CN109251996A - Detect dCAPS label and the application of the low temperature resistant gene C OLD1 genotype of rice - Google Patents
Detect dCAPS label and the application of the low temperature resistant gene C OLD1 genotype of rice Download PDFInfo
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Abstract
The present invention provides a kind of method for detecting the low temperature resistant gene C OLD1 genotype of rice and its dCAPS labels.DCAPS marks upstream primer sequence are as follows: dcaps-F:5'-CCGTCAATCTGCCTTACAGCTATCTGTCAC-3', downstream primer sequence dcaps-R:5'-TGAGCTGCCTTTCCAATGTTTTGAAGGCC-3'.Pcr amplification product further uses restriction enzyme StuI digestion, through 8% polyacrylamide gel electrophoresis, silver staining.It can detect that 196bp band is containing COLD1indGene, not low temperature resistant rice material can detect that the rice of 169bp band is containing COLD1japGene, low temperature resistant rice material.It is identified by the genotype for handing over F2 generation individual to carry out COLD1 3 japonica rice varieties and 4 rice varieties and 98 Xian round-grained rice, it was demonstrated that the label can accurately identify the japonica rice COLD1 of rice COLD1 genejapGenotype, long-grained nonglutinous rice COLD1indGenotype and heterozygous genotypes.Detection method accuracy height, high specificity, there is stronger practicability in marker assisted selection.
Description
Technical field
The present invention relates to a kind of dCAPS labels for detecting the low temperature resistant gene C OLD1 genotype of rice, belong to rice breeding technology
Field.
Background technique
Rice is the most important cereal crops of second-biggest-in-the-world cereal crops and China for being only second to wheat.Rice is
Thermophilic crops, extremely sensitive to temperature change, low temperature can restrict rice yield, cause the rice significantly underproduction, chilling injury
It is world rice main producing region common problem.Wherein the Asia rice region such as Japan, Korea and China is influenced by chilling injury
It is the most serious, it is estimated that China is every year because of about 3,000,000,000~5,000,000,000 kg of the paddy underproduction caused by chilling injury.In addition, low temperature cold
Evil be also influence rice quality an important factor for one of, under different growing low-temperature treatment rice quality the study found that low
Temperature damages to plants caused by sudden drop in temperature the reduction that will lead to the nutriments such as protein in rice, amylopectin content, fat, and mass of 1000 kernel is caused to subtract
Few, sterile grain rate increases.The low temperature resistant mechanism of rice is studied, the low temperature resistant gene of rice is identified, cultivates low temperature resistant rice varieties,
It is an effective way for solving Analysis of Rice Chilling Injury.
Traditional rice breeding method depends on the selection of rice phenotype, takes a long time and vulnerable to environment and human factor
It influences, the efficiency of breeding is very low.With the progress of Modern Molecular Biotechnology, molecular marker assisted selection obtains in rice breeding
To being widely applied, the efficiency of breeding has been significantly increased.In recent years, domestic and foreign scholars to the low temperature resistant mechanism of rice into
A large amount of research is gone, the positioning of low temperature resistant QTL and clone etc. all have made some progress.At present, it has been found that some
QTL relevant to rice growth different times.The seedling stage that Virgilio etc. is reported on No. 4 chromosomes is low temperature resistant
QTL site qCTS4；Koseki etc. reports a seedling stage low temperature resistant QTL site qSCT11 on No. 11 chromosomes of rice；Zhou
Deng located 1 boot stage low temperature resistant QTL site qCTB7 on No. 7 chromosomes of rice；Makoto Kuroki etc. is at rice 8
It located boot stage low temperature resistant QTL site qCTB8 on chromosome；Xiao etc. report one it is low temperature resistant related to rice maturity
QTL site qRC10-2, in addition, there is part cold tolerance gene to be cloned, including qLTG3-1, COLD1, qCTS-9, GSTZ2,
CTB4a etc..Wherein, COLD1 gene encodes the G-protein Signal Regulation factor of a positioning and cell membrane and endoplasmic reticulum, energy and G
Protein alpha subunit RGA interaction activates the GTP enzymatic activity in the channel Ca2+ and G-protein, enhances the cold resistance of rice.Japonica rice COLD1 gene
In a SNP site SNP2 originating from wild rice in China affect the activity of COLD1 gene, there is japonica rice preferably cold-resistant
Property.By japonica rice COLD1japGene, which introduces rice variety, can significantly improve the cold resistance of long-grained nonglutinous rice, in the cold resistance breeding of long-grained nonglutinous rice
In have important application value.
