CN111808984A - SNP (Single nucleotide polymorphism) marker related to hot pepper cytoplasmic male sterility restoring gene, specific primer and application of specific primer - Google Patents
SNP (Single nucleotide polymorphism) marker related to hot pepper cytoplasmic male sterility restoring gene, specific primer and application of specific primer Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a hot pepper cytoplasmic male sterility recovery trait related SNP marker, a specific primer and application thereof. The nucleotide sequence of the SNP marker related to the cytoplasmic male sterility restoring trait of the pepper is shown as SEQ ID No.1, the 25 th base of the sequence shown as the SEQ ID No.1 is C or G, or the nucleotide sequence of the SNP marker is shown as the SEQ ID No.2, and the 24 th base of the sequence shown as the SEQ ID No.2 is C or T. The invention does not need to carry out hot pepper test cross and pollen identification, can judge whether the hot pepper material is a maintainer line or a restorer line only by carrying out DNA detection on hot pepper plants in the seedling stage, and can be widely applied to molecular marker assisted breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to SNP markers and specific primers related to a hot pepper cytoplasmic male sterility restoring gene and application thereof.
Background
The hybrid seeds prepared by utilizing the hot pepper nuclear male sterile line can save the manual castration link, save the manpower and improve the income. But fertile plants which account for about half of the female parent must be removed in the flowering period, which causes waste of fields and the like and influences the yield per unit. The cytoplasmic male sterile line of the pepper is selected as the female parent, the fertility separation problem of the female parent does not need to be considered, the procedure can be simplified, the labor cost can be saved by more than 50 percent, and the purity of the hybrid seeds can be ensured.
The pepper takes fruits as products, good pollination and fertilization are the precondition for the development of pepper fruits under normal conditions, and if the fertilization is poor, the pepper fruits cannot bear or the fruits cannot expand to a normal level, so that the quality and the yield of the pepper are seriously influenced. Therefore, the breeding of the stable restorer line becomes the key of the cytoplasmic male sterility three-line breeding of the pepper. Phenotype selection, backcross and test cross evaluation are required in the transformation process of the restorer line, and the transformation of a stable restorer line with excellent target characters is time-consuming and labor-consuming.
There are multiple cytoplasmic male sterility restorer genes in pepper, and different restorer lines may contain different restorer genes. In the prior art, a large number of molecular markers related to the cytoplasmic male sterility restorer of the pepper are developed by utilizing different restorer line materials, but the molecular markers have the problems of inconsistent marker and phenotype, low applicability and the like, so that the application of the molecular markers related to the cytoplasmic male sterility restorer of the pepper in breeding is limited.
Disclosure of Invention
The purpose of the present invention is to provide SNP markers associated with the cytoplasmic male sterility recovery trait in capsicum.
It is still another object of the present invention to provide KASP-specific primers for the SNP markers.
It is still another object of the present invention to provide the use of the SNP marker.
Still another object of the present invention is to provide a method for screening a cytoplasmic male sterility restorer line of capsicum.
Still another object of the present invention is to provide a test kit for screening cytoplasmic male sterility restorer line of capsicum.
The SNP marker related to the cytoplasmic male sterility restoring trait of capsicum according to the embodiment of the invention has the nucleotide sequence shown as SEQ ID No.1, and the 25 th base of the sequence shown as SEQ ID No.1 is C or G, or,
the nucleotide sequence of the SNP marker S1609 is shown as SEQ ID No.2, and the 24 th base of the sequence shown as SEQ ID No.2 is C or T.
SEQ ID No:1
CAGTTCAATAAAGAAAAAGGCATT[C/G]TGATAAACACCTCTATTCTTTGACGA
SEQ ID No:2
TGGAAAAAAAAATGGGTATTTAG[C/T]TTTCATATCCTCTTTTGATTCTCATG
According to the KASP specific primer of the SNP marker related to the cytoplasmic male sterility recovery trait of the hot pepper, the sequence of the primer is as follows:
specific primers for S1597 are as follows:
the primer A1: 5'-CAGTTCAATAAAGAAAAAGGCATTC-3' is used as a primer,
the primer A2: 5'-CAGTTCAATAAAGAAAAAGGCATTG-3' is used as a primer,
5'-TCGTCAAAGAATAGAGGTGTTTATCA-3' as a back primer; or
Specific primers for S1609 are as follows:
the primer A1: 5'-TGGAAAAAAAAATGGGTATTTAGC-3' is used as a primer,
the primer A2: 5'-TGGAAAAAAAAATGGGTATTTAGT-3' is used as a primer,
rear primer 5'-CATGAGAATCAAAAGAGGATATGAAA-3'.
