CN106399468A - Rice early-heading main-effect QTL molecular markers, identifying method thereof, and applications of molecular markers and identifying method - Google Patents
Rice early-heading main-effect QTL molecular markers, identifying method thereof, and applications of molecular markers and identifying method Download PDFInfo
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Abstract
The invention discloses rice early-heading main-effect QTL molecular markers, an identifying method of the rice early-heading main-effect QTL molecular markers, and applications of the molecular markers and the identifying method. The found rice early-heading main-effect QTL qHD8.3 molecular markers are respectively RM6670, M55, M07, M13 and M15, one pair or a plurality of pairs of primers corresponding to the abovementioned molecular markers are adopted for carrying out PCR amplification, and the amplification product is detected. The invention further provides applications of the molecular markers or the identifying method of the molecular markers in locating and cloning of rice early-heading QTL qHD8.3. With the adoption of the technical scheme, the cloning of the early-heading QTL qHD8.3 can be accelerated, the technology can be applied to the assisted selective breeding, the timely hybridization trans-breeding process is facilitated, and finally, the breeding process is accelerated.
Description
Technical field
The present invention relates to biology field, early heading main effect QTL molecular marker and its authentication method and the application of particularly a kind of Oryza sativa L..
Background technology
As one of most important cereal crops of China or even the world, the raising of its yield has highly important strategic importance for solving the problems, such as following world food to Oryza sativa L..But, existing rice varieties mostly have extremely strong region and season limitation, this have impact on the raising further of the Main Agronomic Characters such as yield and quality to a great extent, the therefore region of improvement rice varieties and seasonal adaptation, cultivate the new rice variety adapting to particular locality ecological environment and cultivation cropping system, become one of main target of current rice breeding.
Heading stage is the Main Agronomic Characters determining rice varieties region and seasonal adaptation, and the high yield and stable yields of rice varieties is played an important role, and is subject to the extensive attention of breeding man for a long time.Rice ear sprouting period mainly determines (Chang TT, Li CC, Vergara BS. (1969) Euphytica, 18 by its photonasty, response to temperature and basic nutrition growth:79-91;Tsai KH.(1985)Rice Genet Newslett,2:77-78;Tsai KH.(1986)In:Rice Genetics.International Rice Research Institute,339-349).Because the light and temperature condition of different regions has very big difference, the improvement of therefore different regions precocity rice varieties has to select different types of early heading QTL to adapt to the light and temperature condition of locality.Although being cloned into many and arabidopsiss florescence DNA homolog gene in current Oryza sativa L., they have very big difference to the Regulation Mechanism at florescence;And, although Oryza sativa L. also can be made to obtain the characteristic of early heading after the morning heading gene overexpression obtaining in Oryza sativa L. with the method for homologous clone, but the difference due to genetic background, leads to these homologous geness still cannot be applied to the cultivating process of precocious new rice variety so far.Therefore, excavation Oryza sativa L. early heading gene, the Poa linn kind to cultivation adaptation South China Double Cropping rice workspace ecological condition and its cross combination, solution breeding practice mid-early maturity are significant with the contradiction that high yield is difficult to take into account.
In terms of the positional cloning research of Heading date gene, up to the present, there is the report of a large amount of heading stage QTLs positioning, but the heading stage QTL being excavated by the method for map based cloning is less, 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. gene is to be cloned by the method for map based cloning, and the clone of these genes is greatly promoted the understanding to Rice Heading mechanism for the people.
Due to the excavation of heading stage QTL, can be deepened us Rice Heading regulated and control network is appreciated and understood by, meanwhile, the molecular marker that it is obtained can be also used for assisted selection.Due to molecular marker assisted selection (Marker-Assisted Selection, MAS) method is the selection that the genotype to kind is carried out, not protected from environmental, therefore MAS Breeding Application, in the improvement of QTL that Oryza sativa L. is early eared, will effectively lift the efficiency of breeding.
Content of the invention
Main effect QTL molecular marker and its authentication method and application the invention provides a kind of Oryza sativa L. early ears, by detection and Oryza sativa L. early heading main effect QTL molecular marker, can determine and have or not early heading channel genes in breeding lines, improve the efficiency of selection of this character, accelerate Breeding progress.
