CN104711353A - Method and application of screening of molecular markers MluI-6 related to watermelon flesh colors - Google Patents
Method and application of screening of molecular markers MluI-6 related to watermelon flesh colors Download PDFInfo
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Abstract
The invention discloses a method and application of screening of molecular markers MluI-6 related to watermelon flesh colors, and belongs to the technical field of molecular marker-assisted breeding. The method provided by the invention comprises the following steps: sequencing different flesh color systems of watermelons; after comparing an obtained sequence with gene sequences related to watermelon flesh colors for analyzing, obtaining a chromosome interval in which the gene sequences related to the watermelon flesh colors are positioned; screening out CAPS mutant sequences in the chromosome interval, designing CAPS molecular marker primers according to the mutant sequences, and then utilizing the CAPS molecular marker primers and watermelon genome DNAs to carry out PCR proliferation; finally, carrying out enzyme digestion on proliferation products, and carrying out verification through electrophoresis. The method has the advantages that assisted selection can be carried out on the watermelon flesh colors in the seedling period of the watermelons, the operation is simple and convenient, the result is explicit, the breeding efficiency is greatly improved, the breeding period is shortened, and the breeding work efficiency is improved, and is suitable for the molecular marker-assisted selection of watermelon lycopene.
Description
Technical field
The present invention relates to screening method and the application of a kind of molecule marker MluI-6 relevant to watermelon flesh color, belong to molecular mark technical field.
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.In recent years, high throughput sequencing technologies fast development, the watermelon genomic data obtained by sequencing technologies is that the assignment of genes gene mapping of watermelon major traits and the exploitation of a large amount of molecule marker provide new approach.Develop a large amount of molecule markers relevant to watermelon Other Main Agronomic Characters and can provide strong theoretical foundation for watermelon breeding is operated in gene aspect, serve traditional breeding method work fast and efficiently.
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.
Summary of the invention
The invention provides screening method and the application of a kind of molecule marker MluI-6 relevant to watermelon flesh color, in order to carry out molecular marker assisted selection to containing Lyeopene and the watermelon plant not containing Lyeopene, the technical scheme of employing is as follows:
The object of the present invention is to provide the screening method of a kind of molecule marker MluI-6 relevant to watermelon flesh color, the method checks order to the watermelon of different pulp colour strain, after again the gene order that gained sequence is relevant to watermelon flesh color being compared, between the chromosomal region obtaining the gene order place relevant to watermelon flesh color, the sequence that there is CAPS sudden change is filtered out in chromosomal region, according to mutant nucleotide sequence design CAPS molecule marker primer, recycling CAPS molecule marker primer and watermelon genomic dna carry out pcr amplification, electrophoresis is utilized to verify after finally cutting amplified production enzyme.
Described method steps is as follows:
1) watermelon of two kinds of different pulp colour strains is chosen;
2) utilize high throughput sequencing technologies determination step 1) selected by the gene order of two kinds of watermelons, the gene order that gained sequence is relevant to watermelon flesh color compares, acquisition exists between the chromosomal region at the gene order place relevant to watermelon flesh color, the base section that sequencing sequence exists SNP mutational site is screened in this interval, the enzyme analyzing SNP site cuts information, obtains the sequence that there is CAPS sudden change;
3) for step 2) sequence of gained, design CAPS molecule marker primer;
4) with watermelon genomic dna for template, utilize step 3) the CAPS molecule marker primer of gained carries out pcr amplification reaction, obtains PCR primer;
5) restriction enzyme is utilized to step 4) PCR primer that obtains carries out endonuclease reaction;
6) agarose gel electrophoresis is utilized to step 5) digestion products that obtains verifies.
Step 1) the watermelon strain of described two kinds of different pulp colours, one is yellow, and another kind is redness.
