CN118308521A - A SNP molecular marker related to rice grain width and grain weight, a primer set and uses thereof - Google Patents

Abstract

The invention discloses a SNP molecular marker, a primer set and a use related to rice grain width and grain weight, and belongs to the technical field of molecular biology. The SNP molecular marker is located at the 1071th base of the GSW8 gene of rice chromosome 8, and its polymorphism is C or T. When the 1071th base is T, the rice is a variety with increased grain width and grain weight. The present invention identified a grain type and grain weight related mutant gsw8-D in the EMS mutagenesis mutant library of the heavy panicle hybrid rice backbone parent Shuhui 498 (R498), and identified the candidate gene GSW8 on chromosome 8; it was found that the 1071th base C to T substitution in the coding region of the gene caused the 357th serine (S) to mutate to phenylalanine (F), thereby producing a dominant large-grain phenotype, and the grain width and thousand-grain weight were significantly increased.

Description

A SNP molecular marker related to rice grain width and grain weight, a primer set and uses thereof

Technical Field

The invention belongs to the technical field of molecular biology, and in particular relates to a SNP molecular marker related to rice grain width and grain weight, a primer set and uses.

Background technique

Rice is the main food crop in my country. More than 60% of the population relies on rice as their staple food. Further increasing rice production is of great significance to ensuring my country's food security. Rice yield is determined by the number of effective panicles per plant, the number of panicles, and the thousand-grain weight. Grain shape, including grain length, grain width, length-to-width ratio, and grain thickness, is a factor that directly affects the thousand-grain weight. Therefore, cloning genes related to grain shape and exploring excellent alleles are important ways to improve grain shape and thousand-grain weight and increase rice yield.

At present, many grain-type-related genes have been cloned, and grain shape is regulated through different signal pathways, including: ubiquitin-proteasome pathway, G protein signal pathway, MAPK signal pathway, plant hormone regulation pathway, and transcription factor pathway (Li et al., 2019; Li et al., 2021; Ren et al., 2023). However, most grain-type genes are cloned through mutants. In addition to regulating grain shape, they often carry other unfavorable traits. Therefore, most grain-type genes are difficult to be directly used in breeding. In addition, although some major QTLs for grain shape have been cloned, most of them are recessive and difficult to be directly used in hybrid rice breeding. Therefore, there is an urgent need to further explore dominant excellent alleles of grain shape, and then apply them to hybrid rice breeding to increase rice yield.

Summary of the invention

In view of the above-mentioned deficiencies in the prior art, the present invention provides a SNP molecular marker, a primer set and uses related to rice grain width and grain weight, providing excellent alleles and molecular markers for improving the grain type and 1000-grain weight of breeding parents, and providing new gene resources for hybrid rice breeding.

To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

A SNP molecular marker related to rice grain width and grain weight, located at the 1071th base of the GSW8 gene on rice chromosome 8, and its polymorphism is C or T. The GSW8 gene has been disclosed in the invention patent with application date of 2021-7-8 and publication number: CN 113388016A: A protein GSW8 for regulating rice grain shape and 1000-grain weight, its encoding gene and application.

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

A kit comprises the above primer set.

A gene chip comprises the above primer set.

The primer set and the kit are used for detecting rice grain width and grain weight.

The primer set and the kit are used in breeding rice varieties with increased grain width and grain weight.

The primer set and the kit are used in cultivating transgenic rice with increased grain width and grain weight, or improving rice germplasm resources.

Application of Shangshui SNP molecular markers in rice breeding.

A method for detecting the above-mentioned SNP molecular marker comprises the following steps:

S1, extracting DNA from the sample to be tested;

S2. Using the sample DNA to be tested as a template, using the primer set of claim 2 or the kit of claim 3 for detection;

S3. If the base at position 1071 of the GSW8 gene on chromosome 8 of the sample to be tested is T, the rice is determined to be a variety with increased grain width and grain weight.

Beneficial effects of the present invention:

The present invention identifies a grain size and grain weight related mutant gsw8-D in an EMS mutagenesis mutant library of heavy panicle hybrid rice backbone parent Shuhui 498 (R498), and identifies a candidate gene GSW8 ( G rain size and grain weight 8) on chromosome 8 by using a MutMap gene positioning method; it is found that the 1071st base C to T substitution in the coding region of the gene causes the 357th serine (S) to phenylalanine (F) mutation, thereby producing a dominant large-grain phenotype, and the grain width and 1000-grain weight are significantly increased.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an analysis of agronomic traits of R498 and gsw8-D; (A) plant type of R498 and gsw8-D at maturity, scale, 10 cm; (B, C) comparison of grain length and grain width of R498 and gsw8-D, scale, 3 mm; (D-G) statistical data of plant height, grain length, grain width and thousand-grain weight of R498 and gsw8-D; P value was calculated using t-test, ** indicates significant difference at 0.01 level;

