CN117604077A - Method for identifying functional marker group of Piz seat series alleles - Google Patents
Method for identifying functional marker group of Piz seat series alleles Download PDFInfo
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 3
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Abstract
The invention belongs to the technical field of functional marker detection of rice blast resistance genes, and discloses a method for identifying a functional marker group of Piz locus series alleles, which selects an allele exon cds2 of the Piz locus as a target sequence for molecular marker development; multiple sequence comparison is carried out among different disease resistance alleles and disease susceptibility genes according to target sequences, and SNP loci for distinguishing different resistance alleles of Pik loci are screened; designing KASP (kaSP) marker primers according to SNP locus sequences, developing functional marker sets for distinguishing Pik locus resistance genes and different resistance alleles, and distinguishing different resistance alleles by using the functional marker sets. The functional marker group developed by the invention is based on the KASP marker technology, can rapidly introduce the resistance allele into excellent breeding materials by directly utilizing a molecular marker assisted selection method, can accurately distinguish different resistance alleles, and has important breeding application value.
Description
Technical Field
The invention belongs to the technical fields of plant pathology, genomics, molecular biology, molecular genetics and rice disease resistance breeding, in particular relates to the technical field of functional marker detection of rice blast resistance genes, and particularly relates to a method for identifying functional marker groups of Piz locus series alleles.
Background
At present, rice is one of the most important food crops in the world. The rice blast caused by the rice blast fungus (Magnap rthe grisea) is the most killing rice disease in the world so far, the average annual reduction yield of the rice caused by the rice blast is about 10-30%, and the serious is 40-50%, and even more, the particles are not harvested. Up to now, the disease cannot be fully and properly controlled in production, and the global climate begins to warm up along with the increase of greenhouse effect, the outbreak of rice blast disease is developed to the trend of being unable to be recovered, and the data result shows that the outbreak area of the rice blast disease in 2010 reaches 6000km 2 2014 is the most serious year of Chinese rice blast, up to 58000km 2 The method comprises the steps of carrying out a first treatment on the surface of the The onset area in 2019 reaches 3333km 2 Therefore, the method effectively prevents the occurrence of rice blast, and minimizes the loss caused by the rice blast is one of the most important tasks of rice production, so that the screening of good varieties resistant to the rice blast is very important. At present, the screening of rice blast resistant varieties is mainly carried out by artificial inoculation identification or natural induction identification, the method is original, the mechanism is not clear enough, the rice blast resistance identification is carried out by the artificial inoculation method, the identification period is longer, the pathogenic bacteria physiological race is lagged, and the problems of uneven pathogen distribution, large annual resistance difference and the like exist by utilizing the natural induction identification method. Therefore, the molecular marker assisted selection method is of great application value in improving the rice blast resistance of the variety.
A plurality of rice blast resistant genes, such as Pizt, pi9, pigm, pi2, pik, pi21, pi54, etc., have been cloned. 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 breeding of rice blast resistance varieties from a traditional breeding method based on artificial phenotype identification to a molecular breeding method based on molecular marker assisted selection.
The resistance gene of Piz locus is the resistance locus with better application effect in breeding at present. The locus contained 7 resistance alleles, i.e., pizz, pi9, pigm, pi2. These resistance genes have different resistance effects, and some resistance alleles have lost the value of breeding applications. Therefore, the accurate identification of different resistant alleles has important breeding application value.
The identification method of the molecular markers of Piz locus resistance genes has been reported in patents and articles, but most of the molecular markers only consider the sequence difference between Pik genes of disease resistant varieties and disease sensitive varieties, but cannot distinguish the difference between different resistance alleles. In particular, the InDel marker methods currently identifying Pigm, pi9 and Pi 2/pizz cannot distinguish between resistance alleles Pi2 and pizz; the InDel labeling method needs to detect polymorphism by using a gel electrophoresis method, is suitable for molecular marker analysis of a small amount of samples, and is not suitable for automatic high-throughput detection of a large amount of samples. Meanwhile, the existing InDel marking method cannot reflect any function information.
