CN108660234A - With the molecular labeling SIsv0362 of millet leaf color gene close linkage - Google Patents

With the molecular labeling SIsv0362 of millet leaf color gene close linkage Download PDF

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CN108660234A
CN108660234A CN201711295539.4A CN201711295539A CN108660234A CN 108660234 A CN108660234 A CN 108660234A CN 201711295539 A CN201711295539 A CN 201711295539A CN 108660234 A CN108660234 A CN 108660234A
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molecular labeling
millet
primer
leaf color
seq
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邹洪锋
蔡伟
崔巨多
肖建军
杨祖荣
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Zhongyuan Huada Agricultural Technology Co Ltd
Shenzhen Huada Millet Industry Ltd By Share Ltd
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Zhongyuan Huada Agricultural Technology Co Ltd
Shenzhen Huada Millet Industry Ltd By Share Ltd
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/13Plant traits

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Abstract

The invention belongs to molecular biology fields, are related to a kind of molecular labeling, and in particular, to a kind of molecular labeling with millet leaf color gene close linkage, the molecular labeling contain SEQ ID NO:Nucleotide sequence shown in 1.The invention further relates to expand purposes, a kind of millet leaf color gene positioning method and a kind of Millet Breeding method of primer, the molecular labeling and the primer of the molecular labeling in the assignment of genes gene mapping of millet leaf color or millet genetic breeding.Present invention finds the molecular labeling SIsv0362 with millet leaf color gene close linkage, the hereditary close linkage distance of the molecular labeling and millet leaf color gene is 1.7cM, millet genomic dna sequence and millet leaf color genetic link are got up, the foundation of millet molecular mark system is more advantageous to.The molecular labeling and molecular labeling amplimer of the present invention can it is easy, quickly, be applied to millet assistant breeding with high throughput, there is important theory and practice directive significance for the hereditary and selection and improvement process of accelerating millet variety.

Description

With the molecular labeling SIsv0362 of millet leaf color gene close linkage
Technical field
The invention belongs to technical field of molecular biology, are related to a kind of molecular labeling, and in particular, to a kind of and millet leaf The molecular labeling SIsv0362 of piece color gene close linkage.The invention further relates to primer, the molecules for expanding the molecular labeling Purposes in the assignment of genes gene mapping of millet leaf color or millet genetic breeding of label and primer, a kind of millet leaf color gene are fixed Position method and a kind of Millet Breeding method.
Background technology
Millet is initiated by important cereal crops and the characteristic drought-resistant crops in China, and seed sprouting only needs to account for its heavy The moisture of amount 26%, and the seed of sorghum, wheat and corn sprouts 40%, 45% and 48% water for being respectively necessary for its weight Point, while millet is higher to the endurance of low soil fertility.Millet is that environmentally friendly crop and diploid self-pollination are made Object, genome about 470M, is highly suitable as genome research object.
Plant leaf color is the general performance of various pigments in chloroplaset, and normal blade Determination of Chlorophyll is dominant, is usually showed For green.Leaf variegation is that the frequency of mutation is higher in higher plant and is easy to the mutant character of identification.So far, rice, Wheat, barley, corn and soybean, cotton, tobacco, corn, sunflower, tomato, cucumber, potato, mulberry tree etc. are nearly all high Leaf color mutant has been had been found that in plant.Its mutator directly or indirectly influences the synthesis of light and pigment, especially chlorophyll And degradation, cause the content of various pigments and ratio to change, so as to cause plant leaf variegation.
Hua Da gene studies institute started to millet genomics research project jointly early in 2009, and complete in 2011 At stage Journal of Sex Research, achievement is in May, 2012 in internationally famous magazine《Nature-biotechnology》(Nature Biotechnology it is delivered online on).Currently, the structure of genetic linkage maps has become the assignment of genes gene mapping, map based cloning and base Because of the important foundation of group function research, and molecular labeling is the basis for building genetic linkage maps.Leaf color is straight It connects and is related to crop photosynthesis efficiency, be the Main Agronomic Characters of high yield and stable yields.Therefore, there is an urgent need to find and millet leaf The molecular labeling of piece color gene close linkage.
