CN106399468B - Rice early heading main effect QTL molecular labeling and its identification method and application - Google Patents

Rice early heading main effect QTL molecular labeling and its identification method and application Download PDF

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CN106399468B
CN106399468B CN201610345177.4A CN201610345177A CN106399468B CN 106399468 B CN106399468 B CN 106399468B CN 201610345177 A CN201610345177 A CN 201610345177A CN 106399468 B CN106399468 B CN 106399468B
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CN106399468A (en
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覃宝祥
李容柏
韦敏益
吴子帅
刘立龙
刘芳
罗继景
邱永福
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Guangxi University
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Abstract

The present invention discloses a kind of rice early heading main effect QTL molecular labeling and its identification method and application, have found rice early heading main effect QTL qHD8.3 molecular labeling, it is RM6670, M55, M07, M13 and M15 respectively, using one or more pairs of carry out PCR amplifications in the corresponding primer of above-mentioned molecular labeling, amplified production is detected;The present invention also provides the application of above-mentioned molecular labeling or molecular marker identification method in the rice early positioning and clone of heading QTL qHD8.3.It can accelerate the clone of morning heading QTL qHD8.3 through the invention, and assisted selection can be applied to, convenient for timely hybridizing transformation, accelerate breeding process.

Description

Rice early heading main effect QTL molecular labeling and its identification method and application
Technical field
The present invention relates to molecular biology field, early heading main effect QTL molecular labeling and its identification of especially a kind of rice Methods and applications.
Background technique
Rice is as one of most important cereal crops in China or even the world, and the raising of yield is for solving the following whole world Food problem has highly important strategic importance.However, mostly there is extremely strong region and seasons for existing rice varieties Limitation, this largely affects further increasing for the Main Agronomic Characters such as yield and quality, therefore improves rice The region of kind and seasonal adaptation cultivate the new rice variety for adapting to given area ecological environment and cultivating cropping system, at For one of the main target of current rice breeding.
Heading stage is the Main Agronomic Characters for determining rice varieties region and seasonal adaptation, to the high yields of rice varieties and Stable yields plays an important role, for a long time by the extensive attention of breeder.Rice ear sprouting period mainly by its photonasty, response to temperature and Basic nutrition growth determines (Chang TT, Li CC, Vergara BS. (1969) Euphytica, 18:79-91;Tsai KH.(1985)Rice Genet Newslett,2:77-78;Tsai KH.(1986)In:Rice Genetics.International Rice Research Institute,339-349).Due to the light temperature item of different regions There are great differences for part, thus the improvement of different regions precocity rice varieties have to select different types of early heading QTL with Adapt to local light and temperature condition.Although being cloned into much genes with arabidopsis florescence DNA homolog in current rice, But there is very big differences for their Regulation Mechanisms to the florescence;Moreover, although the method with homologous clone obtains in rice It so that rice is obtained the characteristic early eared after the early heading gene overexpression obtained, but due to the difference of genetic background, cause These homologous genes can not still be applied to the cultivating process of precocious new rice variety so far.Therefore, rice early heading gene is excavated, It is early in the Poa linn kind for adapting to South China Double Cropping rice workspace ecological condition and its cross combination, solution breeding practice to cultivating The ripe contradiction for being difficult to take into account with high yield is of great significance.
In terms of the positional cloning research of Heading date gene, up to the present, there are a large amount of heading stage QTLs positioning Report, but less by the heading stage QTL that the method for map based cloning is excavated, so far, only Hd1, Hd6, Hd3a, Ehd1, Ehd2, Ghd7, DTH8 and EL1 (Yano M.et al. (2000) Plant Cell, 12:2473-2483; Takahashi,Y.et al.(2001)Proc Natl Acad Sci USA,98:7922-7927;Kojima S.et al. (2002)Plant Cell Physiol,43:1096–1105;Doi K.et al.(2004)Genes Dev,18:926-936; Matsubara K.et al.(2008)Plant Physiol,148(3):1425-1435;Xue WY.et al.(2007)Nat Genet,40:761-767;Wei XJ.et al.(2010)Plant Physiol,153:1747-1758;Dai C,Xue HW. (2010) Embo J, 29 (11): 1916-1927) etc. genes be to be cloned by the method for map based cloning, the clone of these genes It is greatly promoted understanding of the people to Rice Heading mechanism.
