CN105200144A - High-flux molecular marker for identifying pulp color of Cucumis melo L. and special primer for high-flux molecular marker - Google Patents
High-flux molecular marker for identifying pulp color of Cucumis melo L. and special primer for high-flux molecular marker Download PDFInfo
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Abstract
The invention discloses a high-flux molecular marker for identifying pulp color of Cucumis melo L. and a special primer for the high-flux molecular marker. The special primer disclosed by the invention is used for detecting a KASP primer for identifying a CmOr gene of the Cucumis melo L., and the specific nucleotide sequences of the specific primer are show in sequences 1 to 3 in a sequence table. A high-flux molecular marker system, based on a PCR (Polymerase Chain Reaction) SNP (Single Nucleotide Polymorphism) Line platform, which is provided by the invention is used for detecting a CmOr genetype of the Cucumis melo L., the operation flow is fully automatic, and artificial errors are reduced; the high-flux molecular marker is high in analysis flux and is very suitable for simultaneously detecting a large amount of samples. The molecular marker for high-flux detection is designed on the basis of a sequence of a color control gene CmOr of Cucumis melo L. and is applied to transformation breeding of color characters of the Cucumis melo L., so that the time and the labor cost can be greatly saved, the breeding efficiency of assisted selection of the molecular marker is improved, and transformation breeding of the pulp color of the Cucumis melo L. to an excellent skeleton inbred line is accelerated.
Description
Technical field
The invention belongs to biology field, relate to a kind of high-throughput molecule marker for the identification of muskmelon pulp colour and primer special thereof.
Background technology
Muskmelon (CucumismeloL.) is the Important Economic crop of Curcurbitaceae Cucumis muskmelon kind.In the main edible part of muskmelon, endocarp, be referred to as pulp, pulp colour proterties is the important commodity property of muskmelon, and unique color often more gets consumer reception.Muskmelon pulp colour has abundant variation type, mainly contain the mixture colors (WatanabeK of orange, shallow orange, green, white and these colors, SaitoT, HirotaS, TakahashiB, FujishitaN.1991.Carotenoidpigmentsinorange, lightorange, greenandwhitefleshcoloredfruitsofmelon (CucumismeloL.) .JournaloftheJapaneseSocietyforFoodScienceandTechnology, 38:153 – 159.).The diversity of pulp colour makes muskmelon breeding towards the future development of diversification, increases market to the demand of dissimilar muskmelon.
Muskmelon pulp colour is by two Gene Handling: green meat gene (gf) and plain boiled pork gene (wf, to orange pulp in recessive), wherein the green meat gene of plain boiled pork gene pairs has dominant epistatiic.Muskmelon Exocarpium Citri Rubrum meat (Gf) to green meat (gf) for dominant; When carrying gfgf gene, muskmelon pulp is rendered as and presents green meat (Wf-gfgf) or plain boiled pork (wfwfgfgf) (Hughes, M. (1948) TheinheritanceoftwocharactersofCucumismeloandtheirinterr elationship.Proc.Am.Soc.Hortic.Sci.59,399 – 402.; Clayberg, C. (1992) InteractionandlinkagetestoffleshcolorgenesinCucumismeloL .Rep.CucurbitGenet.Coop.15,53.).
In recent years, there is investigator by methods such as genetic map construction, genomic library construction and QTL location, located the gene locus controlling pulp colour.Cuevas etc. (2009) form relevant β-carotene synthetic gene by 1 with pulp colour and navigate to muskmelon the 9th linkage group.Muskmelon collection of illustrative plates that Haral-Beja etc. (2010) build at it the 2nd, 6,8 linkage groups have found 3 QTLs relevant with pulp colour; Wang Xianlei etc. (2011) utilize Japan's " peace No. 2, agriculture " and Xinjiang honey melon " K413 " to hybridize the F2 colony obtained, QTL relevant for pulp colour is navigated in the 9th linkage group, and have found two flanking markers being respectively 12.8cm and 7.4cm with pulp colour genetic distance.Yang Guanghua etc. (2014) also by the assignment of genes gene mapping of pulp colour on Chromosome 9.Because the experiment material of early stage institute is different with mark, the QTL quantity relevant from muskmelon pulp colour proterties navigated to is different with position, can not be corresponding with corresponding karyomit(e), relatively just very difficult between different collection of illustrative plates.In addition muskmelon pulp colour character inheritance background is complicated, adopt traditional hybridization, to backcross and Phenotypic Selection carries out that breeding cycle is long, cost is high, molecular mark can select germ plasm resource from genotype top sieve, greatly improve breeding efficiency, molecular mark has been widely applied in breeding practice, obtains good effect.At present about the molecule marker of muskmelon pulp colour and the distant of target gene, versatility and accuracy are not high, cannot play a role in actual breeding work, this limits to a certain extent and carries out for muskmelon pulp colour correlated character the breeding process improved.Obtain the molecule marker chain with muskmelon pulp colour controlling gene, the functional molecular marker particularly for the crucial mutational site exploitation of target gene utilizes molecule marker to assist the transformation of muskmelon pulp colour proterties, realizes the precondition of germplasm innovation.
