Molecular marker related to rice tiller number regulation gene and application thereof

Abstract

The embodiment of the invention discloses a molecular marker related to a rice tiller number regulation gene and application of the molecular marker. The molecular marker comprises nucleotide single base mutation at the 28427378bp and the 28513517bp on the No.3 chromosome of a rice genome and insertion of two nucleotide bases at the 28428063bp and the 284283477bp, and the molecular markers are SNP328427378A / G, INDEL328428063C / CAT, INDEL3284283477T / TAC and SNP328513517C / T. The molecular marker is characterized in that the nucleotide single base mutation at the 28427378bp and the nucleotide single base insertion at the 28428063bp and the 284283477bp on the No.3 chromosome of the rice genome are shown in the description. The molecular marker provided by the invention can be used for rapidly and accurately detecting genotypes with more tillering numbers of the rice OsTB1 and OsSLR1 genes, and is beneficial to efficient application of the genes in improvement of a rice plant type structure.

Description

Technical Field

[0001] The present invention relates to the technical field of crop genetic breeding, and in particular to a molecular marker related to a rice tiller number regulating gene and its application. Background Art

[0002] Rice is one of the most important food crops in the world, among which the number of tillers and panicles are two key factors affecting rice yield.

[0003] The number of rice tillers determines the number of panicles, which directly affects rice yield and plant type, and is a very important agronomic trait. The ideal number of tillers is of great significance to the formation of an ideal plant type and to ensure the high yield, high quality and stable yield of rice varieties. Therefore, discovering the tiller number-related genes that directly affect the number of panicles per plant and revealing the genetic mechanism of tiller development and the establishment of the effective number of panicles are of great significance for improving the yield level of rice varieties. OsTB1 and OsSLR1 are both key genes that regulate the number of rice tillers and play an important regulatory role in improving the number of rice tillers.

[0004] Since the natural variation sites of many genes often lead to enhanced or weakened gene functions, the excellent alleles can be used for crop molecular breeding. Mining the excellent alleles of key genes that regulate rice tillering 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 alleles of genes that regulate the number of rice tillers, 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 regulating gene and its application. Summary of the invention

[0006] The embodiments of this specification provide a rice tiller number regulating gene-related molecular marker and its application, providing genetic resources for the genetic improvement of rice tillering. 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, to accurately identify the tested rice.

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

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

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

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

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

[0012] The molecular marker INDEL_3_28428347_T / TAC is located at 28428347bp on rice chromosome 3, and has two nucleotide base insertions;

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

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

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

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

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

[0018] The primer pair for detecting the molecular marker SNP_3_28513517_C / T includes specific upstream primers X4F and Y4F and downstream universal primer C4R, and the primer sequences are shown in the sequence listing SEQ ID NOs: 10-12, respectively.

[0019] The present specification also provides a method for detecting multiple genotypes of tiller number of rice genes OsTB1 and OsSLR1, the detection method comprising:

[0020] Using rice genomic DNA as a template, PCR reaction was performed using the primer sequences SEQ ID NO: 1-12 of the present application to detect the genotypes of SNP_3_28427378, INDEL_3_28428063, and INDEL_3_28428347 sites in the rice genomic DNA to be tested;

[0021] If the genotype of the SNP_3_28427378 site of the rice genome to be tested is genotype A, the genotype of the INDEL_3_28428063 site is genotype C, and the genotype of the INDEL_3_28428347 site is genotype T, then the rice to be tested is the OsTB1 genotype with a small number of tillers;

[0022] If the genotype of the SNP_3_28427378 site of the rice genome to be tested is the G genotype, the genotype of the INDEL_3_28428063 site is the CAT genotype, and the genotype of the INDEL_3_28428347 site is the TAC genotype, then the rice to be tested is the OsTB1 genotype with a large number of tillers;

[0023] The genotype of the SNP_3_28513517 site in the genomic DNA of the rice to be tested is detected. If the genotype of the SNP_3_28513517 site in the genome of the rice to be tested is the C genotype, the rice to be tested is an OsSLR1 genotype with a small number of tillers; if the genotype of the SNP_3_28513517 site in the genome of the rice to be tested is the T genotype, the rice to be tested is an OsSLR1 genotype with a large number of tillers.

[0024] The embodiments of the present specification also provide a kit for detecting molecular markers related to the number of rice tillers, wherein the kit comprises primers with primer sequences of SEQ ID NOs: 1-12.

[0025] The present specification also provides a gene chip in an embodiment, wherein the gene chip comprises primers having nucleotide sequences as shown in SEQ ID NO: 1-12 in the sequence listing.

