CN102031259B - Molecular marker SIsv0641 closely linked with millet plant height genes - Google Patents
Molecular marker SIsv0641 closely linked with millet plant height genes Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology and relates to a molecular marker, in particular to a molecular marker closely linked with millet plant height genes. The nucleotide sequence of the molecular marker is shown in SEQ ID NO:1, or the molecular marker is a DNA fragment containing a nucleotide sequence shown in SEQ ID NO:1 in a millet genome. The invention also relates to a primer of the molecular marker, an application of the molecular marker in positioning of millet plant height genes or genetic breeding of millet, a millet plant height gene positioning method and a millet breeding method. The invention discovers a molecular marker SIsv0641 closely linked with millet plant height genes and contacts a millet genome DNA sequence with the millet plant height genes, thereby being more favorable for establishing a millet molecular marker aided breeding system.
Description
Technical field
The invention belongs to biology field, relate to a kind of molecule marker, particularly, relate to a kind of and the closely linked molecule marker of millet plant height gene.The invention still further relates to primer, purposes, a kind of millet plant height gene positioning method and a kind of Millet Breeding method of this molecule marker in the assignment of genes gene mapping of millet plant height or millet genetic breeding of this molecule marker.
Background technology
Millet (Setaria italica L.Beauv.) is the important food crop that originate from China, mainly at the north of China, occupies the status of outbalance in national food production and grain security.Millet is the diploid self pollination crop, and its genome is less, about 470Mb, and these characteristics make it be well suited for object as genome research.
At present, the structure of genetic linkage maps has become the important foundation of the assignment of genes gene mapping, map based cloning and genome structure and functional study.And molecule marker is the basis that makes up genetic linkage maps.Plant height is as the Main Agronomic Characters that is related to high crop yield and stable yields, and the plant type, lodging resistance, Regional Distribution and the output that are directly connected to kind consist of proterties.Therefore, in the urgent need to finding and the closely linked molecule marker of millet plant height gene.
Summary of the invention
The inventor, provides a kind of and the closely linked molecule marker SIsv0641 of millet (Setaria italica L.Beauv.) plant height gene, and following invention is provided thus through a large amount of experiment and unremitting efforts according to whole genome sequence:
One aspect of the present invention relates to a kind of and the closely linked molecule marker SIsv0641 of millet plant height gene, and its nucleotide sequence is shown in SEQ ID NO:1.Wherein add frame and partly be the design of primers section:
520bp
In the present invention, term " with the closely linked molecule marker of millet plant height gene " refers to such molecule marker, and in genetic linkage maps, the genetic linkage of this molecule marker and millet plant height gene distance is less than 2cM or less than 3cM; Perhaps millet greater than 400 samples in, contain this molecule marker in the sample of non-short plant type.
The primer that also relates in one aspect to molecule marker of the present invention of the present invention, its nucleotide sequence is shown in SEQ ID NO:2 and SEQ ID NO:3:
SIsv0641F：CTCACTGCAAGACTCGACCT(SEQ ID NO：2)
SIsv0641R：CGCTTCCCATCCATGCCATT(SEQ ID NO：3)。
Molecule marker of the present invention also can be for containing the dna fragmentation of nucleotide sequence shown in the SEQ ID NO:1; The length of this dna fragmentation is suitable length, but is not particularly limited, for example, and less than 10,000bp, less than 5,000bp, less than 2,000bp, less than 1,500bp, less than 1,200bp, less than 1,000bp or less than 800bp.
In one embodiment of the invention, described molecule marker (dna fragmentation that contains nucleotide sequence shown in the SEQ ID NO:1) is the dna fragmentation of nucleotide sequence shown in the SEQ ID NO:1 in the millet genome, 5 ' the end of the SEQ ID NO:1 that namely comprises and/or the nucleotide sequence beyond the 3 ' end also are the sequences in the millet genome, preferably, be the 5 ' end of SEQID NO:1 in the millet genome and/or the upstream and downstream sequence of 3 ' end.It will be understood by those skilled in the art that as long as amplification or detect this molecule marker in the millet genomic dna, must detect or increase and obtain containing the sequence shown in the SEQ ID NO:1.The length of the 5 ' end of SEQ ID NO:1 and/or the upstream and downstream sequence of 3 ' end is suitable length, be not particularly limited, for example, satisfy the length of molecule marker less than 10,000bp, less than 5,000bp, less than 2,000bp, less than 1,500bp, less than 1,200bp, less than 1,000bp or less than 800bp.
