Summary of the invention
The object of this invention is to provide a kind of and the closely linked molecule marker of millet pollen color gene.
Another object of the present invention is to provide a kind of primer pair that can be used for pcr amplification and the closely linked molecule marker of millet pollen color gene, and the molecule marker obtained by this primer pair amplifies.
Another object of the present invention is to provide the purposes of above-mentioned molecule marker in millet pollen color gene location, detection and millet assistant breeding and the detection method of above-mentioned molecule marker.
Object of the present invention also comprises provides a kind of carrier comprising above-mentioned molecule marker, and the reconstitution cell containing this carrier; A kind of localization method comprising the millet pollen color gene using above-mentioned molecule marker is provided, and uses the millet auxiliary breeding means of described molecule marker.
To achieve these goals, present invention employs following technical scheme:
The invention discloses a kind of and the closely linked molecule marker of millet pollen color gene, described molecule marker contains sequence shown in SeqIDNo.1; Preferred described molecule marker has sequence shown in SeqIDNo.1.
The invention also discloses the primer pair of a kind of amplification and the closely linked molecule marker of millet pollen color gene, the primer 1 of described primer pair is containing sequence shown in SeqIDNo.2, and primer 2 contains sequence shown in SeqIDNo.3; Preferred described primer 1 has sequence shown in SeqIDNo.2, and primer 2 has sequence shown in SeqIDNo.3;
SeqIDNo.2:5’-CAGGATAAACTTCACCAATCG-3’;
SeqIDNo.3:5’-CGAACCATTCATATCTGGCT-3’。
The invention also discloses a kind of with the closely linked molecule marker of millet pollen color gene, described molecule marker be by above-mentioned primer pair with pollen yellow-white or orange millet genomic dna for template obtains through pcr amplification.
The preferred molecule marker obtained by above-mentioned primer pair amplifies contains sequence shown in SeqIDNo.1.
In one embodiment of the invention, described molecule marker (DNA fragmentation containing nucleotide sequence shown in SeqIDNo.1) is the DNA fragmentation of nucleotide sequence shown in SeqIDNo.1 in millet genome, nucleotide sequence beyond the 5 ' end of namely comprised SeqIDNo.1 and/or 3 ' end is also sequence in millet genome, preferably, be 5 ' end and/or 3 ' the upstream and downstream sequence of holding of SeqIDNo.1 in millet genome.As long as this molecule marker that it will be understood by those skilled in the art that amplification or detect in pollen yellow-white or orange millet genomic dna, must detect or increase the sequence obtained containing shown in SeqIDNo.1.5 ' the end of SeqIDNo.1 and/or the length of 3 ' the upstream and downstream sequence of holding are suitable length, be not particularly limited, such as, the length meeting molecule marker is less than 10,000bp, is less than 5,000bp, is less than 2,000bp, be less than 1,500bp, be less than 1,200bp, be less than 1,000bp or be less than 800bp.
In one embodiment of the invention, 5 ' the end of the SeqIDNo.1 that described molecule marker (DNA fragmentation containing nucleotide sequence shown in SeqIDNo.1) comprises and/or 3 ' holds be operably connected artificial sequence and/or control sequence, such as promotor, enhanser, terminator, restriction enzyme site, primer sequence etc.Wherein, term " operationally " is defined as a kind of following conformation in the present invention, and in this conformation, control sequence such as promotor is appropriately placed on a position of SeqIDNo.1, so that the generation of this control sequence polypeptide of instructing SeqIDNo.1 to encode.
The invention also discloses a kind of recombinant vectors, it contains molecule marker of the present invention.Described recombinant vectors can be the expression vector or the cloning vector that are inserted with molecule marker of the present invention.After obtaining above-mentioned recombinant vectors, it will be understood by those skilled in the art that according to different needs, recombinant vectors is transformed in suitable cell, obtain the reconstitution cell containing this recombinant vectors.Therefore, the invention also discloses a kind of reconstitution cell containing described recombinant vectors.
