CN109468410A - The gene molecule marker and its application that a kind of and capsicum genic male sterile gene msc-2 is isolated - Google Patents
The gene molecule marker and its application that a kind of and capsicum genic male sterile gene msc-2 is isolated Download PDFInfo
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Abstract
The invention discloses a kind of gene molecule marker isolated with capsicum genic male sterile gene msc-2 and its applications.The present invention provides the primers of detection capsicum fertility, for the primer pair of energy amplifying specific segment；The specific fragment includes the target sequence of the primer pair of primers F and primer R composition；The primers F is following a1) or a2): a1) single strand dna shown in the sequence 1 of sequence table；A2) by sequence 1 by one or several nucleotide substitution and/or deletion and/or addition and with the DNA molecular with the same function of sequence 1；The primer R is following a3) or a4): a3) single strand dna shown in the sequence 2 of sequence table；A4) by sequence 2 by one or several nucleotide substitution and/or deletion and/or addition and with the DNA molecular with the same function of sequence 2.Using the label developed in the present invention, Rapid identification goes out fertile plant in seedling stage.
Description
Technical field
The invention belongs to field of biotechnology, more particularly to one kind to be divided into capsicum genic male sterile gene msc-2
From gene molecule marker and its application.
Background technique
Capsicum (Capsicum annuumL.) is Solanaceae (Solanaceae) Capsicum herbaceous plant, alias chilly, sea
Green pepper, peppery eggplant, spicy etc., be our people be fond of, one of maximum vegetable species of cultivated area.According to statistics, global capsicum kind
It plants area and is already expired 50,000,000 mu, 37,000,000 tons of annual output, be maximum seasoning crop in the world.China's pepper planting area is super
24,000,000 mu are crossed, 10% or so of 35% or more, the Zhan Quanguo vegetables area of world's capsicum area is accounted for, is only second to the plantation of Chinese cabbage
Area, the economic gross output value of capsicum are more than 70,000,000,000 yuan, the output value and benefit occupy first of vegetable crop (China's Vegetable circulates association,
2017).Pepper hybrid advantage is clearly, and male currently, the capsicum variety overwhelming majority promoted both at home and abroad is the first generation of hybrid
The utilization of infertility can reduce seed costs significantly, and be easy to guarantee the purity of hybrid, be also beneficial to protection intellectual property, institute
With male sterile research has important theoretical and practical significance to the utilization of Pepper hybrid advantage.
Cell Genetic Sterility type (genic male sterility, GMS) and nucleo-cytoplasmic interreaction infertility type are existed simultaneously on capsicum
(cytoplasmic sterility, cytoplasmic male sterility, CMS).The fertility of CMS is common by cytoplasm and cell nucleus gene
Control can be bred as the male sterile line of 100% infertility, using more convenient when hybrid seed produces, but the restoring gene of male parent
It is mainly distributed in small fruit type capsicum (Shen Huolin etc., 1994), restorer is more difficult in large fruit capsicum finds, excellent miscellaneous of breeding
Hand over kind more difficult, and male sterility is often unstable, influences the purity of hybrid.The fertility of capsicum GMS is only by cell nucleus gene
Control, is not influenced by cytoplasm, and what report utilized at present is mostly 1 pair of recessive nuclear gene control, and common self-mating system can make
The fertility full recovery of hybrid, paternal origin is extensive in breeding, and convenient for being bred as excellent Hybrid, and sterility is stablized, raw
It is easy to guarantee that the purity of hybrid, GMS ratio CMS application prospect are broader when producing seed.
So far, the nearly 20 (ms of the capsicum genic male sterile gene that countries in the world scholar has found1--ms15,
msk, msc-1, msc-2), wherein msc-1 and msc-2 is the sterile gene for being found and being utilized by domestic scholars, but these bases
Whether the relationship, gene because between are mutually same unclear.Only ms1 and msc-1 fertility control gene is cloned out at present,
In terms of linked marker exploitation, domestic and international researcher positions 5 ms genes (ms1, ms3, ms8, ms10, msc-1)
Or develop linked marker.Other than msc-1 develops a genetic marker, the molecular labeling of remaining and ms gene linkage is wanted
Linkage distance farther out otherwise label higher operating costs, need to be improved in applicability.So if can develop and ms
Capsicum nuclear male sterility can be improved in breeding apart from the lower molecular labeling of closer use cost in gene linkage
Utilization efficiency reduces breeding breeding cost.
