CN114410819A - Functional marker group for identifying multiple rice blast resistance alleles of rice Pik locus, primer combination and application of functional marker group - Google Patents

Abstract

The invention discloses a functional marker group for identifying a plurality of rice blast resistance alleles at the Pik locus of rice, wherein the functional marker group is a KASP functional marker group and comprises functional markers MPik1, MPik2, MPik3, MPik4 and MPik5, and the rice blast resistance alleles comprise Pi1, Pikm _ TS, PiKs, PiKa, Pik, PiKp and Pi 7. Also discloses a complete set of KASP primer group for detecting the functional marker group and a detection method. By carrying out multi-sequence comparison among different disease-resistant alleles and disease-sensitive genes, SNP loci which can be used for distinguishing different alleles of a Pik locus are screened out, and on the basis, a functional marker group which can be used for distinguishing the Pik locus disease-resistant genes and distinguishing different disease-resistant alleles is successfully developed; the functional marker groups can be used for accurately distinguishing different resistance alleles, and have important breeding application value.

Description

Functional marker group for identifying multiple rice blast resistance alleles of rice Pik locus, primer combination and application of functional marker group

Technical Field

The invention relates to the technical field of molecular biology, in particular to a functional marker group for identifying multiple rice blast resistance alleles of a rice Pik locus, a primer combination and application thereof.

Background

Rice is one of the most important food crops in the world. The rice blast caused by Pyricularia oryzae (Pyricularia oryzae Cav) is the most damaging rice disease in the world so far, and the yield of rice is reduced by about 10% -30% per year due to the rice blast in the world, and is 40% -50% in severe cases, and even no grain is harvested. So far, the disease cannot be properly controlled in production, the global climate begins to become warm along with the aggravation of greenhouse effect, and the outbreak of rice blast disease of rice develops towards the trend of being unobtainable; therefore, screening of good varieties of rice blast resistance is very important for controlling the occurrence of the rice blast from the source. The screening of the rice blast resistant varieties mainly comprises artificial inoculation identification or natural induction identification, the method is original, the mechanism is not clear, the artificial inoculation method is used for the rice blast resistance identification, the identification period is long, the physiological races of pathogenic bacteria are lagged, and the natural induction identification method has the problems of uneven distribution of the pathogenic bacteria, large annual resistance difference and the like. Therefore, the method for improving the rice blast resistance of the variety by using the molecular marker-assisted selection has important application value.

Several rice blast resistance genes have been cloned at present, such as Pizt, Pi9, Pigm, Pi2, Pik, Pi21, Pi54, etc. The cloning of the rice blast resistance gene provides an important basis for molecular marker-assisted breeding of rice blast, and promotes the transition of the cultivation of rice blast resistance varieties from the traditional breeding method based on artificial inoculation identification to the molecular breeding method based on molecular marker-assisted selection.

The resistance gene of the Pik locus is a resistance gene locus with better application effect in breeding at present. The locus contained 7 resistance alleles, i.e., Pi1, Pikm _ TS, PiKs, PiKa, Pik, PiKp, Pi 7. These resistance genes have different resistance effects, and some resistance alleles have lost the value of breeding applications. Therefore, the accurate identification of different resistance alleles has important breeding application value.

Molecular marker identification methods of Pik locus resistance genes have been reported in patent documents and articles, but most of the molecular markers only consider sequence differences between Pik genes of disease-resistant varieties and susceptible varieties, but cannot distinguish differences between different resistance alleles.

Disclosure of Invention

The invention carries out multi-sequence comparison among different rice blast disease-resistant alleles and disease-sensitive genes, screens SNP/Indel sites which can be used for distinguishing different alleles of Pik loci, develops functional marker groups which can be used for distinguishing the Pik locus resistance genes and different resistance alleles on the basis, and can accurately distinguish different resistance alleles by using the functional marker groups, thereby having important breeding application value. Based on this, the invention claims the following technical scheme:

in a first aspect of the invention, there is provided a set of functional markers for identifying a plurality of rice blast resistance alleles at the Pik locus of rice, the set of functional markers being a KASP functional marker set comprising the functional markers MPik1, MPik2, MPik3, MPik4, MPik5, the rice blast resistance alleles comprising Pi1, Pikm _ TS, Piks, Pika, Pik, PiKp and Pi7, the markers MPik1, MPik2, MPik3, MPik4, and MPik5, the correspondence between the genotype and the allele of the SNP/Indel locus is as shown in the following table:

	MPik1	MPik2	MPik3	MPik4	MPik5
						Pi1	T	AA	T	T	T
Pikm_TS	T	AA	T	A	T
						PiKs	T	AA	T	A	C
PiKa	C	AA	G	A	T
						Pik	T	AA	T	A	T
PiKp	C	GC	G	A	C
						Pi7	C	AA	G	A	C

the specific positions of the SNP/Indel sites are shown in the following table:

mark number	Type of molecular marker	Physical location	Differential bases
				MPik_1	SNP	2040 th of the sequence of the Pi1 gene	[T/C]
MPik_2	SNP	Position 8824 of the Pi1 gene sequence	[AA/GC]
				MPik_3	Indel	489 th position of the sequence of the Pi1 gene	[GC/T]
MPik_4	Indel	11284 th position of Pi1 gene sequence	[T/AA]
				MPik_5	SNP	5419 th position of Pi1 gene sequence	[T/C]

Note: the nucleotide sequence of the Pi1 gene in the table is shown in SEQ ID NO. 16.