The low temperature resistant character of rice is typical quantitative character, and vulnerable to the influence of the factors such as environment, traditional breeding technique is logical
Phenotypic Observation is crossed to be difficult accurately to identify it.Molecular Marker Assisted Selection Technology is to educate molecular marking technique with conventional
Kind of means combine, on the basis of functional gene is cloned or is positioned, by with target gene close linkage or point isolated
Son label identifies the individual with specific desired genotype or genotype combination in group to cultivate with excellent target
A kind of technological means of character rice varieties.Compared to traditional breeding method means, it greatly improves the efficiency of breeding and accuracy,
It has been widely used in the breeding practice of various crops.Functional label is according to DNA sequence between target gene allele
Column difference and the molecular labeling that developed, advantage is mainly shown as and isolates with target gene, can accurately identify mesh
Allele is marked, expeditiously the beneficial gene in natural population or breeding population is screened, reduces gene linkage burden.
Digestion amplification polymorphism sequence (Cleaved Amplified Polymorphic Sequence, CAPS) marks
Special PCR primer is designed according to the DNA sequence dna in the site SNPs, then combines PCR reaction product with restriction enzyme
A kind of molecular labeling generated.But it is less that SNP site is located exactly at the case where restriction enzyme site, so being needed on this basis into one
Step improvement.Derivative type digestion amplification polymorphism sequence (derived Cleaved Amplified Polymorphic
Sequence, dCAPS) it is further improved molecular labeling on the basis of CAPS label, its principle is by expanding
Introduce base mismatch in primer, SNP site introduces restriction endonuclease sites, thus enable not iso-allele be distinguished mirror
It is fixed.The assignment of genes gene mapping of dCAPS molecular marking technique, map based cloning, genotype identification etc. are all widely used.
Summary of the invention
The present invention is according to japonica rice COLD1japWith long-grained nonglutinous rice COLD1indThe nucleotide sequence difference of SNP2 in gene devises
A kind of dCAPS label, by handing over F2 generation individual to carry out COLD1's 3 japonica rice varieties and 4 rice varieties and 98 Xian round-grained rice
Genotype is identified, it was demonstrated that the label can accurately identify the japonica rice COLD1 of rice COLD1 genejapGenotype, long-grained nonglutinous rice
COLD1indGenotype and heterozygous genotypes can be used for the molecular mark of COLD1 gene.
Detect the dCAPS label of the low temperature resistant gene C OLD1 genotype of rice, which is characterized in that the sequence of the dCAPS label
It is classified as:
Dcaps-F:5'-CCGTCAATCTGCCTTACAGCTATCTGTCAC-3',
Dcaps-R:5'-TGAGCTGCCTTTCCAATGTTTTGAAGGCC-3'.
The COLD1 assignment of genes gene mapping encodes a G-protein Signal Regulation factor in No. 4 chromosomes, it can be mutual with G-protein α subunit
Make, activates Ca2+The GTPase of channel and G-protein activity, to enhance the cold resistance of rice.Polymorphic site in COLD1 gene
SNP2 is base A in low temperature resistant japonica rice and wild rice in China, and in not low temperature resistant rice variety be base T or C (such as
Shown in Fig. 1), the present invention designs dCAPS according to this base difference and marks.One is introduced in the position of the 25bp of downstream primer
T → A mutation, the position of 27bp are introduced into T → G mutation (position shown in box in Fig. 1), so that COLD1japBase
There is the restriction enzyme site of a StuI (AGGCCT) in the pcr amplification product of cause, and COLD1indThe PCR product of genotype it is identical
Position is TGGCCT or CGGCCT, cannot be identified by StuI digestion.
The present invention also provides application of the dCAPS label in the detection low temperature resistant gene C OLD1 genotype of rice, the labels
It can accurately identify the japonica rice COLD1 of rice COLD1 genejapGenotype, long-grained nonglutinous rice COLD1indGenotype and heterozygous genotypes,
It can be used for the molecular mark of COLD1 gene.
The present invention finally provide detection the low temperature resistant gene C OLD1 genotype of rice method, which is characterized in that including with
Lower step:
(1) it using the genomic DNA of sample to be tested as template, is marked using dCAPS and target sequence is expanded by PCR method；
The sequence of the dCAPS label are as follows:
Dcaps-F:5'-CCGTCAATCTGCCTTACAGCTATCTGTCAC-3',
Dcaps-R:5'-TGAGCTGCCTTTCCAATGTTTTGAAGGCC-3'；
PCR response procedures are as follows: 94 DEG C, 5min；94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 30s, 35 circulations；72 DEG C,
10min；
(2) digestion is carried out to amplified production using restriction enzyme StuI, then digestion products is separated by electrophoresis, so
It is detected afterwards with cma staining；
(3) genotype of COLD1 is determined according to electrophoresis result.
