CN113604603A

CN113604603A - SNP marker linked with rice bacterial leaf blight resistant gene Xa7 and application thereof

Info

Publication number: CN113604603A
Application number: CN202111072351.XA
Authority: CN
Inventors: 贾佩陇; 彭佩; 田冰川; 吴云天; 蒋友如; 唐顺学
Original assignee: Huazhi Biotechnology Co Ltd
Current assignee: Huazhi Biotechnology Co Ltd
Priority date: 2021-09-14
Filing date: 2021-09-14
Publication date: 2021-11-05
Anticipated expiration: 2041-09-14
Also published as: CN113604603B

Abstract

The invention discloses an SNP marker linked with a rice bacterial leaf blight resistant gene Xa7 and application thereof, wherein the SNP marker is OS900223_ K01, the polymorphic site of the SNP marker is positioned at the basic group of the chromosome 27970206 of Nippon No. 6, the polymorphism is G/A, the scheme of the invention utilizes the specific SNP site co-separated from Xa7 and combines with KASP detection technology, solves the technical problem in the traditional molecular marker assisted breeding, does not need agarose gel electrophoresis, can quickly, accurately, efficiently and high-flux identify the Xa7 gene, accelerates the variety breeding process, and has important significance for promoting the Xa7 gene to be widely applied in commercial breeding.

Description

SNP marker linked with rice bacterial leaf blight resistant gene Xa7 and application thereof

Technical Field

The invention belongs to the field of agricultural molecular biology, and particularly relates to an SNP marker linked with a rice bacterial leaf blight resistant gene Xa7 and application thereof.

Background

Stress is one of the important factors affecting crop growth and yield, and is mainly divided into two categories: one is biological stress, such as insect pest, bacterial disease, fungal disease, etc.; one is abiotic stress, mainly due to the environmental impact on the crops during their growth, such as saline-alkali property of the land, temperature, heavy metals, light and water. Biotic stress is particularly severe in crops, and can cause more than half of the global crop yield loss each year, compared to abiotic stress, which is the major cause of crop yield loss caused by pathogenic microorganisms. Improve the disease resistance of crops and effectively reduce the yield loss of the crops after being stressed by organisms. As an important staple grain crop, rice is often influenced by various biotic stresses in the growth process, wherein bacterial leaf blight caused by gram-negative bacteria, namely Xanthomonas oryzae pv. oryzae, Xoo is a bacterial disease with high propagation speed, wide occurrence range and strong mutability, and the growth and yield of the rice are severely limited. Bacterial leaf blight can occur at each stage of rice growth and development, and pathogenic bacteria invade vascular bundles of rice, so that leaves are withered, and normal growth of rice is influenced. At present, bacterial blight is mainly prevented and controlled by chemical reagents to inhibit pathogen infection, but the prevention and control effect is poor, environmental pollution is easily caused, and ecological balance is damaged. Because the occurrence of the bacterial blight disease is influenced by various factors, the chemical reagent is difficult to control the spread of the bacterial blight disease, and the yield loss caused by the disease can be effectively reduced and the disease prevalence can be blocked by breeding the rice variety containing the natural bacterial blight resistance gene.

Related researches show that more than 40 genes for resisting bacterial blight are identified, and most of the genes are dominant genes. Wherein 16 genes such as 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 elucidated. Xa21, the first gene cloned in the map, showed excellent resistance to the most bacterial blight-identifying strains, and was widely used in rice breeding for disease resistance. Xa21 encodes a receptor-like protein kinase consisting of 1025 amino acids and has a structure divided into nine regions: a signal peptide region, an unknown function 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 mainly determined by the activity of two functional domains, LRRs and STK. Xa23 is a bacterial leaf blight resistance gene found in normal wild rice and has broad spectrum resistance. The protein encoded by the gene consists of 113 amino acids, and has 50% of 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 the lack of TALE element combined by AvrXa23 in the promoter of the disease-sensitive gene Xa23, so that the plant shows the disease-sensitive character. Xa21 and Xa23 are bacterial blight resistance genes with broad spectrum resistance. The molecular marker-assisted selection is utilized by breeders to polymerize Xa21 and Xa23 into important rice breeding materials, so that the important rice breeding materials obtain durable bacterial leaf blight resistance, and the breeding efficiency of new resistant varieties is improved. Xa7 is another gene which is widely applied to the breeding of rice bacterial leaf blight resistance besides Xa21 and Xa23, and has the characteristics of broad spectrum, durability, high resistance and the like. Xa7 was derived from rice variety DV85 in Bangladesh and introduced into the susceptible variety IR24 to obtain the bacterial leaf blight resistant near isogenic line IRBB 7. At present, Xa7 has been cloned, and the relevant molecular mechanisms are elucidated. Xa7 mapped to a specific 74kb region that was not present in Nipponbare and gene expression levels were analyzed to determine candidate genes that encoded a protein of 113 amino acids whose function was unknown. The Xa7 promoter contains an AvrXa7 Effector Binding Element (EBE), which is highly similar to the EBE in SWEET14 which plays a role in disease susceptibility, and Xa7 can prevent SWEET14 from being utilized by the blight bacterium, thereby improving the resistance of rice to diseases. AvrXa7 and PthXo3 can activate transcriptional expression of Xa7 and participate in its mediated disease resistance response. The expression of Xa7 can be accelerated by high temperature, and the bacterial leaf blight resistance of rice can be improved. More than 3000 rice material were analyzed and the Xa7 site was found to be predominant in indica. The clone of Xa7 and the research of molecular mechanism are significant for promoting the further application of Xa7 in the breeding of rice bacterial leaf blight resistance.