Summary of the invention
It is an object of the present invention to provide the primers of detection capsicum fertility.
Primer provided by the invention, for the primer pair of energy amplifying specific segment；
The specific fragment includes the target sequence of the primer pair of primers F and primer R composition；
The primers F is following a1) or a2):
A1) single strand dna shown in the sequence 1 of sequence table；
A2) there is phase by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of congenerous；
The primer R is following a3) or a4):
A3) single strand dna shown in the sequence 2 of sequence table；
A4) there is phase by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of congenerous.
Above-mentioned primer is made of the primers F and the primer R.
PCR reagent or kit containing above-mentioned primer are also the scope of protection of the invention.
Mentioned reagent box further includes BsmAI enzyme.
Any one of the application of above-mentioned primer or mentioned reagent box is following b1)-b7):
B1) identify that capsicum to be measured carries allele A and/or allele a；
B2) assisting sifting carries the allele A and does not carry the capsicum variety of the allele a；
B3) assisting sifting carries the allele a and does not carry the capsicum variety of the allele A；
B4) assisting sifting carries the allele a and carries the capsicum variety of the allele A；
B5) whether nuclear male is fertile for auxiliary identification capsicum to be measured；
B6) the capsicum variety of assisting sifting or breeding nuclear male sterility；
B7) assisting sifting or the fertile capsicum variety of breeding nuclear male；
The allele A be following c1) c2) or c3):
C1) DNA molecular shown in sequence 3 in sequence table；
C2) hybridize and DNA molecular with the same function with the c1) DNA sequence dna limited under strict conditions；
C3 the DNA sequence dna) and c1) limited has 90% or more homology and DNA molecular with the same function；
The allele a be following d1) d2) or d3):
D1) DNA molecular shown in sequence 4 in sequence table；
D2) hybridize and DNA molecular with the same function with the d1) DNA sequence dna limited under strict conditions；
D3 the DNA sequence dna) and d1) limited has 90% or more homology and DNA molecular with the same function.
Above-mentioned allele A and/or allele a is also the scope of protection of the invention；
Any one of the application of above-mentioned allele A and/or the allele a are following b5)-b7):
B5) whether nuclear male is fertile for auxiliary identification capsicum to be measured；
B6) the capsicum variety of assisting sifting nuclear male sterility；
B7) the fertile capsicum variety of assisting sifting nuclear male.
Above-mentioned application is, if only containing allele A and without containing allele a or containing allele A and a, for
It is fertile, if only containing allele a and allele A is not contained, for infertility.
Another object of the present invention be to provide a kind of detection capsicum to be measured whether the fertile method of nuclear male.
Method provided by the invention includes the following steps: that the genotype for detecting capsicum to be measured is Msc-2Msc-2, Msc-
2msc-2 or msc-2msc-2,
Genotype is that the capsicum nuclear male of Msc-2Msc-2 or Msc-2msc-2 is fertile, genotype msc-2msc-2
Capsicum nuclear male sterility；
The genotype Msc-2Msc-2 is that capsicum to be measured contains above-mentioned allele A and without containing the allele a；
The genotype Msc-2msc-2 is that capsicum to be measured contains above-mentioned allele A and the allele a；
The genotype msc-2msc-2 is that capsicum to be measured contains above-mentioned allele a and without containing the allele A.
The genotype of above-mentioned detection capsicum to be measured is the method packet of Msc-2Msc-2, Msc-2msc-2 or msc-2msc-2
Include following steps A:
1) PCR amplification is carried out with above-mentioned primer pair capsicum to be measured, obtains amplified production；
2) amplified production described in the digestion of BsmAI enzyme detects digestion products,
If being only the segment of 218bp (sequence 5) containing size in digestion products, capsicum genotype to be measured is Msc-
2Msc-2；
If containing the segment that size is 218bp and 186bp (sequence 6) in digestion products, capsicum genotype to be measured is
Msc-2msc-2；
If only containing the segment of 186bp in digestion products, and not containing the segment of 218bp, then capsicum genotype to be measured is
msc-2msc-2。
The present invention also provides a kind of methods that detection detects the genotype of capsicum to be measured, are claim 9 the method
In step A.