Completing and externally announcing along with muskmelon gene order-checking, particularly based on a large amount of SSR and the SNP marker of the exploitation of muskmelon genomic information, accelerates location and the clone of muskmelon pulp colour correlated character gene.Tzuri etc. (2015) according to forefathers to the Position Research result of pulp colour genes involved in conjunction with muskmelon genomic information, have found the CmOr gene controlling muskmelon pulp colour proterties, the clone of muskmelon pulp colour controlling gene CmOr makes based target gene order development function type molecule marker become possibility, but cut owing to needing to carry out enzyme to PCR primer for SNP site exploitation CAPS (dCAPS) mark of CmOr gene, recycling polyacrylamide gel carries out the steps such as separation detection to target fragment, process is comparatively loaded down with trivial details, the application making the CAPS (dCAPS) for SNP exploitation be marked at the molecular mark aspect of muskmelon pulp colour proterties transformation has certain limitation.
Summary of the invention
An object of the present invention is to provide a kind of KASP primer for detecting muskmelon CmOr gene.
KASP primer for detecting muskmelon CmOr gene provided by the present invention, is made up of primer 1, primer 2 and primer 3; Described primer 1 is the single stranded DNA being followed successively by the 22-49 position of sequence 1 in sequence label A and sequence table from 5 ' end to 3 ' end; Described primer 2 is the single stranded DNA being followed successively by the 22-49 position of sequence 2 in sequence label B and sequence table from 5 ' end to 3 ' end; Described primer 3 is the single stranded DNA of nucleotide sequence as shown in sequence in sequence table 3.
Further, the nucleotides sequence of described sequence label A is classified as the 1-21 position of sequence 1 in sequence table; The nucleotides sequence of described sequence label B is classified as the 1-21 position of sequence 2 in sequence table.
More concrete, described primer 1 is the single stranded DNA of nucleotide sequence as shown in sequence in sequence table 1; Described primer 2 is the single stranded DNA of nucleotide sequence as shown in sequence in sequence table 2.
Second object of the present invention is to provide a kind of test kit for detecting muskmelon CmOr gene.
Test kit for detecting muskmelon CmOr gene provided by the present invention, containing described KASP primer.
Also containing fluorescent probe A, fluorescent probe B, quenching probes A and quenching probes B in described test kit;
The nucleotide sequence of described fluorescent probe A is consistent with the nucleotide sequence of described sequence label A, and 5 ' end connects fluorophor A; The nucleotide sequence of described quenching probes A and the nucleotide sequence reverse complemental of described sequence label A, 3 ' end connects quenching group;
The nucleotide sequence of described fluorescent probe B is consistent with the nucleotide sequence of described sequence label B, and 5 ' end connects fluorophor B; The nucleotide sequence of described quenching probes B and the nucleotide sequence reverse complemental of described sequence label B, 3 ' end connects quenching group.
In the present invention, described fluorophor A is FAM; Described fluorophor B is HEX; Described quenching group is BHQ.
In the present invention, described fluorescent probe A, described fluorescent probe B, described quenching probes A and described quenching probes B are present in KASPV4.02 × MasterMix, wherein said KASPV4.02 × MasterMix is Britain LGC Products, and its catalog number is KBS-1016-002 (being applicable to 96/384 orifice plate) or KBS-1016-011 (being applicable to 1536 orifice plates).
Described KASP primer or described test kit also belong to protection scope of the present invention in following arbitrary middle application:
(A) genotype of detection or auxiliary detection muskmelon CmOr gene;
(B) detection or auxiliary detection muskmelon pulp colour to be measured;
(C) muskmelon pulp colour proterties transformation.
3rd object of the present invention is to provide a kind of genotypic method of detection or auxiliary detection muskmelon CmOr gene.
The genotypic method of detection provided by the present invention or auxiliary detection muskmelon CmOr gene, specifically can to comprise the steps: with the genomic dna of described muskmelon to be measured as template, (described fluorophor A is FAM to the described KASP primer adopting in described test kit; Described fluorophor B is HEX) carry out pcr amplification, gained amplified production is carried out fluorescent signal scanning, Kraken software is adopted to analyze scan-data, according to analytical results according to the genotype of CmOr gene determining described muskmelon to be measured as follows: if the fluorescent signal data of the amplified production of described muskmelon to be measured presents blueness through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is A:A genotype; If the fluorescent signal data of the amplified production of described muskmelon to be measured presents redness through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is G:G genotype; If the fluorescent signal data of the amplified production of described muskmelon to be measured presents green through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is A:G genotype;
Wherein, described A:A genotype is the 323rd Nucleotide of described CmOr gene C DS sequence is the homozygous of A; Described G:G genotype is the 323rd Nucleotide of described CmOr gene C DS sequence is the homozygous of G; Described A:G genotype is the 323rd Nucleotide of described CmOr gene C DS sequence is the heterozygous of A and G.
