CN108774646A - With the relevant SNP marker of the thick character of millet fringe and its detection primer and application - Google Patents

With the relevant SNP marker of the thick character of millet fringe and its detection primer and application Download PDF

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CN108774646A
CN108774646A CN201810340250.8A CN201810340250A CN108774646A CN 108774646 A CN108774646 A CN 108774646A CN 201810340250 A CN201810340250 A CN 201810340250A CN 108774646 A CN108774646 A CN 108774646A
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millet
snp marker
fringe
thick
snp
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赵治海
赵芳
冯小磊
魏玮
张耕耘
王峰
张晓磊
张雅莉
张文英
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ZHANGJIAKOU ACADEMY OF AGRICULTURAL SCIENCES
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Abstract

This application discloses a kind of and relevant SNP marker of the thick character of millet fringe and its detection primer and applications.The application and the relevant SNP marker of the thick character of millet fringe, the SNP marker are located in the bin labels of Article 2 chromosome 31036193bp to 32556243bp, SNP marker and the thick close linkage of millet fringe.The application and the relevant SNP marker of the thick character of millet fringe, it can be used for the molecular mark of the thick character of millet fringe, it can predict that millet fringe is thick by SNP marker detection, early stage identification or screening breeding to realize the thick character of millet fringe provide scientific basis, have important theory and practice directive significance to the hereditary and selection or improvement process of accelerating millet variety.The application detects the primer pair of SNP marker, and parting, the difference of detection SNP expression can be slightly carried out to millet fringe.

Description

With the relevant SNP marker of the thick character of millet fringe and its detection primer and application
Technical field
This application involves Millet Breeding technical fields, more particularly to a kind of and relevant SNP marker of the thick character of millet fringe And its detection primer and application.
Background technology
China is the source area of millet and cultivated area maximum, the highest country of yield, yield account for about the whole world in the world The 80% of total amount.Meanwhile China is also the country that Genetic Resource of Foxtail Millet quantity is most, diversity is most abundant.
The raising of millet yield is that Multiple factors are coefficient as a result, fringe is slightly as an importance of millet fringe portion Shape has close relationship with yield, is the important indicator of single plant yield height, research and the control thick Isoquant character of millet fringe Relevant gene and its position functions etc., for instructing millet genetic breeding to have great importance.
Single nucleotide polymorphism (singlenucleotide polymorphism, SNP) is in genomic level by single DNA sequence polymorphism caused by the variation of nucleotide base, quantity is more in genome, widely distributed, and genetic stability is good. With the development of gene sequencing technology and the reduction of cost, to Different Individual same gene or genetic fragment carry out direct Sequencing and Sequence compares, it will be able to determine base with the presence or absence of variation.Therefore, SNP detections are conducive to Genotyping, are suitable for quickly and advise Modelling screening is unknown or the relationship of known SNP and certain inhereditary feature.
The exploitation of SSR marker is focused primarily upon about the relevant QTL positioning of Quantitative Characters of Foxtail Millet and SNP marker research at present It utilizes etc. by way of and the development and application for being detected the SNP marker of polymorphism using group's self-mating system degeneracy gene order-checking are ground Study carefully and not yet has been reported that.Therefore, carry out the exploitation of Quantitative Characters of Foxtail Millet SNP marker, and establish assist-breeding system, for carrying High millet yield saves breeding cost and is of great significance.
Invention content
The purpose of the application is to provide a kind of new with the relevant SNP marker of the thick character of millet fringe and its detection primer and answer With.
To achieve the goals above, the application uses following technical scheme:
The one side of the application discloses a kind of and relevant SNP marker of the thick character of millet fringe, which is located at second In the bin labels of article chromosome 31036193bp to 32556243bp, also, SNP marker and the thick close linkage of millet fringe.
It should be noted that the application is found that the sequence of one section and the thick close linkage of millet fringe, this section of sequence by research SNP marker in row directly affects the thick character of millet fringe, this section of sequence, that is, Article 2 chromosome 31036193bp to The bin of 32556243bp is marked.
Further investigation revealed that in the bin labels of Article 2 chromosome 31036193bp to 32556243bp, especially It is that the base of SNP marker is C or T, with the thick close linkage of millet fringe.