Invention content
The present inventor provides a kind of and paddy according to millet whole genome sequence according to a large amount of data analysis and experiment The molecular labeling SIsv0362 of sub (Setaria italica L.Beauv.) leaf color gene close linkage, and thus provide Following inventions:
One aspect of the present invention is related to a kind of and millet leaf color gene close linkage molecular labeling SIsv0362, The molecular labeling is SEQ ID NO:Nucleotide sequence shown in 1.It is design of primers section wherein to underline part:
CAGGTCTACTATTCAAGTCCAATCTCTTTTGACGTACAATCCAACTATAACCTAAGGACATTGCGTTTGTAATGTGT TTGGTACTACGATTCGCACCCATCCGGGTGCCGGAAAACAAGACATGTCACGCTTGAATTTACCAAGACATACTTTT TTTTGTAGACGAAAGCATCAAAACAAACTGGTCCGACAATCCCTGCACTTTCCCATTCTTTGCTTATTTCTCCCATC ACCAAGTTGTCTGCACACAGAAAGAGGGAGGAAAGCACAAGACCCAGTCTCCCCAATGCCCCCCATAGACCAAGTTT GGTTAGACAAGCAAGG(SEQ ID NO:1)
In the present invention, term " molecular labeling with millet leaf color gene close linkage " refers to such molecule mark Note, in genetic linkage maps, the genetic linkage distance of the molecular labeling and millet leaf color gene is less than 2cM or is less than 3cM;Or being more than in 400 samples in millet, the molecular labeling is contained in the sample of blade edge colour pattern.Blade edge colour pattern It determines referring to description hereinafter.
The further aspect of the present invention is related to the primer pair of the molecular labeling SIsv0362 of the amplification present invention, the primer pair Primer 1 is to contain SEQ ID NO:Sequence shown in 2, primer 2 are to contain SEQ ID NO:Sequence shown in 3;Preferably, primer 1 is SEQ ID NO:Sequence shown in 2, primer 2 are SEQ ID NO:Sequence shown in 3;
SIsv0362F:5’-CCTTGCTTGTCTAACCAAAC-3’(SEQ ID NO:2)
SIsv0362R:5’-CAGGTCTACTATTCAAGTCC-3’(SEQ ID NO:3)
The molecular labeling of the present invention may be containing SEQ ID NO:The DNA fragmentation of nucleotide sequence shown in 1, the DNA The length of segment is suitable length, but is not particularly limited, for example, less than 10,000bp, it is less than 5,000bp, less than 2, 000bp, it is less than 1,500bp, is less than 1,200bp, is less than 1,000 or is less than 800bp.
In one embodiment of the invention, the molecular labeling (contains SEQ ID NO:Nucleotide sequence shown in 1 DNA fragmentation) it is SEQ ID NO in millet genome:The DNA fragmentation of nucleotide sequence shown in 1, that is, the SEQ ID NO for being included: Nucleotide sequence other than 15 ' ends and/or 3 ' ends is also the sequence in millet genome, it is preferable that in millet genome SEQ ID NO:15 ' ends and/or the upstream and downstream sequence at 3 ' ends.As long as it will be understood by those skilled in the art that amplification or inspection The molecular labeling in millet genomic DNA is surveyed, necessarily can detect or expand to obtain containing SEQ ID NO:Sequence shown in 1 Row.SEQ ID NO:The length of 15 ' ends and/or the upstream and downstream sequence at 3 ' ends is suitable length, is not particularly limited, for example, The length for meeting molecular labeling is less than 10,000bp, is less than 5,000bp, is less than 2,000bp, is less than 1,500bp, is less than 1, 200bp, it is less than 1,000bp or is less than 800bp.
