Molecular markers of rice blast resistance genes and their applications

Abstract

The invention discloses a molecular marker of a rice blast resistance gene, which is a SNP site sequence pairing selected from a gene panel comprising the rice blast resistance genes Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a. The molecular marker can be used to guide rice improvement.

Description

Technical Field

[0001] The invention belongs to the field of gene detection, and in particular relates to a molecular marker of a rice blast resistance gene and an application thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world. More than half of the world's population relies on rice for food. In China, rice cultivation accounts for about 25% of the country's cultivated land, and its output accounts for about 50% of the country's total grain output. Rice is an important strategic resource for my country's food security and sustainable agricultural development. Rice blast is a global rice disease caused by Magnaporthe grisea. Rice blast reduces both rice yield and rice quality, and seriously harms rice yield and quality.

[0003] Breeding rice varieties resistant to rice blast is an effective way to control rice blast. Kongyu 131 and Longjing 31 are the main rice varieties in Heilongjiang, but after a long period of planting, the resistance of rice to rice blast has decreased. In order to improve the resistance of Kongyu 131 and Longjing 31 to rice blast, meet the demand for germplasm in agricultural production, and ensure high and stable yield of rice, it is necessary to improve the varieties of Kongyu 131 and Longjing 31 to improve their resistance to rice blast. Summary of the invention

[0004] We introduced rice blast resistance genes into Kongyu 131 and Longjing 31 by hybridization, screened individuals containing seven rice blast resistance genes, including Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a, by molecular markers linked to rice blast resistance genes, and finally obtained rice blast resistance Kongyu 131 and Longjing 31 through multi-generation hybridization and backcrossing. The results of the above research constitute the basis of the present invention. Specifically, the present invention includes the following technical solutions.

[0005] A molecular marker for a rice blast resistance gene, which is a SNP (Single Nucleotide Polymorphism) site sequence pairing selected from a gene panel comprising the rice blast resistance genes Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a. Specifically, it is a SNP site sequence pairing selected from the following gene panels:

[0006] The SNP sequence for judging the absence / presence of the rice blast resistance gene Pi-i is SEQ ID NO:1 / SEQ ID NO:2, named herein as "S0313_2k"; or SEQ ID NO:3 / SEQ ID NO:4, named herein as "S1879_4k";

[0007] The SNP sequence for judging the absence / presence of the rice blast resistance gene Pi-b is SEQ ID NO:5 / SEQ ID NO:6, named herein as "S0380_300R"; or SEQ ID NO:7 / SEQ ID NO:8, named herein as "S1899-1k";

[0008] The SNP sequence for judging the absence / presence of the rice blast resistance gene Pi-zt is SEQ ID NO:9 / SEQ ID NO:10, named as "Pizt-shy-3" herein; or SEQ ID NO:11 / SEQ ID NO:12, named as "S0268_2" herein;

[0009] The SNP sequence for judging the absence / presence of the rice blast resistance gene Pi-z is SEQ ID NO:13 / SEQ ID NO:14, named herein as "Piz-shy-1"; or SEQ ID NO:15 / SEQ ID NO:16, named herein as "Piz-xy-2";

[0010] The SNP sequence for judging the absence / presence of the rice blast resistance gene Pi-kp is SEQ ID NO:17 / SEQ ID NO:18, named herein as "Pik-shy-4"; or SEQ ID NO:19 / SEQ ID NO:20, named herein as "S0712_6k";

[0011] The SNP sequence SEQ ID NO:21 / SEQ ID NO:22 for judging the absence / presence of the rice blast resistance gene Pi-km, named as "Pik-xy-2" herein;

[0012] The SNP sequence for judging the absence / presence of the rice blast resistance gene Pi-a is SEQ ID NO:23 / SEQ ID NO:24, named herein as "Pia-shy-1"; or SEQ ID NO:25 / SEQ ID NO:26, named herein as "S0346_8k".

[0013] When rice genomic DNA is used as a template to perform PCR amplification of the molecular marker, the primer pairs used are:

[0014] Amplify the SNP sequence SEQ ID NO:1 / SEQ ID NO:2, i.e., "S0313_2k", with forward / reverse primers SEQ ID NO:27 / SEQ ID NO:28 (S0313_2k_F, S0313_2k_R);

[0015] Amplify the SNP sequence SEQ ID NO:3 / SEQ ID NO:4, i.e., "S1879_4k" with forward / reverse primers SEQ ID NO:29 / SEQ ID NO:30 (S1879_4k_F, S1879_4k_R);

[0016] Amplify the SNP sequence SEQ ID NO:5 / SEQ ID NO:6, i.e., "S0380_300R" forward / reverse primers SEQ ID NO:31 / SEQ ID NO:32 (S0380_300R_F, S0380_300R_R);

