CN113604603B - SNP marker linked with rice anti-white leaf spot gene Xa7 and application thereof - Google Patents
SNP marker linked with rice anti-white leaf spot gene Xa7 and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP marker linked with a rice anti-white leaf spot gene Xa7 and application thereof, wherein the SNP marker is OS 900223-K01, a polymorphic site of the SNP marker is positioned at a base of 27970206 bits of a Japanese sunny 6 chromosome, and the polymorphism is G/A.
Description
Technical Field
The invention belongs to the field of agricultural molecular biology, and particularly relates to an SNP marker linked with a rice anti-white leaf blight gene Xa7 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 bacterial leaf blight is affected by various factors, chemical reagent control is difficult to control the spread of bacterial leaf blight, and the yield loss caused by diseases can be effectively reduced and the epidemic of the diseases can be blocked by breeding rice varieties containing natural bacterial leaf blight resistance genes.
Related researches show that more than 40 genes of the identified anti-bacterial wilt 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. Xa21, the first white 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. Xa23 is a white leaf blight resistance gene found in common 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. Xa21 and Xa23 are broad-spectrum resistance genes. The breeder utilizes molecular marker to assist in selection, and the Xa21 and Xa23 are polymerized into important rice breeding materials, so that the rice breeding materials can obtain durable resistance to white leaf blight, and the breeding efficiency of new resistant varieties is improved. Xa7 is another gene which is widely applied to rice white leaf blight resistance breeding besides Xa21 and Xa23, 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 paraisogenic line IRBB7 with resistance to white leaf blight. At present, xa7 has been cloned and the related molecular mechanisms have been elucidated. 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 resistance of rice to white leaf blight. More than 3000 rice material parts were analyzed and found that the Xa7 locus was found to be predominantly present in indica rice. The cloning and molecular mechanism research of Xa7 are of great significance in promoting the further application of Xa7 in rice bacterial leaf blight resistance breeding.
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 Xa7 rice variety with bacterial leaf blight resistance is to use molecular markers co-separated from Xa7 for auxiliary selection, agarose gel electrophoresis is still needed, xa7 genes cannot be identified rapidly and with high flux, and the breeding efficiency of new varieties with bacterial leaf blight resistance 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 SNP marker linked with rice anti-white leaf spot 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.
According to an embodiment of the first aspect of the present invention, the SNP marker is OS900223_K01, and the polymorphism site of the SNP marker is located at the base at 27970206 of Japanese No. 6 chromosome, and the polymorphism is G/A.
The Primer set of the SNP marker according to the second aspect of the invention comprises a specific 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.
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 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 is used in rice genotyping.
According to a third aspect of the present invention, a method for detecting rice anti-white leaf blight gene Xa7 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 Xa7 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 Primer X is detected, the base of the detection site is G, and the rice sample tested is judged to contain no white leaf blight resistance gene Xa7; 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 is judged to contain the white leaf blight resistance gene Xa7; 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 G to A, and the tested rice sample is judged to be Xa7 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 SNP marker according to the fourth aspect of the embodiment of the present invention is the use of the SNP marker in rice breeding.
A rice breeding method comprising the steps of: the genotype of the rice bacterial leaf blight resistance gene Xa7 is detected by using the detection method of the genes, and a rice sample carrying the gene of the rice bacterial leaf blight resistance gene Xa7 is selected for subsequent breeding.
In some embodiments of the invention, the application provides a kit for detecting rice bacterial blight resistance gene Xa7, wherein the kit comprises the primer set.
In some embodiments of the invention, the kit comprises a specific primer having a nucleotide sequence as shown in SEQ ID NO. 1-2; preferably, the primer further comprises a universal primer with a 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 invention, the use provides a gene chip comprising the primer set described above.
In some embodiments of the invention, the gene chip comprises specific primers and nucleotide sequences with nucleotide sequences shown in SEQ ID NO. 1-2; preferably, the universal primer shown in SEQ ID NO.3 is also included.
According to the embodiment of the invention, the SNP marker linked with the rice anti-white leaf spot gene Xa7 and the application thereof have at least the following beneficial effects: the invention utilizes the specific SNP locus coseparated with Xa7 and combines with KASP detection technology to solve the technical problem in the traditional molecular marker assisted breeding, does not need agarose gel electrophoresis, can rapidly, accurately, efficiently and high-flux identify Xa7 genes, accelerates the variety breeding process, and has important significance in promoting the wide application of Xa7 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 molecular marker pattern diagram of OS900223_K01 in an embodiment of the invention;
FIG. 3 is a parting chart of the OS900223_K01 molecular marker in the embodiment of the invention;
FIG. 4 is a diagram showing the typing of the OS900223_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 design process of the molecular marker is as shown in figure 1, the physical position is determined through cloned target gene Xa7, the linkage region of the gene is determined, SNP locus and flanking sequence are extracted, and the marker is screened and tested through designing and synthesizing primer sequences of the marker, specifically as follows:
1 primer design
Related studies indicate that the Xa7 gene is not present in japonica rice variety Nipponbare. To find the gene-linked SNP marker, it was found that there was a mutation in the 27970206 th base of chromosome 6, A in a white leaf blight-resistant variety and G in a white leaf blight-sensitive variety, based on the gene-linked region and the related molecular markers provided in the literature (refer to Japanese genome MSU 7.0). The 50bp flanking sequences before and after the site in Japanese sunny are extracted, compared with the corresponding sequences in the resistant material IRBB7, the sequences are designed by using a BatchPrimer3 primer design website, and SNP sites at other positions in the sequences are replaced by degenerate bases. The label consists of 3 primers, wherein the 5' ends of the 2 specific primers are respectively connected with FAM and HEX fluorescent linker sequences. Primers were commissioned for Invitrogen corporation synthesis. 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 white leaf blight resistance gene Xa7; if only the fluorescence signal corresponding to the Primer Y is detected, the base of the detection site is A, and the test material contains a white leaf blight resistance gene Xa7; 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 G:A, and the test material is heterozygous genotype.