In a kind of realization method of the application, one of SNP marker, the i.e. sites PD-02-348, PD- are specifically disclosed The sites 02-348 are located at the positions sequence 31510348bp of Article 2 chromosome, the sequence such as Seq where the sites PD-02-348 Shown in ID No.1, the sites PD-02-348 be in sequence shown in Seq ID No.1 from 5 ' end the 31st bit base.
It should be noted that the bin labels and millet fringe of Article 2 chromosome 31036193bp to 32556243bp Thick close linkage, wherein may include numerous SNP markers, the sites PD-02-348 be the application a kind of realization method in demonstrate,prove It is real with the relevant SNP marker of the thick character of millet fringe, under the guidance of the application, however not excluded that also other in bin labels SNP marker.
Preferably, the application utilizes composite interval mapping with the relevant SNP marker of the thick character of millet fringe to SNP marker Method, using 5% global significance level, the LOD value of QTL detections is 3.573, and phenotypic variation explanation rate is 3.36%.
The another side of the application discloses a kind of primer pair of the SNP marker of detection the application, and the upstream of the primer pair is drawn Object is sequence shown in Seq ID No.2, and downstream primer is sequence shown in Seq ID No.3;
Seq ID No.2:5'-GAATCGAAGACACGGTGGT-3'
Seq ID No.3:5'-GAAGGCTAGGAGGGAGGTT-3'.
The another side of the application discloses a kind of kit of the SNP marker of detection the application, which includes this The primer pair of application.
Preferably, in the kit of the application further include reagent for PCR amplification.
The another side of the application discloses a kind of method of the SNP marker of detection the application, includes drawing using the application The kit of object pair or the application carries out PCR amplification to millet genomic DNA to be measured, passes through the survey of pcr amplification product Sequence is as a result, obtain the base situation of SNP marker.
The another side of the application discloses a kind of method that prediction millet fringe is thick, includes the primer pair using the application, or The kit of person the application passes through the sequencing knot of pcr amplification product to predicting that the millet genomic DNA of object carries out PCR amplification Fruit obtains the base situation of the SNP marker of the application, predicts that millet fringe is thick according to the base situation of SNP marker.
The SNP marker for disclosing the application on one side again of the application slightly predicts in millet fringe, millet fringe slightly identification in early days or Application in millet molecule assisting sifting breeding.
The primer pair for disclosing the application on one side again of the application or the kit of the application slightly predict in millet fringe, millet Fringe slightly in early days identification or millet molecule assisting sifting breeding in application.
Due to using the technology described above, the advantageous effect of the application is:
The application and the relevant SNP marker of the thick character of millet fringe can be used for the molecular labeling auxiliary of the thick character of millet fringe Breeding can predict that millet fringe is thick by SNP marker detection, to realize early stage identification or the screening breeding of the thick character of millet fringe Scientific basis is provided, there is important theory and practice to instruct meaning the hereditary and selection or improvement process of accelerating millet variety Justice.The application detects the primer pair of SNP marker, and parting, the difference of detection SNP expression can be slightly carried out to millet fringe.
Description of the drawings
Fig. 1 is millet Parent genome dna electrophoresis figure in the embodiment of the present application, wherein the first swimming lane is M swimming lanes, table Show that DL2000marker, the second swimming lane are swimming lane expression male parent " the Ji paddy No.1 " genomic DNA for being labeled as 1, third swimming lane The swimming lane for being labeled as 2 indicates A2 female parent gene groups DNA;
Fig. 2 is that millet Parent genomic DNA is expanded using primer pair 9_2F and 9_2R in the embodiment of the present application PCR product electrophoretogram, wherein the first swimming lane is the swimming lane for marking A9-2, is indicated using maternal " A2 " genomic DNA as template PCR product, the second swimming lane are the swimming lane for being labeled as 39-2, are indicated using male parent " Ji paddy No.1 " genomic DNA as the PCR of template Product, third swimming lane are M swimming lanes, indicate DL2000marker;
Fig. 3 is the comparison knot of the sequence in maternal and male parent SNP marker where the sites PD-02-348 in the embodiment of the present application Fruit is schemed, and the sites PD-02-348 are in the bin labels of Article 2 chromosome 31036193bp to 32556243bp, maternal alkali Base is C, and male parent base is T.
