Method for identifying indica-japonica rice varieties and quantifying indica-japonica rice component proportion in rice
Through genome alignment and PCR sequencing methods of indica and japonica differentiation SNPs data sets, rice varieties types and quantification of indica and japonica components were accurately identified, which solved the inaccuracy and complexity of the traditional methods, and achieved efficient breeding and variety identification.
Patent Information
- Application Number
- CN202510464121.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-14
- Publication Date
- 2025-08-08
AI Technical Summary
In the prior art In rice breeding and variety identification, traditional morphological identification methods are greatly affected by the environment and are inaccurate, and the molecular marking methods are complex in operation or high in cost, making it difficult to accurately identify rice varieties and quantify the proportion of indica and japonica ingredients.
The data set of indica and japonica differentiated SNPs was used, and primers were designed for sequencing through genome comparison and PCR amplification, and the number of indica and japonica SNPs was counted, and the data set of indica and japonica differentiated SNPs was constructed to accurately identify rice varieties and quantify indica and japonica components.
Efficient and accurate identification of rice varieties and quantification of indica and japonica ingredients have been achieved, providing scientific basis for breeding and variety identification, shortening the breeding cycle and reducing planting risks.
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Figure CN120452550A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of molecular biotechnology, and in particular to a method for identifying indica and japonica rice varieties and quantifying the proportion of indica and japonica components in rice. Background Art
[0002] Rice, one of the world's most important food crops, is divided into two main subspecies: indica and japonica. Accurately identifying the type of cultivated rice and quantifying the ratio of its indica to japonica components are crucial for rice breeding and variety identification.
[0003] In rice breeding, understanding the indica and japonica composition of rice varieties helps breeders select parents for hybridization, thereby cultivating new varieties with superior traits. For example, by properly pairing indica and japonica parents, the high-yield characteristics of indica rice can be combined with the high-quality characteristics of japonica rice, improving rice yield and quality. Furthermore, in terms of variety identification, accurately determining the type of rice variety and the ratio of indica and japonica components can effectively prevent counterfeit and substandard seeds from entering the market, protecting farmers' interests.
[0004] However, traditional methods for identifying cultivated rice types and quantifying the ratio of indica to japonica rice components have numerous limitations. Previous methods, which rely on morphological characteristics, are significantly affected by environmental factors and can be difficult to accurately distinguish between varieties with similar morphological characteristics. For example, morphological characteristics such as plant height and leaf shape can vary under different growing environments, leading to inaccurate identification results.
[0005] With the advancement of molecular biology techniques, the use of molecular markers for rice variety identification has become a research hotspot. However, some existing molecular marker methods still need to be improved in terms of accuracy and efficiency. Some methods use a limited number of molecular markers, which cannot fully and accurately reflect the genetic characteristics of rice varieties, resulting in large errors in identification results. Furthermore, some methods are complex and costly, making them difficult to widely apply in practical production.
[0006] Previous technologies for obtaining information on rice variation have had certain shortcomings. Data sources were relatively limited, lacking coverage for a sufficient number of rice samples, resulting in an incomplete understanding of rice genetic variation. Furthermore, early sequencing technologies were limited in data quality and throughput, making it impossible to obtain high-quality, large-scale genomic data, thus hindering in-depth exploration of rice genetic information.
[0007] Therefore, there is an urgent need in this field for a method that can efficiently and accurately utilize indica-japonica differentiation SNPs to assist in identifying cultivated rice types and quantify the proportion of indica-japonica components, so as to overcome the shortcomings of existing technologies and meet the needs of practical work such as rice breeding and variety identification. Summary of the Invention
[0008] In order to solve the above technical problems, the present invention provides a method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice. This method can not only accurately identify the type of rice variety, but also quantify the indica and japonica components of rice, providing an important basis for rice breeding and variety identification. By quantifying the indica and japonica ratio, breeders can accurately understand the genetic composition of the material, better utilize hybrid vigor, shorten the breeding cycle, and accelerate the breeding process. Quantifying the indica and japonica components also helps to more accurately predict the adaptability of new varieties in different ecological environments, scientifically guide breeding, and reduce planting risks; at the same time, this method also provides a scientific basis for germplasm resource protection, variety identification, and intellectual property protection.