In one embodiment of the invention, 5 ' the end and/or 3 ' of the SEQ ID NO:1 that described molecule marker (dna fragmentation that contains nucleotide sequence shown in the SEQ ID NO:1) comprises is held be operably connected artificial sequence and/or control sequence, for example promotor, enhanser, terminator, restriction enzyme site, primer sequence etc.Wherein, term " operationally " is defined as a kind of following conformation in the present invention, in this conformation, control sequence for example promotor is suitably placed on the position of SEQ ID NO:1, so that this control sequence instructs the generation of the polypeptide of SEQ ID NO:1 coding.
Another aspect of the present invention relates to a kind of recombinant vectors, and it contains molecule marker of the present invention.Described recombinant vectors can be expression vector or the cloning vector that is inserted with molecule marker of the present invention.Of the present inventionly also relate in one aspect to a kind of reconstitution cell, it contains this recombinant vectors.
The preparation method who also relates in one aspect to molecule marker of the present invention of the present invention comprises the steps: to use the genomic dna of millet of non-short plant type as template, carries out pcr amplification with above-mentioned primer, and the amplified production that obtains namely contains described molecule marker; Preferably, also comprise the step of pcr amplification product being carried out purifying.In one embodiment of the invention, the millet of described non-short plant type is the non-short plant type of F2 in generation of No. 3, paddy No. 1, confused flour beetle or No. 3 selfings of a confused flour beetle generation.
To those skilled in the art, be appreciated that also can the DNA chemosynthesis method obtain molecule marker of the present invention.
The detection method that also relates in one aspect to described molecule marker of the present invention comprises the steps:
(1) nucleotide sequence according to molecule marker of the present invention designs primer;
(2) genomic dna with detected millet increases as template;
(3) judge whether there is this molecule marker in the amplified production.
For example, can be take the genomic dna of detected millet as template, (SEQID NO:2 and SEQ ID NO:3) carries out pcr amplification with above-mentioned primer, obtains amplified production.The amplified production that obtains can be checked order or gel electrophoresis.
The purposes of molecule marker of the present invention in the assignment of genes gene mapping of millet plant height or detection that also relate in one aspect to of the present invention.
The method that also relates in one aspect to the assignment of genes gene mapping of a kind of millet plant height of the present invention comprises the step of using molecule marker of the present invention.
The purposes of molecule marker of the present invention in the millet assistant breeding that also relate in one aspect to of the present invention.
Of the present inventionly also relate in one aspect to a kind of millet auxiliary breeding means, comprise the step that detects molecule marker of the present invention.
Molecule marker of the present invention can be used in from now on the molecular mark, it will be appreciated by those skilled in the art that, such as by detect whether exist molecule marker of the present invention to screen millet ideotype (for example, can reference, the purposes of dna molecular marker in wheat breeding for disease resistance, east, Gansu Province institute's journal (natural science edition), the 1st phase of the 16th volume April in 2006, P65-69).Detect the non-short plant type that is with molecule marker of the present invention, do not have the short plant type that is of this molecule marker.Described detection can be the method that PCR detects, and particularly, can use above-mentioned molecule marker primer 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 the F2 generation of a paddy No. 1, millet A2 sterile line, No. 3, a confused flour beetle or No. 3 selfings of a confused flour beetle generation.Wherein, the part F2 generation (plant height is about 100cm) that millet A2 sterile line, No. 3 selfing of confused flour beetle produce is short plant type; Another part F2 generation that No. 3, paddy No. 1, confused flour beetle or No. 3 selfing of confused flour beetle produce (plant height be about 130cm and about 150cm) is non-short plant type.
The beneficial effect of the invention
The invention provides and the closely linked molecule marker of millet plant height gene, and millet genomic dna and millet plant height gene are connected, more be conducive to the foundation of millet molecular mark system; The hereditary close linkage distance of described molecule marker and plant height gene is 2.1cM.Molecule marker of the present invention can be applied to the millet assistant breeding easy, quick, high-throughput.
Description of drawings
Fig. 1: molecule marker primer (SEQ ID NO:2 and SEQ ID NO:3) is to the partial results of F2 generation 480 individual plant amplifications.
