CN113584220B

CN113584220B - Rice bacterial leaf blight resistance gene Xa21 assisted breeding SNP molecular marker and application thereof

Info

Publication number: CN113584220B
Application number: CN202111082726.0A
Authority: CN
Inventors: 贾佩陇; 彭佩; 田冰川; 吴云天; 郭铭凯; 唐顺学
Original assignee: Huazhi Biotechnology Co ltd
Current assignee: Huazhi Biotechnology Co ltd
Priority date: 2021-09-15
Filing date: 2021-09-15
Publication date: 2023-04-25
Anticipated expiration: 2041-09-15
Also published as: CN113584220A

Abstract

The invention discloses a rice bacterial leaf blight resistance gene Xa21 assisted breeding SNP molecular marker and application thereof, wherein the SNP molecular marker is OS900226_K01, and is positioned at a base at 24107130 position of chromosome 11 of Shuhui 498, and polymorphism is C/T.

Description

Rice bacterial leaf blight resistance gene Xa21 assisted breeding SNP molecular marker and application thereof

Technical Field

The invention belongs to the field of agricultural molecular biology, and particularly relates to a rice bacterial blight resistance gene Xa21 assisted breeding SNP molecular marker and application thereof.

Background

Stress is one of the important factors affecting crop growth and yield, and is largely divided into two categories: one class is biotic stress, such as insect pests, bacterial diseases, fungal diseases, and the like; the first type is abiotic stress, and is mainly the environmental influence on the crop in the growth process, such as soil salt alkalinity, temperature, heavy metal, illumination, moisture and the like. The impact of biotic stress on crops is particularly severe compared to abiotic stress, which can result in more than half a global crop yield loss per year, with biotic stress caused by pathogenic microorganisms being the primary cause of crop yield loss. The disease resistance of crops is improved, and the yield loss of the crops after being subjected to biological stress can be effectively reduced.

Rice is an important staple food crop, and is often influenced by various biotic stresses in the growth process, wherein bacterial leaf blight caused by gram-negative bacteria xanthomonas oryzae (Xanthomonas oryzae pv. Oryzae, xoo) is a bacterial disease with high transmission speed, wide occurrence range and strong mutation, and severely restricts the growth and yield of rice. Bacterial leaf blight can occur at various stages of rice growth and development, and germs invade vascular bundles of rice, so that leaf blight is caused, and normal growth of the rice is affected. At present, bacterial leaf blight is prevented and treated mainly by chemical reagents to inhibit pathogen infection, but the prevention and treatment effect is poor, environmental pollution is easy to cause, and ecological balance is destroyed. As the bacterial leaf blight is affected by various factors, the chemical reagent control is difficult to control the spread of the bacterial leaf blight, and the yield loss caused by the disease can be effectively reduced and the epidemic of the disease can be blocked by breeding rice varieties containing natural bacterial leaf blight resistance genes. Related researches show that more than 40 bacterial blight resistance genes are identified, and most of the genes are dominant genes. Wherein 16 genes of Xa1, xa2, xa3/Xa26, xa4, xa5, xa7, xa10, xa13, xa14, xa21, xa23, xa25, xa27, xa31, xa41, xa45 and the like are cloned, and related gene functions are also described. Xa23 is a bacterial leaf blight resistance gene found in ordinary wild rice, and has broad-spectrum resistance. The protein encoded by the gene consists of 113 amino acids and has 50% amino acid sequence similarity with the protein encoded by Xa 10. Transcription expression of Xa23 requires specific activation of AvrXa23, and the gene cannot be activated due to lack of TALE element bound by AvrXa23 in the promoter of the susceptibility gene Xa23, so that the plant shows susceptibility. Xa7 is a gene widely applied to rice bacterial leaf blight resistance breeding, and has the characteristics of broad spectrum, durability, high resistance and the like. Xa7 is derived from a rice variety DV85 in Bengalea, and is introduced into a susceptible variety IR24 to obtain a bacterial leaf blight resistance near isogenic line IRBB7.Xa7 maps to a specific 74kb region which is not present in Japanese sunny, and the gene expression level of this region was analyzed and a candidate gene encoding a protein whose function was unknown and which consists of 113 amino acids was determined. The Xa7 promoter contains an AvrXa7 Effector Binding Element (EBE), which is highly similar to the EBE in SWEET14 for disease susceptibility, and Xa7 can prevent SWEET14 from being utilized by bacterial blight bacteria and improve the resistance of rice to diseases. AvrXa7 and PthXo3 activate the transcriptional expression of Xa7 and participate in its mediated disease-resistant response. The high temperature can accelerate the expression of Xa7 and improve the bacterial leaf blight resistance of rice. More than 3000 rice material parts were analyzed and found that the Xa7 locus was found to be predominantly present in indica rice. Xa23 and Xa7 are bacterial leaf blight resistance genes with broad-spectrum resistance, and show higher resistance to various bacterial leaf blight identification bacterial lines. The breeder utilizes molecular marker to assist in selection, and Xa23 and Xa7 are polymerized into important rice breeding materials, so that the rice breeding materials can obtain durable bacterial leaf blight resistance, and the breeding efficiency of new resistant varieties is improved.

