CN104561348B - The detection Oryza sativa L. height grain allelic specific PCR molecular markers of weight - Google Patents
The detection Oryza sativa L. height grain allelic specific PCR molecular markers of weight Download PDFInfo
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- 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
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention provides detection rice grain weight QTL(quantitative trait loci, quantitative trait loci)qTGW1.2aWithqTGW1.2bAllelic 8 the specific PCR labellings of upper Milyang 46 height mass of 1000 kernel, wherein, 7 are used for detectingqTGW1.2a, respectively Wn32837, Wn32886, Wn32893, Wn33185, Wn33186, Wn33304b and Wn33252,1 is used for detectingqTGW1.2b, for Wn34526, it is characterised by its PCR primer.Utilize these 8 labellings that paddy DNA is identified, can detect whether detected materials proceeds to the high mass of 1000 kernel allele of rice varieties Milyang 46 in 2 targeted seat respectively, the most whether having the ability improving rice grain weight, whole detection process operation is simple and accuracy is high.
Description
Technical field
The present invention relates to the detection technique field in rice breeding, particularly detection rice grain weight QTLqTGW1.2aWithqTGW1.2bAllelic 8 the specific PCR labellings of upper Milyang 46 height mass of 1000 kernel.
Background technology
At present, the rate of rise of world population is higher than increases in grain production speed, is short of food and becomes global safety problem.
Oryza sativa L., as the staple food of half population in the world, is improved its yield and is significant to solving Food Security.Oryza sativa L.
Volume increase, after experienced by of short stemization and heterosis utilization, is constantly in slow growth state, nearly 30 years China's Oryza sativa L. new products
Planting yield year increase rate and be only 1-2%, wherein some types is even below 1%.Slowly increasing production the stage at this, mass of 1000 kernel is to yield
Improve and played vital effect.Therefore, in the Breeding Process of new rice variety, the height of mass of 1000 kernel is breeding men
One of focus of attention.The material of the kind that need to improve and high mass of 1000 kernel is often hybridized and obtains by the cultivation of high mass of 1000 kernel kind
, measure more accurate after the seed full maturity such as mensuration need of mass of 1000 kernel, and in Breeding Process, sample to be tested quantity is huge, hinders
Hinder breeding process and strengthened breeding cost.Along with the fast development of molecular marker, by molecular marker assisted selection, utilize
Suitably molecular marker can be identified at rice at whole growth periods and mass of 1000 kernel is played the allele of potentiation in receptor kind
Import, thus from offspring, select the rice varieties of high mass of 1000 kernel.At present, Molecular Marker Assisted Selection Technology is in Oryza sativa L.
Disease resistance, the character such as insect resistace and salt tolerant is extensively applied.
Applicant's early stage navigates to 3 QTL(Wang L-L, et controlling Oryza sativa L. mass of 1000 kernel on Oryza sativa L. the 1st chromosome
al. Dissection of qTGW1.2 to three QTLs for grain weight and grain size in
Rice (Oryza sativa L.). Euphytica, DOI 10.1007/s10681-014-1237-7).At these 3 QTL
Interval, derives from the allele of rice varieties Milyang 46 and is respectively provided with the effect improving mass of 1000 kernel, positioning precision then withqTGW1.2aWithqTGW1.2bFor height.The present invention on this basis, ties according to the sequence of resurveying of rice varieties Milyang 46 and precious Shan 97
Really, forqTGW1.2aWithqTGW1.2bInterval, place carries out sequence alignment, designs sequence mark (STS) labelling, and therefrom selects
Going out can the allelic labelling of specificity identification Milyang 46.The molecular marker that the present invention provides is to rice grain weight QTLqTGW1.2a
WithqTGW1.2bThe upper allelic detection of Milyang 46 potentiation has commonly used value, can be widely applied toqTGW1.2a
WithqTGW1.2bIn upper Milyang 46 potentiation allelic transformation research.
