Method for creating new germplasm of large-grain rice by using rice grain type related gene GW2-SNP molecular marker

Abstract

The invention discloses a method for creating a large-grain rice new germplasm by using a rice grain type related gene GW2-SNP molecular marker, and relates to the technical field of rice breeding, and the method comprises the following steps: S1, carrying out PCR amplification on a DNA sequence of a test material by using a GW2-1SNP marker pair to obtain an amplification product; s2, whether A gene deletion exists at the 2328bp position of the amplification product or not is judged, if yes, it is indicated that the test material has the large grain type characteristic, and if not, the test material has the large grain type characteristic; if not, the test material does not have the large grain shape characteristic; the GW2-1SNP (Single Nucleotide Polymorphism) marker pair is GW2-1SNP-F; and GW2-1SNP-R is adopted. By utilizing the marker, a single plant with a large-grain characteristic can be accurately and quickly screened in a rice seedling stage, and then field planting identification is performed, so that the field workload can be greatly reduced, meanwhile, quick homozygosis of excellent characters is facilitated, and the breeding efficiency is improved.

Description

Technical Field

[0001] The present application relates to the technical field of rice breeding, and in particular to a method for creating new large-grain rice germplasm by using rice grain type-related gene GW2-SNP molecular markers. Background Art

[0002] Single nucleotide polymorphism (SNP) is widely present in the genome of species, including: single base conversion or transversion, single base (fragment) insertion or deletion, etc. A new generation of SNP molecular marker technology has gradually emerged for these polymorphic sites. SNP molecular marker technology is used to develop functional SNP markers for genes that control target traits, associate traits with genotypes, and achieve efficient and precise breeding.

[0003] Rice grain shape has always been the focus of breeders. It is one of the three elements of rice yield and an important indicator of rice appearance quality and cooking and eating quality. GW2 is a major gene that negatively controls grain width. The functional markers reported so far are basically dCAPs or KASPR types, which require subsequent enzyme digestion, sequencing or detection using special instruments, or the process is cumbersome and complicated or the instrument equipment requirements are high, which is limited in large-scale breeding and screening applications.

[0004] For example, CN202410055308.X discloses a combination of SNP molecular marker primers for the rice grain length gene GL6.1 and its application. The SNP molecular marker is KA_061502 (polymorphism is T or C). The rice grain length gene GL6.1 can be quickly and accurately detected by identifying the above molecular markers, and can be used in the molecular breeding of new long-grain japonica rice varieties. However, the sequence can only identify the length, not the width and size of the rice grain, and cannot screen the size of the specific rice grain.

[0005] CN202311457797.3 discloses a PARMS molecular marker, primer set and application of the rice grain type gene GW5. The DNA fingerprint of 280 germplasm resources was determined using a gene chip. A significant site was detected at the 5400007th base of chromosome 5. There is a major gene GW5 that regulates the width of rice grains near this site. The rice narrow-grain type GW5 gene can be quickly and accurately detected in different germplasm resources such as indica rice and japonica rice to determine the width and narrow type of rice grains. However, the specific width and specific size of the rice grains carrying the gene sequence were not determined, and the size of the specific rice grains could not be screened.

[0006] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] In response to the above technical problems, the present application provides a method for creating new germplasm of large-grain rice using rice grain type-related gene GW2-SNP molecular markers. The method can be used for the rapid breeding of rice with large-grain characteristics.

[0008] The present application provides a method for creating a new large-grain rice germplasm using a rice grain type-related gene GW2-SNP molecular marker, comprising the following steps: Step S1: using the GW2-1SNP marker pair to perform PCR amplification on the DNA sequence of the test material to obtain an amplified product; Step S2: Determine whether there is a gene A deletion at 2328 bp of the amplified product. If so, it means that the test material has the large particle type feature; if not, it means that the test material does not have the large particle type feature; The GW2-1SNP marker pair is GW2-1SNP-F; GW2-1SNP-R.

[0009] Preferably, the PCR amplification fragment of GW2-1SNP-F is 401; the variation type of GW2-1SNP-F is A316.

[0010] Preferably, the annealing temperature of GW2-1SNP-F is 59.5°C.

[0011] Preferably, the position of GW2-1SNP-F on the GW2 gene is 2190; the position of GW2-1SNP-R on the GW2 gene is 2737.