Due to the excavation of heading stage QTL, our understanding and understanding to Rice Heading regulated and control network can be deepened, meanwhile, Its molecular labeling obtained can be also used for assisted selection.Due to molecular marker assisted selection (Marker-Assisted Selection, MAS) method is the selection carried out to the genotype of kind, be not protected from environmental, thus MAS Breeding Application in Improvement to rice morning heading QTL, will effectively promote the efficiency of breeding.
Summary of the invention
The present invention provides a kind of early heading main effect QTL molecular labeling and its identification method and the applications of rice, pass through detection With rice morning heading main effect QTL molecular labeling, it can determine that whether there is or not early heading channel genes into breeding lines, improve the character Efficiency of selection, accelerate Breeding progress.
To achieve the above object, the technical solution of the present invention is as follows:
A kind of early heading main effect QTL molecular labeling of rice, is named as qHD8.3, the molecular labeling be respectively RM6670,
M55, M07, M13 and M15, corresponding primer is respectively:
RM6670:
Upstream primer: 5'-TCCGTCGCTGCAAATTAGTCC -3';
Downstream primer: 5'-CCATGTACCTCTATAATGGCAAGACC -3';
M55:
Upstream primer: 5'-CCATTTGGTAGGTCCATCTTACCC -3';
Downstream primer: 5'-CTCCCAAGTGAAGTGCTGTCTGG -3';
M07:
Upstream primer: 5'-GGTGGTCTTGATTCCCTTGT -3';
Downstream primer: 5'-GAAACAGATCAGCCTCACTG -3';
M13:
Upstream primer: 5'-ATATACAAGCGAACTCCTGT -3';
Downstream primer: 5'-TAAGTGCGATGAAACAATGA -3';
M15:
Upstream primer: 5'-TGGCACCATCCTCATTGCTC -3';
Downstream primer: 5'-AGCGTCGTCAGATACATTGG -3';
The acquisition methods of above-described rice morning heading main effect QTL molecular labeling, comprising the following steps:
Using DP30 as donor parents, with rice variety 9311 for receptor parent and recurrent parent, pass through continuous backcross, selfing And M8003 line, in BC4F2In generation, obtains the early heading chromosome segment substitution line CSSL13 for stablizing heredity;Again by substitution line CSSL13 hybridizes with 9311, obtains F1Cenospecies obtains F after selfing2Segregating population selects F2Heading stage shows as morning in group Multiple single plants of phase or advanced stage, plant into F respectively2:3Strain selects F2:3The correspondence F that strain does not separate2Single plant is for chain Analysis, obtains molecular labeling described in claim 1.
The present invention also provides rice early to ear main effect QTL molecular labeling in the rice early positioning and clone of heading QTL Application.
Above-described rice morning heading main effect QTL molecular marker identification method, comprising the following steps:
Using rice material complete genome DNA to be identified as template, using above-described molecular labeling RM6670, M55, One or more pairs of carry out PCR amplifications in M07, M13 and M15 corresponding primer, detect amplified production:
(1) it is expanded using molecular labeling RM6670 primer, if amplifying the amplified fragments of 177bp, shows there is morning The presence of heading main effect QTL;
(2) it is expanded using molecular labeling M55 primer, if amplifying the amplified fragments of 177bp, shows there is early heading The presence of main effect QTL;
(3) it is expanded using molecular labeling M07 primer, if amplifying the amplified fragments of 166bp, shows there is early heading The presence of main effect QTL;
(4) it is expanded using molecular labeling M13 primer, if amplifying the amplified fragments of 43bp, shows there is early heading The presence of main effect QTL;
(5) it is expanded using molecular labeling M15 primer, if amplifying the amplified fragments of 131bp, shows there is early heading The presence of main effect QTL.