Step 1) the described gene order relevant to watermelon flesh color, be positioned at the cds section of lycopene in watermelon beta cyclase gene, the GenBank accession number of this section is ABM90917.1, itself and lycopene in watermelon beta cyclase gene close linkage.
Step 3) described CAPS molecule marker primer, forward primer nucleotide sequence is as shown in SEQ ID NO.1, and reverse primer nucleotide sequence is as shown in SEQ ID NO.2.
Step 4) described PCR primer, clip size is 566bp, and nucleotide sequence is as shown in SEQ ID NO.3.
Step 5) described pcr amplification, adopt TD-PCR, 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 90s, 10 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.
Step 6) described checking, digestion products band is 566bp fragment, containing Lyeopene in watermelon flesh; Digestion products band is 287bp and 279bp two kinds of fragments, not containing Lyeopene in watermelon flesh.
Described method concrete steps are:
1) a light yellow watermelon strain and a red watermelon strain is selected;
2) utilize high throughput sequencing technologies to step 1) selected by two kinds of watermelon strains check order, the sequence of gained sequence with the cds section of the lycopene in watermelon beta cyclase gene obtained from NCBI website is compared, between the chromosomal region obtaining the sequence of the cds section that there is lycopene in watermelon beta cyclase gene, the base section in SNP mutational site is there is at this interval screening sequencing data, the enzyme analyzing SNP site cuts information, obtains the sequence that there is CAPS sudden change;
Described CAPS mutant nucleotide sequence is as shown in SEQ ID NO.3;
3) for step 2) sequence of gained, design CAPS molecule marker primer;
Described CAPS molecule marker primer, forward primer nucleotide sequence is as shown in SEQ ID NO.1, and reverse primer nucleotide sequence is as shown in SEQ ID NO.2;
4) with watermelon genomic dna for template, utilize step 3) the CAPS molecule marker primer of gained carries out TD-PCR amplified reaction, obtains PCR primer; Described PCR primer, clip size is 566bp, and nucleotide sequence is as shown in SEQ ID NO.3;
5) restriction enzyme is utilized to step 4) PCR primer that obtains carries out endonuclease reaction;
6) agarose gel electrophoresis is utilized to step 5) digestion products that obtains verifies.
The inventive method is for the identification of the pulp colour with assisting sifting watermelon.
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 566bp by being limited property restriction endonuclease, the fragment that there is not base mutation site can be cut by being limited property restriction endonuclease, obtain two fragments of 287bp and 279bp, can clearly the watermelon material of pulp colour different in two be distinguished by agarose gel electrophoresis.
Molecule marker MluI-6 of the present invention is Late Cambrian, is not found in the prior art also not named.Instant invention overcomes following technical difficulty: first, the hereditary basis of watermelon is comparatively narrow, has announced available molecule marker limited amount at present and polymorphism between differing materials is extremely low.Secondly, the most of molecule markers announced at present are positioned at the non-coding region of gene, are difficult to these marks to combine closely with correlated character, and the molecule marker that therefore can be used for carrying out watermelon flesh color the assignment of genes gene mapping and molecular marker assisted selection is few.Finally, the present invention genome resurvey order sequenced data basis on develop a large amount of CAPS molecule markers, obtain by the proterties investigation of a large amount of fields and the assignment of genes gene mapping equimolecular mark test CAPS molecule marker can distinguished watermelon flesh color.
Beneficial effect of the present invention:
1) the watermelon genomic data that obtains according to high-flux sequence of the present invention, designs and develops the CAPS molecule marker relevant to watermelon flesh color.Develop CAPS molecule marker, in differing materials, there is versatility, result easy and simple to handle is clear and definite.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.May be used for the molecular marker assisted selection of watermelon high hycopene from now on, and accelerate breeding work efficiency.
2) the CAPS molecule marker of the present invention's acquisition, 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.