[0012] Preferably, the method for obtaining the GW2-1 SNP marker comprises the following steps: Step S11: using the rice variety Oochikara as the parent P1 and the Yunnan Province high-quality japonica rice new variety Shengnong No. 1 as the parent P2, hybridization is performed according to the conventional pedigree method to obtain rice plants 2023GT1-2023GT46 with large-grain traits; Step S2: using young leaves of parent P1, parent P2, and rice plants 2023GT1-2023GT46 as extraction materials, extracting rice genomic DNA of parent P1, parent P2, and rice plants 2023GT1-2023GT46; Step S3: Download the full-length sequence of the Nipponbare GW2 gene from NCBI. According to the sequence information of Nipponbare GW2, the normal grain material should have an A base at 2328 bp in the 4th exon, while the DNA sequence of the obtained 2023GT1~2023GT46 raw materials has an A base missing at this position; Step S4: Design SNP primers using the online primer design website BatchPrimer3 to obtain the GW2-1 SNP marker pair.

[0013] Preferably, the extraction of rice genomic DNA from parent P1, parent P2, and rice plants 2023GT1-2023GT46 comprises the following steps: Take the fresh leaves of each extraction material and put them into a 2 ml test tube, and add 3 small steel balls into each centrifuge tube; Put it in the machine for grinding, add 450ml 2×CTAB after grinding and mix it by inversion, then put it in a 65℃ incubator for 30~40min, and mix it by inversion every 10min; Take a 1.5 ml centrifuge tube, corresponding to the rice sample number, add 750 μL of 95% ethanol, and freeze it in a -80℃ refrigerator. After 30-40 minutes at 65℃, add 500 μL of chloroform and shake well. Place the centrifuge tube with chloroform in a centrifuge and centrifuge for 9 minutes at 13,000 rpm. After centrifugation, take the supernatant and add it to a centrifuge tube frozen in a -80°C refrigerator. Mix by inverting and place it in a 80°C refrigerator for 20 minutes. After freezing for 20 minutes, the samples were centrifuged for 5 minutes (14,000 rpm) to precipitate DNA. After centrifugation, the supernatant was discarded, air-dried, and 50-200 μL ddH2O was added. The extracted DNA was stored in a 4°C refrigerator for subsequent experiments to obtain the DNA sequences of each raw material.

[0014] Preferably, the total system used for PCR amplification is 12.5 µL, including 0.5 µL template DNA, 0.5 µL each of upstream and downstream primers, and 11 µL of gold mix.

[0015] Preferably, the PCR amplification reaction is completed on an Arhat 96 gradient PCR instrument (MP60101), and the reaction procedure is as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15s, annealing at 58.5°C for 15s, extension at 72°C for 30s, and 30 cycles; extension at 72°C for 5 min. After the reaction is completed, the amplified product is obtained and stored in a 4°C refrigerator.

[0016] The beneficial effects of this application include: 1) The present application provides a method for creating new germplasm of large-grain rice using the GW2-SNP molecular marker of the rice grain type-related gene, and a method for creating new germplasm breeding materials of large-grain rice using the SNP molecular marker of the rice grain type-related gene GW2. The SNP molecular marker is GW2-1SNP, and the polymorphism of GW2-1SNP is A or 0. Using the above markers, individual plants with large-grain characteristics can be accurately and quickly screened out during the rice seedling stage, and then field planting and identification can be carried out, which can greatly reduce the workload in the field, and is also conducive to the rapid homozygosity of excellent traits and improve breeding efficiency. It is also an efficient, environmentally friendly method with potential for commercial molecular breeding applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electrophoresis diagram obtained after amplification using the GW2-1SNP marker in Example 1 of the present application; in the figure, P1 is parent P1; P2 is parent P2; GT9 is 2023GT9; GT11 is 2023GT11; GT12 is 2023GT12; GT26 is 2023GT26; GT27 is 2023GT27; GT32 is 2023GT32; GT42 is 2023GT42; GT43 is 2023GT43; GT44 is 2023GT44; M is a marker; it can be seen from the figure that the sequence can be used for the amplification product of PCR sequencing analysis; Figure 2 The sequencing results of each PCR product as shown in Table 2 of Example 1 of the present application, as well as the sequence and alignment diagram of the sequencing results of the Nipponbare-GW2 PCR product at 2328bp; wherein the parent P1 is repeated 3 times, numbered respectively as P1-1, P1-2, and P1-3; the parent P2 is repeated 3 times, numbered respectively as P2-1, P2-2, and P2-3; a, sequence alignment diagram; b, sequence information diagram; the frame in a shows the missing diagram of the A gene sequence in the sequence alignment diagram.

