Specific primer pair for identifying oryza longistaminata, oryza sativa, hybrid offspring of oryza sativa and oryza longistaminata and application thereof

Abstract

This invention relates to the field of agricultural technology, specifically providing a specific primer pair for identifying hybrid offspring of wild rice, cultivated rice, and cultivated rice and wild rice, and its application. Genomic DNA is extracted from rice leaves. Using this DNA as a template and the sequences corresponding to SEQ ID NO:3 and SEQ ID NO:4 as specific primers, PCR amplification is performed to obtain amplified fragments. After electrophoresis, fragments showing only a 146 bp major band are identified as wild rice, while those showing only a 155 bp major band are identified as cultivated rice. Fragments that simultaneously amplify both 155 bp and 146 bp fragments are identified as hybrid offspring of cultivated rice and wild rice. This invention can be applied to molecular marker detection and polymorphism detection in hybrid populations of wild rice and cultivated rice, facilitating rapid screening in distant hybridization breeding and improving breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and plant molecular genetics breeding, and relates to a specific primer pair for identifying hybrid offspring of wild rice with long male reproductive organs, cultivated rice, cultivated rice and wild rice with long male reproductive organs, and their application. Background Technology

[0002] Approximately 27 species of wild rice have been discovered worldwide, with three found in China: common wild rice (Oryza rufipogon Griff.), medicinal wild rice (Oryza officinalis Wall.), and warty wild rice (Oryza meyeriana Nees.). Common wild rice shares the same AA genome as cultivated rice (Oryza sativa L.), indicating a close phylogenetic relationship. Medicinal wild rice (CC genome) and warty wild rice (GG genome) have different chromosome sets from cultivated rice, suggesting a more distant phylogenetic relationship. Common wild rice has played a significant role and achieved remarkable results in rice breeding in my country. To achieve new breakthroughs in rice breeding, selecting wild rice species with non-AA genomes is a novel approach. Wild rice possesses unique genetic traits, such as tall stature, robust stems, high photosynthetic efficiency, high resistance to various pests and diseases, cold tolerance, and tolerance to poor soil conditions. These traits hold promising potential for the development of new rice varieties.

[0003] Jiao et al. (2022) created the salt-tolerant introduction line 'IL363' of Oryza longistaminata using '9311' as the recipient. Their research revealed that Na+ transport in the 'IL363' germplasm may be inhibited or that it possesses a better salt excretion mechanism. Xu et al. (2020) used a chromosome segment replacement line population with 9311 as the recipient parent and common wild rice as the donor parent as experimental materials to screen for excellent salt-tolerant germplasm CSSL23 and CSSL153 at the seedling stage. Ji et al. (2023) discovered a salt-tolerant material, E11-9, by testing the salt tolerance of five Oryza longistaminata varieties. Through hybridization of E11-9 with the local Asian cultivated rice variety Hainan Red Rice and subsequent multi-generational selection, a line with significantly better salt tolerance than Hainan Red Rice was created. These studies indicate that the genetic resources of African Oryza longistaminata have practical application value in improving the salt tolerance of Asian cultivated rice.

[0004] Wild rice is typically crossbred with cultivated rice through distant hybridization, followed by backcrossing and self-pollination to cultivate new varieties. Rapid selection of target materials is crucial during the breeding process. Conventional breeding is time-consuming, usually requiring 3 to 6 generations to obtain homozygous stable lines. However, marker-assisted selection (MAG) can effectively improve breeding efficiency, reducing workload and time by at least half. MAG technology uses molecular markers closely linked to or co-segregating with the target gene to directly screen target DNA regions. Because it is unaffected by environmental factors, it improves the reliability and efficiency of selection. Since cultivated rice and wild rice (Oryza sativa) are distantly related and have significant genomic sequence differences, some SSR molecular markers in cultivated rice cannot be directly applied to wild rice. Therefore, it is necessary to develop molecular markers applicable to medicinal wild rice through simple and rapid methods. Especially in the process of distant hybridization between wild rice and cultivated rice, developing molecular markers that can simultaneously detect both wild rice and cultivated rice is crucial. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing technologies, such as the lack of molecular marker identification for wild rice and the inability to directly apply SSR molecular markers of cultivated rice to wild rice. It provides a combination of molecular marker amplification primers that can simultaneously detect wild rice and cultivated rice, and their application in the detection of hybrid populations and polymorphism detection. It also provides a method for accurately distinguishing wild rice, cultivated rice, and their hybrid offspring, providing an efficient molecular marker-assisted tool for distant hybridization breeding of wild rice and cultivated rice, and improving breeding efficiency.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a specific primer pair for identifying hybrid offspring of wild rice, cultivated rice, and cultivated rice and wild rice, consisting of a forward primer F with the nucleotide sequence shown in SEQ ID NO. 3 and a reverse primer R with the nucleotide sequence shown in SEQ ID NO. 4.

