A SNP molecular marker related to rice grain width and grain weight, primer set and use

Abstract

The application discloses a SNP molecular marker related to grain width and weight of rice, a primer group and purposes, and belongs to the technical field of molecular biology. The SNP molecular marker is located at the 1071th base of a GSW8 gene on the 8th chromosome of rice, and the polymorphism is C or T. When the 1071th base is T, the rice is a variety with increased grain width and weight. In an EMS mutagenized mutant library of a heavy panicle type hybrid rice backbone parent Shu Hui 498 (R498), a grain type and weight related mutant gsw8-D is identified, and a candidate gene GSW8 is identified on the 8th chromosome. It is found that the gene is replaced from C to T at the 1071th base in the coding region, the 357th serine (S) is mutated into phenylalanine (F), and then a dominant large grain phenotype is generated, and the grain width and the thousand-grain weight are significantly increased.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a SNP molecular marker related to rice grain width and weight, a primer set and use. BACKGROUND

[0002] Rice is the main food crop in China, and more than 60% of the population mainly eats rice. Further improving rice yield is of great significance to guaranteeing China's food security. Rice yield is determined by the number of effective panicles per plant, the number of grains per panicle, and the thousand-grain weight. Grain type, including grain length, grain width, length-width ratio, and grain thickness, is a factor that directly affects the thousand-grain weight. Therefore, cloning grain type-related genes and mining excellent alleles are important ways to improve grain type and thousand-grain weight and to improve rice yield.

[0003] Many grain type-related genes have been cloned, which regulate grain type through different signaling pathways, mainly including the ubiquitin-proteasome pathway, the G protein signaling pathway, the MAPK signaling pathway, the plant hormone regulation pathway, and the transcription factor pathway (Li et al., 2019; Li et al., 2021; Ren et al., 2023). However, most grain type genes are cloned through mutants, and in addition to regulating grain type, they often carry other unfavorable traits, so most grain type genes are difficult to be directly applied in breeding. In addition, although some major QTLs for grain type have been cloned, most of them are recessive and difficult to be directly applied in hybrid rice breeding. Therefore, it is urgent to further mine dominant grain type excellent alleles and apply them in hybrid rice breeding to improve rice yield. SUMMARY

[0004] In view of the above problems in the prior art, the application provides a SNP molecular marker related to rice grain width and weight, a primer set and use, which provides excellent alleles and molecular markers for improving grain type and thousand-grain weight of breeding parents, and provides new gene resources for hybrid rice breeding.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the application to solve its technical problems is:

[0006] A SNP molecular marker related to rice grain width and weight is located at the 1070th base of the GSW8 gene on the 8th chromosome of rice. GSW8 The polymorphism of the 1070th base is C or T. GSW8 The GSW8 gene has been disclosed in the application for a protein GSW8 for regulating rice grain type and thousand-grain weight, its coding gene and application, with the application date of July 8, 2021 and the publication number of CN 113388016A.

[0007] A primer set for detecting the above-mentioned SNP molecular marker, the nucleotide sequence of which is shown in SEQ ID NO. 1-4.

[0008] A kit comprising the above-described primer set.

[0009] A gene chip comprising the aforementioned primer set.

[0010] The above primer set and kit are used in the detection of rice grain width and grain weight.

[0011] The above primer sets and kits are used in the breeding of rice varieties with increased grain width and grain weight.

[0012] The above primer sets and kits are used in the cultivation of transgenic rice with increased grain width and weight, or in the improvement of rice germplasm resources.

[0013] Application of SNP molecular markers in rice breeding.

[0014] A method for detecting the above-mentioned SNP molecular markers includes the following steps:

[0015] S1. Extract DNA from the sample to be tested;

[0016] S2. Using the DNA sample to be tested as a template, perform the detection using the above primer set or kit;

[0017] S3. If the sample to be tested is located on the 8th chromosome of rice. GSW8 If the base at position 1070 of the gene is T, then the rice variety is determined to have increased grain width and grain weight.

