A method for identifying indica-japonica hybrid rice based on InDel marker

Abstract

This invention relates to the field of biotype identification technology, and in particular to a method for identifying indica-japonica hybrid rice based on novel InDel markers. This invention screens 10 pairs of novel InDel molecular markers from indica-japonica differentiation QTLs, and combines them with 19 pairs of representative and specific InDel molecular markers to design a 29-pair InDel molecular marker system for detecting indica-japonica rice attributes. This improves the identification capability, allows for further subdivision of indica-japonica rice attributes, and identifies more genetic diversity among varieties.

Description

Technical Field

[0001] This invention relates to the field of biotype identification technology, and in particular to a method for identifying intermediate indica-japonica hybrid rice based on a novel InDel marker. Background Technology

[0002] Indica and japonica rice are important genetic characteristics influencing parental selection, heterosis utilization, and variety improvement in hybrid rice. Over long periods of evolution, indica and japonica rice have developed different genetic backgrounds, resulting in significant differences in ecological adaptability, growth period, plant architecture, yield composition, and hybrid performance. Therefore, accurately identifying the indica / japonica attributes of rice materials is crucial for analyzing the genetic composition of rice germplasm resources, optimizing hybrid combinations, and improving breeding efficiency.

[0003] Currently, methods for identifying the indica and japonica rice traits mainly include morphological identification, physiological and biochemical index identification, and molecular marker-based genetic identification. Among these, morphological and physiological / biochemical identification methods are affected by environmental conditions and the growth stage of the rice, resulting in limited stability and accuracy of the results. With the development of molecular biology techniques, DNA molecular marker-based identification methods have been developed. These methods are unaffected by environmental factors, have short detection cycles, and high repeatability, and are now an important technical means for identifying the indica and japonica rice traits.

[0004] However, existing InDel (insertion and deletion marker) systems for indica and japonica rice attributes are mainly developed based on known indica and japonica differentiation regions. Although they can effectively distinguish between typical indica and japonica rice materials, for indica-japonica hybrid rice materials with complex genetic backgrounds, their genomes contain genetic segments from both indica and japonica sources, which weakens the genetic differences at some loci. For materials in the intermediate state of indica-japonica differentiation, it is difficult to achieve precise differentiation using existing marker systems.

[0005] Patent document CN101323878B discloses a method for identifying indica and japonica rice using DNA insertion or deletion molecular markers. However, this method has limitations in its ability to accurately classify and distinguish indica-japonica hybrid rice materials with complex genetic backgrounds, especially those in an intermediate stage of indica-japonica differentiation. Therefore, developing new indica-japonica molecular markers and methods for more accurately identifying intermediate indica-japonica hybrid rice is crucial. Summary of the Invention

[0006] The purpose of this invention is to introduce 10 new InDel markers obtained from screening genomic regions related to differences between Indica and Japonica rice into the existing InDel marker system, forming a detection system of 29 InDel markers. This system comprehensively analyzes the genetic composition of the tested materials from multiple genetic regions, enabling precise identification of typical Indica rice, Japonica rice, and intermediate types of Indica and Japonica rice with different degrees of susceptibility.

[0007] Another objective of this invention is to screen for 10 newly developed InDel marker sites. These 10 newly developed InDel pairs include: INDEL2, INDEL3, INDEL7, INDEL8, INDEL10, INDEL11, INDEL13, INDEL14, INDEL15, and INDEL21.

[0008] To achieve the above objectives, this invention discloses a method for identifying intermediate indica-japonica hybrid rice based on InDel marker combinations, characterized by comprising the following steps: S1. Extract genomic DNA from the indica-japonica hybrid rice material to be tested; S2. The genomic DNA was amplified by PCR (polymerase chain reaction) using the InDel marker combination to obtain the genotype information of the test material at each InDel site; S3. Determine the InDel genotype of the material to be tested based on the amplification results; S4. Calculate the indica genetic component and / or japonica genetic component of the material to be tested based on the InDel genotype information, and determine the indica-japonica type of the indica-japonica hybrid rice material to be tested based on the calculation results; The differences in the properties of indica and japonica rice stem from the genetic differentiation of genomes formed during long-term evolution, with sequence variations existing in multiple genomic regions among different indica and japonica rice materials. Insertion-deletion variations, due to their stable inheritance, significant polymorphism, and ease of detection among different rice subspecies, can serve as molecular markers reflecting genetic differences between indica and japonica rice. This invention screens indica-japonica difference InDel loci distributed across different genomic regions, constructing a combined detection system containing existing indica-japonica specific InDel markers and newly screened InDel markers. Because the insertion or deletion states at these InDel loci differ among different indica-japonica materials, PCR amplification can generate extended fragments of varying lengths, yielding genotype information for the corresponding InDel loci. While a single InDel locus can only reflect differences in a local genomic region, multiple InDel loci distributed across different chromosomal regions can comprehensively reflect the overall genetic composition of the material. This invention obtains the overall indica-japonica genetic information of the tested material through joint analysis of multiple InDel loci. Based on the genotype information obtained from multiple InDel loci, the genetic similarity between materials is calculated, and cluster analysis is used to form different genetic types based on differences in genetic distance between materials. Because the genetic composition of indica and japonica rice materials differs systematically at multiple InDel loci, different types of indica and japonica rice materials can form corresponding genetic groups in the clustering results, thereby enabling the identification of the indica and japonica attributes of the tested indica-japonica hybrid rice materials.

[0009] Preferably, the InDel loci include 19 pairs of existing InDel markers for identifying indica and japonica rice and 10 pairs of newly developed InDel markers. This invention employs an InDel marker combination consisting of 19 pairs of existing InDel markers for identifying indica and japonica rice and 10 pairs of newly developed InDel markers. The 19 pairs of existing InDel markers for identifying indica and japonica rice cover multiple chromosomal regions, providing basic genetic information on indica and japonica rice materials. The 10 pairs of newly developed InDel markers originate from genomic regions with more pronounced differences between indica and japonica rice, supplementing loci with insufficient genetic information in existing detection systems and further increasing the detection dimensions of genetic differences in indica and japonica rice.