Accompanying drawing explanation
Fig. 1 is that CAPS primer MluI-6 is at LSW-177, COS, F
1and F
2situation is cut for the enzyme in colony;
(the F that 1 ~ 3, LSW-177, COS and hybridization thereof obtain
1for plant DNA by CAPS mark MluI-6 carry out PCR reaction obtain PCR primer fragment (566bp); 4, D2000marker; 5 ~ No. 7 swimming lanes are respectively LSW-177, COS and F
1for the digestion products of PCR primer, No. 5 swimming lane clip size are 566bp, No. 6 swimming lane clip size are 287 and 279bp, No. 7 swimming lanes are that each one of 566bp and 287,279bp is (because 1% agarose resolving power is 100bp, cannot distinguish 287 and 279bp, therefore two bands merge into one on electrophorogram); 8 ~ 17, F
2the digestion products of the watermelon individual plant of red pulp is presented in generation; 18 ~ 25, swimming lane is F
2the digestion products of the watermelon individual plant of light yellow pulp is presented in generation; Marker is D2000marker, and stripe size is from top to bottom followed successively by: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Fig. 2 is the PCR primer of 9 kinds of watermelon materials;
(1, LSW-177; 2, MSW-28; 3, PI179881; 4, PI482255; 5, WM-Clr-1; 6, WM-Clr-2; 7, COS; 8, PI459074; 9, PI186490; Marker is D2000marker, and stripe size is from top to bottom followed successively by: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Fig. 3 is the digestion products of 9 kinds of watermelon materials;
(1, LSW-177; 2, MSW-28; 3, PI179881; 4, PI482255; 5, WM-Clr-1; 6, WM-Clr-2; 7, COS; 8, PI459074; 9, PI186490; 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.
Embodiment 1: the extraction of 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 sequence of the cds section 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 sequence of the cds section 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, Primer 6 software is utilized to design CAPS primer in the sequence that there is CAPS site.Primer length is at 18 ~ 26bp, and annealing temperature is 56 ~ 60 DEG C, and GC content is 40% ~ 60%.Gather the F1 generation of LSW-177, COS and the two hybridization acquisition thereof, F2 for the tender true leaf of children of plant, utilize improved method of CTAB to extract genomic dna.
The forward primer nucleotide sequence of the base sequence of CAPS mark is as shown in SEQ ID NO.1, and reverse primer nucleotide sequence is as shown in SEQ ID NO.2, and PCR primer clip size is 566bp, and nucleotide sequence is as shown in SEQ ID NO.3.Specifically as shown in table 1:
The Primer that the CAPS that table 1 reacts for PCR marks, sequence and expection clip size
The acquisition of embodiment 2:PCR product
The CAPS primer that utilization obtains and genomic dna carry out PCR reaction, PCR reaction system (20 μ L) as shown in table 2:
Table 2 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 90s, 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 566bp that individual plant all amplifies a size, consistent with the clip size of expection.
Embodiment 3: digestion verification is carried out to PCR primer
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 3 that enzyme cuts system: (15.3 μ L)
Table 3 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.COS owing to there is not restriction enzyme site, cut by MluI and obtain 287, the fragment of 279bp.F
1the fragment that codominance feature had both comprised a 566bp length of LSW-177 is revealed in representative, comprises again the fragment of 287 and 279bp length.At the 18 strain F for examination
2for in individual plant, there are 10 strain field Phenotypic Expressions for red, and tomato red content can be detected in pulp organization, digestion products band is 566bp, all the other 8 strains show as non-redness, and in pulp organization, can't detect the content of Lyeopene, and digestion products band is 287,279bp.Resolving power due to 1% sepharose is 100bp, therefore 287 and 279bp two bar segment on gel, be shown as a bar segment.
