SNP (Single Nucleotide Polymorphism) marker for detecting different ripening stages of rice, marker primer and application of SNP marker

Abstract

The invention discloses an SNP marker for detecting different ripening stages of rice, marker primers and application of the SNP marker, the SNP marker is C / T mutation of an Hd17 gene at a second exon 1016 position and T / C mutation of an Hd17 gene at a fourth exon 1673 position, the marker primers capable of identifying the mutation information of the site are developed aiming at the mutation, and the nucleotide sequences of the marker primers are respectively shown as SEQ ID NO.3-8. Through verification, the marker primers can be used for identifying the mutation information of the site. The marker primer developed by aiming at the SNP marker can quickly screen and confirm the SNP locus information of a material, the detection result is accurate, the marker primer can be used for screening germplasm resources, materials of different SNP combinations can be selected for planting according to the requirements of different regions on the ripening stage of rice, the adaptability of variety planting of rice in different regions can be effectively improved, and the application prospect is wide. And assistance is provided for rice molecular breeding work.

Description

Technical Field

[0001] The invention relates to the field of molecular marker identification, and in particular to a SNP marker for detecting different maturity stages of rice, a marker primer and an application thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world. Heading date is an important agronomic trait that determines the adaptability of varieties to regions and seasons. Therefore, the allele mining of heading date genes has important theoretical significance for variety improvement, and the development of functional markers directly reflects its application value. In 2012, Matsubara et al. discovered the rice heading date-related gene Hd17 using the map-based cloning method and located the gene on rice chromosome 6 (Matsubara et al., 2012). In terms of the haplotype study of the gene, the materials studied by Matsubara et al. in 2012 were divided into three haplotypes (the first type is Nipponbare, the second type is Kasalath, and the third type is Koshihikari). Among them, the SNP difference at position 1673 caused the amino acid to change from L to S, causing a change in gene function. They then tested the base variation at this functional site in Asian cultivated rice (indica and japonica) and wild rice materials, and found that a few japonica rice materials were T (consistent with Nipponbare) and most were C; very few indica rice materials were T, and the rest were C; and all wild rice materials were C (consistent with Koshihikari); in other words, a few japonica rice materials had bases consistent with Nipponbare at this functional site, while indica rice and wild rice were basically consistent with Koshihikari (Matsubara et al., 2012).

[0003] According to existing reports, the materials for analyzing the SNP changes of the Hd17 gene are not comprehensive. The SNP (C / T) difference at position 3869 is insufficiently supported as a functional marker for identifying rice varieties or evolution. It is not unique enough and cannot be well applied to variety breeding. In the study of Matsubara et al (2012), it was proposed that the 'Koshihikari' SNP (C) at position 3869 is mainly distributed in indica rice and wild rice, while the 'Nipponbare' SNP (T) is mainly distributed in japonica rice. However, due to the limited number of materials, no definite conclusion can be drawn and there is no obvious difference in geographical distribution. The heading period of rice determines the regional adaptability of rice and affects its yield. The mining of more alleles provides more options for the breeding of excellent rice varieties. At present, the research materials of rice heading period genes are concentrated in Asian cultivated rice and some wild rice, and African rice is not involved. Based on this, this study not only introduces Asian cultivated rice and wild rice, but also introduces African rice materials to broaden the research basis, which will be more comprehensive and convincing for the study of allelic variation and development of functional markers of a gene. In short, there are more and deeper functions of the Hd17 gene to be explored, and the developed functional markers have significant application value in the screening and identification of excellent variety breeding. Summary of the invention

[0004] The purpose of the present invention is to provide a SNP marker and marker primer for detecting different maturity stages of rice. The marker primer can quickly screen and confirm the SNP site information of the material, and can select materials with different SNP combinations for planting according to the requirements of rice maturity in different regions, effectively improve the adaptability of rice varieties planted in different regions, and can be used for screening germplasm resources to provide assistance for rice molecular breeding.

[0005] In order to achieve the above object, the present invention provides an application of a rice SNP marker in detecting different maturity stages of rice, wherein the SNP marker comprises a C / T mutation at position 1016 of the second exon and a T / C mutation at position 1673 of the fourth exon of the rice Hd17 gene.