Among the above, the genotype of capsicum to be measured is the genotype of capsicum msc-2 gene to be measured.
The experiment proves that can use the label developed in the present invention, Rapid identification goes out fertile plant in seedling stage, from
And save in breeding using manually going to pull out fertile plant, a large amount of man power and material can be saved, to improving capsicum male
The breeding efficiency of infertility reduces breeding breeding cost, and the utilization of Pepper hybrid advantage is promoted to have important theory and apply valence
Value.
Detailed description of the invention
Fig. 1 is amplification of the d-CAPs-msc-2 in fertile and sterile pond.
Fig. 2 is verifying of 41 parts of known profile material to d-CAPs-msc-2.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Capsicum nuclear male sterility F2Group 17C772 is to be selfed the hybrid seed of " grinding No. 16 in Ji ", obtained group
Body.Wherein, " grinding No. 16 in Ji " is recorded in the following literature: protecting field Sweet Pepper Varieties " grinding No. 16 in Ji " cultivation technique [J] Modern Agriculture
Village's science and technology, 2015 (2): 80-80.
Dual-purpose Ms line in pepper group 18Q5430 construction method is as follows: (1) " grinding No. 16 in Ji " is the first generation of hybrid, from
Friendship obtains F2Generation, and there is Fertility segregation, in F2Female parent is done with sterile plant in generation, fertile plant does male parent, does multipair strain intermolecular hybrid.
(2) sowing strain intermolecular hybrid combination, can be there are two types of situation, and a kind of groups is all fertile, and a kind of groups will appear the fertility point of 1:1
From；Occur the sterile plant in isolated cross combination with fertility and do female parent, the similar fertile plant of character is done male parent progress strain and is mixed with
It hands over, progeny population fertility remains the segregation ratio of 1:1.(3) through 5 it is more than generation with group's sterile plant and fertile plant strain intermolecular hybrid,
Group's Other Main Agronomic Characters are basicly stable, sterile and fertile than keeping 1:1, that is, have been bred as new male sterile two-purpose line, have numbered
For 18Q5430.
The acquisition of embodiment 1, molecular labeling d-CAPs-msc-2
One, the exploitation of molecular labeling d-CAPs-msc-2
1, the building of target group
This test is with capsicum nuclear male sterility F2Group 17C772 and a male sterile two-purpose line group
18Q5430 is test material, and obtaining one includes 89 single plant capsicum nuclear male sterility F2Group 17C772 and a packet
Male sterile two-purpose line group 18Q5430 containing 1034 single plants, single plant count pollen fertility.
2, molecular markers development
(1) capsicum nuclear male sterility F is taken2The fertile single plant of group 17C772 mixes pond, obtains fertile pond.Each may be used
The single plant number for educating pond is 30 plants.
(2) capsicum nuclear male sterility F is taken2The sterile single plant of group 17C772 mixes pond, obtains sterile pond.Each not
The single plant number for educating pond is 25 plants.
(3) fertile pond and sterile pond are subjected to simplification and resurvey sequence.
Utilize the heavy sequencing result positional candidate gene in fertile pond and sterile pond.
Utilize F2Fertile pond and sterile pond simplify the candidate gene that weight sequencing result is navigated in group, clone candidate base
Because showing that sterile material candidate gene compared with Fertile material has the missing an of base on second exon
Candidate gene is named as allele A (Msc-2) in Fertile material, and nucleotides sequence is classified as sequence 3；
Candidate gene is named as allele a (msc-2) in sterile material, and nucleotides sequence is classified as sequence 4, for by sequence
The sequence that 3 the 535th T are lacked.
