Functional marker primer of rice bacterial blight resistant gene Xa21 and application of functional marker primer

Abstract

The invention provides a functional marker primer of a rice bacterial blight resistant gene Xa21 and application of the functional marker primer. The functional marker consists of a pair of dominant markers for identifying a disease-resistant section and a pair of dominant markers for identifying an infected section; the dominant markers for identifying the disease-resistant section are ZiW-Xa21-RF and ZiW-Xa21-RR, the target sequences of the dominant markers are located in the Xa21 gene, and the nucleotide sequences of the dominant markers are shown as SEQ ID NO.3 and SEQ ID NO.4; the dominant markers for identifying the susceptible regions are ZiW-Xa21-SF and ZiW-Xa21-SR, the target sequences of the dominant markers only specifically exist in susceptible varieties and are located in an Xa21 gene cluster, and the nucleotide sequences of the dominant markers are as shown in SEQ ID NO.5 and SEQ ID NO.6. The invention also provides a method for detecting the genotype of the rice bacterial blight resistant gene Xa21 by using the functional marker primer. The method can be used for rapidly and accurately detecting the genotype of the rice Xa21 at low cost and high throughput. The functional marker primer of the rice bacterial-blight-resistant gene Xa21 can be used for identifying bacterial-blight-resistant rice plants, screening germplasm resources and carrying out rice bacterial-blight-resistant molecular marker assisted breeding.

Description

Technical Field

[0001] Bacterial blight is one of the three major diseases of rice. At present, bacterial blight resistance identification has been included in the evaluation indicators in the variety approval in some provinces in China. In the practice of bacterial blight resistance breeding, the main method used in China is to quickly select rice varieties resistant to bacterial blight by combining the prospects of major dominant resistance genes with the background of bacterial blight resistance. The major dominant bacterial blight resistance genes that have been verified and widely promoted in China are Xa7, Xa21 and Xa23. Xa21 was cloned in the last century, and its donor came from a long-term wild rice variety IRBB21. Due to its strong resistance, Xa21, as a major dominant gene, only needs to exist in one of the parents of the hybrid combination to improve the bacterial blight resistance of the hybrid. Therefore, there is a demand for follow-up breeding of Xa21, especially in the varieties approved in Guangdong Province, China, where the carrier frequency of Xa21 is relatively high.

[0002] The existing markers for identifying the Xa21 gene mainly include the linked codominant marker pTA248 and the dominant marker U1 (Davierwala AP, et al., 2001; Wang GL, et al., 1996). Among them, the most commonly used is the marker pTA248. However, since Xa21 is a gene cluster composed of an effective resistance gene and multiple homologous genes without resistance effect, and the marker pTA248 is not completely linked to Xa21 in this gene cluster, it is easy to make false positive and false negative errors when tracking the Xa21 gene using the pTA248 marker. The dominant marker U1 can only identify whether the Xa21 gene exists, but cannot identify whether the Xa21 gene at the locus is homozygous or heterozygous, which is not conducive to the actual needs of breeding. In addition, the amplified fragment of pTA248 can reach 644-983bp in different varieties, and the amplified fragment of the dominant marker U1 is as long as 1.4kb, which requires a long extension time and a higher DNA template quality during the PCR process, and does not meet the actual needs of simple and fast breeding. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a functional marker primer of a rice bacterial blight resistance gene Xa21 and its application in view of the deficiencies of the above-mentioned prior art. The functional marker primer of the rice bacterial blight resistance gene Xa21 of the present invention can detect the rice Xa21 genotype quickly, accurately, at low cost and with high throughput, which is conducive to the efficient application of the gene in molecular breeding of rice bacterial blight resistance.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: A functional marker primer for a rice bacterial blight resistance gene Xa21, the functional marker primer comprising a primer pair ZiW-Xa21-RF / ZiW-Xa21-RR and a primer pair ZiW-Xa21-SF / ZiW-Xa21-SR; The nucleotide sequence of the primer pair ZiW-Xa21-RF / ZiW-Xa21-RR is shown in SEQ ID NO.3-4; The nucleotide sequences of the primer pair ZiW-Xa21-SF / ZiW-Xa21-SR are shown in SEQ ID NOs. 5-6.

[0005] A functional marker of a rice bacterial blight resistance gene Xa21, the functional marker comprising a functional marker amplified by a primer pair ZiW-Xa21-RF / ZiW-Xa21-RR and a functional marker amplified by a primer pair ZiW-Xa21-SF / ZiW-Xa21-SR; The nucleotide sequence of the primer pair ZiW-Xa21-RF / ZiW-Xa21-RR is shown in SEQ ID NO.3-4; The nucleotide sequences of the primer pair ZiW-Xa21-SF / ZiW-Xa21-SR are shown in SEQ ID NOs. 5-6.