[0010] Preferably, the method for obtaining the 10 pairs of newly developed InDel markers is as follows: S1. Screening for QTL regions with InDel variation among 237 QTL (quantitative trait loci) regions of rice with differences between indica and japonica varieties; S2. Design InDel primers based on the QTL regions with InDel mutations; S3. Perform PCR amplification and detection on typical indica rice and typical japonica rice materials according to the InDel primers; The 10 newly developed InDel pairs were obtained through screening based on differences in amplified fragments. Differences in indica and japonica rice characteristics are genomic-level differences formed during long-term genetic differentiation, and related differential loci are regionally distributed in the rice genome. Molecular variation loci reflecting these genetic differences exist in some genomic regions that control or are associated with indica-japonica rice differentiation characteristics. This invention improves the targeting of candidate InDel loci screening by screening regions with InDel variations from 237 rice QTL regions exhibiting indica-japonica differences, utilizing the genetic difference information contained in the QTL regions. InDel variations may manifest as insertions or deletions between different rice materials. InDel loci located in regions related to indica-japonica differences can serve as candidate molecular markers for distinguishing materials with different indica-japonica genetic backgrounds. Furthermore, specific primers were designed based on the screened InDel variation regions, and PCR amplification detection was performed on typical indica and japonica rice materials, respectively. Typical indica rice and typical japonica rice have different insertion / deletion states at the corresponding InDel sites. After PCR amplification, they can produce extended fragments of different lengths. By comparing the differences in amplification products, InDel sites with stable expansion differences between indica rice and japonica rice materials can be screened.

[0011] Preferably, the 10 pairs of newly developed InDel markers include: INDEL2, INDEL3, INDEL7, INDEL8, INDEL10, INDEL11, INDEL13, INDEL14, INDEL15, and INDEL21.

[0012] Preferably, the InDel primer comprises a nucleotide sequence having Seq ID NO.1-20.

[0013] Preferably, the typical japonica rice materials include Qiuguang, Zaoshajing, and Balali; the typical indica rice materials include Aijiao Nante, Nanning 11, and IR36.

[0014] Preferably, the DNA extraction in S1 includes: Leaf samples were collected from rice plants at the 3-4 leaf stage. After being crushed, DNA extraction solution was added for lysis. The lysate was then extracted with chloroform, centrifuged, and precipitated with ethanol to obtain genomic DNA.

[0015] Preferably, the PCR amplification in S2 includes: The InDel primers were used to amplify the genomic DNA of the test material by PCR to obtain the amplified product of the target InDel fragment. The PCR amplification system included template DNA, DNA polymerase, forward primer, reverse primer, and nuclease-free water. The PCR amplification program was as follows: 35 cycles of amplification were performed after pre-denaturation at 94°C. Each cycle included denaturation at 94°C, annealing at 53°C, and extension at 72°C. After the cycle, an extension at 72°C was performed. The PCR amplification products were separated by agarose gel electrophoresis.

[0016] Preferably, the determination of the InDel genotype of the material to be tested in S3 is as follows: the genotype information of the corresponding InDel locus is obtained based on the presence and type of the electrophoretic band; for samples that do not show an amplified band, PCR amplification and electrophoresis detection are repeated, and if no amplified band is found after repeated detection, it is recorded as a missing allele.

[0017] Preferably, the calculation in S4 is as follows: calculate the indica rice gene frequency Fi and japonica rice sex gene frequency Fj of the test material based on the genotype information of each InDel site, and determine the indica / japonica attribute of the test material according to the classification criteria corresponding to Fi and Fj.

[0018] At each InDel locus, samples whose band positions correspond to those of the typical indica rice material are denoted as homozygous indica genotype (Indica-Indica genotype, II); samples whose band positions correspond to those of the typical japonica rice material are denoted as homozygous japonica genotype (Japonica-Japonica genotype, JJ); and samples exhibiting both typical indica and japonica rice bands are denoted as heterozygous indica-japonica genotype (Indica-Japonica genotype, IJ). The average allele frequencies of indica and japonica rice types for each material are calculated based on the genotype data of each rice sample at all InDel loci (N), using the following formula: Frequency of indica genotype (Fi):

[0019] Frequency of japonica genotype (Fj):

[0020] In the formula, X ii It is a homozygous indica rice genotype (II), X jj The homozygous japonica rice genotype is (JJ), X ij The genotype is IJ, representing the heterozygous genotype of indica-japonica rice. N represents the number of indica-japonica specific InDel primer pairs. Based on the frequency of japonica or indica genes at multiple InDel loci in the tested rice samples, the following gene frequencies were defined: japonica gene frequency Fj≤0.10; indica gene frequency Fi≥0.90; Fj=0.11~0.25; Fj=0.75~0.89; Fj=0.26~0.39; Fj=0.60~0.74; Fj=0.40~0.49; Fj=0.49~0.59; Fj=0.5; Fj=0.50; Fj=0.49. Materials with indica-japonica indices of ~0.59 or Fi of 0.40~0.49, Fj of 0.60~0.75 or Fi of 0.26~0.40, Fj of 0.75~0.89 or Fi of 0.11~0.25, Fj≥0.90 or Fi≤0.10 are respectively classified into nine categories of indica-japonica attribute types: "typical japonica rice", "japonica rice", "partially japonica", "intermediate type", "intermediately indica", "partially indica", "indica rice", and "typical indica rice".

[0021] The beneficial effects of this invention are as follows: This technology improves the distinguishing ability of indica and japonica rice attribute identification. Based on the existing indica-japonica specific InDel marker system, 10 new InDel marker pairs are further screened from QTL regions of rice with indica-japonica differences, and combined with the existing 19 InDel marker pairs to form a detection system containing 29 InDel marker pairs. By increasing the detection sites with information on indica-japonica genetic differences, the detection system can obtain more information reflecting the genetic composition of the tested material, thus improving the distinguishing ability of materials with different indica-japonica genetic backgrounds.

[0022] This technology enhances the ability to identify intermediate indica-japonica hybrid rice materials. By introducing 10 new InDel markers for indica-japonica differences, this invention supplements the existing detection system, further highlighting the differences between materials of different genetic types, thereby improving the ability to identify intermediate indica-japonica hybrid rice materials.