Allelic variation form based on the candidate gene, by the F of capsicum nuclear male sterility2Group is divided into following gene
Type: genotype Msc-2Msc-2 (allele A is homozygous), genotype Msc-2msc-2 (allele A and allele a heterozygosis)
With genotype msc-2msc-2 (allele a is homozygous).
(4) the allelic variation form based on the candidate gene develops molecular labeling d-CAPs-msc-2.
Mark the primer sequence of d-CAPs-msc-2 as follows:
D-CAPs-msc-2F:TTGTATCAATGGCTCATTGGAGATTGCGTCT (sequence 1)；
D-CAPs-msc-2R:AACTCCCTCCCTGGCTACAAATATGTCC (sequence 2).
4, genetic linkage analysis
Using 1034 single plants for screening resulting polymorphic molecular marker d-CAPs-msc-2 analysis 18Q5430 group with
And capsicum nuclear male sterility F2The genotype of group 17C772, and the field fertility phenotype qualification result of group is combined, knot
Fruit shows: molecular labeling d-CAPs-msc-2 is isolated with sterile gene, is a genetic marker.
Two, the method that molecular labeling d-CAPs-msc-2 identifies capsicum fertility to be measured is established
1, the genomic DNA of capsicum to be measured is extracted
Extract the genomic DNA of capsicum to be measured.
2, PCR amplification and digestion
It uses the genomic DNA of capsicum to be measured for template, carries out PCR amplification with the primer of label d-CAPs-msc-2.It receives
Collect amplified production.
The reaction system (10 μ l) of above-mentioned PCR amplification are as follows: 1 μ L of the DNA, (production of Takara company of 10 × Buffer buffer
Product RR001A) 5 μ L, 0.4 dNTPs μ L, archaeal dna polymerase (the product RR001A of Takara company) 0.2 μ L, d-CAPs-msc-2F
Primer and each 0.5 μ L (10uM/L) of d-CAPs-msc-2R primer, with 2.4 μ L ddH2O is by total volume polishing to 20 μ l.
Pcr amplification reaction program: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 35
A circulation；72 DEG C of extension 5min.
0.1 1 μ L (NEB Products #R0529V) of μ L, CutSmart of BsmAI enzyme is added into above-mentioned amplified production, it
55 degree of digestion 3h afterwards.
By 7% native polyacrylamide gel electrophoresis of above-mentioned digestion products, photograph is observed after cma staining.And it is right
Digestion products are sequenced.
As a result as follows:
If containing only the segment that size is 218bp (sequence 5) in digestion products, the capsicum genotype to be measured is Msc-
2Msc-2 (allele A is homozygous), the capsicum to be measured are homozygous fertile；
If containing the segment that size is 218bp and 186bp (sequence 6), the capsicum genotype to be measured in digestion products
For Msc-2msc-2 (allele Aa heterozygous), the capsicum to be measured is that heterozygosis is fertile；
If only containing the segment of 186bp in digestion products, and not containing the segment of 218bp, then capsicum genotype to be measured is
Msc-2msc-2 (allele a is homozygous), the capsicum to be measured are homozygous infertility.
When capsicum to be measured is fertile pond or sterile pond, experimental result is shown in Fig. 1, (M is DNA marker, and F is fertile pond, and S is
Sterile pond).The result shows that molecular labeling d-CAPs-msc-2 has stable polymorphism, and molecular labeling d- between two ponds
CAPs-msc-2 contains the segment that size is 218 and 186bp in fertile pond detection product, contains only in sterile pond DNA detection product
Having size is the segment of 186bp, and test strip is clear, and difference is obvious between two ponds.The genotype in fertile pond is genotype Msc-
2msc-2, the genotype in sterile pond are genotype msc-2msc-2.
The application of embodiment 2, molecular labeling d-CAPs-msc-2
1, genomic DNA is extracted
(being can for the capsicum homozygous inbred lines or kind for extracting 41 parts of known types (Msc-2Msc-2) shown in table 1
Educate) genomic DNA.
Table 1 is 41 parts of capsicum self-mating systems and kind
2, PCR amplification and digestion
PCR amplification and digestion are carried out using the method for embodiment 1, detect digestion products.
