CN106834529A - A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application - Google Patents
A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application Download PDFInfo
- Publication number
- CN106834529A CN106834529A CN201710216627.4A CN201710216627A CN106834529A CN 106834529 A CN106834529 A CN 106834529A CN 201710216627 A CN201710216627 A CN 201710216627A CN 106834529 A CN106834529 A CN 106834529A
- Authority
- CN
- China
- Prior art keywords
- muskmelon
- flesh
- molecular labeling
- primer
- molecular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application, belong to muskmelon molecular marker assisted selection breeding technical field.As shown in SEQ ID NO.1, the bit base m of nucleotide sequence the 640th is G to molecular labeling HF wherein shown in SEQ ID NO.1, then be accredited as the muskmelon of orange flesh.Present invention also offers a kind of method of the primer pair and identification muskmelon orange flesh proterties using the primer pair of a kind of molecular labeling HF, the authentication method can carry out assisted Selection in the Seedling Stage of muskmelon to muskmelon pulp colour, result easy to operate is clear and definite, substantially increase breeding efficiency and shorten the breeding time limit, accelerate breeding work efficiency.Molecular labeling HF and its primer pair and authentication method can be used for muskmelon molecular mark, the identification of muskmelon flesh trait auxiliary and assistant breeding, the screening of muskmelon flesh trait.
Description
Technical field
The present invention relates to a kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application, belong to
Muskmelon molecular marker assisted selection breeding technical field.
Background technology
Molecular marker assisted selection (molecular marker assisted selection, MAS) is with the modern times point
Sub- developing rapidly for biology techniques and the new technology that produces, it can rapidly and accurately analyze the something lost of individuality from molecular level
Composition is passed, so as to realize directly selecting genotype, molecular breeding is carried out.Molecular marker assisted selection breeding be exactly utilize with
The chain DNA marker of crop important character or functional gene improve the modern molecular breeding technique of plant variety, therefore, molecule
Marker assisted selection successfully it is critical only that obtained molecular labeling it is whether mutually chain with proterties to be analyzed and whether
There is versatility between the different materials of same species.
Muskmelon breeding objective is related to the improvement to some economic characters, and to take into account the producer with the aspect of consumer two
The need for.The main target of world today's muskmelon breeding is disease-resistant, quality and characteristic breeding.Muskmelon pulp colour is muskmelon outward appearance
The important indicator that quality is constituted, especially current muskmelon enters dining table, makes the product with gorgeous color frequently as high-grade fruit
Plant and enjoy favor.The rich in nutrition content of muskmelon pulp, except soluble solid (13%-15%) is occupied first of melon sugar content
Outward, it is also yellow containing various mineral matters (calcium, phosphorus, iron etc.) needed by human and beta carotene, lutein, zeaxanthin, violet
Various pigments such as matter, the difference of carotenoid species and its relative amount contained by muskmelon pulp causes many of pulp colour
Sample.Meanwhile, pigment in muskmelon pulp also has the physiological function beneficial to human body, there are some researches show, beta carotene can be with
Damage of the free radical to body is eliminated, with antioxidation, is played the role of for pre- anti-cancer important.Additionally, beta carotene
Vitamin A can be changed into human body, vitamin A is to maintain strength and material necessary to skin normal condition, and shortage then can
Cause scheroma and yctalopia (JA Olson.Carotenoids and human health, 1999,55 (3):207-216；H
Nishino,M Murakoshi,et al.Cancer prevention by carotenoids,2015,483(12):165-
168).Therefore it is current to have the melon variety of different pulp colours by the efficient seed selection of method of molecular marker assisted selection
One of pith of muskmelon breeding work.
With the fast development of DNA molecular marker technology, entered using the molecular labeling with plant related gene close linkage
Row assisted Selection, can greatly improve the efficiency to objective trait screening.In recent years, it is swift and violent with high throughput sequencing technologies
Development, the muskmelon genomic data obtained by sequencing technologies is the assignment of genes gene mapping of Hami melon and a large amount of molecular labelings
Exploitation provides new approach.CAPS molecular labelings are the molecular labelings that polymorphism is produced with restriction enzyme site single base mutation, with
Its distribution is extensively, make a variation stable a new generation's mark as the assignment of genes gene mapping and molecular biology research, CAPS marking operations
Simplicity, is as a result easy to observation and has general applicability between same species.