Accordingly, described A:A genotype refers to muskmelon genomic dna to be measured as template, primer pair CmOr-SNPF/CmOr-SNPR is adopted to carry out homozygous (namely amplified production is single, as shown in sequence 5) that the 162nd of the nucleotide sequence of the DNA chain in pcr amplification gained specific amplification products with sequence shown in primer CmOr-SNPF is A; Described G:G genotype refers to muskmelon genomic dna to be measured as template, primer pair CmOr-SNPF/CmOr-SNPR is adopted to carry out homozygous (namely amplified production is single, as shown in sequence 4) that the 162nd of the nucleotide sequence of the DNA chain in pcr amplification gained specific amplification products with sequence shown in primer CmOr-SNPF is G; Described A:G genotype refers to muskmelon genomic dna to be measured as template, adopt primer pair CmOr-SNPF/CmOr-SNPR to carry out heterozygous that the 162nd of the nucleotide sequence of the DNA chain in pcr amplification gained specific amplification products with sequence shown in primer CmOr-SNPF be A and G (i.e. amplified production two kinds, respectively as sequence 5 and sequence 4 shown).The application of described method in following (1) or (2) also belongs to protection scope of the present invention:
(1) detection or auxiliary detection muskmelon pulp colour to be measured;
(2) muskmelon pulp colour proterties transformation.
In the application, if CmOr gene is A:A genotype or A:G genotype, then pulp colour is orange; If CmOr gene is G:G genotype, then pulp colour is green or white.
The preparation method of described test kit also belongs to protection scope of the present invention.
After the preparation method of described test kit specifically can comprise the steps: described primer 1, described primer 2 and described primer 3 individually to pack, be packaged in same reagent box with described fluorescent probe A, described fluorescent probe B, described quenching probes A and described quenching probes B.
In the present invention, the GenBank accession number of described CmOr gene C DS sequence is KM505046 or KM505047.
In the present invention, described muskmelon specifically optional in following any one: M285, Elizabethan, hybridize gained offspring, green treasured, V é drantais and PI414723 by described M285 and described Elizabethan.
The present invention will based on KASP (KompetitiveAllele-SpecificPCR, competitive ApoE gene) the SNPline genotype tests technology platform of technology, according to the crucial variant sites design primer of target gene, the special coupling of prime end base is utilized to carry out SNP somatotype to target gene.The SNPLine platform of PCR-based is high-throughout molecular marker systems, and operating process is full-automatic, reduces personal errors; Analysis throughput is high, and compatible 96,384,1536 porous plates, can complete 20 to 500000 SNP genotypings every day, is applicable to very much a large amount of sample and detects simultaneously.Based on the molecule marker of muskmelon pulp colour controlling gene CmOr gene order design high throughput testing, and be applied to the transformation of muskmelon pulp colour proterties, can greatly save time and cost of labor, improve the breeding efficiency of molecular marker assisted selection, accelerate the transformation of muskmelon pulp colour objective trait to excellent Inbred Lines.
Accompanying drawing explanation
Fig. 1 utilizes muskmelon pulp colour controlling gene CmOr high-throughput molecular marker analysis 96 hole sample panel SNP genotyping result schematic diagram.Wherein, NTC represents blank (black),? represent due to DNA poor quality or concentration too low, amplified production is not by clear and definite somatotype (pink colour), and G:G be red, and A:G be green, and A:A is blueness.
Fig. 2 is muskmelon pulp colour corresponding to CmOr gene high-throughput molecular marker analysis different genotype.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The genotype of the CmOr gene C DS sequence (GenBank:KM505046 or KM505047) described in following embodiment carries out naming according to the 323rd Nucleotide (SNP site) of CmOr gene C DS sequence.A:A genotype refers to that the 323rd Nucleotide of CmOr gene C DS sequence is the homozygous of A; G:G genotype refers to that the 323rd Nucleotide of CmOr gene C DS sequence is the homozygous of G; A:G genotype refers to that the 323rd Nucleotide of CmOr gene C DS sequence is the heterozygous of A and G.
Muskmelon M285: be Exocarpium Citri Rubrum meat, be preserved in Germplasm Resources of Farm Crop storehouse, Beijing on September 18th, 2015, classification number is 303.1925, and Unified number is 32097, the public can from the preservation from Germplasm Resources of Farm Crop storehouse, Beijing, agricultural and forest science institute Vegetable Research center, Beijing obtains.
The green treasured of muskmelon: be green meat, be recorded in " Liu Qingchen; Li Haiyan; woods Jian Hua. plastic greenhouse Planting Mode of Thin-skin Muskmelon in early spring. Tianjin agriculture and forestry science and technology; 02 phase in 2015 " literary composition, the public can obtain these melon varieties from applicant in Two decades years from the applying date, only uses for repeating related experiment of the present invention.