According to the KASP specific primer of the embodiment of the invention, the 5 'end of the pre-primer A1 is added with FAM fluorescent tag sequence, and the 5' end of the pre-primer A2 is added with HEX fluorescent tag sequence.
According to the KASP specific primer of the embodiment of the invention, the 5 'end of the pre-primer A1 is added with FAM fluorescent tag sequence, and the 5' end of the pre-primer A2 is added with HEX fluorescent tag sequence.
According to the KASP specific primer of the present embodiment, the FAM fluorescent tag sequence is GAAGGTGACCAAGTTCATGCT, HEX fluorescent tag sequence is GAAGGTCGGAGTCAACGGATT.
The invention also provides application of the SNP marker related to the cytoplasmic male sterility recovery character of the pepper, in particular to application in molecular marker assisted breeding of the pepper, screening of the cytoplasmic male sterility recovery line of the pepper and purity assisted identification of pepper hybrids.
The method for screening the cytoplasmic male sterility restorer line of capsicum according to the embodiment of the invention comprises the step of screening a capsicum restoring gene by applying the SNP marker.
According to the method for identifying and screening the hot pepper cytoplasmic male sterility restoring line, provided by the embodiment of the invention, when the base at the SNP marker locus related to the hot pepper cytoplasmic male sterility restoring trait is C: C genotype, the hot pepper material to be detected is a 'restoring line';
when the base at the SNP marker site related to the cytoplasmic male sterility recovery trait of the pepper is G: G or T: T genotype, the pepper material to be detected is a 'maintainer line'.
The invention also provides a detection kit for screening the hot pepper male cytoplasmic male sterile restorer line, and the kit contains the KASP specific primer.
The kit also comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B. The nucleotide sequence of the fluorescent probe A is GAAGGTGACCAAGTTCATGCT, and the 5' end is connected with a fluorescent group A; the nucleotide sequence of the quenching probe A is a reverse complementary sequence of the sequence, and the 3' terminal is connected with a quenching group.
The nucleotide sequence of the fluorescent probe B is GAAGGTCGGAGTCAACGGATT, and the 5' end is connected with a fluorescent group B; the nucleotide sequence of the quenching probe B is a reverse complementary sequence of the sequence, and the 3' terminal is connected with a quenching group.
In the invention, the fluorophore A is a FAM fluorescent label sequence; the fluorescent group B is specifically a HEX fluorescent label sequence; the quenching group is specifically Q.
The invention has the beneficial effects that:
the invention does not need to carry out hot pepper test cross and pollen identification, can judge whether the hot pepper material is a maintainer line or a restorer line only by detecting the DNA of the hot pepper plant in the seedling stage, and can be widely applied to molecular marker assisted breeding. The marker of the invention can be used for large-scale detection by using KASP genotyping technology, thus improving the efficiency and shortening the identification time.
The molecular marker provided by the invention realizes the screening of the pepper restorer gene in the seedling stage, greatly shortens the breeding time and saves manpower and material resources. By using the specific primer of the invention and adopting KASP gene typing technology, the pepper CM334 and Zunla _1 which are published full genome sequence restorers are used as a contrast, the pepper CM334 and Zunla _1 which are restorers have the same genotype and the heterozygous genotype are used as restorers, and the pepper CM334 and Zunla _1 which are restorers have different genotypes are used as maintainers. The molecular marker can be used for screening the pepper restorer line and identifying the purity of hybrid seeds, and the breeding efficiency is improved. The SNP markers S1597 and S1609 of the invention are positioned in the flanking region of the caPPR6 gene of the pepper, and belong to candidate gene closely linked molecular markers.