In one embodiment of the invention, the molecular labeling (contains SEQ ID NO:Nucleotide sequence shown in 1 DNA fragmentation) the SEQ ID NO that are included:15 ' ends and/or 3 ' ends, which are operably connected, artificial sequence and/or control sequence Row, such as promoter, enhancer, terminator, restriction enzyme site, primer sequence etc..Wherein, term " operationally " is in the present invention Defined in be a kind of following conformation, in the conformation, control sequence such as promoter is appropriately placed SEQ ID NO:The one of 1 On a position, so that the control sequence instructs SEQ ID NO:The generation of the polypeptide of 1 coding.
Another aspect of the present invention relates to a kind of recombinant vectors, contain the molecular labeling of the present invention.The recombinant vector Can be the expression vector or cloning vector of the molecular labeling inserted with the present invention.The further aspect of the present invention is related to a kind of heavy Group cell, contains the recombinant vector.
The further aspect of the present invention is related to a kind of method for the molecular labeling preparing the present invention, includes the following steps:It uses The millet genomic DNA of blade edge colour pattern carries out PCR amplification as template, with above-mentioned primer pair, and obtained amplified production contains There is the molecular labeling;Preferably, further include the steps that purifying the product of PCR amplification.In the implementation of the present invention In scheme, the blade edge colour pattern millet is the F that No. 3 selfings of paddy No. 1, a confused flour beetle 3 or confused flour beetle generate2Leaf in generation Piece green type.
To those skilled in the art, it will be understood that can also the chemically synthesized methods of DNA obtain the present invention molecule Label.
The further aspect of the present invention is related to a kind of method of the detection molecular labeling, includes the following steps:
(1) the nucleotide sequence design primer of molecular labeling according to the present invention;
(2) it is expanded using the genomic DNA for being detected millet as template;
(3) judge to whether there is the molecular labeling in amplified production.
Preferably, the primer contains SEQ ID NO respectively to be above-mentioned:2 and SEQ ID NO:3 primer pair.
For example, can be using the genomic DNA of detected millet as template, with above-mentioned primer (SEQ ID NO:2 and SEQ ID NO:3) PCR amplification is carried out, amplified production is obtained, obtained amplified production can be carried out to sequencing or gel electrophoresis.
The further aspect of the present invention is related to the molecular labeling of the present invention or the primer of the molecular labeling in millet leaf color Purposes in the assignment of genes gene mapping or detection.
The further aspect of the present invention is related to the molecular labeling of the present invention or the primer of the molecular labeling in millet assistant breeding In purposes.
The further aspect of the present invention is related to a kind of method of the millet leaf color assignment of genes gene mapping, including uses point of the present invention The step of primer of son label or the molecular labeling.
The further aspect of the present invention is related to a kind of millet auxiliary breeding means, includes the molecular labeling or use of the detection present invention The step of primer of the molecular labeling is detected.
The molecular labeling of the present invention can be used in molecular mark from now on, and those skilled in the art can manage Solution, such as by detecting whether the molecular labeling in the presence of the present invention come whether screen millet leaf color be green (for example, can be with With reference to, purposes of the DNA molecular marker in wheat breeding for disease resistance, Long Dong institutes journal (natural science edition), April the 16th in 2006 Roll up the 1st phase, P65-69).It detects that the millet leaf color of the molecular labeling with the present invention is green, does not have the molecule mark The millet leaf color of note is yellow.The detection can be PCR detection method, specifically, can use the above-mentioned present invention Molecular labeling primer.The detection can also be carried out by the method for sequencing.The millet auxiliary breeding means have quick, letter Just, high-throughput advantage.
In the present invention, specifically, the millet can be paddy No. 1, millet A2 sterile lines, confused flour beetle 3 or open miscellaneous The F that No. 3 selfings of paddy generate2Generation.Wherein, the part F that No. 3 millet A2 sterile lines, confused flour beetle selfings generate2On behalf of blade yellow type; Open the part F that No. 3 selfings of paddy No. 1, confused flour beetle 3 or confused flour beetle generate2On behalf of blade edge colour pattern.
