CN109652577A - SSR molecular marker L08 and application with the high bar QTL close linkage of rice - Google Patents

SSR molecular marker L08 and application with the high bar QTL close linkage of rice Download PDF

Info

Publication number
CN109652577A
CN109652577A CN201810860012.XA CN201810860012A CN109652577A CN 109652577 A CN109652577 A CN 109652577A CN 201810860012 A CN201810860012 A CN 201810860012A CN 109652577 A CN109652577 A CN 109652577A
Authority
CN
China
Prior art keywords
rice
marker
ssr
molecular marker
high bar
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810860012.XA
Other languages
Chinese (zh)
Inventor
姚方印
陈高
张华�
柳絮
宣宁
杨永义
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biotechnology Research Center of Shandong Academy of Agricultural Sciences
Original Assignee
Biotechnology Research Center of Shandong Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biotechnology Research Center of Shandong Academy of Agricultural Sciences filed Critical Biotechnology Research Center of Shandong Academy of Agricultural Sciences
Priority to CN201810860012.XA priority Critical patent/CN109652577A/en
Publication of CN109652577A publication Critical patent/CN109652577A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Botany (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to the SSR molecular marker L08 with the high bar QTL close linkage of rice and apply.With the SSR molecular marker L08 of the high bar QTL close linkage of rice, marker upstream nucleotide sequence is as shown in SEQ ID NO.3, and marker downstream nucleotide sequence is as shown in SEQ ID NO.4.The present invention has filtered out the SSR molecular marker L08 with the high bar QTL close linkage of rice for the first time.The high bar gene of the 1st chromosome of rice can be carried out genetic analysis and finely positioning by above-mentioned SSR marker, can be used for constructing rice dense genetic map and Variety fingerprinting.