M represents marker, and it is 200bp DNA Ladder; Its molecular weight comprises: 4000bp, 3000bp, 2500bp, 2000bp, 1800bp, 1600bp, 1400bp, 1200bp, 1000bp, 800bp, 600bp, 400bp and 200bp.Swimming lane 1-12 is the pcr amplification product of F2 individual plant of the non-short plant type of 12 strains in generation; Swimming lane 13-24 is the pcr amplification product of F2 individual plant of the short plant type of 12 strains in generation.The result shows: stripe size is 520bp in the plant of non-short plant type, and stripe size is less than 520bp in the plant of short plant type.
Explanation about the biomaterial preservation
The present invention relates to following biomaterial:
1. open No. 1 seed of paddy, it is preserved in Chinese Typical Representative culture collection center (CCTCC) on September 6th, 2010, and deposit number is CCTCC NO:P201012, and the preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, and 430072.
2. millet A2 male-sterile seed, it is preserved in Chinese Typical Representative culture collection center (CCTCC) on September 6th, 2010, deposit number is CCTCC NO:P201013, and the preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, 430072.
3. open No. 3 seeds of confused flour beetle, it is preserved in Chinese Typical Representative culture collection center (CCTCC) on September 6th, 2010, and deposit number is CCTCC NO:P201010, and the preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, and 430072.
Embodiment
Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: millet F2 is for the structure of segregating population
Male parent is for opening paddy No. 1: plant type high (non-short plant type, plant height are about 150cm), and anti-Sethoxydin (Sethoxydin is a kind of weedicide), boot leaf is long and narrow, and bristle is red, and clever shell is red, can educate, and the leaf colour cast is green.
Female parent is the A2 sterile line: plant type short (short plant type, plant height are about 100cm), and not anti-Sethoxydin, boot leaf is short and wide, and bristle is green, and clever shell is green, partial sterility, the leaf colour cast is yellow.
Male parent and hybridization of female parent obtain F1 (plant height is about 130cm for No. 3, a confused flour beetle, non-short plant type).
F1 produces F2 for colony for selfing, totally 480 individual plants.480 F2 are carried out the plant height character analysis for individuality, find non-short plant type 360 strains (strain be about high 130cm and 150cm about), short plant type 120 strains (plant height is about 100cm).
Embodiment 2: in Parent and F1 generation, F2, are for the extraction of genes of individuals group DNA
Extract respectively Parent among the embodiment 1, F1 generation and 480 F2 for the genomic dna of individuality with the CTAB method, concrete grammar is as follows:
(1) takes by weighing the fresh blade of 1.0g, shred and put into mortar, with adding 3ml1.5 * CTAB after the liquid nitrogen grinding, grind to form homogenate and change in the centrifuge tube of 15ml, then add 1ml 1.5 * CTAB flushing in the mortar and change in the centrifuge tube again.Behind the mixing in 65 ℃ of water-baths 30 minutes, during frequently slowly shake up.
1.5 * CTAB prescription following (1L) wherein:
CTAB 15g
1M Tris.Cl (pH is 8.0) 75ml
0.5M EDTA 30ml
NaCl 61.4g
Add deionized water and be settled to 1L, adding final concentration before using is the mercaptoethanol of 0.2% (2ml).
(2) to be cooled to room temperature, add equal-volume chloroform/primary isoamyl alcohol (24: 1), mixing gently becomes deep green to subnatant.
(3) 4200rpm is centrifugal 10 minutes, and upper water is moved on to new 15ml centrifuge tube mutually, adds the dehydrated alcohol of 2 times of volume precoolings, mixes static 5 minutes.Place 30 minutes precipitation DNA in-20 ℃.
(4) 4200rpm is centrifugal 10 minutes, discards supernatant, adds 1ml 75% washing with alcohol precipitation 1 time, is inverted the centrifuge tube dry DNA, adds 200 μ l TE dissolving DNAs.
(5) detect genomic dna with 0.8% sepharose.
(6) with the Parent that obtains and F1 generation, F2 for the genomic dna of individuality be stored in-20 ℃ for subsequent use.
Embodiment 3: the preparation of molecule marker
The genomic dna of the male parent of extracting in the embodiment 2, F1 generation or the non-short plant type of F2 in generation is as template, and (SEQ ID NO:2 and SEQ ID NO:3) carries out pcr amplification with the molecule marker primer.
