CN108148920B - Functional molecular marker of hot pepper nuclear male sterility related gene and application thereof - Google Patents
Functional molecular marker of hot pepper nuclear male sterility related gene and application thereof Download PDFInfo
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Abstract
The invention discloses a functional molecular marker of a hot pepper nuclear male sterility related gene and application thereof. The specific primer pair provided by the invention consists of two primers for amplifying specific DNA fragments; the specific DNA fragment has a target sequence of a primer pair consisting of a primer InDel-12F and a primer InDel-12R in a pepper genome; the primer InDel-12F is a single-stranded DNA molecule shown in a sequence 3 of a sequence table, and the primer InDel-12R is a single-stranded DNA molecule shown in a sequence 4 of the sequence table. By applying the molecular marker InDel-12 provided by the invention, the pepper variety with nuclear male sterility can be quickly screened out. The invention has important theoretical significance and economic value.
Description
Technical Field
The invention belongs to the field of plant molecular breeding, and particularly relates to a functional molecular marker of a hot pepper nuclear male sterility related gene and application thereof.
Background
Capsicum (Capsicum annuum L.) is a herbaceous plant of Capsicum of Solanaceae (Solanaceae), and is also called Capsicum annuum, fructus Solani Melongenae, and fructus Capsici, etc., and is one of the vegetable species which is popular for people in China and has the largest cultivation area. According to statistics, the planting area of the pepper in the world is 5000 ten thousand mu, the annual output is 3700 ten thousand tons, and the pepper is the largest seasoning crop in the world. In China, the pepper is planted in more than 2000 ten thousand acres, accounting for 35 percent of the pepper area in the world and 10 percent of the vegetable area in the country, which is next to the planting area of Chinese cabbage in the country. The total economic value of the pepper exceeds 700 million yuan, and the yield and the benefit are the first of vegetable crops (Chinese vegetable circulation society). At present, most of pepper varieties mainly promoted in China are first filial generation, and utilization of pepper heterosis has important economic value and social value.
The hybrid vigor of the pepper is very obvious, and the artificial emasculation can be saved by utilizing the male sterile line for seed production, so that the production cost of hybrid seeds is effectively reduced, the purity of the seeds is ensured, and the intellectual property rights are protected. The naturally mutated male sterility of most vegetable crops has only one genetic type, but a nuclear male sterility type and a nucleoplasm interaction sterility type (namely a cytoplasmic male sterility type) exist on the pepper at the same time, so the pepper is a test material for researching the expression of the male sterility fertility of the plants. The fertility of Cytoplasmic Male Sterility (CMS) of hot pepper is controlled by cytoplasm and cell nucleus genes together, so that a male sterile line with 100% sterility can be bred, the use is convenient, but the restoring genes of the male parent are mainly distributed in small-fruit hot pepper, the restoring line in generally cultivated large-fruit hot pepper is difficult to find, and the breeding of excellent hybrid varieties is difficult. The fertility of pepper nucleus male sterility (GMS) is only controlled by nuclear genes and is not influenced by cytoplasm, most of the current reports are 1 pair of recessive nuclear genes, only male sterility dual-purpose lines can be bred, 50% of fertile plants need to be removed during breeding, but the pepper nucleus male sterility is relatively stable in fertility, the inheritance is simple and convenient to use, and the ordinary inbred lines can fully restore the fertility of hybrids, so that the male parent source in breeding is wide, and the excellent hybrid varieties can be bred conveniently. Therefore, the method has important theoretical and application values for finely positioning the GMS fertility gene of the pepper and analyzing the molecular mechanism of the key gene of nuclear male sterility, improving the breeding efficiency of pepper male sterility, reducing the breeding cost of breeding and promoting the utilization of pepper heterosis.
At present, fine positioning of pepper GMS fertility genes is not carried out at home and abroad, molecular markers which can be practically applied in breeding work are not available, the breeding and transformation work of pepper GMS dual-purpose lines is also a classical backcross breeding method, if two generations in a year are adopted, the period for breeding the dual-purpose lines is as long as 6-7 years, and if GMS related molecular markers can be developed, the breeding and transformation time can be shortened to 3 years, so that the breeding efficiency can be obviously improved.
Disclosure of Invention
The invention aims to solve the technical problem of providing a molecular marker for identifying whether capsicum to be detected is karyon male fertile or karyon male sterile.
In order to solve the above technical problems, the present invention provides a specific primer pair.