Specific implementation mode
This application provides SNP marker, detection primer and its applications with millet fringe slightly relevant bin marked regions.Paddy The sub- thick close linkage SNP marker PD-02-348 of fringe is located at Article 2 chromosome 31036193bp to 32556243bp's In bin labels, it is particularly located at the positions sequence 31510348bp of Article 2 chromosome.
It should be noted that bin refers in Bin mappings (i.e. Bin map), using whole in mapping population in the application Recombination site information obtains the least unit (Recombination bin) for constituting recombination event.Using obtained bin as Label is for subsequent linkage map structure and QTL positioning.
In the application, genetic map (genetic map or linkage map) refers to using recombination fraction between genetic marker as base The linear array figure of relative position between the label of plinth structure.Based on high throughput weight sequencing technologies by mapping population parent and Progeny population is sequenced, and using a certain number of continuous SNP as the foundation for judging filial generation recombination site, obtains each filial generation Full-length genome physics recombinates collection of illustrative plates.Using recombination site information whole in mapping population, the minimum for constituting recombination event is obtained Unit (Recombination bin) and Bin figures (i.e. Bin map), and the bin utilized is used for subsequent company as label Lock map construction and QTL positioning.
The application offer millet male parent " Ji paddy No.1 " and maternal " A2 " material, the F1 generation material of the two hybridization, and from Hand over 441 parts of materials of F2 groups in 13 generations.Each part material is planted in body in Zhangjiakou Area, Hebei Province, and the characters numbers such as record and arrangement fringe are thick According to.After each part material progress degeneracy genome is resurveyed sequence, compares the splicing of reference gene group and complete, obtain SNP polymorphism marks. By the thick trait data of fringe, associated SNP marker is obtained, and SNP marker is verified by cloning and sequencing.
The application is described in further detail below by specific embodiments and the drawings.Following embodiment is only to the application It is further described, should not be construed as the limitation to the application.
One high-flux sequence of embodiment and SNP marker information analysis
This example utilizes millet male parent " Ji paddy No.1 " and maternal " A2 " material, the F1 generation material of the two hybridization, and selfing 441 parts of materials of F2 groups in 13 generations, i.e. recombinant inbred lines (Recombinant inbred lines, abridge RILs).By each part Material is planted in body in Zhangjiakou Area, Hebei Province, and is recorded and arranged fringe and slightly wait trait datas.Each part material progress degeneracy genome is resurveyed After sequence, compares the splicing of reference gene group and complete, obtain SNP marker.By analyzing the thick trait data of fringe, obtain associated SNP marker, and SNP marker is verified by cloning and sequencing.
This example utilizes RADseq methods, extracts the genomic DNA of each individual of sample, amounts to 444 samples, including it In 441 RILs, 2 parents, 1 F1 individual.Quality testing is carried out to the genomic DNA of extraction, enzyme is carried out to qualified DNA It cuts, electrophoresis recycles DNA fragmentation, and carries out cluster preparations plus connector, finally upper machine sequencing.
This example is as follows:
(1) the fresh blades of 1.0g are weighed, shreds and is put into mortar, with 1.5 × CTAB of 3mL are added after liquid nitrogen grinding, are ground into Homogenate is transferred in the centrifuge tube of 15mL, and 1.5 × CTAB of 1mL are then added into mortar rinses, then is transferred in centrifuge tube, mixing Afterwards in 65 DEG C of water-bath 30min, during which slowly shake up frequently.
1.5 × CTAB of wherein 1L is formulated:The Tris.Cl75mL of the pH8.0 of CTAB, 1mol/L of 15g, EDTA 30mL, the NaCl 61.4g of 0.5mol/L, adds deionized water to be settled to 1L;Using it is preceding be added about 2mL mercaptoethanol, Make its final concentration of 0.2%.