[0006] Furthermore, the nucleotide sequence of the functional marker amplified by the primer pair ZiW-Xa21-RF / ZiW-Xa21-RR is shown as SEQ ID NO.1; the nucleotide sequence of the functional marker amplified by the primer pair ZiW-Xa21-SF / ZiW-Xa21-SR is shown as SEQ ID NO.2.

[0007] The use of the above-mentioned functional marker primer in any of the following: 1) Application in the detection of rice bacterial blight resistance gene Xa21; 2) Application in prediction of resistance to rice bacterial blight; 3) Application in rice breeding for resistance to bacterial blight; 4) Application in improving resistance of rice germplasm resources to bacterial blight.

[0008] A method for detecting the genotype of rice bacterial blight resistance gene Xa21 using the above functional marker primers, extracting genomic DNA from rice leaves to be tested as a template, using primer pair ZiW-Xa21-RF / ZiW-Xa21-RR and primer pair ZiW-Xa21-SF / ZiW-Xa21-SR for PCR amplification, and judging whether the rice contains the rice bacterial blight resistance gene Xa21 and the genotype of Xa21 according to the band pattern of the amplified product; The reaction system of the PCR amplification is: PCR Master Mix 1×, ZiW-Xa21-RF 750nM, ZiW-Xa21-RR 750nM, ZiW-Xa21-SF 750nM, ZiW-Xa21-SR 750nM, and the remaining volume is supplemented to 15μL with water; the reaction conditions of the PCR amplification are: 94℃5min; 94℃30s, 60℃30s, 72℃30s, 35 cycles; 72℃10min; If there is only one PCR product band and its length is 113bp, the rice to be tested is a homozygous Xa21 genotype resistant to bacterial blight; if there is only one PCR product band and its length is 288bp, the rice to be tested is a homozygous xa21 genotype susceptible to bacterial blight; if there are two PCR products and their sizes are 113bp and 288bp respectively, the rice to be tested is a heterozygous genotype.

[0009] A method for predicting rice bacterial blight resistance using the functional marker primers, extracting genomic DNA from rice leaves to be tested as a template, performing PCR amplification using primer pair ZiW-Xa21-RF / ZiW-Xa21-RR and primer pair ZiW-Xa21-SF / ZiW-Xa21-SR, and judging whether the rice contains the rice bacterial blight resistance gene Xa21 and the genotype of Xa21 based on the band pattern of the amplified product; The reaction system of the PCR amplification is: PCR Master Mix 1×, ZiW-Xa21-RF 750nM, ZiW-Xa21-RR 750nM, ZiW-Xa21-SF 750nM, ZiW-Xa21-SR 750nM, and the remaining volume is supplemented to 15μL with water; the reaction conditions of the PCR amplification are: 94℃5min; 94℃30s, 60℃30s, 72℃30s, 35 cycles; 72℃10min; If there is only one PCR product band and its length is 113bp, the rice to be tested is a homozygous Xa21 genotype resistant to bacterial blight; if there is only one PCR product band and its length is 288bp, the rice to be tested is a homozygous xa21 genotype susceptible to bacterial blight; if there are two PCR products and their sizes are 113bp and 288bp respectively, the rice to be tested is a heterozygous genotype.

[0010] Compared with the prior art, the present invention has the following advantages: 1. The primer pairs ZiW-Xa21-RF and ZiW-Xa21-RR target the sequence within the Xa21 gene, and the amplified fragment is completely linked to the Xa21 gene with high accuracy; 2. The primer pairs ZiW-Xa21-SF and ZiW-Xa21-SR target the specific sequence interval of susceptible varieties. The sequence colinearity between this interval and the disease-resistant interval of the Xa21 gene is poor, and recombination exchange is not likely to occur. The linkage is tight and the accuracy is high. 3. The target sequence fragment amplified by primer pair ZiW-Xa21-RF and ZiW-Xa21-RR is 113bp, and the target sequence fragment amplified by primer pair ZiW-Xa21-SF and ZiW-Xa21-SR is 288bp. The sizes of the anti-susceptible sequence fragments are greatly different and can be easily distinguished by electrophoresis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic diagram of the colinear relationship of the homologous gene cluster G1 (group 1) at the Xa21 locus of the rice bacterial blight resistance gene among various varieties in Example 1 of the present invention.

[0012] Figure 2 It is a schematic diagram of the colinear relationship of the homologous gene cluster G2 (group 2) at the Xa21 locus of the rice bacterial blight resistance gene among various varieties in Example 1 of the present invention.