For achieving the above object, the technical scheme is that:
A kind of Oryza sativa L. early ears main effect QTL molecular marker, is named as qHD8.3, described molecular marker be respectively RM6670,
M55, M07, M13 and M15, its corresponding primer is respectively:
RM6670:
Forward primer:5'—TCCGTCGCTGCAAATTAGTCC—3';
Downstream primer:5'—CCATGTACCTCTATAATGGCAAGACC—3';
M55:
Forward primer:5'—CCATTTGGTAGGTCCATCTTACCC—3';
Downstream primer:5'—CTCCCAAGTGAAGTGCTGTCTGG—3';
M07:
Forward primer:5'—GGTGGTCTTGATTCCCTTGT—3';
Downstream primer:5'—GAAACAGATCAGCCTCACTG—3';
M13:
Forward primer:5'—ATATACAAGCGAACTCCTGT—3';
Downstream primer:5'—TAAGTGCGATGAAACAATGA—3';
M15:
Forward primer:5'—TGGCACCATCCTCATTGCTC—3';
Downstream primer:5'—AGCGTCGTCAGATACATTGG—3';
The acquisition methods of above-described Oryza sativa L. morning heading main effect QTL molecular marker, comprise the following steps:
With DP30 as donor parents, with rice variety 9311 as receptor parent with recurrent parent, by continuous backcross, selfing and orthoselection, in BC4F2In generation, obtains the morning heading chromosome segment substitution line CSSL13 of stable heredity;Again by substitution line CSSL13 and 9311 hybridization, obtain F1Cenospecies, obtain F after selfing2Segregating population, selects F2In colony, heading stage shows as multiple individual plants in early stage or late period, plants into F respectively2:3Strain, selects F2:3Strain does not have detached corresponding F2Individual plant is used for linkage analysises, obtains the molecular marker described in claim 1.
Main effect QTL molecular marker the positioning and the application in clone in Oryza sativa L. morning heading QTL present invention also offers Oryza sativa L. early ears.
Above-described Oryza sativa L. early heading main effect QTL molecular marker identification method, comprises the following steps:
With rice material complete genome DNA to be identified as template, using in above-described molecular marker RM6670, M55, M07, M13 primer corresponding with M15 one or more pairs of enter performing PCR amplification, amplified production is detected:
(1) being expanded using molecular marker RM6670 primer, if amplifying the amplified fragments of 177bp, being shown the presence having early main effect QTL of earing;
(2) being expanded using molecular marker M55 primer, if amplifying the amplified fragments of 177bp, being shown the presence having early main effect QTL of earing;
(3) being expanded using molecular marker M07 primer, if amplifying the amplified fragments of 166bp, being shown the presence having early main effect QTL of earing;
(4) being expanded using molecular marker M13 primer, if amplifying the amplified fragments of 43bp, being shown the presence having early main effect QTL of earing;
(5) being expanded using molecular marker M15 primer, if amplifying the amplified fragments of 131bp, being shown the presence having early main effect QTL of earing.
Wherein, the reaction system of the above PCR is as follows:
The reaction condition of the above PCR is:94 DEG C of degeneration 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, 35 circulations of amplification, last 72 DEG C extend 10min eventually.
Above-described molecular marker identification method, DNA cloning product detects through polyacrylamide gel electrophoresis, and the DNA band by silver staining colour developing record amplification.
The present invention also aims to providing the application that above-described molecular marker identification method is early eared in kind in breeding rice.
Beneficial effects of the present invention are:
(1) the main effect QTL qHD8.3 molecular marker present invention finds a kind of Oryza sativa L. early ears, with the Oryza sativa L. being previously reported by early heading QTL such as Hd1, Hd6, Hd3a, Ehd1, Ehd2, the differences such as Ghd7 and DTH8, enrich people Rice Heading regulated and control network is appreciated and understood by, also increase the multiformity of Oryza sativa L. morning heading genetic resourceses, provide more choices for cultivating different types of early heading rice varieties;
(3) present invention, by the discovery of Oryza sativa L. morning heading main effect QTL qHD8.3 molecular marker, can accelerate the clone of this morning heading QTL qHD8.3, and can be applied to assisted selection, be easy to timely hybridize transformation, accelerate breeding process;
(2) present invention establishes a kind of Oryza sativa L. early heading main effect QTL qHD8.3 molecular marker identification method, can accurately and rapidly differentiate the morning heading individual plant with qHD8.3, improve the efficiency of selection of this character, accelerate Breeding progress.
Brief description
Fig. 1 is the heading stage phenotype of chromosome segment substitution line CSSL13;Wherein:Fig. 1 a:Difference in 9311 and CSSL13 phenotypes under normal long-day conditions;Fig. 1 b:At the heading stage of each 30 plants of early season in 2014 statistics 9311 and CSSL13, calculate meansigma methodss, A, B represent significant difference under 0.01 level;Fig. 1 c:At in season in evening in 2014 at statistics 9311 and CSSL13 each 30 plants of heading stage, calculate meansigma methodss, C, D represent significant difference under 0.01 level.