Advantageous effect of the invention
The present invention provides the molecular labelings with millet leaf color gene close linkage, and by millet genomic DNA with Millet leaf color genetic link gets up, and is more advantageous to the foundation of millet molecular mark system;The molecule mark Note and the hereditary close linkage distance of leaf color gene are 1.7cM.Molecular labeling, the amplimer of the present invention can be to millet Leaf color gene carries out good parting, and the early stage identification and screening breeding to realize millet leaf color provide molecule and assist skill Art supports, can it is easy, quickly, be applied to millet assistant breeding with high throughput, for accelerate millet variety hereditary and selection and Improvement process has important theory and practice directive significance.
Description of the drawings
Fig. 1:Molecular labeling primer (SEQ ID NO:2 and SEQ ID NO:3) to F2The part knot of 480 single plant amplifications of generation Fruit.Wherein:
Swimming lane 2,8,10,11,12,14,15,17,19,20,21,23,24 is F2The single plant of 13 plants of blade edge colour patterns in generation Pcr amplification product.Swimming lane 1,3~7,9,13,16,18,22 is F2The single plant pcr amplification product of 11 plants of blade yellow types in generation. Swimming lane M is marker, is D2000;Its molecular weight includes 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp.Knot Fruit shows:Stripe size is 324bp in the plant of blade edge colour pattern, and stripe size is 742bp in the plant of blade yellow type.
Specific implementation mode
Embodiment of the present invention is described in detail in the following with reference to the drawings and specific embodiments, but art technology Personnel will be understood that the following example is merely to illustrate the present invention, and should not be construed as the limitation of the present invention.In following embodiments Experimental method be unless otherwise specified to be carried out according to the condition that normal condition or manufacturer suggest.In following embodiments Material, reagent used etc. are unless otherwise specified commercially or the conventional products of acquisition purchased in market.
Embodiment 1:The structure of millet genetic group
Male parent is paddy No. 1:High plant type, leaf green, a kind of anti-Sethoxydin (herbicide), boot leaf is long and narrow, and bristle is red Color, glume is red, fertile.
Female parent is A2 sterile lines:Short plant type, blade yellow, not anti-Sethoxydin, boot leaf short-wide, bristle green, glume are green Color, partial sterility.
Male parent and hybridization of female parent obtain F1(blade edge colour pattern), F1480 F are obtained in selfing2For single plant.To 480 F2It is single Strain carries out leaf color character analysis, finds 360 plants of blade edge colour pattern, 120 plants of blade yellow type.
Embodiment 2:Parent and F1Generation, F2For the extraction of genes of individuals group DNA
Parent, F are extracted respectively with CTAB methods1The 480 millet F obtained in generation and embodiment 12The genome of single plant DNA, the specific method is as follows:
(1) the fresh blades of 1.0g are weighed, shreds and is put into mortar, with 3mL1.5 × CTAB is added after liquid nitrogen grinding, are ground into Homogenate is transferred in the centrifuge tube of 15mL, and 1mL1.5 × CTAB is then added into mortar rinses, then is transferred in centrifuge tube, after mixing In 65 DEG C of water-bath 30min, during which slowly shake up frequently.
Wherein 1.5 × CTAB formulas are following (1L):The 1mol/L Tris.HCl (pH 8.0) of CTAB, 75mL of 15g, 0.5mol/L EDTA of 30mL, the NaCl of 61.4g, add deionized water to be settled to 1L, using the preceding mercaptoethanol that 2ml is added, make The final concentration of 0.2ml/100ml of mercaptoethanol.
(2) it is cooled to room temperature, isometric chloroform/isoamyl alcohol (24 is added:1), gently mixing, until subnatant becomes dark green Color.
(3) 4200rpm centrifuges 10min, and upper strata aqueous phase is moved on to new 15mL centrifuge tubes, adds 2 times of volumes to be pre-chilled anhydrous Ethyl alcohol mixes static 5min.30min, which is placed, in -20 DEG C precipitates DNA.
(4) 4200rpm centrifuges 10min, discards supernatant, 75% ethyl alcohol of 1mL washing precipitation is added 1 time, and it is dry to be inverted centrifuge tube 200 μ L TE dissolving DNAs are added in dry DNA.
(5) genomic DNA is detected with 0.8% Ago-Gel.