[0006] The present invention also provides a SNP marker primer for detecting different maturity stages of rice. The marker primer comprises SNP1016 and SNP1673, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 3 to 5 and SEQ ID NOs. 6 to 8, respectively.

[0007] The present invention also provides a kit for detecting different maturity stages of rice, which comprises marker primers whose nucleotide sequences are shown as SEQ ID NOs. 3 to 5 and SEQ ID NOs. 6 to 8 respectively.

[0008] The marker primers provided by the invention can be used in rice breeding, including identification of rice maturity and screening of rice germplasm resources at different maturity stages.

[0009] The present invention also provides a method for identifying different maturity stages of rice, comprising the following steps:

[0010] (1) extracting genomic DNA from the rice plants to be tested;

[0011] (2) using the extracted genomic DNA as a template and performing PCR amplification using the above-mentioned labeled primers;

[0012] (3) The maturity phenotype of rice is determined by the amplification results. When the genotype of the two SNP loci of the tested rice is TC type, the tested rice is the latest to mature; when it is CC type, the tested rice is medium to mature; when it is CT type, the tested rice is the earliest to mature.

[0013] Preferably, the PCR amplification program is 94°C for 15 min; 94°C for 20 sec, 61-55°C for 60 sec, 94°C for 20 sec, with touch-down of -0.6°C / Cycle, for a total of 10 cycles; 94°C for 20 sec; 57°C for 60 sec, for a total of 28 cycles.

[0014] The present invention has the following advantages:

[0015] The present invention develops and discloses two SNP sites for the first time. Different genes mutated at these sites can lead to phenotypic differences in rice at different maturity stages. The SNP site can be used as a SNP marker. The detection of this marker can be used for the detection and identification of rice maturity phenotypes, which has practical forward-looking application value in rice breeding.

[0016] The present invention discloses marker primers developed for the above two SNP site markers, which can realize the detection and identification of the maturity phenotype of the detected rice material through PCR amplification, and can select materials with different SNP combinations for planting according to the requirements of rice maturity in different regions, effectively improving the adaptability of rice varieties planted in different regions, and can be used for screening of germplasm resources, and has significant application value in rice molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of SNP variation and phenotypic analysis in the coding region of the Hd17 gene in the 14 SSSLs of the present invention are shown.

[0018] Figure 2 It is the two SNP site information of the Hd17 gene in the present invention and the growth phenotype and maturity of the corresponding rice material.

[0019] Figure 3This is the KASP amplification result of the SNP1 (1016) site of 41 materials in the present invention.

[0020] Figure 4 This is the KASP amplification result of the SNP2 (1673) site of 41 materials in the present invention. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0023] Experimental Example 1 Development of SNP markers and marker primers

[0024] The donor parents W01 (Tetep), W03 (Zhong4188), W05 (Zihui 100), W08 (IR64), W10 (Nanyangzhan), W20 (Chenglongshuijingmi), W22 (KhAzAr), W24 (Starbonnet99), W27 (IAPAR9) and HP (O.glaberrima) from all over the country and different countries were hybridized with the recipient parent HJX74 (Huajingxian 74), and then single segment substitution lines (SSSLs) were obtained through multi-generation backcrossing. By designing detection primers for the Hd17 gene, molecular marker detection was used to screen and confirm that 14 SSSSLs carried the Hd17 gene from the backcross lines. The fragment identification results are shown in Table 1.

[0025] Table 1 Information on single segment replacement materials carrying Hd17 gene

[0026]

[0027]

[0028] After comparison, the rice heading period-related gene Hd17 is homologous to the gene encoding the ELF3 protein in Arabidopsis. The sequence of Hd17 in Nipponbare has 2283 bases and encodes 760 amino acids. In addition to Nipponbare, the gene coding sequences of Hd17 of the recipient parent HJX74 and 14 SSSLs were sequenced by designing primers Hd17-1F and Hd17-1R. The specific primer information is shown in Table 2. The coding sequence of Hd17 was aligned and analyzed using ClustalW2 software as well as haplotype analysis.