[0018] The beneficial effects of this invention are:

[0019] This invention identified a grain type and grain weight-related mutant in the EMS-induced mutant library of the heavy-panicle hybrid rice backbone parent Shuhui 498 (R498). gsw8 - D Candidate genes were identified on chromosome 8 using the MutMap gene localization method. GSW8 ( G rain s ize and g rain weight 8 It was discovered that the gene had a C-to-T substitution at position 1070 of the coding region, which resulted in a mutation of serine (S) at position 357 to phenylalanine (F), thereby producing a dominant large grain phenotype with a significant increase in grain width and thousand-grain weight. Attached Figure Description

[0020] Figure 1 It is R498 and gsw8-D Agronomic trait analysis; among which, (A)R498 and gsw8-D Mature plant type, scale bar, 10cm; (B, C) R498 andgsw8-D Grain length and grain width comparison, scale, 3 mm; (D-G) R498 and gsw8-D Statistical analysis of plant height, grain length, grain width and 1000-grain weight; using t t-test P Values, ** represent significant difference at 0.01 level;

[0021] Figure 2 is gsw8-D Genetic analysis and gene mapping; wherein, (A) gsw8-D / R498 F2 population grain width distribution; (B) MutMap gene mapping results; (C) gsw8-D in the candidate gene GSW8 Coding region mutation site schematic diagram; (D) Four-primer detection marker electrophoresis effect diagram developed based on gsw8-D mutation site;

[0022] Figure 3 is gsw8-D Backcross introduction of hybrid rice backbone parent Shu Hui 527 and Hua Zhan;

[0023] Figure 4 is Shu Hui 527 and Hua Zhan background gsw8-D Agronomic trait analysis; wherein, (A) R527 and R527- gsw8-D Mature plant type, scale, 10 cm; (B) R527 and R527- gsw8-D Grain width comparison, scale, 3 mm; (C) HZ and HZ- gsw8-D Mature plant type, scale, 10 cm; (D) HZ and HZ- gsw8-D Grain width comparison, scale, 3 mm; (E-J) R527 and HZ background gsw8-D Statistical analysis of grain length, grain width and 1000-grain weight; using t t-test P Values, ** represent significant difference at 0.01 level;

[0024] Figure 5 Shu Hui 498 background gsw8-D Application in hybrid rice; wherein, (A) Hybrid rice combination mature plant type, scale, 10 cm; (B) Hybrid rice combination grain width comparison, scale, 3 mm; (C) Hybrid rice combination grain number per plant comparison, scale, 3 cm; (D-G) Hybrid rice combination grain length, grain width, 1000-grain weight and field actual grain yield per plant data statistics; using t t-test P Values, ** represent significant difference at 0.01 level.

[0025] Figure 6 Hua Zhan background gsw8-DApplication in hybrid rice. Among them, (A) plant type of hybrid rice combination at mature stage. Scale, 10 cm. (B) comparison of grain width of hybrid rice combination. Scale, 3 mm. (C) comparison of actual grain number per plant of hybrid rice combination. Scale, 3 cm. (D-G) grain length, grain width, 1000-grain weight and actual yield per plant of hybrid rice combination data statistics. Use t Test calculation P Values, ** represent significant difference at 0.01 level. DETAILED DESCRIPTION

[0026] The specific embodiments of the present application are described below to facilitate the understanding of the present application by those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0027] Example 1 Large grain mutant gsw8 - D Identification and phenotype analysis

[0028] The inventors identified a large grain mutant from the EMS mutant library of R498, which was named gsw8-D . Compared with wild type R498, the plant height of mutant gsw8-D was reduced by 9.53% ( Figure 1 A, D). Further, the grain length, grain width and 1000-grain weight of wild type and mutant were measured by automatic grain analysis instrument (Mini 1600, Sichuan Jialeimei Technology Co., Ltd.). It was found that compared with R498, the grain length of mutant gsw8-D had no significant difference ( Figure 1 B, E), the grain width increased by 20.8%, resulting in a significant increase of 8.62% in 1000-grain weight ( Figure 1 C, F-G).