The present invention designs dCAPS molecular labeling by 2 nucleotide sequence difference of COLD1 gene SNP, to 7 rice varieties
COLD1 carry out genotype detection, it is consistent that testing result and sequencing obtain COLD1 progress genotype results.Utilize the label pair
Indica-japonica hybrid F2 is identified for the COLD1 genotype of group, the results showed that japonica rice COLD1japGenotype, heterozygous genotypes
And long-grained nonglutinous rice COLD1indGenotypic segregation ratio meets 1:2:1 (χ 2=1.238, P=0.537 > 0.05).These are the result shows that originally
Invention design dCAPS is labeled as codominant marker, can effectively distinguish pure and mild, heterozygous genotypes, can be applied to rice COLD1 base
The molecular mark of cause works.
Detailed description of the invention
Fig. 1 is COLD1japAnd COLD1indPartial sequence compares and dCAPS marks design diagram.
Fig. 2 is the electrophoretogram that 7 rice varieties COLD1 genotype detections are used for using dCAPS label.
Fig. 3 is 7 rice varieties COLD1 genotype sequencer maps.
Fig. 4 is the electrophoretogram using dCAPS label for 48 F2 for single plant COLD1 genotype detection.
Specific embodiment
Below in conjunction with drawings and examples, the present invention is further explained.
Embodiment 1
1, rice material
Rice material used in the present invention include elegant water 134, Shen Fan 14, Shen Fan 26,9311, imperial Mortopl B, Guanglu ai 4,
Nona Bokra, wherein elegant water 134 is conventional japonica rice kind, Shen Fan 14 and Shen Fan 26 are Restoring Line of Japonica Rice, and imperial Mortopl B is long-grained nonglutinous rice
System is kept, Guanglu ai 4 and Nona Bokra are conventional Indica Rice Cultivars.F2 derives from elegant water 134 for population material and wide land is short by 4
Number hybridization.
2, oryza sativa genomic dna extracts
Each rice varieties take 50mg or so blade, are shredded with scissors, are put into 2mL centrifuge tube, be added 600 μ L1.5 ×
The steel ball of CTAB solution (1.5%CTAB, 75mMTris-HCl, 15mMEDTA, 1.05MNaCl, PH8.0) and a diameter 5mm,
The frequency oscillation of 70Hz grinds 90s on quick beveller；Sample warm bath 20min, addition in 56 DEG C of water-baths after grinding
450 μ L chloroforms, acutely after concussion, 12000r/min is centrifuged 10min；Take 450 μ L supernatants to the centrifuge tube of another 1.5mL
In, the dehydrated alcohol of 900 μ L is added, places 10min in -20 DEG C of refrigerators after mixing, 12000r/min is centrifuged 10min, supernatant is abandoned,
The distilled water dissolving DNA of 200 μ L is added after drying, it is spare to put -20 DEG C of refrigerators.
3, the design of dCAPS label
Polymorphic site SNP2 is base A in low temperature resistant japonica rice and wild rice in China in COLD1 gene, and intolerant to
It is base T or C (as shown in Figure 1) in the rice variety of low temperature, the present invention designs dCAPS according to this base difference and marks.?
The position that the position of the 25bp of downstream primer is introduced into T → A mutation 27bp is introduced into one T → G and is mutated (box in Fig. 1
Shown in position), so that COLD1japThere is the restriction enzyme site of a StuI (AGGCCT) in the pcr amplification product of gene, and
COLD1indThe same position of the PCR product of genotype is TGGCCT or CGGCCT, cannot be identified by StuI digestion.
Design the sequence of dCAPS label are as follows:
Dcaps-F:5'-CCGTCAATCTGCCTTACAGCTATCTGTCAC-3',
Dcaps-R:5'-TGAGCTGCCTTTCCAATGTTTTGAAGGCC-3'.
Pcr amplification product is 196bp, then PCR product StuI digestion, COLD1japThe PCR product of genotype can be cut
It opens, obtains the product of 169bp.
4, the detection method of molecular labeling
50 μ L of PCR system total volume, including 25 μ L 2 × Taq PCR Master Mix, forward and reverse primer (dCAPS
Label) each 1.5 μ L (10 μm of ol/L), 1mL oryza sativa genomic dna (100ng/ μ L)；PCR response procedures are as follows: 94 DEG C, 5min；
94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 30s, 35 circulations；72 DEG C, 10min；The amplification of COLD1 gene specific molecular labeling obtains
The PCR product obtained is after restriction enzyme StuI digestion 2h, the electrophoresis 60V in 8% polyacrylamide denaturing electrophoretic gel
Overnight, it is then detected with cma staining.