[0017] Amplify the SNP sequence SEQ ID NO:7 / SEQ ID NO:8, i.e., "S1899-1k_F" with forward / reverse primers SEQ ID NO:33 / SEQ ID NO:34 (S1899-1k_F, S1899-1k_R);

[0018] Amplify the SNP sequence SEQ ID NO:9 / SEQ ID NO:10, i.e., "Pizt-shy-3" with forward / reverse primers SEQ ID NO:35 / SEQ ID NO:36 (Pizt-shy-3_F, Pizt-shy-3_R);

[0019] Amplify the SNP sequence SEQ ID NO:11 / SEQ ID NO:12, i.e., "S0268_2" with forward / reverse primers SEQ ID NO:37 / SEQ ID NO:38 (S0268_2_F, S0268_2_R);

[0020] Amplify the SNP sequence SEQ ID NO:13 / SEQ ID NO:14, i.e., the forward / reverse primers SEQ ID NO:39 / SEQ ID NO:40 (Piz-shy-1, Piz-shy-1_R);

[0021] Amplify the SNP sequence SEQ ID NO:15 / SEQ ID NO:16, i.e., the forward / reverse primers SEQ ID NO:41 / SEQ ID NO:42 (Piz-xy-2_F, Piz-xy-2_R);

[0022] Amplify the SNP sequence SEQ ID NO:17 / SEQ ID NO:18, i.e., the forward / reverse primers SEQ ID NO:43 / SEQ ID NO:44 (Pik-shy-4_F, Pik-shy-4_R);

[0023] Amplify the SNP sequence SEQ ID NO: 19 / SEQ ID NO: 20, i.e., "S0712_6k" forward / reverse primers SEQ ID NO: 45 / SEQ ID NO: 46 (S0712_6k_F, S0712_6k_R);

[0024] Amplify the SNP sequence SEQ ID NO:21 / SEQ ID NO:22, i.e., "Pik-xy-2" forward / reverse primers SEQ ID NO:47 / SEQ ID NO:48 (Pik-xy-2_F, Pik-xy-2_R);

[0025] Amplify the SNP sequence SEQ ID NO:23 / SEQ ID NO:24, i.e., the forward / reverse primers SEQ ID NO:49 / SEQ ID NO:50 (Pia-shy-1_F, Pia-shy-1_R);

[0026] The forward / reverse primers for amplifying the SNP sequence SEQ ID NO:25 / SEQ ID NO:26, i.e., "S0346_8k", are SEQ ID NO:51 / SEQ ID NO:52 (S0346_8k_F, S0346_8k_R).

[0027] The above-mentioned molecular markers can be used to construct a detection model for determining whether rice has a gene for resistance to rice blast.

[0028] Therefore, the second aspect of the present invention is to provide a detection model for judging whether rice has a rice blast resistance gene, which includes the above-mentioned molecular marker judgment standard, that is, after the rice genomic DNA is PCR amplified using the primers, if the sequence number of the SNP site of a certain rice blast resistance gene detected is an odd number n (n=1-25), then the rice blast resistance gene is absent; on the contrary, if the sequence number of the SNP site of a certain rice blast resistance gene detected is an even number n+1 (2-26), then the rice blast resistance gene is contained. Specifically,

[0029] SEQ ID NO: 1 or SEQ ID NO: 3 indicates that rice does not contain the rice blast resistance gene Pi-i;

[0030] SEQ ID NO: 5 or SEQ ID NO: 7 indicates that rice does not contain the rice blast resistance gene Pi-b;

[0031] SEQ ID NO:9 or SEQ ID NO:11 indicates that rice does not contain the rice blast resistance gene Pi-zt;

[0032] SEQ ID NO: 13 or SEQ ID NO: 15 indicates that rice does not contain the rice blast resistance gene Pi-z;

[0033] SEQ ID NO: 17 or SEQ ID NO: 19 indicates that rice does not contain the rice blast resistance gene Pi-kp;

[0034] SEQ ID NO: 17 or SEQ ID NO: 21 indicates that rice does not contain the rice blast resistance gene Pi-km;

[0035] SEQ ID NO:23 or SEQ ID NO:25 indicates that rice does not contain the rice blast resistance gene Pi-a;

[0036] SEQ ID NO: 2 or SEQ ID NO: 4 indicates that rice contains the rice blast resistance gene Pi-i;

[0037] SEQ ID NO:6 or SEQ ID NO:8 indicates that rice contains the rice blast resistance gene Pi-b;

[0038] SEQ ID NO: 10 or SEQ ID NO: 12 indicates that rice contains the rice blast resistance gene Pi-zt;

[0039] SEQ ID NO: 14 or SEQ ID NO: 16 indicates that rice contains the rice blast resistance gene Pi-z;

[0040] SEQ ID NO: 18 or SEQ ID NO: 20 indicates that rice contains the rice blast resistance gene Pi-kp;

[0041] SEQ ID NO: 18 or SEQ ID NO: 22 indicates that rice contains the rice blast resistance gene Pi-km;

[0042] SEQ ID NO: 24 or SEQ ID NO: 26 indicates that rice contains the rice blast resistance gene Pi-a.