In the second aspect of the present invention, the set of KASP primer sets for detecting the above-described functional marker set is protected and composed of the following (1) to (5):

(1) KASP primer set for detection of MPik 1: the primer 1 is a single-stranded DNA with a tag sequence A and 22 th to 44 th sites of a sequence 1 in a sequence table from a 5 'end to a 3' end, the primer 2 is a single-stranded DNA with a tag sequence B and 22 th to 44 th sites of a sequence 2 in the sequence table from a 5 'end to a 3' end, and the primer 3 is a single-stranded DNA with a nucleotide sequence shown as a sequence 3 in the sequence table;

(2) KASP primer set for detection of MPik 2: the primer 4 is a single-stranded DNA with a tag sequence A and 22 th to 44 th sites of a sequence 4 in a sequence table from a 5 'end to a 3' end, the primer 5 is a single-stranded DNA with a tag sequence B and 22 th to 41 th sites of a sequence 5 in the sequence table from a 5 'end to a 3' end, and the primer 6 is a single-stranded DNA with a nucleotide sequence shown as a sequence 6 in the sequence table;

(3) KASP primer set for detection of MPik 3: the primer 7 is single-stranded DNA of a tag sequence A and 22 th to 42 th positions of a sequence 7 in a sequence table from a 5 'end to a 3' end, the primer 8 is single-stranded DNA of a tag sequence B and 22 th to 41 th positions of a sequence 8 in the sequence table from the 5 'end to the 3' end, and the primer 9 is single-stranded DNA of which the nucleotide sequence is shown as a sequence 9 in the sequence table;

(4) KASP primer set for detection of MPik 4: the primer 10 is a single-stranded DNA which is sequentially provided with a tag sequence A and 22 th to 46 th sites of a sequence 10 in a sequence table from a 5 'end to a 3' end, the primer 11 is a single-stranded DNA which is sequentially provided with a tag sequence B and 22 th to 46 th sites of a sequence 11 in the sequence table from the 5 'end to the 3' end, and the primer 12 is a single-stranded DNA with a nucleotide sequence shown as a sequence 12 in the sequence table;

(5) KASP primer set for detection of MPik 5: the primer 13 is single-stranded DNA of the tag sequence A and the 22 th to 42 th positions of the sequence 13 in the sequence table from the 5 'end to the 3' end, the primer 14 is single-stranded DNA of the tag sequence B and the 22 th to 40 th positions of the sequence 14 in the sequence table from the 5 'end to the 3' end, and the primer 15 is single-stranded DNA of which the nucleotide sequence is shown as the sequence 15 in the sequence table.

Preferably, the tag sequence A is a FAM fluorescent tag sequence, and the tag sequence B is a HEX fluorescent tag sequence.

Preferably, the sequences of the primers 1-15 are sequentially shown as SEQ ID NO. 1-15 in the sequence table.

In the third aspect of the invention, the functional marker group is protected from being applied to identification or auxiliary identification of rice blast resistance allele types of rice Pik loci or rice molecular marker-assisted breeding.

In the fourth aspect of the invention, the KASP primer group is protected to be applied to identification or auxiliary identification of rice blast resistance allele types of rice Pik loci or rice molecular marker-assisted breeding.

In a fifth aspect of the present invention, there is provided a method for identifying multiple rice blast resistance alleles at the Pik locus of rice, comprising the steps of:

1) extracting the genome DNA of a rice sample to be detected;

2) PCR amplification of the extracted genomic DNA using the primer set according to claim 3, detection of SNP/Indel differential sites in the KASP functional marker group in the amplification product, each marker being amplified individually, and determination of whether the Pik locus blast resistance allele of the sample to be tested is any of Pi1, Pikm _ TS, Piks, PiKa, Pik, PiKp, Pi7, based on the correspondence between the genotype and allele of the SNP/Indel site in the functional markers MPik1, MPik2, MPik3, MPik4, MPik5 in the table of claim 1.

In the method, the primer combination is adopted for PCR amplification, a fluorescence detector is adopted for detecting an amplification product, and the genotype of the different loci in the functional markers MPik1, MPik2, MPik3, MPik4 and MPik5 is judged according to a fluorescence signal, wherein the corresponding relationship is shown as the following table:

the PCR reaction system is a 10ul system: contains 1.0ul 10 XPARMS Buffer, 1.0ul dNTP, 1.0ul of each of 3 primers of each group of KASP primers, the concentration of each primer is 4 mol.L^-10.2ul PARMS PCR enzyme, 30-50 ng/ul template DNA1.0ul, 3.8ul ddH₂O。

The PCR reaction program is: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 45s at 72 ℃ and 35 cycles; finally, extension is carried out for 10min at 72 ℃.

The invention has the beneficial effects that:

by carrying out multi-sequence comparison among different disease-resistant alleles and disease-sensitive genes, SNP loci which can be used for distinguishing different alleles of Pik loci are screened out, and on the basis, functional marker groups which can be used for distinguishing the Pik loci resistant genes and distinguishing different resistant alleles are successfully developed. The functional marker groups can be used for accurately distinguishing different resistance alleles, and have important breeding application value.

The functional marker group-based identification method established by the invention can realize accurate identification of different resistance alleles. On the basis, different resistance alleles can be systematically evaluated, and the resistance alleles with stronger effect and playing an important role in breeding are screened out; the functional marker group developed by the invention can also directly utilize a molecular marker-assisted selection method to rapidly introduce resistance alleles into excellent breeding materials; the functional marker group and the identification method can greatly accelerate the identification of resistance allele and the breeding process.

Drawings

FIG. 1 is the results of an alignment of the sequences of 7 different resistance alleles.

FIG. 2 is a graph showing the results of KASP marker detection of 13 rice breeding materials.

FIG. 3 is a graph showing the results of KASP marker detection of 208 rice germplasm resources.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

The experimental procedures in the following examples are conventional unless otherwise specified.

Example 1

(1) Sequence alignment between different alleles and acquisition of characteristic SNP/Indel

Sequence alignments were performed using genomic sequences of different resistance alleles. The different disease-resistant alleles and their GenBank accession numbers are Pi1(HQ606329), Pikm _ TS (AB462256), PiKs (HQ662329), PiKa (AB616659), Pik (HM048900), PiKp (HM035360), Pi7(HQ 660231). The allele of the corresponding rice variety Nipponbare served as a control for the susceptibility allele. The results of the alignment of the sequences of the 7 different resistance alleles are shown in FIG. 1.

The resistance allele of the Pik locus consists of two genes in tandem, Pik-1 and Pik-2, respectively. Wherein Pik-1 has 3 exons and Pik-2 has 2 exons. In order to reflect more functional information about the variant site, the exon sequences of different alleles are further used for sequence alignment.

After sequence comparison, the characteristic variant sequences that can identify different alleles were selected based on the results of gene sequence and exon sequence comparison (table 1) for further molecular marker development.

TABLE 1 characteristic variant sequences that can distinguish between different resistance alleles

Note: in Table 1, 1) hold is the alignment of different allele sequences of Pik, 1_ cds2 and 1_ cds3 are the alignment of multiple exons 2 and 3 of Pik-1; 2_ cds2: PiK-2 exon 2 multiple sequence alignment;

2) var _ s, var _ e: the sequence comparison file comprises variation starting and stopping positions;

3) r200, L200: the upstream and downstream 200bp positions of the variant sequence are used for extracting partial sequences for marking and developing.

