CN104711354B - A kind of and the screening technique of the relevant molecular labeling BsaHI 6 of watermelon flesh color and application - Google Patents
A kind of and the screening technique of the relevant molecular labeling BsaHI 6 of watermelon flesh color and application Download PDFInfo
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Abstract
The invention discloses a kind of and the screening technique of the relevant molecular labeling BsaHI 6 of watermelon flesh color and application, belong to molecular mark technical field.Method provided by the present invention is that the watermelon of different pulp colour strains is sequenced, after gained sequence and the relevant gene order of watermelon flesh color are compared again, obtain and the chromosome interval where the relevant gene order of watermelon flesh color, filtered out in chromosome interval there are CAPS mutation sequence, CAPS molecular labeling primers are designed according to mutant nucleotide sequence, CAPS molecular labeling primers and watermelon genomic DNA is recycled to carry out PCR amplification, finally to being verified after amplified production digestion using electrophoresis.The method of the present invention can carry out assisted Selection in the Seedling Stage of watermelon to watermelon flesh color, and result easy to operate is clear and definite, substantially increases breeding efficiency and shortens the breeding time limit, accelerates breeding work efficiency.Suitable for the molecular marker assisted selection of lycopene in watermelon.
Description
Technical field
The present invention relates to a kind of and the screening technique of the relevant molecular labeling BsaHI-6 of watermelon flesh color and application, belongs to
In molecular mark technical field.
Background technology
Molecular marker assisted selection is the new technology produced with developing rapidly for modern molecular biology technique, it can
Rapidly and accurately to analyze the genetic constitution of individual from molecular level, directly select, divided so as to fulfill to genotype
Sub- breeding.The successful key of molecular marker assisted selection is whether obtained molecular labeling is mutually chain with character to be analyzed
And whether there is versatility between the different materials of same species.
The pulp colour of watermelon is the important indicator that quality of watermelon is formed, in Watermelon Fruit containing lycopene, β-
A variety of pigments such as carrotene, lutein, zeaxanthin, the composition of different pigments and the number of content show watermelon flesh
Different colours.Meanwhile the pigment in watermelon flesh also has the physiological function beneficial to human body, some researches show that lycopene
With efficient antioxidation activity, the incidence of cancer and cardiovascular and cerebrovascular can be effectively reduced, and a kind of feature can be used as
Pigment is widely used in functional food, medicine and cosmetics (Sahin K, Tuzcu M, Sahin N, et al.Nrf2/
HO-1signaling pathway may be the prime target for chemoprevention of
cisplatin-induced nephrotoxicity by lycopene.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).Beta carotene can reduce lipid peroxidation and protect organism not to be destroyed (Olson J
A.Molecular actions of carotenoids.In:Canfield LM(ed).Carotenoids in Human
Health, New Y ork.Academy of Sciences, 1993.156-166), lutein is to safeguarding that it is important that eyesight has
Effect.Therefore it is current watermelon by the variety of watermelon of the efficient selection and breeding difference pulp colour of method of molecular marker assisted selection
The major part of breeding work.
With the fast development of DNA molecular marker technology, using the molecular labeling with plant related gene close linkage into
Row assisted Selection, can greatly accelerate the screening and the assignment of genes gene mapping of major traits, so as to greatly improve breeding selection efficiency.But
It is that the genetic distance of watermelon is more narrow, the available molecular labeling number that document has been announced at present is less and polymorphism is relatively low,
Seriously constrain the assignment of genes gene mapping of watermelon Other Main Agronomic Characters and the exploitation of related molecular marker.In recent years, high throughput sequencing technologies
Grow rapidly, the assignment of genes gene mapping and a large amount of molecule mark of the watermelon genomic data obtained by sequencing technologies for watermelon major traits
The exploitation of note provides new approach.Exploitation can largely be educated with the relevant molecular labeling of watermelon Other Main Agronomic Characters for watermelon
Kind, which is operated in gene aspect, provides strong theoretical foundation, fast and efficiently serves traditional breeding method work.
CAPS molecular labelings are the molecular labelings that polymorphism is produced with restriction enzyme site single base mutation, are had a very wide distribution with it
A new generation's mark as the assignment of genes gene mapping and molecular biology research is stablized in general, variation, and CAPS marking operations are easy, are as a result easy to
Observe and there is general applicability between same species.