The invention also discloses the preparation method of molecule marker of the present invention, comprise the steps: to use the genomic dna of pollen color yellow-white or orange millet as template, carry out pcr amplification with above-mentioned primer pair, the 706bp amplified production obtained is namely containing described molecule marker; Preferably, the step of pcr amplification product being carried out purifying is also comprised.
To those skilled in the art, be appreciated that and also the method for DNA chemosynthesis can obtain molecule marker of the present invention.
The invention also discloses the detection method of described molecule marker, comprise step: according to the nucleotide sequence design primer of above-mentioned molecule marker, with detected millet genomic dna for template increases, and judge whether there is this molecule marker in amplified production.Preferably, described primer is the above-mentioned primer pair respectively containing SeqIDNo.2 and SeqIDNo.3.
Such as, with the genomic dna of detected millet for template, pcr amplification can be carried out with above-mentioned primer (SeqIDNo.2 and SeqIDNo.3), obtains amplified production.The amplified production obtained can be carried out checking order or gel electrophoresis.
The invention also discloses described molecule marker in millet pollen color gene location or the purposes in detecting.
The invention also discloses the method for a kind of millet pollen color gene location, described method comprises the step using molecule marker of the present invention.
The invention also discloses the purposes of described molecule marker in millet assistant breeding.
The invention also discloses a kind of millet auxiliary breeding means, described method comprises the step detecting molecule marker of the present invention or molecular marker primer pair.
Molecule marker of the present invention can be used in molecular mark from now on, it will be appreciated by those skilled in the art that, as by detection whether exist molecule marker of the present invention to screen millet pollen whether containing control pollen color be yellow-white or orange color gene (such as, can reference, the purposes of DNA molecular marker in wheat breeding for disease resistance, Long Dong institute journal (natural science edition), volume the 1st phase April the 16th in 2006, P65-69).Described detection can be the method that PCR detects, and particularly, can use the primer pair of above-mentioned molecule marker of the present invention.Described detection can also be undertaken by sequence measurement.This millet auxiliary breeding means has easy, quick, high-throughout advantage.
In the present invention, particularly, described millet can be a paddy No. 1, millet A2 sterile line, No. 3, a confused flour beetle or the F2 generation of opening confused flour beetle No. 3 selfings generations.Wherein, paddy No. 1 pollen color yellow-white is opened; Millet A2 sterile line pollen color brown; Open confused flour beetle No. 3 pollen colors orange; Open the F2 seville orange flower powder color part yellow-white of confused flour beetle No. 3 selfings generations, part is orange, part is brown.
Owing to adopting above technical scheme, the beneficial effect that the present invention is possessed is:
The invention provides and the closely linked molecule marker of millet pollen color gene, genomic dna sequence and millet pollen color gene connect by this molecule marker, are conducive to the foundation of millet molecular mark system; The hereditary close linkage distance of described molecule marker and millet pollen color gene is 1.2cM.Molecule marker of the present invention and molecule marker amplimer can be applied to Millet Breeding practice and resource and cultivar identification easy, quick, high-throughput.
Embodiment
The invention discloses a kind of primer pair and molecule marker SIsv0553 closely linked with millet pollen color gene.Utilize primer pair of the present invention, with millet genomic dna for template carries out PCR, can obtain and the closely linked molecule marker of millet pollen color gene, this molecule marker is called after molecule marker SIsv0553 in the present invention.It is pointed out that and it will be understood by those skilled in the art that except obtaining except molecule marker of the present invention by above-mentioned pcr amplification, molecule marker of the present invention can also be obtained by chemosynthesis.