Description

SSR molecular marker L08 and application with the high bar QTL close linkage of rice
Technical field
It the present invention relates to the SSR molecular marker L08 with the high bar QTL close linkage of rice and applies, in particular to rice letter Substance complex sequences (SSR, Simple sequence repeats) evaluation of markers and its in the analysis of rice high bar gene genetic and essence Application in fine positioning belongs to DNA molecular marker technology and technical field of molecular biology.
Background technique
SSR (simple sequence repeat, simple repeated sequence), is called microsatellite locus (Microsatellites), be the sequence as made of the repetitive unit tandem sequence repeats of 1-6 nucleotide (Sharma et al., 2007) and a kind of DNA molecular genetic marker of based on PCR (polymerase chain reaction), it is distributed widely in Entire Plant Genome.The SSR motif number of repetition of different materials is different in same species, is guarded relatively with duplicate block two sides Single-copy sequence design primer, expands genome DNA, and the length polymorphism of am-plified fragments can be used as different materials Specific molecular marker.Confirm that SSR is present in eucaryote and most prokaryotes.With RFLP, RAPD, AFLP etc. Molecular labeling is compared, and SSR has the characteristics that polymorphism is high, result repeatability is high, reliable and stable, easy to operate, it has also become heredity Most widely used a kind of codominant marker in analysis and plant breeding (Plieske and Struss, 2001).Tradition The method for obtaining SSR marker need to be expended by a series of experiments such as foundation, screening-gene group library, cloning and sequencing, design of primers Many human and material resources.As gene order-checking is more and more convenient, by the method for developing SSR direct in genomic data label It is employed more and more.It has been developed in many crops such as soybean, corn, rice, wheat, tomato, cotton at present a large amount of SSR primer, in the Genetic diversity evaluation of these crops and other work (Ziekiewicz et al., 1994； Varshney et al.,2005)。
Rice is staple food crop for the survival of mankind, and there are about 1/2 populations using rice as staple food in the whole world.Rice It is also a kind of important strategic goods and materials, accounts for more than half of China's total grain output, ensures Rice Production to ensuring China's grain Safety and agricultural sustainable development have highly important strategic importance.Rice is due to, genetic transformation system smaller with genome Establish improve and with other gramineae plants there are preferable general character the advantages that, have become gramineae plant and unifacial leaf The model plant (Izawa and Shimamoto, 1996) of plant.Currently, having been developed according to Sequencing of Rice Genome result A large amount of SSR markers (McCouch et al., 2002；Saini et al.,2004；Nonoue et al.,2008； Matsubara et al.,2008；Maas et al., 2010), but it is still unsaturated to the exploitation of rice SSR marker.In view of water Rice importance in terms of grain security and agricultural sustainable development develops new specific rice SSR label primer, for building Dense genetic map and Variety fingerprinting are of great significance, for Rice molecular breeding, new varieties, seed resource Evaluation and cultivation technique innovation have important directive significance.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides with the high bar QTL close linkage of rice SSR molecular marker L08 with Using.Above-mentioned SSR molecular marker is to provide polymorphism, makes up the deficiency that current rice SSR marker more lacks, and is rice germplasm Identification research and its Population Genetics provide strong research tool.The present invention provides SSR on 3 pairs of the 1st chromosomes of rice Label, and they are applied in the high bar gene genetic analysis of rice and finely positioning.
Technical solution of the present invention is as follows:
With the SSR molecular marker L06 of the high bar QTL close linkage of rice, which is a pair, marker upstream nucleotide Sequence is as shown in SEQ ID NO.1, and marker downstream nucleotide sequence is as shown in SEQ ID NO.2.
The above-mentioned SSR molecular marker L06 with the high bar QTL close linkage of rice loses in building the 1st chromosome high density of rice Application in blit spectrum and Variety fingerprinting.