[0023] This technology improves the precision of genetic clustering analysis. This invention uses 29 pairs of InDel markers to perform genetic similarity analysis on rice materials, increasing the number of information loci that can be used for genetic classification. This allows intermediate types of materials to be further subdivided into more indica and japonica genetic groups, more accurately reflecting the genetic differences between different rice materials, which is beneficial for the evaluation of rice germplasm resources.

[0024] This technology offers a stable and simple detection method suitable for screening breeding materials. Based on PCR amplification and electrophoresis, this invention obtains InDel locus genotype information, demonstrating stable and reproducible detection processes. Analyzing the genetic composition of the indica and japonica varieties of the target material can assist breeders in parent selection and hybrid material screening, improving the efficiency of material evaluation in rice breeding. Attached Figure Description

[0025] Figure 1 The relative positions of 19 pairs of InDel molecular markers on chromosomes; Figure 2-5 Fj fitting curves and coefficients of determination for 19 sites; Figure 6-8 Changes in the proportions of the three genotypes at 19 loci over 6 years; Figure 9-11 Fit curves and coefficients of determination for heterozygous genotypes; Figure 12-13 Fitting curves and coefficients of determination for homozygous genotypes; Figure 14 The relative positions of 10 pairs of InDel markers on the chromosome; Figure 15 Cluster analysis of genetic similarity of 19 InDel markers; Figure 16 Cluster analysis of genetic similarity of 29 InDel markers. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0027] Example 1: Genetic Characteristics Analysis of Indica-Japonica Hybrid Rice Based on 19 Pairs of InDel Markers The experimental materials selected were all varieties of indica-japonica hybrid rice participating in the regional trials in Zhejiang Province from 2019 to 2024, including the single late-season rice pellet group (hereinafter referred to as single late-season pellet group), the single late-season long-season rice group (hereinafter referred to as single late-season long-season group), and the continuous late-season rice group (hereinafter referred to as continuous late-season group), as well as their corresponding control varieties. The materials were numbered P1-P36, and the variety names and pedigrees are shown in Appendix 1.

[0028] DNA extraction from rice leaf tissue: (1) During the 3 to 4-leaf stage of rice growth and development, select one single plant of the variety to collect fresh leaves, place them in a 2ml centrifuge tube, and add a small steel ball with a diameter of 5mm. (2) Add 500µL of DNA extraction solution to the centrifuge tube and place it in the crusher. Set the frequency to 60Hz and shake for 90s. If the crushing effect is not ideal, repeat the operation. (3) Place the centrifuge tubes in a 65°C water bath for 30 minutes. During this time, the centrifuge tubes need to be shaken several times to thoroughly mix the contents; (4) In a fume hood, add an equal volume of chloroform to the centrifuge tube, seal the tube tightly, shake up and down to mix the liquid in the tube thoroughly, and then centrifuge at 12000 rpm for 10 min. (5) Transfer the supernatant to a new 1.5 ml centrifuge tube and add 75% ethanol solution to the new 1.5 ml centrifuge tube; (6) Centrifuge at 12000 rpm for 8 min. After centrifugation, remove the supernatant and place the centrifuge tube in a ventilated area to dry. Add 520 µL ddH2O (double-distilled water) to dissolve the nucleic acid precipitate. (7) The concentration of the extracted DNA was determined using a protein nucleic acid quantification instrument. After the determination, the DNA sample was stored at -20℃ for subsequent experiments. Primer sequence synthesis: 19 pairs of InDel primers were selected (Table 1), which evenly covered 12 chromosomes. Figure 1 ), synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0029] Table 1. 19 pairs of InDel primers and DNA sequences

[0030] PCR reaction procedure and product separation: The reaction mixture was prepared in 10 µL volumes, and the reagents were purchased from Novoprotein. The target fragment was amplified using a Biometra PCR amplification system. The total reaction volume was 10 µL, including 2 µL template DNA, 5 µL Tap enzyme, 1.5 µL ddH2O, 0.75 µL Limer-F, and 0.75 µL Primer-R. The reaction program was set as follows: 94 °C pre-denaturation for 4 min, 35 cycles, each cycle consisting of denaturation at 94 °C for 30 s; annealing at 53 °C for 30 s; extension at 72 °C for 30 s; and a final extension at 72 °C for 7 min. The amplified products were separated by electrophoresis on a 3% agarose gel at 150 V. DNA samples without electrophoretic bands were repeated for PCR amplification and electrophoresis. Samples without bands after three repetitions were considered as having no bands. Electrophoresis images were captured using a BioRad gel imager, and the results were analyzed using Quantity One electrophoresis image processing software. If a material is repeated three times and still does not appear, it is treated as a missing band (nullallele) and no related statistics are performed.

[0031] Determination of Indica and Japonica rice properties: DNA from 326 rice varieties was amplified using 19 primer pairs. PCR electrophoresis results of the sequenced varieties 9311 and Nipponbare (japonica rice) were used as controls. At each InDel locus, samples with bands identical to those of 9311 were designated as homozygous indica genotype (Indica-Indica genotype, II), samples identical to those of Nipponbare were designated as homozygous japonica genotype (Japonica-Japonica genotype, JJ), and samples showing both 9311 and Nipponbare bands were designated as heterozygous indica-japonica genotype (Indica-Japonica genotype, IJ) (Table 2). The average allele frequencies of indica and japonica types for each material were calculated based on the genotype data of all rice samples at all InDel loci (N), using the following formula: Indica rice gene frequency indica genotype Fi ):

[0032] Frequency of japonica genotype (Fj):

[0033] In the formula, X ii It is a homozygous indica rice genotype (II), X jjThe homozygous japonica rice genotype is (JJ), X ij The genotype is IJ, which is a heterozygous genotype of indica-japonica rice. N is the number of indica-japonica specific InDel primer pairs.

[0034] Table 2. Classification criteria for determining the characteristics of indica and japonica rice based on the frequency of InDel marker genes.