Fig. 2 is F2For individual plant heading stage scattergram.
Fig. 3 is molecular labeling primer (M07 and M13) to F2The partial results figure of 52 evening heading individual plant amplifications of generation;Wherein, Fig. 3 a:The amplification banding pattern to evening heading individual plant for molecular labeling primer M07;Fig. 3 b:The amplification banding pattern to evening heading individual plant for molecular labeling primer M13, the 10th plant is to exchange individual plant;
Fig. 4 is Primary Location figure on the 8th chromosome for the Oryza sativa L. qHD8.3 gene.
Specific embodiment
With reference to embodiment, the invention will be further described, but embodiments of the present invention are not limited to following examples.
Embodiment 1:The screening of molecular marker and positioning
(1) structure of early heading chromosome segment substitution line CSSL13 and replacement fragment analysis
With 9311 as receptor parent, Guangxi common wild-rice DP30 as donor parents, by continuous backcross, selfing and orthoselection, in BC4F2The morning heading chromosome segment substitution line CSSL13 of the stable heredity that generation obtains.
According to gramene website (http://www.gramene.org/) SSR marker announced and Indica obstruct the STS labelling of sequence difference designed, designed, carry out polymorphic detection between to parent DP30 and 9311, detect altogether 408 to the labelling showing obvious polymorphism, using this 408 pairs of polymorphic molecular markers, CSSL13 is carried out with the fragment analysis that replace, result shows, the CSSL13 replacement fragment containing 5 donor parents DP30 altogether, it is located at the 1st respectively, 2, 3, 8, on No. 11 chromosomes, remaining background is all from 9311 (tables 1), the reason presence of these replacement fragments possibly causes its heading stage to make a variation.
The replacement fragment of table 1CSSL13 and its length
Numbering | Chromosome | Replacement fragment | 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 analysises of substitution line CSSL13
Under the field condition of the normal short-day in Nanning (Nanning early season) and normal long-day (Nanning season in evening), CSSL13 does sth. in advance heading 14.2d (Fig. 1 a, Fig. 1 b) and 13.8d (Fig. 1 c) than receptor parent 9311 respectively, t test Analysis show, the heading stage difference between CSSL13 and 9311 has all reached pole significant level.This shows that the characteristic of CSSL13 early heading may be to illumination and temperature-insensitive.Through illumination box strict long-day and short-day, high temperature and K cryogenic treatment (processed with E1 and E2 respectively and represent), as shown in table 2, result shows that the heading stage difference between CSSL13 and 9311 is respectively 14.5d, 14.8d, 14.0d and 13.0d, difference has all reached pole significant level (table 2), has further demonstrated that qHD8.3 is a morning heading QTL insensitive to light temperature.
Table 2 substitution line material C SSL13 and 9311 heading stage natural law under difficult environmental conditions
Note:E1:High temperature long-day environment;E2:Shading treatment, 10h illumination/14h is dark;E3:On 32 DEG C of daytime, at 27 DEG C of night, 12h illumination/12h is dark;E4:On 25 DEG C of daytime, at 20 DEG C of night, 12h illumination/12h is dark.a:Represent that under 0.05 level, difference is not notable
(3) F of CSSL13/93112The structure of segregating population and genetic analyses
(1) hybridized with receptor parent 9311 using early heading chromosome segment substitution line CSSL13, obtain F1Cenospecies, plant F1Cenospecies, selfing obtains F2Segregating population, for genetic analyses.
(2) to F2Segregating population carries out early identification of earing
Drawn according to field investigation record result, result shows, in F2In 412 individual plants of segregating population, heading stage is 76~100d, as shown in Fig. 2 assuming obvious bimodal distribution.With 92d as boundary, colony is divided into early heading and evening heading two big class.The individual plant of early heading has 297 plants, and the evening individual plant of heading has 115 plants.Through analysis shows, the individual plant number of early heading and evening heading meets 3:1 segregation ratio (X2 c=0.94<X2 0.05,1=3.841), this shows that early heading characteristic is controlled CSSL13 by a pair dominant main effect karyogene.
(3) qHD8.3 molecular marker linkage analysises and Primary Location
From F2In 412 individual plants of segregating population, choose 100 plants of early heading individual plants and 100 plants of evenings heading individual plants, individual plant sowing and plantation respectively, the F to these individual plants2:3Strain carries out descendant's detection, finally filters out 76 early heading individual plants of heading stage stable heredity and 52 evenings individual plant of earing is used for the Primary Location of target gene.
