CN104711358A - Molecular marker MboII-33 for indicating and identifying watermelon pulp color and application - Google Patents
Molecular marker MboII-33 for indicating and identifying watermelon pulp color and application Download PDFInfo
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Abstract
The invention discloses a molecular marker MboII-33 for indicating and identifying watermelon pulp color and application and belongs to the technical field of molecular biology. The fragment size of a PCR product amplified by the molecular marker is 524bp, and the nucleotide sequence is as shown in SEQ ID NO.1. The CAPS molecular marker has universality in different materials and can identify watermelons in the watermelon seedling stage, the operation is simple and convenient, and the result is definite. The molecular marker can be used for molecular marker-assisted selection of high lycopene of watermelon in future, and the breeding work efficiency is improved.
Description
Technical field
The present invention relates to molecule marker MboII-33 and the application of a kind of indication and qualification watermelon flesh color, belong to technical field of molecular biology.
Background technology
Molecular marker assisted selection is the new technology produced along with developing rapidly of modern molecular biology technique, and it can analyze individual genetic composition rapidly and accurately from molecular level, thus realizes genotypic direct selection, carries out molecular breeding.The successful key of molecular marker assisted selection is that obtained molecule marker is whether mutually chain with proterties to be analyzed and whether between the differing materials of same species, have versatility.
The pulp colour of watermelon is the important indicator that quality of watermelon is formed, containing multiple pigments such as Lyeopene, β-carotene, xenthophylls, zeaxanthins in Watermelon Fruit, the composition of different pigment and content number make watermelon flesh present different colours.Simultaneously, pigment in watermelon flesh also has the physiological function useful to human body, there are some researches show, Lyeopene has efficient anti-oxidant activity, the sickness rate of cancer and cardiovascular and cerebrovascular can be effectively reduced, and functional food can be widely used in as one is functional pigmented, (Sahin K in medicine and makeup, Tuzcu M, Sahin N, et al.Nrf2/HO-1signalingpathway may be the prime target for chemoprevention of cisplatin-induced nephrotoxicity bylycopene.Food Chem Toxicol, 2010, 48 (10): 2670-2674, Mortensen A, Skibsted L H, Sampson J, et al.Comparative mechanisms and rates of free radical scavenging by carotenoid antioxidants.FEBS Lett, 1997,418 (12): 91-97).β-carotene can reduce not destroyed (Olson J A.Molecular actions of carotenoids.In:Canfield LM (ed) the .Carotenoids in HumanHealth of lipid peroxidation protection organism; New Y ork.Academy of Sciences; 1993.156-166), xenthophylls is to safeguarding that eyesight has important effect.Therefore be the major portion of current watermelon breeding work by the variety of watermelon of the different pulp colour of the efficient seed selection of the method for molecular marker assisted selection.
Along with the fast development of DNA molecular marker technology, utilize and carry out assisted Selection with the closely linked molecule marker of plant genes involved, screening and the assignment of genes gene mapping of major traits can be accelerated greatly, thus greatly improve breeding selection efficiency.But the genetic distance of watermelon is comparatively narrow, the available molecule marker number announced of current document is less and polymorphism is lower, seriously constrains the exploitation of the assignment of genes gene mapping of watermelon Other Main Agronomic Characters and related molecular marker.
Summary of the invention
For providing a CAPS molecule marker, in order to carry out molecular marker assisted selection to containing Lyeopene and the watermelon plant not containing Lyeopene, this invention takes following technical scheme:
One object of the present invention is the molecule marker MboII-33 providing a kind of indication and qualification watermelon flesh color, and the PCR primer clip size that this molecule marker amplifies is 524bp, and nucleotide sequence is as shown in SEQ ID NO.1.。
Described molecule marker is positioned at lycopene in watermelon beta cyclase cds section, and the GenBank accession number of this section is ABM90917.1, with lycopene in watermelon beta cyclase gene close linkage.
The diagnostic primers of described molecule marker is to the nucleotide sequence of forward primer as shown in SEQ ID NO.2, and the nucleotide sequence of reverse primer is as shown in SEQ ID NO.3.
Described molecule marker MboII-33 is applied to the pulp colour of qualification and assisting sifting watermelon.
Described application is after the DNA of extraction testing sample, utilizes diagnostic primers to carrying out pcr amplification, and recycling restriction enzyme carries out enzyme to amplified production and cuts, and finally utilizes electrophoresis to detect digestion products.
