CN108060259A - Detect the specific PCR molecular markers of high grain weight allele on rice grain weight QTLqGW35.5 - Google Patents
Detect the specific PCR molecular markers of high grain weight allele on rice grain weight QTLqGW35.5 Download PDFInfo
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Abstract
The present invention provides detection rice grain weight QTL（Quantitative trait loci, quantitative trait loci）qGW35.57 specific PCRs mark of the upper high mass of 1000 kernel allele of Milyang 46, is respectively Wn35550, Wn35554, Wn35604, Wn35608, Wn35626, Wn35632 and Wn35635, is characterized in that its PCR primer.Paddy DNA is identified using this 7 marks, the high mass of 1000 kernel allele whether detected materials are transferred to rice varieties Milyang 46 in targeted seat can be detected respectively, whether there is the ability for improving rice grain weight, entire detection process is easy to operate and accuracy is high.
Description
First, technical field
The present invention relates to the detection technique fields in rice breeding, particularly detect close on rice grain weight QTL qGW35.5
7 specific PCRs mark of positive 46 high mass of 1000 kernel allele.
2nd, background technology
At present, world population is continuously increased, and cultivated area but gradually decreases, and grain security has become global protrusion and asks
Topic.Rice is the staple food of half population in the world, and it is the important channel for solving Food Security to improve rice yield.Mass of 1000 kernel
It is one of deciding factor of rice yield.In new rice variety selection and breeding, high rice yield is brought up again by increasing grain, is to cultivate
The important means of high-yield rice kind.The cultivation of high mass of 1000 kernel kind often carries out the material of the kind that need to be improved and high mass of 1000 kernel
Hybridization is screened high mass of 1000 kernel strain in offspring and is obtained.The seeds full maturity such as high mass of 1000 kernel strain, need is screened by phenotypic number
After could measure, delay breeding process；And sample to be tested quantity is huge in Breeding Process, and breeding is of high cost.In recent years, molecule
Marker assisted selection is applied more and more widely in rice breeding.For grain weight QTL exploitation molecular labelings, carry out Gao Qian
The molecular marker assisted selection of grain weight kind, can significantly improve efficiency of selection；It and can be in rice at whole growth periods to high grain weight
Importing situation of the allele in rice strain identified, more flexibly and can reduce cost.
The rice grain weight QTL qTGW1.2c that patentee will position early period[1]Two new QTL are decomposed into, wherein,
The allele from Milyang 46 can significantly improve mass of 1000 kernel on qGW35.5 seats.The present invention on this basis, according to rice
Kind Milyang 46 resurveys sequence as a result, carrying out sequence alignment, design insertion and deletion for section where qGW35.5 with precious Shan 97
(InDel) mark, and select can specificity identification Milyang 46 allele mark.Molecular labeling provided by the invention
There is commonly used value to the detection of the high mass of 1000 kernel allele of Milyang 46 on rice grain weight QTL qGW35.5, it can be extensive
Ground is applied in the transformation research of qGW35.5 high mass of 1000 kernel allele.
3rd, the content of the invention
The technical problem to be solved by the present invention is to provide on detection qGW35.5 Milyang 46 high mass of 1000 kernel allele it is special
Property molecular labeling 7.
The present invention uses following technical scheme：Sequence is resurveyed as a result, to qGW35.5 locations according to Milyang 46 and precious Shan 97
Between and its sequence in close linkage section be compared, according to insertion and deletion condition, 12 Indel marks are devised in the section
Note, is verified through laboratory qualification, filters out 7 marks in two kinds with polymorphism.Be respectively designated as Wn35550,
Wn35554, Wn35604, Wn35608, Wn35626, Wn35632 and Wn35635；
The upstream primer sequence of Wn35550 is 5'-AATGAAACTAGGAATGGCTC-3', and the nucleotides sequence is classified as SEQ
ID NO:1 shown nucleotide sequence；
The downstream primer sequence of Wn35550 is 5'-GAAACACAGGATAAATATAGGAA-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 2.
The upstream primer sequence of Wn35554 is 5'-GATAATACAATGGTTGCCAA-3', and the nucleotides sequence is classified as SEQ
ID NO:3 shown nucleotide sequence；
The downstream primer sequence of Wn35554 is 5'-TTTGATAGATTTCATACGGTT-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 4.