Figure 2 shows the genetic analysis and gene localization of gsw8-D; (A) distribution of grain width of gsw8-D/R498 _F2 population; (B) results of MutMap gene localization; (C) schematic diagram of the mutation site of gsw8-D in the coding region of the candidate gene GSW8; (D) electrophoresis effect diagram of the four-primer detection marker developed based on the mutation site of gsw8-D;

FIG3 is a schematic diagram of gsw8-D backcrossing into hybrid rice backbone parents Shuhui 527 and Huazhan;

Figure 4 is an analysis of agronomic traits of gsw8-D under the background of Shuhui 527 and Huazhan; (A) plant type of R527 and R527-gsw8-D at maturity, scale, 10 cm; (B) comparison of grain width of R527 and R527-gsw8-D, scale, 3 mm; (C) plant type of HZ and HZ-gsw8-D at maturity, scale, 10 cm; (D) comparison of grain width of HZ and HZ-gsw8-D, scale, 3 mm; (E-J) statistics of grain length, grain width and thousand-grain weight of gsw8-D under the background of R527 and HZ; P value was calculated using t-test, ** indicates significant difference at the 0.01 level;

Figure 5 is the application of Shuhui 498 background gsw8-D in hybrid rice; (A) hybrid rice combination plant type at maturity, scale, 10 cm; (B) hybrid rice combination grain width comparison, scale, 3 mm; (C) hybrid rice combination single plant actual grain number comparison, scale, 3 cm; (D-G) hybrid rice combination grain length, grain width, 1000-grain weight and actual single plant yield data statistics in the field; t-test was used to calculate P value, ** indicates significant difference at the 0.01 level.

Figure 6 is the application of Huazhan background gsw8-D in hybrid rice. Among them, (A) hybrid rice combination plant type at maturity. Scale, 10cm. (B) hybrid rice combination grain width comparison. Scale, 3mm. (C) hybrid rice combination single plant actual grain number comparison. Scale, 3cm. (D-G) hybrid rice combination grain length, grain width, thousand-grain weight and actual field single plant yield data statistics. The P value was calculated using the t-test, and ** indicates a significant difference at the 0.01 level.

Detailed ways

The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

Example 1 Identification and phenotypic analysis of the large-grain mutant gsw8-D

The inventors identified a large-grain mutant in the EMS mutant library of R498 and named it gsw8-D. Compared with the wild-type R498, the plant height of the mutant gsw8-D was reduced by 9.53% (Figure 1A, D). The grain length, grain width and thousand-grain weight of the wild type and mutant were further measured using an automatic seed analyzer (Mini 1600, Sichuan Jellemei Technology Co., Ltd.). It was found that compared with R498, there was no significant difference in the grain length of the mutant gsw8-D (Figure 1B, E), and the grain width increased by 20.8%, resulting in a significant increase in thousand-grain weight by 8.62% (Figure 1C, F-G).

Example 2 Genetic analysis and gene localization of the large-grain mutant gsw8-D

1. Positioning population construction and genetic analysis

The mutant gsw8-D was crossed with the wild type R498 to obtain _F1 plants, _which were then self-pollinated to obtain the _F2 segregating population. The test found that the grains of the _F1 plants showed a large-grain phenotype similar to that of the mutant gsw8-D, indicating that gsw8-D is a dominant mutation (Figure 2A). Further investigation of the grain width of each plant in the _F2 population revealed that the grain width of the _F2 population showed a typical bimodal distribution, with 563 plants with large-grain phenotypes and 192 plants with small-grain phenotypes, which met the segregation ratio of 3:1 according to the chi-square test. These results indicate that the large-grain phenotype of the mutant gsw8-D is controlled by a single gene with complete dominance ( FIG2A ).

2. Gene positioning

Thirty extreme grain width individuals in the population were selected for mixed pool sequencing and gene localization was performed using the MutMap positioning method (Abe et al., 2012). An obvious linkage segment was identified on chromosome 8 (Figure 2B), in which only one SNP (C1752T) was located in the coding region of the gene, resulting in an amino acid substitution, that is, the serine at position 357 mutated to phenylalanine (Ser357Phe) (Figure 2C), and the gene was named GSW8.

3. Detection marker development and co-segregation validation

Based on the above SNP, a four-primer detection PCR marker 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:

GSW8-I-F sequence: 5'-GCAGAGACATGCTGTTGATTTCTACATATT-3' (SEQ ID NO. 1);

GSW8-I-R sequence: 5'-AATGCTCGTAGTCCAGATTGGAAATAAG-3' (SEQ ID NO. 2);

GSW8-I-F sequence: 5'-TTATCATCAAGATGGGATTTCAATGAAG-3' (SEQ ID NO. 3);

GSW8-I-R sequence: 5′-ATCCGATCATCACTTGATAGATTCCTTT-3′ (SEQ ID NO. 4);