[0043] The above detection model for determining whether rice contains rice blast resistance gene can be input into a computer, a gene detection device such as ABI 3730 sequencer or Illumina sequencer, or a gene sequencing platform in the form of programming or mathematical software package.

[0044] The third aspect of the present invention is to provide a kit for detecting rice blast resistance genes, which comprises primers for detecting the above-mentioned SNP site sequences, especially SEQ ID NOs: 27-52.

[0045] The above-mentioned kit may also include a standard control table for determining whether rice has a rice blast resistance gene.

[0046] Another aspect of the present invention is to provide the use of the above-mentioned molecular markers, detection models, and kits in rice improvement.

[0047] For example, by determining whether rice has a certain rice blast resistance gene, we can guide the introduction of the rice blast resistance gene into rice that lacks the rice blast resistance gene through genetic engineering, or by hybridizing with rice varieties containing the rice blast resistance gene to form transgenic rice or hybrid rice.

[0048] The molecular marker of the rice blast resistance gene constructed by the present invention has been tested in rice varieties Yueguang, Kongyu 131 and Longjing 31, and it is proved that the molecular marker of the rice blast resistance gene constructed by the present invention can accurately determine whether the rice has the rice blast resistance genes Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a, effectively guide the improvement of rice varieties, and is used for rice blast resistance breeding, accelerates the breeding screening speed, screens plants containing the rice blast resistance gene in breeding, and finally breeds rice varieties containing rice blast resistance, which has important economic significance for increasing rice yield and ensuring food supply security. DETAILED DESCRIPTION

[0049] The rice variety Koshihikari contains seven rice blast resistance genes, namely Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a, while Kongyu 131 and Longjing 31 lack these genes. The present invention is based on the whole genome sequencing of the rice varieties Koshihikari, Kongyu 131 and Longjing 31, and then assembles and compares the genome sequences, and finally screens out 13 molecular markers linked to the rice blast resistance genes, and establishes a gene detection panel for rapid and accurate detection of the rice blast resistance genes Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a.

[0050] Gene (detection) panel is a term used after the development of high-throughput gene detection and gene sequencing. It refers to designing probes corresponding to several genes on the same capture chip to capture target DNA for subsequent gene sequencing. In the detection, not only one site or one gene is detected, but multiple sites, multiple genes, and multiple sites are detected at the same time. These sites and genes need to be selected and combined according to a standard, which requires a detection panel.

[0051] The gene panel herein may also be referred to as a gene set, which is a collection or combination of all genes related to rice blast resistance detection, and may also include a probe capture scheme for detecting rice blast resistance genes or rice genome regions.

[0052] The gene panel of the present invention comprises a specific SNP sequence set constructed for seven rice blast resistance genes Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a, and includes 13 molecular markers.

[0053] SNP is a single nucleotide polymorphism, which refers to the polymorphism of nucleic acid sequence caused by the change of a single nucleotide base (including conversion, transversion, and insertion / deletion of a single base, etc.), which is well known in the art.

[0054] By using this gene panel, technicians in this field can easily determine whether the rice hybrid offspring contain the corresponding rice blast resistance genes Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a in the breeding of rice blast-resistant rice. The rice blast resistance of rice plants can be identified at the seedling stage, thereby improving the efficiency of rice breeding and greatly saving time costs, labor costs and land utilization.

[0055] The detection model of the rice blast resistance gene of the present invention is a discrete mathematical model established by using the molecular markers for detecting Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a, i.e., SNP site sequence pairing as variables. For the convenience of description, the model may also be referred to as "rice blast resistance gene model" or "molecular marker model" herein.

[0056] In one embodiment, when the molecular marker of the rice blast resistance gene of the present invention is provided in the form of a kit, the kit may include at least one of the following items in addition to various primers: a carrying tool, whose space is divided into a limited space that can accommodate one or more containers, 96-well plates or strips, the container is, for example, a kit, a medicine bottle, a test tube, and the like, each container containing a separate component for the method of the present invention; an instruction manual, which can be written on a bottle, a test tube and the like, or on a separate piece of paper, or on the outside or inside of a container, for example, a paper with an operation demonstration video APP download window such as a QR code, and the instruction manual can also be in the form of multimedia, such as a CD, a U disk, a network disk, etc.