[0013] Figure 3 It is a linkage relationship diagram of the disease-resistant target sequence and the disease-susceptible target sequence of the functional marker primer of the rice bacterial blight resistance gene Xa21 in Example 1 of the present invention in several representative rice varieties, and its linkage relationship diagram with the G1 and G2 gene clusters in several representative rice varieties.

[0014] Figure 4 It is a schematic diagram of a specific sequence within the R gene and the ZiW-Xa21-RF and ZiW-Xa21-RR markers designed for this sequence.

[0015] Figure 5 It is an electrophoresis diagram of the functional marker primer pair of Example 3 of the present invention and the Xa21 genotype detection of five rice varieties with public genome sequences (IRBB21, Nipponbare, Minghui 63, Zhenshan 97 and R498) and the He9S / Zhenshan 97 hybrid F2 single plant.

[0016] Figure 6 This is an electrophoresis diagram of the functional marker primers of Example 4 of the present invention detecting the Xa21 genotypes of N rice varieties bred by the unit. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0018] Embodiment 1: This example is a schematic diagram of the design of functional marker primers for the rice bacterial blight resistance gene Xa21 (LOC9271075 (NCBI)).

[0019] The reference genomes of the five rice varieties on the NCBI website are Zhonghui 8015 (ZH8015): CP141107.1; Nipponbare (Nip): AP014967.1; Minghui 63 (MH63): CP054686.1; Zhenshan 97 (ZS97): CP056062.1; and R498: CP018167.1.

[0020] The homologous gene cluster of the Xa21 locus is composed of the following two types of linkage groups: G1 and G2, such as Figure 1 and Figure 2 shown.

[0021] The physical positions of the linkage groups of each rice variety on the reference genome are shown in Tables 1 to 5 below.

[0022] Table 1 Table 2 Table 3 Table 4 Table 5 The R gene is the functional gene of Xa21, and the Xa21 gene is carried in the rice variety Zhonghui 8015. ZiW-Xa21-RF and ZiW-Xa21-RR target a part of the sequence within the Xa21 gene, and the target fragment size is 113 bp (SEQ ID NO. 1).

[0023] The S segment is a disease marker targeting sequence (xa21), which is carried by four varieties except Zhonghui 8015. It has no direct homology with the Xa21 gene. ZiW-Xa21-SF and ZiW-Xa21-SR target the S segment, and the size of the segment is 288 bp (SEQID NO. 2).

[0024] The R gene is located between the G1 and G2 linkage groups and has no direct colinearity with the linkage groups of susceptible varieties.

[0025] The S segment was mainly located between the G1 and G2 linkage groups and had no direct colinearity with the linkage groups of disease-resistant varieties.

[0026] Therefore, it can be considered that the R gene and the S fragment can be separated, are unlikely to undergo recombination, have a close linkage relationship, and are suitable for the development of intragenic functional markers.

[0027] There is a specific sequence in the R gene. ZiW-Xa21-RF and ZiW-Xa21-RR designed for this sequence are combined into a pair of dominant markers ( Figure 4 ).

[0028] There are two main approaches to markers for the Xa21 gene in the literature: one is to use the classic pTA248 marker, which is more reliable in being closely linked to the R gene in the linkage group of disease-susceptible varieties. However, it has the problem of large target sequence fragments, long extension time in the amplification program, and the risk of losing linkage after partial recombination; the other is to use a fragment at the end of the G2 linkage group that is highly homologous to the R gene as a target to design a codominant marker (corresponding to fragments G2-1-5, G2-2-8, G2-3-5, G2-4-5). Its advantage is that the designed amplification system is simple. However, it also has the disadvantage of weak linkage relationship. At the same time, the Xa21 gene is a locus that provides a broad-spectrum and high resistance to bacterial blight. In actual breeding, the selection intensity is strong and the selection is frequent, and its recombination probability is high. The classic marker pTA248 is physically closer to the R gene, so in breeding practice, the sequence represented by the G2-15 fragment as the target sequence of the linked marker is more likely to recombine with the Xa21 gene, resulting in false positive or false negative results.

[0029] The ZiW-Xa21 primer combination has been optimized to address the above defects. Its disease resistance sequence target is within the Xa21 gene and is completely co-segregated with the Xa21 gene. Its disease-susceptibility fragment target has no colinear relationship with the Xa21 gene cluster, and there is no possibility of direct recombination. In addition, the physical location is more strongly linked than the classic pTA248 marker. In addition, the resistance and sensitivity target sequence fragments of the ZiW-Xa21 primer combination are small, easy to amplify, and have large differences, which is suitable for large-scale simple breeding.