Above-mentioned and the high bar QTL close linkage of rice SSR molecular marker L06 is in the high bar gene genetic of the 1st chromosome of rice Application in analysis and finely positioning.
With the SSR molecular marker L08 of the high bar QTL close linkage of rice, which is a pair, marker upstream nucleotide Sequence is as shown in SEQ ID NO.3, and marker downstream nucleotide sequence is as shown in SEQ ID NO.4.
The above-mentioned SSR molecular marker L08 with the high bar QTL close linkage of rice loses in building the 1st chromosome high density of rice Application in blit spectrum and Variety fingerprinting.
Above-mentioned and the high bar QTL close linkage of rice SSR molecular marker L08 is in the high bar gene genetic of the 1st chromosome of rice Application in analysis and finely positioning.
With the SSR molecular marker L10 of the high bar QTL close linkage of rice, which is a pair, marker upstream nucleotide Sequence is as shown in SEQ ID NO.5, and marker downstream nucleotide sequence is as shown in SEQ ID NO.6.
The above-mentioned SSR molecular marker L10 with the high bar QTL close linkage of rice loses in building the 1st chromosome high density of rice Application in blit spectrum and Variety fingerprinting.
Above-mentioned and the high bar QTL close linkage of rice SSR molecular marker L10 is in the high bar gene genetic of the 1st chromosome of rice Application in analysis and finely positioning.
Beneficial effect
The present invention has filtered out SSR molecular marker L06, the SSR molecule mark of 3 pairs with the high bar QTL close linkage of rice for the first time Remember L08, SSR molecular marker L10.By to rice Single Segment Substitution Lines in Rice W27-14-1-2-03-24 and recurrent parent HJX74 base It is detected because a group DNA is expanded, and using 6% modacrylic acyl ammonia gel electrophoresis, finds above-mentioned 3 pairs and the high bar of rice The amplified production banding pattern of the SSR molecular marker of QTL close linkage is obvious, and has good polymorphism between kind, in utilization Genetic analysis and finely positioning can be carried out for the high bar gene of rice by stating 3 pairs of SSR markers, can be used for constructing the 1st chromosome of rice Dense genetic map and Variety fingerprinting.
Detailed description of the invention
Fig. 1 is that SSR marker parent's polymorphism screens polyacrylamide gel electrophoresis detection on rice Single Segment Substitution Lines in Rice As a result；
Wherein: 1 is recurrent parent HJX74, and 2 be rice Single Segment Substitution Lines in Rice W27-14-1-2-03-24, and a is that RM315 is more State property screens that polyacrylamide gel electrophoresis detects as a result, b is that RM104 parent polymorphism screens polyacrylamide gel electrophoresis Detection as a result, c be RM529 polymorphism screening polyacrylamide gel electrophoresis detection as a result, d be PSM331 polymorphism screening Polyacrylamide gel electrophoresis detection as a result, e be PSM423 polymorphism screen polyacrylamide gel electrophoresis detection result；
Fig. 2 is rice SSR marker PSM331, and RM1068 is in F2Denaturing polyacrylamide gel electrophoresis in separation in group The result of detection；
Wherein: (a) and (b) is respectively to be replaced to PSM331 and RM1068 in HJX74 (P1) and single slice using SSR marker It is PH-1 (t) gene magnification in W27-14-1-2-03-24 (P2) segregating population as a result, 1-20 is F2Group's individual；
Fig. 3 is the mapping group that recurrent parent HJX74 hybridizes development with rice Single Segment Substitution Lines in Rice W27-14-1-2-03-24 The genetic map of the Plant height gene PH-1 (t) of body positioning；
Fig. 4 is rice SSR marker L06, L08 and L10 in F2Denaturing polyacrylamide gel electrophoresis in separation in group The result of detection；
Wherein: (a), (b) and (c) be respectively using SSR primer pair L06, L08 and L10 in HJX74 (P1) and single slice generation Change is PH-1 (t) gene magnification in W27-14-1-2-03-24 (P2) segregating population as a result, 1-20 is F2Group's individual；
Fig. 5 is Molecular Linkage Map of the dominant tall gene on the 1st the short arm of a chromosome of rice.
Specific embodiment
With reference to the accompanying drawings of the specification and embodiment technical scheme is described further, but the present invention protected The range of shield is without being limited thereto.
Biological material source involved in embodiment is as follows:
Rice Single Segment Substitution Lines in Rice W27-14-1-2-03-24 and recurrent parent HJX74 is purchased from Agricultural University Of South China's agronomy Institute；