The step of described application is as follows:
1) DNA of testing sample is extracted;
2) with the DNA of testing sample for template, utilize diagnostic primers to carrying out pcr amplification, obtain amplified production;
3) restriction enzyme is utilized to step 2) amplified production of gained carries out enzyme and cuts, and obtains digestion products;
4) agarose gel electrophoresis is utilized to step 3) digestion products of gained detects.
Step 2) described diagnostic primers pair, the nucleotide sequence of forward primer is as shown in SEQ ID NO.2, and the nucleotide sequence of reverse primer is as shown in SEQ ID NO.3.
Preferably, step 2) described pcr amplification, adopt touchdown PCR, amplification program is: 94 DEG C of denaturation 7min, 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, often circulation reductions by 0.5 DEG C, and 72 DEG C extend 40s, 30 circulations; 94 DEG C of sex change 30s, 45 DEG C of annealing 30s, 72 DEG C extend 40s, 10 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
Preferably, step 4) described digestion products to be detected, be utilize agarose gel electrophoresis detect enzyme cut after product in the band that whether is 524bp containing size, the sample containing 524bp band, not containing Lyeopene, pulp colour is non-redness; Sample not containing 524bp band, containing Lyeopene, pulp colour is red.
Another object of the present invention is to the preparation method providing a kind of described molecule marker MboII-33, the step of the method is as follows:
1) a light yellow watermelon strain and a red watermelon strain is selected;
2) utilize high throughput sequencing technologies to step 1) selected by the watermelon of two strains check order, and the sequence of gained sequence and lycopene in watermelon beta cyclase gene cds section is compared, acquisition exists between the chromosomal region of the sequence of lycopene in watermelon beta cyclase gene cds section;
3) in step 2) search the base section that there is SNP sudden change in gained chromosomal region, and the enzyme analyzing SNP site cuts information, obtains the sequence that there is CAPS sudden change;
4) for step 3) sequence of gained, design primer;
5) utilize step 4) primer of gained with watermelon genomic dna for template carries out TD-PCR amplification, obtain amplified production;
6) restriction enzyme is utilized to step 5) amplified production of gained carries out digestion verification.
The present invention utilizes CAPS molecule marker primer to obtain the DNA fragmentation relevant to watermelon flesh color by pcr amplification, recycling restriction enzyme carries out endonuclease reaction to the fragment obtained, and the watermelon material due to two kinds of different colours exists the difference of the single base mutation of restriction enzyme site in obtained DNA fragmentation.Therefore, the site that there is single base mutation can not to be cut and to obtain a size be the fragment of 524bp by being limited property restriction endonuclease, the fragment that there is not base mutation site can be cut by being limited property restriction endonuclease, because 166bp and the 510bp place in this sequence all exists restriction enzyme site, therefore after two site enzymes are cut, two fragments of final acquisition 344bp (401-57) and 123bp, can be distinguished the watermelon material of pulp colour different in two clearly by agarose gel electrophoresis.
The CAPS molecule marker that the present invention obtains, can well distinguish in the watermelon material of different pulp colours, known by the result of sequence near molecule marker designation of chromosome being carried out to Blast comparison, this mark and lycopene in watermelon beta cyclase close linkage, therefore, present invention obtains one and efficiently can distinguish molecule marker with presence or absence of different watermelon flesh tomato redness, for the be correlated with location of major gene of lycopene in watermelon from now on lays the foundation.
The beneficial effect that the present invention obtains is as follows:
CAPS molecule marker is the molecule marker producing polymorphism with restriction enzyme site single base mutation, the stable a new generation's mark becoming the assignment of genes gene mapping and molecular biology research of, variation extensive with its distribution range, CAPS marking operation is easy, and result is easy to observation and has general applicability between same species.
The watermelon genomic data that the present invention obtains according to high-flux sequence, designs and develops the CAPS molecule marker relevant to watermelon flesh color.By the method for molecular marker assisted selection, assisted Selection can be carried out to watermelon flesh color at the Seedling Stage of watermelon, substantially increase breeding efficiency shortening the breeding cycle.
In addition, according to the result of data analysis, develop lycopene beta cyclase gene close linkage in CAPS molecule marker and Watermelon Fruit, therefore, present invention obtains one and can be used for differentiating the molecule marker of watermelon flesh whether containing Lyeopene.