[0030] Embodiment 2: This example is a method for detecting the genotype of the bacterial blight resistance gene Xa21 using the functional marker primers of Example 1, comprising the following steps: S1. Extracting genomic DNA from rice leaves to be tested; S2, using the rice leaf genomic DNA extracted in S1 as a template, PCR amplification was performed with primers ZiW-Xa21-RF, ZiW-Xa21-RR, ZiW-Xa21-SF and ZiW-Xa21-SR to obtain PCR products; The total volume of the PCR amplification reaction system is 15 μL: the final concentration of PCR Master Mix is ​​1×, the final concentrations of the four primers ZiW-Xa21-RF (SEQ ID NO.3), ZiW-Xa21-RR (SEQ ID NO.4), ZiW-Xa21-SF (SEQ ID NO.5) and ZiW-Xa21-SR (SEQ ID NO.6) are all 750 nM, and the remaining volume is supplemented to 15 μL with water; The PCR amplification reaction conditions are as follows: hot start at 94°C for 5 min; 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 10 min.

[0031] S3. Separate the PCR products obtained in S2 on 9% polyacrylamide gel and perform silver staining for color development. Record the band pattern on a film observation light and take photos for preservation. If there is only one PCR product band with a length of 113 bp, the rice to be tested is a homozygous Xa21 genotype resistant to bacterial leaf blight; if there is only one PCR product band with a length of 288 bp, the rice to be tested is a homozygous xa21 genotype susceptible to bacterial leaf blight; if there are two PCR products with sizes of 113 bp and 288 bp respectively, the rice to be tested is a heterozygous genotype.

[0032] Embodiment 3: This example uses the functional marker primers obtained in Example 1 to detect the genotype of the rice bacterial blight resistance gene Xa21, and uses the functional marker to detect the Xa21 genotype of five rice varieties with public genome sequences (IRBB21, Nipponbare, Minghui 63, Zhenshan 97 and R498) and the He9S / Zhenshan 97 hybrid F2 individual plants.

[0033] Figure 5This is an electrophoresis diagram of the Xa21 genotype detection of five rice varieties with public genome sequences (IRBB21, Nipponbare, Minghui 63, Zhenshan 97 and R498) and the He9S / Zhenshan 97 hybrid F2 single plant. Lanes 1 to 5 are rice varieties IRBB21, Nipponbare, Minghui 63, Zhenshan 97 and R498, lanes 6 to 28 are He9S / Zhenshan 97 hybrid F2 single plants, and lane M is a marker (Takara Marker DL1000). The original resistance source IRBB21 of Xa21 was amplified to obtain a 113bp disease-resistant specific band, and the susceptible controls Nipponbare, Minghui 63, Zhenshan 97 and R498 were amplified to obtain a 288bp disease-susceptible specific band. In the He9S / Zhenshan97 hybrid F2 plant separation population, three band type combinations were amplified: 113bp, 288bp, 113bp+288bp, and the three band types corresponded to disease-resistant homozygous plants, disease-susceptible homozygous plants, and heterozygous disease-resistant plants, respectively; the field-directed rice bacterial blight resistance phenotype was consistent with the genotype result obtained by the marker. Therefore, the electrophoresis detection results of the present invention are consistent with the published genome data and the identification results of the separation population are accurate, indicating that the functional marker primers of the present invention can accurately type the homozygous and heterozygous genes of Xa21.

[0034] Embodiment 4: To further verify the accuracy of genotyping by functional marker primers, this example uses the functional marker primers obtained in Example 1 to perform PCR amplification and electrophoresis detection on the Xa21 genotypes of 10 rice varieties bred by this unit, namely He 9S, BaS, DaS, RongshengB, FuxingB, JintaiB, JinyanB, Jinhui1131, Jinhui2050 and Jinhui2121; the detection method is consistent with the method described in Example 2.

[0035] Figure 6 The electrophoretic diagram of Xa21 genotype detection of 12 rice varieties. Lanes 1 and 2 are respectively the disease-resistant rice varieties IRBB21 (disease-resistant control) and He9S, and lanes 3 to 12 are respectively the disease-susceptible rice varieties Zhenshan 97A (disease-susceptible control), Rongsheng B, Fuxing B, Jintai B, Jinyan B, Jinhui 1131, Jinhui 2050, Jinhui 2121, BaS, and DaS. The disease-resistant varieties IRBB21 and He9S of Xa21 were amplified to obtain a 113bp disease-resistant specific band, and the 10 disease-susceptible varieties were amplified to obtain a 288bp disease-susceptible specific band. The indirect rice bacterial blight resistance phenotype was consistent with the genotype result obtained by the marker. Therefore, the electrophoretic detection results of the present invention show that the functional marker primers of the present invention can be used to accurately type the homozygous genes of Xa21 of multiple background varieties.