[0035] Based on the Fj values ​​of all varieties, the mean Fj values ​​for each of the 19 loci over 6 years were calculated. A scatter plot of the annual mean Fj values ​​for each locus was then generated using Origin 2024 software. A fitting curve was used to fit the plot, and the coefficient of determination (R²) was selected from the fitted curves. 2 The largest and best-fitting curve is determined by the following criteria: R 2 A value greater than 0.90 indicates an excellent compliance rate; R 2 A value of 0.80~0.90 indicates good compliance; R 2 A score of 0.60 to 0.80 indicates a moderate level of compliance; R 2 A value less than 0.60 indicates a poor fit. Based on the different equations of the fitted curves for each locus, L, S, and P were used to represent linear regression, hyperbolic regression, and parabolic regression, respectively, to visually describe the evolutionary patterns of different loci. Based on the detection results of the indica or japonica bands at the 19 InDel loci, the locus was identified as the homozygous indica genotype Xii, the homozygous japonica genotype Xjj, and the indica-japonica heterozygous genotype Xij. Using the same data processing methods as for the analysis of Fj, loci with an average single genotype ratio exceeding 0.6 were selected, and scatter plots were generated using Origin 2024 software. The remaining steps were consistent with the evolutionary patterns analyzed for Fj.

[0036] Table 3. Statistics on the frequency of japonica rice genes and the classification of indica and japonica rice from 2019 to 2024.

[0037]

[0038] Statistics were compiled on 326 indica-japonica hybrid rice varieties participating in regional rice trials in Zhejiang Province from 2019 to 2024, including varieties in the single-season late-grain, single-season late-long-grain, and consecutive-late-season groups. The frequency of the japonica gene (Fj) in the InDel indica-japonica index was analyzed (Table 3). Compared with 2019, the average Fj decreased from 0.5696 to 0.5387 in 2024; the minimum decreased from 0.4474 to 0.3947; and the maximum decreased from 0.8421 to 0.6944. These results indicate that during this period, the proportion of japonica genetic components in the experimental indica-japonica hybrid rice breeding materials gradually decreased, while the proportion of indica genetic components relatively increased. Based on the indica / japonica rice attribute determination in Table 1, the percentage of each type of variety in the total sample was calculated. The 6-year average showed that the intermediate-japonica type was the most prevalent, accounting for 55.69%, followed by the semi-japonica type at 25.4%. 12.11% of the varieties were intermediate-type, and 6.81% were intermediate-indica-type. These results indicate that the hybrid rice breeding materials generally maintain a predominantly japonica genetic background, but at the same time, they have shown a certain degree of infiltration of indica genetic components. Looking at the changes in the proportions of each type across years, the proportion of the intermediate-to-indica type increased from an average of 6.59% in the first three years to 7.02% in the last three years; the proportion of the intermediate-to-japonica type increased from an average of 47.95% in the first three years to 63.42% in the last three years; the proportion of the intermediate type decreased from an average of 12.66% in the first three years to 11.56% in the last three years; and the proportion of the japonica type decreased from an average of 32.80% in the first three years to 18.00% in the last three years. This indicates that among the tested varieties, the number of intermediate-to-japonica types increased significantly, the number of japonica types decreased significantly, and the number of intermediate and intermediate-to-indica types increased or decreased slightly. These results demonstrate that the 19 pairs of indica-japonica specific InDel markers can effectively reflect the changes in the genetic composition of indica-japonica hybrid rice materials from different years and can be used to analyze the changing trends of indica and japonica genetic components during the breeding process. However, with the continuous hybridization and selection of materials with different genetic backgrounds during the breeding process, the tested materials gradually showed more complex indica and japonica genetic compositions, especially with an increase in the proportion of intermediate-japonica and other intermediate types of materials. This indicates that the existing marker system still needs to be further supplemented with new genetic markers that can reflect the differences between indica and japonica varieties in order to improve the ability to accurately distinguish materials with similar genetic compositions.

[0039] Table 4. Frequency of Japonica type genes at 19 loci in indica-japonica hybrid rice

[0040] Analysis of the Fj values ​​of 19 InDel loci in 326 tested varieties from 2019 to 2024 revealed significant differences in the contribution of different InDel loci to the genetic composition of indica and japonica rice. Based on the six-year average Fj values, the 19 loci were classified as either indica or japonica. The average Fj values ​​of loci R6M44, R12M27, and R9M42 reached 0.8934, 0.8154, and 0.7968, respectively, exhibiting high japonica genetic characteristics and thus classified as japonica-type loci. The average Fj values ​​of loci R7M7, R3M53, R1M37, and R5M30 ranged from 0.6480 to 0.7449, indicating a preference for japonica-type loci. The average Fj values ​​of loci R2M10, R2M50, R3M10, R4M17, and R6M14... The average Fj value of the five loci (R1M7, R5M13, R8M33, R7M37, and R12M10) ranged from 0.4222 to 0.4943, indicating a moderate bias towards indica rice. The average Fj values ​​of the two loci (R10M17 and R11M23) were 0.2734 and 0.1954, respectively, indicating bias towards indica and indica rice. These results indicate that the 19 InDel loci exhibit different genetic biases towards indica and japonica rice in the indica-japonica hybrid rice population. Among them, there were 12 loci of japonica rice type, japonica rice type and intermediate japonica rice type, accounting for 63.16% of all detected loci. This indicates that the current experimental material has a clear japonica rice genetic background. However, different loci show different indica and japonica genetic compositions, indicating that different chromosomal regions were not selected in a consistent manner during the long-term breeding selection process. Some genomic regions tend to retain japonica type genetic components, while some regions tend to introduce or retain indica type genetic components.

[0041] Analysis of the annual trends in Fj values ​​at various InDel loci from 2019 to 2024 revealed distinct genetic evolution patterns at different loci. Specifically, the Fj values ​​at five loci—R1M7, R5M13, R7M37, R11M23, and R12M27—showed a continuous downward trend, decreasing by 27.3%, 21.8%, 24.5%, 23.9%, and 17.3%, respectively. This indicates a gradual decrease in the frequency of the japonica allele at these loci, while the frequency of the indica allele gradually increased, suggesting that these loci may have been influenced by selection of favorable indica genetic components. The Fj value at the R6M14 locus increased by 22.7%, from 0.4205 in 2019 to 0.5070 in 2024. This indicates that the genetic change at this locus followed the opposite direction to the aforementioned loci, with the proportion of japonica genetic components gradually increasing during breeding, demonstrating a selection trend towards japonica alleles. In addition, the Fj values ​​of eight loci, namely R1M37, R2M10, R2M50, R3M10, R4M17, R5M30, R7M7 and R9M42, changed little over the six years, with a change of no more than 5%, indicating that these loci have been relatively stable in the breeding selection process in recent years.