Wherein, the reaction system of PCR described above is as follows:
The reaction condition of PCR described above are as follows: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s expand 35 Circulation, last 72 DEG C extend 10min eventually.
Above-described molecular marker identification method, DNA cloning product is detected through polyacrylamide gel electrophoresis, and is passed through The DNA band of silver staining colour developing record amplification.
The object of the invention is also to provide above-described molecular marker identification methods in breeding rice morning heading product Application in kind.
The invention has the benefit that
(1) present invention finds a kind of rice morning heading main effect QTL qHD8.3 molecular labelings, with the rice being previously reported Early heading QTL such as Hd1, Hd6, Hd3a, Ehd1, Ehd2, Ghd7 and DTH8 etc. are different, enrich people and regulate and control to Rice Heading The understanding and understanding of network also increase the diversity of rice morning heading genetic resources, to cultivate different types of early heading water Rice varieties provide more choices;
(3) present invention can accelerate morning heading QTL by the discovery of rice morning heading main effect QTL qHD8.3 molecular labeling The clone of qHD8.3, and assisted selection can be applied to, convenient for timely hybridizing transformation, accelerate breeding process;
(2) present invention establishes a kind of rice early heading main effect QTL qHD8.3 molecular marker identification method, can it is accurate, Rapidly identify the early heading single plant with qHD8.3, improves the efficiency of selection of the character, accelerate Breeding progress.
Detailed description of the invention
Fig. 1 is the heading stage phenotype of chromosome segment substitution line CSSL13;Wherein: Fig. 1 a: under normal long-day conditions Difference in 9311 and CSSL13 phenotype;Fig. 1 b:2014 early season counts 9311 and CSSL13 each 30 plants of heading stage, calculates flat Mean value, A, B indicate 0.01 horizontal lower significant difference;Fig. 1 c:2014 season in evening counts 9311 and CSSL13 each 30 plants of heading stage, Average value is calculated, C, D indicate 0.01 horizontal lower significant difference.
Fig. 2 is F2For single plant heading stage distribution map.
Fig. 3 is molecular labeling primer (M07 and M13) to F2The partial results figure of 52 evening heading single plant amplifications of generation;Wherein, Fig. 3 a: molecular labeling primer M07 to evening heading single plant amplification banding pattern;Fig. 3 b: molecular labeling primer M13 to evening heading single plant Banding pattern is expanded, the 10th plant is exchange single plant;
Fig. 4 is Primary Location figure of the rice qHD8.3 gene on the 8th chromosome.
Specific embodiment
Below with reference to embodiment, the invention will be further described, but embodiments of the present invention be not limited to Lower embodiment.
Embodiment 1: the screening and positioning of molecular labeling
(1) building of early heading chromosome segment substitution line CSSL13 and replacement fragment analysis
Using 9311 be receptor parent, Guangxi common wild-rice DP30 is donor parents, by continuous backcross, selfing and orient Selection, in BC4F2What generation obtained stablizes the early heading chromosome segment substitution line CSSL13 of heredity.
The SSR marker and Indica announced according to the website gramene (http://www.gramene.org/) obstruct sequence The STS of difference designed, designed is marked, and to polymorphic detection is carried out between parent DP30 and 9311, detects that 408 pairs show altogether The label of obvious polymorphism, carries out replacement fragment analysis to CSSL13 using this 408 pairs of polymorphic molecular markers, the results showed that, Replacement segment of the CSSL13 altogether containing 5 donor parents DP30 is located on the 1st, 2,3,8 and No. 11 chromosome, remaining back Scape is all from 9311 (tables 1), and the presence of these replacement segments may be the reason of causing its heading stage to make a variation.