Xa21, the first bacterial leaf blight resistance gene cloned by the map, shows excellent resistance to most bacterial leaf blight-identified strains and has been widely used in rice disease-resistant breeding. Xa21 encodes a receptor-like protein kinase consisting of 1025 amino acids, the structure of which can be divided into nine regions: a signal peptide region, an unknown functional region, leucine rich repeat regions (LRRs), a charged region, a transmembrane region, a charged region, a membrane proximal region, a serine-threonine kinase region (STK), and a carboxy terminal tail region. The disease resistance of Xa21 is largely dependent on the activity of both the LRRs and STK domains. Along with the cloning of Xa21 genes and the maturation of molecular marker assisted selection technology, the breeding of new rice bacterial blight resistant varieties has great potential. The molecular marker assisted selection technology is applied by a learner to polymerize Xa7 and Xa21 into Minghui 63, so as to improve the resistance of Minghui 63 to bacterial leaf blight; in the related technology, xa4, xa21 and Xa27 are introduced into a restorer line taking Mianhui 725 or 9311 as a background, and a new restorer line XH2431 carrying Xa4, xa21 and Xa27 is bred after hybridization of the two strains; related art Xa4, xa5 and Xa21 were polymerized into the same rice material, wherein the material containing 3 resistance genes exhibited bacterial blight resistance significantly stronger than the material containing 1 or 2 resistance genes, and other agronomic traits of rice were not affected by the resistance genes. The wide application of the molecular marker assisted selection technology accelerates the breeding process of new rice bacterial blight resistant varieties. At present, molecular marker assisted selection is widely applied to breeding of new rice varieties. The commonly used auxiliary breeding molecule markers are RFLP, RAPD, SSR, AFLP, CAPS or dCAPS, the use of the markers requires complicated gel electrophoresis detection, the automation degree is low, the flux is small, the nucleic acid dye used in the detection process can pollute the environment, the product can sometimes be subjected to nonspecific amplification, and the accurate judgment cannot be made by people, so that the detection efficiency is reduced. The breeding of Xa21 bacterial blight-resistant rice varieties is to use molecular markers co-separated from Xa21 for auxiliary selection, agarose gel electrophoresis is still needed, xa21 genes cannot be identified rapidly and with high flux, and the breeding efficiency of new bacterial blight-resistant varieties is limited.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a rice bacterial leaf blight resistance gene Xa21 assisted breeding SNP molecular marker.

The invention also provides a primer group of the SNP molecular marker.

The invention also provides a detection method of the SNP molecular marker.

The invention also provides application of the SNP molecular marker.

According to the SNP molecular marker of the embodiment of the first aspect of the invention, the SNP molecular marker is OS900226_K01, the SNP molecular marker is positioned at the base at 24107130 position of chromosome 11 of Shuhui 498, and the polymorphism is C/T.

The Primer group of the SNP molecular marker according to the second aspect of the invention, wherein the specific Primer comprises a Primer X and a Primer Y, and the nucleotide sequence of the Primer X is shown as SEQ ID NO. 1; the nucleotide sequence of the Primer Y is shown as SEQ ID NO. 2.