Muskmelon Elizabethan: be plain boiled pork, be loaded in " Wang Mingyao. the technical measures of Cultivation in Greenhouse thick-skinned melon Elizabethan. Chinese watermelon and muskmelon; 01 phase in 1996 " literary composition, the public can obtain these melon varieties from applicant in Two decades years from the applying date, only uses for repeating related experiment of the present invention.
Melon variety V é drantais and melon variety PI414723: be Exocarpium Citri Rubrum meat, " BoualemA, FerganyM, FernandezR; etal.ConservedMutationinanEthyleneBiosynthesisEnzymeLead stoAndromonoecyinMelons.Science, 2008Aug8; 321 (5890): 836-8 " on the books in a literary composition, the public can obtain these melon varieties from applicant in Two decades years from the applying date, only uses for repeating related experiment of the present invention.
Embodiment 1, the high-throughput KASP indicia designs of muskmelon pulp colour shape genes involved CmOr and the exploitation of primer special sequence thereof
One, for the extraction trying muskmelon material and genomic dna thereof
A, confession examination material selection
For examination body material: the known Exocarpium Citri Rubrum meat muskmelon M285 (A:A genotype) of the genotype of CmOr gene, the green treasured of green meat muskmelon (G:G genotype), plain boiled pork muskmelon Elizabethan (G:G genotype).Wherein, the genotype sequence verification of the CmOr gene of these three kinds of muskmelons.
The extraction of B, genomic dna
DNA extraction process will be carried out respectively for examination material, obtain for examination material genomic dna;
DNA extraction is with reference to the method (MurrayM of (1980) such as Murray, ThompsonWF.RapidisolationofhighmolecularweightplantDNA [J] .NuclAcidRes, 1980,8:668-673.) basis on improvement form; Concrete steps are as follows:
(1) get 0.3-0.5g young leaflet tablet in 8 platoon pipes, often pipe adds 300 μ lCTAB, puts into 2 steel balls, smashes with Retch instrument.
(2) often manage and add 300 μ lCTAB again, mix rear 65 DEG C of water-bath 60min, put upside down mixing once every 10min.
(3) water-bath is placed on room temperature cooling 10min, adds 300 μ l chloroforms: primary isoamyl alcohol (24:1, volume ratio), fully mixes, the centrifugal 15min of 4500rpm.
(4) get 400 μ l supernatant liquors, add in the Virahol of the 400 μ l precoolings added in advance (in 96 hole depth orifice plates), mix gently.Place 30min for-20 DEG C.
(5) in the centrifugal 30min of 4500rpm, abandon supernatant, the ethanol with 70% washes precipitation 2-3 time, air-dry extremely without ethanol taste under room temperature.
(6) 50-100 μ l (containing 0.5-1 μ lRNase10mg/ml) ddH is added
2o, 37 DEG C of water-bath 30min.
Get 5 μ l samples electrophoresis detection on the sepharose of 1.0%, and with standard λ DNA for contrast, sample concentration is adjusted to unanimously.
Two, the exploitation of the high-throughput molecule marker primer of CmOr gene is specific to
Utilize https: muskmelon CmOr (MELO3C005449) the genomic dna sequence information that //melonomics.net/ announces on the net, design primer increases the specific fragment of Exocarpium Citri Rubrum meat and green meat (plain boiled pork) muskmelon material C mOr gene respectively.
The primer sequence increased for above-mentioned specific fragment is as follows:
CmOr-SNPF：5'-CTCCTTGGTTTTCTTCAT-3'；
CmOr-SNPR：5'-GTAACGAAATCTTGGACAC-3'。
The PCR reaction system (25 μ L) of specific fragment amplification is: 50ng genomic dna, and 2.5 μ L are containing 15mMMgCl
210 × Buffer; 1.0 μ L concentration are the dNTPs of 2.5mM; 1UTaqDNA polysaccharase; 2.0 μ L concentration are the PCR upstream and downstream mix primer of 10 μMs; ddH
2o supplies 25 μ L.Wherein, Taq DNA polymerase and reaction buffer, dNTPs is Beijing Quanshijin Biotechnology Co., Ltd's product.
Pcr amplification reaction program is: stage 1:94 DEG C denaturation 3min; Stage 2:94 DEG C 30s, 52 DEG C of 30s, 72 DEG C of 30s, circulate 30 times altogether; Stage 3:72 DEG C extends 10min; Stage 4:4 DEG C of maintenance.Wherein PCR instrument is the Veriti96wellThermalCycler of AppliedBiosystems company; Pcr amplification product send Sinogenomax Co., Ltd. to check order.