Embodiment 4: utilize the CAPS mark obtained the variety of watermelon of different pulp colour to be carried out to the screening of germ plasm resource
For differentiating the suitability of CAPS mark to other watermelon material developed, we have chosen the watermelon material of the 9 kinds of different colours comprising LSW-177 and COS, and it carries out pcr amplification and digestion verification to utilize this primer pair.The title of these 9 kinds of watermelon materials and color etc. describe as shown in table 4:
Table 49 kinds of variety of watermelon introductions
The genomic dna extracting these 9 kinds of watermelon materials carries out PCR reaction to it, adopts 1% agarose electrophoresis to detect PCR primer, and as shown in Figure 2,9 kinds of materials for examination all amplify the band of a 566bp size to detected result.
Carry out digestion verification to obtained PCR primer, acquired results as shown in Figure 3.
Composition graphs 3 and table 3 can be found out: all detect content of lycopene in 1 ~ No. 6 pulp for examination material, digestion products all presents the band of a 566bp size, and field phenotype is respectively red, pink colour and orange; All Lyeopene do not detected in 7 ~ No. 9 material pulp, PCR primer, after enzyme is cut, all obtains 287, the band of 279bp size, and field phenotype is light yellow and white.
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. the screening method of a molecule marker MluI-6 relevant to watermelon flesh color, it is characterized in that, the watermelon of different pulp colour strain is checked order, after again the gene order that gained sequence is relevant to watermelon flesh color being compared, between the chromosomal region obtaining the gene order place relevant to watermelon flesh color, the sequence that there is CAPS sudden change is filtered out in chromosomal region, according to mutant nucleotide sequence design CAPS molecule marker primer, recycling CAPS molecule marker primer and watermelon genomic dna carry out pcr amplification, electrophoresis is utilized to verify after finally cutting amplified production enzyme.
2. method according to claim 1, it is characterized in that, step is as follows:
1) watermelon of two kinds of different pulp colour strains is chosen;
2) utilize high throughput sequencing technologies determination step 1) selected by the gene order of two kinds of watermelons, the gene order that gained sequence is relevant to watermelon flesh color compares, acquisition exists between the chromosomal region at the gene order place relevant to watermelon flesh color, the base section that sequencing sequence exists SNP mutational site is screened in this interval, the enzyme analyzing SNP site cuts information, obtains the sequence that there is CAPS sudden change;
3) for step 2) sequence of gained, design CAPS molecule marker primer;
4) with watermelon genomic dna for template, utilize step 3) the CAPS molecule marker primer of gained carries out pcr amplification reaction, obtains PCR primer;
5) restriction enzyme is utilized to step 4) PCR primer that obtains carries out endonuclease reaction;
6) agarose gel electrophoresis is utilized to step 5) digestion products that obtains verifies.
3. method according to claim 2, is characterized in that, step 1) the watermelon strain of described two kinds of different pulp colours, one is yellow, and another kind is redness.
4. method according to claim 2, it is characterized in that, step 1) the described gene order relevant to watermelon flesh color, be positioned at the cds section of lycopene in watermelon beta cyclase gene, the GenBank accession number of this section is ABM90917.1, itself and lycopene in watermelon beta cyclase gene close linkage.
5. method according to claim 2, is characterized in that, step 3) described CAPS molecule marker primer, forward primer nucleotide sequence is as shown in SEQ ID NO.1, and reverse primer nucleotide sequence is as shown in SEQ ID NO.2.
6. method according to claim 2, is characterized in that, step 4) described PCR primer, clip size is 566bp, and nucleotide sequence is as shown in SEQ ID NO.3.
7. method according to claim 2, is characterized in that, step 5) described pcr amplification, adopt TD-PCR, 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 90s, 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.
8. method according to claim 2, is characterized in that, step 6) described checking, digestion products band is 566bp fragment, containing Lyeopene in watermelon flesh; Digestion products band is 287bp and 279bp two kinds of fragments, not containing Lyeopene in watermelon flesh.