Through the above analysis, the problems and defects existing in the prior art are as follows:
1) The screening method of rice blast resistant varieties is original at present, and the mechanism is not clear enough; the rice blast resistance identification is carried out by using an artificial inoculation method, the identification period is longer, and the physiological race of pathogenic bacteria is lagged; the natural induction identification method has the problems of uneven pathogen distribution, large annual resistance difference and the like.
(2) The current molecular marker identification method for Piz locus resistance genes only considers sequence differences between Pik genes of disease-resistant varieties and disease-sensitive varieties, and cannot distinguish differences between different resistance alleles.
(3) The current InDel marker methods to identify Pigm, pi9 and Pi 2/pizz do not distinguish between the resistance alleles Pi2 and pizz; the polymorphism detection method by gel electrophoresis is suitable for molecular marker analysis of a small amount of samples, is not suitable for automatic high-throughput detection of a large amount of samples, and can not reflect any functional information.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a method for identifying a functional marker group of Piz locus series alleles, in particular to a functional marker group for identifying a plurality of rice blast resistance alleles of a rice Piz locus and application thereof.
The invention is achieved by a method of identifying a set of functional markers for a Piz locus series allele, comprising: the allelic exon cds2 of the Piz locus is selected as a target sequence for molecular marker development; multiple sequence comparison is carried out among different disease resistance alleles and disease susceptibility genes according to target sequences, and SNP loci for distinguishing different resistance alleles of Pik loci are screened; designing KASP marker primers according to SNP locus sequences, developing functional marker sets for distinguishing Pik locus resistance genes and different resistance alleles, and distinguishing different resistance alleles by using the functional marker sets.
Further, the method of identifying a functional marker panel for the Piz locus series allele comprises the steps of:
step one, selecting a molecular marker development target sequence;
step two, target sequence variation analysis;
thirdly, developing KASP function markers of the characteristic SNP;
and step four, genotyping based on the functional marker group.
Further, the selection of the molecular marker development target sequence in the first step includes:
the different disease resistant allele sequences are Pi2, pi9, pigm and pizz, with the corresponding Nipponbare alleles as controls for the disease resistant alleles. Alleles of the Piz locus include 3 exons, cds1, cds2 and cds3, preferentially selecting the coding region as the target sequence for molecular marker development; sequence comparison is carried out by using sequences of 3 exons cds1, cds2 and cds3 and known rice genome respectively to obtain homologous genes of Piz loci of different varieties, and cds2 is selected as a target sequence for molecular marker development.
Further, the target sequence variation analysis in step two includes:
the coding region variation of different alleles was determined by multiple sequence alignment using the cds2 sequences of Pigm, pizt, pi, pi9 and the susceptibility alleles, japan.
Further, the KASP function marker development of the characteristic SNP in step three includes:
screening SNP loci for identifying different resistance alleles according to target sequence comparison results; and (3) taking 200bp flanking sequences of the SNP locus to carry out KASP (kaSP) marker primer design, and finally obtaining the functional marker set for identifying different alleles of the Pik locus.
Further, the primer sequences of the functional marker sets for identification of different alleles at Pik locus are as follows:
the nucleotide sequence of the primer Piz-521Rt is SEQ ID NO:1, the nucleotide sequence of the primer Piz-521Rc is SEQ ID NO:2, the nucleotide sequence of the primer Piz-521Rg is SEQ ID NO:3, the nucleotide sequence of the primer Piz-521F is SEQ ID NO:4, the nucleotide sequence of the primer Piz-273Ft is SEQ ID NO:5, the nucleotide sequence of the primer Piz-273Fg is SEQ ID NO:6, the nucleotide sequence of the primer Piz-273R is SEQ ID NO:7, the nucleotide sequence of the primer Pigm-375Ra is SEQ ID NO:8, the nucleotide sequence of the primer Pigm-375Rc is SEQ ID NO:9, the nucleotide sequence of the primer Pigm-375F is SEQ ID NO:10, the nucleotide sequence of the primer Pi9-440Fc is SEQ ID NO:11, the nucleotide sequence of the primer Pi9-440Ft is SEQ ID NO:12, and the nucleotide sequence of the primer Pi9-440R is SEQ ID NO:13.