Enzyme involved in embodiment, reagent and kit are ordinary commercial products.
The design of embodiment 1 and the SSR molecular marker of the high bar QTL close linkage of rice
1.1 primer design methods:
The design of molecular labeling is carried out in the chromosome segment for needing to develop microsatellite marker.From (The on the website TIGR Institute of Genome Research) (http://www.tigr.org/tdb/rice) download region PAC/BAC The sequence of clone.TIGR by IRGSP (International Rice Genome Sequencing Project, IRGSP) The PAC/BAC Clonal integration of sequencing has arrived on genetic map, from target area download PAC/BAC clone sequence firstly the need of Searching for microsatellite sequence is carried out using repetitive sequence search software SSRIT (http://www.gramene.org/microsat/) Then rope therefrom selects suitable microsatellite sequence (generally requiring repeat number 8 or more, while avoiding motif is AT), downloading Include a bit of genome sequence (350-500bp) of this microsatellite sequence, finally carries out setting for PCR primer using this sequence Meter.The design of primer is carried out using software Primer Premier 5.0.The primer of design generally requires G/C content in 45-65% Between, Tm value is between 55-65 DEG C, no mispairing, dimer and hairpin structure.
In the chromosomal region for needing to develop deletion polymorphism label, (the The Institute of from the website TIGR Genome Research) (http://www.tigr.org/tdb/rice) download the region OryzasativaLcv.Nipponbare PAC/BAC clone sequence Column.The PAC/BAC needed clone is selected first, each clone excision at the continuous small fragment of 2kb.Then these 2kb are utilized Sequence and rice variety 93-11 carry out sequence alignment, select sequence alignment similitude high and only there is base to lack in some section The sequence of (base deletion is generally more than 5 bases) is lost as candidate sequence, is finally lacked using these candidate sequences in base The both ends design front and back primer of mistake, the product general control amplified is between 100bp to 300bp.It is other by design of primers It is general to require.
The physical positioning of molecular labeling is with the pseudochromosome model (Pseudomolecules) of the IRSGP OryzasativaLcv.Nipponbare established For reference object.Using direct physical positioning method, according to the pseudochromosome model of the IRGSP OryzasativaLcv.Nipponbare delivered by each two parents This molecular labeling with polymorphism directly positions, to be built into molecular linkage map.The specific method is as follows:
Compare tool by the website NCBI BLAST, to the front and back primer sequence of each molecular labeling OryzasativaLcv.Nipponbare gene It is searched in group sequence, to determine on chromosome or the BAC clone where the molecular labeling, is according to resulting result is compared It would know that specific location of the molecular labeling on each chromosome.
According to molecular marker analysis as a result, by the individual of parent's banding pattern with HJX74 and W27-14-1-2-03-24 Assignment 1 and 3 respectively, the individual assignment 2 with parents' banding pattern (heterozygosis banding pattern).The result that microsatellite is analyzed is converted into digital shape Then formula inputs computer to use 3.0 software of MAPMAKER/EXP.Version to target group's heading stage and microsatellite The mask data of label carries out linkage analysis, using Kosambi function by recombination fraction be converted into genetic distance (Centimorgan, CM), the local molecular linkage map of target gene is constructed.
According to the repetitive sequence of clone end, BLASTn software (http://www.ncbi.nlm.nih.gov/blast) Each clone is stitched together, the physical location cloned where new primer is determined, is integrated on genetic map, and between two label of calculating Physical location.
The microsatellite sequence research tool that (http://www.gramene.org) is provided on the website Gramene SSRIT (Simple Sequence Repeat Identification Tool) finds out the microsatellite sequence in BAC sequence, so It is afterwards 200-1000bp with the automatic or manual amplification length of designing of flanking sequence of the Primer Premier 5.0 to microsatellite The upstream and downstream primer pair of left and right, and carry out filtering out 3 pairs of SSR markers, then by the raw limited public affairs of work bioengineering (Shanghai) share Department's synthesis.