[0026] The embodiment of the present specification also provides an application of a molecular marker related to the number of rice tillers in rice breeding, characterized in that the molecular marker related to the number of rice tillers 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 rice genome os-Nipponbare-reference-IRGSp-1.0 sequence as a reference sequence;

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

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

[0030] The molecular marker INDEL_3_28428347_T / TAC is located at 28428347bp on rice chromosome 3, and has two nucleotide base insertions;

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

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

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

[0034] The embodiments of the present specification also provide a rice breeding method, wherein the detection method detects the genotypes of the rice genes OsTB1 and OsSLR1, and selects rice samples carrying the OsTB1 and OsSLR1 genotypes with a large number of tillers for subsequent breeding.

[0035] At least one of the above technical solutions adopted in the embodiments of this specification can achieve the following beneficial effects: using the OsTB1 and OsSLR1 molecular markers of the present invention, the genotypes of rice tillering regulatory genes OsTB1 and OsSLR1 can be quickly and accurately detected in germplasm resources such as indica rice and japonica rice, as well as recombinant inbred line materials after hybridization of different parents. The present invention uses the SNP molecular markers and INDEL molecular markers to perform large-scale identification of breeding populations through KASP technology, accelerate the molecular breeding process, and facilitate the application of tillering regulatory genes OsTB1 and OsSLR1 in breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of this specification or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this specification. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0037] Figure 1 The tillering number of rice varieties with different genotypes of rice genes OsTB1 and OsSLR1;

[0038] Figure 2 This is the application of the molecular markers of the embodiments of the present invention in the identification of tillering number of rice materials. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below in conjunction with the drawings in the embodiments of this specification. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments of this specification, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

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

[0041] Example 1

[0042] The molecular markers of rice genes OsTB1 and OsSLR1 and their applications are obtained by analyzing the tillering number data of 653 core rice germplasm resources to find several polymorphic sites related to tillering number in rice genes OsTB1 and OsSLR1, including 2 SNPs and 2 INDELs. The details are as follows:

[0043] 1. Use the rice reference genome (os-Nipponbare-reference-IRGSp-1.0) sequence to design primers and obtain the molecular markers provided in the examples of this specification.

[0044] 2. Sample testing

[0045] The CTAB method was used to extract rice leaf genomic DNA, and the molecular markers SNP_3_28427378_A / G, INDEL_3_28428063_C / CAT, INDEL_3_28428347_T / TAC, and SNP_3_28513517_C / T were detected using KASP technology. 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 30μM 0.046 100 μM Genotype 1 Primer X 12μM 0.046 100 μM Genotype 2 Primer Y 12μM 0.046 2×KASP mix 5 DNA Template 50ng / ul 5 Ultrapure water To 10 Total volume 10 (ignore 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, annealing temperature decreased by 1°C in 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, the fluorescence data was read at 30°C, and the fluorescence data was plotted as an XY scatter plot with HEX / ROX on the Y axis and FAM / ROX on the X axis. If a large number of fluorescence signals corresponding to primer X were detected in the fluorescence data of the sample amplification product, the detection site was a homozygous OsTB1, OsSLR1 genotype with a small number of tillers; if a large number of fluorescence signals corresponding to primer Y were detected, the detection site was a homozygous OsTB1, OsSLR1 genotype with a large number of tillers; if two fluorescence signals were detected at the same time, the detection site was a heterozygous OsTB1, OsSLR1 genotype.

[0049] 3. Verification of 16 germplasm resources

[0050] 16 rice germplasm resources 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. As can be seen from Table 2, the detection results of 8 materials, including Jiefangxian, Guangluai 15-1, Zhenshan 97, Guang122, Qingsiai 16B, Matatag2, Malayhong and IR64a, were OsTB1 and OsSLR1 genotypes with a large number of tillers, and the detection results of the other 8 materials were OsTB1 and OsSLR1 genotypes with a small number of tillers. Based on the typing results in Table 2, the tiller numbers of these 16 rice germplasm resources were further counted, and the results are as follows Figure 1As shown. Figure 1 As shown, the tillering number of germplasm resources containing OsTB1 and OsSLR1 genotypes with a large number of tillers is higher than that of materials containing OsTB1 and OsSLR1 genotypes with a small number of tillers. It can be seen that the molecular markers described in the present invention can be used for efficient detection of rice OsTB1 and OsSLR1 tillering number genotypes to screen rice OsTB1 and OsSLR1 tillering number germplasm resources.

[0051] It should be noted that a large number of tillers or a small number of tillering data both mean a relatively large number of tillers or a relatively small number of tillers. Specifically, compared with rice varieties in which no molecular marker site variation has occurred, a relatively increased number of tillers is considered to have a large number of tillers. Similarly, compared with rice varieties in which no molecular marker site variation has occurred, a relatively decreased number of tillers is considered to have a small number of tillers.