The specific primer pair provided by the invention can consist of two primers for amplifying specific DNA fragments; the specific DNA fragment has a target sequence of a primer pair consisting of a primer InDel-12F and a primer InDel-12R in a pepper genome;
the primer InDel-12F can be a1) or a2) as follows:
a1) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;
the primer InDel-12R can be a3) or a4) as follows:
a3) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and has the same function as the sequence 4.
The specific primer pair can specifically consist of the primer InDel-12F and the primer InDel-12R.
The function of the specific primer pair can be any one of the following b1) -b 7):
b1) identifying the allele A and/or the allele a carried by the pepper to be detected;
b2) auxiliary screening of pepper varieties carrying the allele A and not carrying the allele a;
b3) auxiliary screening of pepper varieties carrying the allele a and not carrying the allele A;
b4) auxiliary screening of pepper varieties carrying the allele a and carrying the allele A;
b5) the method comprises the following steps of (1) assisting in identifying whether the pepper to be detected is nucleus male fertile;
b6) auxiliary screening of a capsicum variety with nuclear male sterility;
b7) and (3) auxiliary screening of a nuclear male fertile pepper variety.
The specific primer pair is used for amplifying the following molecular marker InDel-12 related to the male fertility of pepper nuclei.
The invention also provides a kit comprising any one of the specific primer pairs.
Conventional reagents for PCR amplification and/or conventional reagents for genome extraction and/or conventional reagents for agarose gel electrophoresis may also be included in the kit.
The function of the kit can be any one of the following b1) -b 7):
b1) identifying the allele A and/or the allele a carried by the pepper to be detected;
b2) auxiliary screening of pepper varieties carrying the allele A and not carrying the allele a;
b3) auxiliary screening of pepper varieties carrying the allele a and not carrying the allele A;
b4) auxiliary screening of pepper varieties carrying the allele a and carrying the allele A;
b5) the method comprises the following steps of (1) assisting in identifying whether the pepper to be detected is nucleus male fertile;
b6) auxiliary screening of a capsicum variety with nuclear male sterility;
b7) and (3) auxiliary screening of a nuclear male fertile pepper variety.
The invention also protects the application of any one of the specific primer pairs or any one of the kits, and the specific primer pair can be any one of the following b1) -b 7):
b1) identifying the allele A and/or the allele a carried by the pepper to be detected;
b2) auxiliary screening of pepper varieties carrying the allele A and not carrying the allele a;
b3) auxiliary screening of pepper varieties carrying the allele a and not carrying the allele A;
b4) auxiliary screening of pepper varieties carrying the allele a and carrying the allele A;
b5) the method comprises the following steps of (1) assisting in identifying whether the pepper to be detected is nucleus male fertile;
b6) auxiliary screening of a capsicum variety with nuclear male sterility;
b7) and (3) auxiliary screening of a nuclear male fertile pepper variety.
The invention also protects a molecular marker InDel-12 related to the male fertility of pepper nuclei, which can be a DNA fragment obtained by using the genome DNA of the pepper to be detected as a template and adopting any one of the specific primer pairs for amplification. The molecular marker InDel-12 can be specifically the allele A and/or the allele a.
The invention also protects the application of the molecular marker InDel-12, which can be any one of the following b5) -b 7):
b5) the method comprises the following steps of (1) assisting in identifying whether the pepper to be detected is nucleus male fertile;
b6) auxiliary screening of a capsicum variety with nuclear male sterility;
b7) and (3) auxiliary screening of a nuclear male fertile pepper variety.
The method for identifying whether the to-be-detected pepper nucleus is male fertile by applying the molecular marker InDel-12 in an auxiliary way comprises the following steps: if the allele A exists in the genome of the pepper to be detected, the pepper to be detected can be subjected to nuclear male fertility; and if the pepper genome to be detected does not have the allele A, the pepper nucleus to be detected is male sterile.
The method for screening the chili variety with nuclear male sterility by applying the molecular marker InDel-12 in an auxiliary way comprises the following steps: and if the genome of the pepper to be detected does not have the allele A, the pepper to be detected is a candidate pepper variety with nuclear male sterility, otherwise, the pepper to be detected is not the candidate pepper variety with nuclear male sterility.
The method for applying the molecular marker InDel-12 to assist in screening the nuclear male fertile pepper variety comprises the following steps: and if the allele A exists in the genome of the pepper to be detected, the pepper to be detected is a candidate karyon male-fertile pepper variety, otherwise, the pepper to be detected is not the candidate karyon male-fertile pepper variety.
