CN108103230A - Detect the specific PCR molecular markers of elongated grain allele on rice grain shape QTLqGL35.1 - Google Patents
Detect the specific PCR molecular markers of elongated grain allele on rice grain shape QTLqGL35.1 Download PDFInfo
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- 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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Abstract
The present invention provides detection rice grain shape QTL（Quantitative trait loci, quantitative trait loci）qGL35.112 specific PCRs mark of upper treasure's 97 elongated allele of Shan, respectively Wn35243, Wn35257, Wn35376, Wn35381, Wn35386, Wn35411, Wn35424, Wn35434, Wn35472, Wn35480, Wn35485 and Wn35496 are characterized in that its PCR primer.Paddy DNA is identified using this 12 marks, the elongated grain allele whether detected materials are transferred to rice varieties treasure Shan 97 in targeted seat can be detected, whether there is the ability for reducing that grain is wide, increases grain length, increases paddy length-width ratio, 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 precious on rice grain shape QTL qGL35.1
12 specific PCRs mark of 97 elongated allele of Shan.
2nd, background technology
With the improvement of living standards, requirement of the people to rice quality is higher and higher.Particle shape is important rice appearance
Quality trait, mainly by grain length, grain is wide and length-width ratio determines.Usually, southern region of China likes edible grain wide small and grain
The rice grown up, i.e. elongated shape rice.Moreover, the wide small rice chalkiness rice rate of grain and chalkiness degree are often lower, market value is more
It is high.But the wide reduction of grain often brings the reduction of grain weight, and yield of brown rice is caused to reduce.This is showed on particle shape QTL
It is fairly obvious.9 control wide QTL of grain have been cloned at present, and wherein on 8, the wide allele of granule can cause grain to drop again
It is low.This gives the challenge of the improvement band of rice grain shape.It selects to develop DNA marker to the grain again particle shape QTL without active effects,
For rice grain shape marker assisted selection, this problem can effectively solve the problem that.
Patentee will control the QTL qTGW1.2c of rice grain shape and grain weight[1]It is decomposed into two new QTL.Wherein,
QGL35.1 only controls particle shape, and to grain weight without remarkable effect.On the QTL seats, the allele from precious Shan 97 can be shown
It writes and reduces wide grain, increase grain length, increases paddy length-width ratio so that rice grain shape becomes elongated.The present invention on this basis, root
Sequence is resurveyed as a result, carrying out sequence alignment for section where qGL35.1 according to rice varieties treasure Shan 97 and Milyang 46, and design is inserted
Enter missing (InDel) mark, and select can 97 allele of specificity identification treasure Shan mark.It is provided by the invention
Molecular labeling has commonly used value to the detection of 97 elongated allele of precious Shan on rice grain shape QTL qGL35.1, can
To be widely used in the transformation of the elongated grain allele of qGL35.1 research.
3rd, the content of the invention
The technical problem to be solved by the present invention is to provide the specificity of 97 elongated allele of precious Shan on detection qGL35.1
Molecular labeling 12.