[0029] Example 2 Genetic analysis and gene location of large grain mutant gsw8 - D

[0030] 1. Construction of mapping population and genetic analysis

[0031] The mutant gsw8-D was crossed with wild type R498 to obtain F1 plants, and F1 self-crossing obtained F2 separation population. Grain examination found that F1 plants showed large grain phenotype similar to mutant gsw8-D , indicating that gsw8-D is a dominant mutation ( Figure 2 ​A). Further investigation into the grain width of individual plants in the F2 population revealed a typical bimodal distribution, with 563 plants exhibiting a large-grain phenotype and 192 plants exhibiting a small-grain phenotype. The chi-square test confirmed a segregation ratio of 3:1 (χ²c = 0.053 < χ²0.05(1) = 3.84), indicating that the mutant... gsw8-D The large-particle phenotype is controlled by a single gene that is completely dominant. Figure 2 A).

[0032] 2. Gene localization

[0033] Thirty plants with extreme grain widths from the population were selected for pooled sequencing. Gene mapping was performed using the MutMap method (Abe et al., 2012), and a clear linkage segment was identified on chromosome 8. Figure 2 B), where only one SNP (C1752T) is located in the coding region of the gene, resulting in a single amino acid substitution, namely, the serine at position 357 is mutated to phenylalanine (Ser357Phe). Figure 2 C), naming the gene GSW8 .

[0034] 3. Detection marker development and co-separation validation

[0035] Based on the above SNPs, a four-primer detection method for PCR markers was developed using the website (http: / / primer1.soton.ac.uk / primer1.html). The four primers were mixed in equal amounts and used. The primer sequences are as follows:

[0036] GSW8-IF sequence: 5'-GCAGAGACATGCTGTTGATTTCTACATATT-3' (SEQ ID NO.1);

[0037] GSW8-IR sequence: 5'-AATGCTCGTAGTCCAGATTGGAAATAAG-3' (SEQ ID NO. 2);

[0038] GSW8-IF sequence: 5'-TTATCATCAAGATGGGATTTCAATGAAG-3' (SEQ ID NO. 3);

[0039] GSW8-IR sequence: 5'-ATCCGATCATCACTTGATAGATTCCTTT-3' (SEQ ID NO.4);

[0040] Leaves were taken from each individual plant in the F2 population, and DNA was extracted using the standard CTAB method. PCR amplification was then performed using the four primers described above. The PCR amplification system (20 μL) included: DNA template: 3 μL, Taq enzyme (5 U / μL): 0.2 μL, dNTPs (2.5 mM): 2 μL, primers (10 mM): 4 μL, and 10× buffer (containing Mg2+). 2+ 25mM): 2 μL, ddH2O: 8.8 μL. PCR amplification conditions were: (1) 94℃ pre-denaturation for 5 min; (2) 94℃ denaturation for 30 s; (3) 56℃ annealing for 30 s; (4) 72℃ extension for 30 s; a total of 35 cycles; (5) 72℃ extension for 10 min; (6) 20℃ extension for 2 min. After PCR amplification, electrophoresis analysis was performed on a 3.5% agarose gel. The electrophoresis results were as follows: Figure 2 As shown in D, the wild type amplified two bands at 151 bp and 292 bp. gsw8-D The mutant strain amplified two bands at 199 bp and 292 bp, while the heterozygous strain amplified three bands at 151 bp, 199 bp, and 292 bp, indicating that the detection marker can accurately distinguish between the two strains. GSW8 Different genotypes can be used for further linkage analysis and backcross identification.

[0041] Analysis after electrophoresis revealed small individual plants GSW8 All were wild-type, while the large-grained phenotype individual plants were all wild-type. gsw8-D Mutant or heterozygous, indicating that the SNP is completely co-segregated with the large-particle phenotype ( Figure 2 A). These results illustrate GSW8 SNP substitution in exon 3 is what causes the production gsw8-D The mutant phenotype is caused by the gene encoding a protein with an unknown function.