Up to the present, the molecular labeling of main application is RFLP, SSR, AFLP, RAPD etc., CAPS points in muskmelon breeding
Son mark is with less.Compared with RFLP, CAPS molecular labelings have easy to operate, polymorphism advantage high；CAPS molecule marks
Stability of the note than RAPD, reliability, repeatability are more preferably；For AFLP, CAPS molecular labeling workloads are small, easy statistics
Band；For SSR, the design of CAPS labeled primers is more easy.In addition, CAPS molecular labelings are to DNA concentration and purity
It is required that it is relatively low, reduce the work difficulty that DNA is extracted.As can be seen here, CAPS marks have huge DEVELOPMENT PROSPECT.This molecular labeling
Invention it is significant for enriching the diversity of muskmelon breeding molecular labeling.
The content of the invention
To provide a kind of molecular labeling that can be used for indicating and identifying muskmelon orange flesh proterties, the invention provides one
Indication and molecular labeling and its primer and the application of identification muskmelon orange flesh proterties are planted, the technical scheme of use is as follows：
It is an object of the invention to provide it is a kind of indicate and identification muskmelon orange flesh proterties molecular labeling HF, described point
As shown in SEQ ID NO.1, the bit base M of nucleotide sequence the 640th is G or A, SEQ to son mark HF wherein shown in SEQ ID NO.1
The bit base m of nucleotide sequence the 640th shown in ID NO.1 is G, then be accredited as the muskmelon of orange flesh, core shown in SEQ ID NO.1
The bit base m of nucleotide sequence the 640th is A, then be accredited as the muskmelon of non-orange flesh.
The present invention also provides the molecular labeling HF in muskmelon molecular mark, the auxiliary identification of muskmelon flesh trait
With the application in assistant breeding, the identification of muskmelon flesh trait auxiliary, the screening of muskmelon flesh trait.
The present invention also provides a kind of primer pair that the molecular labeling HF is detected for PCR, including nucleotide sequence such as SEQ
The anti-sense primer HF-R of sense primer HF-F and nucleotide sequence shown in ID NO.2 as shown in SEQ ID NO.3.
The present invention also provides the primer pair of the molecular labeling HF in muskmelon molecular mark, muskmelon flesh trait
Application in auxiliary identification and assistant breeding, the screening of muskmelon flesh trait.
The present invention also provides a kind of method for identifying muskmelon orange flesh proterties, comprises the following steps：
1) genomic DNA of muskmelon sample to be detected is extracted；
2) with step 1) obtained by muskmelon sample gene group DNA to be detected be template, using the molecule described in claim 3
Marking the primer pair of HF carries out pcr amplification reaction, obtains PCR primer；
3) to step 2) gained PCR primer endonuclease reaction, digestion products agarose are carried out with restriction enzyme Hinf I
Gel electrophoresis is detected；If detection obtains two fragments that length is 640bp and 306bp, muskmelon to be measured has orange fruit
Meat proterties；If detection obtains the single fragment that length is 946bp, muskmelon to be measured has non-orange flesh proterties.
Preferably, step 2) in pcr amplification reaction described in 20 μ L system it is as follows：
Preferably, step 3) described in pcr amplification reaction be TD-PCR amplified reactions.
It is highly preferred that the specific procedure of the TD-PCR amplified reactions is：94 DEG C of predegenerations 7min, 94 DEG C of denaturation 1min,
60 DEG C of annealing 30s, often circulation reduces by 0.5 DEG C, 72 DEG C of extension 90s, 30 circulations；94 DEG C of denaturation 30s, 45 DEG C of anneal 30s, 72 DEG C
Extend 30s, 10 circulations；72 DEG C of extension 10min.
The present invention also provides a kind of kit of the detection muskmelon pulp colour containing described primer pair.
The present invention also provides a kind of application of kit in muskmelon pulp colour is detected.
Evaluation of markers HF of the present invention is located on muskmelon CmOr genes, and the producer at the 640th base
Mutation, A is sported by G, causes amino acid to sport histidine by arginine, improves carotenoid content, especially β-Hu Luo
Foretell cellulose content so that pulp presents orange.