[0057] The present invention has the following advantages in screening rice plants resistant to rice blast. First, compared with the method of identifying the resistance of rice plants by inoculating pathogens in traditional disease-resistant breeding, the experimental results of screening rice plants resistant to rice blast by molecular markers in the present invention are more stable and reliable. The reason is that the molecular marker method is less interfered by environmental factors, so the results are more reliable. Second, the inoculation experiment cycle in the traditional method is long, the cost is high, and the operation process is complicated; while the molecular marker method of the present invention has a short experimental cycle and is easy to operate. Third, the molecular marker identification method can take samples at the seedling stage, extract DNA, and find the SNP corresponding to disease resistance through PCR amplification and sequencing, identify individual plants resistant to rice blast, eliminate other plants, and improve the screening efficiency of individuals resistant to rice blast, which can meet the needs of high-throughput molecular marker-assisted breeding and realize the industrialized molecular breeding of rice blast resistance genes.

[0058] The following is an example of screening and verifying the molecular markers of rice blast resistance genes to illustrate the technical effects of the present invention. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0059] The percentages involved in the embodiments, unless otherwise specified (such as explicitly expressed as volume percentage or ratio), all refer to mass percentages.

[0060] Example 1: Molecular marker screening of rice blast resistance genes

[0061] Instruments: PCR instrument, Qubit fluorescence quantification instrument, ABI 3730 sequencer.

[0062] Rice gene donor varieties: Koshihikari, Kongyu 131 and Longjing 31.

[0063] Molecular marker screening includes the following steps:

[0064] 1) Extracting genomic DNA from the rice plant to be tested;

[0065] 2) using one or more pairs of primers selected from Table 1 below to perform PCR amplification on rice genomic DNA;

[0066] The PCR reaction system for 20 μl is as follows: 2 μl of 10X PCR reaction buffer, 0.8 μl of 25 mM MgSO4, 2 μl of 2 mM dNTP, 1.2 μl each of 5 μM forward primer F and reverse primer R, 20 ng of genomic DNA, 0.44 μl of KOD-Plus polymerase, and ddH2O is added to make up to 20 μl.

[0067] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds, extension at 68°C for 1 minute, for a total of 35 cycles; and incubation at 68°C for 5 minutes.

[0068] 3) SNP molecular markers of rice blast resistance genes. The PCR products were purified and then subjected to sequencing reaction using an ABI3730 sequencer. If the single nucleotide polymorphism site is a SNP of Koshihikari, it indicates that the corresponding rice blast resistance gene is present.

[0069] For example, for the detection method of the SSR molecular marker (S0268_2) of the rice blast resistance gene Pi-zt, S0268_2_F is fluorescently labeled with FAM, and the PCR amplification product is tested for the size of the specific fragment by the ABI 3730 sequencer. If a 332bp specific fragment is amplified, it indicates that the rice blast resistance gene Pi-zt is present. If a 367bp specific fragment is amplified, it indicates that the rice blast resistance gene Pi-zt is not present. Ordinary PCR primers can also be used without fluorescent labeling, and the PCR product is identified by agarose gel electrophoresis to identify the fragment size.

[0070] SSR refers to simple sequence repeats, also known as microsatellite DNA. Its core sequence of tandem repeats is 1-6bp, among which the most common are dinucleotide repeats, namely (CA)n and (TG)n. The core sequence structure of each microsatellite DNA is the same, and the number of repeat units is 10-60. Its high polymorphism mainly comes from the different numbers of tandem repeats.

[0071] The gene sequencing results of three rice varieties, Yueguang, Kongyu 131 and Longjing 31, are shown in Table 1.

[0072] Table 1. Molecular markers, primer pairs, and identified SNP sites of rice blast resistance genes

[0073]

[0074]

[0075] In Table 1, "-F" in the primer names represents forward; "-R" represents reverse.

[0076] A total of 13 molecular markers were obtained for seven rice blast resistance genes, Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, Pi-km and Pi-a.

[0077] Example 2: Breeding of Kongyu 131 for resistance to rice blast

[0078] With the help of whole genome sequencing analysis, the gene panel established in Example 1 can be applied to rice breeding for resistance to rice blast, including the improvement of Heilongjiang main cultivated varieties Kongyu 131 and Longjing 31, by introducing rice blast resistance genes Pi-i, Pi-b, Pi-zt, Pi-z, Pi-kp, km, and Pi-a into the genomes of Kongyu 131 and Longjing 31 to improve the resistance of Kongyu 131 and Longjing 31 to rice blast.

[0079] For example, the donor variety containing the rice blast resistance gene is the rice blast resistant Koshihikari variety, and the recipient variety is Kongyu 131. Our goal is to introduce the rice blast resistance gene from the Koshihikari variety into Kongyu 131, enhance Kongyu 131's resistance to rice blast, and achieve high and stable yields for Kongyu 131.