Accompanying drawing explanation
Fig. 1 is that CAPS primer MboII-33 is at LSW-177, COS, F
1and F
2situation is cut for the enzyme in colony;
(1 ~ No. 3 swimming lane is respectively the F that LSW-177, COS and hybridization thereof obtain
1for plant DNA by CAPS mark MboII-33 carry out PCR reaction obtain PCR primer fragment (524bp), No. 4 swimming lanes are D2000marker, and 5 ~ No. 6 swimming lanes are respectively LSW-177, COS and F
1for the digestion products of PCR primer, No. 5 swimming lane clip size are 344 and 123bp, and No. 6 swimming lane clip size are 524bp, and No. 7 swimming lanes are each one of 524bp and 344,123bp, and 8 ~ No. 16 swimming lanes are F
2present the digestion products of the watermelon individual plant of red pulp in generation, 17 ~ 25 swimming lanes are F
2present the digestion products of the watermelon individual plant of light yellow pulp in generation, Marker is D2000marker, and stripe size is from top to bottom followed successively by: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.
Material therefor, reagent, instruments and methods in following examples, without specified otherwise, be the conventional material in this area, reagent, instruments and methods, all obtain by commercial channel.
The extraction of embodiment 1 design of primers and genomic dna
Select the F that light yellow watermelon strain Cream of Saskatchewan (being called for short " COS ") and red watermelon strain LSW-177 and hybridization thereof obtain
1, F
2be whether there is Lyeopene in experiment material checking CAPS Marker Identification watermelon flesh for colony.Result as shown in Figure 1.
High throughput sequencing technologies is utilized to check order to COS and LSW-177 two watermelon strains, and the cds region sequence of lycopene in watermelon beta cyclase gene is obtained by NCBI website, by the comparison of this sequence with order-checking material, between the chromosomal region obtaining the cds region sequence that there is lycopene in watermelon beta cyclase gene, in this interval, excavate the base section that sequencing data exists SNP mutational site, the enzyme analyzing SNP site cuts information, obtain the sequence that there is CAPS sudden change, the sequence that there is CAPS site designs CAPS primer.Primer length is at 18 ~ 26bp, and annealing temperature is 56 ~ 60 DEG C, and GC content is 40% ~ 60%.Gather the F of LSW-177, COS and the two hybridization acquisition thereof
1generation, F
2the tender true leaf of children for plant, utilizes improved method of CTAB to extract genomic dna.
The acquisition of embodiment 2PCR product
The CAPS primer that utilization obtains and genomic dna carry out PCR reaction, PCR reaction system (20 μ l) as shown in table 1:
Table 1.PCR reaction system
Adopt touchdown PCR (touchdown PCR, TD-PCR) to increase, program is: 94 DEG C of denaturation 7min, 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, often circulation reductions by 0.5 DEG C, and 72 DEG C extend 40s, 30 circulations; 94 DEG C of sex change 30s, 45 DEG C of annealing 30s, 72 DEG C extend 40s, 10 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
Utilize 1% agarose gel electrophoresis to detect obtained PCR primer, detected result as shown in Figure 1, LSW-177, COS, F
1and F
2it is the fragment of 524bp that individual plant all amplifies a size, consistent with the clip size of expection.
Embodiment 3 pairs of PCR primer carry out digestion verification
According to the restriction enzyme operational guidance restriction endonuclease of Thermo, carry out digestion verification to obtained PCR primer, it is as shown in table 2 that enzyme cuts system: (15.3 μ l)
Table 2. endonuclease reaction system
37 DEG C of water-bath enzymes cut through night, and digestion products adopts 1% agarose electrophoresis to detect, and detected result as shown in Figure 1.LSW-177 owing to there is not restriction enzyme site, being not limited property restriction endonuclease cut still in 524bp, COS owing to there is restriction enzyme site, cut by MboII and obtain 344, the fragment of 123bp.F
1the fragment that codominance feature had both comprised a 524bp length of LSW-177 is revealed in representative, comprises again the fragment of 344 and 123bp length.At the 18 strain F for examination
2for in individual plant, there are 9 strain field Phenotypic Expressions for red, and Lyeopene can be detected in pulp organization, digestion products band is 344,122bp, all the other 9 strains show as non-redness, and can't detect Lyeopene in pulp organization, and digestion products band is 524bp.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention; any person skilled in the art; not departing from spirit and scope of the invention; various changes and modification can be done; therefore, what protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. a molecule marker MboII-33 for indication and qualification watermelon flesh color, it is characterized in that, the PCR primer clip size that molecule marker amplifies is 524bp, and nucleotide sequence is as shown in SEQ ID NO.1.