Sequencing result: with the genomic dna of green meat (plain boiled pork) muskmelon for template, adopts primer pair CmOr-SNPF/CmOr-SNPR to carry out the nucleotide sequence of the DNA chain in pcr amplification gained specific amplification products with sequence shown in primer CmOr-SNPF as shown in sequence in sequence table 4; With the genomic dna of Exocarpium Citri Rubrum meat muskmelon material M285 for template, primer pair CmOr-SNPF/CmOr-SNPR is adopted to carry out the nucleotide sequence of the DNA chain in pcr amplification gained specific amplification products with sequence shown in primer CmOr-SNPF as shown in sequence in sequence table 5.Find through the comparison of DNAMAN software the G-A single base mutation site that above-mentioned CmOr gene amplification specific fragment (sequence 4 and sequence 5) exists at 162bp place (the 323rd Nucleotide corresponding to CmOr gene), design high-throughput KASP molecule marker according to this SNP.
KASP labeled primer sequence for high flux screening is as follows:
CmOr-F1：5’-TCCATATGCAAGAAAATTTTGTTTCGA
-3’；
CmOr-F2：5’-TCCATATGCAAGAAAATTTTGTTTCGA
-3’；
CmOr-R：5’-ATAGAGGGACCGGAAACAGTACAAG-3’。
Due to place, above-mentioned G-A single base mutation site in CmOr gene DNA chain just and there is in CmOr gene the DNA chain reverse complemental of sequence shown in upstream primer CmOr-F1 or CmOr-F2, for SNP site, 3 ' of upstream primer CmOr-F1 and CmOr-F2 holds as allelic variation base (T or C of runic underscore part, CmOr-F1 and CmOr-F2 is added corresponding universal linker sequence (fluorescence labels sequence) at 5 ' end, as follows:
CmOr-F1adaptor:5 '-GAAGGTGACCAAGTTCATGCT-3 ' (FAM fluorescence labels sequence);
CmOr-F2adaptor:5 '-GAAGGTCGGAGTCAACGGATT-3 ' (HEX fluorescence labels sequence).
Obtain corresponding CmOr gene high-throughput molecule marker primer sequence:
CmOr_Allele-F1：
5 '-
gAAGGTGACCAAGTTCATGCTtCCATATGCAAGAAAATTTTGTTTCGA
3 ' (sequence 1, wherein underscore part is FAM fluorescence labels sequence);
CmOr_Allele-F2：
5 '-
gAAGGTCGGAGTCAACGGATTtCCATATGCAAGAAAATTTTGTTTCGA
3 ' (sequence 2, wherein underscore part is HEX fluorescence labels sequence);
CmOr-R:5 '-ATAGAGGGACCGGAAACAGTACAAG-3 ' (sequence 3).
Above-mentioned primer is synthesized by Shanghai Sheng Gong company Beijing combining unit.
Embodiment 2, utilize the foundation of the method for high-throughput KASP marker detection muskmelon CmOr gene
One, genomic dna is extracted
See embodiment 1 step one.
Two, pcr amplification
The genomic dna extracted with step one, for template, carries out pcr amplification respectively with the primer special for the KASP mark detecting muskmelon CmOr gene that embodiment 1 is developed, obtains pcr amplification product.
KASP gene type PCR reaction system:
96 orifice plates: 10ng genomic dna, 5 μ lKASPV4.02 × MasterMix, 0.14 μ lKASP72 × assaymix, adds ddH
2o to 10 μ l.
384 orifice plates: 5ngDNA, 2.5 μ lKASPV4.02 × MasterMix, 0.07 μ lKASP72 × assaymix, adds ddH
2o to 5 μ l.
1536 orifice plates: 5ngDNA, 2.5 μ lKASPV4.02 × MasterMix, 0.07 μ lKASP72 × assaymix, adds ddH
2o to 5 μ l.
Wherein, KASPV4.02 × MasterMix is LGC Products, and its kind is KBS-1016-002 for the catalog number of the KASPV4.02 × MasterMix of 96/384 orifice plate; Catalog number for the KASPV4.02 × MasterMix of 1536 orifice plates is KBS-1016-011.KASP2 × MasterMix is by fluorescent probe A, fluorescent probe B, quenching probes A and quenching probes B, and the Taq enzyme of high-fidelity, the compositions such as dNTP.The sequence of fluorescent probe A is 5 '-GAAGGTGACCAAGTTCATGCT-3 ', and 5 ' end connects 1 fluorophor FAM; The sequence of fluorescent probe B is 5 '-GAAGGTCGGAGTCAACGGATT-3 ', and 5 ' end connects 1 fluorophor HEX; The sequence of quenching probes A is 5 '-AGCATGAACTTGGTCACCTTC-3 ', and 3 ' end connects quenching group BHQ; The sequence of quenching probes B is 5 '-AATCCGTTGACTCCGACCTTC-3 ', and 3 ' end connects quenching group BHQ.
It is after 100 μMs that primer CmOr_Allele-F1, CmOr_Allele-F2 and CmOr-R that KASP72 × assaymix is obtained by embodiment 1 are diluted to concentration respectively, by CmOr_Allele-F1 diluent, CmOr_Allele-F2 diluent and CmOr-R diluent and ddH
2o is mixed to get by the volume ratio of 12:12:30:46.