Based on the above analysis, the SNP/Indel sites in the alignment numbers from 10 to 13 in table 1 were further selected for KASP marker development, where the target sequences of both molecular markers are derived from sequence variations of exons.

(2) Development of KASP functional marker for characteristic SNP/Indel

On the basis of obtaining the characteristic variant sequence, further carrying out sequence comparison among different resistance alleles, and screening SNP/Indel sites for identifying the different resistance alleles according to the sequence comparison result. Taking the 200bp flanking sequence of the SNP/Indel site to carry out KASP marked primer design. The set of functional markers finally obtained is shown in table 2.

TABLE 2 functional marker set for differential allelic discrimination of the Pik locus

Comparison document1)

Pi1

Pikm_TS

PiKs

PiKa

Pik

PiKp

Pi7

S2)

KASP tag

Piflag17_260

T

T

T

C

T

C

C

no_hit

MPik1

Piflag14_217

AA

AA

AA

AA

AA

GC

AA

no_hit

MPik2

Piflag16_197

T

T

T

G

T

G

G

no_hit

MPik3

Piflag15_201

T

A

A

A

A

A

A

no_hit

MPik4

Piflag16_215

T

T

C

T

T

C

C

no_hit

MPik5

Note: 1) piflag 17-260, which represents SNP/Indel at the 260 th position in the multi-sequence alignment file Piflag17, and the meanings of the rest alignment files are analogized;

2) no hit-no homologous sequences in the Nipponbare genome.

Designing KASP special labeling primers for SNP/InDel sites in the table 2, wherein each group of KASP primers comprises 2 site specific primers and 1 common primer, the 5 'end of one specific primer is added with a FAM fluorescent label sequence, the 5' end of the other specific primer is added with a HEX fluorescent label sequence, and the primer sequences are shown in the table 3 (the sequences of the primers 1-15 are respectively shown as SEQ ID NO. 1-15 in the sequence table):

TABLE 3 primers specific for KASP markers

Note: the underlined sections in the table are the fluorescent tag sequences.

(3) Functional marker set-based resistance allele identification

Rice leaves are taken to extract DNA by a CTAB method or other DNA extraction methods to obtain template DNA for PCR amplification.

PCR amplification and PCR product detection: synthesizing primers in the functional marker group, and carrying out PCR amplification on the obtained template DNA under the following amplification conditions: 10ul of reaction system: 3 primers containing 1.0ul 10 XPAMS Buffer, 1.0ul dNTP, per group of KASP primers (concentration of each primer is 4 mol. L)^-1) 1.0ul, 0.2ul GTtaq (PARMS PCR enzyme, Wuhan City peptide Biotechnology Co., Ltd., Cat. No. NO53278), 1.0ul template DNA (50ng/ul), 3.8ul ddH each₂And O. The PCR reaction program is: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 45s at 72 ℃ and 35 cycles; finally, extension is carried out for 10min at 72 ℃. By using FAM and HEX as reporter fluorescence and ROX as reference fluorescence, the amplification product can be rapidly detected in a microplate reader and a quantitative PCR (polymerase chain reaction) instrument with the 3 fluorescence detection channels.

And (3) genotype identification: the detected fluorescence signals were converted into the nucleotides at the SNP/Indel sites in the functional marker set in Table 2, the correspondence between the fluorescence signals and the genotypes of the SNP/Indel sites is shown in Table 4, and the correspondence between the genotypes of the functional marker sites and the alleles of the Pik locus is shown in Table 5. Determining which allele of the rice blast resistance gene of the sample is Pi1, Pikm _ TS, PiKs, PiKa, Pik, PiKp and Pi7 according to the genotype identification result of the functional marker group of the 5 markers obtained by detection, and converting a fluorescence signal generated in KASP marker analysis into the nucleotide information of the SNP/InDel locus according to the table 4 when the genomic DNA of the sample to be detected is respectively marked and detected by MPik1, MPik2, MPik3, MPik4 and MPik 5; and (4) integrating the detection results of the 5 molecular markers, and judging the corresponding allelic gene types according to the table 5. Specific positions of SNP/Indel sites are shown in Table 6.

TABLE 4 fluorescent signal and SNP/Indel site genotype data conversion Table

TABLE 5 functional marker locus genotype and Pik locus allele mapping table

Genotype(s)	MPik1	MPik2	MPik3	MPik4	MPik5
						Pi1	T	AA	T	T	T
Pikm_TS	T	AA	T	A	T
						PiKs	T	AA	T	A	C
PiKa	C	AA	G	A	T
						Pik	T	AA	T	A	T
PiKp	C	GC	G	A	C
						Pi7	C	AA	G	A	C
S	-	-	-	-	-

TABLE 6 differential site positions

Mark number	Type of molecular marker	Physical location1)	Differential bases
				MPik_1	SNP	2040 th of the sequence of the Pi1 gene	[T/C]
MPik_2	SNP	Position 8824 of the Pi1 gene sequence	[AA/GC]
				MPik_3	Indel	489 th position of the sequence of the Pi1 gene	[GC/T]
MPik_4	Indel	11284 th position of Pi1 gene sequence	[T/AA]
				MPik_5	SNP	5419 th position of Pi1 gene sequence	[T/C]

Note: 1) the coding sequence of the Pi1 gene is HQ606329:2660-14748, and the specific nucleotide sequence is shown in SEQ ID NO. 16.

The Pik functional marker groups of 13 important breeding materials of rice were identified according to the above method, and the KASP marker detection results are shown in FIG. 2 and the Pik locus allele detection results are shown in Table 7.

Table 7.13 Rice breeding Material Pik locus allele detection results

Name of Rice Material

MPik-1

MPik-2

MPik-3

MPik-4

MPik-5

Alleles

Middle flower 11

C

AA

G

A

C

Pi7

Azucena

-

-

-

-

-

S

IRGC127742

-

-

-

-

-

S

IRGC127652

C

AA

G

A

C

Pi7

26R

-

-

-

-

-

S

Air culture 131

C

AA

T

A

T

Pika

Wanghui No.1

C

AA

G

A

C

Pi7

Chinese character' hui 927

C

AA

G

A

C

Pi7

R498

C

AA

G

A

C

Pi7

R280

C

AA

G

A

C

Pi7

IR64

C

AA

G

A

C

Pi7

N22

C

AA

G

A

-

Pi7

Rice flower fragrance No. 2

T

AA

G

A

C

PiKs

Note: the result S in the column "allele" in the table indicates that the rice variety is a rice blast-susceptible variety.