[0080] 1. Breeding of Kongyu 131 containing the rice blast resistance gene Pi-zt

[0081] First, the Koshihikari variety containing the rice blast resistance gene Pi-zt was hybridized with Kongyu 131. Then, the molecular markers linked to the rice blast resistance gene were used to screen the plants containing the Pi-zt rice blast resistance gene. Afterwards, the rice blast resistance plant was backcrossed with Kongyu 131. The purpose of backcrossing is to restore its background to Kongyu 131. In each generation of backcrossing, we selected plants containing the Pi-zt rice blast resistance gene and backcrossed them with Kongyu 131. After four to five generations of backcrossing, the plant background has basically returned to Kongyu 131. Then, the material was self-pollinated, and then the homozygous individuals containing the Pi-zt rice blast resistance gene were screened by molecular markers, which is the Kongyu 131 containing the rice blast resistance gene Pi-zt that we successfully cultivated. After breeding, the material can be used for field planting.

[0082] 2. Breeding of Kongyu 131 containing the rice blast resistance gene Pi-i

[0083] Kongyu 131 was hybridized with the Koshihikari variety containing the rice blast resistance gene Pi-zt. The subsequent hybridization, backcrossing and self-pollination process was the same as "breeding of Kongyu 131 containing the rice blast resistance gene Pi-zt", with only the rice blast resistance selection marker being different.

[0084] 3. Breeding of Kongyu 131 containing the rice blast resistance gene Pi-ta2

[0085] Kongyu 131 was hybridized with the Koshihikari variety containing the rice blast resistance gene Pi-ta2. The subsequent hybridization, backcrossing and selfing process was the same as "breeding of Kongyu 131 containing the rice blast resistance gene Pi-zt", with only the rice blast resistance selection marker being different.

[0086] 4. Breeding of Kongyu 131 containing the rice blast resistance gene Pi-b

[0087] Kongyu 131 was hybridized with the Koshihikari variety containing the rice blast resistance gene Pi-b. The subsequent hybridization, backcrossing and selfing process was the same as "breeding of Kongyu 131 containing the rice blast resistance gene Pi-zt", with only the rice blast resistance selection marker being different.

[0088] 5. Breeding of Kongyu 131 containing the rice blast resistance gene Pi-kp,km

[0089] Kongyu 131 was hybridized with the Koshihikari variety containing the rice blast resistance gene Pi-kp,km. The subsequent hybridization, backcrossing and selfing process was the same as "breeding of Kongyu 131 containing the rice blast resistance gene Pi-zt", with only the rice blast resistance selection marker being different.

[0090] 6. Breeding of Kongyu 131 containing the rice blast resistance gene Pi-z

[0091] Kongyu 131 was hybridized with the Koshihikari variety containing the rice blast resistance gene Pi-z. The subsequent hybridization, backcrossing and self-pollination process was the same as "breeding of Kongyu 131 containing the rice blast resistance gene Pi-zt", with only the rice blast resistance selection marker being different.

[0092] 7. Breeding of Kongyu 131 containing two rice blast resistance genes Pi-b and Pi-zt

[0093] Through the above breeding process, we obtained Kongyu 131 containing the rice blast resistance gene Pi-b and Kongyu 131 containing the rice blast resistance gene Pi-zt, and hybridized the two. Then, the plants containing both the rice blast resistance genes Pi-b and Pi-zt were screened through molecular markers linked to the rice blast resistance gene, and self-pollinated. The homozygous individuals containing both the rice blast resistance genes Pi-b and Pi-zt were further screened through molecular markers linked to the rice blast resistance gene. The homozygous individuals can be used for field planting after breeding.

[0094] 8. Breeding of Kongyu 131 containing two rice blast resistance genes Pikm and Pi-zt

[0095] Kongyu 131 containing the rice blast resistance gene Pikm and Kongyu 131 containing the rice blast resistance gene Pi-zt were hybridized. Afterwards, the screening process was the same as "breeding of Kongyu 131 containing two rice blast resistance genes Pi-b and Pi-zt".

[0096] 9. Breeding of Kongyu 131 containing two rice blast resistance genes Pi and Pi-zt

[0097] Kongyu 131 containing the rice blast resistance gene Pi and Kongyu 131 containing the rice blast resistance gene Pi-zt were hybridized. Afterwards, the screening process was the same as "breeding of Kongyu 131 containing two rice blast resistance genes Pi-b and Pi-zt".

[0098] 10. Breeding of Kongyu 131 containing two rice blast resistance genes Pikm and Pi-b

[0099] Kongyu 131 containing the rice blast resistance gene Pikm and Kongyu 131 containing the rice blast resistance gene Pi-b were hybridized. Afterwards, the screening process was the same as "breeding of Kongyu 131 containing two rice blast resistance genes Pi-b and Pi-zt".

[0100] 11. Breeding of Kongyu 131 containing two rice blast resistance genes Pi-i and Pi-b

[0101] Kongyu 131 containing the rice blast resistance gene Pi-i and Kongyu 131 containing the rice blast resistance gene Pi-b were hybridized. Afterwards, the screening process was the same as "breeding of Kongyu 131 containing two rice blast resistance genes Pi-b and Pi-zt".