[0042] In summary, the 19 InDel loci exhibited significant differential genetic evolution characteristics between 2019 and 2024. At some loci, japonica-type genes were gradually replaced by indica-type genes, while at others they maintained or increased japonica-type genetic characteristics, and still others remained relatively stable. These results indicate that the breeding process of indica-japonica hybrid rice does not involve uniform selection across the entire genome, but rather targeted selection in different genetic regions according to breeding objectives. Therefore, relying solely on a limited number of traditional InDel loci may be insufficient to fully reflect the subtle genetic differences in complex indica-japonica hybrid rice materials. Further screening of novel InDel markers that can supplement information from different genetic regions is needed to improve the precise identification capabilities of indica-japonica hybrid rice materials.

[0043] Genetic evolution analysis results of the 19 pairs of InDel markers in Example 1: Using the average Fj values ​​of 19 loci, Origin 2024 software was used to plot the Fj fitting curves for the 19 loci from 2019 to 2024, and the equation and coefficient of determination R of the curves were calculated. 2 To investigate the degree of fit of the fitted curve; Depend on Figure 2-5 It can be seen that the coefficient of determination R of the fitting curves at the nine sites, including R1M37, R3M53, and R5M13, is... 2R² values ​​greater than 0.9 indicate high consistency. The R² values ​​at three loci, R1M7, R5M30, and R6M44, are between 0.8 and 0.9, showing good consistency. The R² values ​​at six loci, R2M10, R2M50, and R3M10, are between 0.6 and 0.8, showing moderate consistency. Only one locus, R4M17, has an R² value of 0.41408, indicating poor consistency. Therefore, it can be concluded that the Fj variations at most InDel loci are not random fluctuations but exhibit certain annual evolutionary patterns, reflecting the selection trends in relevant regions during the breeding of indica-japonica hybrid rice.

[0044] Further analysis of the genetic change patterns at different loci based on the type of fitted equations revealed that some loci exhibited a linear decreasing trend, such as R1M7, R7M37, and R12M27, with their Fj values ​​gradually decreasing with increasing years. This indicates that the frequency of japonica alleles at these loci decreased year by year, while the frequency of indica alleles gradually increased, suggesting that these regions were influenced by selection favoring indica genetic components during breeding. Other loci exhibited a hyperbolic increasing trend, such as R3M53 and R6M14, with their Fj values ​​generally increasing. This indicates that the japonica genetic component at these loci has gradually increased in recent years, suggesting that some regions tend to retain or enrich japonica genetic characteristics during breeding selection. In addition, loci such as R5M13, R6M44, R8M33, and R11M23 showed a hyperbolic downward trend, indicating that the frequency of japonica type genes at these loci gradually decreased; while loci such as R1M37, R2M10, R2M50, R3M10, R5M30, and R9M42 showed fluctuating changes, but the overall trend was not obvious, indicating that the genetic composition of these regions has been relatively stable in the breeding process over the past 6 years.

[0045] In summary, Fj fitting analysis of 19 InDel loci indicates that the changes in the genetic composition of indica-japonica rice in different genomic regions during indica-japonica hybrid rice breeding are not synchronous, but rather exhibit significant locus specificity. At some loci, the japonica genetic component gradually decreases, showing a trend of indica gene introgression; at other loci, the japonica genetic component is maintained or increased; and at still others, the genetic composition remains relatively stable. This suggests that there is directional selection targeting different genomic regions during indica-japonica hybrid rice breeding, rather than synchronous changes in the overall indica-japonica genetic background.

[0046] Analysis of homozygous and heterozygous indica and japonica varieties at 19 loci: Based on the amplified bands at 19 InDel loci, the genotypic composition of 326 indica-japonica hybrid rice materials from 2019 to 2024 was analyzed at various loci. Figure 6-8The results showed that among the 19 InDel loci examined, the indica-japonica heterozygous genotype (Xij) accounted for a high proportion at most loci, with a 6-year average of 63.47%, significantly higher than the homozygous japonica genotype (Xjj, 24.66%) and the homozygous indica genotype (Xii, 11.88%). These results indicate that current indica-japonica hybrid rice materials maintain a high proportion of indica-japonica heterozygosity at most indica-japonica specific InDel loci, suggesting that indica and japonica alleles from different genetic backgrounds combine at most loci during indica-japonica hybridization, forming a relatively obvious heterozygous genetic characteristic. Further analysis of the genotype distribution characteristics at different InDel loci revealed significant differences in the ability of each locus to reflect the indica-japonica heterozygosity status. Among them, the proportions of the Xij type at loci R2M10 and R3M10 reached 95.28% and 93.96%, respectively, exhibiting extremely high indica-japonica heterozygous characteristics. The proportions of the Xij type at loci R1M7, R2M50, R4M17, R7M37, R8M38, and R6M14 all exceeded 78%, indicating that these loci exist mainly in a heterozygous state in indica-japonica hybrid rice materials. Further analysis revealed that the proportion of the Xij type at 11 loci exceeded 60%, indicating that these loci can effectively reflect the simultaneous existence of indica and japonica genetic components in indica-japonica hybrid rice materials, and therefore can serve as important detection loci for characterizing indica-japonica heterozygous genetic characteristics. In addition to the above heterozygous loci, some InDel loci showed obvious homozygous genetic characteristics. Among them, the proportions of the Xjj genotype at loci R6M44, R9M42, and R12M27 reached 83.64%, 65.86%, and 60.82%, respectively, showing a high proportion of pure japonica genotypes, indicating that these loci mainly maintain japonica genetic characteristics in the tested materials. The proportion of the Xii genotype at locus R11M23 reached 62.99%, showing obvious pure indica genotype characteristics, indicating that this locus mainly carries indica genetic information. In addition, some loci simultaneously exhibited high proportions of Xii, Xij, and Xjj genotypes, without showing a clear dominant genotype, indicating that these loci have high polymorphism among materials with different genetic backgrounds. These results show that the 19 InDel loci exhibit different genotype distribution characteristics in the indica-japonica hybrid rice population. Some loci mainly reflect the indica-japonica heterozygous state, some mainly retain the indica or japonica homozygous state, while some loci show high genetic polymorphism. Different types of loci collectively constitute the genetic composition analysis system for indica-japonica hybrid rice, reflecting the indica-japonica attributes of the material from multiple perspectives, such as heterozygosity, the proportion of indica-type inheritance, and the proportion of japonica-type inheritance. This also indicates that relying solely on a single type of InDel locus is insufficient to comprehensively describe the genetic composition of complex indica-japonica hybrid rice materials. Therefore, by adding new InDel loci with different genetic characteristics, the information coverage of the detection system and its ability to distinguish intermediate indica-japonica materials can be further improved.