The detection results in table 7 are consistent with the known allele types corresponding to various rice varieties, and the accuracy and reliability of Pik locus rice blast resistance genotyping of rice by using the KASP functional marker group of the invention are verified.

Example 2, Pik functional marker panel identification of 208 rice resources

A Pik function marker group of 208 rice resources was identified by the method of example 1, the KASP marker test results are shown in FIG. 3, the Pik locus allele test results are shown in Table 8, where S represents susceptible disease and R represents resistant disease; part of the genomic DNA of the rice material was detected as a susceptible allele of the Pik locus using the 5 markers of the present invention, but the phenotype was resistance, probably because these materials contained a rice blast resistance gene other than the Pik locus.

TABLE 8.208 Rice breeding materials Pik locus allele test results

Sequence listing

<110> Tianjin City academy of agricultural sciences

<120> functional marker group, primer combination and application thereof for identifying multiple rice blast resistance alleles at rice Pik locus

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-1Ft

<400> 1

gaaggtgacc aagttcatgc tgatcggaac tggagaggat aatt 44

<210> 2

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-1Fc

<400> 2

gaaggtcgga gtcaacggat tgatcggaac tggagaggat aatc 44

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-1R

<400> 3

aggcatacca taacacatgt tcac 24

<210> 4

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-2Rt

<400> 4

gaaggtgacc aagttcatgc ttctcactgc aactttcagc tgtt 44

<210> 5

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-2Rc

<400> 5

gaaggtcgga gtcaacggat tcactgcaac tttcagctgg c 41

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-2F

<400> 6

gcaggttcgc gcttgatc 18

<210> 7

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-3Rc

<400> 7

gaaggtgacc aagttcatgc tatggaaggc tatccttggc ac 42

<210> 8

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-3Ra

<400> 8

gaaggtcgga gtcaacggat ttggaaggct atccttggca a 41

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-3F

<400> 9

cggcacaggc ttccagatg 19

<210> 10

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-4Ft

<400> 10

gaaggtgacc aagttcatgc tcaaagaaag gattcttatc ccaagt 46

<210> 11

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-4Fa

<400> 11

gaaggtcgga gtcaacggat tcaaagaaag gattcttatc ccaaga 46

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-4R

<400> 12

agccctgtca actgatgaag g 21

<210> 13

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-5Ft

<400> 13

gaaggtgacc aagttcatgc tgtttctgga ggtcagccaa gt 42

<210> 14

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-5Fc

<400> 14

gaaggtcgga gtcaacggat tttctggagg tcagccaagc 40

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer MPik-5R

<400> 15

tgaaattcac atatggattt cacc 24

<210> 16

<211> 12089

<212> DNA

<213> Artificial sequence

<220>

<223> coding sequence of Pi1 gene

<400> 16

ctagctagta gtttctgttt gaatttcaat atctgctact cgatcttggg tttcctcttc 60

aattttagtc gctcggccgc aggtaggaca cctgccatca agaagaatcc cttttccctt 120

gtagtcatct cgaacagcag ggagattatc atgctgtgcc tgtgcaggct cagtatcgat 180

cccactgctg cccgcaatgt tttcactgct cccgtgtgac tcagttgata tctccttata 240

cccagcattg atgaggaggg tgatcggccg gttgggatgc tcctcggctt ctttcttcac 300

gacgtcaatg gtggcgctga tgccaggggc gtcgctcttg taccatggcg agcaccgaaa 360

gaccaccttt tggaggctct tcaggttggt gatgccgatg gcatcgttgc ttgctgggcc 420

ggcgtagaac ttgaactcga gaagcttgag gttgggcatg gcgtcttcat ggaaggctat 480

ccttggcacc cggctgtcga cacggaagct ctccagcatc tggaagcctg tgccgttgat 540

ggttatgggt tgtcttggta gggcctcgaa cctcagcacg agcgtctgca ggttgggcat 600

ctcggcgagg aacttttggt catcatcctc aagcttgtgg cagagcctga tgtctaggca 660

agaaagaacg cggaagtggt ctttgatcat cttcgggata cgcatatgac tgccgggaac 720

tttgaatatc ccaacaaggg gaggtccgaa acgatcgatg atcgcgatgg acagaacctc 780

cctgcatttt gcacgcgtag cccctggaat accgttgatc agagcttcgc agacgccttc 840

gggcaaccgc acgccatcgc cactgtcacc ggcaagcacg cgcaacgatt gggagatctg 900

gccagcttgc catggcagct ctctcaccct cgtgtttctc acgtccagag tctgtagatg 960

attcagctcc ccaatctcct ttgggagctc cctaacccca gtgctcctca cgcacataat 1020

cttgagattc tgcagcttgc caacctgcgg cgggagctca ctgattggag tgttcctcac 1080

gtccagagtg tgcagatgct gcagctcccg tatctgcagt gggagctcag tgatgtccgt 1140