At present, molecular marker assisted selection is widely applied to breeding of new rice varieties. Common molecular markers for assisting breeding are RFLP, RAPD, SSR, AFLP, CAPS or dCAPS, the markers need complicated gel electrophoresis detection, the automation degree is low, the flux is low, nucleic acid dye used in the detection process can pollute the environment, products can be subjected to nonspecific amplification sometimes, accurate judgment cannot be made by people, and the detection efficiency is reduced. The Xa7 bacterial-leaf-blight-resistant rice variety breeding is assisted by a molecular marker co-separated from Xa7, agarose gel electrophoresis is still needed, the Xa7 gene cannot be identified quickly and in high flux, and the breeding efficiency of a new bacterial-leaf-blight-resistant variety is limited.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides an SNP marker linked with the rice bacterial leaf blight resistant gene Xa 7.

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

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

The invention also provides application of the SNP marker.

The SNP marker according to an embodiment of the first aspect of the invention, which is OS900223_ K01, has a polymorphic site at the base at position 27970206 of chromosome 6 of Nipponbare, and has a polymorphism of G/A.

The Primer set for SNP labeling described above according to an embodiment of the second aspect of the present invention includes specific primers including Primer X and Primer Y, wherein the nucleotide sequence of 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 primer set further comprises a universal primer nucleotide sequence as shown in SEQ ID No. 3.

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

In some embodiments of the invention, the primer set is used for detecting rice genotypes.

According to the third aspect of the invention, the method for detecting the rice bacterial leaf blight resistant gene Xa7 comprises the following steps:

s1, extracting genome DNA from rice leaves;

and S2, detecting the 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 the rice to be detected contains the Xa7 gene according to the polymorphism of the detection site.

In some embodiments of the invention, only the fluorescence signal corresponding to Primer X is detected, and the base at the detection site is G, the rice sample to be tested is judged not to contain the bacterial blight resistance gene Xa 7; if only the fluorescent signal corresponding to the Primer Y is detected, the base of the detection site is A, and the rice sample to be tested contains the bacterial leaf blight resistance gene Xa 7; and if the fluorescence signals corresponding to the Primer X and the Primer Y are detected simultaneously, the base of the detection site is G: A, and the tested rice sample is judged to be Xa7 heterozygous genotype.

In some embodiments of the present invention, in step S1, the simplified CTAB method (cetyl trimethyl ammonium bromide method) is used to extract genomic DNA from rice.

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

The application of the SNP marker according to the fourth aspect of the invention is the application of the SNP marker in rice breeding.

A rice breeding method comprises the following steps: the genotype of the rice bacterial leaf blight resistant gene Xa7 is detected by the gene detection method, and a rice sample carrying the rice bacterial leaf blight resistant gene Xa7 is selected for subsequent breeding.

In some embodiments of the invention, the application is to provide a kit for detecting a rice bacterial blight-resistant gene Xa7, and the kit comprises the primer set.

In some embodiments of the invention, the kit comprises a specific primer having a nucleotide sequence shown in SEQ ID No. 1-2; preferably, the primer also comprises a universal primer with the nucleotide sequence shown as SEQ ID NO. 3.

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

In some embodiments of the present invention, the application is to provide a gene chip, wherein the gene chip comprises the primer set.

In some embodiments of the invention, the gene chip comprises a specific primer and a nucleotide sequence, wherein the nucleotide sequence is shown as SEQ ID NO. 1-2; preferably, the primer also comprises a universal primer shown in SEQ ID NO. 3.