[0102] 12. Breeding of Kongyu 131 containing two rice blast resistance genes Pi-i and Pikm

[0103] Kongyu 131 containing the rice blast resistance gene Pi-i and Kongyu 131 containing the rice blast resistance gene Pikm were hybridized. Afterwards, the screening process was the same as "breeding of Kongyu 131 containing two rice blast resistance genes Pi-b and Pi-zt".

[0104] 13. Breeding of Kongyu 131 containing three rice blast resistance genes Pikm, Pi-zt, Pi-b

[0105] Through the above breeding process, we obtained Kongyu 131 containing two rice blast resistance genes Pikm and Pi-zt, and then hybridized it with Kongyu 131 containing rice blast resistance gene Pi-b. Among the hybrid individuals, the molecular markers linked to the rice blast resistance gene were used to screen out plants containing the rice blast resistance genes Pikm, Pi-zt, and Pi-b at the same time, and self-pollinated. The molecular markers linked to the rice blast resistance gene were further screened to obtain homozygous individuals containing the rice blast resistance genes Pikm, Pi-zt, and Pi-bt at the same time. The homozygous individuals can be used for field planting after breeding.

[0106] Example 3: Breeding of Longjing 31 for resistance to rice blast

[0107] The donor variety containing the rice blast resistance gene is the rice blast resistant Koshihikari variety, and the recipient variety is Longjing 31. Our goal is to introduce the rice blast resistance gene from the Koshihikari variety into Longjing 31, enhance the resistance of Longjing 31 to rice blast, and achieve high and stable rice yields.

[0108] 1. Cultivation of Longjing 31 containing the rice blast resistance gene Pi-i by molecular marker screening linked to the rice blast resistance gene

[0109] First, the Koshihikari variety containing the rice blast resistance gene Pi-i was hybridized with Longjing 31. Then, the molecular markers linked to the rice blast resistance gene were used to screen the plants containing the Pi-i rice blast resistance gene. Afterwards, the rice blast resistant plant was backcrossed with Longjing 31. The purpose of backcrossing is to restore its background to Longjing 31. In each generation of backcrossing, we selected plants containing the Pi-i rice blast resistance gene and backcrossed them with Longjing 31. After four to five generations of backcrossing, the plant background has basically returned to Longjing 31. Then, the material was self-pollinated, and then the homozygous individuals containing the Pi-i rice blast resistance gene were screened by molecular markers, which is the Longjing 31 containing the rice blast resistance gene Pi-i that we successfully cultivated. After breeding, the material can be used for field planting.

[0110] 2. Cultivation of Longjing 31 containing the rice blast resistance gene Pi-b by molecular marker screening linked to the rice blast resistance gene

[0111] The Koshihikari variety containing the rice blast resistance gene Pi-b was hybridized with Longjing 31. Afterwards, the backcrossing and selfing process was the same as the "Cultivation of Longjing 31 containing the rice blast resistance gene Pi-i using the method of molecular marker screening linked to the rice blast resistance gene", with only the molecular markers for rice blast resistance screening being different.

[0112] 3. Cultivation of Longjing 31 containing the rice blast resistance gene Pi-zt by molecular marker screening linked to the rice blast resistance gene

[0113] The Koshihikari variety containing the rice blast resistance gene Pi-zt was hybridized with Longjing 31. Afterwards, the backcrossing and selfing process was the same as the "Cultivation of Longjing 31 containing the rice blast resistance gene Pi-i using the method of molecular marker screening linked to the rice blast resistance gene", with only the molecular markers for rice blast resistance screening being different.

[0114] 4. Breeding Longjing 31 containing the rice blast resistance gene Pi-z by molecular marker screening linked to the rice blast resistance gene

[0115] The Koshihikari variety containing the rice blast resistance gene Pi-z was hybridized with Longjing 31. Afterwards, the backcrossing and selfing process was the same as the "Cultivation of Longjing 31 containing the rice blast resistance gene Pi-i using the method of molecular marker screening linked to the rice blast resistance gene", with only the molecular markers for rice blast resistance screening being different.

[0116] 5. Cultivation of Longjing 31 containing the rice blast resistance gene Pi-kp,km by molecular marker screening linked to the rice blast resistance gene

[0117] The Koshihikari variety containing the rice blast resistance gene Pi-kp,km was hybridized with Longjing 31. Afterwards, the backcrossing and selfing process was the same as "Cultivating Longjing 31 containing the rice blast resistance gene Pi-i using the method of molecular marker screening linked to the rice blast resistance gene", with only the molecular markers for rice blast resistance screening being different.