The PCR reaction system is as follows:
Sterilized water 20.2 μ l
10*Buffer (contains Mg
2+) 2.5 μ l
dNTPs(25mM) 0.15μl
Taq enzyme (5U/ μ l) 0.15 μ l
Forward primer 0.5 μ l
Reverse primer 0.5 μ l
Template 1.0 μ l
Cumulative volume 25 μ l
The PCR response procedures is as follows:
94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 40 seconds, and moved 35 circulations; Last 72 ℃ were extended 3 minutes.Pcr amplification product can be 4 ℃ of preservations.
With the amplified production purifying, obtain molecule marker.Check order behind the purifying, the result is shown in SEQ IDNO:1.
To those skilled in the art, be appreciated that also and can obtain by the method for DNA chemosynthesis this molecule marker.
Embodiment 4: the preliminary screening of molecule marker primer
Male parent is carried out de novo order-checking (60X contig N50:22K, scaffold N50:320K; Total size:400Mb), the female parent order (10X) of resurveying.
According to the Parent sequencing data, utilize SOAP software (SOAP2.20 for example, can download from http://soap.genomics.org.cn/, also can use other sequence alignment software) sequence difference between the Parent relatively, then based on the sequence of difference, in male parent 5 ' end and about 50bp position, 3 ' the end outside, choose at random the length about 20bp, with primerpremier software Random Design primer, designed altogether 1105 pairs of primers, the sequence (SEQ ID NO:2-41) of part primer wherein has been shown in the following table 1.
Table 1:1105 is to the part primer in the random primer
The genomic dna in the Parent that extracts in the embodiment 2 respectively and F1 generation is as template, carries out pcr amplification with 1105 pairs of primers of design, and is similar among other condition of PCR reaction system and program and the embodiment 3.
The PCR product is carried out agarose electrophoresis detect, to detect validity and the polymorphism of primer.Refer in this validity whether amplified production is arranged; Polymorphism refers to that the clip size of amplified production is variant between Parent.
Carry out the screening of primer according to following screening criteria: Parent and F1 all have amplified production, and the Parent amplified production all only has a distinct banding pattern and big or small variant, F1 shows as the heterozygosis banding pattern of Parent banding pattern, and two bands of male parent and maternal banding pattern are namely arranged.
The selection result: from 1105 pairs of primers of Random Design, filter out 616 pairs of primers.
Embodiment 5: millet F2 is for the structure of genetic linkage maps
480 individualities with 616 pairs of molecule marker primers F 2 colonies that obtain among the embodiment 4 carry out respectively the PCR detection, the genomic dna of template used 480 individualities for the F2 colony that makes among the embodiment 2, other composition and the PCR program of PCR system are identical with embodiment 4.
The PCR product is carried out agarose gel electrophoresis, and wherein molecule marker primer (SEQ ID NO:2 and SEQ ID NO:3) to 480 individual partial results that increase as shown in Figure 1.
Whole electrophoresis result are carried out data statistic analysis, concrete grammar is as follows: be a that is designated as of male parent type with F2 colony individual plant amplified band, amplified band is the b that is designated as of maternal type, amplified band contains the h that is designated as of male parent type and maternal type simultaneously, all do not have be designated as-, finally obtain the genotype data of 616 pairs of primer amplifications of 480 individualities of F2 colony.Such as, the data of 480 individualities that obtain with first pair of primer are a, b, and h ,-, b ... totally 480 data, the data that obtain 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 MapMaker 3.0 softwares (Constructing genetic maps withMAPMAKER/EXP 3.0, S Lincoln, M Daly, E Lander-Cambridge, MA:Whitehead Institute, 1992) carry out the genetic linkage maps drafting, obtain genetic linkage map.The genetic linkage map subscript understands that the position of 616 pairs of primers reaches and the genetic distance of plant height gene.
According to the plant height phenotype of 480 individualities, similar to the male parent type proterties a (non-short plant type) that is designated as, similar to the maternal type proterties b (short plant type) that is designated as, proterties occupy and is designated as h (non-short plant type) between male parent and the female parent.Obtain the phenotypic data of 480 individualities, the phenotypic data of 480 individualities compares with the genotype data of 480 individualities that obtain before, and similar Gao Ze represents this mark and plant height proterties close linkage.
According to the above results with the plant height assignment of genes gene mapping on genetic linkage maps.
