla is a widely compatible semi-glutinous indica-japonica hybrid restorer line.

[0016] Preferably, molecular marker detection of broad-compatibility genes, semi-glutinous genes, and restorer genes is performed on F1 generation plants, specifically including:

[0017] DNA was extracted from rice plant leaves using the CTAB method.

[0018] Using InDel-labeled primers InDel-S5 n The extracted DNA was subjected to rice broad-affinity site S5. n Molecular detection of genes, forward primer InDel-S5 n -F sequence is 5'-CTTGCTTAATCAACCCATTTCC-3'; reverse primer InDel-S5 n The -R sequence is 5'-TTCTCCGATCCGAGCCAAA-3';

[0019] KASP markers were used to analyze the extracted DNA for the semi-glutinous gene Wx. mp Molecular detection, forward primer 1 Wx mp - The F1 sequence is 5'-GAAGGTGACCAAGTTCATGCTAGGTTTTTCCATTGCTACAAGCA-3', with forward primer 2 Wx mp - The F2 sequence is 5'-GAAGGTCGGAGTCAACGGATTAGGTTTTTCCATTGCTACAAGCG-3', and the reverse primer is Wx. mp The -R sequence is 5'-AACTAATGATGACTCCACCTTCTCCA-3';

[0020] Molecular detection of the male sterility restoration gene Rf1a in japonica rice was performed using the InDel-labeled primer InDel-Rf1a. The forward primer InDel-Rf1a-F sequence was 5'-CTGATGATCGAGGAGGAGGTA-3', and the reverse primer InDel-Rf1a-R sequence was 5'-TAACGCGTCTTCCATCCTACT-3'.

[0021] Preferably, the extraction of DNA from rice plant leaves using the CTAB method includes:

[0022] (1) Take 2-3cm of rice seedling leaves, cut the leaves into small sections, put them into a 2ml centrifuge tube, add 1-2 steel balls, freeze in liquid nitrogen for 60 seconds, and immediately put them into a tissue grinder. Grind the sample into fine powder at a frequency of 60Hz for 50 seconds.

[0023] (2) Add 800 μl of preheated 2% CTAB extraction buffer, immediately vortex vigorously to mix, and place the centrifuge tube in a 65°C water bath for 30-60 minutes. During this period, gently invert and mix several times every 10-15 minutes to ensure complete lysis.

[0024] (3) Remove the centrifuge tube, cool it to room temperature, add 400 μl of chloroform:isoamyl alcohol (24:1), gently and thoroughly invert and mix for 1-2 minutes to form an emulsion, centrifuge at 12000 rpm for 12 minutes at room temperature, and the liquid will separate into layers. The upper layer is the aqueous phase containing DNA.

[0025] (4) Carefully pipette 400 μl of the upper aqueous phase and transfer it to a new 1.5 ml centrifuge tube. Add 0.7 times the volume of pre-cooled isopropanol and gently invert and mix several times until you see white filamentous or flocculent DNA precipitate. Centrifuge at 12000 rpm for 10 minutes at 4°C and discard the supernatant.

[0026] (5) Add 1 ml of pre-cooled 70% ethanol to the DNA precipitate, gently invert and rinse the precipitate, centrifuge at ≥12000 rpm for 5 minutes at 4℃, carefully discard the supernatant; open the tube cap, let it evaporate and dry naturally in a clean bench for 15-30 minutes, add 100-200 μl of ultrapure water to dissolve the DNA according to the amount of precipitate, and store at -20℃.

[0027] Preferably, in step (2), the 2% CTAB extraction buffer is obtained by preheating 2% CTAB, 100mM Tris-HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4M NaCl at 65°C.

[0028] Preferably, the primer InDel-S5 is used. n The extracted DNA was subjected to rice broad-affinity site S5. n Molecular testing of genes includes:

[0029] First, the extracted rice genomic DNA was amplified by PCR; second, the InDel-labeled primers InDel-S5 were used. n The DNA of the rice germplasm resources or composite materials to be tested was amplified by adding it to the PCR reaction system. Finally, an agarose gel with a mass concentration of 1.5% was prepared, and GelRed with a volume ratio of 0.5% was added. The sample was loaded onto the gel for electrophoresis and observed under a gel imaging system to obtain the detection results.

[0030] Preferably, choose the broad affinity S5. n Homozygous or heterozygous genes include:

[0031] If amplification of the rice genomic DNA reveals only a single characteristic band of 390 bp, then it is a broad-compatibility S5. n Homozygous for a gene locus;

[0032] If the rice genomic DNA is amplified and only a single characteristic band of 526 bp is observed, then it does not contain the broad-affinity S5. n Gene loci;

[0033] If the amplified rice genomic DNA shows two characteristic bands at 390 bp and 526 bp, it indicates that it is a broadly compatible S5 variety. n Heterozygotes at gene loci.

[0034] Rice broad-compatibility site S5 n In the molecular detection of genes, the PCR reaction system is 15 μL, specifically including: 1.0 μL of 50 ng / μL DNA, and primers InDel-S5. n -F and InDel-S5 n Add 0.5 μL of each of the -R and 7.5 μL of 2xPCR Master Mix, and bring the total volume to 15 μL with ddH2O.

[0035] The PCR reaction procedure included: pre-denaturation at 94℃ for 3 min; followed by denaturation at 94℃ for 30 s, annealing of the Rf1a gene at 55℃ for 15 s, extension at 72℃ for 30 s, for 30 cycles; then extension at 72℃ for 5 min, followed by cooling at 10℃.