[0118] 6. Breeding Longjing 31 containing the rice blast resistance gene Pi-a using molecular marker screening linked to the rice blast resistance gene

[0119] The Koshihikari variety containing the rice blast resistance gene Pi-a was hybridized with Longjing 31. Afterwards, the backcrossing and selfing process was the same as the "Cultivation of Longjing 31 containing the rice blast resistance gene Pi-i using the method of molecular marker screening linked to the rice blast resistance gene", with only the molecular markers for rice blast resistance screening being different.

[0120] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Sequence Listing <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> Molecular markers of rice blast resistance genes and their applications <130> SHPI2010672 <160> 52 <170> SIPOSequenceListing 1.0 <210> 1 <211> 32 <212> DNA <213> Oryza sativa L. <400> 1 gcaaacctta tgattttcat ggaaattcta ca 32 <210> 2 <211> 32 <212> DNA <213> Oryza sativa L. <400> 2 gcaaacctta tgattttcat ggaaattcta cg 32 <210> 3 <h2 style=";text-align:left;direction:ltr"><211> 39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaacacatga ttctaacata aatacaagtg taaaatata 39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaacacatga ttctaacata aatacaagtg taaaatatg 39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 28<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctacatatgt caagaattac tgcaactt 28<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 28<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctacatatgt caagaattac tgcaactc 28<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccgtacagtt acacacgtaa tcca 24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccgtacagtt acacacgtaa tccc 24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 9<h2 style=";text-align:left;direction:ltr"> <211> 40 <212> DNA <213> Oryza sativa L. <400> 9 aattttgata aaggatacaa gcacagtata gctcaatctt 40 <210> 10 <211> 40 <212> DNA <213> Oryza sativa L. <400> 10 aattttgata aaggatacaa gcacagtata gctcaatctc 40 <210> 11 <211> 367 <212> DNA <213> Oryza sativa L. <400> 11 tcgtgtcatg agctgtactc cctccgtcaa aaaataaaa aaaagaaaa aaggcaaacc 60 tgattccgt gtccaatatt tgaccatccg tcttatttaa aaaaattata aaaaaaatta 120 aaaagacaag tcacgcataa aatattaatt atgttttatc atctaacaac aatgaaaata 180 caaattataa aaaaaattca tataaaacgg acagttaaaa ttggacacga aaacgcaggg 240 tttgtttttt tttttttt acggagggag ttcgggatga acacatgaaa gtctctttcc 300 agagagttaa aatggccatc attgtagatg tggacatcaa aaaggtgtct gatcaaaaag 360 gtgtctg 367 <210> 12 <211> 332<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cagacacctt tttgatgtcc acatctacaa tgatggccat tttaactctc tggaaagaga 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctttcatgtg ttcatcccgt aatctctccg tcaaaaaaaa acaaaccctg cgtttccgtg 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tctaatttta actatctgtc ttatatgaaa ctttttataa tttatatttt cattgttgtt 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agatgataag acataattaa tattttatgc gtgacttgtc tttttaattt ttttcataat 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttttttaaat aagacggacg gtcaaatatt ggacacggaa attaaggttt gtcttttttt 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttttgacgga gggaggacag ctcatgacac ga 332<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> actgtaacag taagaatgag gaagactggt acaataggt 39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 14<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 14<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> actgtaacag taagaatgag gaagactggt acaataggc 39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Oryza sativa L.<h2 style=";text-align:left;direction:ltr"> <400> 15 ggctttcagt gtgacaggag ttaatttcc ttctattaac ttgtt <210> 16 <211> 45 <212> DNA <213> Oryza sativa L. <400> 16 ggctttcagt gtgacaggag ttaatttcc ttctattaac ttgtg <210> 17 <211> 45 <212> DNA <213> Oryza sativa L. <400> 17 gggacaaccc gtgatgccat agataccgag ttcaccacag aagat <210> 18 <211> 45 <212> DNA <213> Oryza sativa L. <400> 18 gggacaaccc gtgatgccat ttcaccacag 45. gggacaaccc gtgatgccat <210> 19 <211> 29 <212> DNA <213> Oryza sativa L. <400> 19 gatgataata tcggagagta agtgagcta <210> 20 <211> 29 <212> DNA <213> Oryza sativa L. <400> 20 gatgataata tcggagagta agtgagctg <210> 21 <211> 43 <212> DNA <213> Oryza sativa L. <400> 21 agagactgta tcacttgcat ccccgaatac agttagagat cgg 43 <210> 22 <211> 43 <212> DNA <213> Oryza sativa L. <400> 22 agagactgta tcacttgcat ccccgaatac agttagagat cga 43 <210> 23 <211> 34 <212> DNA <213> Oryza sativa L. <400> 23 cacaaaagaa ttgtttgtcc tatacaaggc ctgt 34 <210> 24 <211> 34 <212> DNA <213> Oryza sativa L. <400> 24 cacaaaagaa ttgtttttcc tatacaaggc ctgt 34 <210> 25 <211> 27 <212> DNA <213> Oryza sativa L. <400> 25 tgcaaattct tggttgtgat ctagaac 27 <210> 26 <211> 27 <212> DNA <213> Oryza sativa L. <400> 26 tgcaaattct tggttgtgat ctagaag 27 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence() <400> 27 tgggtgaatt catggcactg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence() <400> 28 caaaaagccc tccagcaacg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence() <400> 29 ggacggtggc ttcctattcg 20 <210> 30 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 30 tcgcaagaca agtcatcaca gg 22 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence() <400> 31 gctggaggag ggatacgcag 20 <210> 32 <211> twenty one <212> DNA <213> Artificial Sequence() <400> 32 cgcgaatgcc gtctctaatt c 21 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence() <400> 33 ccgggccgag ttttgttctt 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence() <400> 34 cgtgtcccag cgatgagttg 20 <210> 35 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 35 tgttttacca aagttggctg tg 22 <210> 36 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 36 attcgttgct ctgaaatggt tc 22 <210> 37 <211> twenty four <212> DNA <213> Artificial Sequence() <400> 37 cagacacctt tttgatcaga cacc 24 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence() <400> 38 tcgtgtcatg agctgtcctc 20 <210> 39 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 39 caactgtcaa ttccaaggtc aa 22 <210> 40 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 40 catcaagttc tctcgcacac tc 22 <210> 41 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 41 gtctttgttt tggatcgcta gg 22 <210> 42 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 42 catacgctct ctttcagcct tt 22 <210> 43 <211> twenty three <212> DNA <213> Artificial Sequence() <400> 43 caccttgtta tatccagcct acc 23 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence() <400> 44 gatgccagcg gtgtcaat 18 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence() <400> 45 gaaacggagg ctgactggtg 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence() <400> 46 gatctgaccg tggaggttcg 20 <210> 47 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 47 ggcaaaaact tgatcttggt tc 22 <210> 48 <211> twenty two <212> DNA <213> Artificial Sequence() <400> 48 cagaactcag gaaggtgctt ct 22 <210> 49 <211> twenty four <212> DNA <213> Artificial Sequence() <400> 49 gggtctgtac tgattatgca ctgt 24 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence() <400> 50 acggcgagag taatggcata 20 <210> 51 <211> twenty one <212> DNA <213> Artificial Sequence() <400> 51 cgtgcttgga tttttgtaag c 21 <210> 52 <211> twenty one <212> DNA <213> Artificial Sequence() <400> 52 gcatccagct gcacatttcc c 21