The allele A and/or the allele a also belong to the protection scope of the invention.
The invention also protects the application of the allele A and/or the allele a, which can be any one of the following b5) -b 7):
b5) the method comprises the following steps of (1) assisting in identifying whether the pepper to be detected is nucleus male fertile;
b6) auxiliary screening of a capsicum variety with nuclear male sterility;
b7) and (3) auxiliary screening of a nuclear male fertile pepper variety.
The invention also provides a method for detecting whether the capsicum to be detected is nuclear male fertile, which comprises the following steps: detecting whether the genotype of the pepper to be detected is genotype MsMs, genotype Mmsms or genotype MsMs, wherein the pepper nucleus with the genotype MsMs or the genotype Mmsms is male fertile, and the pepper nucleus with the genotype MsMs is male sterile;
the genotype MsMs indicates that the pepper to be detected contains the allele A and does not contain the allele a; the genotype Msms is that the pepper to be detected contains the allele A and the allele a; the genotype msms is that the pepper variety to be tested contains the allele a and does not contain the allele A.
In the above method, the "detecting whether the genotype of the pepper to be detected is the genotype MsMs, the genotype MsMs or the genotype MsMs" may be K1) or K2):
K1) taking the genome DNA of the pepper to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair to obtain a PCR amplification product; if the PCR amplification product has 176bp fragments and does not have 169bp fragments, the genotype of the pepper to be detected is genotype MsMs, if the PCR amplification product has 176bp and 169bp fragments and the genotype of the pepper to be detected is genotype Mmsms, if the PCR amplification product has 169bp fragments and does not have 176bp fragments, the genotype of the pepper to be detected is genotype MsMs;
K2) taking the genome DNA of the pepper to be detected as a template, carrying out PCR amplification by adopting the specific primer pair, and then carrying out sequencing; if the PCR amplification product contains a nucleotide sequence shown by a sequence 5 in the sequence table and does not contain a nucleotide sequence shown by a sequence 6 in the sequence table and the genotype of the pepper to be detected is genotype Msms, if the PCR amplification product contains a nucleotide sequence shown by a sequence 5 in the sequence table and a nucleotide sequence shown by a sequence 6 in the sequence table and the genotype of the pepper to be detected is genotype Msms, if the PCR amplification product contains a nucleotide sequence shown by a sequence 6 in the sequence table and does not contain a nucleotide sequence shown by a sequence 5 in the sequence table and the genotype of the pepper to be detected is genotype MsMs.
The invention also provides a method for detecting the allele A and/or the allele a carried by the capsicum to be detected, which can be G1) or G2):
G1) taking the genome DNA of the pepper to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair to obtain a PCR amplification product; if the PCR amplification product has 176bp fragments and does not have 169bp fragments, the pepper to be detected carries the allele A and does not carry the allele a, if the PCR amplification product has 176bp and 169bp fragments, the pepper to be detected carries the allele A and the allele a, and if the PCR amplification product has 169bp fragments and does not have 176bp fragments, the pepper to be detected carries the allele a and does not carry the allele A;
G2) taking the genome DNA of the pepper to be detected as a template, carrying out PCR amplification by adopting the specific primer pair, and then carrying out sequencing; if the PCR amplification product contains a nucleotide sequence shown by a sequence 5 in the sequence table and does not contain a nucleotide sequence shown by a sequence 6 in the sequence table and the pepper to be detected carries the allele A and does not carry the allele a, if the PCR amplification product contains a nucleotide sequence shown by a sequence 5 in the sequence table and a nucleotide sequence shown by a sequence 6 in the sequence table and the pepper to be detected carries the allele A and the allele a, if the PCR amplification product contains a nucleotide sequence shown by a sequence 6 in the sequence table and does not contain a nucleotide sequence shown by a sequence 5 in the sequence table and the pepper to be detected carries the allele a and does not carry the allele A.
Any of the above alleles A may be c1) or c2) or c3) or c4) as follows:
c1) DNA molecule shown in sequence 1 in the sequence table;
c2) DNA molecule shown in sequence 5 in the sequence table;
c3) a DNA molecule which hybridizes with the DNA sequence defined by c1) or c2) under strict conditions and has the same function;
c4) DNA molecule with more than 90% homology with the DNA sequence limited by c1) or c2) and the same function.