The upstream primer sequence of Wn35604 is 5'-AAGATTTTAAGATCGCTTT-3', and the nucleotides sequence is classified as SEQ
ID NO:5 shown nucleotide sequence；
The downstream primer sequence of Wn35604 is 5'-CTATATACTTTCTCCGTCCC-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 6.
The upstream primer sequence of Wn35608 is 5'-CACGTTTTCCTACGAATGGTC-3', and the nucleotides sequence is classified as
SEQ ID NO:7 shown nucleotide sequence；
The downstream primer sequence of Wn35608 is 5'-TTATCTCATGGTCTAGTGGC-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 8.
The upstream primer sequence of Wn35626 is 5'-ATACCTGTCGGCAGATATCGC-3', and the nucleotides sequence is classified as
SEQ ID NO:9 shown nucleotide sequence；
The downstream primer sequence of Wn35626 is 5'-GCGCCGAAACGTAATCAGA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 10.
The upstream primer sequence of Wn35632 is 5'-AAAACACCTTACGATATGCGAT-3', and the nucleotides sequence is classified as
SEQ ID NO:11 shown nucleotide sequence；
The downstream primer sequence of Wn35632 is 5'-CCTTTAGTCTCGGACCATGC-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 12.
The upstream primer sequence of Wn35635 is 5'-ACGAACAATTAAAGTTGGGTA-3', and the nucleotides sequence is classified as
SEQ ID NO:13 shown nucleotide sequence；
The downstream primer sequence of Wn35635 is 5'-ACGGATACTACAACATGAGTGA-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 14.
The PCR amplification system of 7 Indel marks is consistent.Amplification system is Tris-HCL (pH 8.8) 33.5mM, (NH4)2SO48.0mM, MgCl21.5mM, TWEEN-20 0.05%, dNTPs 0.2mM, upstream and downstream primer each 3.3ng/ μ l, Taq
2.0 unit of archaeal dna polymerase, masterplate DNA 50ng；PCR reaction conditions are other consistent in addition to annealing temperature, 94 DEG C 2 of denaturation temperature
Minute；94 DEG C of denaturation temperature 45 seconds, 55 DEG C of annealing temperature 45 seconds, 72 DEG C 1 minute, 30 cycles；72 DEG C 8 minutes.
4th, illustrate
Fig. 1 Indel mark the testing result of Wn35608.
M：Molecular weight compares；P1：Precious Shan 97；P2：Milyang 46；1-16：Sample to be tested
The mass of 1000 kernel distribution of two sets of near isogenic lines two places plantations of Fig. 2.
A. near isogenic lines L1 groups
B. near isogenic lines L2 groups
5th, specific embodiment
It is explained further the present invention with reference to embodiments, but embodiment is not the present invention any type of limit
It is fixed.Experimental method in following embodiments is conventional method unless otherwise specified.Experiment material used in following embodiments
Material, reagent etc., are commercially available unless otherwise specified.
The exploitation of Milyang 46 specific molecular marker on 1 rice mass of 1000 kernel QTL qGW35.5 of embodiment
It is precious to the parent of QTL target groups using sequence alignment program DNASTAR MegAlign modules (Lasergene)
Shan 97 and the Milyang 46 base sequence in section where mass of 1000 kernel QTL qGW35.5 are compared, according to 2 kinds in comparison
The insertion shown or deletion segment develop 12 using primer-design software Oligo 7.0 and are respectively used to detection qGW35.5
The Indel marks of synergy allele Milyang 46 specific fragment.By following step identify these exploitation mark whether in treasure
There is specificity between Shan 97 and Milyang 46, and whether can accurately be identified in segregating population and carry Milyang 46 equipotential base
Cause.Detection method is discussed in detail so that Indel marks Wn35608 as an example in the present invention：
1.DNA Trace bio-elements
(1) precious Shan 97, Milyang 46 and 16 single-strain seeds selected at random in segregating population are respectively placed in advance
The culture dish of label, 30 DEG C germinate 7 days.
(2) 2~3cm of each culture dish seedling leaves of clip is cut into the fragment of 0.5cm long, is put into 2.0mL centrifuge tubes.
(3) 450 μ l DNA extracting solutions and a steel ball are added in, is ground using tissue grinder instrument.