[0018] Figure 3 This is a comparison diagram of the grains of the large-grain strains (2023GT11, 2023GT26) obtained in Example 1 of the present application and the grains of the normal strain (parent P2); wherein a is the grains of the large-grain strain 2023GT11, b is the grains of the large-grain strain 2023GT26; c is the grains of the normal strain P2; Figure 4 These are actual measurement photos of the grains of the normal strain P2 and the large-grain strain (2023GT11) obtained in Example 1 of the present application; wherein column a shows 5 grains of the normal strain P2; column b shows 5 grains of the large-grain strain; as can be seen from the figure, the grains of the large-grain strain are overall twice the size of the grains of the normal strain P2. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0020] Example Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0021] Example 1 1. Extraction of rice DNA 48 test rice materials were collected; the 48 test rice materials included: parent P1, parent P2, and numbers 2023GT1~2023GT46.

[0022] Sources of the 48 tested rice materials: Parent P1 is Oochikara (Oryza sativa L., cv. Oochikara, transliterated as Ochi Jialuo in Chinese), a japonica rice variety introduced from Japan to my country in the 1990s (for details, see Hao Zaibin. Anatomical observation of the crown root of the rice lateral root-less mutant RM109 [J]. Journal of Nuclear Agricultural Sciences, 2002, (01): 15-19.); Parent P2 is a new high-quality japonica rice variety in Yunnan Province, Shengnong No. 1, Dian Shen Dao No. 2019019. The two parental materials were hybridized according to the conventional pedigree method in the Songming Greenhouse of Yunnan Academy of Agricultural Sciences in May 2017. The hybridization method was carried out according to the commonly used operations in this field. In 2018, they entered the systematic breeding nursery. Every year, excellent individual plants were selected according to the comprehensive field performance and planted in the next year. After 5 years of continuous planting, 46 plant lines with stable large-grain traits (abbreviated as: large-grain materials) were obtained in 2023, numbered 2023GT1~2023GT46.

[0023] Figure 3 a is the grain of the large-grain strain 2023GT11; Figure 3 b shows the grains of the large-grain strain 2023GT26; Figure 3 c is the grain of the normal strain P2. As can be seen from the figure, the grain size of the large-grain strains 2023GT11 and 2023GT26 is 1 to 2 times larger than that of the parent P2. Figure 4 Column a in the middle shows 5 grains (from 2023GT9, 2023GT11, 2023GT12, 2023GT26 and 2023GT27) of the normal line (parent P2); Figure 4 Column b in the middle shows grains from five large-grain strains; as can be seen from the figure, the grains from the large-grain strains are twice the size of the grains from the normal strain P2, indicating that the grains of 2023GT9, 2023GT11, 2023GT12, 2023GT26, and 2023GT27 have significant large-grain characteristics.

[0024] The young leaves of the 48 tested rice materials were used as extraction materials, and the rice genomic DNA was extracted using the improved CTAB method. The DNA extraction process of each extraction material was as follows: 1) Mark the test tube number, take each extraction raw material fresh leaf and put it into a 2ml test tube, and add 3 small steel balls into each centrifuge tube; 2) Grind in a machine, add 450 ml of 2×CTAB and mix thoroughly by inversion, then place in a 65°C incubator for 30-40 minutes (invert and mix thoroughly every 10 minutes); 3) Take a 1.5 ml centrifuge tube, corresponding to the rice sample number, add 750 μL of 95% ethanol, and freeze it in a -80°C refrigerator. After freezing at 65°C for 30-40 minutes, add 500 μL of chloroform and shake well; 4) Place the centrifuge tube with chloroform in a centrifuge and centrifuge for 9 minutes at 13,000 rpm. After centrifugation, take the supernatant and add it to a centrifuge tube frozen in a -80°C refrigerator. Invert and mix well, then place it in a 80°C refrigerator for 20 minutes. 5) After freezing for 20 minutes, place in a centrifuge and centrifuge for 5 minutes (14,000 rpm) to precipitate DNA. After centrifugation, discard the supernatant, air-dry, and add 50-200 μL ddH2O. The extracted DNA is stored in a 4°C refrigerator for subsequent experiments to obtain the DNA sequence of each sample.

