CN105385778A - Primers and method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput - Google Patents
Primers and method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput Download PDFInfo
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Abstract
The invention relates to primers and a method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput. The primers comprise the specific primer of which the nucleotide sequence is shown as SEQ ID No.1 and SEQ ID No.2 and a universal prime of which the nucleotide sequence is shown as SEQ ID No.3. According to the primers and method, the allelic variation condition of the AhFAD2B genes can be detected and identified in high throughput, the maximum sample quantity of one-time detection can reach 1536, the detection cost is low, rapidness and accuracy are achieved, and the efficiency and accuracy of peanut breeding selection are greatly improved.
Description
Technical field
The present invention relates to primer and the method for high throughput testing peanut AhFAD2B gene SNP gene type, belong to technical field of molecular biology.
Background technology
Peanut is one of large oil crops in the world four, and the content of its seed lipid acid and composition are the important indicators weighing peanut quality.From Oil stability angle, the peanut of high gas oil ratio/linoleic acid ratio (O/L ratio) and goods not easily oxidative rancidity thereof, shelf-lives is longer.Research shows, the high gas oil ratio peanut after curing is than conventional oil acid content peanut extended pot life 8 times (Mozingoetal., 2004).Oleic acid is of value to maintenance good cholesterol high-density lipoprotein (HDL) (HDL) level, can strengthen insulin sensitivity, and improve some inflammation (MesaGarciaetal., 2006; Vassiliouetal., 2009).The industrial saturation ratio improving grease usually through hydrogenation, to improve the stability of grease, this often introduces trans double bond and generates trans fatty acid in carbochain, and long-term edible meeting increases the probability suffering from cardiovascular and cerebrovascular disease greatly.Therefore, the high gas oil ratio content of Ecological Property of Peanut Seeds and high O/L ratio just become the major objective of peanut grease genetic improvement.
FAD2 is the key enzyme controlling plant seed oleic acid, linoleic acid content, and its catalysis oleic acid produces linolic acid at carbon 12 desaturations.Peanut is allotrtraploid (2n=4X=40; genome AABB); AhFAD2A and AhFAD2B is positioned at the non-allelic genes in different genes group; these two gene pairs oleic acid, linoleic acid contents all have contribution; these two genetic transcriptions are suppressed or enzyme is lived reduces, and seed can show high gas oil ratio proterties.Some high gas oil ratio mutant (as F435,8-2122, M2-225, MF) (Nordenetal., 1987 that the U.S. is obtained by chemomorphosis and spontaneous mutation screening the eighties in last century; Ashri, 1988), use till today as parent always.These kinds or strain seed oleic acid content are all about 80%.The hereditary basis analyzing F435 and Derivative line high gas oil ratio proterties thereof finds, 1,442nt place, AhFAD2B gene coding region A base is inserted, and causes proteins encoded premature termination.For the SNP difference of said mutation body and Derivative line AhFAD2B gene, develop multiple mark for detecting and detection method in recent years, as enzyme cuts amplification polymorphism ordination (CAPS, cleavedamplifiedpolymorphicsequence), allele specific pcr (AS-PCR, Allele-SpecificPCR), sequencing, quantitative real-time PCR etc., thus realize carrying out assisted Selection (Jungetal., 2000a to high gas oil ratio peanut breeding; Chuetal., 2007; Chuetal., 2009; Chenetal., 2010).Current China has also cultivated several high gas oil ratio peanut varieties, the flower cultivated as Shandong peanut institute educates 32 (Shandong Province's authorizations in 2009), flower educates 51 and Hua Yu 52, Kaifeng, Henan Province agricultural and forest science research institute opens agriculture H03-3, opens agriculture 61, opens agriculture 176, opens 17-15, and Hebei province spends Ji No. 11, No. 13 etc.These kinds are identical in the mutational site at 442nt place with mutant F435AhFAD2B gene.Identical mark and detection method can be used.