The leaves of each individual plant in the above _F2 population were taken for individual plant extraction using the conventional CTAB method, and PCR amplification was performed using the above four primers. 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, 10×Buffer (containing Mg ²⁺ , 25 mM): 2 μL, ddH ₂ O: 8.8 μL. The PCR amplification conditions were: (1) 94°C pre-denaturation for 5 min; (2) 94°C denaturation for 30 s; (3) 56°C annealing for 30 s; (4) 72°C extension for 30 s; a total of 35 cycles; (5) 72°C extension for 10 min; (6) 20°C extension for 2 min. After PCR amplification, 3.5% agarose gel electrophoresis analysis was performed. The electrophoresis effect is shown in Figure 2D. The wild type amplified two bands of 151 bp and 292 bp, the gsw8-D mutant amplified two bands of 199 bp and 292 bp, and the heterozygous type amplified three bands of 151 bp, 199 bp and 292 bp, indicating that the detection marker can accurately distinguish different genotypes of GSW8 and can be used for further linkage analysis and backcross identification.

After electrophoresis, it was found that the GSW8 of small-grained plants was wild type, while the large-grained plants were all gsw8-D mutant or heterozygous, indicating that the SNP was completely co-segregated with the large-grained phenotype (Figure 2A). These results indicate that the SNP substitution in exon 3 of GSW8 is the cause of the gsw8-D mutant phenotype, and the gene encodes an expression protein with unknown function.

Example 3 Agronomic characteristics of gsw8-D in the background of hybrid rice main parents Shuhui 527 and Huazhan

The gsw8-D mutant in the Shuhui 498 background was backcrossed with the hybrid rice backbone parents Shuhui 527 (R527) and Huazhan (HZ) for five generations, respectively. The backcross schematic diagram is shown in Figure 3. Using the four-primer detection marker developed for gsw8-D, R527-gsw8-D in the R527 background and HZ-gsw8-D in the HZ background were identified (Figure 4).

Compared with wild-type R527, R527-gsw8-D had a reduced plant height (Figure 4A), no significant difference in grain length, but a significant increase in grain width of 21.52%, resulting in a 7.43% increase in 1000-grain weight (Figure 4B, E-G). Compared with wild-type HZ, HZ-gsw8-D had a reduced plant height (Figure 4C), no significant difference in grain length, but a significant increase in grain width of 27.73%, resulting in a 29.90% increase in 1000-grain weight (Figure 4D, H-J). These results indicate that the gsw8-D allele has a significant effect on improving the grain shape and 1000-grain weight of the backbone parent of hybrid rice, and has important breeding potential.

Example 4 Application of gsw8-D in hybrid rice breeding

Based on the above results, gsw8-D is completely dominant and can significantly increase grain width and 1000-grain weight in different backgrounds of hybrid rice backbone parents R498, R527 and HZ. In order to test whether gsw8-D is valuable in hybrid rice breeding, R498 and gsw8-D were tested with the sterile line Quanxiang 1A (QX1A), and it was found that the QX1A/gsw8-D combination had a slightly lower plant height than the QX1A/R498 combination, and there was no significant difference in grain length, but the grain width increased by 15.12%, the 1000-grain weight increased by 10.73%, and the actual yield per plant in the field increased by 12.30% (Figure 5).

In addition, HZ and HZ-gsw8-D were tested with the sterile line Quan 9311A (Q9311A), and it was found that the plant height of the Q9311A/HZ-gsw8-D combination was slightly lower than that of the Q9311A/HZ combination, and there was no significant difference in grain length, but the grain width increased by 18.60%, and the 1000-grain weight increased by 23.28%. Finally, the actual single-plant yield in the field increased by 14.86% (Figure 6).

In summary, the gsw8-D allele is of great value in hybrid rice breeding and can significantly increase the yield of hybrid rice.

Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims (9)

1. A SNP molecular marker related to rice grain width and grain weight, which is characterized in that the SNP molecular marker is positioned at 1071 base of GSW8 gene of rice 8 chromosome, and the polymorphism is C or T.

2. A primer group for detecting the SNP molecular marker according to claim 1, wherein the nucleotide sequence of the primer group is shown as SEQ ID NO. 1-4.

3. A kit comprising the primer set of claim 2.

4. A gene chip comprising the primer set according to claim 2.

5. The use of the primer set of claim 2 and the kit of claim 3 for detecting rice grain width and grain weight.

6. The use of the primer set of claim 2 and the kit of claim 3 for breeding rice varieties with increased grain width and grain weight.

7. Use of the primer set of claim 2 and the kit of claim 3 for breeding transgenic rice with increased grain width and grain weight or for improving rice germplasm resources.

8. Use of the SNP molecular marker of claim 1 in rice breeding.

9. A method for detecting the SNP molecular marker of claim 1, comprising the steps of:

S1, extracting DNA of a sample to be detected;

S2, detecting by using the primer set of claim 2 or the kit of claim 3 by taking DNA of a sample to be detected as a template;

s3, if the base of the sample to be detected at 1071 position of the GSW8 gene of the 8 th chromosome of the rice is T, judging that the rice is a variety with increased grain width and grain weight.