[0025] 2. Primer design The full-length sequence of the Nipponbare GW2 gene was downloaded from NCBI. According to the sequence information of Nipponbare GW2, the normal grain-type material should have an A base at 2328bp in the 4th exon, while the large grain-type material of the obtained 2023GT1~2023GT46 samples had an A base missing at this position.

[0026] Therefore, we explored SNP sites near the base deletion site in exon 4 and designed SNP primers using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / ). The designed primers were provided by Qingke Biotechnology. The primer information is shown in Table 1: Table 1: Information of two pairs of SNP primers designed at the 4th exon site of rice GW2 gene Note a: The marker position refers to the position of the designed primers on the GW2 gene.

[0027] GW2 is a cloned major gene that negatively controls grain width, and its phenotype is reflected in its influence on rice grain width and grain weight. The gene symbol is LOC_Os02g14720, with a total length of 6965 bp, 8 exons, and a cDNA length of 1634 bp.

[0028] 3. PCR amplification PCR amplification was performed using the extracted DNA of 48 tested rice materials as templates. The PCR reaction system was as follows: total system 12.5 µL, template DNA 0.5 µL, upstream and downstream primers 0.5 µL each, gold mix 11 µl (Gold Mix (TSE102) of Beijing Qingke Biotechnology Co., Ltd.). The PCR reaction was completed on an Arhat 96 gradient PCR instrument (MP60101). The reaction procedure was as follows: pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 15 s, annealing at 58.5 °C for 15 s, extension at 72 °C for 30 s, and 30 cycles; extension at 72 °C for 5 min. After the reaction, the amplified product was obtained and stored in a refrigerator at 4 °C.

[0029] 4. Electrophoresis and sequencing analysis Take 5µl of the amplified products of parent P1, parent P2, 2023GT9, 2023GT11, 2023GT12, 2023GT26, 2023GT27, 2023GT32, 2023GT42, 2023GT43, and 2023GT44, and perform electrophoresis on high-purity low-electrosmotic Agarose gel, and perform gel imaging with a gel imager (JY045-3C). Read the neat, clear, and non-diffuse bands on the gel plate ( Figure 1 ), check the corresponding PCR, and send the remaining PCR to Qingke Company for PCR product sequencing.

[0030] 5. Validation of SNP markers in populations 5.1 Verification of population material creation process: Parent P1 is Oochikara, a japonica rice variety introduced from Japan in the 1990s (for details, see Hao Zaibin. Anatomical observation of the crown root of the rice lateral root-less mutant RM109 [J]. Journal of Nuclear Agricultural Sciences, 2002, (01): 15-19.); Parent P2 is Shengnong 1, a new high-quality japonica rice variety in Yunnan Province. The two parental materials were hybridized in the Songming greenhouse of Yunnan Academy of Agricultural Sciences in May 2017 according to the conventional pedigree method. The hybridization method was carried out according to the commonly used operation in this field. In 2018, they entered the systematic breeding nursery. Every year, excellent single plants were selected according to the comprehensive performance in the field and planted in the next year. After 5 years of continuous planting, 46 stable plant lines were obtained in 2023, numbered 2023GT1-2023GT46.

[0031] 5.2 Verification According to the sequence information of Nipponbare GW2, the SNP marker designed in the 4th exon, after PCR amplification and sequencing, the normal grain material should have an A base at 2328bp, while the large grain material has an A base missing at this position. The SNP marker published by the present invention was used for detection and verification in 48 test materials. It can be seen that at 2328bp, the F6 individual strain with large grain characteristics has an A base missing, as shown in Table 2. The PCR test results of the samples are as follows Figure 2 As shown, Figure 2 a is the sequence alignment diagram obtained by PCR detection of each sample in Table 2; b is the sequence information diagram obtained by PCR detection of each sample in Table 2. 2023GT9, 2023GT11, 2023GT12, 2023GT26 and 2023GT27 have an A deletion at 2328 bp, while 2023GT42, 2023GT43 and 2023GT44 include an A base at this site, the same as Nipponbare-GW2 and parents P1 and P2.

[0032] Subsequently, the above 8 representative hybrid materials and parents were transplanted into the field for field planting. After the natural growth cycle in the field was completed, 3 plants of each sample were taken back to the room for seed testing and analysis. The results are shown in Table 2. The data in Table 2 are the average values ​​of the 3 samples. The 5 lines (2023GT9, 2023GT11, 2023GT12, 2023GT26 and 2023GT27) with large-grain traits detected at the seedling stage had significantly higher panicle length, grain weight and grain width than the lines without large-grain loci detected after rice maturity (Table 2, Figure 3 ).