Above detection method only fluorescence quantifying PCR method can realize high throughput testing, TaqMan probe method is very effective for the detection of AhFAD2B gene 442 InDel differences, but because the fluorescently-labeled probe of the method is gene-specific probe, testing cost is relatively high.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of primer and method of high throughput testing peanut FAD2B gene SNP gene type are provided.The present invention devises specificity amplification primer and the universal primer of AhFAD2B gene, the TouchdownPCR method that connected applications condition is strict, establishes the method for application LGC high-throughput marker detection detection of platform peanut AhFAD2B Allelic Variation.
The present invention is achieved through the following technical solutions:
A primer for high throughput testing peanut AhFAD2B gene SNP gene type, comprising:
Special primer, nucleotide sequence is as shown in SEQIDNo.1 and SEQIDNo.2;
Universal primer, nucleotide sequence is as shown in SEQIDNo.3.
SEQ ID No .1 is by 21 based compositions, and this sequence is the reverse complementary sequence of AhFAD2B gene ORF district from 5 ' end 441-461 bit base; SEQIDNo.2 is by 22 based compositions, and this sequence is the reverse complementary sequence of AhFAD2B gene ORF district from 5 ' end 442-463 bit base; SEQ ID No .3 is by 22 based compositions, and this sequence is that AhFAD2B gene ORF district is from 5 ' end 415-436 bit base sequence.Allele-1tail and Allele-2tail is connected to respectively at the 5 ' end of SEQIDNo.1 and SEQIDNo.2, these two to add tailer sequence identical with the sequence of the universal fluorescent probe in LGC company MasterMix detection kit respectively, 5 ' end of probe marked FAM and HEX fluorophor respectively, and this probe is researched and developed product by LGC company and sold.
A method for high flux screening AhFAD2B Allelic Variation, comprises the steps:
I () to get sample the DNA of product, after 12000g is centrifugal, dries 20 ~ 35min for 65 DEG C, obtained genomic dna;
(ii) with the obtained genomic dna of step (i) for template, the PCR reaction system of preparation containing FAM quenching group and HEX quenching group, carries out pcr amplification, fluorescence intensity, reading of data; The nucleotide sequence of primer is as shown in SEQIDNo.1, SEQIDNo.2, SEQIDNo.3;
(iii) to the data analysis of step (ii), when the fluorescence only having FAM probe being detected, then the 442nd isozygotys for A base deletion, i.e. wild-type genotype, is designated as BB; When the fluorescence only having HEX probe being detected, then the 442nd A base is inserted and is isozygotied, i.e. mutated-genotype, is designated as bb; When the fluorescence of FAM probe and HEX probe being detected, then the 442nd is heterozygote, is designated as Bb.
Preferred according to the present invention, in described step (ii), the PCR reaction system containing FAM quenching group and HEX quenching group is made up of the MasterMix containing FAM quenching group and HEX quenching group of LGC Company, genomic dna and primer;
Preferred further, the PCR reaction system in described step (ii) is 1 μ L, and component is as follows:
Preferred according to the present invention, in described step (ii), pcr amplification reaction condition is as follows:
94 DEG C of denaturation 15min;
The first step amplified reaction, 94 DEG C of sex change 20s, 61 DEG C-55 DEG C annealing, 61 DEG C-55 DEG C extend 60s, 10 TouchDown circulations, each circulation reduction by 0.6 DEG C;
Second step amplified reaction, 94 DEG C of sex change 20s, 55 DEG C of annealing, 55 DEG C extend 60s, 26 circulations.
Preferred according to the present invention, in described step (ii), the temperature of fluorescence intensity is less than 40 DEG C.
Preferred according to the present invention, in described step (ii), digital independent uses LGC company SNPviewer software to carry out.
Preferred according to the present invention, in described step (iii), interpretation of result uses LGC company SNPviewer software to carry out.This software can the disappearance of high throughput testing AhFAD2B gene 442ntA base or insertion.