(6) by obtained Parent, F1Generation and 480 millet F2The genomic DNA of single plant be stored in -20 DEG C it is spare.
Embodiment 3:The preparation of molecular labeling
With extracted in embodiment 2 male parent, F1Generation or F2The genomic DNA of blade edge colour pattern in generation is template, to divide Sub- labeled primer (SEQ ID NO:2 and SEQ ID NO:3) PCR amplification is carried out.
A.PCR reaction systems:25 μ L systems, the content of each component substance are respectively:1 μ L template DNAs, 0.5 μ L primers Fs, 0.5 μ L primers R, 0.15 μ L dNTPs (10mM), 0.15 μ L Taq archaeal dna polymerases (5U/ μ L), 2.5 10 × PCR of μ L Buffer、ddH225 μ L of O polishings, mixing, centrifugation.
B. amplification program:94 DEG C of pre-degenerations 5 minutes;94 DEG C are denaturalized 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 40 seconds, fortune 35 cycles of row;Last 72 DEG C extend 3 minutes, and pcr amplification product can be preserved at 4 DEG C.
C. amplified production is purified, obtains molecular labeling.It is sequenced after purification, as a result such as SEQ ID NO:Shown in 1.
To those skilled in the art, it will be understood that the molecule mark can also be obtained by the chemically synthesized methods of DNA Note.
Embodiment 4:The preliminary screening of molecular labeling amplimer
De novo sequencings (60X contig N50 are carried out to male parent:22K, scaffold N50:320K;Total size:400Mb), female parent carries out resurveying sequence (10X).
According to Parent sequencing data, using SOAP softwares, (such as SOAP2.20, can be from http:// Soap.genomics.org.cn/ is downloaded, and can also use other sequence alignment programs) sequence that compares between Parent is poor It is different, it is then based on the sequence of difference, at the end of male parent 5 ' and 3 ' the end outside positions about 50bp, randomly selects the sequence of 20bp or so length Row, with primer premier software Random Design primers, 1105 pairs of primers altogether list portion therein in table 1 below Separate sequence (the SEQ ID NO of object:2-41).
Table 1:Part primer in 1105 pairs of random primers
Parent and F to be extracted in embodiment 2 respectively1The genomic DNA in generation is template, with 1105 pairs of primers of design PCR amplification is carried out, the other conditions of PCR reaction systems and program are similar with embodiment 3.
PCR product is subjected to agarose electrophoresis detection, with the validity and polymorphism of detection primer.Refer in this validity Whether there is amplified production;Polymorphism refers to that the clip size of amplified production between Parent is variant.
The screening of primer is carried out according to following screening criteria:Parent and F1There is amplified production, and Parent expands Increasing production object only has an apparent banding pattern and size variant, F1It is presented with the heterozygosis banding pattern of Parent banding pattern, that is, has male parent With two bands of maternal banding pattern.
The selection result:616 pairs of primers are filtered out from 1105 pairs of primers of Random Design.
Embodiment 5:Millet F2Structure for genetic linkage maps and the assignment of genes gene mapping
(1) genetic map construction
Using the 616 pairs of molecular labeling primers filtered out in embodiment 4 to F2480 individuals of group carry out PCR respectively Detection, it is template used for F obtained in embodiment 22480 individual genomic DNAs of group, other ingredients of PCR system And PCR programs are same as Example 4.
To PCR product into row agarose gel electrophoresis, wherein molecular labeling primer (SEQ ID NO:2 and SEQ ID NO: 3) as shown in Figure 1 to the partial results of 480 individual amplifications.
Whole electrophoresis results are subjected to data statistic analysis, the specific method is as follows:By F2Group's single plant amplified band is father This type is denoted as a, and amplified band is the b that is denoted as of maternal type, and amplified band is denoted as h containing male parent type and maternal type simultaneously, all It is not no be denoted as-, finally obtain F2The genotype data of 480 individual 616 pair primer amplifications of group.For example, with first pair 480 individual data that primer obtains are a, b, h ,-, totally 480 data, the data obtained with second pair of primer are b ... ... B, a, h, a ,-... ... totally 480 data, totally 616 pairs of primers count respectively, gained is the F2The genotype data of group.