Any one of the above alleles a may be d1) or d2) or d3) or d4) as follows:
d1) DNA molecule shown in sequence 2 in the sequence table;
d2) DNA molecule shown in sequence 6 in the sequence table;
d3) DNA molecules which hybridize under stringent conditions with the DNA sequences defined under d1) or d2) and have the same function;
d4) DNA molecule with more than 90% homology with the DNA sequence defined by d1) or d2) and the same function.
The specific step of assisting in identifying whether the capsicum to be detected is nuclear male fertile can be assisting in identifying whether the capsicum to be detected is nuclear male fertile.
The invention discloses a gene related to hot pepper nuclear male fertility and an allelic variation form thereof: allele a and allele a. On the basis, a specific primer pair for identifying the allele A and the allele a and a relation between an allelic variation sequence and whether the pepper nucleus is male fertile or not are provided. Pepper nuclei of genotype MsMs or genotype Mmsms are male fertile, and pepper nuclei of genotype MsMs are male sterile; the genotype MsMs indicates that the pepper to be detected contains the allele A and does not contain the allele a; the genotype Msms is that the pepper to be detected contains the allele A and the allele a; the genotype msms is that the pepper variety to be tested contains the allele a and does not contain the allele A. By applying the molecular marker provided by the invention, the pepper material with nucleus male fertility can be quickly screened out, so that the breeding pace of new pepper varieties is accelerated. The invention has important theoretical significance and economic value.
Drawings
FIG. 1 shows the results of the experiment A in step two of example 1.
FIG. 2 shows the experimental results of step one in example 2.
FIG. 3 shows the results of the experiment in step two of example 2.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The pepper cell nucleus sterile dual-purpose lines 09C460AB and 09C460A are both described in the following documents: liu et al, cloning and expression analysis of candidate genes related to fertility of pepper GMS [ J ]. Chinese agricultural science 2014, 47(16):3264-3276.0.
Example 1 development of molecular marker InDel-12 and polymorphism detection
First, development of molecular marker InDel-12
1. Counting pollen fertility of each individual plant in the positioned group
A pepper nucleus sterile dual-purpose line 09C460AB is used as a test material (fertile plants and sterile plants are near isogenic lines), and a positioning population consisting of 1110 single plants is obtained. And respectively counting and positioning the pollen fertility of each individual plant in the population.
2. Development of molecular marker InDel-12
(1) And (4) mixing the fertile individual plants of the positioned groups obtained in the step (1) to obtain a fertile pool. The number of the individual plants in each fertile pond is 30.
(2) And (3) mixing the sterile single plants of the positioned population obtained in the step (1) to obtain a sterile pool. The number of the individual plants in each sterile pool is 30.
(3) Each fertile or sterile pool is re-sequenced.
And (4) positioning candidate genes by using the re-sequencing results of the fertile pool and the sterile pool. Based on a large amount of sequence analysis, the inventor of the invention finds that two allelic variation forms exist in the candidate gene in the pepper nucleus sterile dual-purpose line 09C460 AB: one is shown as a sequence 1 in a sequence table (named as allele A), and the other is shown as a sequence 2 in the sequence table (named as allele a). Compared to allele A, allele a has a 7bp deletion. The 7bp deletion nucleotide sequence is 5 '-AAACTAA-3'.
Based on two allelic variation forms of the candidate gene, the pepper nucleus sterile dual-purpose line 09C460AB is divided into two genotypes: genotype Msms (allele a heterozygous with allele a) and genotype Msms (allele a homozygous).
(4) Based on two allelic variation forms of the candidate gene, a molecular marker InDel-12 is developed.
The nucleotide sequence of the amplified molecular marker InDel-12 is shown as follows:
primer InDel-12F: 5'-GCCTGAAGTTCAAGTGGCTC-3' (SEQ ID NO: 3 in the sequence Listing);
primer InDel-12R: 5'-CCACAGATGCAGTAACAAGCA-3' (SEQ ID NO: 4 of the sequence Listing).
Second, polymorphism detection
A. Polymorphism detection
1. Extracting the genome DNA of the pepper to be detected by adopting an improved CTAB method.
2. And (3) performing PCR amplification by using the genome DNA in the step (1) as a template and using a primer pair consisting of the primer InDel-12F and the primer InDel-12R in the step (I) to obtain a PCR amplification product.