In some embodiments of the invention, the 5 'end of Primer X is linked to a FAM fluorescent sequence and the 5' end of Primer Y is linked to a HEX fluorescent sequence.

In some embodiments of the invention, the primer set further comprises a universal primer nucleotide sequence as set forth in SEQ ID NO. 3.

In some embodiments of the invention, the primer set is used in rice genotyping.

According to a third aspect of the present invention, a method for detecting bacterial leaf blight resistance gene Xa21 of rice, the method comprises the steps of:

s1, extracting genome DNA from rice leaves;

s2, detecting a target site of the sample by using the genomic DNA extracted in the step S1 as a template and using the primer group, and judging whether Xa21 genes are contained in the rice to be detected according to the polymorphism of the detection site.

In some embodiments of the invention, only the fluorescent signal corresponding to the Primer X is detected, the base of the detection site is T, and the rice sample to be tested is judged to contain the bacterial leaf blight resistance gene Xa21; if only the fluorescent signal corresponding to the Primer Y is detected, the base of the detection site is C, and the rice sample tested is judged to contain no bacterial leaf blight resistance gene Xa21; if fluorescent signals corresponding to the Primer X and the Primer Y are detected at the same time, the base of the detection site is T to C, and the tested rice sample is judged to be Xa21 heterozygous genotype.

In some embodiments of the invention, in step S1, genomic DNA is extracted from rice using a simplified CTAB method (cetyl trimethylammonium bromide method).

In some embodiments of the invention, in step S2, SNP sites are detected using the KASP (competitive allele-specific PCR) technique.

The use of the above-described SNP molecular marker according to the fourth aspect of the embodiment of the invention is the use of the SNP molecular marker in rice breeding.

A rice breeding method comprising the steps of: the detection method of the gene is used for detecting the bacterial leaf blight resistance gene Xa21 of rice, and rice samples carrying the bacterial leaf blight resistance gene Xa21 of rice are selected for subsequent breeding.

In some embodiments of the invention, the application provides a kit for detecting rice bacterial blight resistance gene Xa21, wherein the kit comprises specific primers with nucleotide sequences shown as SEQ ID NO.1 and 2 and universal primers with nucleotide sequences shown as SEQ ID NO. 3.

In some embodiments of the invention, the kit is used for rice breeding.

In some embodiments of the invention, the use provides a gene chip comprising specific primers with nucleotide sequences shown in SEQ ID NO.1 and 2 and universal primers with nucleotide sequences shown in SEQ ID NO. 3.

The rice bacterial leaf blight resistance gene Xa21 assisted breeding SNP molecular marker has at least the following beneficial effects: according to the scheme, the molecular marker is utilized, and the KASP technology is applied to detect the Xa21 gene in the rice material, so that whether the rice contains the bacterial blight resistance gene Xa21 can be rapidly and accurately detected. The scheme of the invention utilizes the specific SNP locus coseparated with Xa21 and combines with KASP detection technology to solve the technical problem in the traditional molecular marker assisted selection, and can rapidly, accurately, efficiently and high-throughput identify Xa21 genes without agarose gel electrophoresis, thereby accelerating variety breeding process and having important significance for promoting the wide application of Xa21 genes in commercial breeding.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a molecular marker development flow chart in an embodiment of the invention;

FIG. 2 is a diagram showing the typing of the OS900226_K01 molecular marker in the embodiment of the invention;

FIG. 3 is a diagram showing the typing of the OS900226_K01 molecular marker in the embodiment of the invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

The embodiment of the invention comprises the following steps: the molecular marker is designed by determining the physical position of a cloned target gene Xa21 as shown in figure 1, obtaining SNP loci through sequence comparison analysis, extracting SNP loci and flanking sequences, designing and synthesizing primer sequences of markers, and screening the markers, wherein the method comprises the following steps:

1 primer design

According to the information in published literature, the Xa21 gene sequence is located in chromosome 11 region 24103808-24108095 of shuhui 498, which region is subjected to SNP locus mining using the resequencing data of Xa21 donor material and non-donor material. Determining 24107130 th base of chromosome 11 of Shuhui 498 to be co-separated with Xa21 gene, extracting flanking sequences of 50bp before and after the SNP locus, and designing a primer by using BatchPrimer 3. The tag consisted of 3 primers, wherein the 5' ends of 2 specific primers were ligated to FAM and HEX fluorescent linker sequences, respectively, 1 universal primer, primers were commissioned by Invitrogen company.