Application of the 2.2 rice SSR markers in Plant Height of Rice gene PH-1 (t) accurate positioning
Polymorphic screening is carried out by the SSR marker announced, the high bar assignment of genes gene mapping of rice will be controlled in the 1st chromosome of rice Region (Fig. 1, Fig. 2) between short long-armed PSM331 and RM1068 two labels, specific embodiment is as follows:
Polymorphic screening is carried out according to the SSR marker announced is (as shown in table 1), finds RM315, PSM331, PSM423, RM104, RM529 totally 5 SSR markers have between two parents it is polymorphic.
According to molecular marker analysis as a result, using MAPMAKER (EXP3.0b) software carry out molecular data analysis, and Calculate the genetic distance between label.Plant height gene PH-1 (t) is positioned on the 1st chromosome, the microsatellite positioned at long-armed side The genetic distance of label RM1068, PSM423, RM104 and PH-1 (t) is 0.5cM, 0.9cM, 8.9cM, is located at another The genetic distance of microsatellite marker PSM331, RM472, RM315 and PH-1 (t) of side are respectively 1.0cM, 3.7cM, 6.4cM (figure 3)。
SSR marker on 1 Single Segment Substitution Lines in Rice of table
In order to further position target gene, according to the SSR marker announced between PSM331 and RM1068,10 pairs are chosen (as shown in table 2) expands recombinant strain, wherein RM11964, RM11966, and RM11977 has polymorphic, use between two parents This 3 pairs labels expand the recombinant strain between PSM331 and RM1068, analyze its genotype and phenotype, will be high Stalk gene PH-1 (t) is positioned between RM11977 and RM1068, physical distance 68kb.
SSR marker between table 2 PSM331 and RM1068
In order to further be accurately positioned target gene, according to known rice whole genome sequence, in the 1st chromosome of rice Find microsatellite sequence in BAC sequence between upper RM11977 and RM1068, devise 10 pairs of new SSR markers altogether, with this 10 SSR marker detection primer expands parent, filters out 3 pairs from 10 pairs of SSR markers, all has polymorphism (Fig. 4).Point It is not named as SSR molecular marker L06, SSR molecular marker L08, SSR molecular marker L10.By further to RM11977 and Remaining recombinant strain is analyzed between RM1068.Analysis finds 1 plant height stalk plant, and 1 plant of semi-dwarf mutant plant displaying target gene exists The upstream of SSR molecular marker L06, and 3 plant height stalk plant then displaying target gene in the downstream of primer RM11977.Thus final Gene PH-1 (t) is positioned between RM11977 and L06, physical distance 23kb.
The marker upstream nucleotide sequence of SSR molecular marker L06 is as shown in SEQ ID NO.1, marker downstream nucleotides sequence Column are as shown in SEQ ID NO.2；The marker upstream nucleotide sequence of SSR molecular marker L08 is as shown in SEQ ID NO.3, downstream Labeled nucleotide sequence is as shown in SEQ ID NO.4；The marker upstream nucleotide sequence of SSR molecular marker L10 such as SEQ ID Shown in NO.5, marker downstream nucleotide sequence is as shown in SEQ ID NO.6 (as shown in table 3).
New SSR marker between table 3 RM11977 and RM1068
The application of embodiment 2 rice SSR molecular marker L06, SSR molecular marker L08, SSR molecular marker L10
2.1 oryza sativa genomic dnas extract
Rice genome total DNA is extracted using improvement TPS simplified method, the specific steps are as follows:
1, take 1~2, top spire in each single plant of tillering regularity, save with it is spare in -80 DEG C of refrigerators；
2, it takes the rice leaf of 2~4cm long to be put into 1.5ml centrifuge tube when extracting DNA, is put into liquid nitrogen, grind, add TPS Extract 900ml, 75 DEG C of 30~60min of water-bath；
3,12000rpm is centrifuged 10min, and honest and upright and thrifty 500ml is transferred to new 1.5ml centrifuge tube in absorption；
4, pre- cold isopropanol is added or dehydrated alcohol is filled it up with.4 DEG C overnight, and 12000rpm is centrifuged 10min；
5, supernatant is abandoned, it is drying precipitated, add 150 μ l aqua sterilisas, 4 DEG C are spare.
The TPS extract component is as follows: 100mM Tris-HCL (pH8.0), 10mM EDTA (pH8.0), 1MKCL.
2.2 PCR amplification
PCR amplification carries out in PE9700 type thermal cycler.20 μ l reaction system components are as follows:
The 1 μ l of DNA profiling of 2 × Hifi PCR Mix 10 μ l, 50~100ng, 1 μ l of forward primer, 1 μ l of reverse primer, ddH27 μ l of O, overall reaction system are 20 μ l.