Beneficial effect
1, the present invention is directed to disappearance and the insertion of 442 the A bases in closely-related AhFAD2BORF district with arachic acid linoleic acid content, select the section that GC content is higher, devise Auele Specific Primer respectively, to guarantee the accuracy of gene type;
2, the present invention is when designing primer, considers the difference of allelic variation site sequence, by the 3 ' end of 1 of Classification Identification base difference design at two Auele Specific Primers, is guaranteed the specificity increased by TouchDownPCR;
3, the present invention high throughput testing can differentiate AhFAD2B alleles variation situation, and one-time detection sample size reaches as high as 1536, and testing cost is low, fast, accurately, substantially increases efficiency and the accuracy of peanut breeding selection.
Accompanying drawing explanation
Fig. 1 is that in embodiment 2, part KASP method detects each genotype point bunch distribution plan in AhFAD2B sample;
Fig. 2 is that in embodiment 2, TaqMan probe method detects each genotype point bunch distribution plan in AhFAD2B;
Embodiment
Below in conjunction with embodiment and Figure of description, technical scheme of the present invention is further elaborated, but institute of the present invention protection domain is not limited thereto.
In following embodiment, all methods are ordinary method if no special instructions.The synthesis of the primer, probe completes by gene limited-liability company.
Embodiment 1, for detecting the design of the primer of AhFAD2B gene 442 A insertion mutations
There is wild-type disappearance in AhFAD2B gene group sequence ORF district the 442nd bit base sequence and saltant type inserts A base two kinds of sequences, causes the aminoacid sequence of encoding to be saltant type coding frameshit premature termination by wild-type complete protein mutation of encoding.According to the position in said mutation site, design KASP allelic differences primer is (as table 1, SEQIDNo:1, SEQIDNo:2), the diversity sequence of AhFAD2B gene is made to be positioned at 3 ' end of primer, and 5 ' end is connected to Allele-1tail and Allele-2tail respectively, these two to add tailer sequence identical with the sequence of the universal fluorescent probe in LGC company MasterMix detection kit, and 5 ' end of probe marked FAM and HEX fluorophor (this probe sequence is LGC house journal) respectively.According to AhFAD2B gene group sequence ORF district, design universal primer (as table 1, SEQIDNo.3).
Table 1. detects the primer information of AhFAD2B allele sudden change
SEQ ID No .1 is by 21 based compositions, and this sequence is the reverse complementary sequence of AhFAD2B gene ORF district from 5 ' end 441-461 bit base; SEQIDNo.2 is by 22 based compositions, and this sequence is the reverse complementary sequence of AhFAD2B gene ORF district from 5 ' end 442-463 bit base;
SEQ ID No .3 is by 22 based compositions, and this sequence is the forward sequence of AhFAD2B gene ORF district from 5 ' end 415-436 bit base.
Allelotrope primer 5 ' end is connected to Allele-1tail and Allele-2tail respectively, and these two add tailer sequence and are connected with the probe identification of FAM luminophore and HEX luminophore respectively, detects 442 A base deletions or insertion respectively.