[0052] Table 2. Typing 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 tillering materials

[0055] The number of germplasm resources in natural populations was expanded, and the genotyping of rice tillering regulatory genes OsTB1 and OsSLR1 was performed using the molecular markers described in the present invention. The tillering number and yield data under normal conditions and salt stress (0.4%) were further analyzed. The results are as follows: Figure 2 As shown, the tiller number and yield of the genotype materials containing OsTB1 and OsSLR1 with a large number of tillers under normal conditions and salt stress (0.4%) conditions are higher than those of the genotype materials containing OsTB1 and OsSLR1 with a small number of tillers. 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 number of tillers and salt tolerance and their subsequent breeding applications.

[0056] By using the OsTB1 and OsSLR1 molecular markers of the present invention, the genotypes of the rice tillering regulatory genes OsTB1 and OsSLR1 can be quickly and accurately detected in germplasm resources such as indica rice and japonica rice, as well as 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, the molecular breeding process can be accelerated, and the application of the tillering regulatory genes OsTB1 and OsSLR1 in breeding can be facilitated.

[0057] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0058] The above is only an embodiment of this specification and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A molecular marker related to the number of rice tillers, characterized in that: The molecular markers include: SNP_3_28427378_A / G, INDEL_3_28428063_C / CAT, INDEL_3_28428347_T / TAC, SNP_3_28513517_C / T; The molecular marker uses the rice genome os-Nipponbare-reference-IRGSp-1.0 sequence as a reference sequence; The molecular marker SNP_3_28427378_A / G is located at 28427378bp on chromosome 3 of rice, and has a nucleotide single base mutation A / G; The molecular marker INDEL_3_28428063_C / CAT is located at 28428063bp on chromosome 3 of rice, and has two nucleotide base insertions; The molecular marker INDEL_3_28428347_T / TAC is located at 28428347bp on rice chromosome 3, and has two nucleotide base insertions; The molecular marker SNP_3_28513517_C / T is located at 28513517bp on chromosome 3 of rice, and has a nucleotide single base mutation C / T.

2. The molecular marker according to claim 1, characterized in that 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 and Y1F and downstream universal primer C1R, and the primer sequences are shown in the sequence table SEQ ID NO: 1-3 respectively; The primer pair for detecting the molecular marker INDEL_3_28428063_C / CAT includes specific upstream primers X2F and Y2F and downstream universal primer C2R, and the primer sequences are shown in the sequence table SEQ ID NO: 4-6 respectively; The primer pair for detecting the molecular marker INDEL_3_28428347_T / TAC includes specific upstream primers X3F and Y3F and downstream universal primer C3R, and the primer sequences are shown in the sequence table SEQ ID NO: 7-9 respectively; The primer pair for detecting the molecular marker SNP_3_28513517_C / T includes specific upstream primers X4F and Y4F and downstream universal primer C4R, and the primer sequences are shown in the sequence listing SEQ ID NOs: 10-12, respectively.

3. A method for detecting multiple genotypes of tiller number of rice genes OsTB1 and OsSLR1, characterized in that: The detection method comprises: Using rice genomic DNA as a template, using the primers described in claim 2 to perform a PCR reaction to detect the genotype of SNP_3_28427378, INDEL_3_28428063, and INDEL_3_28428347 sites in the rice genomic DNA to be tested; If the genotype of the SNP_3_28427378 site of the rice genome to be tested is genotype A, the genotype of the INDEL_3_28428063 site is genotype C, and the genotype of the INDEL_3_28428347 site is genotype T, then the rice to be tested is the OsTB1 genotype with a small number of tillers; If the genotype of the SNP_3_28427378 site of the rice genome to be tested is the G genotype, the genotype of the INDEL_3_28428063 site is the CAT genotype, and the genotype of the INDEL_3_28428347 site is the TAC genotype, then the rice to be tested is the OsTB1 genotype with a large number of tillers; The genotype of the SNP_3_28513517 site in the genomic DNA of the rice to be tested is detected. If the genotype of the SNP_3_28513517 site in the genome of the rice to be tested is the C genotype, the rice to be tested is an OsSLR1 genotype with a small number of tillers; if the genotype of the SNP_3_28513517 site in the genome of the rice to be tested is the T genotype, the rice to be tested is an OsSLR1 genotype with a large number of tillers.

4. A kit for detecting the molecular markers related to the rice tillering number according to claim 1, characterized in that: The kit comprises the primers according to claim 2.

5. A gene chip, characterized in that: The gene chip includes primers whose nucleotide sequences are shown in SEQ ID NO: 1-12 in the sequence table.

6. Application of a molecular marker related to rice tillering number in rice breeding, characterized in that: The molecular marker related to the rice tillering number is as described in claim 1.

7. The use according to claim 6, characterized in that The application is used for screening rice varieties with a large number of rice tillers.

8. The use according to claim 7, characterized in that The application is further used for screening salt-tolerant rice varieties.

9. A rice breeding method, characterized in that: The genotypes of the rice genes OsTB1 and OsSLR1 are detected according to the detection method of claim 3, and rice samples carrying the OsTB1 and OsSLR1 genotypes with a large number of tillers are selected for subsequent breeding.

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