The present invention is expanded by PCR using CAPS molecular labeling primers and obtains the DNA fragmentation related to muskmelon pulp colour,
Recycle restriction enzyme carries out endonuclease reaction to the fragment for obtaining, by two different colors of muskmelon material is being obtained
DNA fragmentation in there is the difference of restriction enzyme site single base mutation.Site accordingly, there exist single base mutation will not be restricted
Restriction endonuclease cuts and obtains a size for the fragment of 946bp, and the fragment in the absence of base mutation site can be by restriction enzyme
Digestion is opened, and obtains two fragments of 640bp and 306bp, can clearly by different pulp in two by agarose gel electrophoresis
The muskmelon material of color makes a distinction.
Beneficial effect of the present invention：
1st, the present invention designs and develops the CAPS related to muskmelon pulp colour according to the muskmelon genomic data for obtaining
Molecular labeling, develop CAPS molecular labelings has versatility in different materials, obtains one and can be used to differentiate muskmelon really
The molecular labeling of meat color.The inventive method can carry out assisted Selection to muskmelon pulp colour in the Seedling Stage of muskmelon, operate
Easy result clearly, substantially increases breeding efficiency and shortens the breeding time limit, accelerates breeding work efficiency.
2nd, the CAPS molecular labelings that the present invention is obtained, can be carried out well in the muskmelon material of different pulp colours
Distinguish, therefore, present invention obtains the molecular labeling that can efficiently distinguish different muskmelon pulp colours.
3rd, the present invention can judge pulp colour in the Muskmelon Seedlings phase (heart stage of three leaf one), and the time is about 20 days；And routine is educated
Kind needing fruit full maturity can just be judged that the time is about 90 days to 140 days, it can be seen that, can be effective using the present invention
Shorten breeding time, accelerate breeding speed.Specificity of the invention is good, and accuracy rate is 83.3%.
Brief description of the drawings
Fig. 1 is digestion situations of the CAPS marks HF in MR-1 (orange flesh material) and X191 (green flesh material)；
(0, D2000marker Plus；1-2 swimming lanes are respectively the DNA of X191, MR-1 plant, and to mark HF to enter performing PCR by CAPS anti-
Answer obtained PCR primer (946bp)；No. 3 swimming lanes are the digestion products of X191, and clip size is 946bp；No. 4 swimming lanes are MR-
1 digestion products, clip size is 640bp and 306bp；Marker is D2000marker Plus, and stripe size is from top to bottom
It is followed successively by：5000bp、3000bp、2000bp、1000bp、750bp、500bp、250bp、100bp).Fig. 2 is 70 kinds of muskmelon materials
PCR primer；
Fig. 3 is 70 kinds of digestion qualification results of muskmelon material；
In Fig. 2 and Fig. 3：0, Marker is D2000marker Plus, and stripe size is from top to bottom followed successively by：5000bp、
3000bp、2000bp、1000bp、750bp、500bp、250bp、100bp；1, X073；2, X024；3, M4-53；4, X006；5,
X206；6, X021；7, X200；8, X011；9, X121；10, X190；11, X198；12, X176；13, M4-72；14, X237；15,
X047；16, M4-2；17, X194；18, X161；19, M4-123；20, M4-4；21, M4-133；22, X157；23, M4-129；
24, X149；25, M4-1；26, X222；27, X088；28, X101；29, X167；30, M4-127；31, X095；32, X136；33,
X071；34, M4-126；35, X156；36, X045；37, X058；38, X089；39, X239；40, X109；41, M4-83；42,
M4-5；43, X201；44, M4-61；45, X015；46, X193；47, M4-58；48, X205；49, X210；50, X233；51,
X053；52, M4-90；53, M4-115；54, X177；55, X197；56, M4-18；57, X072；58, M4-70；59, M1-101；
60, M1-33；61, M1-20；62, X033；63, M1-96；64, X208；65, X208；66, X039；67, X023；68, X038；
69, X150；70, X206.
Specific embodiment
With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Embodiment 1：The exploitation of molecular labeling and its primer
The present embodiment is that experiment material develops CAPS molecules from orange muskmelon strain MR-1 and green muskmelon strain X191
Mark.MR-1 and X191 muskmelon strains are sequenced using high throughput sequencing technologies, and muskmelon is obtained by GenBank websites
CmOr coding sequences, by the sequence and the comparison of sequencing material, have obtained muskmelon CmOr gene coding regions and there is SNP
The interval of mutation, in the interval, excavates the base section that sequencing data has SNP mutation site, analyzes the digestion of SNP site
There is the sequence of CAPS mutation, i.e. CAPS molecular labelings HF in information, acquisition.