Summary of the invention
Present invention solves the technical problem that and there is provided detectionqTGW1.2aWithqTGW1.2bUpper Milyang 46 height mass of 1000 kernel etc.
The specific molecular marker 8 of position gene, wherein, detectionqTGW1.2a7, detectionqTGW1.2b1.
The present invention is by the following technical solutions: according to Milyang 46 and the heavy sequencing result of precious Shan 97, rightqTGW1.2aWithqTGW1.2bThe sequence that place is interval and close linkage is interval is compared, and according to inserting and deletion condition, sets at this section
Count 12 STS labellings, verified through laboratory qualification, filter out 8 labellings in two kinds with polymorphism.
For detectingqTGW1.2aUpper Milyang 46 height mass of 1000 kernel is allelic is marked with 7, is respectively designated as
Wn32837, Wn32886, Wn32893, Wn33185, Wn33186, Wn33304b and Wn33252, particular sequence is as follows:
The forward primer sequence of Wn32837 is 5'-CGTCCTGCGTTTCGACATGACT-3', and described nucleotides sequence is classified as
Nucleotide sequence shown in SEQ ID NO:1；
The downstream primer sequence of Wn32837 is 5'-TATTTCTACCACTCCAAGCACCA-3', described nucleotide sequence
For the nucleotide sequence shown in SEQ ID NO:2；
The forward primer sequence of Wn32886 is 5'-CGTGTTACTAACCCAGTCAACTCAGG-3', described nucleotides sequence
It is classified as the nucleotide sequence shown in SEQ ID NO:3；
The downstream primer sequence of Wn32886 is 5'-CAATAGTAGCAGCTAAATTGGCGTTC-3', described nucleotides sequence
It is classified as the nucleotide sequence shown in SEQ ID NO:4；
The forward primer sequence of Wn32893 is 5'-TTCCGATTTTGTTTTCTTGGTCCC-3', described nucleotide sequence
For the nucleotide sequence shown in SEQ ID NO:5；
The downstream primer sequence of Wn32893 is 5'-GCCGCACATTTATTACCTTAATCCTG-3', described nucleotides sequence
It is classified as the nucleotide sequence shown in SEQ ID NO:6；
The forward primer sequence of Wn33185 is 5'-GGAGGATAACGCAAGCAAACTTGA-3', described nucleotide sequence
Shown nucleotide sequence for SEQ ID NO:7；
The downstream primer sequence of Wn33185 is 5'-CCATAGCTTGTAAAAGACAGTGCC-3', described nucleotide sequence
For the nucleotide sequence shown in SEQ IDNO:8；
The forward primer sequence of Wn33186 is 5'-AGTTAGTGCAGCAATCTACGTCCT-3', described nucleotide sequence
Shown nucleotide sequence for SEQ ID NO:9；
The downstream primer sequence of Wn33186 is 5'-AGCTCCTCCAGTGACGATACGG-3', and described nucleotides sequence is classified as
Nucleotide sequence shown in SEQ IDNO:10；
The forward primer sequence of Wn33304b is 5'-AAGCAACATAACTTATTCTACTC-3', described nucleotide sequence
Shown nucleotide sequence for SEQ ID NO:11；
The downstream primer sequence of Wn33304b is 5'-ACGACATCCACTTCGCACC-3', and described nucleotides sequence is classified as
Nucleotide sequence shown in SEQ IDNO:12；
The forward primer sequence of Wn33252 is 5'-GCATGTATCAAAGATTCGATGAGA-3', described nucleotide sequence
Shown nucleotide sequence for SEQ ID NO:13；
The downstream primer sequence of Wn33252 is 5'-TGAAAACTCATGGCTACGCT-3', and described nucleotides sequence is classified as
Nucleotide sequence shown in SEQ IDNO:14.