9. method according to claim 1, it is characterized in that, concrete steps are:
1) a light yellow watermelon strain and a red watermelon strain is selected;
2) utilize high throughput sequencing technologies to step 1) selected by two kinds of watermelon strains check order, the sequence of gained sequence with the cds section of the lycopene in watermelon beta cyclase gene obtained from NCBI website is compared, between the chromosomal region obtaining the sequence of the cds section that there is lycopene in watermelon beta cyclase gene, the base section in SNP mutational site is there is at this interval screening sequencing data, the enzyme analyzing SNP site cuts information, obtains the sequence that there is CAPS sudden change;
3) for step 2) sequence of gained, design CAPS molecule marker primer; Described CAPS molecule marker primer, forward primer nucleotide sequence is as shown in SEQ ID NO.1, and reverse primer nucleotide sequence is as shown in SEQ ID NO.2;
4) with watermelon genomic dna for template, utilize step 3) the CAPS molecule marker primer of gained carries out TD-PCR amplified reaction, obtains PCR primer; Described PCR primer, clip size is 566bp, and nucleotide sequence is as shown in SEQ ID NO.3;
5) restriction enzyme is utilized to step 4) PCR primer that obtains carries out endonuclease reaction;
6) agarose gel electrophoresis is utilized to step 5) digestion products that obtains verifies.
10. the method described in claim 1-9, is characterized in that, for the identification of the pulp colour with assisting sifting watermelon.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148526A (en) * | 2016-07-11 | 2016-11-23 | 北京市农林科学院 | A kind of molecular marker Hf1 Indel relevant to watermelon flesh hardness and application thereof |
| CN106755357A (en) * | 2016-12-05 | 2017-05-31 | 江苏省农业科学院 | Differentiate CAPS molecule labelling methods and the application of atropurpureus striped pericarp tomato |
| CN111549172A (en) * | 2020-06-12 | 2020-08-18 | 中国农业科学院郑州果树研究所 | Watermelon leaf posterior green gene linkage site and CAPS marker |
| CN113151563A (en) * | 2021-05-12 | 2021-07-23 | 东北农业大学 | Method, primer pair and kit for identifying watermelon pulp color and application of kit |
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) |
-
2015
- 2015-03-17 CN CN201510117027.3A patent/CN104711353B/en active Active
Patent 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) |
Non-Patent Citations (3)
| Title |
|---|
| HAEJEEN BANG ET AL.: "Development of a codominant CAPS marker for allelic selection between canary yellow and red watermelon based on SNP in lycopene b-cyclase (LCYB) gene", 《MOL BREEDING》 * |
| 刘传奇 等: "西瓜遗传图谱构建及果实相关性状QTL 分析", 《中国农业科学》 * |
| 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》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148526A (en) * | 2016-07-11 | 2016-11-23 | 北京市农林科学院 | A kind of molecular marker Hf1 Indel relevant to watermelon flesh hardness and application thereof |
| CN106148526B (en) * | 2016-07-11 | 2019-11-12 | 北京市农林科学院 | One kind molecular labeling Hf1-Indel relevant to watermelon flesh hardness and its application |
| CN106755357A (en) * | 2016-12-05 | 2017-05-31 | 江苏省农业科学院 | Differentiate CAPS molecule labelling methods and the application of atropurpureus striped pericarp tomato |
| CN106755357B (en) * | 2016-12-05 | 2020-08-07 | 江苏省农业科学院 | CAPS molecular marking method for identifying purple black striped peel tomatoes and application |
| CN111549172A (en) * | 2020-06-12 | 2020-08-18 | 中国农业科学院郑州果树研究所 | Watermelon leaf posterior green gene linkage site and CAPS marker |
| CN111549172B (en) * | 2020-06-12 | 2023-02-28 | 中国农业科学院郑州果树研究所 | Watermelon leaf posterior green gene linkage site and CAPS marker |
| CN113151563A (en) * | 2021-05-12 | 2021-07-23 | 东北农业大学 | Method, primer pair and kit for identifying watermelon pulp color and application of kit |
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