gtttctcacg tccagaatcc gcagatgctt cagctctcca atctcttgcg gaagttcact 1200

gatccgagtg ctccccacat acaaaatctc caaatgcttc agcttcctca tctcctgagg 1260

gagctttcgg atccgcgttc ccttgagacc aaggtacctc actctgaggc ttaactgttc 1320

acatatgccc tgcagatgag aatcctgtat atccttatta tcttcaaggt ccaacactcg 1380

caagcgctta aacatataga aggggattcc cttgggtcgc gccacgtcac caaggacgac 1440

aaggctgcaa gtgtgtgaca catccatact catgcttgac aagcagtcca ctggataccc 1500

cccctgaaga gacagccggc gaatcaacct tggggaggat gcactagtac tagtatcaga 1560

tcccagaccc aagcatgtta aaaaattgta ctccttggac ttgcatctca ggaaggccag 1620

catcacgggg tggatctcat agtaattata actgttgttg tctccgtgct gcgtaatcca 1680

tcctctgtta ataagctcgc caaagtaacc ttctgcttct ttctcttccg acacaaatcc 1740

ttccgcaatc cacctcctga ccagacgacc cctttcgatt ctgttagacc aatggtatgc 1800

actacagtac aacaacaaag tcctcagata aagaggaaga tggttataac caaggcataa 1860

actctccgcc aatggttgta aggatgggat gtccaaaata ccatcctcta tgtgtctcaa 1920

atccctacat tttttcactt cagcaccacc taattcttct atctctccaa ccaatgctga 1980

cgacagccaa ataagtgcta aaggcatacc ataacacatg ttcacaatat catagcatga 2040

attatcctct ccagttccga tcctgttgcc tgccccagac tttgttgcta tcccccaaga 2100

caacgaccaa gcatcattat tatctagatc cccaacttcg tagacaaaaa catcattgtc 2160

atcagtgtgg cacttctcag ctattgaatt aagacgagta gtcatgatta ttctaccacc 2220

cagatcattc ttgggaatgg actttctgat gacttcccat tcttcccaat gccaaatgtc 2280

atcgattaca atgagatacc tataaaagtc gagaagagta ataagcctca atagcatatt 2340

agtattacat tagcatatat gcatagagct aacaatttca cagatgtgag tccactttga 2400

gatgaacagt cagctgatat atattagttt cgtcatctgg ttgtattttg cttttgttga 2460

aatttaactt gcagttccta ggagtcgtgt tctgaatctt gagtatttcc ggtctgacgg 2520

cattgttttc atgacctgat cctagagctt ttatggtcag tagagaagaa atggggcagc 2580

taacatttgt ttgcgatctc cataacaagt tctttttttt cgccatgcag gctgaagtaa 2640

ccctggccag aatttattaa gcacaaggag tttacaagtt acatagatcg gcaacgatgt 2700

tgctgaggaa agcactgtgt gtctcagagg gaccggtcaa gaaatcaaat tgatgtttct 2760

tttcattatt tacagaacct gtattgaggg ttggaaatgt atatagcatt atatttccta 2820

cacttaagtg cactgtacgg tgtgaaataa ttgtgtatat gatttaattc gtttctctta 2880

ttggtgaatt tttggagatt gcttgcattt tccagtattt actgtattga atgatgttga 2940

aatagttgaa tcaatataaa tttagtaaaa aattaaatgt gctaataagt tactgcaaca 3000

agtctaaccc gttgcatcag gttatagcta tagaacatgc ttgtcgcact gaattattgc 3060

cacaaaaatg aatgtgtgga aataggctat ttatgaatag gtctgtgttg ggaaatctta 3120

ttgcagcatg gtacaaactg ttgcaataaa tacctatgac atatcaagtt gttgcaatag 3180

caatgcaaaa ccattgtaat agcacctttt tatgcaacca tcggaaaatc attgcaatag 3240

cctattacca cgggatttat tgctacggct cccaatggat tcaattatgt cacaataggt 3300

acatatcaca acgattttca gcttttgcaa tgaaaatttc cgttgcaaga aatatatctc 3360

cttgcagtca gccatggcca cctaggacat agccaatctc caagagattg agatccttta 3420

tgcccagtct ccaccatagt ccaatggcag gcatagcttg tcccaagcca gcaagcaatg 3480

gcttcccgaa tccctaacta gttccagaac ttgttctagt tgtttttttt tcttgtggtt 3540

gttctatgct ttgttgttca taaccttaaa gcatgggtgt ttttactgtc ctcgagagag 3600

gtactggaag gtgcctttac tctctaaaaa aacatttata gtcagaccag cacaccgttg 3660

gctggttcat gtatagactg gtcaaaacct aaaaggagcg aatgtttgtt ttgtagaagt 3720

ttcttatagt gtgtgtatat atggctcaat tggcctaatt aaattatata attttgtcca 3780

attatatgtt caggacttcg agagaacaaa tattgggatt aaatttaatg ccacatgtcc 3840

attgatacga aagttgtctc actactactt tggatgcatg gggcatgggt tacagatatg 3900

cccccaaaag tttttgaaaa aatatcaagt atatacgtca aagaacacac atgtatacag 3960

tgtgcactaa aataaaagcc cagagttcta gtctagtttt tctttacata aatggcaaat 4020

actgattttc ctttaaatgt tttcatattt gtgatactgg ttgttatgaa ggtaaaagaa 4080

catgacatat ttttaatgat cttgagggct atcatatttt tatagtggca aatgctttta 4140

tactgttctt gaagacataa cagtcaggaa gtcttcacag tgctaactca tgatattggc 4200

tctctctctc tctctcgttc ttgttgttgt aaggcaggaa ctctatttca ttaataaaaa 4260

gttgaagtac aatcttttat aagcagtcta caagagaatg gagagattgt tagtagctta 4320

cttaacacat agctgagcag cgacaagtgc tctccagcga caacagcatg gcggcaacag 4380

actcgttatc gctagcgggg cataggttgt ttgggcagca tgaaaccagt gcttggcgac 4440

aacaaagcag cagactagtt ccatcccctc ccttcgctca cggcgaaaca ggggagcttt 4500

tgtggtagtt gcaatggcag tgagagggca tggcagggga agagcgggct agctctgcat 4560

ttccctgttt tctgaagcaa ggaggaaaag cttgaaactt aatcttatcg tcttttcttt 4620

ttggtcgttt gtttttcgtg tttctcagct ttgagatttg gagtgtatgc acttgtgtaa 4680

cctttggctt tctatataga ggccagattt ctattcatta tcttaaaaaa aaaagatgac 4740

cactccctgt ggatttacaa atactgcaag cctttgcaag atcagtacca tcacgagtaa 4800