[0008] Secondly, this invention provides the application of the specific primer pair described above in identifying hybrid offspring of wild rice, cultivated rice, and cultivated rice and wild rice with long male reproductive organs. The specific steps of the application are as follows:

[0009] Step 1: Extract genomic DNA from the rice germplasm resources to be identified;

[0010] Step 2: Using the genomic DNA extracted in Step 1 as a template, perform PCR amplification using the specific primer pair described above;

[0011] The PCR amplification system is 10 μL, containing 50 ng of DNA, 0.5 μL each of forward primer F and reverse primer R, 5 μL of 2×Taq PCR Master Mix, and the remaining volume is supplemented with ddH2O.

[0012] The PCR amplification program is as follows: pre-denaturation at 94℃ for 10 min; denaturation at 94℃ for 30 sec, annealing at 55℃ for 30 sec, extension at 72℃ for 30 sec, for a total of 35 cycles; and finally extension at 72℃ for 10 min, followed by cooling and storage at 16℃.

[0013] Step 3, Electrophoresis detection: The PCR amplification products were detected by 8% polyacrylamide gel electrophoresis. The gel formulation and electrophoresis operation were performed according to the standard method. After electrophoresis, the products were fixed and colored, and the band characteristics were observed and recorded.

[0014] A third aspect of the present invention provides a method for distinguishing between wild rice with long male reproductive organs and cultivated rice, the method comprising the following steps:

[0015] 1. Genotype detection: Following steps one to three in the above application, perform PCR amplification and electrophoresis detection on the genomic DNA of the rice to be tested;

[0016] 2. Result Interpretation: Statistical analysis of the genotype band characteristics of the rice samples:

[0017] If a 155 bp band is amplified, it is identified as cultivated rice;

[0018] If a 146 bp band is amplified, it is identified as wild rice with long male reproductive function;

[0019] If both 155 bp and 146 bp bands are amplified simultaneously, it is identified as a hybrid offspring of wild rice and cultivated rice.

[0020] The PCR amplification products were detected by 8% polyacrylamide gel electrophoresis. The gel formulation and electrophoresis operation were performed according to standard methods. After electrophoresis, the products were fixed and colored. The band characteristics were observed and recorded: if there was only one 155 bp electrophoretic band, the rice to be identified was cultivated rice; if there were two electrophoretic bands of 146 bp and 155 bp, the rice to be identified was a hybrid offspring of cultivated rice and wild rice with long male tassels.

[0021] The beneficial effects of this invention are:

[0022] 1. High specificity: The specific primer pairs provided by this invention can accurately distinguish between cultivated rice such as indica rice and japonica rice and wild rice with long male tassels, and can also accurately identify the hybrid offspring of the two. The bands are clear and highly recognizable, avoiding the subjectivity and error of traditional identification methods.

[0023] 2. Simple and efficient operation: The PCR amplification reaction system is simple and easy to prepare, the reaction conditions are mild and easy to control, the 8% polyacrylamide gel electrophoresis detection process is mature, the whole detection process is short and the identification results can be obtained quickly.

[0024] 3. High breeding application value: It can be directly applied to the molecular marker-assisted selection of distant hybrids between wild rice and cultivated rice, which can effectively shorten the breeding cycle, reduce the breeding workload of 3-6 generations, and reduce the working time and cost by at least half. It provides key technical support for breeding new cultivated rice varieties with excellent traits of wild rice (such as salt tolerance and stress resistance).

[0025] 4. Wide applicability: The primer combination and detection method are not affected by environmental factors and are applicable to different genotypes of long-male wild rice, cultivated rice and hybrid offspring. They can be widely used in molecular biology detection, plant genetics and breeding and other fields. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the molecular marker screening method of the present invention.

[0027] Figure 2 This is a schematic diagram showing the sequence alignment of indica conventional rice, japonica conventional rice, and long-male wild rice.

[0028] Figure 3 This is a schematic diagram illustrating the specific primer pairs used in this invention for identifying cultivated rice, wild rice, and hybrid offspring.

[0029] Figure 4 A schematic diagram of the breeding process for cultivated rice with long-male wild rice lineage through recurrent selection. Detailed Implementation

[0030] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected. Example

[0031] This embodiment provides the design, synthesis, and screening of molecular markers, specifically including the following:

[0032] 1. Development of molecular marker amplification primer pairs capable of simultaneously detecting wild rice and cultivated rice.