The response procedures of KASP gene type pcr amplification reaction is:
Stage 1:94 DEG C denaturation 15min; Stage 2:94 DEG C 20s, 61-55 DEG C of (each cycle down 0.6 DEG C) 1min, circulates 10 times altogether; Stage 3:94 DEG C 20s, 55 DEG C of 1min, circulate 26 times altogether.Wherein PCR water-bath thermal cycling is the Hydrocycler16-32 high throughput thermally recycle system, is applicable to 96,384 and 1536 orifice plates.
Experiment arranges the blank not adding template DNA in reaction system simultaneously, and each PCR plate arranges 2 blanks.
Three, the fluorescent scanning of pcr amplification product
Adopt two-way singly exciting to read plate instrument PHERAstar and scan pcr amplification product, FAM excitation wavelength is 485nm, and emission wavelength is 520nm, HEX excitation wavelength is 528nm, emission wavelength is 560nm, and system reference fluorescent ROX excitation wavelength is 575nm, and emission wavelength is 610nm.
Each pcr amplification product sample arranges at least 3 repetitions.
Four, allelic gene typing
Adopt Kraken
tM(concrete operation method is with reference to Kraken to reading the analysis of plate instrument PHERAstar scan-data for software
tMsoftware document, the public can directly buy from LGC company, see network address http://www.lgcgroup.com/products/genotyping-software/kraken/#.V hcaT9Kl8_M), according to analytical results according to the concrete genotype determining muskmelon CmOr gene to be measured as follows: the genotype being aggregated in the sample of the display blueness close to X-axis is the allelotype connecting FAM fluorescence labels sequence, the genotype be aggregated in close to the sample of the display redness in Y-axis is the allelotype connecting HEX fluorescence labels sequence, the genotype of the sample that middle display is green is two kinds of allelic heterozygous, display pink colour sample may due to DNA poor quality or concentration too low, amplified production is not by clear and definite somatotype, the sample of lower left corner display black is blank.
Specifically, as follows: if the fluorescent signal data of the amplified production of described muskmelon to be measured presents blueness through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is A:A genotype; If the fluorescent signal data of the amplified production of described muskmelon to be measured presents redness through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is G:G genotype; If the fluorescent signal data of the amplified production of described muskmelon to be measured presents green through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is A:G genotype.
Embodiment 3, the checking of high-throughput KASP marker detection muskmelon CmOr gene and the application in breeding
One, for examination material
Comprise for examination body material: with Exocarpium Citri Rubrum meat melon variety M285 (A:A genotype) for donor parents, plain boiled pork melon variety Elizabethan (G:G genotype) is recurrent parent, carries out backcross transformation to muskmelon pulp colour proterties.M285 and Elizabethan's hybridization obtain corresponding F1 generation, and F1 generation selfing obtains F2 colony, and F1 and recurrent parent Elizabethan backcross and obtain BC colony.Select Exocarpium Citri Rubrum meat melon variety PI414723 (A:A genotype) and V é drantais (A:A genotype), the contrast that the green treasured of green meat melon variety (G:G genotype), parent Elizabethan (G:G genotype) and M285 (A:A genotype) screen as molecule marker aid mark.The genotype sequence verification of the CmOr gene of contrast muskmelon.
Above BC, F
2for segregating population in the autumn in 2013 Hainan cultivate, collect seed, in April, 2014 nursery in greenhouse, be colonizated in Vegetable Research Centre, Beijing Academy of Agriculture and Forest Sciences's folium ilicis chinensis test base, wherein male parent, female parent, F
1, PI414723, V é drantais, green treasured, each field planting 20 strain, BC, F
2colony's field planting 200 strain.Spacing in the rows 60cm, line-spacing 120cm, adopt the mode of hanging climing cultivation, and single climing training, stays single melon, conveniently field management.
Two, high-throughput KASP marker detection
On the one hand, adopt the embodiment of the present invention 1 to develop in high-throughput KASP marker detection step one genotype of the CmOr gene supplying examination muskmelon, concrete operations, see embodiment 2, obtain the genotype of each CmOr gene for examination muskmelon.On the other hand, after pollinating 60 days, melon checking pulp colour phenotype is opened.And then the goodness of fit of statistics high-throughput KASP marker detection genotype results and pulp colour phenotypic results.According to existing report, if CmOr gene is A:A genotype or A:G genotype, then pulp colour is orange, if CmOr gene is G:G genotype, then pulp colour is green or white (see " GalilTzuri, XiangjunZhou, NoamChayut, HuiYuan, VitalyPortnoy, AyalaMeir, UziSa ' ar, FabianBaumkoler, MichaelMazourek, EfraimLewinsohn, ZhangjunFei, ArthurA.Schaffer, LiLi, JosephBurger, NuritKatzirandYaakovTadmor (2015) A ' golden ' SNPinCmOrgovernsthefruitfleshcolorofmelon (Cucumismelo) ThePlantJournal, 82, 267 – 279 " literary composition.