The content of the invention
The present invention provides a kind of and relevant molecular labeling BsaHI-6 of watermelon flesh color screening technique, to right
Molecular marker assisted selection is carried out containing lycopene and the watermelon plant for not containing lycopene, the technical solution of use is such as
Under：
It is an object of the invention to provide a kind of and relevant molecular labeling BsaHI-6 of watermelon flesh color screening side
Method, this method are that the watermelon of different pulp colour strains is sequenced, then gained sequence and watermelon flesh color is relevant
After gene order is compared, acquisition and the chromosome interval where the relevant gene order of watermelon flesh color, are contaminating
Filtered out in colour solid section there are the sequence of CAPS mutation, CAPS molecular labeling primers are designed according to mutant nucleotide sequence, are recycled
CAPS molecular labeling primers and watermelon genomic DNA carry out PCR amplification, finally to being tested after amplified production digestion using electrophoresis
Card.
The method 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 two kinds of watermelons gene order, by gained sequence with west
The relevant gene order of melon pulp colour is compared, and obtains and there is gene order relevant with watermelon flesh color place
Chromosome interval, base section of the sequencing sequence there are SNP mutation site is screened in the section, analyzes the enzyme of SNP site
Cut information, sequence of the acquisition there are CAPS mutation；
3) sequence obtained by step 2), designs CAPS molecular labeling primers；
4) using watermelon genomic DNA as template, it is anti-to carry out PCR amplification using the CAPS molecular labeling primers obtained by step 3)
Should, obtain PCR product；
5) PCR product obtained using restriction enzyme to step 4) carries out endonuclease reaction；
6) digestion products obtained using agarose gel electrophoresis to step 5) are verified.
The watermelon strain of the described two different pulp colours of step 1), one kind is yellow, and another kind is red.
It is lycopene in watermelon beta cyclase gene with the relevant gene order of watermelon flesh color described in step 1)
Cds region sequences, itself and lycopene in watermelon beta cyclase gene close linkage.
Step 3) the CAPS molecular labeling primers, forward primer nucleotide sequence reversely draw as shown in SEQ ID NO.1
Thing nucleotide sequence is as shown in SEQ ID NO.2.
Step 4) the PCR product, clip size 1874bp, nucleotide sequence is as shown in SEQ ID NO.3.
Step 5) the PCR amplification, using TD-PCR, program is：94 DEG C of pre-degeneration 7min, 94 DEG C of denaturation 30s, 60 DEG C are moved back
Fiery 30s, often circulation reduce by 0.5 DEG C, 72 DEG C of extension 90s, 30 circulations；94 DEG C of denaturation 30s, 45 DEG C of annealing 30s, 72 DEG C extend
90s, 10 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.
Step 6) the verification, digestion products band are two kinds of fragments of 1350bp and 524bp, contain tomato in watermelon flesh
Red pigment；Digestion products band is a kind of fragments of 1874bp, and lycopene is free of in watermelon flesh.
The method concretely comprises the following steps：
1) a light yellow watermelon strain and a red watermelon strain are selected；
2) two kinds of watermelon strains selected by step 1) are sequenced using high throughput sequencing technologies, by gained sequence with from
The cds region sequences of the lycopene in watermelon beta cyclase gene obtained on NCBI websites are compared, and obtain that there are watermelon tomato
The chromosome interval of the cds region sequences of red pigment beta cyclase gene, in section screening sequencing data, there are SNP mutation site
Base section, analyzes the digestion information of SNP site, sequence of the acquisition there are CAPS mutation；
The CAPS mutant nucleotide sequences are as shown in SEQ ID NO.3；
3) mutant nucleotide sequence obtained by step 2), designs CAPS molecular labeling primers；
The CAPS molecular labeling primers, forward primer nucleotide sequence is as shown in SEQ ID NO.1, reverse primer nucleosides
Acid sequence is as shown in SEQ ID NO.2；
4) using watermelon genomic DNA as template, TD-PCR expansions are carried out using the CAPS molecular labeling primers obtained by step 3)
Increase reaction, obtain PCR product；The PCR product, clip size 1874bp, nucleotide sequence is as shown in SEQ ID NO.3；
5) PCR product obtained using restriction enzyme to step 4) carries out endonuclease reaction；
6) digestion products obtained using agarose gel electrophoresis to step 5) are verified.
The method of the present invention is used to identify and the pulp colour of assisting sifting watermelon.
The present invention is obtained using CAPS molecular labeling primers by PCR amplification and the relevant DNA fragmentation of watermelon flesh color,
Restriction enzyme is recycled to carry out endonuclease reaction to the fragment of acquisition, since two different colors of watermelon material is being obtained
DNA fragmentation in there are restriction enzyme site single base mutation difference.Site accordingly, there exist single base mutation will not be limited
Property restriction endonuclease cut and obtain the fragment that size is 1874bp, the fragment there is no base mutation site can be by restricted interior
Enzyme cutting is cut, and obtains two fragments of 1350bp and 524bp, can be clearly by different fruits in two by agarose gel electrophoresis
The watermelon material of meat color distinguishes.