The replacement segment and its length of table 1CSSL13
Number Chromosome Replace segment Length (kb)
SL1 1 RM486-RM11865-RM315 1778
SL2 2 RM7423-M9-RM13429-RM1920 6765
SL3 3 M11-RM15288-RM411 3850
SL4 8 RM6670-M55-M07-M13-M15 5713
SL5 11 M230-RM6680 1792
(2) the Genetic characteristics specificity analysis of substitution line CSSL13
Under the normal short-day in Nanning (Nanning early season) and the field condition of normal long-day (Nanning season in evening), CSSL13 points Do not ear ahead of time 14.2d (Fig. 1 a, Fig. 1 b) and 13.8d (Fig. 1 c) than receptor parent 9311, t test Analysis show CSSL13 and Heading stage difference between 9311 has all reached extremely significant level.This shows that the characteristic that CSSL13 is early eared may be to illumination and temperature It spends insensitive.(table is handled by illumination box stringent long-day and short-day, high temperature and low-temperature treatment with E1 and E2 respectively Show), as shown in table 2, the results showed that the heading stage difference between CSSL13 and 9311 be respectively 14.5d, 14.8d, 14.0d and 13.0d, difference have all reached extremely significant horizontal (table 2), have further demonstrated that qHD8.3 is an early pumping insensitive to light temperature Fringe QTL.
The heading stage number of days of table 2 substitution line material C SSL13 and 9311 under difficult environmental conditions
Note: E1: high temperature long-day environment;E2: shading treatment, 10h illumination/14h are dark;E3: 32 DEG C of daytime, night 27 DEG C, 12h illumination/12h is dark;E4: on 25 DEG C of daytime, 20 DEG C of night, 12h illumination/12h is dark.A: 0.05 horizontal lower difference is indicated It is not significant
(3) F of CSSL13/93112The building and genetic analysis of segregating population
(1) hybridized using early heading chromosome segment substitution line CSSL13 with receptor parent 9311, obtain F1Cenospecies, kind Plant F1Cenospecies, selfing obtain F2Segregating population is used for genetic analysis.
(2) to F2Segregating population carries out early heading identification
Result is recorded according to field investigation to draw, the results showed that, in F2In 412 single plants of segregating population, heading stage is 76~100d, as shown in Fig. 2, apparent bimodal distribution is presented.Using 92d as boundary, group is divided into early heading and evening heading two is big Class.The single plant early eared has 297 plants, and the evening single plant of heading has 115 plants.Through analysis shows, the single plant number of early heading and evening heading Mesh meets the segregation ratio (X of 3:12 c=0.94 < X2 0.05,1=3.841), this shows that CSSL13 early ears characteristic by a pair of dominant The control of main effect karyogene.
(3) qHD8.3 molecular labeling linkage analysis and Primary Location
From F2In 412 single plants of segregating population, 100 plants of early heading single plants and 100 plants of evening heading single plants are chosen, it is single respectively Strain sowing and plantation, to the F of these single plants2:3Strain carries out descendant's detection, and finishing screen selects 76 morning for stablizing heredity at heading stage Heading single plant and 52 evenings heading single plants are used for the Primary Location of target gene.