tagcaagagc ttcagtgata agggagttgt ttccacatca taaaaccatt ggcctgtcac 4860

aaaggagggg cctgccccat ttctgtatct atttactagg tagaataaag taatgctaat 4920

taaaaaaagt ttggcatatc ttgatatata cctcatcgag gacagaaaaa aaaaacccaa 4980

cattatatgg acatatagcc tgttcacttt aatttgggaa gccggattaa ggacaagcta 5040

gataagagat ataagatatt gcttactttt tgttgaggag gaaagctgat atgttgtcga 5100

tgagatgttg ttgaagagct ctcccggtcc cactcccacc atatggtgtg ctaagtgtat 5160

cagttactcc tagttgtgct tgagcgaaaa tgtctgcaag agtctctgtt agattgggac 5220

tggggctgga acttggagag attgatgcga aaacccggca ttggaactgc gttcccaatg 5280

catgatatag tactcgggca atcgttgttt tgcctccccc tggcaatcca aggatgcaaa 5340

ttgttttgac ctcgtgaaat tcacatatgg atttcaccgg cgcaagcatc gccgttatct 5400

ccttcacatc ttcctttact tggctgacct ccagaaacat cgcagggccc accttcttcc 5460

ggagcgcaga gaccagattg atggagtcaa tgccatcacc gaccaccaca acatcgtctc 5520

ttaggtcacc ggcgattgca accgagtgca ctccaaccgt acttgcaacc aatgacattg 5580

cttttgtacg gctcttatcg tccaccatgg gaatcttgaa cacgattttt tgctgcaaca 5640

aaaaatgaag tacttccaag ttgaagaatc aagagaaaaa gtaaacaaat ttattggcag 5700

cgccatccaa ggaagcgaag ctatttgtga ggaatcgaga tccaccggta ccatttcccc 5760

tcccattatc ctgaggggga catgaggttt tcgcttgcgc catcgtgaag catcactgct 5820

cccttgttcc agcaactcga tgagctcttc cttacgtttg tgcatcccca ccagccctgc 5880

atctggtggc ggcaagccag ctcgacggtg taccgacgac ggcggaaggc tcgccgccgg 5940

tagcttccag tcagagaagc gattggagac acgctcctca actcttgcct tgagctcacc 6000

gaaagggctt gcgcttcttc gctgctcgaa attgagctcc aagaagtcgt cgacggcatc 6060

atccaggtcg taggacagct cccggacctc cgcaatcaag ccatccttgc acgccgcatc 6120

gtcatccccc atcctcccca agatatttgg ggtgaggaga gagtgcacgg cctccagctc 6180

ggatctgatg aactctgcgt cgctccggct cccctccaga agattgcact ccccgtcgtc 6240

cagcaaagcg gccagcttca ctagcacggg cgccaaggcc cccgtggctg cggttacggc 6300

catggcagcc gcctccatcc ctactcgcgc ctcgatcggc ggctcggccg atgccctttt 6360

gcgttgagtg ctactctctc acctcctgac tctccgactc cgagtgtaat tagagcaagg 6420

ttaacagtat agccaactag tagcttcaat ttatttatag ccaatctaat agcttattca 6480

tacaagtact atactattaa tacctggtcc cacctgtcat acacacactg cgtcttggag 6540

tctgtgctac agttggctac aaatctatag cccgctaccc ttctctctcc ttatttatct 6600

ccttaaaata tgtttgcagt tggcttatag cctgctattg taactgctct taaagtggtg 6660

gtatttttct cggtcctctc gcaatgggtt agagaactgc tttcacataa ttagttcctt 6720

tgttctctag tataagggat tttaacattt tactttgtac tcccttcatt ctaaattgat 6780

ctatatatag tttttttagg ttattcctaa atgatctata tatttgtgtt catttattaa 6840

gtctattcgt tatttgtgtg cattggagta aatggacatt gatgcatgct tccatgcaca 6900

caagtattta taacccacat gcaatatctt gatttgctat tggctaggaa atattgggga 6960

tgatgcatgc attgagtttg ttgttagagt aaatataata tgaaagagtt attagttttt 7020

cttggtcttg gtgtacctat aaaatacgta gatcaattta gaatggaggg agtacgatcc 7080

ttcgttttat taaaaaaaag tttgaaaatt attatttaat tttcttttac ttattttatt 7140

atctaaaatc ttttaagcac aactttttat ttttattatt tgcataattt ttttaaaata 7200

agataaatgg ttaaatagga ctccctctat tccataatat aaggcacaac cacttttctt 7260

agatgtttca taatataagg catgcatgca tataggcaat taactatgac ctctttttta 7320

ttaaattatt attttttaac atcctctaca ctcatgttct ctgattctat tggatgcatg 7380

cattgtattt attaggatgt tctaaactac aagataataa taattatttt cttggtgttt 7440

gggttagagg tggttatgac ttatatttta taatggaggg agtagtacaa gtataatgtt 7500

aaagaattta atattatatt aaaggacgga ggaaatatta gggagaaacg gcatgaccac 7560

gagtcagtca acgagtctgc gaatgcagtg cagtattatt ggcctccgcc caaaagcctc 7620

acccccaagg ccccaatctt cccaccgcgc cgctgtcccc ccctccgacg gcggccacca 7680

tatcccccaa tcttcctccc gttggtcgct ggctggcctg gccagctctg gtcggaggag 7740

ggttcagttc catgggtgcg tcccctgccc atccctccac ttctactttc gtcttgagat 7800

ctcgccgttt ctgatctcct tgccatggca accagttgat ttggctgcca agatcgtccg 7860

tgctttgcaa aggtgaacaa tctttttctt cttgggcttt cactttgttg tgttaataga 7920

ctcagcgccg tcgcaacttg caaataaaaa ggttcagatc ggaaaatgga aataccgtac 7980

acagtagtct atagagaatc gactagtatt tcctgactgt aagtaaggaa gttgtaggta 8040

ccatgttctg atgattatct ttgaagcttg gagggggaca aaaagttggg agagaaacaa 8100

gaatgtattt gttcacttga ttgggggcag taggtaaaat ctcagcgtta ggaatttgca 8160

cctctttatt aagtttgact aaatttatag aaaaaaatta gcaacactta aaacaccaaa 8220

ttagtttcat tgaatccaac attgaatata ttttgataat atgtttactt tgtgttcaaa 8280

atgttactat atttttctat aaatttgatc aaatttcaat aagtttgact agaaaaaaaa 8340

gtcaaaccga cttataatat gaatataaat ggagggagta gattataaat tatctttatt 8400

ataatacttt gagtgtacaa acgaatgaat aaagtttaca aagttgaaaa gttattttta 8460