Muskmelon CmOr gene orders are located between No. 9 chromosome 5207122-5214439.The nucleotides sequence of molecular labeling HF
As shown in SEQ ID NO.1, the bit base m of sequence the 640th is G or A to row wherein shown in SEQ ID NO.1, shown in SEQ ID NO.1
The bit base m of sequence the 640th is G, and muskmelon flesh trait is presented as orange, and the bit base M of sequence the 640th is shown in SEQ ID NO.1
A, then muskmelon flesh trait be presented as non-orange.
CAPS primers are designed in the sequence that there is CAPS sites using the softwares of Primer 6.Primer length is 18bp-
26bp, annealing temperature is 56-60 DEG C, and G/C content is 40%-60%.
The forward primer HF-F nucleotide sequences of the base sequence of CAPS molecular labelings HF as shown in SEQ ID NO.2, instead
It is specific as shown in table 1 to primer HF-R nucleotide sequences as shown in SEQ ID NO.3：
Table 1 is used for Primer, sequence and the expected clip size of the CAPS molecular labelings HF of PCR reactions
Embodiment 2：The extraction of genomic DNA and PCR react
Collection MR-1, X191 muskmelon material colony leaves extract genomic DNA using the CTAB methods of improvement.
Utilize obtained CAPS primers and genomic DNA to enter performing PCR to react, PCR reaction systems (20 μ as shown in table 2
L)：
The PCR reaction systems of table 2
Expanded using touchdown PCR (touchdown PCR, TD-PCR), program is：94 DEG C of predegeneration 7min, 94 DEG C
Denaturation 1min, 60 DEG C of annealing 30s, often circulation reduces by 0.5 DEG C, 72 DEG C of extension 90s, 30 circulations；94 DEG C of denaturation 30s, 45 DEG C are moved back
Fiery 30s, 72 DEG C of extension 30s, 10 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.
Embodiment 3：Digestion verification is carried out to PCR primer
Restriction enzyme operating guidance according to Hinf I carries out endonuclease reaction, and the PCR primer to being obtained carries out digestion
Checking, digestion system is as shown in table 3：(15.3μL)
The endonuclease reaction system of table 3
37 DEG C of water-bath digestion 3-4h, digestion products are detected that testing result is as shown in Figure 1 using 1% agarose electrophoresis.
X191 (green) because in the absence of restriction enzyme site, it is still 946bp not cut by restriction enzyme, in MR-1 (orange) due to
There is restriction enzyme site, cut by Hinf I and obtain 640bp and the fragment of 306bp.Result is as shown in Figure 1.
Embodiment 4：The sieve of germ plasm resource is carried out to the melon variety of different pulp colours using the CAPS marks for being obtained
Choosing
To differentiate applicability of the developed CAPS marks to other muskmelon materials, we select 70 parts with orange, green
The muskmelon material of color and white flesh, using the primer pair, it enters performing PCR amplification and digestion verification.Table 4 is 70 parts of muskmelon materials
Title and the description such as color.
4 70 kinds of melon variety introductions of table
Method according to embodiment 2 extracts genomic DNA and PCR reacts, and enters according to the enzymatic cleavage methods in embodiment 3
Row digestion verification, identifies 70 kinds of flesh traits of muskmelon material, and specific method is as follows：
1) genomic DNA in 70 kinds of muskmelon samples to be detected is extracted respectively；
2) respectively with step 1) obtained by 70 kinds of muskmelon sample gene group DNA to be detected be template, using molecular labeling HF
Primer pair (implement 1 in HF-R and HF-F) carry out pcr amplification reaction, obtain PCR primer；
3) to step 2) gained PCR primer endonuclease reaction, 1% fine jade of digestion products are carried out with restriction enzyme Hinf I
Sepharose electrophoresis is detected；If detection obtains two fragments that length is 640bp and 306bp, muskmelon to be measured has orange
Color flesh trait；If detection obtains the single fragment that length is 946bp, muskmelon to be measured has non-orange flesh proterties.