If the segment for being only 218bp containing size in digestion products, the capsicum genotype to be measured is Msc-2Msc-2
(homozygous), the capsicum to be measured are homozygous fertile；
If the segment for being 218bp and 186bp containing size in digestion products, the capsicum genotype to be measured is Msc-
2msc-2 (heterozygous), the capsicum to be measured are that heterozygosis is fertile；
If only containing the segment of 186bp in digestion products, and not containing the segment of 218bp, then capsicum genotype to be measured is
Msc-2msc-2, the capsicum to be measured are homozygous infertility.
The capsicum homozygous inbred lines of 41 parts of known types (Msc-2Msc-2) or the genotype call results such as table 1 of kind
With Fig. 2 (F: fertile pond；S: sterile pond, behind be followed successively by 41 parts of selfing based materials) shown in, it can be seen that 41 parts of selfing based materials
Genotype be Msc-2Msc-2, be it is fertile, this is identical as known genotype and fertility, it was demonstrated that uses d-CAPs-msc-2
The success rate of label verifying is 100%；Show that the applicability of this label is very strong, can use this note pair in breeding practical application
Fertility carries out Rapid identification.
Sequence table
<110>China Agricultural University
<120>a kind of gene molecule marker isolated with capsicum genic male sterile gene msc-2 and its application
<160>6
<170> PatentIn version 3.5
<210> 1
<211> 31
<212> DNA
<213>artificial sequence
<400> 1
ttgtatcaat ggctcattgg agattgcgtc t 31
<210> 2
<211> 28
<212> DNA
<213>artificial sequence
<400> 2
aactccctcc ctggctacaa atatgtcc 28
<210> 3
<211> 2007
<212> DNA
<213>artificial sequence
<400> 3
atggtgaagg agatgaagga gatgccgact ttagatctaa gcggatcgaa aaaaaggaag 60
aggaatataa atgagaaggt gtttaagttc aagaattttg gtgaacaagg gtttcctata 120
gagtttatag ggtgcaattt tgagcaaaat gttaagcttc ttttggaatt tgcacaacaa 180
gaaaatgtta gtatttggtc atttcagcta gaagttcata gacatccacc aatgcatgtg 240
gttctctttg ttgttgaaga acaagttgag ttgtccctca atcccaattg caagcattgt 300
caatatatag gctggggcaa ccatctaatg tgcaacaaga agtaccattt catgttgccc 360
acaaaggaca caattgcagc ttgtgtagag ggaggcctaa aaaacaatat tggtggagaa 420
aatagaagta agttgaattt gatagaaata gagggtcata tgatgcatgg tgtgtttcac 480
tctaatggtt ttgggcattt gctttgtatc aatggctcat tggagattgc ttcttctgac 540
ttgcctggcc actctatcat ggacttttgg gatcgccttt gcattggact tggtgcaagg 600
aaagtgagct taagagatgt ctcaacaaag aaaggcatgg atctcaggct actcaacaca 660
gtagcctatg gtgagccatg gtttgggcga tggggttaca aatttggccg tggaagcttt 720
ggtgtaactc aagaaacata ccaaagtgca atcaatgcca tacaaaacat gccattagcc 780
ttattggcac atcatgtagg ggtcataaat attaatgaga tattaacggt gttatcgagg 840
taccaaatgt tatctggtca ttcattagtc acactttgtg atgccattca tttcatgttg 900
gagctcaaat cgaggattcc gaaggaaagt aaccttacct catgttatcc ggggttattg 960
gttgacacca cttgtaggtg gtcacctaaa cgcgttgaga tggctattag ggttgttgtg 1020
gaagccctaa aaagggccga gtcacgttgg gtgtctaggc aagaggttcg tgatgctgcg 1080
cgtgcctata ttggtgatac agggctccta gatttcgtgc ttaagtcatt gggaaatcat 1140
attgttggaa agtacttggt tcgtcgatgc ttaaatccag tgaccaaagt tttggaatac 1200