[0036] Preferably, the extraction of the DNA using KASP markers for the semi-glutinous gene Wx is... mp Molecular detection includes:

[0037] PCR amplification of extracted rice genomic DNA, selecting the semi-glutinous gene wxmp wx mp homozygous or dark endosperm mutant gene Wx mp wx mp The hybrid; the three molecular marker primers Wx mp -F1、Wx mp -F2 and Wx mp -R is added to the same PCR reaction system to amplify the DNA of the sample to be tested. After the amplification is completed, the instrument captures the fluorescence signal to obtain the raw data. The raw data is then analyzed using the instrument's built-in CFXMaestro software to obtain specific genotyping data.

[0038] Preferred, select the semi-glutinous rice gene wx mp wx mp homozygous or dark endosperm mutant gene Wx mp wx mp Heterozygotes are identified by processing the raw data using CFX Maestro software, including filtering, noise reduction, and fluorescence signal standardization, and determining the genotype based on the fluorescence signal ratio. If only FAM signal is detected, it is denoted as Wx. mp Allele homozygosity; if only VIC signal is detected, it indicates that the allele is not Wx. mp If both signals are present, it indicates Wx mp Allele heterozygosity.

[0039] Half-glutinous rice gene Wx mp In the molecular detection, the PCR reaction system was 10 μL, specifically including: 1.0 μL of 50 ng / μL DNA, and primer Wx. mp -F1 0.5μL, Wx mp -F2 0.5μL, Wx mp - R 0.5μL, KASP Master mix 5μL (premixed reagent, containing buffer, dNTPs enzyme, etc.), add ddH2O to 10μL;

[0040] The PCR reaction program included: 95℃ pre-denaturation for 3 min; followed by 95℃ denaturation for 20 s, 65℃ annealing for 60 s, and 72℃ extension for 30 min, for a total of 40 cycles.

[0041] Preferably, the molecular detection of the male sterility restoration gene Rfla in japonica rice using the InDel-labeled primer InDel-Rfla includes:

[0042] First, the extracted rice genomic DNA was amplified by PCR. Second, the InDel-labeled primer InDel-Rfla was added to the PCR reaction system to amplify the DNA of rice germplasm resources or breeding populations. Finally, an agarose gel with a mass concentration of 1.5% was prepared, and GelRed with a volume ratio of 0.5% was added. The sample was loaded onto the gel and electrophoresed, and the results were observed under a gel imaging system to obtain the detection results.

[0043] Preferably, homozygous or heterozygous individuals for the Rfla gene include:

[0044] If the rice genomic DNA is amplified and only a single characteristic band of 1,145 bp is found, then it is a homozygote of the Rfla gene.

[0045] If the rice genomic DNA is amplified and there is only a single characteristic band of 571 bp, then it does not contain the Rfla gene locus.

[0046] If the rice genomic DNA is amplified and two characteristic bands of 1,145 bp and 571 bp are present simultaneously, it indicates that the rice is a heterozygote of the Rfla gene.

[0047] In the molecular detection of the male sterility restorer gene Rfla in japonica rice, the PCR reaction system was 15 μL, specifically including: 1.0 μL of 50 ng / μL DNA, 0.5 μL each of primers InDel-Rfla-F and InDel-Rfla-R, 7.5 μL of 2xPCR Master Mix, and ddH2O added to 15 μL.

[0048] The PCR reaction procedure included: pre-denaturation at 94℃ for 3 min; followed by denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min 20 s, for 30 cycles; then extension at 72℃ for 5 min, followed by cooling at 10℃.

[0049] Preferably, in S04, the male parent plants corresponding to the selected superior combinations are subjected to anther culture, and after chromosome doubling, those with a 390bp characteristic band are selected. n The homozygous genotype and the homozygous genotype RflaRfla with a 1,145 bp characteristic band, and the KASP fluorescence signal showed Wx mp Wx mp A homozygous single plant.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a breeding method based on three F1 generation indica-japonica semi-glutinous restorer lines. By screening through a specific molecular marker rapid detection method, a combination of target gene aggregation is obtained. Combined with anther culture, homozygotes are quickly obtained. The effective aggregation of wide-compatibility genes, semi-glutinous genes and restorer line genes is achieved in indica-japonica hybrid rice restorer lines, which accelerates the breeding process and the rapid application of semi-glutinous hybrid japonica rice in production.

[0051] The molecular markers used in the method of this invention for auxiliary selection are all PCR markers. Since it does not involve DNA sequencing, restriction endonucleases, etc., it is simple to operate, low in cost, and more efficient and faster. Furthermore, it can quickly and accurately target the broad-affinity site S5 at each generation of seedlings during the selection process. n , semi-glutinous gene Wx mp Screening for different genotypes of the restorer gene Rf1a greatly improves the predictability of breeding work.

[0052] The method of this invention can obtain a variety containing the broad-compatibility locus S5 in a single hybridization by crossing it with indica-japonica varieties. n , semi-glutinous gene Wx mp Materials containing the three target genes Rf1a and the restoration gene can control fertility (Rf1a, S5) in early generations. n ) and quality (Wx mp By precisely screening key genes, the blindness and lag of phenotypic selection in traditional breeding are avoided, greatly improving selection efficiency and accuracy. Combined with anther culture technology, homozygous and stable DH lines can be obtained in a very short time (one generation), significantly simplifying the breeding process and accelerating the breeding and application cycle of semi-glutinous indica-japonica hybrid rice varieties.