The present invention uses following technical scheme：Sequence is resurveyed as a result, to qGL35.1 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, 15 Indel are devised in the section
Mark, is verified through laboratory qualification, filters out 12 marks in two kinds with polymorphism.It is respectively designated as
Wn35243、 Wn35257、Wn35376、Wn35381、Wn35386、Wn35411、Wn35424、Wn35434、Wn35472、
Wn35480, Wn35485 and Wn35496；
The upstream primer sequence of Wn35243 is 5'-CCGGGATGATACATTAAACCAA-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 1；
The downstream primer sequence of Wn35243 is 5'-CAGAAATGGCCTAAATCCAC-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 2；
The upstream primer sequence of Wn35257 is 5'-CACCCATATATACTGGCAAT-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 3；
The downstream primer sequence of Wn35257 is 5'-CCCTTCCGAAATAACCCAA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 4；
The upstream primer sequence of Wn35376 is 5'-CCACATAAAGCCCAAACCAA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 5；
The downstream primer sequence of Wn35376 be 5'-TTTACATAATTAATACGGGATTGC-3', the nucleotide sequence
For SEQ ID NO:Nucleotide sequence shown in 6；
The upstream primer sequence of Wn35381 is 5'-CCAGGCTGTAGGAATCCCAA-3', and the nucleotides sequence is classified as SEQ
ID NO:7 shown nucleotide sequence；
The downstream primer sequence of Wn35381 be 5'-ACGACCGAGTTTAGTTCATAGCTTT-3', the nucleotide sequence
For SEQ ID NO:Nucleotide sequence shown in 8；
The upstream primer sequence of Wn35386 is 5'-CATGCAATGTGCCATTCC-3', and the nucleotides sequence is classified as SEQ
ID NO:9 shown nucleotide sequence；
The downstream primer sequence of Wn35386 is 5'-GTGTCAGCCTTAATCGTTC-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 10；
The upstream primer sequence of Wn35411 is 5'-CACTATGCTTCTATAGCTGT-3', and the nucleotides sequence is classified as SEQ
ID NO:11 shown nucleotide sequence；
The downstream primer sequence of Wn35411 be 5'-ATTTACAAAGTTTAAGAAACCCAT-3', the nucleotide sequence
For SEQ ID NO:Nucleotide sequence shown in 12；
The upstream primer sequence of Wn35424 be 5'-AGGTAAAACTTTTATATTATGCAA-3', the nucleotide sequence
For SEQ ID NO:13 shown nucleotide sequence；
The downstream primer sequence of Wn35424 is 5'-TGTAACCCTACATCCC-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 14.
The upstream primer sequence of Wn35434 is 5'-TTCTACTAGGATACGTCCCAT-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 15；
The downstream primer sequence of Wn35434 is 5'-CTGGAGTTGAAGCTGCACAT-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 16；
The upstream primer sequence of Wn35472 is 5'-TATTTAAAGCCGCCCACGACA-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 17；
The downstream primer sequence of Wn35472 is 5'-TTCTTGAAATGCGGATGCTCCA-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 18；
The upstream primer sequence of Wn35480 is 5'-TCCGATGCAAACATTCACT-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 19；
The downstream primer sequence of Wn35480 is 5'-CATCGCGTCCTCGTCTTCC-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 20；
The upstream primer sequence of Wn35485 is 5'-CCATTAGGCGGTCTCCAAT-3', and the nucleotides sequence is classified as SEQ
ID NO:21 shown nucleotide sequence；
The downstream primer sequence of Wn35485 is 5'-CCACTAGCGAGCTACTTAGGAA-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 22；
The upstream primer sequence of Wn35496 is 5'-CTCAGATCACTGGAGGTCAA-3', and the nucleotides sequence is classified as SEQ
ID NO:23 shown nucleotide sequence；
The downstream primer sequence of Wn35496 is 5'-AAGTTGTGAAATTTATGGGTCA-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 24；
The PCR amplification system of 12 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, each 3.3ng/ μ l of upstream and downstream primer,
2.0 unit of Taq archaeal dna polymerases, masterplate DNA 50ng；PCR reaction conditions are other consistent in addition to annealing temperature, denaturation temperature