Further, the genotyping based on the functional marker group in the fourth step includes:
(1) Taking rice leaves, and extracting DNA by using CTAB or other DNA extraction methods;
(2) PCR amplification and PCR product detection: after synthesizing the primer in the functional mark group, carrying out PCR amplification; wherein the amplification conditions were 10. Mu.L of a system containing 1.0. Mu.L of 10 XPRMS Buffer, 1.0. Mu.L of dNTPs, 3 kinds of primers1.0μL 4mol·L -1 0.2 mu L PARMS PCR enzyme, 1.0 mu L template DNA,3.8 mu L ddH 2 O; the PCR reaction procedure was carried out at 94℃for 5min,35 cycles of 94℃for 30s,58℃for 30s,72℃for 45s, and 72℃for 10min; FAM and HEX are adopted as report fluorescence, ROX is adopted as reference fluorescence, and amplification products are rapidly detected in an enzyme-labeled instrument with 3 fluorescence detection channels and a quantitative PCR instrument;
(3) Genotyping: the alleles are determined from the genotyping results of the functional marker sets of the markers.
It is another object of the present invention to provide a set of functional markers identifying the allele of the Piz locus series comprising the Pizt, pi2, pi9, pigm, S and the susceptibility allele obtained by performing the method of identifying a set of functional markers of the Piz locus series; wherein, alm-273 of pizz is TG, alm-521 is AGGCGGC, alm-440 is T, alm-375 is GGAATG; pi2 is CT alm-273, alm-521 is AGGCGGC, alm-440 is T, alm-375 is GGAATG; alm-273 of Pi9 is CT, alm-521 is CTCTTTGC, alm-440 is C, alm-375 is GGAATG; alm-273 of Pigm is CT, alm-521 is AGGCAGC, alm-440 is T, alm-375 is ATAATT; alm-273 of S is CT, alm-521 is AGGCAGC, alm-440 is T, alm-375 is GGAATG.
It is another object of the present invention to provide a molecular marker suitable for automated detection of a large number of samples, comprising a set of functional markers identifying alleles of the Piz locus series.
It is another object of the present invention to provide a gene coding region-based molecular marker comprising a set of functional markers identifying alleles of the Piz locus series.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
first, aiming at the technical problems in the prior art and the difficulty of solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:
the resistance allele at the Piz locus is a resistance gene commonly used in rice breeding. There are 4 resistance alleles known to date, pizt, pigm, pi and Pi2. Existing molecular marker techniques do not allow for accurate discrimination between the several resistance alleles. The method based on the functional marker group can realize accurate identification of different resistance alleles. On the basis, the functional marker group provided by the invention can be used for carrying out systematic evaluation on different resistance alleles, and the resistance alleles with stronger effect and capable of playing an important role in breeding are screened out. The functional marker group developed by the invention is based on a KASP (KASP-mediated isothermal amplification) marker technology, which is a molecular marker technology suitable for high-throughput automatic detection, and a molecular marker assisted selection method can be directly utilized to rapidly introduce a resistance allele into an excellent breeding material.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
the invention performs multi-sequence comparison between different disease-resistant alleles and disease-sensitive genes, screens SNP loci which can be used for distinguishing different alleles of Pik loci, and develops functional marker sets which can be used for distinguishing the resistance genes of Pik loci and different resistance alleles on the basis. The invention can accurately distinguish different resistance alleles by utilizing the functional marker group, and has important breeding application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for identifying functional marker sets for the Piz locus series alleles according to an embodiment of the invention;
FIG. 2 is a schematic diagram of a cluster analysis result based on Piz gene variation according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of the result of a multiple sequence alignment of different alleles cds2 provided by an embodiment of the invention;
FIG. 4A is a KASP signature of Piz locus- -Piz-373 of 13 rice breeding materials provided by an embodiment of the invention;
FIG. 4B is a KASP signature of Piz locus Pi9-440 of 13 rice breeding materials provided by an embodiment of the invention;
FIG. 4C is a KASP signature of Piz locus- -Piz-521RtRc of 13 rice breeding materials provided by the embodiment of the invention;
FIG. 4D is a KASP signature of Piz locus- -Piz-521RtRg of 13 rice breeding materials provided by the embodiment of the invention;
FIG. 4E is a KASP signature of Piz locus Pigm-373 for 13 rice breeding materials provided by an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In view of the problems with the prior art, the present invention provides a method for identifying functional marker sets for the Piz seat series alleles, which is described in detail below with reference to the accompanying figures.