Primer pair of the present invention contains sequence shown in ordered list SeqIDNo.2 and SeqIDNo.3 respectively,
SeqIDNo.2:5’-CAGGATAAACTTCACCAATCG-3’;
SeqIDNo.3:5’-CGAACCATTCATATCTGGCT-3’。
Those skilled in the art know, in sequence shown in above-mentioned SeqIDNo.2 and SeqIDNo.3,1 ~ 10 base can be increased respectively at its 5 ' end or 3 ' end, the base type increased also can be determined according to basepairing rule according to the base type in region that millet genomic dna matches with SeqIDNo.2 and SeqIDNo.3, the primer pair obtained thus substantially identical with the amplified production of SeqIDNo.2 and SeqIDNo.3 (DNA sequence dna between upstream and downstream primer is identical).Therefore, the above-mentioned 5 ' end or 3 ' at SeqIDNo.2 and SeqIDNo.3 is held to increase by 1 ~ 10 base respectively and can increase and is obtained the primer pair of basic same DNA fragment, includes in primer pair of the present invention.In the embodiment that the present invention is concrete, primer pair of the present invention is preferably sequence shown in SeqIDNo.2 and SeqIDNo.3.
The present invention, by paternal pollen yellow-white and the brown homozygote hybridization of maternal pollen, obtains F1 generation (No. 3, confused flour beetle), then produces F2 for colony with F1 generation selfing, totally 480 individual plants.Preferred employing SV molecular markers development method, first carries out denovo order-checking (60XcontigN50:22K, scaffoldN50:320K to male parent; Totalsize:400Mb), female parent is resurveyed sequence (10X); Then according to Parent sequencing data, sequence difference between the SOAP comparison Parent utilizing Hua Da independent development, respectively in 5 ' end and 3 ' the about 50bp position, end outside of male parent diversity sequence, the primer of the Design of length diversity sequence amplification of random selecting about 20bp, devises 1105 pairs of primers according to different diversity sequences.Increase for template with the DNA of Parent and F1, from 1105 pairs of primers, filter out 616 to the primer with polymorphism and validity.Adopt the 616 pairs of primers developed, PCR detection is carried out to 480 individualities of F2 colony, and carries out data statistic analysis, carry out genetic map drafting with MapMaker3.0 software, obtain molecule marker closely linked with pollen color gene of the present invention, and amplimer.Adopt the male parent sequence that the primer pair amplifies filtered out obtains, the molecule marker SIsv0553 namely in the present invention.Character analysis is carried out to F2 individuality, and according to gene character data and phenotype trait data, millet pollen color gene is positioned on genetic map.
Also by reference to the accompanying drawings the present invention is described in further detail below by specific embodiment.Following examples are only further detailed the present invention, should not be construed as limitation of the present invention.
Embodiment 1: millet F2 is for the structure of segregating population
Male parent: anti-Sethoxydin, plant type is high, and boot leaf is long and narrow, and bristle is red, and clever shell is red, and can educate, leaf colour cast is green, pollen yellow-white, and heading stage is late period.Male parent is paddy No. 1 seed.
Maternal: not anti-Sethoxydin, plant type is short, and boot leaf is short and wide, and bristle is green, and clever shell is green, partial sterility, and leaf colour cast is yellow, and pollen is brown, and heading stage is early stage.Female parent is millet A2 male-sterile seed.
F2 informative population: male parent and hybridization of female parent obtain F1 generation (F1 pollen color is orange), and F1 selfing obtains F2.Wherein F1 is a confused flour beetle No. 3 seeds.Altogether F2 for individual plant 480 strain, wherein, pollen color be yellow-white have 120 strains, orange has 240 strains, and brown has 120 strains.
Above-mentioned paddy No. 1 seed, millet A2 male-sterile seed and confused flour beetle No. 3 seeds can see Chinese patent application " molecule marker SIsv0372s closely linked with millet anti-herbicide gene ", publication number CN101974521A, date of publication on February 16th, 2011.
Embodiment 2: Parent and F1 generation, F2 are for the extraction of genes of individuals group DNA
Extract the genomic dna of the Parent in embodiment 1, F1 generation and 480 F2 generation individualities respectively by CTAB method, concrete grammar is as follows:
(1) take the fresh blade of 1.0g, shred and put into mortar, add 3mL1.5 × CTAB with after liquid nitrogen grinding, grind to form homogenate and proceed in the centrifuge tube of 15mL, in mortar, then add 1mL1.5 × CTAB flushing proceed to again in centrifuge tube.In 65 DEG C of water-bath 30min after mixing, period slowly shakes up frequently.