1, above-mentioned high-throughput molecule marker system anlysis parents, F1 and F is utilized
2colony's individual plant genotype, with Germplasm Resources of Cucumis Melo L material PI414723, V é drantais of known type, green treasured, M285, Elizabethan for contrast, sample segment detected result as shown in Figure 1, according to the labeled primer of the crucial variant sites design of the G-A of CmOr gene, F2 colony individual plant is carried out SNP somatotype and obtain A:A, A:G and G:G tri-kinds of genotype, A:A and A:G genotype corresponds to Exocarpium Citri Rubrum meat phenotype, the green meat of G:G genotype (plain boiled pork) phenotype.The sample of lower left corner display black is the blank of each PCR plate, the genotype being aggregated in the sample of the display blueness close to X-axis is the allelotype (A:A) connecting FAM fluorescence labels sequence, the genotype be aggregated in close to the sample of the display redness in Y-axis is the allelotype (G:G) connecting HEX fluorescence labels sequence, the genotype of the sample that middle display is green is two kinds of allelic heterozygous (A:G), display pink colour sample may due to DNA poor quality or concentration too low, amplified production is not by clear and definite somatotype.
Result shows: all SNP genotyping result for examination melon variety of CmOr gene high-throughput KASP Markers for Detection of the present invention are all consistent with pulp colour trait phenotypes, and CmOr gene is the genotypic muskmelon pulp colour of A:A and A:G is orange; CmOr gene is G:G genotypic muskmelon pulp colour is green or white, and rate of accuracy reached is to 100%.Fig. 2 is the representative of the muskmelon pulp colour of CmOr gene high-throughput molecular marker analysis different genotype.
In addition, in order to further proved invention institute supplying method is to the accuracy of the genotypic detected result of CmOr gene, contriver is also random has carried out sequence verification to each CmOr gene for examination muskmelon, and the 323rd Nucleotide that it is its CmOr gene C DS sequence of the genotypic sample of A:A that result confirms through CmOr gene high-throughput KASP Markers for Detection of the present invention is the homozygous of A really; The 323rd Nucleotide being its CmOr gene of the genotypic sample of G:G through CmOr gene high-throughput KASP Markers for Detection of the present invention is the homozygous of G really; The 323rd Nucleotide being its CmOr gene of the genotypic sample of A:G through CmOr gene high-throughput KASP Markers for Detection of the present invention is the heterozygous of A and G really.
2, pass through the genotype to parents, F1, F2, BC1, BC2 different groups CmOr gene and phenotypic data statistic analysis, find that the high-throughput molecular marker analysis result of CmOr gene and phenotype investigation result coincide rate all up to 100% (see table 1).In the process of backcross transformation, along with the increase of the algebraically that backcrosses, fruit quality and appearance character level off to the recurrent parent of transformation gradually, when transformation is to the recurrent parent of fruit quality and appearance character closely transformation, further BC colony is carried out selfing, by high-throughput molecule marker, to the screening of CmOr gene, (wherein BC colony retains detection genotype is the plant of A:G, BC colony self progeny retains the plant of genotype A:A), Exocarpium Citri Rubrum meat (A:A genotype) Muskmelon Inbred Line meeting objective trait can have been cultivated.
Table 1CmOr gene high-throughput molecular marker gene type analysis result and phenotype investigate the rate of coincideing
Claims (10)
1. for detecting the KASP primer of muskmelon CmOr gene, it is characterized in that: described KASP primer is made up of primer 1, primer 2 and primer 3; Described primer 1 is the single stranded DNA being followed successively by the 22-49 position of sequence 1 in sequence label A and sequence table from 5 ' end to 3 ' end; Described primer 2 is the single stranded DNA being followed successively by the 22-49 position of sequence 2 in sequence label B and sequence table from 5 ' end to 3 ' end; Described primer 3 is the single stranded DNA of nucleotide sequence as shown in sequence in sequence table 3.
2. KASP primer according to claim 1, is characterized in that: the nucleotides sequence of described sequence label A is classified as the 1-21 position of sequence 1 in sequence table; The nucleotides sequence of described sequence label B is classified as the 1-21 position of sequence 2 in sequence table.
3. KASP primer according to claim 1 and 2, is characterized in that: described primer 1 is the single stranded DNA of nucleotide sequence as shown in sequence in sequence table 1; Described primer 2 is the single stranded DNA of nucleotide sequence as shown in sequence in sequence table 2.
4. for detecting the test kit of muskmelon CmOr gene, it is characterized in that: described test kit contains arbitrary described KASP primer in claim 1-3.
5. test kit according to claim 4, is characterized in that: also containing fluorescent probe A, fluorescent probe B, quenching probes A and quenching probes B in described test kit;
The nucleotide sequence of described fluorescent probe A is consistent with the nucleotide sequence of described sequence label A, and 5 ' end connects fluorophor A; The nucleotide sequence of described quenching probes A and the nucleotide sequence reverse complemental of described sequence label A, 3 ' end connects quenching group;
The nucleotide sequence of described fluorescent probe B is consistent with the nucleotide sequence of described sequence label B, and 5 ' end connects fluorophor B; The nucleotide sequence of described quenching probes B and the nucleotide sequence reverse complemental of described sequence label B, 3 ' end connects quenching group.