Primer is respectively SSR molecular marker detection primer L06, SSR molecular marker detection primer L08, SSR molecular marker inspection Survey primer L10.
PCR response procedures are as follows:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of extension 1min, 35 recycle；72 DEG C are prolonged Stretch 5min.
The separation of 2.3 polyacrylamide gel electrophoresises and polymorphic detection
The polyacrylamide denaturant gel electrophoresis that PCR product mass percent is 6%, basic operation includes following three Step:
Glue: weighing 9.6g urea, adds 10 × tbe buffer liquid of 45ml distilled water and 8ml, keeps urea complete with glass rod stirring Then fully dissolved is added 40% acrylamide solution of 12ml, stirs evenly, 10% Ammonium Persulfate 98.5 of 0.8ml and 35 μ l TEMED are added, It pours into after stirring evenly in the glass plate sealed with Ago-Gel, is laid flat after filling immediately, it is made to be inclined at 10 ° or so with desktop Angle.It is inserted into comb, standing 1~2h keeps gelling solid.
Above-mentioned 10 × tbe buffer liquid component is as follows:
108g Tris-HCl, 55g boric acid, 7.44g EDTA add distilled water to dissolve, and are settled to 1000ml；
Above-mentioned 40wt% acrylamide solution component is as follows:
38g acrylamide, 2g N, N '-methylene-bisacrylamide, add water to be settled to 100ml；
Point sample: after glue completely solidifies, carefully extracting comb, and holding loading wells as far as possible is complete.Glass plate is fixed on vertical On straight electrophoresis tank, suitable 1 × TBE electrophoretic buffer is added；Electrophoretic buffer repeated flushing loading wells is taken, removing is extra not to be had The substance of solidification；Pcr amplification product is taken, 4 μ l loading buffers are added in every pipe PCR product, with microsyringe (purchase after mixing From Shanghai Medicine Laser Instrument Plant) take 3~4 μ l to inject loading wells.
Above-mentioned loading buffer component is as follows, is weight percentage:
0.25% bromophenol blue, 0.25% dimethylbenzene cyanogen, 50% glycerol, excess water.
Electrophoresis: adjust voltage to 250V, electrophoresis time about 3~4h.
After electrophoresis, glass plate is removed from electrophoresis tank, takes out gel, rinsed twice in distilled water；It is transferred to 0.1wt%AgNO3It is dyed in solution, the jog 10min on shaking table；Then gel is transferred in distilled water and is rinsed twice；Most After be transferred in developing solution and develop the color, be transferred in tap water and save after coloring, record banding pattern result or gel imaging.
Developing solution component is as follows:
6g sodium hydroxide, 0.076g sodium tetraborate, 1.6ml formaldehyde, add water to be settled to 400ml.
Rice SSR marker PSM331, RM1068, L06, L08 and L10 it can be seen from above-mentioned experimental result (Fig. 1~4) Amplified production banding pattern it is obvious, and between kind have good polymorphism, using the present invention design SSR molecular marker L06, SSR molecular marker L08, SSR molecular marker L10, which expand F2 grade segregating population, is positioned at water for the dominant Plant height gene Between rice the 1st chromosome long arm RM1068 and L06, physical distance 23kb.It is annotated using SSR marker and by rice genome System (RiceGAAS) has carried out predictive genes and functional analysis to the chromosomal region of 23kb between RM1068 and L06, at this In region discovery contain 5 ORF, wherein ORF3 is related with the synthesis of gibberellin, initial guess its be target gene PH-1 (t) (Fig. 5).The high bar gene of SSR molecular marker primer pair rice designed using the present invention carries out genetic analysis and finely positioning, together When can also be used for rice building dense genetic map and Variety fingerprinting.
Sequence table
<110>Biotechnology Research Center, Shandong Academy of Agricultural Sciences
<120>with the SSR molecular marker L08 of the high bar QTL close linkage of rice and application
<141> 2018-07-31
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence ()
<400> 1
aataagacgg atggtcaaac g 21
<210> 2
<211> 21
<212> DNA
<213>artificial sequence ()
<400> 2
gcccaacgac aagtcaaaaa g 21
<210> 3
<211> 19
<212> DNA
<213>artificial sequence ()
<400> 3
taacggggct tcctctcct 19
<210> 4
<211> 19
<212> DNA
<213>artificial sequence ()
<400> 4
tcttcccaaa cccaaccat 19
<210> 5
<211> 19
<212> DNA
<213>artificial sequence ()
<400> 5
catgttgcgc atttccttt 19
<210> 6
<211> 19
<212> DNA
<213>artificial sequence ()
<400> 6
ggctggctgg ttcctagtc 19