[0009] To this end, the present invention provides the following technical solutions:
[0010] In a first aspect, the present invention provides, in an optional embodiment, a method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice, comprising the following steps:
[0011] S1: The genome data of the indica and japonica rice sample populations were aligned with the reference genome Nipponbare to obtain the SNPs sites of each sample. Then, after filtering out the SNPs sites with missing values, the SNPs with a fixed frequency of ≥98% and a linkage disequilibrium rate r in the indica and japonica rice sample populations were selected. 2 The indica-japonica differentiation SNPs with a % score < 0.5 and located in the gene coding region were constructed as the indica-japonica differentiation SNPs dataset;
[0012] S2: Based on the indica-japonica differentiation SNPs dataset, primers are designed for the specified fragments of the test rice, PCR amplification and sequencing are performed, and the genotypes of the corresponding sites in the indica-japonica differentiation SNPs dataset are extracted. The number or ratio of indica and japonica SNPs in the test rice are counted respectively, thereby determining the ratio of indica rice components to japonica rice components in the test rice.
[0013] Preferably, the indica-japonica differentiation SNPs dataset contains 624 SNPs sites.
[0014] Preferably, the number of the indica rice sample group is 212, and the number of the japonica rice sample group is 96. The software used for the sequence alignment is MUMMER. The method for sequence alignment comprises: using the "nucmer" program in the MUMMER software package to align the genomes of all samples with the reference genome Nipponbare to generate a preliminary alignment result; then using the "delta-filter" program in the MUMMER software package to optimize the preliminary alignment result; finally, based on the optimized comparison result, using the "show-snps" program in the MUMMER software package and setting the '-C-I' parameter to identify the SNP sites between the sample and the reference genome. When using the "delta-filter" program in the MUMMER software package, it is necessary to set the parameter "-1" to filter out mutually unique matching regions to obtain high-precision alignment results.
[0015] Preferably, when determining the ratio of indica rice components to japonica rice components in the rice to be tested, the number of effective sites of indica and japonica SNPs in the rice to be tested needs to be greater than 436.
[0016] Preferably, the calculation formula for determining the ratio of indica rice components to japonica rice components in the rice to be tested is:
[0017] Indica rice component = the number of effective indica SNPs in the tested rice / (the number of effective indica SNPs in the tested rice + the number of effective japonica SNPs in the tested rice) × 100%;
[0018] Japonica rice component = the number of effective japonica SNPs in the rice to be tested / (the number of effective indica SNPs in the rice to be tested + the number of effective japonica SNPs in the rice to be tested) × 100%.
[0019] Preferably, after determining the ratio of indica rice components to japonica rice components in the rice to be tested, the method further includes determining the indica and japonica varieties of the rice to be tested;
[0020] The method for determining the indica and japonica rice varieties to be tested is:
[0021] If the indica component is >90%, the sample to be tested is considered to be pure indica rice; if the japonica component is >90%, the sample to be tested is considered to be pure japonica rice; if the indica component is >70% and <90%, the sample to be tested is considered to be indica rice containing japonica components; if the japonica component is >70% and <90%, the sample to be tested is considered to be japonica rice containing indica components; if the indica component is >50% and <70% or the japonica component is >50% and <70%, the sample to be tested is considered to be indica-japonica hybrid rice.
[0022] Compared with the prior art, the present invention has one of the following beneficial effects:
[0023] 1. The method provided by the present invention can not only accurately identify the type of rice varieties, but also quantify the indica and japonica components of rice, providing an important basis for rice breeding and variety identification. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is a flow chart of the method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice in Example 1 of the present invention;
[0025] Figure 2 This is a genotype heat map of 624 indica-japonica differentiation sites in rice varieties Shuhui 527, Longjing 4302A, Jiahe 212A, and Shuohui 70 in Example 1 of the present invention;
[0026] Figure 3 This is a schematic diagram of the results of quantifying the ratio of indica and japonica components in varieties Shuhui 527, Longjing 4302A, Jiahe 212A, and Shuohui 70 in Example 1 of the present invention;
[0027] Figure 4 These are the results of indica and japonica variety identification of Shuohui 70 using the indica and japonica differentiation dataset provided by the present invention in Example 1 of the present invention and the results of indica and japonica variety identification of Shuohui 70 using the rice variety identification method provided by the prior art in Example 2. DETAILED DESCRIPTION
[0028] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0031] Example 1
[0032] See also Figure 1 This embodiment provides a method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice, comprising the following steps:
[0033] (1) The genome data of 212 indica rice and 96 japonica rice sample groups were sequenced using MUMMER software to obtain SNPs. The genomes of all rice samples were aligned with the reference genome Nipponbare using the "nucmer" program in the MUMMER software package to generate preliminary alignment results. The preliminary alignment results were then optimized using the "delta-filter" program in the MUMMER software package, and the parameter "-1" was used to filter out mutually unique matching regions to obtain high-precision alignment results. Based on the optimized alignment results, the "show-snps" program in the MUMMER software package was finally used with the '-C-I' parameter to identify the SNPs between the sample and the reference genome.