(4) 450 μ l chloroform extract liquors are added in, cover tightly lid, turn upside down mixing.
(5) 11,000rpm centrifuges 2 minutes to clear split-phase.400 μ l of Aspirate supernatant, are transferred to new 1.5ml centrifuge tubes
In, abandon pipette tips
(6) absolute ethyl alcohol of 800 μ l precoolings is added in, covers tightly lid, turn upside down mixing.- 20 DEG C are placed 30 minutes.
(7) 11,000rpm are centrifuged 3 minutes and are invested centrifugation bottom of the tube to precipitation, abandon supernatant.
(8) wash precipitation 2 times with 70% ethyl alcohol, 1.5ml centrifuge tubes are inverted on paper, spontaneously dry.
(9) 1/10 × TE buffer solutions precipitation of 100 μ l is added in.
(10) 2 μ l is taken to carry out PCR amplification.
2.PCR is expanded and detection
Amplification system is Tris-HCL (pH 8.8) 33.5mM, (NH4)2SO48.0mM, MgCl21.5mM, TWEEN-
200.05%, dNTPs 0.2mM, each 2.0 unit of 3.3ng/ μ l, Taq archaeal dna polymerase of upstream and downstream primer, masterplate DNA 50ng；
PCR reaction conditions are other consistent in addition to annealing temperature, 94 DEG C of denaturation temperature 2 minutes；94 DEG C of denaturation temperature 45 seconds, annealing temperature
55 45 seconds, 72 DEG C 1 minute, 30 cycle；72 DEG C 8 minutes.
2 μ l PCR products is taken to be splined on 6% non-denaturing polyacrylamide gel (PAGE)；Electrode is connected, in the constant electricity of 100V
When pressure electrophoresis 3 is small, power supply is closed；Remove gel, silver staining colour developing.
As shown in Figure 1, on Wn35608 seats, precious Shan 97 and Milyang 46 show polymorphism, in segregating population, sample
Product 1,9 and 12 are in that precious Shan 97 is homozygous, and 2,3,4,6,8,10,11,13,14 and 16 be in heterozygous, remaining individual is pure in Milyang 46
Mould assembly illustrates that Indel marks can be as the specific mark of detection Milyang 46 control rice mass of 1000 kernel QTL qGW35.5.Pass through
This method section finishing screen where qGW35.5 selects 7 molecular labelings with specificity.
The verification of the 2 high mass of 1000 kernel allele of application specific molecular markers for identification Milyang 46 of embodiment
1 near isogenic lines is built
The molecular labeling established using embodiment 1 hands over the precious Shan 97/ // treasure's Shan 97//treasure's Shan 97/ of combination close from Indica Xian
Individual is screened in the Gao Dai groups of sun 46, has developed 2 sets of near isogenic lines groups, has been respectively designated as L1 and L2, this Liang Tao group
It is separated in qGW35.5 sections, remaining background section is consistent.L1 groups contain 97 type near isogenic lines of precious Shan 20, Milyang 46 type
Near isogenic lines 27.L2 groups contain 97 type near isogenic lines of precious Shan 28, Milyang 46 type near isogenic lines 34.
2 phenotypic evaluations
In summer in 2017 2 sets of near isogenic lines were planted in China Paddy Rice Inst of Hangzhou, Zhejiang province city proving ground.Often
A strain presses RANDOMIZED BLOCK DESIGN, sets 2 repetitions, each 10 single plants of strain kind.After maturation, intermediate 5 plants are taken to measure thousand
Weight.
3 results and analysis
Statistic analysis result shows in L1 and L2 groups, compared with being in 97 type strain of precious Shan with qGW35.5 sections, carries
0.40g and 0.44g has been respectively increased in the mass of 1000 kernel average value of the strain of Milyang 46 type allele.Two sets of different near isogenic lines
Qtl analysis result confirm that the importing of Milyang 46 allele can improve mass of 1000 kernel, and effect is big between different groups
Low height is consistent.
Embodiment 2 confirms molecular labeling provided by the invention to the high mass of 1000 kernel of Milyang 46 on rice grain weight QTL qGW35.
The detection reliability of allele is high, can be applied to the high mass of 1000 kernel allele of Milyang 46 on mass of 1000 kernel QTL qGW35.5
In transformation research.