Claims

1. A molecular marker for a rice blast resistance gene, which is the following SNP site sequence pairing: The SNP sequence for judging the absence / presence of the rice blast resistance gene Pi-i is SEQ ID NO: 1 / SEQ ID NO: 2; or SEQ ID NO: 3 / SEQ ID NO:

4.

2. The molecular marker according to claim 1, characterized in that When the PCR amplification of the molecular markers was performed using rice genomic DNA as a template, the primer pairs used were: Forward / reverse primers SEQ ID NO: 27 / SEQ ID NO: 28 for amplifying SNP sequence SEQ ID NO: 1 / SEQ ID NO: 2; The forward / reverse primers for amplifying the SNP sequence SEQ ID NO: 3 / SEQ ID NO: 4 are SEQ ID NO: 29 / SEQ ID NO:

30.

3. The molecular marker according to claim 2, characterized in that Used to construct a detection model to determine whether rice contains rice blast resistance genes.

4. A method for constructing a detection model for determining whether rice has a rice blast resistance gene, characterized in that: Using the molecular marker as described in claim 2 as the judgment standard, that is, using the primers to perform PCR amplification on rice genomic DNA, if the sequence number of the SNP site of a certain rice blast resistance gene detected is an odd number, then the rice blast resistance gene is absent; on the contrary, if the sequence number of the SNP site of a certain rice blast resistance gene detected is an even number, then the rice blast resistance gene is contained.

5. The method according to claim 4, characterized in that The detection model is input into a computer, a gene detection device or a gene sequencing platform in the form of programming or a mathematical software package.

6. A kit for detecting a rice blast resistance gene, characterized in that: Comprising primers for detecting the SNP site sequence as claimed in claim 1 or 2.

7. The kit according to claim 6, characterized in that Also included is a standard control table for determining whether rice has the rice blast resistance gene.

8. Use of the molecular marker according to any one of claims 1 to 3, or the kit according to claim 6 or 7 in rice improvement.

9. The use according to claim 8, characterized in that By determining whether rice has a rice blast resistance gene, the rice blast resistance gene is introduced into rice lacking the rice blast resistance gene through genetic engineering, or by hybridizing with a rice variety containing the rice blast resistance gene, transgenic rice or hybrid rice is formed.

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