PCR reaction (20. mu.L): 1. mu.L of template (containing genomic DNA 100ng), 2. mu. L, dNTPs (product of Takara Co.) and 10 XBuffer (product of Takara Co.) 0.8. mu. L, DNA 0.4. mu.L of polymerase, 0.2. mu.L each of primer InDel-12F (10. mu. mol/L) and primer InDel-12R (10. mu. mol/L) and ddH2O 15.4μL。
And (3) PCR reaction conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 32 cycles; 5min at 72 ℃.
3. Taking the PCR amplification product (2 mu L) obtained in the step 2, carrying out 7% non-denaturing polyacrylamide gel electrophoresis detection, carrying out silver nitrate staining, and judging according to the staining result as follows:
if the banding pattern of the PCR amplification product is banding pattern A (a banding is shown and is 176bp), the genotype of the hot pepper to be detected is genotype MsMs;
if the banding pattern of the PCR amplification product is banding pattern B (two banding bands are displayed, and the banding bands are 176bp and 169bp respectively), the genotype of the hot pepper to be detected is genotype Msms;
if the banding pattern of the PCR amplification product is banding pattern C (one banding is shown and 169bp is shown), the genotype of the pepper to be detected is genotype msms.
When the pepper to be detected is a fertile pond or a sterile pond, the experimental result is shown in figure 1(M is a DNA marker, F is the fertile pond, and S is the sterile pond). The result shows that the molecular marker InDel-12 has stable polymorphism between two pools, and the band amplified by the molecular marker InDel-12 is clear, and the difference between the two pools is obvious. The genotype of the fertile pool is genotype Msms, and the genotype of the sterile pool is genotype Msms.
B. Polymorphism detection two
1. The same as 1 in the step A.
2. The same as step A2.
3. Sequencing the PCR amplification product obtained in the step 2, and judging according to a sequencing result as follows:
if the PCR amplification product contains the nucleotide sequence shown as the sequence 5 in the sequence table and does not contain the nucleotide sequence shown as the sequence 6 in the sequence table, the genotype of the pepper to be detected is the genotype MsMs;
if the PCR amplification product contains a nucleotide sequence shown as a sequence 5 in the sequence table and a nucleotide sequence shown as a sequence 6 in the sequence table, the genotype of the pepper to be detected is genotype Msms;
and if the PCR amplification product contains the nucleotide sequence shown as the sequence 6 in the sequence table and does not contain the nucleotide sequence shown as the sequence 5 in the sequence table, the genotype of the pepper to be detected is the genotype msms.
And when the pepper to be detected is a fertile pond or a sterile pond, the obtained experimental result is completely consistent with the experimental result in the first polymorphism detection.
The results show that the molecular marker InDel-12 developed in the first step has higher polymorphism. The molecular marker can be used for distinguishing pepper materials containing allele A homozygosis, pepper materials containing allele a homozygosis and pepper materials containing allele A and allele a heterozygosis.
Third, genetic linkage analysis
Analyzing the genotypes of 1110 individual plants of the positioning population by using a molecular marker InDel-12, and performing genetic linkage analysis by combining the field fertility phenotype identification result of the positioning population.
The result shows that the molecular marker InDel-12 and the hot pepper nuclear male sterile gene are coseparated and are a gene marker. Detecting the genotype of the pepper to be detected by adopting a molecular marker InDel-12, and then judging as follows:
if the genotype is genotype MsMs or genotype Mmsms, the nucleus of the pepper to be detected can be male-fertile;
and if the genotype is the genotype msms, the nuclear male sterility of the pepper to be detected is detected.
Example 2 application of molecular marker InDel-12
One, application one
The pepper to be tested was 46 selfed lines and varieties of pepper of which the known genotype was the genotype MsMs (see Table 1). All selfed lines and varieties of pepper are publicly available from the horticultural college of Chinese agricultural university.
TABLE 1
1. The pepper to be tested was genotyped according to the first polymorphism detection method in example 2.
2. And (3) planting the pepper sample to be detected in a field, performing conventional field management, and observing pollen fertility after the pepper sample is mature.
The experimental results of step 1 are shown in FIG. 2(M is DNA marker, F is fertile pool, S is sterile pool, and the rest is 46 parts of pepper inbred line and variety). The result shows that the banding patterns of the PCR amplification product of the pepper to be detected are all banding patterns A (one banding is shown and is 176bp), and the genotype of the pepper to be detected is genotype MsMs; predicting the nuclear male fertility of the pepper to be detected.