[0047] Evolutionary analysis of homozygous and heterozygous indica and japonica rice at 19 InDel loci: The evolution of the three genotypes at different loci over 6 years was analyzed using Origin 2024 software. The heterozygous genotype ratios at 11 Xij genotype loci, the homozygous japonica genotype ratios at 3 Xjj genotype loci, and the homozygous indica genotype ratio at 1 Xii genotype locus were plotted. Fitting curves were generated over 6 years, and the coefficient of determination R was calculated. 2 .according to Figure 9-11 We can obtain the analysis results for 11 Xij types and 4 R sites. 2 Those with a value above 0.9 have excellent concordance, namely R1M7, R2M10, R8M33, and R5M30; the three sites R 2 The concordance was good between 0.8 and 0.9 for R1M37, R2M50, and R3M53; the R values ​​at the two sites were... 2 The concordance was moderate between 0.6 and 0.8 for R5M13, R7M37, R3M10, and R4M17; the R values ​​for the other two sites were... 2 Below 0.6, the fit is poor. Using the same method as the Fj fitting curve, in the Xij-type fitting curve, the R1M37 and R2M10 sites show an increasing S1-type hyperbolic change. The R2M50, R4M17, and R7M37 sites show a decreasing S2-type hyperbolic change. The R8M33 site shows an S3-type curve change. The R1M7, R3M10, R3M53, R5M13, and R5M30 sites show a P-type parabolic change. Based on... Figure 12 It can be seen that, for the three Xjj-type fitting results, the R9M42 site R 2 The value was 0.92157, indicating excellent agreement, and the change showed a bicurve fluctuation but with no significant increase or decrease, characteristic of the S3 type. The R12M27 locus... 2 The R² value was 0.64197, indicating moderate agreement and a P-shaped change with a significantly decreasing parabolic curve. The R² value at the R6M44 site was below 0.6, indicating poor agreement. Based on... Figure 13 It can be seen that the fitting results of the Xii type, R11M23 site R 2The coefficient of determination (CCD) was 0.9945, indicating excellent agreement and a hyperbolic decreasing S1-type change. Analysis of the inter-year fitting curves and CCDs for the three genotypes showed that most Xij genotype sites had a high heterozygous gene ratio, but overall showed a decreasing evolutionary trend, with a few showing an increasing or fluctuating trend. The Xjj genotype showed varying trends, with both increasing and decreasing. The Xii genotype showed an increasing evolutionary trend. These results indicate that the homozygous and heterozygous genotypes of the 19 InDel loci exhibited different trends across different years, reflecting differences in the direction and degree of breeding selection in different genomic regions. Therefore, it is necessary to further supplement the InDel markers with novel genetic information to improve the ability to distinguish the genetic composition of indica-japonica hybrid rice materials, especially intermediate-type materials.

[0048] Example 2: Design of 10 pairs of new InDel: Published QTLs related to the differentiation between indica and japonica rice were retrieved from the NCBI database, resulting in 237 QTLs associated with this differentiation. These QTLs cover various traits related to rice fertility, yield, growth period, and plant type. The genomic sequences of each relevant QTL were located in the National Rice Data Center, using the indica rice reference genome (Indica 9311) and the japonica rice reference genome (Nipponbare). The genomic locations corresponding to each QTL were searched in NCBI BLAST, and sequence alignment was performed. InDel sites with insertion or deletion lengths greater than or equal to 15 bp were selected. The selected InDel sequences were imported into the sequence editing and analysis tool, Edit Seq software, along with the corresponding sequences from Nipponbare and 9311, to locate the target InDel site. Using the InDel site as the center, forward and reverse primers were designed approximately 150 bp to either side of the InDel site to ensure that the PCR amplification product completely covered the InDel site. Adjust primer parameters to make the lengths and annealing temperatures of the forward and reverse primers similar, and control the GC content within the normal range. The design parameters include: PCR amplification product length of 100–300 bp, annealing temperature of 55–65℃, and primer GC content of 40%–60%. Complete primer design.

[0049] DNA extraction from rice leaf tissue, primer sequencing and synthesis, PCR reaction procedure and product separation, and determination of indica and japonica rice characteristics were the same as in Example 1. Figure 14 As shown, 10 new InDel marker pairs were obtained, namely INDEL2, INDEL3, INDEL7, INDEL8, INDEL10, INDEL11, INDEL13, INDEL14, INDEL15, and INDEL21. The newly screened 10 InDel molecular marker pairs and their DNA sequences are shown in Table 5.

[0050] Table 5. Newly screened 10 pairs of InDel molecular markers and DNA sequences

[0051] Example 3: Identifying Indica and Japonica rice properties using 19 pairs and 29 pairs of InDel markers: The existing 19 pairs of InDel markers were used to identify the indica / japonica properties of 107 rice varieties. The detection method in Example 1 was used to determine the indica / japonica properties of the 107 rice varieties. Then, 10 newly developed InDel marker pairs were added to the existing 19 pairs of InDel markers, forming a combination of 29 InDel marker pairs. The same detection and indica / japonica property determination methods were then used to re-identify the 107 materials. (Details of the 107 hybrid rice varieties are shown in Appendix Table 2).

[0052] Table 6. Comparison of the results of determining the indica and japonica rice attributes using 19 pairs and 29 pairs of InDel markers.

