Molecular Markers Related to Genes Regulating Rice Tillering Number and Their Applications

Abstract

This embodiment of the specification discloses molecular markers related to rice tiller number regulation genes and their applications. The molecular markers include single nucleotide mutations at positions 28,427,378 bp and 28,513,517 bp on chromosome 3 of the rice genome, and two nucleotide insertions at positions 28,428,063 bp and 28,428,347 bp. The molecular markers are respectively: SNP_3_28427378_A / G, INDEL_3_28428063_C / CAT, INDEL_3_28428347_T / TAC, SNP_3_28513517_C / T. Using the molecular markers provided by the present invention, genotypes with more tiller numbers of rice OsTB1 and OsSLR1 genes can be detected quickly and accurately, which is beneficial to the efficient application of these genes in improving the plant type structure of rice.

Description

Technical Field

[0001] This specification relates to the technical field of crop genetic breeding, and particularly to a molecular marker related to a rice tiller number regulation gene and its application. Background Art

[0002] Rice is one of the most important food crops in the world. Among them, the tiller number and the panicle number are two key factors affecting rice yield.

[0003] The rice tiller number determines the panicle number and directly affects rice yield and plant type. It is a very important agronomic trait. An ideal tiller number is of great significance for forming an ideal plant type and ensuring high yield, good quality and stable yield of rice varieties. Therefore, exploring the tiller number-related genes that directly affect the panicle number per plant and revealing the genetic mechanism of tiller development and the establishment of effective panicle number is of great significance for improving the yield per unit of rice varieties. Both OsTB1 and OsSLR1 are key genes regulating rice tiller number and play an important regulatory role in improving rice tiller number.

[0004] Since natural variation sites of many genes often lead to enhanced or weakened gene functions, and the excellent allelic variations among them can be used for crop molecular breeding, mining the excellent allelic variations of key genes regulating rice tiller development can lay an important foundation for increasing rice yield. Molecular marker technology can achieve rapid and high-energy screening of excellent traits, so it is widely used in crop genetic improvement. Rice tillering is a quantitative trait controlled by multiple loci. It is urgent to mine the excellent allelic variations of genes regulating rice tiller number, so as to use molecular markers to aggregate excellent alleles, improve rice tillering and increase rice yield.

[0005] Based on this, the embodiments of this specification provide a molecular marker related to a rice tiller number regulation gene and its application. Summary of the Invention

[0006] The embodiments of this specification provide a molecular marker related to a rice tiller number regulation gene and its application, providing gene resources for rice tiller genetic improvement. This specification also provides primer sets and detection methods corresponding to SNP molecular markers and INDEL molecular markers for rice tiller number-related genes OsTB1 and OsSLR1 respectively, so as to accurately identify the rice to be tested.

[0007] To solve the above technical problems, the embodiments of this specification are implemented as follows:

[0008] This embodiment of the specification provides a molecular marker related to the tiller number of rice, and the molecular marker includes SNP_3_28427378_A / G, INDEL_3_28428063_C / CAT, INDEL_3_28428347_T / TAC, SNP_3_28513517_C / T;

[0009] The molecular marker uses the rice genome os-Nipponbare-reference-IRGSp-1.0 sequence as the reference sequence;

[0010] The molecular marker SNP_3_28427378_A / G is located at the 28427378bp on chromosome 3 of rice, and there is a single nucleotide mutation A / G;

[0011] The molecular marker INDEL_3_28428063_C / CAT is located at the 28428063bp on chromosome 3 of rice, and there are 2 nucleotide base insertions;

[0012] The molecular marker INDEL_3_28428347_T / TAC is located at the 28428347bp on chromosome 3 of rice, and there are 2 nucleotide base insertions;

[0013] The molecular marker SNP_3_28513517_C / T is located at the 28513517bp on chromosome 3 of rice, and there is a single nucleotide mutation C / T.

[0014] Furthermore, the detection primers of the molecular marker include:

[0015] The primer pair for detecting the molecular marker SNP_3_28427378_A / G includes the specific upstream primers X1F, Y1F and the downstream universal primer C1R, and the primer sequences are shown in SEQ ID NO:1-3 of the sequence list respectively;

[0016] The primer pair for detecting the molecular marker INDEL_3_28428063_C / CAT includes the specific upstream primers X2F, Y2F and the downstream universal primer C2R, and the primer sequences are shown in SEQ ID NO:4-6 of the sequence list respectively;

[0017] The primer pair for detecting the molecular marker INDEL_3_28428347_T / TAC includes the specific upstream primers X3F, Y3F and the downstream universal primer C3R, and the primer sequences are shown in SEQ ID NO:7-9 of the sequence list respectively;

[0018] The primer pairs for detecting the molecular marker SNP_3_28513517_C / T include specific upstream primers X4F, Y4F and a downstream universal primer C4R, and the primer sequences are shown in SEQ ID NO:10-12 of the sequence listing respectively.