Wherein 1.5 × CTAB formula following (1L):
Add deionized water and be settled to 1L, before using, add the mercaptoethanol that final concentration is 0.2% (2ml).
(2) to be cooled to room temperature, add equal-volume chloroform/primary isoamyl alcohol (24: 1), mix gently, become deep green to subnatant.
(3) the centrifugal 10min of 4200rpm, moves on to new 15mL centrifuge tube mutually, adds the dehydrated alcohol of 2 times of volume precoolings, mix static 5min by upper water.Place 30min in-20 DEG C and precipitate DNA.
(4) the centrifugal 10min of 4200rpm, discards supernatant, adds 1mL75% washing with alcohol and precipitates 1 time, is inverted centrifuge tube dry DNA, adds 200 μ LTE dissolving DNAs.
(5) genomic dna is detected with the sepharose of 0.8%.
(6) the genomic dna individual Parent obtained and F1 generation, F2 generation is stored in-20 DEG C for subsequent use.
Embodiment 3: the preparation of molecule marker
With the male parent extracted in embodiment 2, F1 generation or the genomic dna in F2 generation for template, with molecule marker amplimer, pcr amplification is carried out to (SeqIDNo.2 and SeqIDNo.3).
PCR reaction system is as follows:
PCR response procedures is as follows:
94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds, 60 DEG C of annealing 30 seconds, 72 DEG C extend 40 seconds, run 35 circulations; Last 72 DEG C extend 3 minutes.Pcr amplification product can 4 DEG C of preservations.
Obtain molecule marker through above-mentioned amplification procedure, preferably after amplification, amplified production is carried out purification process.Check order after purifying, result is as shown in SeqIDNo.1.
To those skilled in the art, be appreciated that and also can obtain this molecule marker by the method for DNA chemosynthesis.
Embodiment 4:SV molecular markers development
Male parent: denovo checks order, 60XcontigN50:22K, scaffoldN50:320K; Totalsize:400Mb; Maternal: resurvey sequence 10X.
According to Parent sequencing data, utilize SOAP software (the such as SOAP2.20 of Hua Da independent development, can download from http://soap.genomics.org.cn/, also other sequence alignment program can be used) compare sequence difference between Parent, then based on the sequence of difference, with the primer of primerpremier software design amplification diversity sequence; Based on different diversity sequences, devise altogether 1105 pairs of primers.Part primer sequence (SeqIDNo.2-SeqIDNo.41) has wherein been shown in table 1 below
Part primer in table 11105 pair random primer
Respectively with the Parent extracted and the genomic dna of F1 generation for template, carry out pcr amplification with design 1105 pairs of primers.
PCR reaction system (25 μ L):
PCR response procedures: 94 DEG C of denaturation 5min; Then enter 35 circulations: 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 40s; After loop ends, 72 DEG C extend 3min; 4 DEG C of preservations.
PCR primer electrophoresis detection: the sepharose 120v electrophoresis 25min of 1.2%, EB dye 10min, according to glue and record.
The validity of primer and polymorphism: refer to whether have amplified production in this validity, polymorphism refers to that between Parent, the clip size of amplified production is variant.
The screening of primer is carried out: Parent and F1 all have amplified production according to following screening criteria, and Parent amplified production all only has a distinct banding pattern and size is variant, F1 shows as the heterozygosis banding pattern of Parent banding pattern, namely has two bands of male parent and maternal banding pattern.
The selection result: according to above-mentioned screening criteria, filters out 616 pairs of primers from 1105 pairs of primers of design.
Embodiment 5: genetic map construction and the assignment of genes gene mapping
(1) genetic map construction
Carry out PCR detection with the molecule marker that 616 couple of exploitation has a polymorphism to 480 of F2 colony individualities, template used is 480 of obtained F2 colony individual genomic dnas.