For detectingqTGW1.2bUpper Milyang 46 height mass of 1000 kernel is allelic is marked with 1, named Wn34526, tool
Body sequence is as follows:
The forward primer sequence of Wn34526 is 5'-TCATCATCTGTTCGTGGGTT-3', and described nucleotides sequence is classified as
The shown nucleotide sequence of SEQ ID NO:15；
The downstream primer sequence of Wn34526 is 5'-TTGAAATATAAACTCTATACAATCTGCT-3', described nucleotide
Sequence is the nucleotide sequence shown in SEQ IDNO:16.
The PCR amplification system of 8 STS labellings is consistent.Amplification system is, Tris-HCL(pH 8.8) 33.5 mM, (NH4)2SO4 8.0 mM, MgCl21.5 mM, TWEEN-20 0.05%, dNTPs 0.2 mM, each 3.3 ng/ l of upstream and downstream primer,TaqArchaeal dna polymerase 2.0 unit, masterplate DNA 50 ng；Other is consistent in addition to annealing temperature for PCR reaction condition, denaturation temperature
Spend 94 DEG C 2 minutes；Denaturation temperature 94 DEG C 45 seconds, (Wn33815, Wn33252 and Wn34526 are 50 to annealing temperature 50-55 DEG C
DEG C, remaining 5 labelling is all 55 DEG C) 45 seconds, 72 DEG C 1 minute, 30 circulations；72 DEG C 8 minutes.
Accompanying drawing explanation
The testing result of Fig. 1 STS labelling Wn32886.
M: molecular weight compares；P1: precious Shan 97；P2: Milyang 46；1 ~ 16: testing sample
Fig. 2 two overlaps the mass of 1000 kernel distribution of NIL two places plantation.
A.2014 Lingshui, spring in year HainanqTGW1.2a
B.2014 Lingshui, spring in year HainanqTGW1.2b
C.2014 summer in year Fuyang, ZhejiangqTGW1.2a
D.2014 summer in year Fuyang, ZhejiangqTGW1.2b。
Detailed description of the invention
The present invention is explained further below in conjunction with embodiment, but the present invention is not done any type of limit by embodiment
Fixed.Experimental technique in following embodiment, if no special instructions, is conventional method.Experiment material used in following embodiment
Material, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1 Oryza sativa L. mass of 1000 kernel QTLqTGW1.2aWithqTGW1.2bThe exploitation of upper Milyang 46 specific molecular marker
Utilize sequence alignment program DNASTAR MegAlign module (Lasergene) precious to the parent of QTL target group
Shan 97 and Milyang 46 are at mass of 1000 kernel QTLqTGW1.2aWithqTGW1.2bThe base sequence in interval, place is compared, according to 2
Insertion that kind shows in comparison or deletion segment, application primer-design software Oligo 7.0 develops 12 and uses respectively
In detectionqTGW1.2aWithqTGW1.2bThe STS labelling of potentiation allele Milyang 46 specific fragment.Identified by following step
Whether what these were developed marks whether to there is specificity between precious Shan 97 and Milyang 46, and can accurately reflect in segregating population
Make and carry Milyang 46 allele.The present invention is discussed in detail detection method as a example by STS labelling Wn32886:
1.DNA Trace bio-element
(1) single-strain seed of precious Shan 97, Milyang 46 and 16 random chooses in segregating population is respectively placed in advance
The culture dish of label, 30 DEG C germinate 7 days.
(2) clip each culture dish seedling leaves 2～3 cm, is cut into the fragment of 0.5 cm length, puts in 2.0 mL centrifuge tubes.
(3) adding 450 μ l DNA extraction liquid and steel balls, application tissue beveller is ground.
(4) add 450 μ l chloroform extracts, cover tightly lid, mixing of turning upside down.
(5) 11,000 rpm are centrifuged 2 minutes to clear split-phase.Aspirate supernatant 400 μ l, proceeds to 1.5 new ml centrifuge tubes
In, abandon rifle head
(6) add the dehydrated alcohol of 800 μ l pre-coolings, cover tightly lid, mixing of turning upside down.Place 30 minutes for-20 DEG C.