The detection in embodiment 2, peanut AhFAD2B gene SNP mutational site
Loci detection is carried out with the primer designed according to AhFAD2B allele difference and LGC company test kit MasterMix probe
The F1 generation hybrid that to comprise common oleic acid peanut (genotype aaBB) for examination peanut material be maternal and high gas oil ratio peanut (genotype aabb) is paternal hybrid, F2 are detected for segregating population according to the primer of AhFAD2B allele difference design and MasterMix probe with in embodiment 2, simultaneously with the common oleic acid peanut of female parent and high gas oil ratio peanut for contrast, concrete grammar is as follows:
I () is for the extraction of examination peanut sample genomic dna
The plant genome DNA provided by TIANGEN company extracts test kit (catalog number (Cat.No.): DP-305), and extracts for examination peanut sample genomic dna according to test kit specification sheets, and concrete steps are as follows:
1. in liquid nitrogen, grind fresh or-20 DEG C of freezing specimen materials (noting: sample will be ground to powder fast, fully) of 100mg;
2. ground powder is transferred to rapidly preliminary election and 700 μ l are housed, (before experiment, in the GP1 of preheating, mercaptoethanol is added in the centrifuge tube of the damping fluid GP1 of 65 DEG C of preheatings, its final concentration is made to be 0.1wt%), after putting upside down mixing rapidly, centrifuge tube is placed on 65 DEG C of water-baths 20 minutes, puts upside down centrifuge tube in water-bath process with biased sample for several times;
3. add 700 μ l chloroforms, fully mix, the centrifugal 5min of 12000rpm; Note: be rich in the plant tissue of polyphenol or starch if extract, can before the 3rd step, with phenol: chloroform/1:1 carries out equal-volume extracting;
4. carefully previous step gained upper strata aqueous phase is proceeded to a new centrifuge tube, add the damping fluid GP2 of 700 μ l, fully mix;
5. the liquid of mixing is proceeded to adsorption column CB3, the centrifugal 30s of 12000rpm, discards waste liquid;
6. in adsorption column CB3, add 500ul damping fluid GD (whether preoperation inspection adds dehydrated alcohol), the centrifugal 30s of 12000rpm, discards waste liquid;
7. in adsorption column CB3, add 600ul rinsing liquid PW (whether preoperation inspection adds dehydrated alcohol), the centrifugal 30s of 12000rpm, outwells waste liquid;
8. repeating step 7.;
9. put back in collection tube by adsorption column CB3, the centrifugal 2min of 12000rpm, outwells waste liquid.Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material; Attention: the object of this step is removed by rinsing liquid remaining in adsorption column, in rinsing liquid, the residual of dehydrated alcohol can affect follow-up endonuclease reaction experiment;
10. be put into by adsorption column in a clean centrifuge tube, to the unsettled dropping in adsorption film mid-way appropriate 50-200 μ l elution buffer TE (pH value is between 7.0-8.5), room temperature places 2min.The centrifugal 2min of 12000rpm collects DNA solution;
Finally use content and the purity of UV spectrophotometer measuring DNA, institute test sample product A260/280 is between 1.8 ~ 2.0 in result display, shows that extracted DNA purity is higher, by obtained DNA after 12000g is centrifugal, dry 30min for 65 DEG C, obtained genomic dna;
(ii) with the obtained genomic dna of step (i) for template, preparation PCR reaction system
The nucleotide sequence of PCR primer is as shown in SEQIDNo.1, SEQIDNo.2, SEQIDNo.3, and MasterMix fluorescent probe is LGC reagent kit product;
The DNA extracted with step (i) is template, detects AhFAD2B gene group sequence ORF district the 442nd bit base sequence InDel equipotential difference with the primer in embodiment 1 table 1.
KASPPCR reaction is carried out in LGCSNPlineXL water-bath PCR instrument, and total reaction system is 1 μ L.
Comprise 10 ~ 50ng genomic dna in PCR reaction system, 0.5 μ LMatsermix, 0.013 μ L allelotrope primer and universal primer mixture, all the other are supplied with deionized water.After application of sample, low-speed centrifugal, sealer.
The KASPPCR reaction system of table 2AhFAD2B gene SNP somatotype
Pcr amplification reaction condition is as follows:
94 DEG C of denaturation 15min;
The first step amplified reaction, 94 DEG C of sex change 20s, 61 DEG C-55 DEG C annealing, 61 DEG C-55 DEG C extend 60s, 10 TouchDown circulations, each circulation reduction by 0.6 DEG C;
Second step amplified reaction, 94 DEG C of sex change 20s, 55 DEG C of annealing, 55 DEG C extend 60s, 26 circulations.
After pcr amplification terminates, when temperature of reaction is down to less than 40 DEG C, reading of data, digital independent uses LGC company SNPviewer software to carry out.