As shown in FIG. 1, the method for identifying the functional marker group of the Piz locus series alleles provided by the embodiment of the invention comprises the following steps:
s101, selecting an allele exon cds2 of a Piz locus as a target sequence for molecular marker development;
s102, carrying out multi-sequence comparison between different disease resistance alleles and disease susceptibility genes according to a target sequence, and screening SNP loci for distinguishing different resistance alleles of Pik loci;
s103, designing KASP (KASP sequence-related fragment) marking primers according to SNP locus sequences, and developing functional marking groups for Pik locus resistance gene differentiation and different resistance allele differentiation.
The embodiments of the present invention provide for distinguishing all alleles of the Piz locus including Pigm, pi9, pi2, pizt and the susceptibility alleles. The embodiment of the invention also develops a molecular marker suitable for the automatic detection of a large number of samples, and simultaneously develops a molecular marker based on a gene coding region.
The method for identifying the functional marker group of the Piz locus series alleles provided by the embodiment of the invention specifically comprises the following steps:
1) Selection of molecular marker development target sequences
Different disease resistant allele sequences are Pi2 (DQ 352453), pi9 (DQ 285630), pigm (KU 904633), pizz (DQ 352040). The corresponding Japanese allele served as a control for the susceptibility allele.
The allele at the Piz locus has 3 exons. In order to make the mutation site reflect more functional information, we prefer to select the coding region as the target sequence for molecular marker development. In order to make the developed markers suitable for detection of more varieties, the invention respectively uses sequences of 3 exons (cds 1, cds2 and cds 3) and 38 known rice genomes to carry out sequence comparison, so as to obtain homologous genes of Piz loci of different varieties. Analysis shows that: 38 varieties contain cds1 and cds2 sequences, part of the varieties have the cds3 sequence deleted, and the variation in cds2 is more (see figure 2), so the invention selects cds2 as a target sequence for molecular marker development.
2) Target sequence variation analysis
Multiple sequence alignment was performed using the cds2 sequences of Pigm, pizt, pi, pi9 and the susceptibility alleles, and coding region variations of different alleles were found (see fig. 3). 1 different colors than grey represent primers for different PCR amplified fragments; com represents a KASP-labeled common primer; rev, representing the reverse complement on the basis of the given sequence; grey represents the detected target mutation site;