[0034] (2) After filtering out SNPs with missing values, the SNPs with a fixed frequency of ≥98% and a linkage disequilibrium rate r in the indica and japonica rice sample populations were 2 The differential SNPs with a p-value < 0.5 and located in the coding regions of genes were constructed as the Indica-Japonica differentiation SNP dataset, which contains 624 loci.
[0035] (4) According to the indica-japonica differentiation SNPs dataset, primers were designed for Shuhui 527, Longjing 4302A, Jiahe 212A, and Shuohui 70, and the designated fragments were PCR amplified and sequenced. SNPs were identified using Nipponbare as the reference genome. The genotypes of the corresponding sites of the rice varieties Shuhui 527, Longjing 4302A, Jiahe 212A, and Shuohui 70 were extracted according to the indica-japonica differentiation SNPs dataset obtained in step (2). The number of indica and japonica SNPs in the genotype determination results of the above rice varieties was counted respectively. That is, there were 624 indica SNPs in Shuhui 527 and 624 indica SNPs in Longjing 4302A. There are 623 indica SNPs and 1 japonica SNPs. In Jiahe 212A, there are 624 japonica SNPs, and in Shuohui 70, there are 104 indica SNPs and 520 SNPs, so as to determine the ratio of indica and japonica components in the tested rice. That is, in Shuhui 527, the indica component is 100% and the japonica component is 0%. In Longjing 4302A, the indica component is 99.84% and the japonica component is 0.16%. In Jiahe 212A, the indica component is 0% and the japonica component is 100%. In Shuohui 70, the indica component is 16.67% and the japonica component is 83.33%. The results are shown in Figure 2-4 .
[0036] Example 2
[0037] Taking Shuohui 70 as an example, this variety is a japonica rice with indica ancestry. Its genetic background is mainly japonica components, but contains a small amount of indica components. The indica-japonica differentiation data set provided by this patent was used to measure Shuohui 70. For detailed steps, please refer to Example 1. The results showed that its japonica ancestry ratio was 83.33%, which is highly consistent with its known genetic background. The 957-SNP site (i.e., patent CN113265485A, a method for predicting the degree of indica-japonica differentiation of rice, SNP site combination and its application) was used to measure Shuohui 70. The results showed that the japonica ancestry ratio of this variety was only 56.8%, which was significantly lower than the actual situation of this variety. The 40-SNP site method (i.e., literature Zheng, XH, Ye, JH, Cheng, CP, Wei, XH, Ye, XF and Yang, YL, 2021. Xian-geng identification by SNP markers in Oryza sativa L.) was used to determine the japonica ancestry of Shuohui 70. The results showed that the japonica ancestry ratio of this variety was only 50%, which was significantly lower than the actual situation of this variety. This result shows that the 40-SNP site and 957-SNP site determination methods have a large deviation in identifying the indica and japonica component ratios of Shuohui 70 and cannot accurately reflect its genetic background (see Figure 4 ).
[0038] Therefore, the indica-japonica differentiation dataset provided by this patent can more comprehensively and effectively cover the indica-japonica differentiation sites in the rice genome, thereby more accurately reflecting the proportion of indica-japonica components in the variety, especially for identifying rice varieties with mixed bloodlines, and has wide applicability and reliability.