[0053] The results showed that the original 19 InDel markers identified 5 typical indica rice varieties, 30 indica rice varieties, 9 varieties with a slight indica hull, 8 varieties with a moderate indica hull, 10 varieties with an intermediate phenotype, 35 varieties with a moderate japonica hull, and 10 varieties with a moderate japonica hull. The results of the 29 InDel marker tests showed that the number of typical indica rice varieties remained at 5, the number of indica rice varieties increased from 30 to 31, the number of varieties with a slight indica hull decreased from 9 to 7, the number of varieties with a moderate indica hull remained at 8, the number of varieties with an intermediate phenotype decreased significantly from 10 to 2, the number of varieties with a moderate japonica hull increased from 35 to 41, and the number of varieties with a moderate japonica hull increased from 10 to 13. These results indicate that, while maintaining a largely consistent overall classification framework for indica and japonica rice attributes, adding 10 newly developed InDel marker pairs can further differentiate some materials in the original 19-marker system that were in an intermediate state of indica-japonica differentiation into types such as intermediate-japonica or semi-japonica, thereby reducing the number of intermediate-type materials whose indica-japonica attributes are difficult to determine. This demonstrates that the 10 new InDel marker pairs can supplement the genetic difference information provided by the original 19 marker pairs, improving the ability to further distinguish materials with similar indica-japonica genetic backgrounds and in an intermediate state of indica-japonica differentiation. The 10 new InDel marker pairs do not simply increase the number of detection sites, but rather supplement the genetic identification information of intermediate-type materials provided by the original 19 InDel marker pairs, enabling further classification of some materials whose indica-japonica attributes could not be further determined.

[0054] Cluster analysis of 19 InDel pairs and 29 InDel pairs: The UPGMA method was used to cluster the tested materials based on genetic similarity. First, PCR amplification was performed on the rice materials using 19 or 29 pairs of InDel primers, followed by electrophoresis to obtain banding information at different InDel loci for each material. Then, the electrophoresis results were converted into digital data. Each pair of InDel primers corresponded to one locus, and each polymorphic band represented one allele; the presence of a band at the same migration location was recorded as "1", the absence of a band as "0", and the absence of a band as "9". Next, the genetic similarity coefficient GS and genetic distance GD between two materials were calculated based on the banding data.

[0055] Calculation formula:

[0056] Where Mx and My represent the total number of fragments in the two materials, and Mxy represents the number of common fragments between the two materials; then GD is calculated based on GS. Based on the genetic similarity coefficient between different materials, UPGMA (unweighted group average method) is used for clustering, gradually grouping genetically similar materials into the same group, and finally forming a tree-like clustering result, which is used to observe the genetic relationship and genetic differentiation between materials.

[0057] Test results: Nineteen pairs of InDel primers were used to perform PCR amplification and detection on 107 rice varieties. The results are as follows: Figure 15 As shown, the calculated genetic similarity coefficient was R=0.6888. Cluster analysis of the tested materials using the unweighted average method revealed two main genetic groups, I and II. Group I contained 43 materials, further subdivided into 5 subgroups. The materials in Group I exhibited typical indica, indica, and slightly indica characteristics, with no clearly japonica-type materials. Subgroups i and ii contained 22 and 8 materials respectively, mainly composed of typical indica and indica rice; subgroups iii and iv contained 5 and 7 materials respectively, mainly composed of indica and slightly indica-type materials; subgroup v contained only 1 material slightly indica-type material. The above results indicate that the genetic clustering results obtained based on 19 pairs of InDel markers are basically consistent with the results of the indica and japonica attribute determination, suggesting that the marker combination can reflect the genetic relationship between indica-type materials well.

[0058] Main group II contains 64 materials, further divided into 8 subgroups. Subgroup VII mainly consists of materials with a slight tendency towards japonica varietal, while also including a small number of intermediate and slightly indica varietal materials. Subgroups IX and X mainly consist of materials with a slight tendency towards japonica and a slight tendency towards japonica varietal, and their clustering results are largely consistent with the indica-japonica varietal attribute determination results. However, subgroups VI and VII contain materials with a slight tendency towards indica, a slight tendency towards indica, an intermediate tendency, a slight tendency towards japonica, and a slight tendency towards japonica. The clustering of materials with different indica-japonica attributes within the same genetic subgroup indicates that the genetic information obtained based on 19 pairs of InDel markers has limited ability to distinguish materials with similar indica-japonica genetic backgrounds. In summary, the 19 InDel markers can effectively distinguish materials with a high degree of indica-japonica differentiation. The clustering results are generally consistent with the attribute judgment results based on the indica-japonica index. However, hybrid rice materials in the intermediate state of indica-japonica differentiation cannot be further distinguished.

[0059] according to Figure 16 It can be seen that PCR amplification was performed on 107 rice varieties using 29 pairs of InDel markers. The genetic similarity coefficient calculated based on the amplified band information was R=0.64. Cluster analysis of the tested materials using the unweighted average method divided the 107 materials into two main genetic groups, I and II. Main group I contains 43 varieties, further subdivided into 7 subgroups. Within main group I, subgroup i contains 13 varieties, whose indica-japonica characteristics are mainly typical indica and indica rice; subgroup ii contains 18 varieties, mainly exhibiting indica rice characteristics; and subgroup iii contains 8 varieties, mainly including indica rice and materials with a partial indica rice morphology. Furthermore, subgroups iv, v, vi, and vi each contain only one variety, corresponding to the indica type Zhongzheyou 8, the semi-indica type Liangyou 16 and A Liangyou 87, and the intermediate semi-indica type Zhongliangyou 879, respectively. This result indicates that after cluster analysis using 29 InDel marker combinations, the materials in main group I still predominantly exhibit an indica genetic background, which is largely consistent with the attribute determination results obtained based on the indica-japonica index.

[0060] Group II comprises 64 varieties, further divided into 11 subgroups. Subgroup IX contains 24 varieties, mainly composed of intermediate-to-japonica and semi-japonica types, including 15 intermediate-to-japonica and 6 semi-japonica types; Subgroup X contains 3 varieties, all of which are semi-japonica; Subgroup Z contains 19 varieties, of which 15 are intermediate-to-japonica, 1 is intermediate, and 3 are intermediate-to-indica; Subgroups IX and IXIII each contain 4 varieties, mainly of intermediate-to-japonica and semi-japonica types, with a small amount of intermediate-to-indica type material; Subgroup IXIV contains 4 varieties, including 2 intermediate-to-indica types and 1 intermediate-to-japonica and 1 semi-japonica type. In addition, some materials formed their own subgroups due to their unique genetic characteristics, including the indica-biased Zheheyou 290, the intermediate indica-biased Huazhongyou 68, and the intermediate japonica-biased Jiafengyou 913 and Yongyou 112; Jiaheyou 7245 and Yongyou 181 clustered into the same subgroup, exhibiting intermediate and intermediate japonica types, respectively. Comparing the two results, both marker systems were able to classify the 107 materials into two main genetic groups, and the number of materials in each main group remained consistent. Therefore, the addition of 10 InDel marker pairs did not change the overall classification trend of the original indica-japonica genetic structure, but rather added new genetic difference information based on the original 19 marker pairs, improving the ability to distinguish genetic relationships among the materials.