(iii) analyze the detected result of step (ii), analyze and use LGC company SNPviewer software to carry out, when the fluorescence only having HEX probe being detected, then the 442nd isozygotys for the insertion of A base, i.e. mutated-genotype, is designated as bb; When the fluorescence only having FAM probe being detected, then the 442nd A base deletion is isozygotied, i.e. wild-type genotype, is designated as BB; When the fluorescence of HEX probe and FAM probe being detected, then the 442nd is heterozygote, is designated as Bb.
For AhFAD2B gene the 442nd bit base InDel equipotential Difference test, use two pairs of fluorescent probes (what recognition allele primer 5 ' was held respectively adds tailer sequence), the probe indicating FAM mates completely with dominant allele (B), and another fluorescent probe indicating HEX mates (b) completely with Recessive alleles.
During the experimental design of allelotrope discriminatory analysis, respectively with without Template Controls, known only containing dominant allele B, Recessive alleles b, determine that heterozygosis peanut varieties containing aobvious Recessive alleles (Bb) is for standard.Unknown sample will be divided into following 3 classes: 1. dominant homogeneous: only containing dominant homogeneous gene BB in sample; 2. allozygote: only containing recessive homozygous gene bb in sample; 3. heterozygote: simultaneously containing allelotrope B and allelotrope b in sample.
Result judges: if only there is FAM fluorescent signal obviously to raise, then sample is dominant homogeneous; If only there is HEX fluorescent signal (can detect HEX fluorophor) obviously to raise, then sample is allozygote; Two kinds of fluorescent signals all raise, then sample is heterozygote Bb.For the ease of judging, digital independent and interpretation of result use LGC company SNPviewer software to carry out.
For verifying feasibility of the present invention and accuracy, 91 unknown gene type samples (numbering 1-91) are chosen in laboratory, for improving experiment accuracy with credible, with Shandong spend 14 (BB) with flower educate 32 (bb) as put bunch demarcation, adopt TaqMan probe detection method and KASP-PCR detection method to carry out detection validation respectively, analyze the consistence of two kinds of method detected results.
The detected result of detection method different from comparing sample segment added up by table 3, table 4
Table 3. different detection method sample segment detected result Statistical Comparison
The comparison of the different detection method of table 4. idiostatic ectopic sites
A. experimental period is according to completing 500 sample detection estimations; B. the different cycles calculates according to using different detecting instruments with cost
As can be seen from the above results, KASP detected result and TaqMan probe detected result more than 95% are consistent, and TaqMan probe detected result is more accurate.But TaqMan probe method testing cost is too high, KASP testing cost is lower, therefore, the KASP high throughput testing system of the AhFAD2BSNP gene type that the present invention sets up, by make in peanut breeding process large group screening, the excavation of molecule marker, the Fine Mapping of important character, become more economical, efficient, simple, accurately.
Claims (8)
1. a primer for high throughput testing peanut AhFAD2B gene SNP gene type, is characterized in that, comprising:
Special primer, nucleotide sequence is as shown in SEQIDNo.1 and SEQIDNo.2;
Universal primer, nucleotide sequence is as shown in SEQIDNo.3.
2. a method for high flux screening AhFAD2B Allelic Variation, is characterized in that, comprises the steps:
I () to get sample the DNA of product, after 12000g is centrifugal, dries 20 ~ 35min for 65 DEG C, obtained genomic dna;
(ii) with the obtained genomic dna of step (i) for template, the PCR reaction system of preparation containing FAM quenching group and HEX quenching group, carries out pcr amplification, fluorescence intensity, reading of data; The nucleotide sequence of primer is as shown in SEQIDNo.1, SEQIDNo.2, SEQIDNo.3;
(iii) to the data analysis of step (ii), when the fluorescence only having FAM probe being detected, then the 442nd isozygotys for A base deletion, i.e. wild-type genotype, is designated as BB; When the fluorescence only having HEX probe being detected, then the 442nd A base is inserted and is isozygotied, i.e. mutated-genotype, is designated as bb; When the fluorescence of FAM probe and HEX probe being detected, then the 442nd is heterozygote, is designated as Bb.