[0029] Table 2 Sequencing primer information of Hd17

[0030]

[0031] The SNP variation and phenotypic analysis results of the Hd17 gene coding region in 14 SSSLs were obtained by sequence comparison. Figure 1 As shown, it can be seen that there are 4 haplotypes in 14 SSSLs. Matsubara K et al. (Matsubara K, Natural variation in Hd17, a homolog of Arabidopsis ELF3 that is involved in rice photoperiodic flowering. Plant Cell Physiol. 2012) proposed that the T / C mutation at position 1673 of the Hd17 gene in exon 4 caused the amino acid L to mutate to S, which caused the change in gene function and led to the difference in heading period phenotype. In addition, the comparison of genotype and phenotype found that the C / T mutation at position 1016 of exon 2 caused the amino acid to mutate from P to L, which also affected the change in its function. The changes in the two positions of SNP1016 and SNP1673 and the different combinations of bases (TC, CC, CT) determine the functional and phenotypic differences of Hd17.

[0032] By extracting genomic DNA from 31 parental materials (donor parents collected from different regions by the College of Agriculture of South China Agricultural University) that had been resequenced and spliced ​​at the tillering stage, the Hd17 gene was amplified to obtain the SNP information of the 1016 and 1673 sites for comparison analysis. The results are shown in Table 3, which shows that among these 31 parental materials, according to the base changes at the 1016 and 1673 positions, there are also these three different combinations (TC, CC, CT).

[0033] Table 3 Results of SNP variation analysis in the coding region of the Hd17 gene of 31 resequencing data

[0034]

[0035]

[0036] At the same time, the coding region sequences of the Hd17 gene of six wild rice species (O.rufipogon, O.meridionalis, O.nivara, O.barthii, O.glumipatula and O.longistaminata) were sequenced and sequence comparison analysis was performed. It was found that there were only two combination types of Hd17 in the six wild rice species based on the differences in sites 1016 and 1673, namely TC and CC types, as shown in Table 4 for details.

[0037] Table 4 SNP analysis of Hd17 in 6 wild rice species

[0038]

[0039]

[0040] In order to more fully analyze the differences in the gene sequence of Hd17 in different rice varieties, 3000 published SNPs of the Hd17 gene of cultivated rice were downloaded from the website RFGB (https: / / www.rmbreeding.cn / ). Haplotype analysis was performed on these 3000 rice SNP data. After removing some controversial results, 2733 valid data were used for sequence analysis. According to the base differences at sites 1016 and 1673, there are also three types of base compositions: TC, CC and CT, as shown in Table 5.

[0041] Table 5 SNP analysis of Hd17 in 2733 cultivated rice accessions

[0042]

[0043] In summary, combined with the two loci information of the Hd17 gene and the growth phenotype and maturity of the corresponding rice materials, see Figure 2 ,in, Figure 2 A in the figure is a schematic diagram of Hd17 locus information of rice with different genotypes. Figure 2 B in the figure is the phenotype of rice with different genotypes. It can be seen that the function of Hd17 is determined by the different base compositions of the two sites 1016 and 1673. The TC type is the latest maturing (named Hd17-1), and its heading period is about 81 days; the CC type is the middle maturing (named Hd17-2), and its heading period is about 79 days; the CT type is the earliest maturing (named Hd17-3), and its heading period is about 76 days.

[0044] According to the base variation of these two sites, two sets of marker primers were developed and designed for tracking the corresponding SNP markers of Hd17 gene, as shown in Table 6, where SNP1 and SNP2 correspond to sites 1016 and 1673, respectively.