[0042] Example 3: Hybrid rice backbone parents Shuhui 527 and Huazhan under background gsw8 - D Agronomic traits investigation

[0043] The background of Shu Hui 498 gsw8-D The mutant was backcrossed for 5 generations with the hybrid rice backbone parents Shuhui 527 (R527) and Huazhan (HZ), respectively. The backcross diagram is shown in the figure. Figure 3 As shown. Using the above-mentioned... gsw8-D The developed four-primer detection markers were used to identify R527- in the R527 background. gsw8-D And HZ- in the HZ background gsw8-D ( Figure 4 ).

[0044] Compared to wild-type R527, R527-gsw8-D Plant height decreased ( Figure 4 A) There was no significant difference in grain length, but the grain width increased significantly by 21.52%, resulting in a 7.43% increase in thousand-grain weight. Figure 4 B, EG). Compared to wild-type HZ, HZ- gsw8-D Plant height decreased ( Figure 4 C), there was no significant difference in grain length, but the grain width increased significantly by 27.73%, resulting in a 29.90% increase in thousand-grain weight. Figure 4 D, HJ). These results indicate that... gsw8-D Alleles have a significant effect on improving the grain shape and thousand-grain weight of hybrid rice backbone parents, and have important breeding potential.

[0045] Example 4 gsw8 - D Application in hybrid rice breeding

[0046] Based on the above results, gsw8-D It was completely dominant, and it significantly increased grain width and thousand-grain weight under different backgrounds, including the core parents of hybrid rice R498, R527, and HZ. To test... gsw8-D Whether it has value in hybrid rice breeding, and whether it can be compared with R498 and gsw8-D Pairing was performed with the sterile line Quanxiang 1A (QX1A) and it was found that QX1A / gsw8-D Compared to the QX1A / R498 combination, the plant height of this combination was slightly lower, and there was no significant difference in grain length, but the grain width increased by 15.12%, the thousand-grain weight increased by 10.73%, and the final actual yield per plant in the field increased by 12.30%. Figure 5 ).

[0047] In addition, HZ and HZ- gsw8-D Pairing was performed with the sterile line Quan 9311A (Q9311A) and it was found that Q9311A / HZ- gsw8-D Compared to the Q9311A / HZ combination, this combination had slightly lower plant height and no significant difference in grain length, but increased grain width by 18.60%, increased thousand-grain weight by 23.28%, and ultimately increased actual yield per plant in the field by 14.86%. Figure 6 ).

[0048] In conclusion, gsw8-D Alleles are of great value in hybrid rice breeding and can significantly increase the yield of hybrid rice.

[0049] Finally, it should be noted that the above specific embodiments 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 examples, 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 claims of the present invention.

Claims

1. A primer set for detecting SNP molecular markers related to rice grain width and grain weight, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO. 1-4; the SNP molecular marker is located on rice chromosome 8. GSW8 At the 1070th base of the gene, the polymorphism is either C or T.

2. A reagent kit, characterized in that, Includes the primer set as described in claim 1.

3. A gene chip, characterized in that, Includes the primer set as described in claim 1.

4. The use of the primer set of claim 1 or the kit of claim 2 in detecting rice grain width and grain weight, if the sample to be tested is located on rice chromosome 8. GSW8 If the base at position 1070 of the gene is T, then the rice variety is determined to have increased grain width and grain weight.

5. The use of the primer set of claim 1 or the kit of claim 2 in breeding rice varieties with increased grain width and grain weight, provided that the sample to be tested is located on rice chromosome 8. GSW8 If the base at position 1070 of the gene is T, then the rice variety is determined to have increased grain width and grain weight.

6. A method for detecting the SNP molecular marker of claim 1, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA of the sample to be tested as a template, perform detection using the primer set described in claim 1 or the kit described in claim 2; S3. If the sample to be tested is located on the 8th chromosome of rice. GSW8 If the base at position 1070 of the gene is T, then the rice variety is determined to have increased grain width and grain weight.

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