(7) 11,000 rpm are centrifuged 3 minutes and invest bottom centrifuge tube to precipitation, abandon supernatant.
(8) precipitate 2 times by 70% washing with alcohol, 1.5 ml centrifuge tubes are inverted on paper, natural drying.
(9) 1/10 × TE buffer solution precipitation of 100 μ l is added.
(10) take 2 μ l and carry out PCR amplification.
2. PCR amplification and detection
Amplification system is, Tris-HCL(pH 8.8) 33.5 mM, (NH4)2SO4 8.0 mM, MgCl21.5 mM,
TWEEN-20 0.05%, dNTPs 0.2 mM, each 3.3 ng/ l of upstream and downstream primer, TaqArchaeal dna polymerase 2.0 unit, mould
Version DNA 50 ng；Other is consistent in addition to annealing temperature for PCR reaction condition, denaturation temperature 94 DEG C 2 minutes；Denaturation temperature 94 DEG C
45 seconds, annealing temperature 50-55 DEG C (Wn33815, Wn33252 and Wn34526 are 50 DEG C, and remaining 4 labelling is all 55 DEG C) 45
Second, 72 DEG C 1 minute, 30 circulations；72 DEG C 8 minutes.
Take 2 μ l PCR primer and be splined on 6% non-denaturing polyacrylamide gel (PAGE)；Connect electrode, in 100V constant voltage
Lower electrophoresis 3 hours, closes power supply；Taking off gel, silver staining develops the color.
As it is shown in figure 1, on Wn32886 seat, precious Shan 97 and Milyang 46 show polymorphism, in segregating population, sample
Product 3,4,7,8,10,11,12 and 14 are homozygous in Milyang 46, and 13 and 16 in heterozygous, and remaining individuality is homozygous in precious Shan 97, says
This STS labelling bright can control Oryza sativa L. mass of 1000 kernel QTL as detection Milyang 46qTGW1.2aSpecific mark.By the method
Filtering out 8 eventually and have specific molecular marker, wherein, 7 are used for detecting Milyang 46 and control Oryza sativa L. mass of 1000 kernel QTLqTGW1.2a, 1 detection Milyang 46 controls Oryza sativa L. mass of 1000 kernel QTLqTGW1.2b。
The embodiment 2 application specific molecular markers for identification Milyang 46 allelic checking of height mass of 1000 kernel
1 NIL builds
Use the molecular marker that embodiment 1 is set up, hand over the precious Shan of combination 97/ // precious Shan 97//precious Shan 97/ close from Indica Xian
Screening individuality in the Gao Dai colony of sun 46, establishes 2 set NIL colonies, exists respectivelyqTGW1.2aWithqTGW1.2bInterval
Separating, remaining background is interval consistent.1st is enclosed withinqTGW1.2aSeparate, containing precious Shan 97 type and Milyang 46 type NIL each 39
Individual；2nd is enclosed withinqTGW1.2bSeparate, containing precious Shan 97 type and each 42 of Milyang 46 type NIL.
2 phenotypic evaluation
Spring 2014 winter in 2013 and summer in 2014 are respectively at Lingshui county of Hainan Province and Fuyang City of Zhejiang Province China Water
Rice institute proving ground plantation NIL, each strain is planted by randomized block design, is arranged 2 repetitions, each strain
8 individual plants of system kind, take middle 5 strains and measure mass of 1000 kernel after maturation.
3 results and analysis
Statistic analysis result shows,qTGW1.2aInterval, compared with precious Shan 97 type strain, carries Milyang 46 type equipotential base
The strain of cause averagely can improve mass of 1000 kernel 0.52 g, averagely can improve 0.44 g in Fuyang in Lingshui；?qTGW1.2bInterval, with
Precious Shan 97 type strain is compared, and carries the allelic strain of Milyang 46 type and averagely can improve mass of 1000 kernel 0.30 g in Lingshui, in richness
Rising tone all can improve 0.31 g(Fig. 2).Two places test all confirms that the allelic importing of Milyang 46 can improve mass of 1000 kernel, and
Effect-size two places are highly consistent.