Embodiment 6: with the checking of the closely linked molecule marker of millet plant height
1. on the basis of the genetic linkage maps that embodiment 5 makes, according to the genetic linkage distance of millet plant height gene, in the hereditary close linkage distance with millet plant height gene be 2.1cM location positioning molecule marker primer (SEQ ID NO:2 and SEQ ID NO:3), and find corresponding male parent sequence location, sequence between the upstream and downstream primer is molecule marker, and its nucleotide sequence is shown in SEQ ID NO:1.
2. in addition, in the electrophoresis to the pcr amplification product of 480 individualities in F2 generation in embodiment 5, amplification for molecule marker primer (SEQ ID NO:2 and SEQ ID NO:3) is: the amplified production of 360 non-short plant types all has the band of 520bp size, and the pcr amplification product of 120 short plant types does not all have the band (the part amplification as shown in Figure 1) of 520bp.And prove that through checking order the sequence of the fragment of this 520bp is identical with SEQ ID NO:1.
As seen, molecule marker of the present invention (SEQ ID NO:1) is and the closely linked molecule marker of millet plant height gene.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (11)
- One kind with the closely linked molecule marker of millet plant height gene, its nucleotide sequence is shown in SEQ ID NO:1.
- 2. recombinant vectors, it contains molecule marker claimed in claim 1.
- 3. reconstitution cell, it contains recombinant vectors claimed in claim 2.
- 4. the primer of molecule marker claimed in claim 1, its nucleotide sequence is shown in SEQ ID NO:2 and SEQ ID NO:3.
- 5. the preparation method of molecule marker claimed in claim 1 comprises the steps:Use the genomic dna of millet of non-short plant type as template, carry out pcr amplification with primer claimed in claim 4;Wherein, the millet of described non-short plant type is that preserving number is that paddy No. 1, the preserving number of CCTCC NO:P201012 is No. 3, the confused flour beetle of CCTCC NO:P201010 or the non-short plant type of F2 in generation that No. 3 selfing of confused flour beetle produces.
- 6. preparation method according to claim 5, it also comprises the step of the product of pcr amplification being carried out purifying.
- 7. the detection method of molecule marker claimed in claim 1 comprises the steps:(1) according to claim 1 the nucleotide sequence design primer of molecule marker;(2) genomic dna with detected millet increases as template;(3) judge whether there is this molecule marker in the amplified production.
- 8. molecule marker claimed in claim 1 is in the assignment of genes gene mapping of millet plant height or the purposes in detecting; Wherein, described millet is that preserving number is that paddy No. 1, the preserving number of CCTCC NO:P201012 is No. 3, the confused flour beetle of CCTCC NO:P201010 or the non-short plant type of F2 in generation that No. 3 selfing of confused flour beetle produces.
- 9. the method for the millet plant height assignment of genes gene mapping comprises that right to use requires the step of 1 described molecule marker; Wherein, described millet is that preserving number is that paddy No. 1, the preserving number of CCTCC NO:P201012 is No. 3, the confused flour beetle of CCTCC NO:P201010 or the non-short plant type of F2 in generation that No. 3 selfing of confused flour beetle produces.
- 10. the purposes of molecule marker claimed in claim 1 in the millet assistant breeding.
- 11. a millet auxiliary breeding means comprises that test right requires the step of 1 described molecule marker.
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| CN102220321B (en) * | 2011-06-02 | 2012-12-26 | 深圳华大基因科技有限公司 | Molecular marker SI sv0057 closely linked with millet fertility gene |
| CN108660232A (en) * | 2017-12-08 | 2018-10-16 | 深圳华大小米产业股份有限公司 | A kind of molecular labeling SIsv0338 and application with millet Plant height gene close linkage |
| CN108660230A (en) * | 2017-12-08 | 2018-10-16 | 深圳华大小米产业股份有限公司 | A kind of molecular labeling SIsv0163 with millet anti-imazethapyr herbicide resistance gene close linkage |
| CN108660237A (en) * | 2017-12-19 | 2018-10-16 | 深圳华大小米产业股份有限公司 | A kind of molecular labeling SIsv1099 and application with millet Plant height gene close linkage |
| CN108715901B (en) * | 2018-04-16 | 2022-03-29 | 张家口市农业科学院 | SNP marker related to millet plant height character and detection primer and application thereof |
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