Using the polymorphic molecular marker with genetic background screened are stablized at these heading stages with the single plant of heredity Linkage analysis is carried out, as a result, it has been found that, there are chain, two labels by the molecular labeling RM6670 and M15 on qHD8.3 and No. 8 chromosome Locate 11 and 5 plants of exchange single plants of each appearance, and mark 11 plants on the 5 plants of exchange single plants and label RM6670 on M15 to be different from, Show that target gene is likely located between label RM6670 and M15;For the position for further reducing simultaneously hard objectives gene, continue Using label RM6670 and M15 between three couples of molecular labelings M55, M07 and M13, to stablize heredity early, late heading single plant into Row PCR amplification and electrophoresis detection, the results showed that, also there are 6 plants, 2 plants, 1 plant of exchange list respectively in molecular labeling M55, M07 and M13 Strain, and 6 plants of exchange single plants on molecular labeling M55 are included in 11 plants of exchange single plants of molecular labeling RM6670, molecule mark 2 plants of exchange single plants on M07 are remembered in 6 plants of exchange single plants of molecular labeling M55, and 5 plants of exchanges on molecular labeling M15 are single Strain contains 1 plant of exchange single plant on molecular labeling M13, and not with the exchange single plant on molecular labeling RM6670, M55, M07 It repeating, it was confirmed that the gene is located on the 8th chromosome, and is positioned between molecular labeling M07 and M13, as shown in Figure 3a, point For sub- labeled primer M07 to the amplification banding pattern of evening heading single plant, the 20th plant is exchange single plant, as shown in Figure 3b, molecular labeling primer For M13 to the amplification banding pattern of evening heading single plant, the 10th plant is exchange single plant;As shown in connection with fig. 4, physical distance is about 1.6Mb.
Above-mentioned linkage analysis and Primary Location process, used DNA extraction method and PCR reaction condition are respectively as follows:
A. donor parents, receptor parent and F are extracted with conventional CTAB method2The DNA of each single plant of segregating population;
The reaction system of B.PCR is as follows:
PCR reaction condition are as follows: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s expand 35 circulations, finally 72 DEG C extend 10min eventually.
DNA cloning product is detected through polyacrylamide gel electrophoresis, is developed the color by silver staining and is recorded the DNA band of amplification.
By the above process it can be seen that can effectively be opened using the molecular labeling of these and early heading main effect QTL qHD8.3 The early heading QTL molecular marker assisted selection breeding of exhibition;It can use big segregating population simultaneously, accelerate the essence to early heading QTL Fine positioning and map based cloning.
(4) F2:3Strain offspring screens analysis
Using the molecular labeling M07 and M13 of acquisition, in F2:376 heterozygosis single plant building F have been screened in strain offspring3It is fixed Position group, heterozygosis single plant divide single plant harvest plantation, and the separation of heading stage phenotype occurs in each strain, and early heading and evening take out The segregation ratio of fringe single plant meets the ratio of 3:1, and screening accuracy rate reaches 100%.Illustrate the molecule using qHD8.3 new gene It is effective for marking M07 and M13 to carry out the marker assisted selection of rice ear sprouting period.
(5) interpretation of result
The above results show using RM6670, M55, M07, M13 and M15 can early heading main effect QTL carries out molecule to rice Label, corresponding primer such as table 3:
The primer and its sequence that table 3 positions
2 molecular marker identification method of embodiment
Using rice material complete genome DNA to be identified as template, using above-described molecular labeling RM6670, M55, One or more pairs of carry out PCR amplifications in M07, M13 and M15 corresponding primer, detect amplified production.
(1) donor parents, receptor parent and F are extracted with conventional CTAB method2The DNA of each single plant of segregating population;
(2) reaction system of PCR is as follows:
PCR reaction condition are as follows: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s expand 35 circulations, finally 72 DEG C extend 10min eventually.
DNA cloning product is detected through polyacrylamide gel electrophoresis, is developed the color by silver staining and is recorded the DNA band of amplification.