The molecular labeling BsaHI-6 of the present invention is to find first, is not found also not ordered in the prior art
Name.The present invention overcomes following technical difficulty：First, the hereditary basis of watermelon is more narrow, has announced available point at present
Sub- marker number is limited and polymorphism between different materials is extremely low.Secondly, the most of molecular labelings announced at present are located at
The noncoding region of gene, it is difficult to which these marks are combined closely with correlated traits, therefore can be used to carry out watermelon flesh color
The molecular labeling of the assignment of genes gene mapping and molecular marker assisted selection is fewer and fewer.Finally, the present invention is in genome weight sequencing data
On the basis of develop substantial amounts of CAPS molecular labelings, pass through substantial amounts of field character investigation and assignment of genes gene mapping equimolecular mark test
Obtain the CAPS molecular labelings that can be distinguished to watermelon flesh color.
Beneficial effect of the present invention：
1) present invention is designed and developed and the relevant CAPS of watermelon flesh color according to obtained watermelon genomic data
Molecular labeling, develop CAPS molecular labelings has a versatility in different materials, and with lycopene beta-ring in Watermelon Fruit
Change enzyme gene close linkage, obtain one can be used for differentiate watermelon flesh whether the molecular labeling containing lycopene.This hair
Bright method can carry out assisted Selection in the Seedling Stage of watermelon to watermelon flesh color, and result easy to operate is clear and definite, greatly improves
Breeding efficiency shortens the breeding time limit, accelerates breeding work efficiency.Suitable for the molecular marker assisted selection of lycopene in watermelon.
2) the CAPS molecular labelings that the present invention obtains, can carry out well in the watermelon material of different pulp colours
Distinguish, after carrying out Blast comparisons to sequence near molecular labeling designation of chromosome, the mark and lycopene in watermelon
Beta cyclase close linkage, therefore, present invention obtains one can efficiently distinguish what different watermelon flesh tomato red whether there is
Molecular labeling.
Brief description of the drawings
Fig. 1 is CAPS primers BsaHI-6 in LSW-177, COS and F2For the digestion situation in colony；
(1~2, the DNA of LSW-177, COS plant marks BsaHI-6 to carry out PCR reactions and obtains PCR product by CAPS
Fragment (1874bp)；3, D2000marker；4~No. 5 swimming lanes are respectively the digestion products of LSW-177, COS；No. 4 swimming lane fragments
Size is 1350bp and 524bp；No. 5 swimming lane clip sizes are 1874bp；6~14, F2The watermelon list of red pulp is presented in generation
The digestion products of strain；15~20, swimming lane F2The digestion products of the watermelon single plant of light yellow pulp are presented in generation；Marker is
D2000marker, stripe size are from top to bottom followed successively by：2000bp、1000bp、750bp、500bp、250bp、100bp).
Fig. 2 is the PCR product 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
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 extraction of design of primers and genomic DNA
Select light yellow watermelon strain Cream of Saskatchewan (referred to as " COS ") and red watermelon strain LSW-
177 and its hybridization obtain F1, F2 for colony for experiment material verify CAPS Marker Identification watermelon flesh in whether there is tomato
Red pigment.The results are shown in Figure 1.
Two watermelon strains of COS and LSW-177 are sequenced using high throughput sequencing technologies, and are obtained by NCBI websites
The cds region sequences of lycopene in watermelon beta cyclase gene are taken, by the sequence and the comparison of sequencing material, have been obtained in the presence of west
The chromosome interval of the cds region sequences of melon lycopene beta cyclase gene, in the section, excavating sequencing data, there are SNP
The base section in mutational site, analyzes the digestion information of SNP site, obtains there are the sequence of CAPS mutation, soft using Primer6
Part there are in the sequence in CAPS sites design CAPS primers.Primer length is in 18~26bp, and annealing temperature is 56~60 DEG C, GC
Content is 40%~60%.Collection LSW-177, COS and its F1 generation of the two hybridization acquisition, F2 are for the young tender true leaf of plant, profit
Genomic DNA is extracted with the CTAB methods of improvement.