[0045] Table 6 SNP functional markers and marker primers of Hd17

[0046]

[0047]

[0048] Experimental Example 2 Application of labeled primers

[0049] In order to detect the effect of the SNP markers obtained above, the functional markers SNP1 (site 1016) and SNP2 (site 1673) were selected as examples for testing. The KASP platform of LGC Company was used to perform SNP high-throughput detection on the above 14 SSSLs and 27 parental materials. The PCR amplification program of KASP is shown in Table 7. The mutations of each material at sites 1016 and 1673 of the Hd17 gene were analyzed. The SNP conditions of all materials at sites 1016 and 1673 of Hd17 are shown in Table 8. The SNP1 amplification results of all materials are shown in Table 9. Figure 3 As shown, the black dot in the lower left corner is the negative control, and the upper left corner and lower right corner are homozygous. The upper left corner indicates that the base at position 1016 of the second exon is T, and the lower right corner indicates that the base is C. The amplification results of SNP2 are shown in Figure 4 As shown, the black dot in the lower left corner is the negative control, and the upper left corner and lower right corner are homozygous. The upper left corner indicates that the 1673 position of the fourth exon is base T, and the lower right corner indicates base C.

[0050] Table 7 PCR amplification program

[0051]

[0052] Table 8 SNPs of Hd17 at 1673 locus in different materials

[0053]

[0054]

[0055] As can be seen from Table 8, the amplification effect of the SNP marker is very good. Through the amplification identification of the marker primer, it can directly reflect the amplification of different materials at this site and directly distinguish the SNP mutation of each material at this site. At position 1016 of exon 2, in the SSSL materials of donors W1 and W3, and in the materials of donors W1, W2, W3, W4, W9, W11, W12, W13, W15, and W17, the base is T, and the base at this site of the other materials tested is C, which is consistent with the above Figure 1 The sequencing results in are consistent. At position 1673 of exon 4, from the results in Table 8, it can be seen that the base in the SSSL materials with donors W8, W10, W20, and W22, and the materials with donors HJX74, W7, W8, W10, W18, W19, and W20 is T, while the base in the other materials tested is C at this position, which is consistent with the above Figure 1 At the same time, after statistics, this result is consistent with the growth phenotype of the corresponding material, that is, the maturity result.

[0056] In summary, the Hd17 marker developed and designed by the present invention is usable. The SNP marker primers developed and designed at the same time can play a good identification role in the detection of experimental materials, can quickly screen and confirm the SNP site information of the material, and the detection results are accurate, which can be used for the screening of germplasm resources and provide assistance for rice molecular design breeding. By analyzing a large amount of materials to obtain a combination of two different SNPs, rice varieties with different maturity periods can be created: TC type is the latest, CC type is medium-mature, and CT type is the earliest. Materials with different SNP combinations can be selected for planting according to the requirements of rice maturity in different regions, which will effectively improve the adaptability of rice varieties planted in different regions.

[0057] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. An application of a rice SNP marker in detecting different maturity stages of rice, characterized in that: The SNP marker includes a C / T mutation at position 1016 of the second exon and a T / C mutation at position 1673 of the fourth exon of the rice Hd17 gene.

2. A SNP marker primer for detecting different maturity stages of rice, characterized in that: The marker primers include SNP1016 and SNP1673, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 3 to 5 and SEQ ID NOs. 6 to 8, respectively.

3. A kit for detecting different maturity stages of rice, characterized in that: The kit comprises the labeled primer as claimed in claim 2.

4. Use of the labeled primer as claimed in claim 2 in rice breeding.

5. The use according to claim 4, characterized in that: The application includes the identification of rice maturity and the screening of rice germplasm resources at different maturity stages.

6. A method for identifying rice at different maturity stages, characterized in that: The following steps are included: (1) extracting genomic DNA from the rice plants to be tested; (2) using the extracted genomic DNA as a template and performing PCR amplification using the labeled primers described in claim 2; (3) The maturity phenotype of rice is determined by the amplification results. When the genotype of the two SNP loci of the tested rice is TC type, the tested rice is the latest to mature; when it is CC type, the tested rice is medium to mature; when it is CT type, the tested rice is the earliest to mature.

7. The identification method according to claim 6, characterized in that: The PCR amplification program is 94°C for 15 min; 94°C for 20 sec, 61-55°C for 60 sec, with touch-down of -0.6°C / Cycle, for a total of 10 cycles; 94°C for 20 sec; 57°C for 60 sec, for a total of 28 cycles.

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