tgtttagagg acatatctaa agcatttcct aaacaagatc aaggttttag ggttaatgac 1260
tcaaaaggga aacaacaata caaaatcaca tgggtacaac ttatgaagga catatacttc 1320
ttgtacaaga atattttaaa agaggacaag ggattaatgt ccaattatac gggcgtctta 1380
gctacaattc cagcagcttc tagaataatc ctagacacga agtacttcct caaagaatac 1440
aaagggatag aggcggattc aagaattgaa gtagacaaat ccaagattta ctgtgcgatt 1500
atgttggcaa ccaaggatgg atttggagta gaagaaaaag taatgacccc atttgagtgc 1560
ttcacattaa gaaaagatgt cacatttgat gagctcaaaa ttgaagtgga aaaaactttt 1620
ggggctattt atcggggact aaggaacttt gctacaagat caattaacaa tttgatgagc 1680
ccgattaatg gatcagaatt ggtttttaat gttatgaaac cagggagcaa agttgtccta 1740
ggaggggtga taatgtcaat tgatcatcat cataataata acaatattaa tggagggata 1800
tttgaaggaa ttaagaataa tattattgtg gattgtcttt gtgggactaa agatgaagaa 1860
gatggagaaa ggatggtttc atgtgatatt tgtgaagttt ggcagcacac tagatgtgtt 1920
aatatcccaa atcatgaaga aattccagac atatttcttt gtaataagtg tgagcaagat 1980
attttacaat ttccttcatt accttag 2007
<210> 4
<211> 2006
<212> DNA
<213>artificial sequence
<400> 4
atggtgaagg agatgaagga gatgtcgact ttagatctaa gcggatcgaa aaaaaggaag 60
aggaatataa atgagaaggt gtttaagttc aagaattttg gtgaacaagg gtttcctata 120
gagtttatag ggtgcaattt tgagcaaaat gttaagcttc ttttggaatt tgcacaacaa 180
gaaaatgtta gtatttggtc atttcagcta gaagttcata gacatccacc aatgcatgtg 240
gttctctttg ttgttgaaga acaagttgag ttgtccctca atcccaattg caagcattgt 300
caatatatag gctggggcaa ccatctaatg tgcaacaaga agtaccattt catgttgccc 360
acaaaggaca caattgcagc ttgtgtagag ggaggcctaa aaaacaatat tggtggagaa 420
aatagaagta agttgaattt gatagaaata gagggtcata tgatgcatgg tgtgtttcac 480
tctaatggtt ttgggcattt gctttgtatc aatggctcat tggagattgc ttctctgact 540
tgcctggtca ctctatcatg gacttttggg atcgcctttg cattggactt ggtgcaagga 600
aagtgagctt aagagatgtc tcaacaaaga aaggcatgga tctaaggcta ctcaacacag 660
tagcctatgg tgagccatgg tttgggcgat ggggttacaa atttggccgt ggaagctttg 720
gtgtaactca agaaacatac caaagtgcaa tcaatgccat acaaaacatg ccattagcct 780
tattggcaca tcatgtaggg gtcataaata ttaatgagat attaacggtg ttatcgaggt 840
accaaatgtt atctggtcat tcattagtca cactttgtga tgccattcat ttcatgttgg 900
agctcaaatc gaggattccg aaggaaagta accttacctc atgttatccg gggttattgg 960
ttgacaccac ttgtaggtgg tcacctaaac gcgttgagat ggctattagg gttgttgtgg 1020
aagccctaaa aagggccgag tcacgttggg tgtctaggca agaggttcgt gatgctgctc 1080
gtgcctatat tggtgataca gggctcctag atttcgtgct taagtcattg ggaaatcata 1140
ttgttggaaa gtacttggtt cgtcgatgct taaatccagt gaccaaagtt ttggaatact 1200
gtttagagga catatctaaa gcatttccta aacaagatca aggttttagg gttaatgact 1260
caaaagggaa acaacaatac aaaatcacat gggtacaact tatgaaggac atatacttct 1320
tgtacaagaa tattttaaaa gaggacaagg gattaatgtc caattatacg ggcgtcttag 1380
ctacaattcc agcagcttct agaataatcc tagacacgaa gtacttcctc aaagaataca 1440
aagggataga ggcggattca agaattgaag tagacaaatc caagatttac tgcgcgatta 1500
tgttggcaac caaggatgga tttggagtag aagaaaaagt aatgacccca tttgagtgct 1560
tcacattaag aaaagatgtc acatttgatg agctcaaaat tgaagtggaa aaaacttttg 1620
gggctattta tcggggacta aggaactttg ctacaagatc aattaacaat ttgatgagcc 1680
cgattactgg atcagaattg gtttttaatg ttatgaaacc agggagcaaa gttgtcctag 1740
gaggggtgat aatgtcaatt gatcatcatc ataataataa caatattaat ggagggatat 1800