FIG. 3 sequence alignment uses sequences
>NIP_cds2
TAGTAGAGCAATCAAAGAGCTGGGGCAGTTAAGCAAGCT
GAGGAAATTAGGTGTGACAAC
AAACGGGTCGACAAAGGAAAAATGTAAGATACTTTATGC
AGCCATTGAGAAGCTCTCTTC
CCTCCAATCTCTCCATGTGGATGCTGTGTTATTCTCAGGTA
TTATTGGAACACTTGAGTG
CCTAGATTCTATTTCATCTCCTCCTCCCCTACTAAGGACAC
TCAGGTTGAATGGAAGTCT
TGAAGAGATGCCTAACTGGATTGAGCAGCTCACTCACCTG
AAGAAGTTCGACTTACGGAG
GAGTAAACTAAAGGAAGGTAAAACCATGCTGATACTTGG
GGCATTGCCCAACCTCATGGT
CCTTTATCTTTATCGGAATGCTTACCTTGGGGAGAAGCTAG
TATTCAAAACGGGAGCATT
CCCAAATCTTAGAACACTTTGTATTTACGAATTGGATCAG
CTAAGAGAGATCAGATTTGA
GGACGGCAGCTCACCCCTGTTGGAAAAGATAGAAATAGG
CAAGTGCAGGTTGGAATCTGG
GATTATTGGTATCATTCACCTTCCAAAGCTCAAGGAGATT
CCAATTACATACGGAAGTAA
AGTGGCTGGGCTTGGTCAGCTGGAGGGAGAAGTGAACAC
ACACCCAAATCGCCCCGTGCT
GCTAATGTACAGTGACCGAAGGTATCACGACCTGGGGGCTG
>DQ352453
TAGTAGAGCAATCAAAGAGCTGGGGCAGTTAAGCAAGCT
GAGGAAATTAGGTGTGACAAC
AAACGGGTCGACAAAGGAAAAATGTAAGATACTTTATGCAGCCATTGAGAAGCTCTCTTC
CCTCCAATCTCTCCATGTGGATGCTGCaggaatctcagatgg---TGG
AACACTTGAGTG
CCTAGATTCTATTTCATCTCCTCCTCCCCTACTGAGGACAC
TCGTGTTGGATGGAATTCT
TGAGGAGATGCCTAACTGGATTGAGCAGCTCACTCACCTG
AAGAAGATCTACTTATTGAG
GAGCAAACTAAAGGAAGGTAAAACCATGCTGATACTTGGGG
CACTGCCCAACCTCATGGT
CCTTCATCTTTATCGGAATGCTTACCTTGGGGAGAAGCTA
GTATTCAAAACAGGAGCATT
CCCAAATCTTAGAACACTTTGGATTTATGAATTGGATCAG
CTAAGAGAGATCAGATTTGA
GGACGGCAGCTCACCCCTGTTGGAAAAGATAGAAATAGG
CGAGTGCAGGTTGGAATCTGG
GATTACTGGTATCATTCACCTTCCAAAGCTCAAGGAGATT
CCAATTAGATACGGAAGTAA
AGTGGCTGGGCTTGGTCAGCTGGAGGGAGAAGTGAACGC
ACACCCAAATCGCCCCGTGCT
GCTAATGTACAGTGACCGAAGGTATCACGACCTGGGGGCTG
>DQ285630
TAGTAGAGCAATCAAAGAGCTGGGGCACTTAAGCAAGTT
GAGGAAATTAGGTGTGATAAC
AAAAGGCTCGACAAAGGAAAAATGTAAGATACTTTATGCAGCCATTGAGAAGCTCTCTTC
CCTCCAATCTCTCTATGTGAATGCTGCGTTATTATCAGA---T
ATTGAAACACTTGAGTG
CCTAGATTCTATTTCATCTCCTCCTCCCCTACTGAGGACAC
TCGGGTTGAATGGAAGTCT
TGAAGAGATGCCTAACTGGATTGAGCAGCTCACTCACCTG
AAGAAGATCTACTTATTGAG
GAGCAAACTAAAGGAAGGTAAAACCATGCTGATACTTGGGGCATTGCCCAACCTCATGGT
CCTTTATCTTTATTGGAATGCTTACCTTGGGGAGAAGCTAGTATTCAAAACGGGAGCATT
CCCAAATCTTAGAACACTTcGTATTTACGAATTGGATCAGCTAAGAGAGATGAGATTTGA
GGATGGCAGCTCACCCCTGTTGGAAAAGATAGAAATctcttgcTGCAGGTTGGAATCAGG
GATTATTGGTATCATTCACCTTCCAAGGCTCAAGGAGATTTCACTTGAATACAAAAGTAA
AGTGGCTAGGCTTGGTCAGCTGGAGGGAGAAGTGAACACACACCCAAATCGCCCCGTGCT
GCgAATGGACAGTGACCGAAGGGATCACGACCTGGGGGCTG
>KU904633
TAGTAGAGCAATCAAAGAGCTGGGGCAGTTAAGCAAGTTGAGGAAATTAGGTGTGATAAC
AAAAGGCTCGACAAAGGAAAAATGTAAGATACTTTATGCAGCCATTGAGAAGCTCTCTTC
CCTCCAATATCTCTATGTGAATGCTGCGTTATTATCAGA---TATTGAAACACTTGAGTG
CCTAGATTCTATTTCATCTCCTCCTCCCCTACTAAGTACACTCAGGTTGAATGGAAGTCT