(2) it after the completion of water-bath, is cooled to room temperature, isometric chloroform/isoamyl alcohol is added, gently mixing, until subnatant becomes Bottle green;Wherein the volume ratio of chloroform/isoamyl alcohol is chloroform:Isoamyl alcohol is 24:1.
(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 50 μ L TE dissolving DNAs are added in dry DNA.
(5) concentration for detecting DNA, is used in combination water to be adjusted to 20ng/ μ L.
(6) it using PstI enzymes to carry out digestion, interrupts genomic DNA, the endonuclease reaction system of PstI enzymes is 30 μ L, including: 20 μ L of genomic DNA, 1 μ L of PstI enzymes, 10 × PstI buffer solutions, 3 μ L, 6 μ L of Nuclease-free water.Endonuclease reaction body After system prepares, in 37 DEG C of constant-temperature incubations at least 1 hour.
(7) adjunction head reacts, and sequence measuring joints are added on the genomic DNA fragment interrupted, and the system of adjunction head reaction is 40 μ L, including:30 μ L of PstI digestion products, 1 μ L of sequence measuring joints, 10 × connection buffer 1 μ L, 2 μ L of T4DNA ligase, rATP 0.4μL、ddH2O 5.6μL。
(8) each sample respectively takes 1 μ L reaction products, is added in a new centrifuge tube, one group, every group of every 12 samples Total volume be 12 μ L.
(9) 300-700bp size segments are cut after dyeing with 3% recycling gel electrophoresis 1h, EB.It is carried out with QIAquick Kit Glue purification recycles, and recovery product is dissolved in the EB solution of 30 μ L.
(10) PCR reactions are carried out, PCR amplification, PCR are carried out to the segment for making sure to keep in mind recycling using the universal primer of sequence measuring joints 25 μ L of reaction system, including:10 × buffer, 2.5 μ L, the 0.25 μ L of dNTPs of 25mM, 5U/ μ L 0.25 μ L of high fidelity enzyme, It is 10 μM of 0.5 μ L of sequence measuring joints forward primer, 10 μM of 0.5 μ L of sequence measuring joints reverse primer, 20 μ L of gel extraction product, sterile 1 μ L of water.
PCR reaction conditions are 94 DEG C of pre-degeneration 5min, are recycled subsequently into 10:94 DEG C are denaturalized 30 seconds, 60 DEG C of annealing 30 Second, 72 DEG C extend 40 seconds, after circulation terminates 72 DEG C extension 3min.
(11) library is built in magnetic beads for purifying, completion.
Specifically purification process is:First, 1.2 times of volume magnetic beads are added into PCR product, stand 10min.Then, it is placed in It is adsorbed on magnetic frame, removes supernatant.Then, 500 μ L, 70% ethanol wash is added twice.After being evaporated on xeothermic instrument, 15 μ L are added EB solution dissolves 5min.Finally, it is adsorbed on magnetic frame, transfer supernatant is in 1.5mL centrifuge tubes.
(12) detection reaches upper machine sequencing after machine standard.
As a result:Digestion is carried out to 444 samples and builds library sequencing, obtains 75.99Gb initial data, average each individual 171.54Mb.Sequencing sequence is compared onto reference gene group, average comparison rate 86.326%, average coverage rate 8.226% is put down Sequencing depth be 3.655 ×.
Wherein, 444 samples include 441 RILs, 2 parents, 1 F1 individual;Reference gene group, that is, the Yugu of this example Genome can obtain Yugu genome sequences from following Internet address hffp:
https://www.ncbi.nlm.nih.gov/genome/?Term=Setaria+italica+ (foxtail+ millet)。
By sequencing and information analysis, there are 33771 polymorphic SNP markers between acquisition parent.According to the side of window sliding Method, it is a window to choose several SNP, slides a SNP every time and determines the genotype of each window and each individual Site is exchanged, bin genotype and bin figures are generated.According to bin genotype datas, with MSTMap software building genetic maps, 2022 A bin is navigated on 9 chromosomes, then genetic map data is imported into MapChart softwares, integrates a genome heredity Linkage map.