5, result and analysis
With dCAPS label to elegant water 134, Shen Fan 14, Shen Fan 26,9311, imperial Mortopl B, Guanglu ai 4 and Nona Bokra
COLD1 genotype Deng 7 rice varieties is detected.The electrophoresis result of PCR product shows that 7 rice varieties can expand
The DNA fragmentation of 196bp size out.By PCR product StuI digestion, digestion products carry out electrophoresis detection, as a result, it has been found that elegant water
134, the PCR product of 3 japonica rice varieties such as Shen Fan 14 and Shen Fan 26 can be cut by StuI, and electrophoresis showed goes out the band of 169bp,
And the PCR product of 4 rice varieties such as 9311, imperial Mortopl B, Guanglu ai 4 and Nona Bokra cannot be cut by StuI, electricity
Swimming still shows the band (Fig. 2) of a 199bp.In order to verify the reliability of dCAPS Marker Identification result, to this 7 rice product
Kind 2 site of COLD1 gene SNP is sequenced, and sequencing result shows 3 japonica rice varieties such as elegant water 134, Shen Fan 14 and Shen Fan 26
The site SNP2 is a base A in COLD1 gene, and 4 long-grained nonglutinous rices such as 9311, imperial Mortopl B, Guanglu ai 4 and Nona Bokra
2 site of COLD1 gene SNP of kind is a base T (as shown in Figure 3).The result one of sequencing result and dCAPS label detection
It causes.
Embodiment 2
It is marked to the F2 obtained using elegant water 134 with Guanglu ai 4 as parents using the dCAPS of COLD1 for group
It is detected.Detection method is same as Example 1.From F2 for randomly choosing 96 plants of rice in group, to the genotype of COLD1 into
Row detection, testing result discovery, have in F2 group respectively 25 wild type (196bp) single plants, 41 heterozygous mutants (196bp,
169bp) single plant, 30 homozygous mutant (169bp) single plants, F2 group intermediate keng rice COLD1japGenotype, heterozygous genotypes and
Long-grained nonglutinous rice COLD1indGenotypic segregation ratio meets 1:2:1 (χ 2=1.238, P=0.537 > 0.05) (as shown in Figure 4).Show this hair
The dCAPS label of bright design can accurately judge the japonica rice COLD1 of rice individual COLD1 genejapGenotype, long-grained nonglutinous rice
COLD1indGenotype and heterozygous genotypes, the molecular mark suitable for COLD1 gene.
Sequence table
<110>Academy of Agricultural Sciences, Shanghai City
<120>dCAPS label and the application of the low temperature resistant gene C OLD1 genotype of rice are detected
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ccgtcaatct gccttacagc tatctgtcac 30
<210> 2
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tgagctgcct ttccaatgtt ttgaaggcc 29
Claims (3)
1. detecting the dCAPS label of the low temperature resistant gene C OLD1 genotype of rice, which is characterized in that the sequence of the dCAPS label
Are as follows:
Dcaps-F:5'-CCGTCAATCTGCCTTACAGCTATCTGTCAC-3',
Dcaps-R:5'-TGAGCTGCCTTTCCAATGTTTTGAAGGCC-3'.
2. application of the label of dCAPS described in claim 1 in the detection low temperature resistant gene C OLD1 genotype of rice.
3. the method for detecting the low temperature resistant gene C OLD1 genotype of rice, which comprises the following steps:
(1) it using the genomic DNA of sample to be tested as template, is marked using dCAPS and target sequence is expanded by PCR method；It is described
The sequence of dCAPS label are as follows:
Dcaps-F:5'-CCGTCAATCTGCCTTACAGCTATCTGTCAC-3',
Dcaps-R:5'-TGAGCTGCCTTTCCAATGTTTTGAAGGCC-3'；
PCR response procedures are as follows: 94 DEG C, 5min；94 DEG C, 30s, 60 DEG C, 30s, 72 DEG C, 30s, 35 circulations；72 DEG C, 10min；
(2) digestion is carried out to amplified production using restriction enzyme StuI, then polyacrylamide gel is carried out to digestion products
Electrophoretic separation, is then detected with cma staining；
(3) genotype of COLD1 is determined according to electrophoresis result.
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Cited By (2)
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| CN110540987A (en) * | 2019-08-29 | 2019-12-06 | 毕节市农业科学研究所 | Design and detection method of new functional marker of rice low temperature resistance gene COLD1 |
| CN111334604A (en) * | 2020-04-01 | 2020-06-26 | 上海市农业科学院 | PCR/LDR molecular marker and method for identifying low temperature resistant gene COLD1 genotype of rice |
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| CN110540987A (en) * | 2019-08-29 | 2019-12-06 | 毕节市农业科学研究所 | Design and detection method of new functional marker of rice low temperature resistance gene COLD1 |
| CN111334604A (en) * | 2020-04-01 | 2020-06-26 | 上海市农业科学院 | PCR/LDR molecular marker and method for identifying low temperature resistant gene COLD1 genotype of rice |
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