The forward primer nucleotide sequence of the base sequence of the CAPS marks is as shown in SEQ ID NO.1, reverse primer
Nucleotide sequence is as shown in SEQ ID NO.2, and PCR product clip size is 1874bp, nucleotide sequence such as SEQ ID NO.3 institutes
Show.It is specific as shown in table 1：
Table 1 is used for Primer, sequence and the expected clip size of the CAPS marks of PCR reactions
Embodiment 2：The acquisition of PCR product
PCR reactions, PCR reaction systems (20 μ as shown in table 2 are carried out using the CAPS primers and genomic DNA obtained
L)：
2 PCR reaction systems of table
Expanded using touchdown PCR (touchdown PCR, TD-PCR), program is：94 DEG C of pre-degeneration 7min, 94 DEG C
30s, 60 DEG C of annealing 30s are denatured, 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 annealing
30s, 72 DEG C of extension 90s, 10 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.
The PCR product obtained is detected using 1% agarose gel electrophoresis, testing result is as shown in Figure 1, LSW-
177th, COS and F2Single plant amplifies the fragment that a size is 1874bp, consistent with expected clip size.
Embodiment 3：Digestion verification is carried out to PCR product
According to the restriction enzyme operating guidance restriction endonuclease of Thermo, to the PCR product obtained
Digestion verification is carried out, digestion system is as shown in table 3：(15.3μL)
3 endonuclease reaction system of table
37 DEG C of water-bath digestions are stayed overnight, and digestion products are detected using 1% agarose electrophoresis, and testing result is as shown in Figure 1.
COS is since there is no restriction enzyme site, and it is still 1874bp not cut by restriction enzyme, since there are digestion in LSW-177
Site, is cut by BsaHI and obtains the fragment of 1350,524bp.In 15 plants of F for examination2For in single plant, there is 9 plants of field phenotypes
Red is shown as, and tomato red content can be detected in pulp organization, digestion products band is 1350,524bp, remaining 6
Strain shows as non-red, and can't detect the content of lycopene in pulp organization, and digestion products band is 1874bp.
Embodiment 4：The screening of germ plasm resource is carried out to the variety of watermelon of different pulp colours using the CAPS marks obtained
To differentiate applicability of the developed CAPS marks to other watermelon materials, we have chosen including LSW-177 and
The watermelon material of 9 kinds of different colours including COS, using the primer pair, it carries out PCR amplification and digestion verification.This 9 kinds of watermelons
The descriptions such as the title and color of material are as shown in table 4：
49 kinds of variety of watermelon introductions of table
The genomic DNA for extracting this 9 kinds of watermelon materials carries out PCR reactions to it, and PCR is produced using 1% agarose electrophoresis
Thing is detected, and testing result for 9 kinds of materials of examination as shown in Fig. 2, amplify the band of a 1874bp size.
Digestion verification is carried out to the PCR product obtained, acquired results are as shown in Figure 3.
It can be seen that with reference to Fig. 3 and table 3：Lycopene content, enzyme are detected in the pulp of 1~No. 6 material to be tested
Cut product and obtain the 1350, band of 524bp sizes, field phenotype is respectively red, pink colour and orange；7~No. 9 material pulp
In be not detected by lycopene, PCR product shows the band of a 1874bp size after digestion, and field phenotype is shallow
Yellow and white.
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 change and modification, therefore, guarantor of the invention
Shield scope should be subject to what claims were defined.
Claims (4)
1. it is a kind of identification watermelon flesh whether the method containing lycopene, it is characterised in that step is as follows：
1) using watermelon genomic DNA as template, pcr amplification reaction is carried out using CAPS molecular labeling primers, obtains PCR product；
The PCR product, clip size 1874bp, nucleotide sequence is as shown in SEQ ID NO.3；The CAPS molecular labeling primers
Forward primer nucleotide sequence as shown in SEQ ID NO.1, reverse primer nucleotide sequence is as shown in SEQ ID NO.2；
2) PCR product obtained using restriction enzyme to step 1) carries out endonuclease reaction；
3) digestion products obtained using agarose gel electrophoresis to step 2) are verified；PCR product can be cut open, and be
Red pulp, is yellow pulp if it cannot be cut open.
2. according to the method described in claim 1, it is characterized in that, the step 1) PCR amplification, using TD-PCR, program is：
94 DEG C of pre-degeneration 7min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 0.5 DEG C of often circulation reduction, 72 DEG C extend 90s, 30 circulations；
94 DEG C of denaturation 30s, 45 DEG C of annealing 30s, 72 DEG C of extension 90s, 10 circulate；72 DEG C of extension 10min, 4 DEG C of preservations.
3. according to the method described in claim 1, it is characterized in that, the step 3) verification, digestion products band are 1350bp
With two kinds of fragments of 524bp, contain lycopene in watermelon flesh；Digestion products band is a kind of fragments of 1874bp, watermelon flesh
In be free of lycopene.
4. application of any methods of claim 1-3 in the pulp colour with assisting sifting watermelon is identified.
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