Claims

1. A rice bacterial blight resistance gene XOt The functional marker primer is characterized by: The functional marker primers include primer pair ZiW-Xa21-RF / ZiW-Xa21-RR and primer pair ZiW-Xa21-SF / ZiW-Xa21-SR; The nucleotide sequence of the primer pair ZiW-Xa21-RF / ZiW-Xa21-RR is shown in SEQ ID NO.3-4; The nucleotide sequence of the primer pair ZiW-Xa21-SF / ZiW-Xa21-SR is shown in SEQ ID NO.5~6.

2. A rice bacterial blight resistance gene XOt A functional tag, characterized in that: The functional markers include functional markers amplified by primer pair ZiW-Xa21-RF / ZiW-Xa21-RR and functional markers amplified by primer pair ZiW-Xa21-SF / ZiW-Xa21-SR; The nucleotide sequence of the primer pair ZiW-Xa21-RF / ZiW-Xa21-RR is shown in SEQ ID NO.3-4; The nucleotide sequence of the primer pair ZiW-Xa21-SF / ZiW-Xa21-SR is shown in SEQ ID NO.5~6.

3. The functional label according to claim 2, characterized in that: The nucleotide sequence of the functional marker amplified by the primer pair ZiW-Xa21-RF / ZiW-Xa21-RR is shown in SEQ ID NO.1; the nucleotide sequence of the functional marker amplified by the primer pair ZiW-Xa21-SF / ZiW-Xa21-SR is shown in SEQ ID NO.

2.

4. Use of the functional marker primer according to claim 1 in any of the following: 1) Genes for resistance to bacterial blight in rice XOt Application in detection; 2) Application in prediction of resistance to rice bacterial blight; 3) Application in rice breeding for resistance to bacterial blight; 4) Application in improving rice germplasm resources’ resistance to bacterial blight.

5. A method for detecting rice bacterial blight resistance gene using the functional marker primers described in claim 1 XOt A genotyping method, characterized in that: Extract the genomic DNA of the rice leaves to be tested as a template, use the functional marker primers to perform PCR amplification, and judge whether the rice contains the rice bacterial blight resistance gene based on the band pattern of the amplified product XOt as well as XOt genotype.

6. The method according to claim 5, characterized in that: The reaction system of the PCR amplification is: PCR MasterMix 1×, ZiW-Xa21-RF 750nM, ZiW-Xa21-RR 750nM, ZiW-Xa21-SF 750nM, ZiW-Xa21-SR750nM, and the remaining volume is made up to 15μL with water; the reaction conditions of the PCR amplification are: 94℃ 5min; 94℃ 30s, 60℃ 30s, 72℃ 30s, 35 cycles; 72℃ 10min.

7. The method according to claim 6, characterized in that: If there is only one PCR product band with a length of 113 bp, the rice to be tested is homozygous for resistance to bacterial blight. XOt Genotype; if there is only one PCR product band with a length of 288 bp, the rice to be tested is homozygous for bacterial blight xa21 Genotype: If there are two PCR products with sizes of 113 bp and 288 bp respectively, the rice to be tested has a heterozygous genotype.

8. A method for predicting resistance to rice bacterial blight using the functional marker primers of claim 1, characterized in that: Extract the genomic DNA of the rice leaves to be tested as a template, use the functional marker primers to perform PCR amplification, and judge whether the rice contains the rice bacterial blight resistance gene based on the band pattern of the amplified product XOt as well as XOt genotype.

9. The method according to claim 8, characterized in that: The reaction system of the PCR amplification is: PCR MasterMix 1×, ZiW-Xa21-RF 750nM, ZiW-Xa21-RR 750nM, ZiW-Xa21-SF 750nM, ZiW-Xa21-SR750nM, and the remaining volume is made up to 15μL with water; the reaction conditions of the PCR amplification are: 94℃ 5min; 94℃ 30s, 60℃ 30s, 72℃ 30s, 35 cycles; 72℃ 10min.

10. The method according to claim 8, characterized in that: If there is only one PCR product band with a length of 113 bp, the rice to be tested is homozygous for resistance to bacterial blight. XOt Genotype; if there is only one PCR product band with a length of 288 bp, the rice to be tested is homozygous for bacterial blight xa21 Genotype: If there are two PCR products with sizes of 113 bp and 288 bp respectively, the rice to be tested has a heterozygous genotype.

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