TGAAGAGATGCCTAACTGGATTGAGCAGCTCACTCACCTG
AAGAAGTTCTACTTACGGAG
GAGCAAACTAAAGGAAGGTAAAACCATGCTGATACTTGG
GGCACTGCCCAACCTCATGTT
CCTTTCTCTTTATCATAATTCTTATCTTGGGGAGAAGCTAG
TATTCAAAACGGGAGCATT
CCCAAATCTTAGAACACTTTGTATTTACGAATTGGATCAG
CTAAGAGAGATCAGATTTGA
GGACGGCAGCTCACCCCTGTTGGAAAAGATAGAAATAGG
CAAGTGCAGGTTGGAATCTGG
GATTATTGGTATCATTCACCTTCCAAAGCTCAAGGAGATT
CCAATTACATACGGAAGTAA
AGTGGCTGGGCTTGGTCAGCTGGAGGGAGAAGTGAACGCAC
ACCCAAATCGCCCCGTGCT
GCTAATGGACAGTGACCGAAGGTATCACGACCTGGGGGCTG
>DQ352040
TAGTAGAGCAATCAAAGAGCTGGGGCAGTTAAGCAAGCT
GAGGAAATTAGGTGTGACAAC
AAACGGGTCGACAAAGGAAAAATGTAAGATACTTTATGC
AGCCATTGAGAAGCTCTCTTC
CCTCCAATCTCTCCATGTGGATGCTGTGTTATTCTCAGGTA
TTATTGGAACACTTGAGTG
CCTAGATTCTATTTCATCTCCTCCTCCCCTACTGAGGACAC
TCGTGTTGGATGGAATTCT
TGAGGAGATGCCTAACTGGATTGAGCAGCTCAtgCACCTGA
AGAAGATCTACTTATTGAG
CAGCAAACTAAAGGAAGGTAAAACCATGCTGATACTTGG
GGCACTGCCCAACCTCATGGT
CCTTCATCTTTATCGGAATGCTTACCTTGGGGAGAAGCTA
GTATTCAAAACAGGAGCATT
CCCAAATCTTAGAACACTTTGGATTTATGAATTGGATCAG
CTAAGAGAGATCAGATTTGA
GGACGGCAGCTCACCCCTGTTGGAAAAGATAGAAATAGG
CGAGTGCAGGTTGGAATCTGG
GATTACTGGTATCATTCACCTTCCAAAGCTCAAGGAGATT
CCAATTAGATACGGAAGTAA
AGTGGCTGGGCTTGGTCAGCTGGAGGGAGAAGTGAACGC
ACACCCAAATCGCCCCGTGCT
GCTAATGTACAGTGACCGAAGGTATCACGACCTGGGGGCTG
3) Development of KASP functional markers featuring SNPs SNP sites that can be used to identify different resistance alleles were screened based on the above target sequence alignment (see table 1). And (3) taking 200bp flanking sequences of the SNP locus to carry out KASP (kaSP) marker primer design, and finally obtaining the functional marker sets for identifying different alleles.
TABLE 1 functional marker sets for identification of different alleles at Pik loci
The primer sequences of the functional marker group provided by the embodiment of the invention are as follows:
Piz-521Rt：
GAAGGTGACCAAGTTCATGCTCCCAGATTCCAACCTGCACTT
FAM
Piz-521Rc：
GAAGGTCGGAGTCAACGGATTCCCAGATTCCAACCTGCACTC
HEX
Piz-521Rg：
GAAGGTCGGAGTCAACGGATTTTCCAACCTGCACTGCAAGAG
HEX
Piz-521F：CCCTGTTGGAAAAGATAGAAATAGG
Piz-273Ft：GAAGGTGACCAAGTTCATGCTCTAACTGGATTGAGCAGCTCAC T FAM
Piz-273Fg：GAAGGTCGGAGTCAACGGATTCTAACTGGATTGAGCAGCTCAT G HEX
Piz-273R：GCATGGTTTTACCTTCCTTTAGTTT
Pigm-375Ra：
GAAGGTGACCAAGTTCATGCTCTTCTCCCCAAGGTAAGCATTA
FAM
Pigm-375Rc：
GAAGGTCGGAGTCAACGGATTCTTCTCCCCAAGGTAAGCATTC
HEX
Pigm-375F：CTAAAGGAAGGTAAAACCATGCTG
Pi9-440Fc：GAAGGTGACCAAGTTCATGCTGCATTCCCAAATCTTAGAACAC TTC FAM
Pi9-440Ft：GAAGGTCGGAGTCAACGGATTGCATTCCCAAATCTTAGAACAC TTT HEX
Pi9-440R：CCTCAAATCTCATCTCTCTTAGCTG
4) Genotyping based on functional marker sets
The specific implementation steps are as follows:
1. the rice leaves are extracted with DNA by CTAB or other DNA extraction methods.