Bibliography
1.Wang L-L,et al.Dissection of qTGW1.2to three QTLs for grain weight
and grain size in rice(Oryza sativa L.).Euphytica,DOI 10.1007/s10681-014-
1237-7
Claims (1)
1. 7 specific PCRs mark of the high mass of 1000 kernel allele of Milyang 46 is included on detection rice grain weight QTL qGW35.5
Upstream primer sequence and downstream primer sequence, PCR mark title be respectively Wn35550, Wn35554, Wn35604,
The primer of Wn35608, Wn35626, Wn35632 and Wn35635, particular sequence are as follows：
The upstream primer sequence of Wn35550 is 5'-AATGAAACTAGGAATGGCTC-3', and the nucleotides sequence is classified as SEQ ID
NO:1 shown nucleotide sequence；
The downstream primer sequence of Wn35550 is 5'-GAAACACAGGATAAATATAGGAA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 2.
The upstream primer sequence of Wn35554 is 5'-GATAATACAATGGTTGCCAA-3', and the nucleotides sequence is classified as SEQ ID
NO:3 shown nucleotide sequence；
The downstream primer sequence of Wn35554 is 5'-TTTGATAGATTTCATACGGTT-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 4.
The upstream primer sequence of Wn35604 is 5'-AAGATTTTAAGATCGCTTT-3', and the nucleotides sequence is classified as SEQ ID
NO:5 shown nucleotide sequence；
The downstream primer sequence of Wn35604 is 5'-CTATATACTTTCTCCGTCCC-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 6.
The upstream primer sequence of Wn35608 is 5'-CACGTTTTCCTACGAATGGTC-3', and the nucleotides sequence is classified as SEQ ID
NO:7 shown nucleotide sequence；
The downstream primer sequence of Wn35608 is 5'-TTATCTCATGGTCTAGTGGC-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 8.
The upstream primer sequence of Wn35626 is 5'-ATACCTGTCGGCAGATATCGC-3', and the nucleotides sequence is classified as SEQ ID
NO:9 shown nucleotide sequence；
The downstream primer sequence of Wn35626 is 5'-GCGCCGAAACGTAATCAGA-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 10.
The upstream primer sequence of Wn35632 is 5'-AAAACACCTTACGATATGCGAT-3', and the nucleotides sequence is classified as SEQ
ID NO:11 shown nucleotide sequence；
The downstream primer sequence of Wn35632 is 5'-CCTTTAGTCTCGGACCATGC-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 12.
The upstream primer sequence of Wn35635 is 5'-ACGAACAATTAAAGTTGGGTA-3', and the nucleotides sequence is classified as SEQ ID
NO:13 shown nucleotide sequence；
The downstream primer sequence of Wn35635 is 5'-ACGGATACTACAACATGAGTGA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 14.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154471A (en) * | 2011-01-17 | 2011-08-17 | 南京农业大学 | Molecular marking method for major quantitative trait loci(QTL) for rice grain length |
| CN103725675A (en) * | 2012-10-15 | 2014-04-16 | 王纪年 | Molecular tagging method for paddy rice |
| CN104561348A (en) * | 2015-01-26 | 2015-04-29 | 中国水稻研究所 | Specific PCR molecular markers for detecting rice high-grain weight alleles |
| CN107058493A (en) * | 2017-01-13 | 2017-08-18 | 中国水稻研究所 | A kind of specific PCR molecular markers and method for detecting the wide allele of rice grain |
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- 2018-01-24 CN CN201810068095.9A patent/CN108060259B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154471A (en) * | 2011-01-17 | 2011-08-17 | 南京农业大学 | Molecular marking method for major quantitative trait loci(QTL) for rice grain length |
| CN103725675A (en) * | 2012-10-15 | 2014-04-16 | 王纪年 | Molecular tagging method for paddy rice |
| CN104561348A (en) * | 2015-01-26 | 2015-04-29 | 中国水稻研究所 | Specific PCR molecular markers for detecting rice high-grain weight alleles |
| CN107058493A (en) * | 2017-01-13 | 2017-08-18 | 中国水稻研究所 | A kind of specific PCR molecular markers and method for detecting the wide allele of rice grain |
Non-Patent Citations (1)
| Title |
|---|
| 裔传灯等: "水稻粒宽基因ＧＳ５的功能标记开发和单倍型鉴定", 《中国水稻科学》 * |
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