Claims (3)

1. the SSR molecular marker L08 with the high bar QTL close linkage of rice, which is a pair, marker upstream nucleotides sequence Column are as shown in SEQ ID NO.3, and marker downstream nucleotide sequence is as shown in SEQ ID NO.4.
2. high in building the 1st chromosome of rice with the SSR molecular marker L08 of the high bar QTL close linkage of rice described in claim 1 Application in density genetic map and Variety fingerprinting.
3. with the SSR molecular marker L08 of the high bar QTL close linkage of rice in the high bar base of the 1st chromosome of rice described in claim 1 Because of the application in genetic analysis and finely positioning.
CN201810860012.XA 2016-02-04 2016-02-04 SSR molecular marker L08 and application with the high bar QTL close linkage of rice Pending CN109652577A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810860012.XA CN109652577A (en) 2016-02-04 2016-02-04 SSR molecular marker L08 and application with the high bar QTL close linkage of rice

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610079379.9A CN105648064B (en) 2016-02-04 2016-02-04 With the SSR molecular marker and application of the high bar QTL close linkage of rice
CN201810860012.XA CN109652577A (en) 2016-02-04 2016-02-04 SSR molecular marker L08 and application with the high bar QTL close linkage of rice

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN201610079379.9A Division CN105648064B (en) 2016-02-04 2016-02-04 With the SSR molecular marker and application of the high bar QTL close linkage of rice

Publications (1)

Publication Number Publication Date
CN109652577A true CN109652577A (en) 2019-04-19

Family

ID=56489379

Family Applications (3)

Application Number Title Priority Date Filing Date
CN201810860011.5A Pending CN108841993A (en) 2016-02-04 2016-02-04 SSR molecular marker L10 and application with the high bar QTL close linkage of rice
CN201810860012.XA Pending CN109652577A (en) 2016-02-04 2016-02-04 SSR molecular marker L08 and application with the high bar QTL close linkage of rice
CN201610079379.9A Expired - Fee Related CN105648064B (en) 2016-02-04 2016-02-04 With the SSR molecular marker and application of the high bar QTL close linkage of rice

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201810860011.5A Pending CN108841993A (en) 2016-02-04 2016-02-04 SSR molecular marker L10 and application with the high bar QTL close linkage of rice

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201610079379.9A Expired - Fee Related CN105648064B (en) 2016-02-04 2016-02-04 With the SSR molecular marker and application of the high bar QTL close linkage of rice

Country Status (1)

Country Link
CN (3) CN108841993A (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107142317A (en) * 2017-06-02 2017-09-08 中国水稻研究所 A kind of method excavated and verify the Plant Height of Rice allele with cumulative effect

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003180362A (en) * 2001-12-17 2003-07-02 National Institute Of Agrobiological Sciences Method for selecting gramineous plant
JP2003319782A (en) * 2002-05-02 2003-11-11 Hokuren Federation Of Agricult Coop:The Method for identifying cultivar of rice plant
CN101368181A (en) * 2008-03-31 2009-02-18 山东省农业科学院高新技术研究中心 Numerator mark concatenated with rice purple pericladium gene PSH1 (t), acquiring method and application thereof
KR20110082973A (en) * 2010-01-13 2011-07-20 충남대학교산학협력단 Fine qtl mapping and identification of rice blast resistance gene
CN102199596A (en) * 2008-11-20 2011-09-28 广西壮族自治区农业科学院 SSR marker BYL8 of brown planthopper resistant gene site bph20
CN103911442A (en) * 2014-03-13 2014-07-09 南宁益谱检测技术有限公司 Rice fingerprint constructing method based on SSR marking and capillary electrophoresis technology
CN103966335A (en) * 2014-05-22 2014-08-06 山东农业大学 Method of developing SNP (single nucleotide polymorphism)-SSR (simple sequence repeat) molecular makers closely linked with SNP by utilizing SNP

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974620B (en) * 2010-08-31 2013-02-13 中国水稻研究所 Method for positioning quantitative trait loci (QTL) of rice
CN103848906B (en) * 2012-12-05 2021-06-25 浙江大学 Rice high temperature resistance related gene OsZFP, screening marker and separation method thereof
KR101493980B1 (en) * 2013-10-08 2015-02-17 대한민국 Indel marker for discrimination of rice cultivar
CN103820444A (en) * 2014-03-25 2014-05-28 扬州大学 Molecular markers of main effect QTL (Quantitative Trait Locus) qPH6 locus of plant height of rice and application thereof
CN103981183B (en) * 2014-05-25 2016-09-07 江苏省农业科学院 SSR marker that on No. 5 chromosomes, aphelenchoides besseyi Resistance QTL relevant to LRGPP is chain and application thereof
CN103981181B (en) * 2014-05-25 2016-09-07 江苏省农业科学院 SSR marker chain with aphelenchoides besseyi Resistance QTL on No. 3 chromosomes and application thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003180362A (en) * 2001-12-17 2003-07-02 National Institute Of Agrobiological Sciences Method for selecting gramineous plant
JP2003319782A (en) * 2002-05-02 2003-11-11 Hokuren Federation Of Agricult Coop:The Method for identifying cultivar of rice plant
CN101368181A (en) * 2008-03-31 2009-02-18 山东省农业科学院高新技术研究中心 Numerator mark concatenated with rice purple pericladium gene PSH1 (t), acquiring method and application thereof
CN102199596A (en) * 2008-11-20 2011-09-28 广西壮族自治区农业科学院 SSR marker BYL8 of brown planthopper resistant gene site bph20
KR20110082973A (en) * 2010-01-13 2011-07-20 충남대학교산학협력단 Fine qtl mapping and identification of rice blast resistance gene
CN103911442A (en) * 2014-03-13 2014-07-09 南宁益谱检测技术有限公司 Rice fingerprint constructing method based on SSR marking and capillary electrophoresis technology
CN103966335A (en) * 2014-05-22 2014-08-06 山东农业大学 Method of developing SNP (single nucleotide polymorphism)-SSR (simple sequence repeat) molecular makers closely linked with SNP by utilizing SNP