[0019] The embodiments of this specification also provide a method for detecting the tiller number genotypes of rice genes OsTB1 and OsSLR1. The detection method includes:

[0020] Using rice genomic DNA as a template, performing PCR reactions with the primer sequences SEQ ID NO:1-12 of this application to detect the genotypes of SNP_3_28427378, INDEL_3_28428063, and INDEL_3_28428347 loci in the genomic DNA of the rice to be tested;

[0021] If the genotype of the SNP_3_28427378 locus in the genome of the rice to be tested is the A genotype, the genotype of the INDEL_3_28428063 locus is the C genotype, and the genotype of the INDEL_3_28428347 locus is the T genotype, then the rice to be tested is the OsTB1 genotype with fewer tillers;

[0022] If the genotype of the SNP_3_28427378 locus in the genome of the rice to be tested is the G genotype, the genotype of the INDEL_3_28428063 locus is the CAT genotype, and the genotype of the INDEL_3_28428347 locus is the TAC genotype, then the rice to be tested is the OsTB1 genotype with more tillers;

[0023] Detect the genotype of the SNP_3_28513517 locus in the genomic DNA of the rice to be tested. If the genotype of the SNP_3_28513517 locus in the genome of the rice to be tested is the C genotype, the rice to be tested is the OsSLR1 genotype with fewer tillers; if the genotype of the SNP_3_28513517 locus in the genome of the rice to be tested is the T genotype, the rice to be tested is the OsSLR1 genotype with more tillers.

[0024] The embodiments of this specification also provide a kit for detecting molecular markers related to rice tiller number. The kit includes primers with the primer sequences SEQ ID NO:1-12.

[0025] The embodiments of this specification also provide a gene chip, and the gene chip includes primers with nucleotide sequences shown in SEQ ID NO:1-12 of the sequence listing.

[0026] The embodiments of this specification also provide an application of a molecular marker related to the tiller number of rice in rice breeding, which is characterized in that the molecular marker related to the tiller number of rice includes: SNP_3_28427378_A / G, INDEL_3_28428063_C / CAT, INDEL_3_28428347_T / TAC, SNP_3_28513517_C / T;

[0027] The molecular marker uses the os-Nipponbare-reference-IRGSp-1.0 sequence of the rice genome as the reference sequence;

[0028] The molecular marker SNP_3_28427378_A / G is located at the 28427378bp on chromosome 3 of rice, with a nucleotide single-base mutation A / G;

[0029] The molecular marker INDEL_3_28428063_C / CAT is located at the 28428063bp on chromosome 3 of rice, with 2 nucleotide base insertions;

[0030] The molecular marker INDEL_3_28428347_T / TAC is located at the 28428347bp on chromosome 3 of rice, with 2 nucleotide base insertions;

[0031] The molecular marker SNP_3_28513517_C / T is located at the 28513517bp on chromosome 3 of rice, with a nucleotide single-base mutation C / T.

[0032] Furthermore, the application is used to screen rice varieties with a large tiller number.

[0033] Furthermore, the application is further used to screen salt-tolerant rice varieties.

[0034] The embodiments of this specification also provide a rice breeding method. The genotypes of rice genes OsTB1 and OsSLR1 are detected by the detection method, and rice samples carrying the genotypes of OsTB1 and OsSLR1 with a large tiller number are selected for subsequent breeding.

[0035] The above at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects: By using the OsTB1 and OsSLR1 molecular markers of the present invention, the genotypes of the rice tiller regulation genes OsTB1 and OsSLR1 can be quickly and accurately detected in germplasm resources such as indica rice and japonica rice, as well as in recombinant inbred line materials after hybridization of different parents. By using the SNP molecular markers and INDEL molecular markers of the present invention, the breeding population can be identified on a large scale through the KASP technology, accelerating the molecular breeding process and facilitating the application of the tiller regulation genes OsTB1 and OsSLR1 in breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of this specification or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in this specification. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 Tillering numbers of rice varieties with different genotypes of the rice genes OsTB1 and OsSLR1;

[0038] Figure 2 Application of the molecular markers in the embodiments of the present invention in the identification of tillering numbers of rice materials. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] In order to enable those skilled in the art to better understand the technical solutions in this specification, the following will clearly and completely describe the technical solutions in the embodiments of this specification in conjunction with the drawings in the embodiments of this specification. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments of this specification, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0040] The experimental methods used in the embodiments of this specification are all conventional methods unless otherwise specified. The experimental materials used are all commercially available unless otherwise specified.