The Xa21 resistance gene detection can be carried out on rice materials with high flux by using a molecular marker primer based on the KASP reaction principle and designed by single base difference of the anti-influenza material. If the PCR product only detects a fluorescence signal corresponding to the Primer X, the base of the detection site is T, and the test material contains a white leaf blight resistance gene Xa21; if only the fluorescence signal corresponding to the Primer Y is detected, the base of the detection site is C, and the test material does not contain the white leaf blight resistance gene Xa21; if the fluorescence signals corresponding to the Primer X and the Primer Y are detected at the same time, the base of the detection site is T:C, and the test material is heterozygous genotype.

Primer design of Table 1OS900226_K01

2 sample detection

DNA extraction: extracting genome DNA from rice leaves by adopting a simplified CTAB method, comprising the following steps:

(1) Taking about 30mg of blades to 1.3mL of a 96-well plate, placing the blades in a freeze dryer, and vacuumizing for 12 hours or more;

(2) After vacuumizing, adding two steel balls into each hole by using a bead divider, covering a silica gel film, grinding for 1min in a high-flux grinding instrument, immediately separating in a deep-hole plate centrifuge, and centrifuging the ground tissue to the bottom of the hole;

(3) Adding 700 mu L of CTAB extracting solution into each hole by using a pipetting workstation TECAN, shaking and uniformly mixing, placing into a 65 ℃ water bath kettle for warm bath for about 1-1.5h, taking 1.3mL of 96-well plates on a vortex oscillator for shaking for several times every 20 min;

(4) Taking out 1.3mL 96-well plate after the warm bath is finished, placing the 96-well plate into a deep-well plate refrigerated centrifuge, and centrifuging at 4000rpm for 10min;

(5) Transferring 380 mu L of supernatant in each well to a new 1.3mL 96-well plate by using a pipetting workstation TECAN, adding equal volume chloroform, mixing uniformly upside down, standing for 2min, placing in a deep-hole plate refrigerated centrifuge, centrifuging at 4000rpm for 10min;

(6) After centrifugation, 250. Mu.L of supernatant is extracted by a pipetting workstation TECAN to 0.8mL of 96-well plate with 250. Mu.L of isopropanol added in advance, and the mixture is uniformly mixed by vortex oscillation and placed in a refrigerator at the temperature of minus 20 ℃ for precipitation for 1 hour or more;

(7) Taking out 0.8mL of the 96-well plate, placing the 96-well plate in a deep-hole plate refrigerated centrifuge, centrifuging at 4000rpm for 15min;

(8) Discarding the supernatant, adding 250 μL of 70% ethanol into each well by using a pipetting workstation TECAN, oscillating for several times on a vortex oscillator, centrifuging for 15min at 5000 rpm;

(9) Discarding the supernatant, and placing in a 65 ℃ oven for 30min to dry;

(10) 200. Mu.L of sterilized ultrapure water was added to each well, and the mixture was left at room temperature overnight for dissolution.

KASP reaction test: the KASP response test was performed on a Douglas Arraytape genotyping platform. The amplification system used for the PCR amplification reaction was 0.8. Mu.L: 20ng of template DNA, 100. Mu.M of each of the two specific primers was added to the mixture after drying, 0.0013. Mu.L of each of the two specific primers, 0.0033. Mu.L of each of the 100. Mu.M universal primers, 0.3945. Mu.L of each of the 2 XKASP Master Mix, and 0.3996. Mu.L of ultrapure water. The PCR amplification is completed in a water bath thermal cycler under the following conditions: pre-denaturation at 94℃for 15min; the first step of amplification reaction, denaturation at 94 ℃ for 20s, annealing at 65-57 ℃ and extension for 60s,10 cycles, wherein the annealing and extension temperature in each cycle is reduced by 0.8 ℃; the second amplification step was performed by denaturation at 94℃for 20s, annealing at 57℃and extension for 60s for 30 cycles. After the reaction is completed, the fluorescent data of KASP reaction products are read by using an Arrayape scanning system, and the result of fluorescent scanning is automatically converted into a pattern.