The SNP marker linked with the rice bacterial leaf blight resistant gene Xa7 and the application thereof according to the embodiment of the invention have at least the following beneficial effects: the invention utilizes the specific SNP locus co-separated from Xa7 and combines the KASP detection technology, solves the technical problem in the traditional molecular marker assisted breeding, does not need agarose gel electrophoresis, can quickly, accurately, efficiently and high-flux identify the Xa7 gene, accelerates the variety breeding process, and has important significance for promoting the wide application of the Xa7 gene 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 following figures and examples, in which:

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

FIG. 2 is a molecular scale chart of OS900223_ K01 in an example of the present invention;

FIG. 3 is a typing chart of the molecular marker OS900223_ K01 in the example of the present invention;

FIG. 4 is a typing chart of the molecular marker OS900223_ K01 in the example of the present invention.

Detailed Description

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

The embodiment of the invention is as follows: a design process of the molecular marker comprises the following steps of determining a physical position through a cloned target gene Xa7, determining a linkage region of the gene, extracting SNP sites and flanking sequences, designing and synthesizing a primer sequence of the marker, and then screening and testing the marker, wherein the design process of the molecular marker comprises the following steps:

1 primer design

Related research shows that the Xa7 gene is not present in Nipponbare of japonica rice variety. In order to find the SNP marker linked to this gene, it was found that there was a mutation at the 27970206 th base of chromosome 6, A in a strain resistant to bacterial blight and G in a strain sensitive to bacterial blight, based on the gene-linked region and related molecular markers provided in the literature (see Japanese fine genome MSU 7.0). Extracting flanking sequences of 50bp around the site in Nipponbare, comparing corresponding sequences in the resistant material IRBB7, designing primers by using a BatchPrimer3 primer design website, and replacing SNP sites at other positions in the sequences by degenerate bases. The label consists of 3 primers, wherein 5' ends of 2 specific primers are respectively connected with FAM and HEX fluorescent linker sequences. The primers were synthesized by Invitrogen corporation. If only the fluorescence signal corresponding to the Primer X is detected by the sample amplification product, the base of the detection site is G, and the test material does not contain the bacterial leaf blight resistance gene Xa 7; if only the fluorescence signal corresponding to Primer Y is detected, the base of the detection site is A, and the test material contains a bacterial blight resistance gene Xa 7; if the fluorescence signals corresponding to the Primer X and the Primer Y are detected simultaneously, the base of the detection site is G: A, and the test material is in a heterozygous genotype.

Primer design for OS900223_ K01 of Table 1

2 sample detection

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

(1) taking about 30mg leaves to 1.3mL 96-well plates, placing in a freeze dryer, and vacuumizing for 12h or more;

(2) after the vacuum pumping is finished, adding two steel balls into each hole by using a ball separator, covering a silica gel film, grinding for 1min in a high-flux grinder, instantly separating in a deep-hole plate centrifuge after grinding, and centrifuging the ground tissues to the bottom of the hole;

(3) adding 700 μ L CTAB extract into each well with a pipetting workstation TECAN, shaking, mixing, placing in a 65 deg.C water bath kettle, warm bathing for 1-1.5h, and taking 1.3mL 96-well plate out of the vortex oscillator every 20min, and oscillating for several times;

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

(5) transferring 380 mu L of supernatant per well to a new 1.3mL 96-well plate by using a liquid transfer workstation TECAN, adding equal volume of chloroform, standing for 2min after reversing and mixing uniformly, placing in a deep-well plate refrigerated centrifuge, and centrifuging at 4000rpm for 10 min;

(6) after centrifugation, extracting 250 mu L of supernatant into a 0.8mL 96-well plate which is added with 250 mu L of isopropanol in advance by using a liquid transfer workstation TECAN, oscillating and mixing uniformly in a vortex, and putting the plate in a refrigerator at the temperature of 20 ℃ below zero for precipitation for 1 hour or more;

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

(8) discarding the supernatant, adding 250 μ L70% ethanol into each well with a pipetting workstation TECAN, oscillating several times on a vortex oscillator at 5000rpm, and centrifuging for 15 min;

(9) discarding the supernatant, and drying in a 65 ℃ oven for 30 min;

(10) add 200. mu.L of sterilized ultrapure water to each well and dissolve overnight at room temperature for use.

KASP reaction test: the KASP reaction assay was performed on a Douglas Arraytape genotyping platform. The amplification system used for the PCR amplification reaction was calculated at 0.8. mu.L: 20ng of template DNA, after drying, 100. mu.M of each of two specific primers 0.0013. mu.L, 100. mu.M of a universal primer 0.0033. mu.L, 2 XKASP Master Mix 0.3945. mu.L, and 0.3996. mu.L of ultrapure water were added. The conditions for completing Touchdown PCR reaction by PCR amplification in a water bath thermal cycler are as follows: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20s, annealing at 65-57 ℃ and extending for 60s for 10 cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.8 ℃; the second amplification reaction, denaturation at 94 ℃ for 20s, annealing at 57 ℃ and extension for 60s, 30 cycles. And after the reaction is finished, the fluorescence data of the KASP reaction product is read by utilizing an Arraytape scanning system, and the result of the fluorescence scanning is automatically converted into a graph.