aatactattc cgataaacct gtatatagag aacttgtgaa aaaaaacacc gtttgaaatg 8520

tgtctgtgat aatctataag aggaacgggg cctaaatata ttcctttttt cctacgaaaa 8580

tgcaagaagt attgcctaat atattgatag agctgaagtt ttaaagtaaa atgaaacatt 8640

tgcatgaggc agcactggac aaaaagcgct tgtgaaaaaa atatgttcca aatattatta 8700

attcattcat ggtatggtct ttattttcat tttcccccct tatccatggg caggacacgg 8760

aaattctcag caggttcgcg cttgatctga actgtctgtg gctcatactc tcttgtgctt 8820

gcaacagctg aaagttgcag tgagaagtac agagaacaag atggagttgg tggtaggtgc 8880

ttccgaagcc accatgaaat ctctcttggg caagctgggc aatcttctag cccaggagta 8940

tgctctcatc agcggtatcc gtggtgacat ccagtacatc aatgacgagc ttgccagcat 9000

gcaggccttc ctccgtgatc tcagcaacgt gccagagggt cacagtcatg gccaccggat 9060

gaaggactgg atgaagcaga tccgagacat cgcctatgat gttgaggact gtatcgatga 9120

ctttgcccac cgcctccctc aggattccat cagcgatgcc aaatggtcct tcctactcac 9180

aaaaatctat gaactatgga catggtggcc acgtcgtgtg attgcttcca acattgccca 9240

actcaaggta cgggcacaac agatcgcaga tcgacgtagt agatacggag tgaacaaccc 9300

agaacacctt gacagtagca gcagtgccag gacccgtgct gtcaattacg aaattgctga 9360

gtatcaggtc acaagccctc agatcattgg tataaaggag cctgtgggga tgaagacggt 9420

catggaggag cttgaggttt ggttaactaa tcctcaagct gaaaatgggc aagctgttct 9480

gtccatagtc ggttttggag gtgtgggaaa gactaccatt gccacagcat tgtacagaaa 9540

agtcagtgaa aaatttcagt gccgggcatc agtagctgtg tctcagaact atgaccaagg 9600

caaagtcctc aatagtattc tgagtcaagt cagcaatcag gagcagggca gcagcacaac 9660

aattagtgag aaaaagaacc tcacctcagg cgctaagagc atgttgaaga cagccctgtc 9720

actgctcaga ggtaattgta tatgtcagcc agaaaatgat ggaaaccctg ataatacacc 9780

aatcaggctg caggaaacaa cggacgatga tcaaaacccc agaaaactgg aacagctcct 9840

ggccgaaaag aggtaccttt ttttgtaaat aaaattgctt tgcttatctg taaattaact 9900

tactcatccc actctaaatc taatgtttat ttccttctat acacagcaca actccatctt 9960

ttgaatgggt tttatttttc tcacttgtgc tcattttttt ttatcatctc tgcagttata 10020

tcctcttgat tgatgacatt tggtctgccg aaacatggga gagtatcaga tcgattttgc 10080

ctaaaaataa taaaggcggt agaataatag tgactacaag atttcaagct gttggttcaa 10140

catgctcccc tcttgaaact gatcgtttgc atacagttga ttttctcacc gatgacgagt 10200

cccaaaactt attcaataca agtatttgtg aatcaaagat aagaaaagat agcaacaaag 10260

tagacgagca agtccctgag gaaatatgga aaatatgtgg gggattgcct ttggccatag 10320

tcagcatggc tggtcttgtc gcctgcaacc caaggaaagc ctgctgcgat tggagtaaac 10380

tttgcaaatc attatttcca gagcaagaaa ctcctcttac cctcgatggt gttacaagga 10440

tactggattg ttgttacaat gatttgcctg cggatctgaa gacttgctta ttgtacttga 10500

gtatatttcc gaagggttgg aaaattagta ggaaacgttt gtcccggcga tggatagctg 10560

aaggttttgc taatgagaag caagggttaa cccaggaaag agttgcagag gcatacttta 10620

atcaactcac aagaaggaac ttagtacgtc ccatggagca tggcagcaat gggaaggtaa 10680

aaacgtttca agttcatgac atggttcttg aatacatcat gtccaaatca atcgaagaga 10740

attttattac tgtggttggt ggacactggc agatgactgc accaagcaat aaagtccgtc 10800

gactgtcgat gcaaagcagt ggatccaatc gtggaagttc aacaaaaggc ctgaacttgg 10860

ctcaagtgag atcactgacg gtgtttggga acctgaacca tgtgccattc cattcattca 10920

actatgggat aatacaggtg ctggatcttg aggactggaa gggtttgaaa gagagacata 10980

tgacggagat atgtcaaatg cttttactca agtatttgag catccgacga acagaaattt 11040

ccaaaattcc ctccaagatt cagaaacttg agtacttgga aactcttgac ataagggaga 11100

catatgtcag ggacctgcct aagtcaatag tccagctaaa acggatcatt agcatacttg 11160

gagggaataa aaacacacgg aaggggctga ggttgcctca agaaaaaagt aagaagccaa 11220

ttaaaaaccc gtcgcctcaa ggaaaaacaa aggagcccgc aaagaaagga ttcttatccc 11280

aagtaaaagg taaaggcgca atgaaagcac tccgtgtact gtcagggatt gagattgttg 11340

aggaatcatc agaagtagct gcaggccttc atcagttgac agggctaagg aagcttgcca 11400

tatacaagct caatataaca aagggtggtg ataccttcaa acaattacag tcctccattg 11460

agtaccttgg cagctgtggt ctgcagactc tggccatcaa tgatgagaat tctgaattta 11520

tcaactcact gggcgacatg cccgcgcctc caagatatct tgtcgccctt gagctgtctg 11580

gcaagttgga gaagctaccc aagtggatca ccagcatcac tactctcaac aagctaacca 11640

tatctgtaac agttcttagg actgaaactt tggagatcct ccacatttta ccttcattgt 11700

tttccctcac cttcgccttt tcacttagtg cagcgaagca ggatcaggac ataataaagg 11760

acatccttga gaataataaa ttggacagtg atggggaaat cgtcattcca gctgaaggat 11820

tcaagagtct taagctgctt cgcttctttg cacctttagt gccgaagctc agctttttgg 11880

acaagaatgc aatgccagca ctcgaaatca ttgaaatgcg gtttaaagac ttcgaaggtc 11940

tatttggcat cgaaatcctt gaaaatctcc gtgaggtgca tctcaaagtt agtgatgggg 12000

cagaagcaat aaccaagttc cttgtaaatg atttgaaggt taatactgag aaaccaaaag 12060

tatttgttga tggcatcgtc actgcatga 12089

Claims (10)