[0041] Example 1

[0042] Molecular markers of the rice genes OsTB1 and OsSLR1 and their applications. These molecular markers were obtained by analyzing the tillering number data of 653 core rice germplasm resources, and several polymorphic sites related to tillering numbers in the rice genes OsTB1 and OsSLR1 were found, including 2 SNPs and 2 INDELs. Specifically as follows:

[0043] 1. Primer design was carried out using the rice reference genome (os-Nipponbare-reference-IRGSp-1.0) sequence to obtain the molecular markers provided in the examples of this specification.

[0044] 2. Sample detection

[0045] Genomic DNA of rice leaves was extracted by the CTAB method. For the molecular markers SNP_3_28427378_A / G, INDEL_3_28428063_C / CAT, INDEL_3_28428347_T / TAC, and SNP_3_28513517_C / T, the KASP technology was used for detection. The KASP reaction system is shown in Table 1.

[0046] Table 1. KASP reaction system

[0047] Final concentration Volume (μl) 100 μM Universal Primer C 30 μM 0.046 100 μM Genotype 1 Primer X 12 μM 0.046 100 μM Genotype 2 Primer Y 12 μM 0.046 2×KASP mix 5 DNA Template 50 ng / ul 5 Ultra-pure water To 10 Total volume 10 (neglecting primer volume)

[0048] The PCR amplification conditions were as follows: 95°C for 15 minutes; 95°C for 10 seconds, 65°C for 60 seconds, with the annealing temperature decreasing by 1°C for each cycle, for a total of 10 cycles; 95°C for 10 seconds, 57°C for 60 seconds, for a total of 38 cycles. After the reaction was completed, fluorescence data was read at 30°C. The fluorescence data was used to create an XY scatter plot with the Y-axis as HEX / ROX and the X-axis as FAM / ROX. If a large amount of fluorescence signal corresponding to primer X was detected in the fluorescence data of the sample amplification product, the detected locus was the genotype of OsTB1 and OsSLR1 with few tillers; if a large amount of fluorescence signal corresponding to primer Y was detected, the detected locus was the genotype of OsTB1 and OsSLR1 with many tillers; if both fluorescence signals were detected simultaneously, the detected locus was the heterozygous genotype of OsTB1 and OsSLR1.

[0049] 3. Verification of 16 germplasm resource materials

[0050] Sixteen rice germplasm resource materials were used to verify the molecular markers: SNP_3_28427378_A / G, INDEL_3_28428063_C / CAT, INDEL_3_28428347_T / TAC, and SNP_3_28513517_C / T. The molecular marker detection results are shown in Table 2. It can be seen from Table 2 that the detection results of a total of 8 materials such as Jiefangxian, Guanglu'ai 15-1, Zhenshan 97, Guang122, Qingsi'ai 16B, Matatag2, Malay Red, and IR64a were the genotypes of OsTB1 and OsSLR1 with many tillers, and the detection results of the other 8 materials were the genotypes of OsTB1 and OsSLR1 with few tillers. Based on the genotyping results in Table 2, the tiller numbers of these 16 rice germplasm resource materials were further counted, and the results were as Figure 1As shown in Figure 1 As shown in Figure 1 , the tiller number of germplasm resource materials with OsTB1 and OsSLR1 genotypes having a large tiller number is higher than that of materials with OsTB1 and OsSLR1 genotypes having a small tiller number. Thus, it can be seen that the molecular markers described in the present invention can be used for the efficient detection of the tiller number genotypes of rice OsTB1 and OsSLR1, so as to screen germplasm resource materials with a large tiller number of rice OsTB1 and OsSLR1.

[0051] It should be noted that both a large tiller number and a small tiller number refer to a relatively large or relatively small tiller number. Specifically, compared with a rice variety in which no molecular marker locus variation occurs, if the tiller number is relatively increased, it is considered to have a large tiller number. Similarly, compared with a rice variety in which no molecular marker locus variation occurs, if the tiller number is relatively decreased, it is considered to have a small tiller number.

[0052] Table 2. Genotyping results of molecular markers of OsTB1 and OsSLR1 genes in 16 germplasm resources

[0053]

[0054] 4. Application of the molecular markers of the present invention in screening rice tiller materials

[0055] After expanding the number of germplasm resources in the natural population and genotyping the tiller regulation genes OsTB1 and OsSLR1 of rice using the molecular markers described in the present invention, further analysis was carried out using the tiller number and yield data under normal conditions and salt stress (0.4%) conditions. The results are as Figure 2 As shown, the tiller number and yield of genotype materials with a large tiller number of OsTB1 and OsSLR1 under normal conditions and salt stress (0.4%) conditions are both higher than those of materials with a small tiller number of OsTB1 and OsSLR1. Thus, it can be seen that the molecular markers described in the present invention can be used for the identification of rice materials with a large tiller number and salt tolerance and their subsequent breeding applications.