With 3.0 softwares of MapMaker (Constructing genetic maps with MAPMAKER/EXP 3.0, S Lincoln,M Daly,E Lander-Cambridge,MA:Whitehead Institute, 1992) carry out genetic linkage map Spectrum is drawn, and genetic linkage map is obtained.The position of 616 pairs of primers and the heredity with leaf color gene are designated on genetic linkage map Distance.
(2) assignment of genes gene mapping
It is similar with male parent type character to be denoted as a (leaf color green type) according to 480 individual leaf color phenotypes, Similar with maternal type character to be denoted as b (leaf color yellow type), character occupy and is denoted as h between male parent and female parent, obtains 480 The phenotypic data of individual, 480 individual phenotypic datas are compared with the 480 individual genotype datas obtained before Compared with similar Gao Ze represents the label and leaf color character close linkage.
According to the above results by the leaf color assignment of genes gene mapping on genetic linkage maps.
Embodiment 6:With the verification of the molecular labeling of millet leaf color gene close linkage
1. made from embodiment 5 on the basis of genetic linkage maps, even according to the heredity with millet leaf color gene Distance is locked, in the hereditary close linkage with millet leaf color gene apart from the location determination for being 1.7cM molecular labeling primer (SEQ ID NO:2 and SEQ ID NO:3), and corresponding male parent sequence location is found, the sequence between upstream and downstream primer is Molecular labeling, nucleotide sequence such as SEQ ID NO:Shown in 1.
2. in embodiment 5 to F2In 480 individual pcr amplification product electrophoresis in generation, molecular labeling primer (SEQ ID NO:2 and SEQ ID NO:3) amplification is:The amplified production of 453 blade edge colour patterns has the item of 324bp sizes Band, and the pcr amplification product of 27 blade yellow types has the band of 742bp, and the nucleotide of the 324bp is proved through sequencing Sequence and SEQ ID NO:1 is identical.
As it can be seen that molecular labeling (the SEQ ID NO of the present invention:1) it is the molecule with millet leaf color gene close linkage Label.
Although the specific implementation mode of the present invention has obtained detailed description, it will be understood to those of skill in the art that root According to all introductions having disclosed, these embodiments can be carry out various modifications and be replaced, these change the present invention's Within protection domain.The full scope of the present invention is given by the appended claims and any equivalents thereof.
Sequence table
<110>Shenzhen Hua Da millet industry limited liability company
Central Plains Hua Da agricultural science and technology Co., Ltd
<120>With the molecular labeling SIsv0362 of millet leaf color gene close linkage
<130> P2017-1-0173.CN
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tctgcacaca gaaagaggga ggaaagcaca agacccagtc tccccaatgc cccccataga 300
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<210> 23
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 23
catgacaagg atcgtcacag 20
<210> 24
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 24
ccaagtgtgt atgcgacaag 20
<210> 25
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 25
ccagcttgct taaggtatca 20
<210> 26
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 26
gcagtgtgtt tctttcatgg 20
<210> 27
<211> 21
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 27
gcaatggttg atagatacga t 21
<210> 28
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 28
ttcgttggct gtagcgttga 20
<210> 29
<211> 21
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 29
gaagaaatca ccaacataac c 21
<210> 30
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 30
caggacatcg ccatggtact 20
<210> 31
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 31
atgaagcgag caagtgaact 20
<210> 32
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 32
tggattatgt ggagccatgt 20
<210> 33
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 33
gcattggtct tcttccaagg 20
<210> 34
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 34
gtttctgcgc taatctgatc 20
<210> 35
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 35
ccgaaatggt ggcaattgct 20
<210> 36
<211> 21
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 36
gagtatgtcg accgtagtac t 21
<210> 37
<211> 21
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 37
gtgatggatt tgcttgtcac t 21
<210> 38
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 38
caggagcttt tgacagtgag 20
<210> 39
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 39
aggtacgctt cccttgtcat 20
<210> 40
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 40
cctacacacc gctataaacc 20
<210> 41
<211> 20
<212> DNA
<213>Artificial synthesized (Artificial Sequence)
<400> 41
atcgtgtacc cagctccgtt 20

Claims (10)

1. a kind of and millet leaf color gene close linkage molecular labeling, it is characterised in that:The molecular labeling contains SEQ ID NO:Nucleotide sequence shown in 1;Preferably, the molecular labeling is SEQ ID NO:Nucleotide sequence shown in 1.