ttgaaggaat taagaataat attattgtgg attgtctttg tgggactaaa gatgaagaag 1860
atggagaaag gatggtttca tgtgatattt gtgaagtttg gcagcacact agatgtgtta 1920
atatcccaaa tcatgaagaa attccagaca tatttctttg taataagtgt gagcaagata 1980
ttttacaatt tccttcatta ccttag 2006
<210> 5
<211> 218
<212> DNA
<213>artificial sequence
<400> 5
ttgtatcaat ggctcattgg agattgcttc ttctgacttg cctggtcact ctatcatgga 60
cttttgggat cgcctttgca ttggacttgg tgcaaggttt gttattctct tcatttcatt 120
ttaacttatg tcacaatgtt taaagtttga caaacaaaga attttaaaat ttatggtctt 180
acacttgttg ggacatattt gtagccaggg agggagtt 218
<210> 6
<211> 186
<212> DNA
<213>artificial sequence
<400> 6
ctgacttgcc tggtcactct atcatggact tttgggatcg cctttgcatt ggacttggtg 60
caaggtttgt tattctcttc atttcatttt aacttatgtc acaatgttta aagtttgaca 120
aacaaagaat tttaaaattt atggtcttac acttgttggg acatatttgt agccagggag 180
ggagtt 186
Claims (10)
1. the primer of capsicum fertility is detected, for the primer pair of energy amplifying specific segment；
The specific fragment includes the target sequence of the primer pair of primers F and primer R composition；
The primers F is following a1) or a2):
A1) single strand dna shown in the sequence 1 of sequence table；
A2 sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 1
The DNA molecular of energy；
The primer R is following a3) or a4):
A3) single strand dna shown in the sequence 2 of sequence table；
A4 sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 2
The DNA molecular of energy.
2. primer according to claim 1, it is characterised in that: the primer is made of the primers F and the primer R.
3. containing the PCR reagent or kit of primer as claimed in claim 1 or 2.
4. kit according to claim 3, it is characterised in that: the kit further includes BsmAI enzyme.
5. the application of primer as claimed in claim 1 or 2 or the kit of claim 3 or 4 is following b1)-b7) in
It is any:
B1) identify that capsicum to be measured carries allele A and/or allele a；
B2) assisting sifting carries the allele A and does not carry the capsicum variety of the allele a；
B3) assisting sifting carries the allele a and does not carry the capsicum variety of the allele A；
B4) assisting sifting carries the allele a and carries the capsicum variety of the allele A；
B5) whether nuclear male is fertile for auxiliary identification capsicum to be measured；
B6) the capsicum variety of assisting sifting or breeding nuclear male sterility；
B7) assisting sifting or the fertile capsicum variety of breeding nuclear male；
The allele A be following c1) c2) or c3):
C1) DNA molecular shown in sequence 3 in sequence table；
C2) hybridize and DNA molecular with the same function with the c1) DNA sequence dna limited under strict conditions；
C3 the DNA sequence dna) and c1) limited has 90% or more homology and DNA molecular with the same function；
The allele a be following d1) d2) or d3):
D1) DNA molecular shown in sequence 4 in sequence table；
D2) hybridize and DNA molecular with the same function with the d1) DNA sequence dna limited under strict conditions；
D3 the DNA sequence dna) and d1) limited has 90% or more homology and DNA molecular with the same function.