[0053] The method of this invention is highly targeted. By designing three F1 generations for directional selection and continuous screening, the restorer line has been made to have broad compatibility, strong resilience and excellent semi-glutinous eating quality. It is an ideal parent for formulating new combinations of high-yield, high-quality and high-seed-filling indica-japonica hybrid rice, and has significant production and application value. Attached Figure Description

[0054] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 This is the S5 plant of Yongyou 2640 / Ninghui 346 F1 in the embodiment of the present invention. nA schematic diagram of molecular gene detection;

[0056] Figure 1 In the middle: M: DNA molecular weight standard, 100-2000bp; 1: 02428; 2: Nipponbare; 3-24: F1 partial single plants, of which samples 5, 9, 10, 14, and 19 are S5. n The genes are homozygous, and samples 6-8, 13, 16, 17, 21, and 23 are S5. n Heterozygous, samples 3, 4, 11, 12, 15, 18, 20, 22, and 24 do not contain S5. n Gene.

[0057] Figure 2 This is a schematic diagram of molecular detection of the restorer gene in Yongyou 2640 / Ninghui 346 F1 plants in an embodiment of the present invention;

[0058] Figure 2 In the middle: M: DNA molecular weight standard, 100-2000bp; 1: Ninghui 346; 2: Nipponbare; 3-24: F1 individual plants, of which samples 3-9, 13, 14, and 20-24 are homozygous for the Rf1a gene, and samples 10-12 and 15-19 are heterozygous for the Rf1a gene.

[0059] Figure 3 The Wx plant of Yongyou 2640 / Ninghui 346 F1 in this embodiment of the invention. mp A schematic diagram of KASP gene detection.

[0060] Figure 3 In the diagram, the circular points near the horizontal axis carrying the A allelic variation are those containing Wx. mp Homozygous loci, those with square spots near the vertical axis carrying the G allele are those without the Wx allele. mp Homozygous loci, the triangular locus between the horizontal and vertical axes carries A / G alleles and contains Wx. mp The heterozygotes at the loci, with the two rhomboid sites near the starting position of the horizontal axis serving as blank controls with added water.

[0061] Figure 4 This is plant S5 of Jia 58A / Yongyou 2640 / Ninghui 346 F1-DH0 in the embodiment of the present invention. n A schematic diagram of molecular detection;

[0062] Figure 4 In the middle: M: DNA molecular weight standard, 100-2000bp; 1: 02428; 2: Nipponbare; 3-24: F1 partial single plants, of which samples 8, 10, 11, 19, 21, 23, and 24 are S5. n Genotype homozygous, 3, 6, 7, and 15 are S5. nHeterozygous genotypes, 4, 5, 9, 12-14, 16-18, 20, and 22, excluding S5. n Gene.

[0063] Figure 5 This is the Jia 58A / Yongyou 2640 / Ninghui 346 F1-DH1 plant S5 in the embodiment of the present invention. n A schematic diagram of molecular detection;

[0064] Figure 5 In the middle: M: DNA molecular weight standard, 100-2000bp; 1: 02428; 2: Nipponbare; 3-24: F1 partial single plants, of which samples 5-10, 14, and 24 are S5. n Homozygous genotype, 3, 4, 11-13, 15-23 excluding S5 n Gene.

[0065] Figure 6 This is a schematic diagram of molecular detection of the restorer gene in the Jia58A / Yongyou2640 / Ninghui346 F1-DH1 plant in an embodiment of the present invention.

[0066] Figure 6 In the middle: M: DNA molecular weight standard, 100-2000bp; 1: Ninghui 346; 2: Nipponbare; 3-24: F1 individual plants, of which samples 3-8, 12-16, and 20-24 are homozygous for the Rf1a gene, and samples 9-11 and 17-19 do not contain the Rf1a gene.

[0067] Figure 7 The embodiment of this invention is the Jia 58A / Yongyou 2640 / Ninghui 346 F1-DH1 plant Wx mp A schematic diagram of KASP gene detection.

[0068] Figure 7 In the diagram, the circular points near the horizontal axis carrying the A allelic variation are those containing Wx. mp Homozygous loci, those with square spots near the vertical axis carrying the G allele are those without the Wx allele. mp Homozygous loci, the triangular locus between the horizontal and vertical axes carries A / G alleles and contains Wx. mp The heterozygotes at the loci, with the two rhomboid spots near the starting position of the horizontal axis serving as the blank control with added water.

[0069] Figure 8 This is a pedigree chart of the widely compatible semi-glutinous indica-japonica hybrid restorer line in this embodiment of the invention. Detailed Implementation

[0070] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0071] This invention provides a breeding method based on three F1 generation indica-japonica semi-glutinous restorer lines, comprising the following steps:

[0072] S01: Contains the heterozygous Rf1arf1a, which contains the male sterility restoration gene, and the broad-affinity S5. n The heterozygous indica-japonica hybrid rice variety Yongyou 2640 (F1) was used as the donor parent, and the rice was bred with the homozygous Rf1aRf1a allele containing the male sterility restorer gene and the Wx allele containing low amylose content in the endosperm. mp Wx mp The homozygous semi-glutinous japonica restorer line Ninghui 346 was used as the recipient parent to obtain the F1 generation population.

[0073] S02: The F1 generation population was initially screened based on key agronomic traits such as plant type and ear type to obtain individual plants with excellent comprehensive traits. Using marker-assisted selection technology, the F1 generation plants obtained from phenotypic screening were further genotype-screened to determine if the male sterility restorer gene Rfla was homozygous. S5 n and Wx mp Plants with excellent combined traits due to heterozygous genes;

[0074] The molecular markers for wide-compatibility genes, semi-glutinous genes, and restorer genes were detected in different F1 generation plants, including:

[0075] 1. DNA extraction from rice plant leaves using the CTAB method; including:

[0076] (1) Take 2-3cm of rice seedling leaves, cut the leaves into small sections, put them into a 2ml centrifuge tube, add 1-2 steel balls, freeze in liquid nitrogen for 60 seconds, and immediately put them into a tissue grinder. Grind the sample into fine powder at a frequency of 60Hz for 50 seconds.