[0033] The genome sequences of 12 chromosomes of indica conventional rice, japonica conventional rice, and long-male wild rice were downloaded from the NCBI database. DNAMAN was used for sequence alignment to identify differentially expressed sites. Primers were designed upstream and downstream of these sites using Primer Premier5, and the primers were synthesized by Platinum Biotech (Hainan) Co., Ltd. The sequences are shown below:

[0034]

[0035] 2. Molecular marker screening and validation

[0036] (1) Extraction of parental DNA

[0037] This invention first uses the CTAB method to extract DNA from rice leaves. The specific method is as follows: The DNA extraction method is based on the traditional CTAB method (Rogers and Bendich, 1985), with slight modifications. A 3 cm rice leaf is placed in a sterilized 2 mL centrifuge tube, a 6 mm steel ball is added, and the tissue is disrupted using a cell disruptor. Then, CTAB extraction is performed. Finally, 200 μL of sterile water (ddH2O) is added to dissolve the air-dried DNA sample, and it is set aside for later use. After the DNA is completely dissolved, 2 μL of the sample is taken and the nucleic acid OD value (OD260 / OD280) and nucleic acid concentration are determined using a UV spectrophotometer (Nanodrop 2000). The DNA sample is then diluted to 50 ng / μL for later use.

[0038] (2) PCR amplification detection

[0039] PCR was performed using Biomiga's 2×PCR premix (containing Mg). 2+ 5 μL of Taq DNA Polymerase; 2.5 mM dNTPs; 10×PCR Buffer, 1 μL of primers (containing 0.5 μL each of forward and reverse primers), 1 μL of template DNA, and ddH2O to make up to 10 μL. The PCR amplification program was the standard SSR program (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 5 min).

[0040] (3) Detection of molecular markers by 8% polyacrylamide gel electrophoresis

[0041]

[0042] After assembling the electrophoresis tank, prepare the gel buffer and slowly pour it between the glass clips to prevent air bubbles. Then insert the 52-well comb and let it stand for at least 30 minutes. Pour 1*TBE buffer into the electrophoresis tank to cover the comb, and gently pull the comb out vertically. Load the PCR products onto the gel. After loading, connect the wires and maintain a constant voltage of 160-180V for electrophoresis for 60-80 minutes. After electrophoresis, disassemble the electrophoresis tank, wash the gel twice with ddH2O, and then immerse it in the fixative solution (0.8 g AgNO3, diluted to 500 ml with ddH2O) and gently shake for 8 minutes. Discard the fixative solution and wash the gel three more times with ddH2O. Then immerse it in the chromogenic solution and gently shake for 6 minutes. Discard the chromogenic solution and rinse once with tap water. At this point, the bands should be clearly visible (chromogenic solution: 10 g NaOH, diluted to 500 ml with ddH2O, and 3 g formaldehyde). ml); then gently wrap the film with plastic wrap to prevent tearing, place it on a film observation lamp to count the stripes and take photos for storage.

[0043] (4) Molecular marker screening analysis

[0044] Test results as follows Figure 1 As shown, from left to right, the rice varieties are: perennial rice, wild rice with long male reproductive organs E11-3, wild rice with long male reproductive organs E11-5, wild rice with long male reproductive organs E11-9, indica rice variety Yandao 3931, indica rice variety Yuxiangyouzhan, indica rice variety Huazhan, japonica rice variety Zhonghua 11, wild rice with long male reproductive organs E11-3, wild rice with long male reproductive organs E11-5, and wild rice with long male reproductive organs E11-9. The figure shows that the molecular marker LS02 exhibits polymorphism across indica / japonica / wild rice with long male reproductive organs. Samples were sent for sequencing.

[0045] Sequencing results revealed the following LS02 sequences: japonica rice (SEQ ID NO:13), indica rice (SEQ ID NO:14), and wild rice (SEQ ID NO:15). Alignment results are as follows: Figure 2 As shown. Example

[0046] (1) Detection of offspring from distant hybridization of rice with molecular marker-specific primer pairs, as detailed below:

[0047] One japonica rice variety, Zhonghua 11, one indica rice variety, 9311, three wild rice varieties (E11-3, E11-5, and E11-9), and germplasm resources and hybrid offspring were selected. The SSR primer pair used was the LS02 primer developed in Example 1. Genotypes of each sample were statistically analyzed. LS02 molecular marker PCR amplification was performed. If the tested rice amplified a 155 bp band, it was identified as cultivated rice; if it amplified a 146 bp band, it was identified as wild rice; if both 155 bp and 146 bp bands were amplified simultaneously, it was identified as a hybrid offspring of cultivated rice and wild rice. Figure 3 It can be seen that the LS02 molecular marker parent can amplify the corresponding band, and the hybrid offspring can also amplify the band patterns of both parents (red box). Therefore, it can be directly applied to molecular marker-assisted selection breeding of distant hybrid offspring.