Drawings
FIG. 1 shows the amplification results of KASP marker S1597 in hybrid combinations, blue dots for individuals of the same genotype as the restorer parent (C: C genotype), green dots for maintainer 77013(G: G genotype), and red dots for heterozygous individuals (C: G genotype);
FIG. 2 shows the amplification results of KASP marker S1609 in hybrid combinations, blue dots for individuals of the same genotype as the restorer parent (C: C genotype), green dots for maintainer 77013(T: T genotype), and red dots for heterozygous individuals (C: T genotype);
FIG. 3 shows the results of the amplification of KASP marker S1597 in 332 pepper fertility segregating population materials. The blue point is a homozygous fertile single plant (C: C genotype), the green point is a homozygous sterile single plant (G: G genotype), and the red point is a heterozygous fertile single plant (C: G genotype);
FIG. 4 shows the results of the amplification of KASP marker S1609 in 332 pepper fertility segregating population materials. The blue point is homozygous fertile single plant (C: C genotype), the green point is homozygous sterile single plant (T: T genotype), and the red point is heterozygous fertile single plant (C: T genotype).
Detailed Description
The pepper material comprises the following components: 77013A, 77013, 0601M, 83-60, IVF2014032, H3, CM334, perennial and pepper hybrid "Xiyangyang" F6A population of isolated material.
Example 1 acquisition of SNP sites associated with cytoplasmic Male sterility Recover trait in Capsicum annuum and determination of corresponding KASP primers
1. Test materials
Test materials of pepper CM334, perennial, IVF2014032, H3, 0601M and its hybrid F1 generation with 77013A were used.
2. Phenotypic identification
And (3) adopting a test cross method to identify the recovering phenotype of each pepper material, and judging fertility by observing whether the anthers of the flowers at different parts of 10 single plants of the F1 generation have pollen shed or not by naked eyes in the growing season of the materials. For plants that are difficult to determine by the naked eye, the presence of pollen was further observed by the acetic acid magenta staining method. And after the growing season is over, judging the fertile and sterile phenotypes according to the existence of fruits on the plants and the existence of seeds in the fruits.
The results show that each individual plant of the 5 filial combined progeny has a large amount of pollen to spill out, each individual plant has normal fruit bearing and normal fruit development, and the 5 male parent materials are cytoplasmic male sterile fertility restoring materials.
3. Determination of SNP (Single nucleotide polymorphism) sites related to cytoplasmic male sterility recovery traits of hot peppers and corresponding KASP (Kasp primer)
(1) Genome re-sequencing and molecular marker design
The 77013 and 0601M genomes were re-sequenced, CM334 was used as reference genome, SNP sites of the application were obtained by screening, and corresponding KASP primers were designed.
Each pair of KASP molecular markers contains 3 primer sequences: a1, A2 and C.
Wherein, A1 and A2 are only the differences of the SNP sites at the ends, and different fluorescent tag sequences are respectively added at the 5' ends of A1 and A2:
5'-GAAGGTGACCAAGTTCATGCT-3' (FAM fluorescent tag sequence);
5'-GAAGGTCGGAGTCAACGGATT-3' (HEX fluorescent tag sequence).
C is a reverse complementary sequence.
The KASP labeled PCR amplification system comprises a DNA template, a primer mixture, a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, high-fidelity Taq enzyme, dNTP and the like. The primer mixture includes primers A1, A2, and C. The sequence of the fluorescent probe A is 5'-GAAGGTGACCAAGTTCATGCT-3', and the 5 ' end is connected with 1 fluorophore FAM; the sequence of the fluorescent probe B is 5'-GAAGGTCGGAGTCAACGGATT-3', and the 5 ' end is connected with 1 fluorophore HEX; the sequence of the quenching probe A is 5'-AGCATGAACTTGGTCACCTTC-3', and the 3 ' terminal is connected with a quenching group Q; the sequence of the quenching probe B is 5'-AATCCGTTGACTCCGACCTTC-3', and the 3 ' terminal is connected with a quenching group Q.
TABLE 1 KASP marker S1597 and S1609 nucleotide sequence information
The specific primers S1597 and S1609 developed by the present application can specifically identify the cytoplasmic male sterility restorer of pepper, while the primers S1598 and S1608 cannot achieve the technical purpose of the present application.