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
于欣: ""一个水稻高秆基因的遗传分析及精细定位"", 《万方学位论文》 *

Also Published As

Publication number Publication date
CN105648064A (en) 2016-06-08
CN105648064B (en) 2019-02-22
CN108841993A (en) 2018-11-20

Similar Documents

Publication Publication Date Title
CN105331717B (en) Watermelon InDel molecular labeling and its application
CN107400715A (en) The exploitation and its application of the special chemoattractant molecule mark of Thinopyrum ponticum and probe
CN105219880A (en) OncidiumLuridum belongs to EST-SSR labeled primer and application thereof
CN111471790B (en) Molecular marker closely linked with wheat grain filling rate QTL QGfr. sicau-7D.1 and application thereof
CN110512018A (en) PCR primer, kit and its application for the screening of wild cabbage self-compatibility material
CN106434944A (en) Application of SNP molecular marker closely linked to aphid resistance gene of prunus persica
CN109735648B (en) Method for screening wheat with different thousand grain weights and special kit thereof
AU2020103461A4 (en) Molecular marker of rice amylose content micro-control gene SSIIIb and application thereof
CN109517922A (en) Barley P3G and C3G synthesize InDel molecular labeling and its application of main effect QTL compact linkage
CN111304355B (en) InDel molecular marker closely linked with rice heading stage gene and application
CN105648064B (en) With the SSR molecular marker and application of the high bar QTL close linkage of rice
CN112695124A (en) Phalaenopsis SSR molecular marker primer composition and application thereof
KR101410329B1 (en) Primer set, method and kit for selecting TSWV-resistant pepper cultivar
CN109517921A (en) Barley P3G and C3G synthesize InDel molecular labeling and its application of main effect QTL compact linkage
KR101426466B1 (en) Complete sequencing of Chloroplast genomes of Panax ginseng-derived Maker, DNA primer sets and Kits for discrimination of Panax ginseng cultivars and Panax species and uses thereof
CN112680542B (en) Universal SSR molecular marker primer composition for orchidaceae plants and application of universal SSR molecular marker primer composition
Dida Molecular Markers in Breeding of Crops: Recent Progress and Advancements
CN105525026B (en) With the SSR molecular marker and application of rice purple leaf sheath QTL close linkage
KR101137799B1 (en) Specific primers for discriminating Suhan strains in Pleurotus ostreatus, and uses thereof
Meng et al. Differential gene expression between cross-fertilized and self-fertilized kernels during the early stages of seed development in maize
KR101736670B1 (en) Primer sets for identification of Phalaenopsis and composition of marker comprising the same
KR100984169B1 (en) Primer set, method and kit for selecting TMV-resistant pepper cultivar
KR101735244B1 (en) Marker for discriminating bolting time in radish and uses thereof
KR101795937B1 (en) Primer set KSLG-CP001 for identification of Phalaenopsis and composition of marker comprising the same
CN111321237A (en) SSR marker-based efficient breeding kit for &#39;Hui&#39; self-fruitful progeny

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20190419

WD01 Invention patent application deemed withdrawn after publication