[0048] (2) The breeding process of cultivated rice with long-male wild rice lineage through recurrent selection is as follows:

[0049] This embodiment conducts a breeding experiment on wild rice with long male reproductive organs. Wild rice E11-9 is used as the male parent, and a recipient with excellent agronomic traits, such as R3261, is used for hybridization, backcrossing, and self-pollination. During this process, molecular markers are used for gene and genetic prospect selection, ultimately obtaining a restorer line with a homozygous mutant gene in the R3261 background. The technical route for hybridization and breeding is as follows: Figure 4 As shown, the specific implementation steps are as follows:

[0050] 1. F1 is obtained by crossing cultivated rice with the recipient parent, such as wild rice E11-9, as the male parent.

[0051] 2. Use F1 as the female parent and the recipient parent, such as R3261, to backcross and obtain BC1F1.

[0052] 3. Plant BC1F1 and use primer pair LS02 to detect genotypes. Select heterozygous genotypes, that is, PCR amplification products that show both donor and recipient genotype bands.

[0053] 4. Use a set of molecular markers (including but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, and SCAR type markers) with polymorphism between the donor and recipient recurrent parents and evenly distributed to identify the genetic background of the single plants selected in step 3, and select plants with high genotype similarity to the recurrent parents (e.g., greater than 88% similarity, or 2% selection rate).

[0054] 5. Use the plants selected in step 4 and the recipient parent, such as R3261, to backcross and obtain BC2F1.

[0055] 6. Plant BC2F1, repeat steps 3 and 4, select plants with heterozygous genotypes and high genetic background recovery rate (e.g., greater than 98%, or 2% selection rate), and harvest them from crossbred BC2F2.

[0056] 7. Plant BC2F2, repeat steps 3 and 4, select the heterozygous plants with the highest genetic background homozygosity, and harvest them from the cross BC2F3. The homozygous plants that segregate from the offspring of BC2F3 are the restorer lines containing the long-male wild rice donor gene in the R3261 background.

[0057] The above example uses R3261 as a breeding example, but it is not limited to R3261 and can be any rice material.

Claims

1. The application of specific primer pairs in identifying hybrid offspring of wild rice, cultivated rice, and cultivated rice and wild rice, characterized in that, The specific primer pair consists of forward primer F with nucleotide sequence SEQ ID NO:3 and reverse primer R with nucleotide sequence SEQ ID NO:4; the male-growing wild rice is male-growing wild rice E11-3, male-growing wild rice E11-5, and male-growing wild rice E11-9; the cultivated rice is japonica rice variety Zhonghua 11, indica rice variety 9311, indica rice variety Yandao 3931, indica rice variety Yuxiangyouzhan, and indica rice variety Huazhan; If only one 146 bp electrophoretic band is found when PCR amplification is performed using the specific primer pair, the rice to be identified is wild rice with long male reproductive function. If there is only one 155 bp electrophoretic band, then the rice to be identified is cultivated rice. If there are two electrophoretic bands of 146 bp and 155 bp, then the rice to be identified is a hybrid offspring of cultivated rice and wild rice with long male reproductive organs.

2. A method for distinguishing between wild rice and cultivated rice with long male reproductive organs, characterized in that, Includes the following steps: Step 1: Extract genomic DNA from the rice germplasm resources to be identified; Step 2: Using the genomic DNA extracted in Step 1 as a template, perform PCR amplification using the specific primer pair described in claim 1; Step 3: Detect PCR amplification products: If there is only one 146 bp electrophoresis band, the rice to be identified is wild rice with long male reproductive function. If there is only one 155 bp electrophoretic band, then the rice to be identified is cultivated rice. If there are two electrophoretic bands of 146 bp and 155 bp, then the rice to be identified is a hybrid offspring of cultivated rice and wild rice with long male tassels. The wild rice varieties with long male reproductive function are wild rice E11-3, wild rice E11-5, and wild rice E11-9; the cultivated rice varieties are japonica rice variety Zhonghua 11, indica rice variety 9311, indica rice variety Yandao 3931, indica rice variety Yuxiangyouzhan, and indica rice variety Huazhan.

3. The method according to claim 2, characterized in that, The PCR amplification system described in step two is 10 μL, which contains 50 ng of DNA, 0.5 μL each of the forward primer F and the reverse primer R, 5 μL of 2×Taq PCR Master Mix, and the remaining volume is supplemented with ddH2O.

4. The method according to claim 2, characterized in that, The PCR amplification program described in step two is as follows: pre-denaturation at 94℃ for 10 min; denaturation at 94℃ for 30 sec, annealing at 55℃ for 30 sec, extension at 72℃ for 30 sec, for a total of 35 cycles; and finally extension at 72℃ for 10 min, followed by cooling and storage at 16℃.

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