TABLE 2KASP detection reaction System

3 mark type data

The results of the KASP reaction verification of the donor material containing the Xa21 gene and the material not containing the Xa21 gene using the label OS900226_k01 are shown in table 3, according to the above detection method. The result shows that 1 part of material is detected as a base T at a test site and is a Xa21 gene homozygous donor; 3 parts of materials are detected at a test site, wherein the detection result is base T to C, and the base T is Xa21 gene heterozygous donor; the detection result of 12 parts of material without Xa21 gene at the test site is base C, the parting chart is shown in figure 2, and the result shows that the molecular marker of the OS900226_K01 can be normally parting.

Table 3OS900226_K01 scoring type results

Material name	Description of materials	Detection result
			IRBB7	Xa7 donor	C
DV85	Xa7 donor	C
			Hua Hui 1437	Xa7, xa21 donors	T:C
Hua Hui 1337	Xa7, xa21 donors	T:C
			IRBB21	Xa21 donor	T
Zhonghui 8015	Xa21 donor	T:C
			Hua Hui 7620	Pi9, xa23 donor	C
CBB23	Xa23 donor	C
			R900	Disease-sensitive material	C
Wide-spectrum 63-4S	Disease-sensitive material	C
			IR24	Disease-sensitive material	C
C815S	Disease-sensitive material	C
			Tianfeng A	Disease-sensitive material	C
R207	Disease-sensitive material	C
			Shuhui 527	Disease-sensitive material	C
Minghui 86	Disease-sensitive material	C

4. Detection of specificity and practicality of molecular markers

To detect the specificity and practicality of the markers of the present invention, the markers OS900226_k01 were validated using 68 parts of diverse parental material. Among 68 diversity parent materials, xa21 donor materials of known genotypes, other anti-white leaf blight gene donor materials, core rice breeding materials, conventional rice materials are included. The labeling typing result is shown in figure 3, 1 part of material is detected as a base T at a test site and is an Xa21 gene homozygous donor (labeled red in a typing graph); 1 part of material is detected at a test site as a base T: C, which is an Xa21 gene heterozygous donor (marked purple in a parting chart); the remaining 66 parts of material were tested at the test site as base C, which is a material that did not contain the Xa21 gene (blue in the typing pattern). The marker OS900226_K01 has high specificity when detecting Xa21 gene locus, and can accurately and efficiently identify whether Xa21 genes are contained in rice materials.

The reagent and the consumable material matched with the Douglas Arraytape genotyping platform used in the invention are purchased from LGC company in England.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

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Claims

1. The detection method of the bacterial leaf blight resistance gene Xa21 of rice is characterized by comprising the following steps:

s1, extracting genome DNA from rice;

s2, carrying out polymorphism detection on SNP molecular markers in the genomic DNA extracted in the step S1 by using the genomic DNA extracted in the step S1 as a template and utilizing a primer group, and judging whether Xa21 genes are contained in the rice to be detected according to the detection polymorphism of the SNP molecular markers; the Primer group comprises a specific Primer and a universal Primer, wherein the specific Primer comprises a Primer X and a Primer Y, and the nucleotide sequence of the Primer X is shown as SEQ ID NO. 1; the nucleotide sequence of the Primer Y is shown as SEQ ID NO. 2; the nucleotide sequence of the universal primer is shown as SEQ ID NO. 3; if only the fluorescence signal corresponding to the Primer X is detected, the base of the detection site is T, and the test material contains a white leaf blight resistance gene Xa21; if only the fluorescence signal corresponding to the Primer Y is detected, the base of the detection site is C, and the test material does not contain the white leaf blight resistance gene Xa21; the SNP molecular marker is OS900226_K01, is positioned at the base at 24107130 position of chromosome 11 of Shuhui 498, and has C/T polymorphism.

2. A rice breeding method for resisting bacterial leaf blight is characterized by comprising the following steps: the method according to claim 1, wherein the rice sample carrying the rice bacterial blight resistance gene Xa21 is selected for subsequent breeding.