[0039] Application Examples
[0040] After identifying the ratio of indica to japonica components in the rice to be tested, the indica-japonica typing of the rice to be tested is determined. If the rice is pure japonica or contains indica components, the rice to be tested is bred using the same breeding methods as japonica rice. If the rice is pure indica or contains japonica components, the rice to be tested is bred using the same breeding methods as indica rice. If the rice is an indica-japonica hybrid, the heterosis of the rice to be tested is utilized to determine its parental origins, conduct systematic breeding, and discover superior recombinant types. This method can quickly determine the indica-japonica typing of rice based on early (seedling) leaf samples, avoiding misjudgments that can occur when relying solely on phenotypes. This can shorten the breeding cycle and accelerate the breeding process.
[0041] Although the principles of the present invention have been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent transformations and simple substitutions based on the technical solution of the present invention fall within the scope of protection of the present invention.
Claims
1. A method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice, characterized in that: The following steps are involved: S1: The genome data of the indica and japonica rice sample populations were aligned with the reference genome Nipponbare to obtain the SNPs sites of each sample. Then, after filtering out the SNPs sites with missing values, the SNPs with a fixed frequency of ≥98% and a linkage disequilibrium rate r in the indica and japonica rice sample populations were selected. 2 The indica-japonica differentiation SNPs with a % score < 0.5 and located in the gene coding region were constructed as the indica-japonica differentiation SNPs dataset; S2: Based on the indica-japonica differentiation SNPs dataset, primers are designed for the specified fragments of the test rice, PCR amplification and sequencing are performed, and the genotypes of the corresponding sites in the indica-japonica differentiation SNPs dataset are extracted. The number or ratio of indica and japonica SNPs in the test rice are counted respectively, thereby determining the ratio of indica rice components to japonica rice components in the test rice.
2. The method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice according to claim 1, characterized in that: The indica-japonica differentiation SNPs dataset contains 624 SNPs sites.
3. The method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice according to claim 1, characterized in that: The number of the indica rice sample groups is 212, and the number of the japonica rice sample groups is 96.
4. The method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice according to claim 1, characterized in that: The software used for sequence alignment is MUMMER.
5. The method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice according to claim 4, characterized in that: The method for sequence alignment comprises: The genomes of all samples were aligned with the reference genome Nipponbare using the "nucmer" program in the MUMMER software package to generate preliminary alignment results; The preliminary alignment results were then optimized using the "delta-filter" program in the MUMMER software package; Finally, based on the optimized comparison results, the "show-snps" program in the MUMMER software package was used with the '-C-I' parameter set to identify the SNP sites between the sample and the reference genome.
6. The method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice according to claim 5, characterized in that: When using the "delta-filter" program in the MUMMER software package, you need to set the parameter "-1" to filter out mutually unique matching regions to obtain high-precision alignment results.
7. The method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice according to claim 1, characterized in that: When determining the ratio of indica rice components to japonica rice components in the tested rice, the number of effective sites of indica and japonica SNPs in the tested rice must be greater than 436.
8. The method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice according to claim 1, characterized in that: The calculation formula for determining the ratio of indica rice components to japonica rice components in the rice to be tested is: Indica rice component = the number of effective indica SNPs in the tested rice / (the number of effective indica SNPs in the tested rice + the number of effective japonica SNPs in the tested rice) × 100%; Japonica rice component = the number of effective japonica SNPs in the rice to be tested / (the number of effective indica SNPs in the rice to be tested + the number of effective japonica SNPs in the rice to be tested) × 100%.
9. The method for identifying indica and japonica rice varieties and quantifying the ratio of indica and japonica components in rice according to claim 8, characterized in that: After determining the ratio of indica rice components to japonica rice components in the rice to be tested, the method also includes determining the indica and japonica varieties of the rice to be tested; The method for determining the indica and japonica rice varieties to be tested is: If the indica component is >90%, the sample to be tested is considered to be pure indica rice; if the japonica component is >90%, the sample to be tested is considered to be pure japonica rice; if the indica component is >70% and <90%, the sample to be tested is considered to be indica rice containing japonica components; if the japonica component is >70% and <90%, the sample to be tested is considered to be japonica rice containing indica components; if the indica component is >50% and <70% or the japonica component is >50% and <70%, the sample to be tested is considered to be indica-japonica hybrid rice.