Using the polymorphic molecular marker with genetic background screening, linkage analysises are carried out to the individual plant of these heading stages stable heredity, it is found that, molecular marker RM6670 and M15 on qHD8.3 and No. 8 chromosome exists chain, two mark are each 11 and 5 plants of exchange individual plants, and 11 plants on the 5 plants of exchange individual plants and labelling RM6670 on labelling M15 are different from, show that target gene is likely located between labelling RM6670 and M15;For reducing the position of simultaneously hard objectives gene further,Continue with three couples of molecular marker M55 between labelling RM6670 and M15、M07 and M13,Morning to stable heredity、Evening heading individual plant enters performing PCR amplification electrophoresis detection,Result shows,In molecular marker M55、M07 and M13 also has 6 plants respectively、2 plants、1 plant of exchange individual plant,And 6 plants of exchange individual plants on molecular marker M55 are included in 11 plants of exchange individual plants of molecular marker RM6670,2 plants of exchange individual plants on molecular marker M07 are included in 6 plants of exchange individual plants of molecular marker M55,5 plants of exchange individual plants on molecular marker M15 contain 1 plant of exchange individual plant on molecular marker M13,And and molecular marker RM6670、M55、Exchange individual plant on M07 does not repeat,Confirm that this gene is located on the 8th chromosome,And it is positioned between molecular marker M07 and M13,As shown in Figure 3 a,The amplification banding pattern to evening heading individual plant for molecular labeling primer M07,20th plant is to exchange individual plant,As shown in Figure 3 b,The amplification banding pattern to evening heading individual plant for molecular labeling primer M13,10th plant is to exchange individual plant;In conjunction with shown in Fig. 4, physical distance is about 1.6Mb.
Above-mentioned linkage analysises and Primary Location process, the DNA extraction method being adopted and PCR reaction condition are respectively:
A. donor parents, receptor parent and F are extracted with conventional CTAB method2The DNA of each individual plant of segregating population;
The reaction system of B.PCR is as follows:
PCR reaction condition is:94 DEG C of degeneration 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, 35 circulations of amplification, last 72 DEG C extend 10min eventually.
DNA cloning product detects through polyacrylamide gel electrophoresis, by the DNA band of silver staining colour developing record amplification.
By said process it can be seen that can effectively carry out early heading QTL molecular marker assisted selection breeding using these and the early molecular marker earing main effect QTL qHD8.3;The finely positioning to early heading QTL and map based cloning can be accelerated using big segregating population simultaneously.
(4) F2:3Strain offspring screening analysis
Using molecular marker M07 and M13 obtaining, in F2:3Screen 76 heterozygosis individual plants in strain offspring and build F3Target group, heterozygosis individual plant divides individual plant to harvest plantation, and each strain all occurs in that the separation of heading stage phenotype, and the segregation ratio of early heading and evening heading individual plant meets 3:1 ratio, its screening rate of accuracy reached to 100%.Illustrate that the marker assisted selection carrying out rice ear sprouting period using molecular marker M07 and M13 of qHD8.3 new gene is effective.
(5) interpretation of result
The above results show, using RM6670, M55, M07, M13 and M15 can early heading main effect QTL carries out molecular marker to Oryza sativa L., its corresponding primer such as table 3:
The primer of table 3 positioning and its sequence
Embodiment 2 molecular marker identification method
With rice material complete genome DNA to be identified as template, using in above-described molecular marker RM6670, M55, M07, M13 primer corresponding with M15 one or more pairs of enter performing PCR amplification, amplified production is detected.
(1) donor parents, receptor parent and F are extracted with conventional CTAB method2The DNA of each individual plant of segregating population;
(2) reaction system of PCR is as follows:
PCR reaction condition is:94 DEG C of degeneration 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, 35 circulations of amplification, last 72 DEG C extend 10min eventually.
DNA cloning product detects through polyacrylamide gel electrophoresis, by the DNA band of silver staining colour developing record amplification.
(3) interpretation of result
(1) expanded using molecular marker RM6670 primer, if amplifying the amplified fragments of 177bp, showing the presence having early main effect QTL of earing, if the amplified fragments of 168bp can be amplified, showing the amplified fragments having parent 9311;
(2) expanded using molecular marker M55 primer, if amplifying the amplified fragments of 177bp, showing the presence having early main effect QTL of earing, if the amplified fragments of 169bp can be amplified, showing the amplified fragments having parent 9311;
(3) expanded using molecular marker M07 primer, if amplifying the amplified fragments of 166bp, showing the presence having early main effect QTL of earing, if the amplified fragments of 150bp can be amplified, showing the amplified fragments having parent 9311;
(4) expanded using molecular marker M13 primer, if amplifying the amplified fragments of 43bp, showing the presence having early main effect QTL of earing, if the amplified fragments of 52bp can be amplified, showing the amplified fragments having parent 9311;
(5) expanded using molecular marker M15 primer, if amplifying the amplified fragments of 131bp, showing the presence having early main effect QTL of earing, if the amplified fragments of 117bp can be amplified, showing the amplified fragments having parent 9311.