[0077] (2) Add 800 μl of preheated 2% CTAB extraction buffer, and immediately vortex vigorously to mix. Place the centrifuge tube in a 65°C water bath for 30-60 minutes, gently inverting and mixing several times every 10-15 minutes to ensure complete lysis. The 2% CTAB extraction buffer is prepared by preheating 2% CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl at 65°C.

[0078] (3) Remove the centrifuge tube, cool it to room temperature, add 400 μl of chloroform:isoamyl alcohol (24:1), gently and thoroughly invert and mix for 1-2 minutes to form an emulsion, centrifuge at 12000 rpm for 12 minutes at room temperature, and the liquid will separate into layers. The upper layer is the aqueous phase containing DNA.

[0079] (4) Carefully pipette 400 μl of the upper aqueous phase and transfer it to a new 1.5 ml centrifuge tube. Add 0.7 times the volume of pre-cooled isopropanol and gently invert and mix several times until you can see white filamentous or flocculent DNA precipitate. Centrifuge at 12000 rpm for 10 minutes at 4°C and discard the supernatant.

[0080] (5) Add 1 ml of pre-cooled 70% ethanol to the DNA precipitate, gently invert and rinse the precipitate, centrifuge at ≥12000 rpm for 5 minutes at 4℃, carefully discard the supernatant; open the tube cap, let it evaporate and dry naturally in a clean bench for 15-30 minutes, add 100-200 μl of ultrapure water to dissolve the DNA according to the amount of precipitate, and store at -20℃.

[0081] 2. Using InDel-labeled primers InDel-S5 n The extracted DNA was subjected to rice broad-affinity site S5. n Molecular detection of genes, forward primer InDel-S5 n -F sequence is 5'-CTTGCTTAATCAACCCATTTCC-3'; reverse primer InDel-S5 n The -R sequence is 5'-TTCTCCGATCCGAGCCAAA-3', and its detection methods include:

[0082] First, PCR amplification was performed on the extracted rice genomic DNA, selecting samples containing the broad-affinity site S5. n Single plants with a characteristic band of 390 bp; secondly, InDel-labeled primers InDel-S5 n The DNA of the rice germplasm resources or composite materials to be tested was amplified by adding it to the PCR reaction system. Finally, an agarose gel with a mass concentration of 1.5% was prepared, and GelRed with a volume ratio of 0.5% was added. The sample was loaded onto the gel for electrophoresis and observed under a gel imaging system to obtain the detection results.

[0083] Among them, the Guangqinhe S5 was selected. n Homozygous or heterozygous genes include:

[0084] If amplification of the rice genomic DNA reveals only a single characteristic band of 390 bp, then it is a broad-compatibility S5. n Homozygous for a gene locus;

[0085] If the rice genomic DNA is amplified and only a single characteristic band of 526 bp is observed, then it does not contain the broad-affinity S5. n Gene loci;

[0086] If the amplified rice genomic DNA shows two characteristic bands at 390 bp and 526 bp, it indicates that it is a broadly compatible S5 variety. n Heterozygotes at gene loci.

[0087] Rice broad-compatibility site S5 n In the molecular detection of genes, the PCR reaction system is 15 μL, specifically including: 1.0 μL of 50 ng / μL DNA, and primers InDel-S5. n -F and InDel-S5 n Add 0.5 μL of each of the -R and 7.5 μL of 2xPCR Master Mix, and bring the total volume to 15 μL with ddH2O.

[0088] The PCR reaction procedure included: pre-denaturation at 94℃ for 3 min; followed by denaturation at 94℃ for 30 s, annealing at 55℃ for 15 s, extension at 72℃ for 30 s, for 30 cycles; then extension at 72℃ for 5 min, followed by cooling at 10℃.

[0089] 3. Use KASP markers to analyze the extracted DNA for the semi-glutinous gene Wx. mp Molecular detection, forward primer 1 Wx mp - The F1 sequence is 5'-GAAGGTGACCAAGTTCATGCTAGGTTTTTCCATTGCTACAAGCA-3', with forward primer 2 Wx mp - The F2 sequence is 5'-GAAGGTCGGAGTCAACGGATTAGGTTTTTCCATTGCTACAAGCG-3', and the reverse primer is Wx. mp The -R sequence is 5'-AACTAATGATGACTCCACCTTCTCCA-3'; its detection methods include:

[0090] PCR amplification of extracted rice genomic DNA, selecting the semi-glutinous gene Wx mp homozygous or heterozygous; using three molecular marker primers Wx mp -F1、Wx mp -F2 and Wx mp -R is added to the same PCR reaction system to amplify the DNA of the sample to be tested. After the amplification is completed, the instrument captures the fluorescence signal to obtain the raw data. The raw data is then analyzed using the instrument's built-in CFX Maestro software to obtain specific genotyping data.

[0091] Half-glutinous rice gene Wx mp In the molecular detection, the PCR reaction system was 10 μL, specifically including: 1.0 μL of 50 ng / μL DNA, and primer Wx. mp -F1 0.5μL, Wx mp-F2 0.5μL, Wx mp - R 0.5μL, KASP Master mix 5μL (premixed reagent, containing buffer, dNTPs enzyme, etc.), add ddH2O to 10μL;

[0092] The PCR reaction program included: 95℃ pre-denaturation for 3 min; followed by 95℃ denaturation for 20 s, 65℃ annealing for 60 s, and 72℃ extension for 30 min, for a total of 40 cycles.