Using constructed millet dense genetic map, (CIM) is analyzed using composite interval mapping, property thick to millet fringe Shape phenotype carries out qtl analysis.QTL detections use 5% global significance level, according to 500 permutation tests as a result, determining that fringe is thick The critical LOD value of trait phenotypes data qtl analysis, analysis obtain and the thick relevant sections prediction bin of fringe and SNP site information.
The results show that it is located at 210.44 centimorgans (cM) of Article 2 chromosome with the relevant SNP marker of the thick character of millet fringe, 172nd bin to the 173rd bin (i.e. chr2_bin172-chr2_bin173), that is, Article 2 chromosome In the bin labels of 31036193bp to 32556243bp;Wherein, the sites PD-02-348 are located at the sequence of Article 2 chromosome The positions 31510348bp;The base of these and the relevant SNP marker of the thick character of millet fringe is all C or T.Made using compound section Figure method, using 5% global significance level, the LOD value of QTL detections is 3.573, and phenotypic variation explanation rate is 3.36%, additivity Effect value (Additive effect, A) is -0.0966.
Two SNP marker of embodiment is verified
According to the bin of prediction labels and SNP site, reference gene group is compared, relevant range gene order is obtained, chosen 300bp or so before and after SNP site designs and develops SNP marker primer, and PCR expansions are carried out as template using male parent and female parent material DNA Increase.Selection primer amplification is normal, the amplified production of the compound prediction size of pcr amplification product carries out gel extraction, and is sequenced, Selection male parent and female parent material amplification gene sequence have the labeled primer of SNP site difference.Using F1 generation and F2 for material as masterplate, Carry out SNP marker primer PCR amplification and result sequencing, selection meet Genotyping and with the relevant labeled primer of phenotypic character.
First, the sequencing result after being recycled according to PCR product selects male parent and female parent material amplification gene sequence SNP The label of point difference.It is as follows:
(1) according to step (1) to (4) in embodiment one, male parent and female parent gene group DNA are extracted respectively with CTAB methods.
(2) with 0.8% agarose gel electrophoresis Detection and Extraction male parent and female parent gene group DNA, testing result such as Fig. 1 It is shown, wherein the second swimming lane is swimming lane expression male parent " the Ji paddy No.1 " genomic DNA for being labeled as 1, and third swimming lane marks Maternal " A2 " genomic DNA is indicated for 2 swimming lane.It can be seen that male parent and female parent gene group DNA are extracted successfully.
(3) by the male parent of extraction and female parent gene group DNA be stored in -20 DEG C it is spare.
(4) respectively using the male parent of extraction and female parent gene group DNA as template, using primer pair 9_2F and 9_2R, to gene Group DNA is expanded.The sense primer 9_2F of primer pair is sequence shown in Seq ID No.2, and downstream primer 9_2R is Seq ID Sequence shown in No.3;
Seq ID No.2:5'-GAATCGAAGACACGGTGGT-3'
Seq ID No.3:5'-GAAGGCTAGGAGGGAGGTT-3'.
25 μ L of PCR reaction systems, including:The Taq enzyme of 10 × buffer, 2.5 μ L, the 0.15 μ L of dNTPs of 25mM, 5U/ μ L 0.15 μ L, 10 μM of 0.5 μ L of sense primer, 10 μM of 0.5 μ L of downstream primer, 1.0 μ L of genomic DNA, 20.2 μ L of sterile water.
PCR reaction conditions are 94 DEG C of pre-degeneration 5min, are recycled subsequently into 35:94 DEG C are denaturalized 30 seconds, 60 DEG C of annealing 30 Second, 72 DEG C extend 40 seconds, after circulation terminates 72 DEG C extension 3min.
In 4 DEG C of preservations after pcr amplification product is purified.Each pcr amplification product takes part to carry out 1% agarose gel electrophoresis Detection, the results are shown in Figure 2.Fig. 2 is the PCR product electrophoretogram that primer pair 9_2F and 9_2R are expanded.The result of Fig. 2 is aobvious Show, the primer pair of design can go out to meet expected target fragments to male parent and maternal genomic DNA amplification.