6. allele A and/or allele a；
The allele A be following c1) c2) or c3):
C1) DNA molecular shown in sequence 3 in sequence table；
C2) hybridize and DNA molecular with the same function with the c1) DNA sequence dna limited under strict conditions；
C3 the DNA sequence dna) and c1) limited has 90% or more homology and DNA molecular with the same function；
The allele a be following d1) d2) or d3):
D1) DNA molecular shown in sequence 4 in sequence table；
D2) hybridize and DNA molecular with the same function with the d1) DNA sequence dna limited under strict conditions；
D3 the DNA sequence dna) and d1) limited has 90% or more homology and DNA molecular with the same function.
7. the application of allele A described in claim 6 and/or the allele a, for any in following b5)-b7)
Kind:
B5) whether nuclear male is fertile for auxiliary identification capsicum to be measured；
B6) the capsicum variety of assisting sifting nuclear male sterility；
B7) the fertile capsicum variety of assisting sifting nuclear male.
8. a kind of detection capsicum to be measured whether the fertile method of nuclear male, include the following steps: the base for detecting capsicum to be measured
Because type be Msc-2Msc-2, Msc-2msc-2 or msc-2msc-2,
Genotype is that the capsicum nuclear male of Msc-2Msc-2 or Msc-2msc-2 is fertile, and genotype is the peppery of msc-2msc-2
Green pepper nuclear male sterility；
The genotype Msc-2Msc-2 is that capsicum to be measured contains allele A described in claim 6 and without containing described etc.
Position gene a；
The genotype Msc-2msc-2 is that capsicum to be measured contains allele A described in claim 6 and the allele
a；
The genotype msc-2msc-2 is that capsicum to be measured contains allele a described in claim 6 and without containing described etc.
Position Gene A.
9. according to the method described in claim 8, it is characterized by: the genotype of the detection capsicum to be measured is Msc-2Msc-
2, the method for Msc-2msc-2 or msc-2msc-2 includes the following steps A:
1) PCR amplification is carried out with primer pair as claimed in claim 1 or 2 capsicum to be measured, obtains amplified production；
2) amplified production described in the digestion of BsmAI enzyme detects digestion products,
If the segment for being only 218bp containing size in digestion products, capsicum genotype to be measured is Msc-2Msc-2；
If the segment for being 218bp and 186bp containing size in digestion products, capsicum genotype to be measured is Msc-2msc-2；
If only containing the segment of 186bp in digestion products, and not containing the segment of 218bp, then capsicum genotype to be measured is msc-
2msc-2。
10. a kind of method that detection detects the genotype of capsicum to be measured is the step A in claim 9 the method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110029189A (en) * | 2019-05-13 | 2019-07-19 | 中国农业大学 | The molecular labeling and application that a kind of and celery cell matter male sterility gene isolates |
| CN111518944A (en) * | 2020-05-29 | 2020-08-11 | 中国农业科学院蔬菜花卉研究所 | Chili cytoplasmic male sterility restoring gene related InDel marker D6-26, specific primer and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108148920A (en) * | 2017-12-19 | 2018-06-12 | 中国农业大学 | The functional form molecular labeling of capsicum nuclear male sterility related gene and its application |
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2019
- 2019-01-11 CN CN201910025786.5A patent/CN109468410A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108148920A (en) * | 2017-12-19 | 2018-06-12 | 中国农业大学 | The functional form molecular labeling of capsicum nuclear male sterility related gene and its application |
Non-Patent Citations (1)
| Title |
|---|
| JEONG ET AL.: "Fine mapping of the genic male-sterile ms1 gene in Capsicum annuum L", 《THEORETICAL AND APPLIED GENETICS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110029189A (en) * | 2019-05-13 | 2019-07-19 | 中国农业大学 | The molecular labeling and application that a kind of and celery cell matter male sterility gene isolates |
| CN111518944A (en) * | 2020-05-29 | 2020-08-11 | 中国农业科学院蔬菜花卉研究所 | Chili cytoplasmic male sterility restoring gene related InDel marker D6-26, specific primer and application thereof |
| CN111518944B (en) * | 2020-05-29 | 2022-05-31 | 中国农业科学院蔬菜花卉研究所 | Chili cytoplasmic male sterility restoring gene related InDel marker D6-26, specific primer and application thereof |
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