1. A functional marker panel for identifying multiple rice blast resistance alleles of the rice Pik locus, characterized by: the functional marker set is a KASP functional marker set and comprises functional markers MPik1, MPik2, MPik3, MPik4 and MPik5, the rice blast resistance alleles comprise Pi1, Pikm _ TS, Piks, PiKa, Pik, PiKp and Pi7, and the corresponding relations of the SNP/Indel locus genotypes and the alleles detected by the markers MPik1, MPik2, MPik3, MPik4 and MPik5 are shown in the following table:

MPik1 MPik2 MPik3 MPik4 MPik5 Pi1 T AA T T T Pikm_TS T AA T A T PiKs T AA T A C PiKa C AA G A T Pik T AA T A T PiKp C GC G A C Pi7 C AA G A C

the specific positions of the SNP/Indel sites are shown in the following table:

mark number Type of molecular marker Physical location Differential bases MPik_1 SNP 2040 th of the sequence of the Pi1 gene [T/C] MPik_2 SNP Position 8824 of the Pi1 gene sequence [AA/GC] MPik_3 Indel 489 th position of the sequence of the Pi1 gene [GC/T] MPik_4 Indel 11284 th position of Pi1 gene sequence [T/AA] MPik_5 SNP 5419 th position of Pi1 gene sequence [T/C]

The nucleotide sequence of the Pi1 gene in the table is shown in SEQ ID NO. 16.

2. A set of KASP primer sets for detecting the functional marker set of claim 1, wherein: consists of the following (1) to (5):

(1) KASP primer set for detection of MPik 1: the primer 1 is a single-stranded DNA with a tag sequence A and 22 th to 44 th sites of a sequence 1 in a sequence table from a 5 'end to a 3' end, the primer 2 is a single-stranded DNA with a tag sequence B and 22 th to 44 th sites of a sequence 2 in the sequence table from a 5 'end to a 3' end, and the primer 3 is a single-stranded DNA with a nucleotide sequence shown as a sequence 3 in the sequence table;

(2) KASP primer set for detection of MPik 2: the primer 4 is a single-stranded DNA with a tag sequence A and 22 th to 44 th sites of a sequence 4 in a sequence table from a 5 'end to a 3' end, the primer 5 is a single-stranded DNA with a tag sequence B and 22 th to 41 th sites of a sequence 5 in the sequence table from a 5 'end to a 3' end, and the primer 6 is a single-stranded DNA with a nucleotide sequence shown as a sequence 6 in the sequence table;

(3) KASP primer set for detection of MPik 3: the primer 7 is a single-stranded DNA with a tag sequence A and 22 th to 42 th positions of a sequence 7 in a sequence table from a 5 'end to a 3' end, the primer 8 is a single-stranded DNA with a tag sequence B and 22 th to 41 th positions of a sequence 8 in the sequence table from the 5 'end to the 3' end, and the primer 9 is a single-stranded DNA with a nucleotide sequence shown as a sequence 9 in the sequence table;

(4) KASP primer set for detection of MPik 4: the primer 10 is a single-stranded DNA which is sequentially provided with a tag sequence A and 22 th to 46 th sites of a sequence 10 in a sequence table from a 5 'end to a 3' end, the primer 11 is a single-stranded DNA which is sequentially provided with a tag sequence B and 22 th to 46 th sites of a sequence 11 in the sequence table from the 5 'end to the 3' end, and the primer 12 is a single-stranded DNA with a nucleotide sequence shown as a sequence 12 in the sequence table;

(5) KASP primer set for detection of MPik 5: the primer 13 is single-stranded DNA of the tag sequence A and the 22 th to 42 th positions of the sequence 13 in the sequence table from the 5 'end to the 3' end, the primer 14 is single-stranded DNA of the tag sequence B and the 22 th to 40 th positions of the sequence 14 in the sequence table from the 5 'end to the 3' end, and the primer 15 is single-stranded DNA of which the nucleotide sequence is shown as the sequence 15 in the sequence table.

3. A KASP primer set according to claim 2, wherein: the tag sequence A is a FAM fluorescent tag sequence, and the tag sequence B is a HEX fluorescent tag sequence.

4. A KASP primer set of claim 3, wherein: the sequences of the primers 1-15 are sequentially shown as SEQ ID NO. 1-15 in the sequence table.

5. Use of the functional marker set of claim 1 for identifying or assisting in identifying the type of rice blast resistance alleles at the Pik locus of rice or in molecular marker assisted breeding of rice.

6. Use of the KASP primer set of any one of claims 2 to 4 for identifying or assisting in identifying the rice blast resistance allele type of the rice Pik locus or in rice molecular marker-assisted breeding.

7. A method for identifying multiple rice blast resistance alleles at the Pik locus of rice, comprising the steps of:

1) extracting the genome DNA of a rice sample to be detected;

2) PCR amplification of the extracted genomic DNA using the primer set according to claim 3, detection of SNP/Indel sites in the KASP functional marker set in the amplification product, each marker amplified individually, and determination of which of Pi1, Pikm _ TS, Piks, Pika, Pik, PiKp and Pi7 the Pik locus rice blast resistance allele of the sample to be tested is due to the correspondence between the genotype and the allele of the SNP/Indel site in the functional markers MPik1, MPik2, MPik3, MPik4 and MPik5 in the table of claim 1.

8. The method of claim 7, wherein: the primer combination of claim 4 is used for PCR amplification, a fluorescence detector is used for detecting the amplification product, and the genotype of the differential loci in the functional markers MPik1, MPik2, MPik3, MPik4 and MPik5 is judged according to the fluorescence signal, and the corresponding relationship is shown as the following table:

9. the method of claim 8, wherein:

the PCR reaction system is a 10ul system: contains 1.0ul 10 XPARMS Buffer, 1.0ul dNTP, 1.0ul of each of 3 primers of each group of KASP primers, the concentration of each primer is 4 mol.L^-10.2ul PARMS PCR enzyme, 30-50 ng/ul template DNA1.0ul, 3.8ul ddH₂O。

10. The method of claim 9, wherein: the PCR reaction program is: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 45s at 72 ℃ and 35 cycles; finally, extension is carried out for 10min at 72 ℃.

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