And 3, the field experiment in the step 3 shows that the pollen fertility of the hot pepper to be detected is fertile.
The result shows that whether the pepper to be detected is nucleus male fertile or not is detected by adopting the molecular marker InDel-12 provided by the invention, and the detection result is completely consistent with the field experiment result.
Second, apply second
Hybridizing with 09C460A as female parent and Shanghai Yuan C (described in the following documents: Shibo, molecular marker assisted selection of hot pepper cytoplasmic male sterility restoring gene, Master graduate paper of Chinese university of agriculture, 2014) as male parent to obtain hybrid F1(ii) a Then hybridized to F1Selfing to obtain 510F2And (4) a group. The 510F2The population was used as the pepper to be tested, and the following experiment was performed.
1. The pepper to be tested was genotyped according to the first polymorphism detection method in example 2.
Part of the experimental results are shown in FIG. 3(M is DNA marker, F is fertile pool, S is sterile pool, and the rest is part F2A population). The results showed 510F2The genotype of the population is genotype MsMs, genotype Mmsms or genotype MsMs, the cell nucleus of the pepper to be tested (380 strains in total) with the genotype MsMs or the genotype Mmsms is male fertile, and the cell nucleus of the pepper to be tested (130 strains in total) with the genotype MsMs is male sterile.
2. And (3) planting the pepper to be detected in a field, performing conventional field management, and observing pollen fertility after the pepper is mature.
The results are shown in Table 2.
TABLE 2
| Fertile plant | Sterile plant | Desired separation ratio | χ2-test |
| 380 | 130 | 3:1 | 0.065 |
The result shows that whether the pepper to be detected is nucleus male fertile or not is detected by adopting the molecular marker InDel-12 provided by the invention, and the detection result is completely consistent with the field experiment result.
The results show that the pepper nucleus male sterility of genotype, genotype MsMs or genotype Mmsms of the pepper to be detected is detected. Therefore, by using the molecular marker InDel-12 developed in example 1, a nuclear male fertile pepper variety or a nuclear male sterile pepper variety can be screened.
<110> university of agriculture in China
Functional molecular marker of pepper nuclear male sterility related gene and application thereof
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 651
<212> DNA
<213> Pepper (Capsicum annuum L.)
<400> 1
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gttaaagaat acaaatccaa gaaccttaat gctgagagaa agaggcgtca aaaacttagt 180
gaaaggctac ttgaattgcg ctcattagtc ccaaacatta caaatatgac aaaagaaacc 240
ataatcactg atgccatcac ttacattagg gagctacaaa caaatgtgga ctacctaagc 300
gagcagcttc ttgaaatgga ggcaactcat gcagaacaat tggagacaaa aaatgaagtg 360
attatcgata ctgcagagga tatgggtaaa tggggcatag agcctgaagt tcaagtggct 420
cacattggtc caactaagct ttggataaaa atagtctgcc agaagaaaag aggtggattt 480
actaaactaa tggagacaat gaatgttctt ggatttgatc ttaatgacac cagtgtcact 540
gcctccagag gagctctgct tgttactgca tctgtggagg tggttcgcgg tggactaaat 600
gaagctaatc aaatcagaga gatcttgctg gagatcatcc gcggaatcta g 651
<210> 2
<211> 644
<212> DNA
<213> Pepper (Capsicum annuum L.)