[0093] 4. Molecular detection of the male sterility restoration gene Rf1a in japonica rice was performed using InDel-labeled primers InDel-Rf1a. The forward primer InDel-Rf1a-F sequence is 5'-CTGATGATCGAGGAGGAGGTA-3', and the reverse primer InDel-Rf1a-R sequence is 5'-TAACGCGTCTTCCATCCTACT-3'. The detection method includes:

[0094] First, PCR amplification was performed on extracted rice genomic DNA, selecting single plants with the 1,145 bp characteristic band Rf1a homozygous genotype in the restorer line gene. Second, InDel-labeled primers InDel-Rfla were added to the PCR reaction system to amplify the DNA of rice germplasm resources or breeding populations. Finally, a 1.5% agarose gel was prepared, and 0.5% GelRed was added. The gel was loaded onto the gel for electrophoresis, and the results were observed under a gel imaging system.

[0095] Among them, homozygous or heterozygous individuals who selected the restorer gene Rf1a include:

[0096] If the rice genomic DNA is amplified and only a single characteristic band of 1,145 bp is found, then it is a homozygote of the Rfla gene.

[0097] If the rice genomic DNA is amplified and there is only a single characteristic band of 571 bp, then it does not contain the Rfla gene locus.

[0098] If the rice genomic DNA is amplified and two characteristic bands of 1,145 bp and 571 bp are present simultaneously, it indicates that the rice is a heterozygote of the Rfla gene.

[0099] In the molecular detection of the male sterility restorer gene Rfla in japonica rice, the PCR reaction system was 15 μL, specifically including: 1.0 μL of 50 ng / μL DNA, 0.5 μL each of primers InDel-Rfla-F and InDel-Rfla-R, 7.5 μL of 2xPCR Master Mix, and ddH2O added to 15 μL;

[0100] The PCR reaction procedure included: pre-denaturation at 94℃ for 3 min; followed by denaturation at 94℃ for 30 s, annealing of the Rf1a gene at 55℃ for 30 s, extension at 72℃ for 1 min 20 s, for 30 cycles; then extension at 72℃ for 5 min, followed by cooling at 10℃.

[0101] S03: The target plants obtained from S02 screening were mated with the japonica semi-glutinous male-sterile line Jia 58A to generate new F1 hybrids. Molecular marker detection was performed on the F1 hybrids generated by the mating groups, and those containing S5 were selected. n Gene combinations were combined, and related agronomic traits were examined to screen for F1 combinations with excellent overall performance traits, including good resilience and high combining ability. Molecular marker detection was performed on the F1 plants obtained from the second hybridization, and S5 plants exhibiting both 390bp and 526bp characteristic bands were selected. n F1, a hybrid combination with heterozygous genotypes.

[0102] S04: The superior combinations obtained from S03 were subjected to anther culture, and chromosome doubling was performed to obtain doubled haploid (DH) plants, resulting in genetically stable lines. Molecular markers were used to differentiate Rfla and S5. n and Wx mp The gene underwent genotyping and obtained the genotype Wx. mp Wx mp S5 n S5 n RflaRfla's indica-japonica cross-linked restoration material;

[0103] Among them, all individual plants within the selected strain contained Wx mp S5 n The combination of the three genes Rfla and anther culture was performed as follows:

[0104] (1) For young spikelets with a distance of 5-8 cm between the flag leaf and the cusp of the main stem and in the single-nucleus stage, retain 5 cm of spikelet stem;

[0105] (2) Disinfect with 75% alcohol. Wrap the ears of grain in a moist gauze, put them in a sealed bag, and place them in the dark at 5±1℃ for 5-8 days.

[0106] (3) In a clean bench, remove the middle spikelets from the spikelet, immerse the spikelets in 75% (v / v) ethanol solution for 30-60 seconds, transfer them to 2.0-2.5% sodium hypochlorite solution and soak for 15-20 minutes, gently shaking them during the process. Rinse with sterile distilled water 3-4 times, 1-2 minutes each time, to thoroughly remove residual disinfectant and dry the surface of the spikelets with sterile filter paper.

[0107] (4) Gently peel the anthers from the spikelets with sterile forceps and dissecting needles, and inoculate the anthers directly onto the surface of the induction medium; Induction medium: use modified M8 as the basic medium, with the following components added: 2,4-dichlorophenoxyacetic acid (2,4-D): 2.0 mg / L, naphthaleneacetic acid (NAA): 2.5 mg / L, kinetin (KT): 1.0 mg / L, sucrose: 40 g / L, agar powder: 6 g / L, and adjust the pH to 5.8;

[0108] (5) Seal the inoculated culture dish with sealing film and incubate in the dark at a temperature of 25±1℃ for 25-30 days. When the callus diameter reaches 1-2 mm, transfer it to differentiation medium.

[0109] (6) Place it in a light incubator with a light cycle of 14 hours of light / 10 hours of darkness and a temperature of 25±1℃. After 15-30 days of culture, green buds begin to appear on the surface of the callus tissue, which then differentiate into small plantlets.

[0110] (7) When the differentiated green seedlings grow to 5-10cm in height, wash the roots, soak them in 0.1% colchicine solution for 4-6 hours, rinse with sterile water, and then transplant.