(3) interpretation of result
(1) it is expanded using molecular labeling RM6670 primer, if amplifying the amplified fragments of 177bp, shows there is morning The presence of heading main effect QTL shows the amplified fragments for having parent 9311 if the amplified fragments of 168bp can be amplified;
(2) it is expanded using molecular labeling M55 primer, if amplifying the amplified fragments of 177bp, shows there is early heading The presence of main effect QTL shows the amplified fragments for having parent 9311 if the amplified fragments of 169bp can be amplified;
(3) it is expanded using molecular labeling M07 primer, if amplifying the amplified fragments of 166bp, shows there is early heading The presence of main effect QTL shows the amplified fragments for having parent 9311 if the amplified fragments of 150bp can be amplified;
(4) it is expanded using molecular labeling M13 primer, if amplifying the amplified fragments of 43bp, shows there is early heading The presence of main effect QTL shows the amplified fragments for having parent 9311 if the amplified fragments of 52bp can be amplified;
(5) it is expanded using molecular labeling M15 primer, if amplifying the amplified fragments of 131bp, shows there is early heading The presence of main effect QTL shows the amplified fragments for having parent 9311 if the amplified fragments of 117bp can be amplified.
Rice varieties are detected based on above-mentioned 5 molecular labelings, it may be verified that whether contain the presence of early heading main effect QTL, The breeding that can carry out early rice varieties of earing, to accelerate Breeding progress.
Sequence table
<110>Guangxi University
<120>rice early heading main effect QTL molecular labeling and its identification method and application
<160>10
<210>1
<211>21
<212>DNA
<213>artificial sequence
<221>molecular labeling RM6670 upstream primer
<400>1
TCCGTCGCTGCAAATTAGTCC 21
<210>2
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<400>2
CCATGTACCTCTATAATGGCAAGACC 26
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<400>3
CCATTTGGTAGGTCCATCTTACCC 24
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<400>4
CTCCCAAGTGAAGTGCTGTCTGG 23
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GGTGGTCTTGATTCCCTTGT 20
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GAAACAGATCAGCCTCACTG 20
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ATATACAAGCGAACTCCTGT 20
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TAAGTGCGATGAAACAATGA 20
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TGGCACCATCCTCATTGCTC 20
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AGCGTCGTCAGATACATTGG 20

Claims (7)

  1. The acquisition methods of main effect QTL molecular labeling 1. a kind of rice is early eared, it is characterised in that the following steps are included:
    Using DP30 as donor parents, with rice variety 9311 for receptor parent and recurrent parent, by continuous backcross, it is selfed and determines To selection, in BC4F2In generation, obtains the early heading chromosome segment substitution line CSSL13 for stablizing heredity;Again by substitution line CSSL13 with 9311 hybridization, obtain F1Cenospecies obtains F after selfing2Segregating population selects F2Heading stage shows as early stage or advanced stage in group Multiple single plants, plant into F respectively2:3Strain selects F2:3The correspondence F that strain does not separate2Single plant is used for linkage analysis, obtains Following rice morning heading main effect QTL molecular labeling:
    It is RM6670, M55, M07, M13 and M15 respectively, corresponding primer is respectively:
    RM6670:
    Upstream primer: 5'-TCCGTCGCTGCAAATTAGTCC -3';
    Downstream primer: 5'-CCATGTACCTCTATAATGGCAAGACC -3';
    M55:
    Upstream primer: 5'-CCATTTGGTAGGTCCATCTTACCC -3';
    Downstream primer: 5'-CTCCCAAGTGAAGTGCTGTCTGG -3';
    M07:
    Upstream primer: 5'-GGTGGTCTTGATTCCCTTGT -3';
    Downstream primer: 5'-GAAACAGATCAGCCTCACTG -3';
    M13:
    Upstream primer: 5'-ATATACAAGCGAACTCCTGT -3';
    Downstream primer: 5'-TAAGTGCGATGAAACAATGA -3';
    M15:
    Upstream primer: 5'-TGGCACCATCCTCATTGCTC -3';
    Downstream primer: 5'-AGCGTCGTCAGATACATTGG -3'.
  2. 2. rice as described in claim 1 early positioning and clone of the heading main effect QTL molecular labeling in rice morning heading QTL In application.