(2) Screening for polymorphic SNP markers
Polymorphism screening was performed using 6 different hybridization combinations. After the KASP PCR amplification reaction, reading needs to be carried out by using a Roche480 fluorescence quantitative instrument for KASP genotyping, and fluorescence data are read. The X-axis is FAM (465-510) channel, and the Y-axis is HEX (533-580) channel. Determining the specific genotype of the gene to be detected according to the analysis result as follows: the genotype of the sample showing blue color close to the X axis is the allele linked to the FAM fluorescent tag sequence, the genotype of the sample showing green color close to the Y axis is the allele linked to the HEX fluorescent tag sequence, the genotype of the sample showing red color in the middle is the heterozygote of the two alleles, and the genotype of the sample showing black color in the lower left corner is represented by H2O substituted blank for template.
The results of phenotypic and genotypic determinations for the KASP primer combinations S1597 and S1609 are shown in Table 2 and FIGS. 1 and 2.
FIG. 1 shows the amplification of KASP marker S1597 in a hybridization combination. The blue spots are molecular marker genotypes (C: C genotype) of the restorer lines (CM334, perennial, H3, 0601M), the green spots are molecular marker genotypes (G: G genotype) of 77013A, 77013, 83-60, IVF2014032 and 77013A × IVF2014032, and the red spots are F: G genotype1Generations (77013A × CM334, 77013A × perennial, 77013A × H3, 77013A × 0601M, 83-60 × H3) molecular marker genotypes (C: G genotypes).
FIG. 2 shows the amplification of KASP marker S1609 in the hybridization combination. The blue spots are molecular marker genotypes (C: C genotype) of restorer lines (CM334, perennial, H3, 0601M), the green spots are molecular marker genotypes (T: T genotype) of 77013A, 77013, 83-60, IVF2014032 and 77013A × IVF2014032, and the red spots are F: T genotype1Generations (77013A × CM334, 77013A × perennial, 77013A × H3, 77013A × 0601M, 83-60 × H3) molecular marker genotypes (C: T genotypes).
The molecular marker shows that the S1597 and S1609 markers have consistent typing, and can be used for screening the pepper restorer line. Restorer IVF2014032 is not phenotypic consistent with the molecular markers because there are multiple restorer genes in pepper and IVF2014032 may not contain the restorer genes for markers S1597 and S1609.
TABLE 2 molecular markers S1597 and S1609 in the parents and F of Capsicum annuum1Generation of typing situations
The results show that the genotypes of the molecular markers S1597 and S1609 are consistent with the phenotypes thereof, and the molecular markers can be used for SNP molecular marker assisted selection of the hot pepper cytoplasmic male sterility restoring material.
S1598 and S1608 markers are only paired with 77013A and CM334 and their hybrid F1Accurate energy-generation typingNo distinction can be made between other restorer parents and their F1The S1598 and S1608 markers are less representative and cannot be used as molecular marker typing and aid selection for natural population restorer lines of pepper.
The polymorphism of the SNP site is that 25 sites of the sequence shown by SEQ ID NO.1 are C or G; or
The polymorphism of the SNP site is that the 24 th site of the sequence shown by SEQ ID NO.2 is C or T.
When the pepper is a 'maintainer line', the SNP loci are G or T genotypes, namely the 25 th position of the nucleotide sequence shown by SEQ ID NO.1 in the pepper genome is G homozygote or the 24 th position of the nucleotide sequence shown by SEQ ID NO.2 is T homozygote;
when the pepper is a 'restorer' line, the SNP loci are C: C genotypes, namely the 25 th site of the sequence shown by SEQ ID NO.1 in the pepper genome is homozygote of C or the 24 th site of the sequence shown by SEQ ID NO.2 is homozygote of C.
Example 2 selection of cytoplasmic Male sterility restorer homozygous Material from Capsicum fertility segregating population Using markers S1597 and S1609
Selecting pepper hybrid 'Xiyangyang' F6The generation segregating material population, including 332 individuals, was PCR amplified using KASP markers. The results are shown in table 3, fig. 3 and 4.