[0111] The successfully doubled plants (DH0 generation) were transplanted to a greenhouse for self-pollination to produce DH1 generation plants, which then influenced the S5 generation. n Wx mp Molecular marker detection was performed on the three genes Rfla, and lines with excellent comprehensive agronomic traits, good disease resistance, high yield, and good combining ability were selected as widely compatible semi-glutinous indica-japonica hybrid restorer lines.

[0112] The technical effects of the present invention will be further described below with reference to specific embodiments.

[0113] Example 1: Parental Selection

[0114] Ninghui 346 is a japonica rice restorer line developed by the Institute of Food Crops, Jiangsu Academy of Agricultural Sciences. This restorer line features large panicles, abundant grains, strong restoring ability, plentiful pollen, excellent rice quality, and significant hybrid vigor. It contains Rf1a and Wx. mp The gene belongs to the semi-glutinous restorer line.

[0115] Yongyou 2640 is a three-line hybrid japonica rice variety bred by Ningbo Seed Co., Ltd. It was approved in Jiangsu Province in 2015, with the approval number Su Shen Dao 201507. This variety has a compact plant type, strong stems, large panicles with many grains, good late-stage color change, and contains S5. n It contains the Rf1a gene and belongs to the non-semi-glutinous type variety.

[0116] Both of the above varieties (lines) are publicly known and available materials, which can be provided by the Jiangsu Academy of Agricultural Sciences.

[0117] II. Molecular Marker Development:

[0118] 1) Rice broad-compatibility site S5 n Development of InDel gene markers:

[0119] According to the research results of Chen et al. (Proceedings of the National Academy of Sciences, 2008, 105(32): 11436-11441), and based on the S5 of the broad affinity material 02428 n Sequence information, including the S5 locus of the Nipponbare japonica rice genome, was confirmed by multiple sequence comparison analysis using Bioedit software. n A 136bp deletion exists. Primers were designed on both sides of the deletion site using Primer5 and named InDel-S5. n Forward primer InDel-S5 n -F sequence is 5'-CTTGCTTAATCAACCCATTTCC-3'; reverse primer InDel-S5 n The -R sequence is 5'-TTCTCCGATCCGAGCCAAA-3', and the Guangqinhe japonica rice 02428 contains S5. n The amplified product size was 390 bp for the site and 526 bp for Nipponbare rice, with an annealing temperature of 55℃. This primer was successfully amplified in Nipponbare, Guangxinhe and Japonica rice 02428, and Yongyou 2640, with products matching the expected size.

[0120] 2) Semi-glutinous gene Wx mp Development of KASP molecular markers:

[0121] Based on the shared G-to-A single base difference at position 473 (exon 4) of the coding region of the Wx site in Kanto 194 and Milky Queen, the genomic DNA sequence of the Wx gene from Nipponbare was obtained. A 150 bp sequence of genomic DNA upstream and downstream of this SNP region was designed as a KASP molecular marker. Two allele-specific forward primers were used, with 3' terminal bases corresponding to the two alleles of the SNP and 5' ends carrying FAM and VIC tag sequences, respectively. A common reverse primer was used, located downstream of the SNP site. Its primer sequence is as follows:

[0122] Forward primer 1 carries a FAM tag and allele A:

[0123] Wx mp-F1:5'-GAAGGTGACCAAGTTCATGCTAGGTTTTTCCATTGCTACAAGCA-3'

[0124] Forward primer 2 carries a VIC tag and an allele G.

[0125] Wx mp -F2:5'-GAAGGTCGGAGTCAACGGATTAGGTTTTTCCATTGCTACAAGCG-3'

[0126] Reverse universal primer:

[0127] Wx mp -R:5'-AACTAATGATGACTCCACCTTCTCCA-3'.

[0128] 3) The extraction of genomic DNA from rice plants was performed using the CTAB method (Lu Yangjiang, Chinese Journal of Rice Science, 1992, (01):47-48), with modifications and simplifications. The specific steps are as follows:

[0129] Take 2-3 cm of rice seedling leaves, cut them into small segments, and place them in a 2 ml centrifuge tube along with 1-2 steel balls. Quickly freeze in liquid nitrogen for 60 seconds, then immediately place in a tissue homogenizer and homogenize at 60 Hz for 50 seconds to obtain a fine powder. Add 800 μl of preheated 2% CTAB extraction buffer and immediately vortex vigorously to mix. Incubate the centrifuge tube in a 65°C water bath for 30-60 minutes, gently inverting and mixing every 10-15 minutes to ensure complete lysis. Remove the centrifuge tube and cool to room temperature. Add 400 μl of chloroform:isoamyl alcohol (24:1) and gently and thoroughly invert and mix for 1-2 minutes to form an emulsion. Centrifuge at 12000 rpm for 12 minutes at room temperature until the liquid separates into layers. The upper layer is an aqueous phase containing DNA. Carefully pipette 400 μl of the upper aqueous phase into a new 1.5 ml centrifuge tube, add 0.7 times the volume of pre-chilled isopropanol, and gently invert several times to mix until a white, filamentous or flocculent DNA precipitate is visible. Centrifuge at 12,000 rpm for 10 minutes at 4°C and discard the supernatant. Add 1 ml of pre-chilled 70% ethanol to the DNA precipitate, gently invert to rinse the precipitate, and centrifuge at ≥12,000 rpm for 5 minutes at 4°C. Carefully discard the supernatant. Open the tube cap and allow it to air dry in a clean bench for 15-30 minutes. Depending on the amount of precipitate, add 100-200 μl of ultrapure water to dissolve the DNA and store at -20°C.