Embodiment 2 confirms that molecular marker that the present invention provides is to rice grain weight QTL qTGW1.2aWithqTGW1.2bIn close
Sun 46 potentiation allelic detection reliability is high, can apply to mass of 1000 kernel QTLqTGW1.2aWithqTGW1.2bMiddle potentiation etc.
In the transformation research of position gene Milyang 46.
<110>China Paddy Rice Inst
<120>detection controls the allelic specific PCR molecular markers of rice grain weight
<160> 14
<170> PatentIn 3.3
<210> 1
<211> 22
<212> DNA
<213>Oryza sativa L.
<400> 1
cgtcctgcgt ttcgacatga ct
<210> 2
<211> 23
<212> DNA
<213>Oryza sativa L.
<400> 2
tatttctacc actccaagca cca
<210> 3
<211> 26
<212> DNA
<213>Oryza sativa L.
<400> 3
cgtgttacta acccagtcaa ctcagg
<210> 4
<211> 26
<212> DNA
<213>Oryza sativa L.
<400> 4
caatagtagc agctaaattg gcgttc
<210> 5
<211> 24
<212> DNA
<213>Oryza sativa L.
<400> 5
ttccgatttt gttttcttgg tccc
<210> 6
<211> 26
<212> DNA
<213>Oryza sativa L.
<400> 6
gccgcacatt tattacctta atcctg
<210> 7
<211> 24
<212> DNA
<213>Oryza sativa L.
<400> 7
ggaggataac gcaagcaaac ttga
<210> 8
<211> 24
<212> DNA
<213>Oryza sativa L.
<400> 8
ccatagcttg taaaagacag tgcc
<210> 9
<211> 24
<212> DNA
<213>Oryza sativa L.
<400> 9
agttagtgca gcaatctacg tcct
<210> 10
<211> 22
<212> DNA
<213>Oryza sativa L.
<400> 10
agctcctcca gtgacgatac gg
<210> 11
<211> 23
<212> DNA
<213>Oryza sativa L.
<400> 11
aagcaacata acttattcta ctc
<210> 12
<211> 19
<212> DNA
<213>Oryza sativa L.
<400> 12
acgacatcca cttcgcacc
<210> 13
<211> 24
<212> DNA
<213>Oryza sativa L.
<400> 13
gcatgtatca aagattcgat gaga
<210> 14
<211> 20
<212> DNA
<213>Oryza sativa L.
<400> 14
tgaaaactca tggctacgct
<210> 15
<211> 20
<212> DNA
<213>Oryza sativa L.
<400> 15
tcatcatctg ttcgtgggtt
<210> 16
<211> 28
<212> DNA
<213>Oryza sativa L.