And 2, PCR amplification and PCR product detection. Primers in the functional marker set were synthesized and then PCR amplified. The amplification conditions were: 10. Mu.L of the system containing 1.0. Mu.L of 10 XPRMS Buffer, 1.0. Mu.L of dNTPs, 1.0. Mu.L of 4 mol.L of 3 kinds of primers -1 0.2 mu L PARMS PCR enzyme, 1.0 mu L template DNA,3.8 mu L ddH 2 O. The PCR reaction was run at 94℃for 5min, then 35 cycles of 94℃for 30s,58℃for 30s,72℃for 45s, and finally 72℃for 10min. By FAM and HEX are used as report fluorescence, and ROX is used as reference fluorescence, and amplified products can be rapidly detected in an enzyme-labeled instrument with 3 fluorescence detection channels and a quantitative PCR instrument.
3. And (5) genotyping. Alleles were determined based on genotyping of the functional marker set of 5 markers.
The resistance allele at the Piz locus is a resistance gene commonly used in rice breeding. There are 4 resistance alleles known to date, pizt, pigm, pi and Pi2. Existing molecular marker techniques do not allow for accurate discrimination between the several resistance alleles. The method based on the functional marker group can realize accurate identification of different resistance alleles. On the basis, the functional marker group provided by the invention can be used for carrying out systematic evaluation on different resistance alleles, and the resistance alleles with stronger effect and capable of playing an important role in breeding are screened out. The functional marker group developed by the invention is based on a KASP (KASP-mediated isothermal amplification) marker technology, which is a molecular marker technology suitable for high-throughput automatic detection, and a molecular marker assisted selection method can be directly utilized to rapidly introduce a resistance allele into an excellent breeding material.
Example 1: functional marker set identification result of 13 parts of rice breeding material
13 rice breeding material Piz locus KASP markers are shown in Table 2 and FIGS. 4A to 4E.
TABLE 2 identification of Piz locus alleles of 13 Rice breeding Material
Example 2: identification results of 208 rice resource Piz functional marker sets 310 rice breeding material Piz functional marker sets are shown in Table 3.
Table 3 identification results of Piz functional marker group of 310 parts of Rice Breeding Material
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The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (10)
1. A method of identifying a set of functional markers for a Piz locus set allele, comprising: the allelic exon cds2 of the Piz locus is selected as a target sequence for molecular marker development; multiple sequence comparison is carried out among different disease resistance alleles and disease susceptibility genes according to target sequences, and SNP loci for distinguishing different resistance alleles of Pik loci are screened; designing KASP (kaSP) marker primers according to SNP locus sequences, developing functional marker sets for distinguishing Pik locus resistance genes and different resistance alleles, and distinguishing different resistance alleles by using the functional marker sets.
2. The method of identifying a functional marker set for a Piz seat series allele according to claim 1, comprising the steps of:
step one, selecting a molecular marker development target sequence;
step two, target sequence variation analysis;
thirdly, developing KASP function markers of the characteristic SNP;
and step four, genotyping based on the functional marker group.
3. The method of identifying functional marker panel alleles of the Piz locus series of claim 2 wherein the selection of molecular marker development target sequences in step one comprises:
the different disease resistant allele sequences are Pi2, pi9, pigm and pizz, and corresponding Japanese alleles are used as the control of the disease resistant alleles; alleles of the Piz locus include 3 exons, cds1, cds2 and cds3, preferentially selecting the coding region as the target sequence for molecular marker development; sequence comparison is carried out by using sequences of 3 exons cds1, cds2 and cds3 and known rice genome respectively to obtain homologous genes of Piz loci of different varieties, and cds2 is selected as a target sequence for molecular marker development.
4. The method of identifying a functional marker panel for a Piz locus series allele according to claim 2, wherein the analysis of the target sequence variation in step two comprises:
the coding region variation of different alleles was determined by multiple sequence alignment using the cds2 sequences of Pigm, pizt, pi, pi9 and the susceptibility alleles, japan.