Agarose gel electrophoresis is carried out to PCR primer, obtains the result of molecular marker primer pair 480 individuality amplification.
Whole electrophoresis result is carried out data statistic analysis, concrete grammar is as follows: by F2 colony individual plant amplified band be male parent type be designated as a, amplified band be maternal type be designated as b, amplified band is designated as h simultaneously containing male parent type and maternal type, band Fuzzy or disappearance be designated as-, be equivalent to shortage of data, finally obtain the genotype data of 616 pairs of individual primer amplifications of F2 colony 480.Such as, 480 that obtain with pair of primers individual data are a, b, h ,-, b ... totally 480 data, the data obtained with second pair of primer are b, a, h, a ,-... totally 480 data, totally 616 pairs of primers are added up respectively, and gained is the genotype data of this F2 colony.
With MapMaker3.0 software (ConstructinggeneticmapswithMAPMAKER/EXP3.0, SLincoln, MDaly, ELander-Cambridge, MA:WhiteheadInstitute, 1992) carry out genetic linkage maps drafting, obtain genetic linkage map.Position and and the genetic distance of millet pollen color gene of 616 pairs of primers can be determined from the genetic linkage map that this obtains.
(2) assignment of genes gene mapping
According to 480 individual pollen color phenotype, similar to male parent type proterties is designated as a (pollen yellow-white), similar to maternal type proterties is designated as b (pollen is brown), and proterties occupy and is designated as h (pollen is orange) between male parent and female parent.Obtain 480 individual phenotypic datas, 480 individual phenotypic datas and 480 that obtain before individual genotype datas are compared, similar Gao Ze represents this mark and pollen color trait close linkage, and is positioned on genetic linkage maps by pollen color gene.
Embodiment 6: with the checking of the closely linked molecule marker of millet pollen color gene
1. on the basis of the obtained genetic linkage maps of embodiment 5, according to the genetic linkage distance with millet pollen color gene, the position being 1.2cM in the hereditary close linkage distance with millet pollen color gene determines molecule marker primer (SeqIDNo.2 and SeqIDNo.3), and find corresponding male parent sequence location, sequence between upstream and downstream primer is molecule marker, and its nucleotide sequence is as shown in SeqIDNo.1.
SeqIDNo.2:5’-CAGGATAAACTTCACCAATCG-3’;
SeqIDNo.3:5’-CGAACCATTCATATCTGGCT-3’。
2. in addition, in the electrophoresis to 480 of F2 generation individual pcr amplification products in embodiment 5, amplification for molecule marker primer (SeqIDNo.2 and SeqIDNo.3) is: pollen color yellow-white or orange plant amount to about 360 strains (the wherein plant of pollen color yellow-white about 120 strain, plant about 240 strain that pollen color is orange), their amplified production all has the band of 706bp size, and the pcr amplification product of the plant of about 120 strain pollen color brown does not all have the band of 706bp (part amplification as shown in Figure 1).And prove that through order-checking the sequence of the fragment of this 706bp is identical with SeqIDNo.1.
Visible, molecule marker of the present invention (SeqIDNo.1) is and the closely linked molecule marker of millet pollen color gene.
Embodiment 7: molecular marker clone
The fragment of the 706bp obtained that increases in embodiment 6 is cloned in pMD18-T carrier, obtains recombinant vectors.This recombinant vectors is transformed in e. coli jm109, chooses mono-clonal, cultivate and obtain reconstitution cell.Plasmid is extracted from reconstitution cell, described plasmid and recombinant vectors, adopt M13 universal primer (sequence information is with reference to TaKaRa goods catalogue) to check order to cloned sequence, result shows, containing molecule marker of the present invention (SeqIDNo.1) in recombinant vectors.The steps such as above-mentioned clone, conversion, cultivation, plasmid extraction are with reference to " the Molecular Cloning: A Laboratory guide third edition ", and Huang Peitang etc. translate, and Science Press publishes in September, 2002.
Above content is in conjunction with concrete embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.