Above-mentioned steps 2) in obtain PCR primer PCR primer is detected using 1% agarose electrophoresis, testing result
As shown in Fig. 2 70 kinds of materials for trying amplify a band for 946bp sizes.
Above-mentioned steps 3) in digestion verification result it is as shown in Figure 3.
Be can be seen that with reference to Fig. 3 and Biao 4：PCR primer product after digestion obtains 640bp and the bands of 306bp two, field
Phenotype is respectively orange or shallow orange；PCR primer shows a band for 946bp sizes after digestion, and field phenotype is green
Color or white.
Can be analyzed with table 4 with reference to Fig. 3 and have that 67 kind digestion results are consistent with field flesh trait, coincidence rate reaches
95.7%；The kind 18 with orange flesh proterties can be wherein inquired about in table 4, clearly there are two product of band in Fig. 3
15 are planted, coincidence rate reaches 83.3%, wherein incongruent 3 kinds are X021, X088, X089.Therefore, using present invention side
Method can be good at whether identification muskmelon has orange flesh proterties, and the inventive method specificity is good, and accuracy rate is high.
Authentication method of the invention can be when not influenceing subsequent growth optimal with the Muskmelon Seedlings of different times as material
Phase is for material extraction DNA with the Muskmelon Seedlings phase (heart stage of three leaf one).
Pulp colour can be judged in the Muskmelon Seedlings phase (heart stage of three leaf one) using authentication method of the invention, the time is about 20
My god；And conventional breeding needs the fruit full maturity just to be judged that the time is about 90 days to 140 days, it can be seen that, using this
Invention can effectively shorten breeding time, accelerate breeding speed.
Embodiment 5
The present embodiment provides a kind of kit of the detection muskmelon pulp colour containing described primer pair, and the kit can
For detection muskmelon pulp colour, wherein comprising the primer pair as shown in SEQ ID NO.2 and SEQ ID NO.3.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this
The people of technology, is not departing from spirit and scope of the invention, can do various changes and modification, therefore, guarantor of the invention
What shield scope should be defined by claims is defined.
Sequence table
<110>Northeast Agricultural University
<120>A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 946
<212> DNA
<213>Molecular labeling HF
<400> 1
ttactgttgg agcacttgtt tcacttccag gttgaattct ggtaaagaac ggcagaagtt 60
gagctcgagg tggcgatggc gatccatggc ctcggattct actgactctt cttcttcttc 120
ttcttttgct ccgtctgttg aatctgatcc ttcggataaa acttcagcca ggttggtgaa 180
ttcttgcttc tggtacctct gtatctggtt gtggttgaag cattttgagg caacgtctag 240
gaagtgaaat gaatgttggt ttgttgtgtt tgatgttgat ttacgtgatg gaattgtaaa 300
atgactggat agtttcttgt gttctttact ttttgagcac tgctacttga aaatgctcgt 360
gttatggttg aaggtttgga ttggattgat tggtcgtttt tgtcgtgatg atttgaagaa 420
tttgttatca gttcaatatt gcgaatacca taagttctgt tcttatccta caaacttttt 480
tatgcttatt aagtcggcca tctccttggt tttcttcatg tgacaattag gaataattta 540
tctgtaaatt aaatgttaac ttaacatgat tttatgcagt ttttgtatca tagagggacc 600
ggaaacagta caagattttg caaaaatgga actgcaagam attcaggaga acattcgaag 660
tcgtcgaaac aaaattttct tgcatatgga agaggtgaga cttagtggac attttgcgta 720
gtagtacgga ctattctcat gaactgattg attattcgcg catatgaaat tttaagtttt 780
tagatgtgtc caagatttcg ttaccttgaa aaatccttac ccagccaaag ccgtcatttg 840
gggggaccac tgccaagtat tagcattatt tttctgtgat gagagtgata aatatgtgtc 900
agacacttgg atttgggcta ttgatccttc ttcatcgttc tctatc 946
<210> 2
<211> 19
<212> DNA
<213> HF-F
<400> 2
ttactgttgg agcacttgt 19
<210> 3
<211> 21
<212> DNA
<213> HF-R
<400> 3
gatagagaac gatgaagaag g 21
Claims (10)
1. it is a kind of indicate and identification muskmelon orange flesh proterties molecular labeling HF, it is characterised in that the molecular labeling HF is such as
Shown in SEQ ID NO.1, the bit base m of nucleotide sequence the 640th is G wherein shown in SEQ ID NO.1, then be accredited as orange flesh
Muskmelon.