2. molecule marker MboII-33 described in claim 1, is characterized in that, is positioned at lycopene in watermelon beta cyclase cds section, and the GenBank accession number of this section is ABM90917.1, with lycopene in watermelon beta cyclase gene close linkage.
3. molecule marker MboII-33 described in claim 1, is characterized in that, the diagnostic primers of described molecule marker is to the nucleotide sequence of forward primer as shown in SEQ ID NO.2, and the nucleotide sequence of reverse primer is as shown in SEQ ID NO.3.
4. molecule marker MboII-33 described in claim 1, is characterized in that, is applied to the pulp colour of qualification and assisting sifting watermelon.
5. apply described in claim 4, it is characterized in that, being after the DNA of extraction testing sample, utilizing diagnostic primers to carrying out pcr amplification, recycling restriction enzyme carries out enzyme to amplified production and cuts, and finally utilizes electrophoresis to detect digestion products.
6. apply described in claim 4, it is characterized in that, step is as follows:
1) DNA of testing sample is extracted;
2) with the DNA of testing sample for template, utilize diagnostic primers to carrying out pcr amplification, obtain amplified production;
3) restriction enzyme is utilized to step 2) amplified production of gained carries out enzyme and cuts, and obtains digestion products;
4) agarose gel electrophoresis is utilized to step 3) digestion products of gained detects.
7. apply described in claim 6, it is characterized in that, step 2) described diagnostic primers pair, the nucleotide sequence of forward primer is as shown in SEQID NO.2, and the nucleotide sequence of reverse primer is as shown in SEQ ID NO.3.
8. apply described in claim 6, it is characterized in that, step 2) described pcr amplification, adopt touchdown PCR, amplification program is: 94 DEG C of denaturation 7min, 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, often circulation reductions by 0.5 DEG C, and 72 DEG C extend 40s, 30 circulations; 94 DEG C of sex change 30s, 45 DEG C of annealing 30s, 72 DEG C extend 40s, 10 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
9. apply described in claim 6, it is characterized in that, step 4) described digestion products to be detected, whether contain the band that size is 524bp in the product after utilizing agarose gel electrophoresis detection enzyme to cut, sample containing 524bp band, not containing Lyeopene, pulp colour is non-redness; Sample not containing 524bp band, containing Lyeopene, pulp colour is red.
10. a preparation method of molecule marker MboII-33 described in claim 1, it is characterized in that, step is as follows:
1) a light yellow watermelon strain and a red watermelon strain is selected;
2) utilize high throughput sequencing technologies to step 1) selected by the watermelon of two strains check order, and the sequence of gained sequence and lycopene in watermelon beta cyclase gene cds section is compared, acquisition exists between the chromosomal region of the sequence of lycopene in watermelon beta cyclase gene cds section;
3) in step 2) search the base section that there is SNP sudden change in gained chromosomal region, and the enzyme analyzing SNP site cuts information, obtains the sequence that there is CAPS sudden change;
4) for step 3) sequence of gained, design primer;
5) utilize step 4) primer of gained with watermelon genomic dna for template carries out TD-PCR amplification, obtain amplified production;
6) restriction enzyme is utilized to step 5) amplified production of gained carries out digestion verification.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194444A (en) * | 2013-04-11 | 2013-07-10 | 北京市农林科学院 | SNP (single nucleotide polymorphism) site and CAPS (cleaved amplified polymorphic sequence) mark interlocked with citrullus lanatus fruit bitter taste gene Bt (bitterness) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103194444A (en) * | 2013-04-11 | 2013-07-10 | 北京市农林科学院 | SNP (single nucleotide polymorphism) site and CAPS (cleaved amplified polymorphic sequence) mark interlocked with citrullus lanatus fruit bitter taste gene Bt (bitterness) |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "EF183521.1", 《GENBANK》 * |
| HAEJEEN BANG等: "Development of a codominant CAPS marker for allelic selection between canary yellow and red watermelon based on SNP in lycopene β-cyclase (LCYB) gene", 《MOL BREEDING》 * |
| 惠伯棣等: "红和黄瓤西瓜中类胡萝卜素含量和组成比较", 《食品科学》 * |
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