5. The method for identifying a functional marker panel for the Piz locus series alleles according to claim 2, wherein the KASP functional marker development of the characteristic SNP in step three comprises:
screening SNP loci for identifying different resistance alleles according to target sequence comparison results; and (3) taking 200bp flanking sequences of the SNP locus to carry out KASP (kaSP) marker primer design, and finally obtaining the functional marker set for identifying different alleles of the Pik locus.
6. The method for identifying functional marker sets for Piz locus series alleles according to claim 5 wherein the primer sequences for the functional marker sets for Pik locus different allele identification are as follows:
the nucleotide sequence of the primer Piz-521Rt is SEQ ID NO:1, the nucleotide sequence of the primer Piz-521Rc is SEQ ID NO:2, the nucleotide sequence of the primer Piz-521Rg is SEQ ID NO:3, the nucleotide sequence of the primer Piz-521F is SEQ ID NO:4, the nucleotide sequence of the primer Piz-273Ft is SEQ ID NO:5, the nucleotide sequence of the primer Piz-273Fg is SEQ ID NO:6, the nucleotide sequence of the primer Piz-273R is SEQ ID NO:7, the nucleotide sequence of the primer Pigm-375Ra is SEQ ID NO:8, the nucleotide sequence of the primer Pigm-375Rc is SEQ ID NO:9, the nucleotide sequence of the primer Pigm-375F is SEQ ID NO:10, the nucleotide sequence of the primer Pi9-440Fc is SEQ ID NO:11, the nucleotide sequence of the primer Pi9-440Ft is SEQ ID NO:12, and the nucleotide sequence of the primer Pi9-440R is SEQ ID NO:13.
7. The method of identifying functional marker sets for Piz locus series alleles according to claim 2 wherein the genotyping based on functional marker sets in step four comprises:
(1) Taking rice leaves, and extracting DNA by using CTAB or other DNA extraction methods;
(2) PCR amplification and PCR product detection: after synthesizing the primer in the functional mark group, carrying out PCR amplification; wherein the amplification conditions were 10. Mu.L of a system containing 1.0. Mu.L of 10 XPRMS Buffer, 1.0. Mu.L of dNTPs, and 1.0. Mu.L of 4 mol.L of 3 kinds of primers -1 0.2 mu L PARMS PCR enzyme, 1.0 mu L template DNA,3.8 mu L ddH 2 O; the PCR reaction procedure was carried out at 94℃for 5min,35 cycles of 94℃for 30s,58℃for 30s,72℃for 45s, and 72℃for 10min; FAM and HEX are adopted as report fluorescence, ROX is adopted as reference fluorescence, and amplification products are rapidly detected in an enzyme-labeled instrument with 3 fluorescence detection channels and a quantitative PCR instrument;
(3) Genotyping: the alleles are determined from the genotyping results of the functional marker sets of the markers.
8. A set of functional markers for identifying an allele of the Piz locus series obtained by performing the method for identifying a set of functional markers for the allele of the Piz locus series of any one of claims 1-7, wherein the set of functional markers for identifying the allele of the Piz locus series comprises pizz, pi2, pi9, pigm, S and a susceptibility allele; wherein, alm-273 of pizz is TG, alm-521 is AGGCGGC, alm-440 is T, alm-375 is GGAATG; pi2 is CT alm-273, alm-521 is AGGCGGC, alm-440 is T, alm-375 is GGAATG; alm-273 of Pi9 is CT, alm-521 is CTCTTTGC, alm-440 is C, alm-375 is GGAATG; alm-273 of Pigm is CT, alm-521 is AGGCAGC, alm-440 is T, alm-375 is ATAATT; alm-273 of S is CT, alm-521 is AGGCAGC, alm-440 is T, alm-375 is GGAATG.
9. A molecular marker suitable for automated detection of a plurality of samples, comprising a set of functional markers for identifying alleles of the Piz locus series according to claim 8.
10. A molecular marker based on a coding region of a gene, wherein the molecular marker based on a coding region of a gene comprises a set of functional markers for identifying alleles of the Piz locus sequence according to claim 8.
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