2. a kind of recombinant vector, it is characterised in that:Contain molecular labeling described in claim 1.
3. a kind of recombinant cell, it is characterised in that:Contain the recombinant vector described in claim 2.
4. a kind of primer pair of amplification molecular labeling described in claim 1, it is characterised in that:The primer 1 of the primer pair is Contain SEQ ID NO:Sequence shown in 2, primer 2 are to contain SEQ ID NO:Sequence shown in 3;Preferably, primer 1 is SEQ ID NO:Sequence shown in 2, primer 2 are SEQ ID NO:Sequence shown in 3.
5. a kind of method preparing molecular labeling described in claim 1, includes the following steps:Use blade edge colour pattern millet Genomic DNA carries out PCR amplification as template, with the primer pair described in claim 4;Preferably, further include by PCR amplification The step of product is purified;Specifically, the blade edge colour pattern millet is paddy No. 1, a confused flour beetle 3 or opens confused flour beetle 3 certainly Hand over the F generated2Blade edge colour pattern in generation.
6. a kind of method that test right requires the molecular labeling described in 1, includes the following steps:It is according to claim 1 The nucleotide sequence design primer of molecular labeling;Genomic DNA to be detected millet is expanded as template;Judge amplification It whether there is the molecular labeling in product;Preferably, the primer is the primer pair described in claim 4.
7. the primer pair of the molecular labeling described in molecular labeling described in claim 1 or claim 4 is in millet leaf color Purposes in the assignment of genes gene mapping or detection.
8. the primer pair of the molecular labeling described in molecular labeling described in claim 1 or claim 4 is in millet assistant breeding In purposes.
9. a kind of method of the millet leaf color assignment of genes gene mapping, it is characterised in that:The method includes using described in claim 1 Molecular labeling or claim 4 described in molecular labeling primer pair the step of.
10. a kind of millet auxiliary breeding means, it is characterised in that:The method includes test rights to require the molecule mark described in 1 The step of remembering or being detected with the primer pair of the molecular labeling described in claim 4.
CN201711295539.4A 2017-12-08 2017-12-08 With the molecular labeling SIsv0362 of millet leaf color gene close linkage Pending CN108660234A (en)

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CN113046458A (en) * 2019-12-27 2021-06-29 深圳华大三生园科技有限公司 Molecular marker closely linked with millet leaf angle gene, detection method and application
CN113278719A (en) * 2020-02-20 2021-08-20 深圳市华大农业应用研究院 Molecular marker SIsv0523 closely linked with millet leaf angle gene

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CN102154285A (en) * 2011-03-24 2011-08-17 深圳华大基因科技有限公司 Molecular marker SIsv0737 closely linked with millet leaf color gene
CN102690811A (en) * 2011-03-24 2012-09-26 深圳华大基因科技有限公司 Molecular marker SIsv1363 closely linked with Setaria italica L. Beauv. leaf color gene
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Publication number Priority date Publication date Assignee Title
CN113046458A (en) * 2019-12-27 2021-06-29 深圳华大三生园科技有限公司 Molecular marker closely linked with millet leaf angle gene, detection method and application
CN113046458B (en) * 2019-12-27 2023-05-23 深圳华大三生园科技有限公司 Molecular marker closely linked with millet leaf included angle gene, detection method and application
CN113278719A (en) * 2020-02-20 2021-08-20 深圳市华大农业应用研究院 Molecular marker SIsv0523 closely linked with millet leaf angle gene
CN113278719B (en) * 2020-02-20 2024-01-30 深圳市华大农业应用研究院 Molecular marker SIsv0523 closely linked with millet leaf angle gene

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