2. molecular labeling HF described in claim 1 is identified and auxiliary in muskmelon molecular mark, muskmelon flesh trait auxiliary
The application helped in breeding, the identification of muskmelon flesh trait auxiliary, the screening of muskmelon flesh trait.
3. the primer pair of molecular labeling HF described in a kind of claim 1, it is characterised in that including nucleotide sequence such as SEQ ID
The anti-sense primer HF-R of sense primer HF-F and nucleotide sequence shown in NO.2 as shown in SEQ ID NO.3.
4. the primer pair of molecular labeling HF described in claim 3 is aided in muskmelon molecular mark, muskmelon flesh trait
Application in identification and assistant breeding, the screening of muskmelon flesh trait.
5. it is a kind of identify muskmelon orange flesh proterties method, it is characterised in that comprise the following steps：
1) genomic DNA of muskmelon sample to be detected is extracted；
2) with step 1) obtained by muskmelon sample gene group DNA to be detected be template, using the molecular labeling described in claim 3
The primer pair of HF carries out pcr amplification reaction, obtains PCR primer；
3) to step 2) gained PCR primer endonuclease reaction, digestion products Ago-Gel are carried out with restriction enzyme Hinf I
Electrophoresis is detected；If detection obtains two fragments that length is 640bp and 306bp, muskmelon to be measured has orange flesh
Shape；If detection obtains the single fragment that length is 946bp, muskmelon to be measured has non-orange flesh proterties.
6. method according to claim 5, it is characterised in that step 2) in pcr amplification reaction described in 20 μ L system such as
Under：
7. method according to claim 5, it is characterised in that step 3) described in pcr amplification reaction be TD-PCR amplifications
Reaction.
8. method according to claim 7, it is characterised in that the specific procedure of the TD-PCR amplified reactions is：94℃
Predegeneration 7min, 94 DEG C of denaturation 1min, 60 DEG C of annealing 30s, often circulation reduces by 0.5 DEG C, 72 DEG C of extension 90s, 30 circulations；94℃
Denaturation 30s, 45 DEG C of annealing 30s, 72 DEG C of extension 30s, 10 circulations；72 DEG C of extension 10min.
9. a kind of primer pair containing described in claim 3 detection muskmelon pulp colour kit.
10. application of the kit described in claim 9 in muskmelon pulp colour is detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710216627.4A CN106834529A (en) | 2017-04-05 | 2017-04-05 | A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710216627.4A CN106834529A (en) | 2017-04-05 | 2017-04-05 | A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106834529A true CN106834529A (en) | 2017-06-13 |
Family
ID=59141230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710216627.4A Pending CN106834529A (en) | 2017-04-05 | 2017-04-05 | A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106834529A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517917A (en) * | 2017-09-19 | 2019-03-26 | 天津大学 | It is a kind of for identifying the SNP marker primer and molecule labelling method of muskmelon pulp colour |
| CN112662803A (en) * | 2021-01-22 | 2021-04-16 | 河南农业大学 | CAPS molecular marker co-separated from melon reticulate pattern character and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711355A (en) * | 2015-03-17 | 2015-06-17 | 东北农业大学 | Molecular marker KpnI-1 for indicating and identifying watermelon pulp color and application |
| CN104711356A (en) * | 2015-03-17 | 2015-06-17 | 东北农业大学 | Molecular marker MboII-37 for indicating and identifying watermelon pulp color and application |
| CN105200144A (en) * | 2015-10-22 | 2015-12-30 | 北京市农林科学院 | High-flux molecular marker for identifying pulp color of Cucumis melo L. and special primer for high-flux molecular marker |
| CN105802961A (en) * | 2014-12-29 | 2016-07-27 | 深圳华大农业与循环经济科技有限公司 | Molecular marker and use thereof |
-
2017
- 2017-04-05 CN CN201710216627.4A patent/CN106834529A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802961A (en) * | 2014-12-29 | 2016-07-27 | 深圳华大农业与循环经济科技有限公司 | Molecular marker and use thereof |
| CN104711355A (en) * | 2015-03-17 | 2015-06-17 | 东北农业大学 | Molecular marker KpnI-1 for indicating and identifying watermelon pulp color and application |
| CN104711356A (en) * | 2015-03-17 | 2015-06-17 | 东北农业大学 | Molecular marker MboII-37 for indicating and identifying watermelon pulp color and application |