94 DEG C 2 minutes；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 Wn35496.
M：Molecular weight compares；P1：Precious Shan 97；P2：Milyang 46；1~20：Sample to be tested
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 precious 97 elongated allele-specific molecular marks of Shan on 1 rice grain shape QTL qGL35.1 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 rice grain shape QTL qGL35.1 are compared, according to 2 kinds than
The insertion shown in or deletion segment develop 15 for detecting using primer-design software Oligo 7.0
The Indel marks of elongated 97 specific fragments of grain allele treasure Shan of qGL35.1.The mark of these exploitations is identified by following step
Whether note has specificity between precious Shan 97 and Milyang 46, and carrying treasure whether can accurately be identified in segregating population
97 allele of Shan.Detection method is discussed in detail so that Indel marks Wn35496 as an example in the present invention：
1.DNA Trace bio-elements
(1) precious Shan 97, Milyang 46 and 20 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-20
0.05%, dNTPs 0.2mM, each 2.0 unit of 3.3ng/ μ l, Taq archaeal dna polymerase of upstream and downstream primer, 50 ng of masterplate DNA；
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
Degree 55 DEG C 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 the figure, on Wn35496 seats, precious Shan 97 and Milyang 46 show polymorphism, in segregating population, sample
2nd, 3,4,6,13,14,19 and 20 is homozygous in Milyang 46, and 1,7,8,9,10,11,15,16 and 18 be in heterozygous, remaining individual
It is homozygous in precious Shan 97, illustrate that Indel marks can control the special mark of rice grain shape QTL qGL35.1 as the precious Shan 97 of detection
Note.By this method, the section finishing screen where qGL35.1 selects 12 molecular labelings with specificity.
The verification of 2 application specific molecular markers for identification treasure Shan of embodiment, 97 long grain allele
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 46,2 sets of near isogenic lines groups is built, is respectively designated as Q2 and B1.Often set group exists
QGL35.1 sections separate, remaining background section is consistent.Q2 groups contain precious 97 type near isogenic lines of Shan 27, and Milyang 46 type is near etc.
Gene line 25；B1 groups contain 97 type near isogenic lines of precious Shan 36；Milyang 46 type near isogenic lines 35.
2 phenotypic evaluations
Near isogenic lines Q2 and B1 is planted in Hainan Province Lingshui respectively at spring -2017 years winters in 2016 and summer in 2017
County and China Paddy Rice Inst of Hangzhou, Zhejiang province city proving ground.Each strain sets 2 repetitions, often by RANDOMIZED BLOCK DESIGN
A 10 single plants of strain kind.Intermediate 5 plants are taken to measure wide grain length, grain, length-width ratio, mass of 1000 kernel after maturation.
3 results and analysis
Statistic analysis result shows to be planted in the Q2 groups of Lingshui, is in Milyang 46 type strain phase with qGL35.1 sections
Than the grain of the strain of carrying 97 type allele of precious Shan is wide averagely to reduce 0.012mm, and grain length improves 0.058mm, length and width
Than adding 0.03.It is planted in the near isogenic lines B1 in Hangzhou, carries the wide reduction of grain of the strain of 97 type allele of precious Shan
0.013mm, grain length improve 0.061mm, and length-width ratio adds 0.03.In 2 sets of near isogenic lines precious 97 type strain of Shan and
The mass of 1000 kernel of Milyang 46 type strain there are no significant difference.Two places experiment all confirms that the importing of precious 97 type allele of Shan can be with
Raising grain length, reduction grain are wide, increase paddy length-width ratio so that particle shape becomes elongated, does not make significant difference again to grain but.
Embodiment 2 confirms detection reliability of the molecular labeling provided by the invention to precious 97 elongated allele of Shan
Height can be applied in the transformation research of the elongated grain allele treasure Shans 97 of rice grain shape QTL qGL35.1.