TABLE 2 reaction System for KASP detection

	Final concentration	Volume (μ L)
			100μM Primer C	0.42μM	0.0033μL
100μM Primer X	0.17μM	0.0013μL
			100μM Primer Y	0.17μM	0.0013μL
2x KASP Master Mix	1×	0.3945μL
			Ultrapure water		0.3996μL
DNA (Dry)		20ng-50ng
			Total volume		0.8μL

3 marking typing data

According to the above detection method, KASP reaction verification was performed on the donor material containing Xa7 gene and the material not containing Xa7 gene using the marker OS900223_ K01, and the results are shown in Table 3.

Table 3 OS900223_ K01 labeled typing results

Name of Material	Description of the materials	The result of the detection
			IRBB7	Xa7 donors	A
DV85	Xa7 donors	A
			Huahui 1437	Xa7, Xa21 donors	A
Huahui 1337	Xa7, Xa21 donors	A
			IRBB21	Xa21 donors	G
Zhonghui 8015	Xa21 donors	G
			Huahui 7620	Pi9, Xa23 donors	G
CBB23	Xa23 donors	G
			R900	Pathogenic material	G
Accounts for 63-4S	Pathogenic material	G
			IR24	Pathogenic material	G
C815S	Pathogenic material	G
			Tianfeng A	Pathogenic material	G
R207	Pathogenic material	G
			Shuhui 527	Pathogenic material	G
Minghui 86	Pathogenic material	G

As can be seen from Table 3, the results of the detection of 4 parts of the materials including IRBB7, DV85, Huahui 1437 and Huahui 1337 at the test site are base A, the results of the detection of 12 parts of the materials without Xa7 gene at the test site are base G, and the typing map is shown in FIG. 2, which indicates that the molecular marker OS900223_ K01 can be typed normally.

4 specificity and Utility verification

The marker OS900223_ K01 was validated using 2F 1 populations and their parents and 68 diverse parent materials. The results of typing the 2F 1 populations and their parent materials are shown in FIG. 3, from which it can be seen that the donor parent material has a base A at the test site and contains the Xa7 gene (blue in the typing scheme); the detection result of the receptor parent material at the test site is a base G, and the receptor parent material does not contain Xa7 gene (marked as red in a typing graph); the detection result of the hybrid population F1 at the test site is base G: A, and the hybrid population belongs to a heterozygote (the mark of a typing graph is purple).

The 68 diversified parent materials comprise Xa7 donor materials with known genotypes, other donor materials for resisting bacterial leaf blight genes, core rice breeding materials and conventional rice materials. The results of typing them with OS900223_ K01 marker are shown in FIG. 4, and 6 materials have base A at the test site and contain Xa7 gene; 62 parts of the material have a base G detected at a test site and do not contain Xa7 gene. Therefore, the marker OS900223_ K01 has high specificity in detecting the Xa7 gene locus, and can accurately and efficiently identify whether the rice material contains the Xa7 gene.

Reagents and consumables for the Douglas Arraytape genotyping platform used in the present invention were purchased from LGC, Inc. in the United kingdom.

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 those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

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Claims

1. An SNP marker linked with a rice bacterial blight resistance gene Xa7, characterized in that the SNP marker is OS900223_ K01, the polymorphic site of the SNP marker is located at the base of chromosome 27970206 of Nipponbare 6, and the polymorphism is G/A.

2. A Primer set for detecting the SNP marker of claim 1, wherein the Primer set includes specific primers including Primer X and Primer Y; 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.

3. The primer set of claim 2, wherein the primer set further comprises a universal primer nucleotide sequence as set forth in SEQ ID No. 3.

4. The Primer set of claim 2, wherein the Primer X is linked to a FAM fluorescent sequence at the 5 'end and the Primer Y is linked to a HEX fluorescent sequence at the 5' end.

5. Use of the primer set according to any one of claims 2 to 4 in rice genotype detection.

6. A method for detecting a rice bacterial leaf blight resistant gene Xa7 is characterized by comprising the following steps:

s1, extracting genome DNA from rice;

s2, using the genomic DNA extracted in the step S1 as a template, detecting a sample target site by using the primer group of any one of claims 2-4, and judging whether the rice to be detected contains the Xa7 gene according to the polymorphism of the detection site.

7. Use of the SNP marker according to claim 1 or the primer set according to any one of claims 2 to 4 for breeding rice.

8. A rice breeding method is characterized by comprising the following steps: the detection method according to claim 6, wherein the rice sample carrying the bacterial leaf blight resistance gene Xa7 is selected for subsequent breeding.

9. A kit comprising the primer set according to any one of claims 2 to 4.

10. A gene chip comprising the primer set according to any one of claims 2 to 4.