| CN105200144A (en) * | 2015-10-22 | 2015-12-30 | 北京市农林科学院 | High-flux molecular marker for identifying pulp color of Cucumis melo L. and special primer for high-flux molecular marker |
Non-Patent Citations (1)
| Title |
|---|
| GALIL TZURI ET AL.: ""A ‘golden’ SNP in CmOr governs the fruit flesh color of melon (Cucumis melo)"", 《THE PLANT JOURNAL》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517917A (en) * | 2017-09-19 | 2019-03-26 | 天津大学 | It is a kind of for identifying the SNP marker primer and molecule labelling method of muskmelon pulp colour |
| CN112662803A (en) * | 2021-01-22 | 2021-04-16 | 河南农业大学 | CAPS molecular marker co-separated from melon reticulate pattern character and application |
| CN112662803B (en) * | 2021-01-22 | 2023-07-25 | 河南农业大学 | CAPS molecular marker co-separated from melon reticulate pattern character and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106868132B (en) | SNP molecular marker related to contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil and application thereof | |
| US11572594B2 (en) | Characteristic sequence, labeled primer and identification method of Carya illinoensis variety Davis | |
| CN105925721B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character | |
| CN104946640B (en) | The specificity labeled primers and detection method of the long woods of oil tea breeding No. 18 | |
| CN103045588B (en) | Molecular marker of major QTL (Quantitative Trait Locus) of soybean seed protein content and application thereof | |
| CN110305978A (en) | SNP site and its versatility molecular labeling, acquisition methods and application of a kind of and pepper fruit towards tight association | |
| CN108754017A (en) | A kind of auxiliary detects CAPS labels and its application of soybean grease content height | |
| CN104711353B (en) | A kind of and the screening technique of the relevant molecular labeling MluI-6 of watermelon flesh color and application | |
| CN104711356B (en) | A kind of molecular marker MboII-37 and application indicated and identify watermelon flesh color | |
| CN103436533B (en) | Molecular marker closely linked with major gene locus of grain weight of wheatear as well as acquiring method and application of molecular marker | |
| CN107881256A (en) | For identifying single nucleotide polymorphism site, primer pair, kit and the application of Peach fruits hardship benevolence/sweet tea benevolence character | |
| CN107630103A (en) | A kind of CAPS molecule labelling methods for identifying rice varieties and application | |
| CN106834529A (en) | A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application | |
| CN102041310B (en) | Method for detecting rose cockscomb character | |
| CN110055351B (en) | Molecular marker BanII-1 related to melon powdery mildew resistance and application thereof | |
| CN104711354B (en) | A kind of and the screening technique of the relevant molecular labeling BsaHI 6 of watermelon flesh color and application | |
| CN116144819B (en) | SNP molecular marker closely linked with main effect QTL of pumpkin pulp carotenoid and application of SNP molecular marker | |
| CN114182042B (en) | SNP (Single nucleotide polymorphism) marker related to brown skin/green skin of Chinese pear and application thereof | |
| CN104711357B (en) | A kind of molecular labeling MboII 40 for indicating and identifying watermelon flesh color and application | |
| CN109652589A (en) | Characteristic sequence, labeled primer and the identification method of thin shell mountain pecan Peach cultivars Gloria Grande | |
| CN108165652A (en) | For the specific molecular marker TGMI001 of Chinese torreya seedling stage sex identification | |
| CN107881255A (en) | A kind of double dominant markers of precise Identification onion S/N cytogene types and its application | |
| CN109652428B (en) | Characteristic sequence, labeled primer and identification method of apocarya variety Sioux | |
| CN104711355B (en) | A kind of molecular labeling KpnI 1 for indicating and identifying watermelon flesh color and application | |
| CN104975010B (en) | The molecular specificity labeled primers and detection method of the long woods of oil tea breeding No. 21 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170613 |
|
| RJ01 | Rejection of invention patent application after publication |