<400> 2
atggtcgtaa ttaagtgtgg taaaatggaa ttccccaaga gcccatttga tgattcacac 60
atgtctgaag aaagggaagt aagagggaga acagataaaa ggaagacaat tgatggtgaa 120
gttaaagaat acaaatccaa gaaccttaat gctgagagaa agaggcgtca aaaacttagt 180
gaaaggctac ttgaattgcg ctcattagtc ccaaacatta caaatatgac aaaagaaacc 240
ataatcactg atgccatcac ttacattagg gagctacaaa caaatgtgga ctacctaagc 300
gagcagcttc ttgaaatgga ggcaactcat gcagaacaat tggagacaaa aaatgaagtg 360
attatcgata ctgcagagga tatgggtaaa tggggcatag agcctgaagt tcaagtggct 420
cacattggtc caactaagct ttggataaaa atagtctgcc agaagaaaag aggtggattt 480
acttggagac aatgaatgtt cttggatttg atcttaatga caccagtgtc actgcctcca 540
gaggagctct gcttgttact gcatctgtgg aggtggttcg cggtggacta aatgaagcta 600
atcaaatcag agagatcttg ctggagatca tccgcggaat ctag 644
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 3
gcctgaagtt caagtggctc 20
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 4
ccacagatgc agtaacaagc a 21
<210> 5
<211> 176
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 5
gcctgaagtt caagtggctc acattggtcc aactaagctt tggataaaaa tagtctgcca 60
gaagaaaaga ggtggattta ctaaactaat ggagacaatg aatgttcttg gatttgatct 120
taatgacacc agtgtcactg cctccagagg agctctgctt gttactgcat ctgtgg 176
<210> 6
<211> 169
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 6
gcctgaagtt caagtggctc acattggtcc aactaagctt tggataaaaa tagtctgcca 60
gaagaaaaga ggtggattta cttggagaca atgaatgttc ttggatttga tcttaatgac 120
accagtgtca ctgcctccag aggagctctg cttgttactg catctgtgg 169
Claims (4)
1. The application of the specific primer pair or the kit comprising the specific primer pair is any one of the following b1) -b 7):
b1) identifying the allele A and/or the allele a carried by the pepper to be detected;
b2) auxiliary screening of pepper varieties carrying the allele A and not carrying the allele a;
b3) auxiliary screening of pepper varieties carrying the allele a and not carrying the allele A;
b4) auxiliary screening of pepper varieties carrying the allele a and carrying the allele A;
b5) the method comprises the following steps of (1) assisting in identifying whether the pepper to be detected is nucleus male fertile;
b6) auxiliary screening of a capsicum variety with nuclear male sterility;
b7) auxiliary screening of nuclear male fertile pepper varieties;
the allele a is c2 as follows:
c2) DNA molecule shown in sequence 5 in the sequence table;
the allele a is d2 as follows:
d2) DNA molecule shown in sequence 6 in the sequence table;
the specific primer pair consists of a primer InDel-12F and a primer InDel-12R;
the primer InDel-12F is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;
the primer InDel-12R is a single-stranded DNA molecule shown in a sequence 4 of a sequence table.
2. The use of a reagent for detecting the genotype of the allele A and the allele a according to claim 1, which is any one of the following b5) -b 7):
b5) the method comprises the following steps of (1) assisting in identifying whether the pepper to be detected is nucleus male fertile;
b6) auxiliary screening of a capsicum variety with nuclear male sterility;
b7) and (3) auxiliary screening of a nuclear male fertile pepper variety.
3. A method for detecting whether the pepper to be detected is nuclear male fertile or not comprises the following steps: detecting whether the genotype of the pepper to be detected is genotype MsMs, genotype Mmsms or genotype MsMs, wherein the pepper nucleus with the genotype MsMs or the genotype Mmsms is male fertile, and the pepper nucleus with the genotype MsMs is male sterile;
the genotype MsMs are determined for the pepper containing the allele A as defined in claim 1 and not containing the allele a; the genotype Msms is that the pepper to be tested contains the allele A and the allele a as defined in claim 1; the genotype msms is that the pepper variety to be tested contains the allele a in claim 1 and does not contain the allele A.
4. A method for detecting that pepper to be detected carries the allele A and/or the allele a in claim 1, which comprises the steps of carrying out PCR amplification by using the specific primer pair in claim 1 and then carrying out sequencing by using the genomic DNA of the pepper to be detected as a template;
if the PCR amplification product contains a nucleotide sequence shown by a sequence 5 in the sequence table and does not contain a nucleotide sequence shown by a sequence 6 in the sequence table and the pepper to be detected carries the allele A and does not carry the allele a, if the PCR amplification product contains a nucleotide sequence shown by a sequence 5 in the sequence table and a nucleotide sequence shown by a sequence 6 in the sequence table and the pepper to be detected carries the allele A and the allele a, if the PCR amplification product contains a nucleotide sequence shown by a sequence 6 in the sequence table and does not contain a nucleotide sequence shown by a sequence 5 in the sequence table and the pepper to be detected carries the allele a and does not carry the allele A.
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| CN109468410A (en) * | 2019-01-11 | 2019-03-15 | 中国农业大学 | The gene molecule marker and its application that a kind of and capsicum genic male sterile gene msc-2 is isolated |
| CN109593878A (en) * | 2019-01-31 | 2019-04-09 | 绿亨科技股份有限公司 | Molecular labeling and its application with capsicum genic male sterile gene close linkage |
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