FIG. 3 shows the result of the amplification of KASP marker S1597 in 332 pepper fertility segregating population materials. The blue point is a homozygous fertile single plant (C: C genotype), the green point is a homozygous sterile single plant (G: G genotype), and the red point is a heterozygous fertile single plant (C: G genotype);
FIG. 4 shows the result of the amplification of KASP marker S1609 in 332 pepper fertility segregating population materials. The blue point is a homozygous fertile single plant (C: C genotype), the green point is a homozygous sterile single plant (T: T genotype), and the red point is a heterozygous fertile single plant (C: T genotype);
the result shows that when the single plant in the test segregation population is fertile, the SNP loci corresponding to the molecular markers S1597 and S1609 are C: C homozygous genotype or C: G and C: T heterozygous genotype; when the single plant in the test segregation population is sterile, the SNP loci corresponding to the molecular markers S1597 and S1609 are homozygous genotypes G: G and T: T, respectively. Thus, the markers S1597 and S1609 of the present invention can be used to select materials containing a cytoplasmic male sterility homozygous restorer gene for pepper from segregating populations.
TABLE 3 typing of molecular markers S1597 and S1609 in isolated populations of Capsicum annuum
Sequence listing
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Claims (10)
1. The SNP marker related to the cytoplasmic male sterility recovery trait of the capsicum is characterized in that the nucleotide sequence of the SNP marker is shown as SEQ ID No.1, wherein the 25 th base of the sequence shown as the SEQ ID No.1 is C or G, or,
the nucleotide sequence of the SNP marker is shown as SEQ ID No.2, wherein the 24 th base of the sequence shown as SEQ ID No.2 is C or T.
2. The KASP specific primer for the SNP marker related to cytoplasmic male sterility restoration shape of capsicum according to claim 1,
specific primers for S1597 having a nucleotide sequence shown in SEQ ID No.1 are as follows:
the primer A1: 5'-CAGTTCAATAAAGAAAAAGGCATTC-3' is used as a primer,
the primer A2: 5'-CAGTTCAATAAAGAAAAAGGCATTG-3' is used as a primer,
5'-TCGTCAAAGAATAGAGGTGTTTATCA-3' as a back primer; or
Specific primers for S1609 having a nucleotide sequence shown in SEQ ID No.2 are as follows:
the primer A1: 5'-TGGAAAAAAAAATGGGTATTTAGC-3' is used as a primer,
the primer A2: 5'-TGGAAAAAAAAATGGGTATTTAGT-3' is used as a primer,
rear primer 5'-CATGAGAATCAAAAGAGGATATGAAA-3'.
3. The KASP-specific primer of claim 2, wherein the 5 'end of the pre-primer A1 is added with FAM fluorescent tag sequence and the 5' end of the pre-primer A2 is added with HEX fluorescent tag sequence.
4. The KASP-specific primer of claim 2, wherein the 5 'end of the pre-primer A1 is added with FAM fluorescent tag sequence and the 5' end of the pre-primer A2 is added with HEX fluorescent tag sequence.
5. The KASP-specific primer of claim 4, wherein said FAM fluorescent tag sequence is GAAGGTGACCAAGTTCATGCT, HEX fluorescent tag sequence is GAAGGTCGGAGTCAACGGATT.
6. The use of the SNP marker associated with cytoplasmic male sterility recovery trait in capsicum according to claim 1.
7. The use of the SNP marker associated with cytoplasmic male sterility recovery trait in capsicum annuum according to claim 1 in molecular marker assisted breeding of capsicum annuum.
8. A method for screening a cytoplasmic male sterility restorer line of capsicum which comprises the step of screening a cytoplasmic male sterility restorer gene of capsicum using the SNP marker related to the cytoplasmic male sterility restoration trait of capsicum according to claim 1.
9. The method for identifying and screening the cytoplasmic male sterility restorer line of pepper according to claim 8, wherein when the base at the SNP marker site related to the cytoplasmic male sterility restoration trait of pepper is C: C genotype, the pepper material to be tested is the "restorer line";
when the base at the SNP marker site related to the cytoplasmic male sterility recovery trait of the pepper is G: G or T: T genotype, the pepper material to be detected is a 'maintainer line'.
10. A kit for detecting a cytoplasmic male sterile restorer line of capsicum which comprises the KASP specific primer of claim 2.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010857815.7A CN111808984A (en) | 2020-08-24 | 2020-08-24 | SNP (Single nucleotide polymorphism) marker related to hot pepper cytoplasmic male sterility restoring gene, specific primer and application of specific primer |
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Application publication date: 20201023 |