3. method as claimed in claim 2, it is characterized in that, in described step (ii), the PCR reaction system containing FAM quenching group and HEX quenching group is made up of the MasterMix containing FAM quenching group and HEX quenching group of LGC Company, genomic dna and primer.
4. method as claimed in claim 3, it is characterized in that, the PCR reaction system in described step (ii) is 1 μ L, and component is as follows:
5. method as claimed in claim 2, it is characterized in that, in described step (ii), pcr amplification reaction condition is as follows:
94 DEG C of denaturation 15min;
The first step amplified reaction, 94 DEG C of sex change 20s, 61 DEG C ~ 55 DEG C annealing, 61 DEG C ~ 55 DEG C extend 60s, 10 TouchDown circulations, each circulation reduction by 0.6 DEG C;
Second step amplified reaction, 94 DEG C of sex change 20s, 55 DEG C of annealing, 55 DEG C extend 60s, 26 circulations.
6. method as claimed in claim 2, it is characterized in that, in described step (ii), the temperature of fluorescence intensity is less than 40 DEG C.
7. method as claimed in claim 2, is characterized in that, in described step (ii), digital independent uses LGC company SNPviewer software to carry out.
8. method as claimed in claim 2, is characterized in that, in described step (iii), interpretation of result uses LGC company SNPviewer software to carry out.
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| CN105969879A (en) * | 2016-06-17 | 2016-09-28 | 山东省农业科学院生物技术研究中心 | High-throughput primer set and detection method for detection of AhFAD2A gene mutation site typing |
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| CN113557956A (en) * | 2021-07-28 | 2021-10-29 | 青岛农业大学 | Construction method of peanut genetic population |
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| CN105969879A (en) * | 2016-06-17 | 2016-09-28 | 山东省农业科学院生物技术研究中心 | High-throughput primer set and detection method for detection of AhFAD2A gene mutation site typing |
| CN105969879B (en) * | 2016-06-17 | 2020-02-07 | 山东省农业科学院生物技术研究中心 | Primer group for high-throughput detection of AhFAD2A gene mutation site typing and detection method |
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| CN108753803B (en) * | 2018-07-16 | 2019-04-23 | 河北省农林科学院粮油作物研究所 | A kind of high oleic acid peanut mutated gene AhFAD2B-814 and application |
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| CN108753803A (en) * | 2018-07-16 | 2018-11-06 | 河北省农林科学院粮油作物研究所 | A kind of high oleic acid peanut mutator AhFAD2B-814 and application |
| CN108893551B (en) * | 2018-07-16 | 2021-09-14 | 河北省农林科学院粮油作物研究所 | Molecular marking method for detecting high oleic acid content of peanuts and application |
| CN109593876A (en) * | 2019-01-25 | 2019-04-09 | 河北省农林科学院粮油作物研究所 | The KASP label serotype specific primer group and its application of high throughput detection AhFAD2B gene mutation site |
| CN110923354A (en) * | 2019-12-13 | 2020-03-27 | 山东省花生研究所 | Method for detecting high-oleic-acid peanuts and application thereof |
| CN110923354B (en) * | 2019-12-13 | 2022-07-01 | 山东省花生研究所 | Method for detecting high-oleic-acid peanuts and application thereof |
| CN113557956A (en) * | 2021-07-28 | 2021-10-29 | 青岛农业大学 | Construction method of peanut genetic population |
| CN113557956B (en) * | 2021-07-28 | 2022-04-15 | 青岛农业大学 | Construction method of peanut genetic population |
| CN118086564A (en) * | 2024-03-25 | 2024-05-28 | 吉林农业大学 | Molecular marker of QTL locus qlinol.17 related to peanut high-oil and high-oleic acid traits, primer combination and application |
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