Table 1 primer design for OS900223_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 2 KASP detection reaction System
| Final concentration | Volume (mu 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 (drying) | 20ng-50ng | |
| Total volume of | 0.8μL |
3 mark type data
The results of the KASP reaction verification of the donor material containing the Xa7 gene and the material not containing the Xa7 gene using the label OS900223_k01 according to the above detection method are shown in table 3.
Table 3 OS900223_K01 scoring type results
| Material name | Description of materials | Detection result |
| IRBB7 | Xa7 donor | A |
| DV85 | Xa7 donor | A |
| Hua Hui 1437 | Xa7, xa21 donors | A |
| Hua Hui 1337 | Xa7, xa21 donors | A |
| IRBB21 | Xa21 donor | G |
| Zhonghui 8015 | Xa21 donor | G |
| Hua Hui 7620 | Pi9, xa23 donor | G |
| CBB23 | Xa23 donor | G |
| R900 | Disease-sensitive material | G |
| Wide-spectrum 63-4S | Disease-sensitive material | G |
| IR24 | Disease-sensitive material | G |
| C815S | Disease-sensitive material | G |
| Tianfeng A | Disease-sensitive material | G |
| R207 | Disease-sensitive material | G |
| Shuhui 527 | Disease-sensitive material | G |
| Minghui 86 | Disease-sensitive material | G |
As can be seen from Table 3, the detection result of 4 parts of materials in total of IRBB7, DV85, hua Hui 1437 and Hua Hui 1337 at the test site is base A, the detection result of 12 parts of materials without Xa7 gene at the test site is base G, the parting chart is shown in figure 2, and the result shows that the OS900223_K01 molecular marker can be normally parting.
4 specificity and practicality verification
The markers OS900223_k01 were validated using 2F 1 populations and their parents and 68 diversity parent materials. The typing results of the 2F 1 groups and the parent materials thereof are shown in figure 3, and the result of detection of the donor parent materials at the test site is base A, and Xa7 genes are contained (the typing graph is marked blue); the detection result of the receptor parent material at the test site is base G, and the receptor parent material does not contain Xa7 genes (the parting chart is marked red); the detection result of the hybridization group F1 at the test site is a base G: A, belonging to heterozygote (marked purple in the typing graph).
The 68 parts of diversity parent materials comprise Xa7 donor materials with known genotypes, other anti-white leaf blight gene donor materials, core rice breeding materials and conventional rice materials. The result of typing the sample by using the OS900223_K01 marker is shown in figure 4, and the detection result of 6 parts of the sample at the test site is base A and contains Xa7 genes; the detection result of 62 parts of materials at the test site is base G, and the Xa7 gene is not contained. Therefore, the marker OS900223_K01 has high specificity when detecting the Xa7 gene locus, and can accurately and efficiently identify whether the Xa7 gene is contained in the rice material.
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.
Sequence listing
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<120> SNP marker linked with rice anti-white leaf spot gene Xa7 and application thereof
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Claims (2)
1. Rice anti-white leaf blight geneXa7Is characterized in that the method comprises the following steps:
s1, extracting genome DNA from rice;
s2, using the genome DNA extracted in the step S1 as a template, detecting polymorphism of SNP molecular markers in the genome DNA extracted in the step S1 by using a primer group, and judging whether rice to be detected contains the SNP molecular markers according to the detection polymorphism of the SNP molecular markersXa7A gene; 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 G, and the rice to be detected does not contain the white leaf blight resistance gene Xa7; if only the fluorescence signal corresponding to the Primer Y is detected, the base of the detection site is A, and the rice to be detected contains a white leaf blight resistance gene Xa7; the SNP marker is OS 900223-K01, the polymorphic site of the SNP marker is positioned at the base at 27970206 of Japanese 6 chromosome, and the polymorphism is G/A.
2. A rice breeding method for resisting bacterial leaf blight is characterized by comprising the following steps: the detection method according to claim 1, wherein the bacterial leaf blight resistance gene is selectedXa7And carrying out subsequent breeding on the rice sample of the gene.
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