6. test kit according to claim 5, is characterized in that: described fluorophor A is FAM; Described fluorophor B is HEX; Described quenching group is BHQ.
7. in claim 1-3 in arbitrary described KASP primer or claim 4-6 arbitrary described test kit in following arbitrary middle application:
(A) genotype of detection or auxiliary detection muskmelon CmOr gene;
(B) detection or auxiliary detection muskmelon pulp colour to be measured;
(C) muskmelon pulp colour proterties transformation.
8. the genotypic method of a detection or auxiliary detection muskmelon CmOr gene, comprise the steps: with the genomic dna of described muskmelon to be measured as template, the described KASP primer in test kit described in claim 6 is adopted to carry out pcr amplification, gained amplified production is carried out fluorescent signal scanning, Kraken software is adopted to analyze scan-data, according to analytical results according to the genotype of CmOr gene determining described muskmelon to be measured as follows: if the fluorescent signal data of the amplified production of described muskmelon to be measured presents blueness through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is A:A genotype, if the fluorescent signal data of the amplified production of described muskmelon to be measured presents redness through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is G:G genotype, if the fluorescent signal data of the amplified production of described muskmelon to be measured presents green through Kraken software analysis in gained somatotype dendrogram, then the CmOr gene of described muskmelon to be measured is A:G genotype,
Described A:A genotype is the 323rd Nucleotide of described CmOr gene C DS sequence is the homozygous of A; Described G:G genotype is the 323rd Nucleotide of described CmOr gene C DS sequence is the homozygous of G; Described A:G genotype is the 323rd Nucleotide of described CmOr gene C DS sequence is the heterozygous of A and G.
9. the application of method described in claim 8 in following (1) or (2):
(1) detection or auxiliary detection muskmelon pulp colour to be measured;
(2) muskmelon pulp colour proterties transformation.
10. the preparation method of test kit described in claim 5 or 6, after comprising the steps: described primer 1, described primer 2 and described primer 3 individually to pack, be packaged in same reagent box with described fluorescent probe A, described fluorescent probe B, described quenching probes A and described quenching probes B.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834529A (en) * | 2017-04-05 | 2017-06-13 | 东北农业大学 | A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application |
| CN109609687A (en) * | 2019-02-02 | 2019-04-12 | 北京市农林科学院 | KASP labeled primer for detecting watermelon blight resistance combines and its application |
| CN109762921A (en) * | 2019-01-28 | 2019-05-17 | 中国农业科学院蔬菜花卉研究所 | For detecting the SNP marker and its application of cucumber pulp colour |
| CN110923357A (en) * | 2019-12-31 | 2020-03-27 | 浙江省农业科学院 | Bottle gourd core molecular marker set developed based on KASP technology and application thereof |
| CN116179741A (en) * | 2022-10-21 | 2023-05-30 | 上海市农业科学院 | Molecular marker of melon monoscopic flower A gene CmACS7 and application thereof |
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2015
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| Title |
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| GALIL TZURI等: "A ‘golden’ SNP in CmOr governs the fruit flesh color of melon (Cucumis melo)", 《THE PLANT JOURNAL》 * |
| HE C等: "SNP genotyping ：the KASP assay", 《METHODS MOL BIOL》 * |
| KASSA SEMAGN等: "Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement", 《MOL BREEDING》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834529A (en) * | 2017-04-05 | 2017-06-13 | 东北农业大学 | A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application |
| CN109762921A (en) * | 2019-01-28 | 2019-05-17 | 中国农业科学院蔬菜花卉研究所 | For detecting the SNP marker and its application of cucumber pulp colour |
| CN109762921B (en) * | 2019-01-28 | 2022-10-28 | 中国农业科学院蔬菜花卉研究所 | SNP (Single nucleotide polymorphism) marker for detecting color of cucumber pulp and application thereof |
| CN109609687A (en) * | 2019-02-02 | 2019-04-12 | 北京市农林科学院 | KASP labeled primer for detecting watermelon blight resistance combines and its application |
| CN109609687B (en) * | 2019-02-02 | 2022-10-04 | 北京市农林科学院 | KASP marker primer combination for detecting watermelon fusarium wilt resistance and application thereof |
| CN110923357A (en) * | 2019-12-31 | 2020-03-27 | 浙江省农业科学院 | Bottle gourd core molecular marker set developed based on KASP technology and application thereof |
| CN110923357B (en) * | 2019-12-31 | 2022-04-19 | 浙江省农业科学院 | Bottle gourd core molecular marker set developed based on KASP technology and application thereof |
| CN116179741A (en) * | 2022-10-21 | 2023-05-30 | 上海市农业科学院 | Molecular marker of melon monoscopic flower A gene CmACS7 and application thereof |
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