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. 12 specific PCRs mark of 97 elongated allele of precious Shan is included on detection rice grain shape QTLqGL35.1
Upstream primer sequence and downstream primer sequence, PCR mark entitled Wn35243, Wn35257, Wn35376, Wn35381,
The primer of Wn35386, Wn35411, Wn35424, Wn35434, Wn35472, Wn35480, Wn35485 and Wn35496, specific sequence
Row are as follows：
The upstream primer sequence of Wn35243 is 5'-CCGGGATGATACATTAAACCAA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 1；
The downstream primer sequence of Wn35243 is 5'-CAGAAATGGCCTAAATCCAC-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 2；
The upstream primer sequence of Wn35257 is 5'-CACCCATATATACTGGCAAT-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 3；
The downstream primer sequence of Wn35257 is 5'-CCCTTCCGAAATAACCCAA-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 4；
The upstream primer sequence of Wn35376 is 5'-CCACATAAAGCCCAAACCAA-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 5；
The downstream primer sequence of Wn35376 is 5'-TTTACATAATTAATACGGGATTGC-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 6；
The upstream primer sequence of Wn35381 is 5'-CCAGGCTGTAGGAATCCCAA-3', and the nucleotides sequence is classified as SEQ ID
NO:7 shown nucleotide sequence；
The downstream primer sequence of Wn35381 is 5'-ACGACCGAGTTTAGTTCATAGCTTT-3', and the nucleotides sequence is classified as
SEQ ID NO:Nucleotide sequence shown in 8；
The upstream primer sequence of Wn35386 is 5'-CATGCAATGTGCCATTCC-3', and the nucleotides sequence is classified as SEQ ID
NO:9 shown nucleotide sequence；
The downstream primer sequence of Wn35386 is 5'-GTGTCAGCCTTAATCGTTC-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 10；
The upstream primer sequence of Wn35411 is 5'-CACTATGCTTCTATAGCTGT-3', and the nucleotides sequence is classified as SEQ ID
NO:11 shown nucleotide sequence；
The downstream primer sequence of Wn35411 is 5'-ATTTACAAAGTTTAAGAAACCCAT-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 12；
The upstream primer sequence of Wn35424 is 5'-AGGTAAAACTTTTATATTATGCAA-3', and the nucleotides sequence is classified as SEQ
ID NO:13 shown nucleotide sequence；
The downstream primer sequence of Wn35424 is 5'-TGTAACCCTACATCCC-3', and the nucleotides sequence is classified as SEQ ID NO:
Nucleotide sequence shown in 14.
The upstream primer sequence of Wn35434 is 5'-TTCTACTAGGATACGTCCCAT-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 15；
The downstream primer sequence of Wn35434 is 5'-CTGGAGTTGAAGCTGCACAT-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 16；
The upstream primer sequence of Wn35472 is 5'-TATTTAAAGCCGCCCACGACA-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 17；
The downstream primer sequence of Wn35472 is 5'-TTCTTGAAATGCGGATGCTCCA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 18；
The upstream primer sequence of Wn35480 is 5'-TCCGATGCAAACATTCACT-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 19；
The downstream primer sequence of Wn35480 is 5'-CATCGCGTCCTCGTCTTCC-3', and the nucleotides sequence is classified as SEQ ID
NO:Nucleotide sequence shown in 20；
The upstream primer sequence of Wn35485 is 5'-CCATTAGGCGGTCTCCAAT-3', and the nucleotides sequence is classified as SEQ ID
NO:21 shown nucleotide sequence；
The downstream primer sequence of Wn35485 is 5'-CCACTAGCGAGCTACTTAGGAA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 22；
The upstream primer sequence of Wn35496 is 5'-CTCAGATCACTGGAGGTCAA-3', and the nucleotides sequence is classified as SEQ ID
NO:23 shown nucleotide sequence；
The downstream primer sequence of Wn35496 is 5'-AAGTTGTGAAATTTATGGGTCA-3', and the nucleotides sequence is classified as SEQ
ID NO:Nucleotide sequence shown in 24.
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| CN109811076A (en) * | 2019-03-08 | 2019-05-28 | 浙江大学 | For identifying molecular labeling and its application of the elongated particle shape of rice |
| CN112342311A (en) * | 2020-11-27 | 2021-02-09 | 中国水稻研究所 | Method for verifying QTL (quantitative trait locus) of rice grain shape and grain weight |
| CN113046461A (en) * | 2021-04-07 | 2021-06-29 | 中国水稻研究所 | Specific DNA marker for identifying long grain allele on rice grain length QTL qGL5-27.3 and application thereof |
| CN113046461B (en) * | 2021-04-07 | 2022-04-29 | 中国水稻研究所 | Specific DNA marker for identifying long grain allele on rice grain length QTL qGL5-27.3 and application thereof |
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