[0033] Table 2 Field test characteristics of parents and test materials This suggests that in large-scale breeding, this SNP marker can be used for large-scale indoor screening at the seedling stage, and the individual plants with the A base at the site can be screened out and only the individual plants with the base 0 at the 2328bp site can be retained, so as to achieve rapid homozygosity of large-grain genetic materials.

[0034] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for creating new large-grain rice germplasm using rice grain type-related gene GW2-SNP molecular markers, characterized in that: The following steps are involved: Step S1: using the GW2-1SNP marker pair to perform PCR amplification on the DNA sequence of the test material to obtain an amplified product; Step S2: Determine whether there is a gene A deletion at 2328 bp of the amplified product. If so, it means that the test material has the large particle type feature; if not, it means that the test material does not have the large particle type feature; The GW2-1SNP marker pair is GW2-1SNP-F; GW2-1SNP-R.

2. The method according to claim 1, characterized in that The PCR amplified fragment of GW2-1SNP-F is 401; the variant type of GW2-1SNP-F is A316.

3. The method according to claim 1, characterized in that The annealing temperature of GW2-1SNP-F is 59.5°C.

4. The method according to claim 1, characterized in that The position of GW2-1SNP-F on the GW2 gene is 2190; the position of GW2-1SNP-R on the GW2 gene is 2737.

5. The method according to claim 1, characterized in that The GW2-1 SNP marker acquisition method includes the following steps: Step S11: using the rice variety Oochikara as the parent P1 and the Yunnan Province high-quality japonica rice new variety Shengnong No. 1 as the parent P2, hybridization is performed according to the conventional pedigree method to obtain rice plants 2023GT1-2023GT46 with large-grain traits; Step S2: using young leaves of parent P1, parent P2, and rice plants 2023GT1-2023GT46 as extraction materials, extracting rice genomic DNA of parent P1, parent P2, and rice plants 2023GT1-2023GT46; Step S3: Download the full-length sequence of the Nipponbare GW2 gene from NCBI. According to the sequence information of Nipponbare GW2, the normal grain material should have an A base at 2328 bp in the 4th exon, while the DNA sequence of the obtained 2023GT1~2023GT46 raw materials has an A base missing at this position; Step S4: Design SNP primers using the online primer design website BatchPrimer3 to obtain the GW2-1 SNP marker pair.

6. The method according to claim 5, characterized in that The method of extracting rice genomic DNA from parent P1, parent P2, and rice plants 2023GT1-2023GT46 comprises the following steps: Take the fresh leaves of each extraction material and put them into a 2 ml test tube, and add 3 small steel balls into each centrifuge tube; Put it in the machine for grinding, add 450ml 2×CTAB after grinding and mix it by inversion, then put it in a 65℃ incubator for 30~40min, and mix it by inversion every 10min; Take a 1.5 ml centrifuge tube, corresponding to the rice sample number, add 750 μL of 95% ethanol, and freeze it in a -80℃ refrigerator. After 30-40 minutes at 65℃, add 500 μL of chloroform and shake well. Place the centrifuge tube with chloroform in a centrifuge and centrifuge for 9 minutes at 13,000 rpm. After centrifugation, take the supernatant and add it to a centrifuge tube frozen in a -80°C refrigerator. Mix by inverting and place it in a 80°C refrigerator for 20 minutes. After freezing for 20 minutes, the samples were centrifuged for 5 minutes (14,000 rpm) to precipitate DNA. After centrifugation, the supernatant was discarded, air-dried, and 50-200 μL ddH2O was added. The extracted DNA was stored in a 4°C refrigerator for subsequent experiments to obtain the DNA sequences of each raw material.

7. The method according to claim 1, characterized in that The total system used for PCR amplification was 12.5 µL, including 0.5 µL template DNA, 0.5 µL each of upstream and downstream primers, and 11 µL of gold mix.

8. The method according to claim 1, characterized in that The PCR amplification reaction was completed on an Arhat 96 gradient PCR instrument. The reaction procedure was as follows: pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 15 s, annealing at 58.5 °C for 15 s, and extension at 72 °C for 30 s for 30 cycles; extension at 72 °C for 5 min. After the reaction, the amplified product was obtained and stored in a 4 °C refrigerator.

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