<400> 16
ttgaaatata aactctatac aatctgct
Claims (2)
1. detect the specific PCR molecular markers of rice varieties Milyang 46 height mass of 1000 kernel allele qTGW1.2a, comprise 7 spies
The primer of the opposite sex labelling Wn32837, Wn32886, Wn32893, Wn33185, Wn33186, Wn33304b and Wn33252 is each 1 right:
The forward primer sequence of Wn32837 is 5'-CGTCCTGCGTTTCGACATGACT-3', and described nucleotides sequence is classified as SEQ
Nucleotide sequence shown in ID NO:1；
The downstream primer sequence of Wn32837 is 5'-TATTTCTACCACTCCAAGCACCA-3', and described nucleotides sequence is classified as SEQ
Nucleotide sequence shown in ID NO:2；
The forward primer sequence of Wn32886 is 5'-CGTGTTACTAACCCAGTCAACTCAGG-3', and described nucleotides sequence is classified as
Nucleotide sequence shown in SEQ ID NO:3；
The downstream primer sequence of Wn32886 is 5'-CAATAGTAGCAGCTAAATTGGCGTTC-3', and described nucleotides sequence is classified as
Nucleotide sequence shown in SEQ ID NO:4；
The forward primer sequence of Wn32893 is 5'-TTCCGATTTTGTTTTCTTGGTCCC-3', and described nucleotides sequence is classified as SEQ
Nucleotide sequence shown in ID NO:5；
The downstream primer sequence of Wn32893 is 5'-GCCGCACATTTATTACCTTAATCCTG-3', and described nucleotides sequence is classified as
Nucleotide sequence shown in SEQ ID NO:6；
The forward primer sequence of Wn33185 is 5'-GGAGGATAACGCAAGCAAACTTGA-3', and described nucleotides sequence is classified as SEQ
The shown nucleotide sequence of ID NO:7；
The downstream primer sequence of Wn33185 is 5'-CCATAGCTTGTAAAAGACAGTGCC-3', and described nucleotides sequence is classified as SEQ
Nucleotide sequence shown in IDNO:8；
The forward primer sequence of Wn33186 is 5'-AGTTAGTGCAGCAATCTACGTCCT-3', and described nucleotides sequence is classified as SEQ
The shown nucleotide sequence of ID NO:9；
The downstream primer sequence of Wn33186 is 5'-AGCTCCTCCAGTGACGATACGG-3', and described nucleotides sequence is classified as SEQ
Nucleotide sequence shown in IDNO:10；
The forward primer sequence of Wn33304b is 5'-AAGCAACATAACTTATTCTACTC-3', and described nucleotides sequence is classified as SEQ
The shown nucleotide sequence of ID NO:11；
The downstream primer sequence of Wn33304b is 5'-ACGACATCCACTTCGCACC-3', and described nucleotides sequence is classified as SEQ
Nucleotide sequence shown in IDNO:12；
The forward primer sequence of Wn33252 is 5'-GCATGTATCAAAGATTCGATGAGA-3', and described nucleotides sequence is classified as SEQ
The shown nucleotide sequence of ID NO:13；
The downstream primer sequence of Wn33252 is 5'-TGAAAACTCATGGCTACGCT-3', and described nucleotides sequence is classified as SEQ
Nucleotide sequence shown in IDNO:14.
2. detect the specific PCR molecular markers of rice varieties Milyang 46 height mass of 1000 kernel allele qTGW1.2b, comprise 1 spy
The primer 1 of opposite sex labelling Wn34526 is right:
The forward primer sequence of Wn34526 is 5'-TCATCATCTGTTCGTGGGTT-3', and described nucleotides sequence is classified as SEQ ID
The shown nucleotide sequence of NO:15；
The downstream primer sequence of Wn34526 is 5'-TTGAAATATAAACTCTATACAATCTGCT-3', described nucleotide sequence
For the nucleotide sequence shown in SEQ IDNO:16.
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| CN107760794A (en) * | 2017-10-24 | 2018-03-06 | 中国水稻研究所 | Detect the specific PCR molecular markers of the weak response to temperature allele of qHd1 of rice varieties treasure Shan 97 |
| CN108103230B (en) * | 2018-01-24 | 2021-03-23 | 中国水稻研究所 | Specific PCR molecular marker for detecting long and thin grain allele on rice grain shape QTLqGL35.1 |
| CN108060259B (en) * | 2018-01-24 | 2021-03-23 | 中国水稻研究所 | Specific PCR molecular marker for detecting high grain weight allele on rice grain weight QTLqGW35.5 |
| CN108179221A (en) * | 2018-02-28 | 2018-06-19 | 中国水稻研究所 | Detect the specific molecular marker of high mass of 1000 kernel allele on rice mass of 1000 kernel QTL qTGW10.2a |
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