[0130] 4) PCR amplification and electrophoresis detection:

[0131] The PCR reaction system was 15 μL, specifically including: 1.0 μL of 50 ng / μL DNA, and primers InDel-S5. n -F and InDel-S5 n Add 0.5 μL of each of -R and 7.5 μL of 2xPCR Master Mix, and bring the total volume of ddH2O to 15 μL. Prepare a 1.5% agarose gel, add 0.5% GelRed, load the gel onto the gel for electrophoresis, and observe the results using a gel imaging system.

[0132] The KASP reaction system is 10 μL, specifically including: 1.0 μL of 50 ng / μL DNA, and primer Wx. mp -F1 0.5μL, Wx mp -F2 0.5μL, Wx mp -R 0.5μL, KASP Master mix 5μL (premixed reagent, containing buffer, dNTPs enzyme, etc.), add ddH2O to 10μL.

[0133] III. The breeding process of widely compatible semi-glutinous indica-japonica hybrid restorer lines, refer to... Figure 8 :

[0134] 1) In the summer of 2023, using Ninghui 346 as the maternal parent in Nanjing, the restorer gene genotype was Rf1aRf1a, and the Wx locus gene was Wx. mp Wx mp Allelic type; using the indica-japonica hybrid rice Yongyou 2640 as the male parent, the restorer gene genotype is Rf1arf1a, and the S5 locus is S5. n The hybrid type yields more than 300 F1 seeds from crosses between the two.

[0135] 2) In the winter of 2023, the aforementioned F1 generation population was planted in Hainan. Phenotypic analysis was performed on the F1 generation population based on agronomic traits to obtain individual plants with excellent comprehensive traits. Using molecular marker-assisted selection technology, homozygous RflaRfla type individual plants with a 1145bp characteristic band were selected for the restorer gene; S5 plants with only a 390bp characteristic band were selected for the S5 locus. n Homozygous, or heterozygous with two characteristic bands at 390 bp and 526 bp; the KASP genotyping result for the Wx site is Wx. mp Homozygous or heterozygous single plants. Finally, 40-50 single plants with excellent overall traits were selected, and cross-crossed with the above-mentioned excellent single plants using the japonica semi-glutinous male-sterile line Jia 58A as the female parent to obtain the F1 combination of the repeated cross (…). Figures 1-3 ).

[0136] 3) In the summer of 2024, F1 plants of the above-mentioned combinations were planted in Nanjing. Key agronomic traits such as plant type, ear type, and seed setting rate were examined. Plants with two characteristic bands at the S5 locus (390bp and 526bp) were selected. Ten F1 combinations with good recovery ability, high combining ability, and excellent overall varietal traits were screened out. Anther culture was performed on the superior combinations to produce an F1-DH0 generation of 200 plants. The successfully doubled plants were transplanted to a greenhouse for self-pollination and seed harvesting of F1-DH1. Figure 4 ).

[0137] 4) In the winter of 2024, F1-DH1 generation populations were planted in Hainan, and molecular markers were used to target Rfla and S5. n and Wx mp Genotyping was performed on the genes. Homozygous RflaRfla type single plants with a 1145bp characteristic band were selected for the restorer gene, and S5 loci with only a 390bp characteristic band were selected. n Homozygous type, and the KASP genotyping result of Wx site selection is Wx mp Homozygous single plants. Twenty plants with superior comprehensive traits were selected and crossbred with different types of sterile lines, such as Nanjing 06A and Nanjing 9008A, to obtain F1 (…). Figures 5-7 ).

[0138] 5) In the summer of 2025, these testcross F1 combinations were planted for field comparative trials. Their growth period, plant height, ear type, and seed setting rate (recovery rate) were examined. Ultimately, one combination with a recovery rate of over 85%, ideal plant type, and excellent overall traits was selected, resulting in the widely compatible indica-japonica semi-glutinous restorer line Ninghui 82.

[0139] The above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent solutions made using the contents of the present invention specification, whether directly or indirectly applied to other related technical fields, are similarly within the patent protection scope of the present invention.

Claims

1. A breeding method based on three F1 generation indica-japonica semi-glutinous restorer lines, characterized in that, Includes the following steps: S01: Contains the heterozygous Rf1arf1a, which contains the male sterility restoration gene, and the broad-affinity S5. n A heterozygous indica-japonica hybrid rice variety (F1) was used as the donor parent, along with the homozygous Rf1aRf1a allele containing the male sterility restorer gene and the homozygous Wx allele containing the low amylose content in the endosperm. mp Wx mp The semi-glutinous japonica restorer line was used as the recipient parent for hybridization to obtain the F1 generation population; S02: Select individual plants with excellent agronomic traits from the F1 generation population. Using marker-assisted selection, further genotypic screening is performed on the F1 plants obtained from phenotypic screening to obtain homozygous male sterility restorer gene Rfla. S5 n and Wx mp Plants with excellent combined traits due to heterozygous genes; S03: The target plants obtained from S02 screening were mated with semi-glutinous japonica sterile lines to generate new F1 hybrids. Molecular marker detection was performed on the F1 hybrids generated by the mating groups, and those containing S5 were selected. n The combination of genes was examined, and related agronomic traits were investigated to screen out F1 combinations with good resilience, high combining ability, and excellent overall performance traits. S04: The superior combination obtained from S03 was subjected to anther culture, and chromosome doubling was performed to obtain doubled haploid plants, resulting in genetically stable lines. Molecular markers were used to differentiate Rfla and S5. n and Wx mp The gene underwent genotyping and obtained the genotype Wx. mp Wx mp S5 n S5 n RflaRfla is a semi-glutinous, wide-affinity indica-japonica crossbred restorer line.