Detecting rice varieties based on above-mentioned 5 molecular markers it may be verified that whether containing the presence of early main effect QTL of earing, the selection-breeding of early rice varieties of earing being carried out, thus accelerating Breeding progress.
Claims (8)
1. a kind of Oryza sativa L. early ear main effect QTL molecular marker it is characterised in that:Described molecular marker is RM6670, M55, M07, M13 and M15 respectively, and its corresponding primer is respectively:
RM6670:
Forward primer:5'—TCCGTCGCTGCAAATTAGTCC—3';
Downstream primer:5'—CCATGTACCTCTATAATGGCAAGACC—3';
M55:
Forward primer:5'—CCATTTGGTAGGTCCATCTTACCC—3';
Downstream primer:5'—CTCCCAAGTGAAGTGCTGTCTGG—3';
M07:
Forward primer:5'—GGTGGTCTTGATTCCCTTGT—3';
Downstream primer:5'—GAAACAGATCAGCCTCACTG—3';
M13:
Forward primer:5'—ATATACAAGCGAACTCCTGT—3';
Downstream primer:5'—TAAGTGCGATGAAACAATGA—3';
M15:
Forward primer:5'—TGGCACCATCCTCATTGCTC—3';
Downstream primer:5'—AGCGTCGTCAGATACATTGG—3'.
2. the acquisition methods of Oryza sativa L. morning heading main effect QTL molecular marker as claimed in claim 1 are it is characterised in that comprise the following steps:With DP30 as donor parents, with rice variety 9311 as receptor parent with recurrent parent, by continuous backcross, selfing and orthoselection, in BC4F2In generation, obtains the morning heading chromosome segment substitution line CSSL13 of stable heredity;Again by substitution line CSSL13 and 9311 hybridization, obtain F1Cenospecies, obtain F after selfing2Segregating population, selects F2In colony, heading stage shows as multiple individual plants in early stage or late period, plants into F respectively2:3Strain, selects F2:3Strain does not have detached corresponding F2Individual plant is used for linkage analysises, obtains the molecular marker described in claim 1.
3. Oryza sativa L. as claimed in claim 1 early ears application in positioning and the clone that Oryza sativa L. early ears QTL for the main effect QTL molecular marker.
4. a kind of authentication method of Oryza sativa L. morning heading main effect QTL molecular marker is it is characterised in that comprise the following steps:
With rice material complete genome DNA to be identified as template, using in the primer corresponding with M15 of molecular marker RM6670, M55, M07, the M13 described in claim 1 one or more pairs of enter performing PCR amplification, amplified production is detected:
(1) being expanded using molecular marker RM6670 primer, if amplifying the amplified fragments of 177bp, being shown the presence having early main effect QTL of earing;
(2) being expanded using molecular marker M55 primer, if amplifying the amplified fragments of 177bp, being shown the presence having early main effect QTL of earing;
(3) being expanded using molecular marker M07 primer, if amplifying the amplified fragments of 166bp, being shown the presence having early main effect QTL of earing;
(4) being expanded using molecular marker M13 primer, if amplifying the amplified fragments of 43bp, being shown the presence having early main effect QTL of earing;
(5) being expanded using molecular marker M15 primer, if amplifying the amplified fragments of 131bp, being shown the presence having early main effect QTL of earing.
5. Oryza sativa L. as claimed in claim 4 early ear main effect QTL molecular marker authentication method it is characterised in that:
The reaction system of described PCR is as follows:
.
6. Oryza sativa L. as claimed in claim 4 early ear main effect QTL molecular marker authentication method it is characterised in that:
PCR reaction condition:94 DEG C of degeneration 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s, 35 circulations of amplification, last 72 DEG C extend 10min eventually.
7. Oryza sativa L. as claimed in claim 4 early ear main effect QTL molecular marker authentication method it is characterised in that:
DNA cloning product detects through polyacrylamide gel electrophoresis, and the DNA band by silver staining colour developing record amplification.
8. the application that early heading main effect QTL molecular marker identification method is early eared in kind in breeding rice of the Oryza sativa L. as described in any one of claim 4~7.
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