  3. The identification method of main effect QTL molecular labeling 3. a kind of rice is early eared, it is characterised in that the following steps are included:
    Using rice material complete genome DNA to be identified as template, using described in claim 1 molecular labeling RM6670, One or more pairs of carry out PCR amplifications in M55, M07, M13 and M15 corresponding primer, detect amplified production:
    (1) it is expanded using molecular labeling RM6670 primer, if amplifying the amplified fragments of 177bp, shows there is early heading The presence of main effect QTL;
    (2) it is expanded using molecular labeling M55 primer, if amplifying the amplified fragments of 177bp, shows there is early heading main effect The presence of QTL;
    (3) it is expanded using molecular labeling M07 primer, if amplifying the amplified fragments of 166bp, shows there is early heading main effect The presence of QTL;
    (4) it is expanded using molecular labeling M13 primer, if amplifying the amplified fragments of 43bp, shows there is early heading main effect The presence of QTL;
    (5) it is expanded using molecular labeling M15 primer, if amplifying the amplified fragments of 131bp, shows there is early heading main effect The presence of QTL.
  4. The identification method of main effect QTL molecular labeling 4. rice as claimed in claim 3 is early eared, it is characterised in that:
    The reaction system of the PCR is as follows:
    2.5 μ L of primer 4pmol/ μ L
    dNTP 2.5mmol/L 2.0μL
    1.0 μ L of DNA profiling 25ng/ μ L
    0.2 μ L of Taq archaeal dna polymerase 5U/ μ L
    10 × PCR buffer, 1.0 μ L
    ddH2O 3.3μL。
  5. The identification method of main effect QTL molecular labeling 5. rice as claimed in claim 3 is early eared, it is characterised in that:
    PCR reaction condition: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s expand 35 circulations, and last 72 DEG C Extend 10min eventually.
  6. The identification method of main effect QTL molecular labeling 6. rice as claimed in claim 3 is early eared, it is characterised in that:
    DNA cloning product is detected through polyacrylamide gel electrophoresis, and is developed the color by silver staining and recorded the DNA band of amplification.
  7. 7. as early heading main effect QTL molecular marker identification method is early in breeding rice for the described in any item rice of claim 3 ~ 6 Application in kind of earing.
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CN106834524B (en) * 2017-03-31 2020-03-17 湖南杂交水稻研究中心 Positioning of rice panicle length major QTLPL6-5 and molecular marker linked with major QTLPL6-5
CN108456744B (en) * 2018-05-15 2021-08-06 中垦种业股份有限公司 Molecular marker of valuable rice storage-tolerant QTL and breeding application
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CN114350836B (en) * 2021-12-30 2023-12-12 中国水稻研究所 QTL qHD1b for promoting rice heading and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN104357453A (en) * 2014-09-03 2015-02-18 中国科学院东北地理与农业生态研究所 Hd2/Hd4 genes promoting advanced heading of paddy rice
CN104611442A (en) * 2015-02-05 2015-05-13 宁夏农林科学院 Primer composition for identifying Ningxia rice varieties and application of primer composition

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN104357453A (en) * 2014-09-03 2015-02-18 中国科学院东北地理与农业生态研究所 Hd2/Hd4 genes promoting advanced heading of paddy rice
CN104611442A (en) * 2015-02-05 2015-05-13 宁夏农林科学院 Primer composition for identifying Ningxia rice varieties and application of primer composition

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Development and mapping of 2240 new SSR markers for rice (Oryza sativa L);McCouch SR et al;《DNA Res》;20021231;第9卷(第6期);199-207
The map-based sequence of the rice genome;International rice genome sequencing project;《Nature》;20050811;第436卷(第7052期);793-800
宁夏水稻品种微卫星标记数据库的建立;马静 等;《植物遗传资源学报》;20160128;第17卷(第2期);226-232
普通野生稻苗期耐冷型QTL的鉴定与分子定位;郑加兴 等;《中国水稻科学》;20110110;第25卷(第1期);52-58

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