2. The breeding method as described in claim 1, characterized in that, Molecular marker detection of wide-compatibility genes, semi-glutinous genes, and restorer genes was performed on F1 generation plants, specifically including: DNA was extracted from rice plant leaves using the CTAB method. Using InDel-labeled primers InDel-S5 n The extracted DNA was subjected to rice broad-affinity site S5. n Molecular detection of genes, forward primer InDel-S5 n -F sequence is 5'-CTTGCTTAATCAACCCATTTCC-3'; reverse primer InDel-S5 n The -R sequence is 5'-TTCTCCGATCCGAGCCAAA-3'; KASP markers were used to analyze the extracted DNA for the semi-glutinous gene Wx. mp Molecular detection, forward primer 1 Wx mp - The F1 sequence is 5'-GAAGGTGACCAAGTTCATGCTAGGTTTTTCCATTGCTACAAGCA-3', with forward primer 2 Wx mp - The F2 sequence is 5'-GAAGGTCGGAGTCAACGGATTAGGTTTTTCCATTGCTACAAGCG-3', and the reverse primer is Wx. mp The -R sequence is 5'-AACTAATGATGACTCCACCTTCTCCA-3'; Molecular detection of the male sterility restoration gene Rf1a in japonica rice was performed using the InDel-labeled primer InDel-Rf1a. The forward primer InDel-Rf1a-F sequence was 5'-CTGATGATCGAGGAGGAGGTA-3', and the reverse primer InDel-Rf1a-R sequence was 5'-TAACGCGTCTTCCATCCTACT-3'.

3. The breeding method as described in claim 2, characterized in that, The use of InDel-labeled primers InDel-S5 n The extracted DNA was subjected to rice broad-affinity site S5. n Molecular testing of genes includes: First, the extracted rice genomic DNA was amplified by PCR; second, the InDel-labeled primers InDel-S5 were used. n The DNA of the rice germplasm resources or composite materials to be tested was amplified by adding it to the PCR reaction system. Finally, an agarose gel with a mass concentration of 1.5% was prepared, and GelRed with a volume ratio of 0.5% was added. The sample was loaded for electrophoresis and observed under a gel imaging system to obtain the detection results. The PCR reaction system consisted of 15 μL, specifically including: 1.0 μL of 50 ng / μL DNA, and primers InDel-S5. n -F and InDel-S5 n Add 0.5 μL of each of the -R and 7.5 μL of 2xPCR Master Mix, and bring the total volume of ddH2O to 15 μL. The PCR reaction procedure included: pre-denaturation at 94℃ for 3 min; followed by denaturation at 94℃ for 30 s, annealing of the Rf1a gene at 55℃ for 15 s, extension at 72℃ for 30 s, for 30 cycles; then extension at 72℃ for 5 min, followed by cooling at 10℃.

4. The breeding method as described in claim 2, characterized in that, The extraction of DNA using KASP markers was analyzed to detect the semi-glutinous gene Wx. mp Molecular detection includes: PCR amplification of extracted rice genomic DNA, selecting the semi-glutinous gene Wx mp homozygous or dark endosperm mutant gene Wx mp The hybrid; the three molecular marker primers Wx mp -F1、Wx mp -F2 and Wx mp -R is added to the same PCR reaction system to amplify the DNA of the sample to be tested. After amplification, the instrument captures the fluorescence signal to obtain the raw data. The raw data is then analyzed using the instrument's built-in CFX Maestro software to obtain specific genotyping data. The PCR reaction system consisted of 10 μL of 50 ng / μL DNA and primer Wx. mp -F1 0.5μL, Wx mp -F2 0.5μL, Wx mp -R 0.5μL, KASP Master mix 5μL, add ddH2O to 10μL; The PCR reaction procedure included: pre-denaturation at 95℃ for 3 min; followed by denaturation at 95℃ for 20 s, annealing at 65℃ for 60 s, extension at 72℃ for 30 s, for 40 cycles.

5. The breeding method as described in claim 2, characterized in that, The molecular detection of the male sterility restoration gene Rfla in japonica rice using the InDel-labeled primer InDel-Rfla includes: First, the extracted rice genomic DNA was amplified by PCR. Second, the InDel-labeled primer InDel-Rfla was added to the PCR reaction system to amplify the DNA of rice germplasm resources or breeding populations. Finally, an agarose gel with a mass concentration of 1.5% was prepared, and GelRed with a volume ratio of 0.5% was added. The sample was loaded onto the gel and electrophoresed, and the results were observed under a gel imaging system to obtain the detection results. The PCR reaction system consisted of 15 μL, specifically including: 1.0 μL of 50 ng / μL DNA, 0.5 μL each of primers InDel-Rfla-F and InDel-Rfla-R, 7.5 μL of 2xPCR Master Mix, and ddH2O added to bring the total volume to 15 μL. The PCR reaction procedure included: pre-denaturation at 94℃ for 3 min; followed by denaturation at 94℃ for 30 s, annealing of the Rf1a gene at 55℃ for 30 s, extension at 72℃ for 1 min 20 s, for 30 cycles; then extension at 72℃ for 5 min, followed by cooling at 10℃.

6. The breeding method as described in claim 1, characterized in that, In S04, the male parent plants corresponding to the selected superior combinations were subjected to anther culture, and after chromosome doubling, homozygous genotype S5 with a 390bp characteristic band was selected. n The genotype RflaRfla, exhibiting a homozygous genotype with a characteristic band of 1,145 bp, and showing a Wx fluorescence signal as detected by KASP, was also observed. mp Wx mp A homozygous single plant.

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