This example respectively survey by the amplified production of the male parent to primer pair 9_2F and 9_2R amplification and female parent gene group DNA Sequence, sequencing sequence sequence, the PD-02- as shown in Seq ID No.1 of the male parent amplified production that primer 9_2F and 9_2R are expanded 348 sites be in sequence shown in Seq ID No.1 from 5 ' end the 31st bit base.Male parent amplified production and maternal amplified production Sequencing sequence comparison result is as shown in figure 3, wherein the base of background mark ash indicates the base in the sites PD-02-348, maternal base For C, male parent base is T.
Then, it is screened according to sample fringe raw data:Male parent fringe is slightly 7cm, and maternal fringe is slightly 5cm.In 441 parts of samples, most Small value 3cm, maximum value 9cm;In the thick 50 minimum samples of fringe, sample number identical with maternal SNP is more than 75%;Fringe is slightly maximum 50 samples in, sample number identical with male parent SNP be more than 75%.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen Specific implementation please is confined to these explanations.For those of ordinary skill in the art to which this application belongs, it is not taking off Under the premise of conceiving from the application, a number of simple deductions or replacements can also be made.
SEQUENCE LISTING
<110>Academy of Agriculture, Zhangjiakou City's (high and cold crop research institute in Hebei province)
<120>With the relevant SNP marker of the thick character of millet fringe and its detection primer and application
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<170> PatentIn version 3.3
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aactttcaca atatccaaga tataccatta cttacatttt tgtgatgtat ctcaaatttt 180
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Claims (10)

1. a kind of and relevant SNP marker of the thick character of millet fringe, it is characterised in that:The SNP marker is located at Article 2 chromosome In the bin labels of 31036193bp to 32556243bp, the SNP marker and the thick close linkage of millet fringe.
2. SNP marker according to claim 1, it is characterised in that:The base of the SNP marker is C or T.
3. SNP marker according to claim 1 or 2, it is characterised in that:The SNP marker includes the sites PD-02-348, The sites PD-02-348 are located at the positions sequence 31510348bp of Article 2 chromosome, the sites the PD-02-348 place Sequence as shown in Seq ID No.1, the sites PD-02-348 be in sequence shown in Seq ID No.1 from 5 ' end the 31st Bit base.
4. SNP marker according to claim 3, it is characterised in that:SNP marker is used using composite interval mapping method The LOD value of 5% global significance level, QTL detections is 3.573, and phenotypic variation explanation rate is 3.36%.
5. a kind of test right requires the primer pair of 1-4 any one of them SNP markers, it is characterised in that:The primer pair Sense primer is sequence shown in Seq ID No.2, and downstream primer is sequence shown in Seq ID No.3;
Seq ID No.2:5'-GAATCGAAGACACGGTGGT-3'
Seq ID No.3:5'-GAAGGCTAGGAGGGAGGTT-3'.
6. a kind of test right requires the kit of 1-4 any one of them SNP markers, it is characterised in that:In the kit Including the primer pair described in claim 5;Preferably, in the kit further include reagent for PCR amplification.
7. a kind of method that test right requires 1-4 any one of them SNP markers, it is characterised in that:Including being wanted using right The kit described in primer pair or the claim 6 described in 5 is sought, PCR amplification is carried out to millet genomic DNA to be measured, is led to The sequencing result for crossing pcr amplification product obtains the base situation of the SNP marker.
8. a kind of method that prediction millet fringe is thick, it is characterised in that:Include using the primer pair described in claim 5, Huo Zhequan Profit requires the kit described in 6, to predicting that the millet genomic DNA of object carries out PCR amplification, passes through the survey of pcr amplification product Sequence is as a result, obtain the base situation of claim 1-4 any one of them SNP markers, according to the base situation of the SNP marker Predict that millet fringe is thick.
9. slightly being predicted in millet fringe according to claim 1-4 any one of them SNP marker, millet fringe slightly identification or millet in early days Application in molecule assisting sifting breeding.
10. kit described in primer pair according to claim 5 or claim 6 is slightly predicted in millet fringe, millet fringe Application in thick early stage identification or millet molecule assisting sifting breeding.
CN201810340250.8A 2018-04-16 2018-04-16 With the relevant SNP marker of the thick character of millet fringe and its detection primer and application Pending CN108774646A (en)

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Citations (1)

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Application publication date: 20181109