[0056] Using the OsTB1 and OsSLR1 molecular markers of the present invention, the genotypes of the rice tiller regulation genes OsTB1 and OsSLR1 can be quickly and accurately detected in germplasm resources such as indica rice and japonica rice, as well as in recombinant inbred line materials after hybridization of different parents. By using the SNP molecular markers and INDEL molecular markers of the present invention, the breeding population can be identified on a large scale through the KASP technology, accelerating the molecular breeding process and facilitating the application of the tiller regulation genes OsTB1 and OsSLR1 in breeding.

[0057] Each embodiment in this specification is described in a progressive manner. For the identical or similar parts among the embodiments, reference can be made to each other, and the differences between each embodiment and other embodiments are emphasized. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and for the relevant parts, reference can be made to the partial description of the method embodiment.

[0058] The above description is only for the embodiments of this specification and is not intended to limit this application. For those skilled in the art, various modifications and changes can be made to this application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the scope of the claims of this application.

Claims

1. A rice gene OsTB1 , OsSLR1 A detection method for the genotype with a large number of tillers, characterized in that The detection method includes: Using rice genomic DNA as a template, performing PCR reactions with detection primers of molecular markers to detect the genotypes of SNP_3_28427378, INDEL_3_28428063, INDEL_3_28428347 loci, and SNP_3_28513517_C / T in the genomic DNA of the rice to be tested. Among them, the molecular markers use the Os-Nipponbare-reference-IRGSp-1.0 sequence of the rice genome as a reference sequence. The detection primers of the molecular markers include: The primer pair for detecting the molecular marker SNP_3_28427378_A / G includes specific upstream primers X1F, Y1F and downstream universal primer C1R, and the primer sequences are shown in SEQ ID NO:1-3 in the sequence listing respectively; The primer pair for detecting the molecular marker INDEL_3_28428063_C / CAT includes specific upstream primers X2F, Y2F and downstream universal primer C2R, and the primer sequences are shown in SEQ ID NO:4-6 in the sequence listing respectively; The primer pair for detecting the molecular marker INDEL_3_28428347_T / TAC includes specific upstream primers X3F, Y3F and downstream universal primer C3R, and the primer sequences are shown in SEQ ID NO:7-9 in the sequence listing respectively; The primer pair for detecting the molecular marker SNP_3_28513517_C / T includes specific upstream primers X4F, Y4F and downstream universal primer C4R, and the primer sequences are shown in SEQ ID NO:10-12 in the sequence listing respectively; If the genotype of the SNP_3_28427378 locus of the rice genome to be tested is the A genotype, the genotype of the INDEL_3_28428063 locus is the C genotype, and the genotype of the INDEL_3_28428347 locus is the T genotype, then the rice to be tested has few tillers OsTB1 Genotype; If the genotype of the SNP_3_28427378 locus of the rice genome to be tested is the G genotype, the genotype of the INDEL_3_28428063 locus is the CAT genotype, and the genotype of the INDEL_3_28428347 locus is the TAC genotype, then the rice to be tested has a large number of tillers OsTB1 Genotype; Detect the genotype of the SNP_3_28513517 locus in the genomic DNA of the rice to be tested. If the genotype of the SNP_3_28513517 locus in the genomic DNA of the rice to be tested is the C genotype, the rice to be tested has a small number of tillers OsSLR1 genotype; if the genotype of the SNP_3_28513517 locus in the genomic DNA of the rice to be tested is the T genotype, the rice to be tested has a large number of tillers OsSLR1 genotype.

2. Use of a molecular marker related to the number of rice tillers in screening rice breeding with a large number of rice tillers and salt tolerance, characterized in that, The molecular markers related to the tiller number of the rice include: SNP_3_28427378_A / G, INDEL_3_28428063_C / CAT, INDEL_3_28428347_T / TAC, SNP_3_28513517_C / T; The molecular markers use the Os-Nipponbare-reference-IRGSp-1.0 sequence of the rice genome as a reference sequence; The molecular marker SNP_3_28427378_A / G is located at the 28427378bp on chromosome 3 of the rice, with a nucleotide single-base mutation A / G; The molecular marker INDEL_3_28428063_C / CAT is located at the 28428063bp on chromosome 3 of the rice, with 2 nucleotide base insertions; The molecular marker INDEL_3_28428347_T / TAC is located at the 28428347bp on chromosome 3 of the rice, with 2 nucleotide base insertions; The molecular marker SNP_3_28513517_C / T is located at the 28513517bp on chromosome 3 of the rice, with a nucleotide single-base mutation C / T.

3. A rice breeding method for screening rice with a large number of tillers and salt tolerance, characterized in that, Detect the genotypes of rice genes according to the detection method described in claim 1 OsTB1 and OsSLR1 , and select rice samples carrying genotypes with a large number of tillers OsTB1 and OsSLR1 for subsequent breeding.

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