[0061] Compared to 19 InDel marker pairs, using 29 InDel marker pairs further subdivided the original 5 subgroups into 7 subgroups in main group I, indicating that the addition of 10 InDel marker pairs can further identify genetic differences within indica-type materials. In main group II, the 19 InDel marker pairs clustered 64 materials, including 10 intermediate-type materials; while with 29 InDel marker pairs, main group II still contains 64 materials, but the number of intermediate-type materials has decreased to 2. Some materials that were previously difficult to further classify have been further distinguished into intermediate-japonica type or other indica-japonica attribute types. This shows that the addition of 10 InDel marker pairs can effectively supplement the insufficient genetic information in the indica-japonica transition region of the existing 19 marker pairs, and improve the ability to identify materials with similar indica-japonica genetic backgrounds.

[0062] The above embodiments demonstrate that this invention combines 10 newly developed InDel marker pairs with 19 existing InDel marker pairs for indica-japonica rice identification, forming a 29-pair InDel marker system. This improves the distinguishing ability for indica-japonica rice attribute identification, enhances the precision of genetic cluster analysis, and strengthens the identification ability for intermediate indica-japonica hybrid rice materials. The 29-pair InDel marker combination of this invention has a higher distinguishing ability for indica-japonica rice attributes and can be used for the fine classification of indica-japonica hybrid rice materials and the screening of breeding materials.

[0063] It should be noted that the above embodiments and comparative examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the present invention.

[0064] sequence list

[0065] Appendix 1. Indica-japonica hybrid rice characteristics in regional trials in Zhejiang Province, 2019-2024.

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[0070] Appendix 2 Results of the determination of indica and japonica rice characteristics of 107 varieties using 19 pairs of InDel markers and 29 pairs of InDel markers

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[0072] .

Claims

1. A method for identifying indica-japonica hybrid rice based on InDel marker combinations, characterized in that, Includes the following steps: S1. Extract genomic DNA from the indica-japonica hybrid rice material to be tested; S2. PCR amplification of the genomic DNA using InDel marker combinations to obtain genotype information of the test material at each InDel locus; S3. Determine the InDel genotype of the material to be tested based on the amplification results; S4. Calculate the indica genetic component and / or japonica genetic component of the material to be tested based on the InDel genotype information, and determine the indica-japonica type of the indica-japonica hybrid rice material to be tested based on the calculation results.

2. The method for identifying indica-japonica hybrid rice based on InDel marker combinations according to claim 1, characterized in that, The InDel sites include 19 pairs of existing InDel markers for identifying indica and japonica rice and 10 pairs of newly developed InDel markers.

3. The method for identifying indica-japonica hybrid rice based on InDel marker combinations according to claim 2, characterized in that, The method for obtaining the 10 pairs of newly developed InDel tags is as follows: S1. Screening for QTL regions with InDel variation among 237 QTL regions of rice with differences between indica and japonica varieties; S2. Design InDel primers based on the QTL regions with InDel mutations; S3. Perform PCR amplification and detection on typical indica rice and typical japonica rice materials according to the InDel primers; The 10 newly developed InDel pairs were obtained by screening based on differences in amplified fragments.

4. The method for identifying indica-japonica hybrid rice based on InDel marker combinations according to claim 2, characterized in that, The 10 pairs of newly developed InDel tags include: INDEL2, INDEL3, INDEL7, INDEL8, INDEL10, INDEL11, INDEL13, INDEL14, INDEL15, and INDEL21.

5. The method for identifying intermediate indica-japonica hybrid rice based on InDel marker combinations according to claim 4, characterized in that, The newly developed InDel primers include those with the nucleotide sequence Seq ID NO.1-20.

6. The method for identifying indica-japonica hybrid rice based on InDel marker combinations according to claim 3, characterized in that, The typical japonica rice materials include Qiuguang, Zaoshajing, and Balali; the typical indica rice materials include Aijiao Nante, Nanning 11, and IR36.

7. The method for identifying indica-japonica hybrid rice based on InDel marker combinations according to claim 1, characterized in that, The DNA extraction of S1 includes: Leaf samples were collected from rice plants at the 3-4 leaf stage. After being crushed, DNA extraction solution was added for lysis. The lysate was then extracted with chloroform, centrifuged, and precipitated with ethanol to obtain genomic DNA.

8. The method for identifying indica-japonica hybrid rice based on InDel marker combinations according to claim 1, characterized in that, The PCR amplification in S2 includes: The InDel primers were used to amplify the genomic DNA of the test material by PCR to obtain the amplified product of the target InDel fragment. The PCR amplification system included template DNA, DNA polymerase, forward primer, reverse primer, and nuclease-free water. The PCR amplification program was as follows: 35 cycles of amplification were performed after pre-denaturation at 94°C. Each cycle included denaturation at 94°C, annealing at 53°C, and extension at 72°C. After the cycle, an extension at 72°C was performed. The PCR amplification products were separated by agarose gel electrophoresis.

9. The method for identifying indica-japonica hybrid rice based on InDel marker combinations according to claim 1, characterized in that, The determination of the InDel genotype of the material to be tested in S3 is as follows: the genotype information of the corresponding InDel locus is obtained based on the presence and type of the electrophoretic band; for samples that do not show an amplified band, PCR amplification and electrophoresis detection are repeated, and if no amplified band is found after repeated detection, it is recorded as a missing allele.

10. The method for identifying indica-japonica hybrid rice based on InDel marker combinations according to claim 1, characterized in that, The calculation in S4 is as follows: calculate the indica rice gene frequency Fi and japonica rice sex gene frequency Fj of the test material based on the genotype information of each InDel site, and determine the indica and japonica attributes of the test material according to the classification criteria corresponding to Fi and Fj.

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