CN110129472A - Water lily DNA fingerprinting and its primer and construction method - Google Patents
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- 241000209490 Nymphaea Species 0.000 title claims abstract description 101
- 235000016791 Nymphaea odorata subsp odorata Nutrition 0.000 title claims abstract description 94
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- 238000001962 electrophoresis Methods 0.000 claims abstract description 6
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- 239000007795 chemical reaction product Substances 0.000 claims abstract description 4
- 230000003321 amplification Effects 0.000 claims description 8
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- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 5
- 238000004925 denaturation Methods 0.000 claims description 5
- 230000036425 denaturation Effects 0.000 claims description 5
- 241000607479 Yersinia pestis Species 0.000 claims description 3
- 230000006378 damage Effects 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 239000000975 dye Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000005498 polishing Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000001228 spectrum Methods 0.000 claims description 3
- 240000002853 Nelumbo nucifera Species 0.000 claims description 2
- 235000006508 Nelumbo nucifera Nutrition 0.000 claims description 2
- 235000006510 Nelumbo pentapetala Nutrition 0.000 claims description 2
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 230000002068 genetic effect Effects 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 7
- 230000007812 deficiency Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 41
- 230000035772 mutation Effects 0.000 description 10
- 241000894007 species Species 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 238000002372 labelling Methods 0.000 description 6
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 3
- 240000006665 Nymphaea gigantea Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000009412 basement excavation Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000209477 Nymphaeaceae Species 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000000739 chaotic effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention relates to water lily DNA fingerprinting and its primers and construction method, belong to field of biotechnology, the construction method of water lily DNA fingerprinting provided by the invention, specifically includes the following steps: 1) water lily DNA is extracted；2) ISSR-PCR amplified reaction and electrophoresis detection: being expanded using ISSR primer pair water lily DNA, and obtained ISSR-PCR reaction product is detected with 1.8% agarose gel electrophoresis；ISSR primer is using one of UBC840, UBC841, UBC811, UBC834, UBC835, UBC843, UBC844, UBC845, UBC873, UBC880 or a variety of；3) DNA fingerprinting constructs.The band that water lily ISSR-PCR reaction system established by the present invention amplifies is reproducible, and polymorphism is high, compensates for the domestic deficiency studied water lily Genetic Diversity of Germplasm and molecular linkage map.It can be used as the foundation of corresponding water lily primary germplasm identification and classification by the water lily DNA fingerprinting that this method constructs, while also providing the guarantee in molecular engineering level for the taxonomic identification and intellectual property protection of new germ plasm and new varieties.
Description
Technical field
The invention belongs to field of biotechnology, specifically, are related to water lily DNA fingerprinting and its primer and building side
Method.
Background technique
Water lily is Nymphaeceae (Nymphaeaceae) Nymphaea (Nymphaea L.) perennial herb Perennial Flowers.Water lily
Germ plasm resource is the material base and water lily research field especially water lily new varieties for developing water lily Fresh Cutting flower, edible production
The basis of breeding and biotechnology research.China is still lack of standardization in terms of the production of flower variety resource and operation at present, and kind is drawn
The phenomenon that kind is chaotic or kind is faked happens occasionally, and therefore, carrying out water lily cultivar resources identification is particularly important.Traditional sleeps
Lotus cultivar resources identification method relies primarily on phenotypic characteristic, although rapid and convenient, phenotypic character is affected by environment larger, mirror
Other error rate is higher, and water lily variety source expands year by year in addition, and the similarity of kind is also higher and higher, causes through traditional table
Type identification method is increasingly difficult to.
Though water lily is in the starting stage in China's research, development is swift and violent, and water lily research at this stage, which is concentrated mainly on, introduces a fine variety
It cultivates, on reproduction breeding, functional activity, physiological and biochemical analysis and breeding of new variety, to water lily Genetic Diversity of Germplasm
Research with molecular linkage map then rare report.
Summary of the invention
In order to solve the problems, such as background technique, the present invention provides water lily DNA fingerprinting and its primers and structure
Construction method is carried out the inhereditary feature analysis and DNA fingerprinting building of water lily germ plasm resource, is educated for water lily taxonomic identification, hybridization
Kind, the excavation of functional gene and utilization, map based cloning etc. technical support is provided, be conducive to make full use of water lily germ plasm resource and hair
It digs, formulate out the excellent water lily new germ plasm of character, water lily breeding research and innovation are had a very important significance.
To achieve the above object, the present invention is achieved through the following technical solutions:
The primer of the described acquisition water lily DNA fingerprinting, using UBC840, UBC841, UBC811, UBC834,
One of UBC835, UBC843, UBC844, UBC845, UBC873, UBC880 or a variety of.
The construction method of the water lily DNA fingerprinting, specifically includes the following steps:
1) water lily DNA is extracted；
2) it ISSR-PCR amplified reaction and electrophoresis detection: is expanded, is obtained using ISSR primer pair water lily DNA
ISSR-PCR reaction product is detected with 1.8% agarose gel electrophoresis；The ISSR primer using UBC840, UBC841,
One of UBC811, UBC834, UBC835, UBC843, UBC844, UBC845, UBC873, UBC880 or a variety of；
3) DNA fingerprinting constructs: establishing finger-print according to the stripe information that every primer is presented: with water lily germplasm money
Source number is file, is row with the site number of every bands of a spectrum；Wherein the site has amplified band to be expressed as grey, i.e. the position
It is " 1 " in point 0-1 matrix, it is colourless which does not have amplified band to be expressed as, i.e., the site is " 0 " in 0-1 matrix, obtains not
With the DNA fingerprinting table of water lily kind.
Preferably, water lily DNA is extracted using complete, no disease and pests harm, heavy water spire undeployed；Water lily DNA sample
Concentration is 50ng/uL.
Preferably, ISSR-PCR amplification reaction system are as follows: using the reaction system of 20ul, including 2 × Es Taq
Master Mix (containing dyestuff) 10uL, water lily DNA and ISSR primer each 1uL, the ddH of 8uL2O polishing.
Preferably, ISSR-PCR amplification program are as follows: (1) 94 DEG C of initial denaturation 1min, (2) 94 DEG C of denaturation 45s, (3) 46-
55 DEG C of annealing 45s, (4) 72 DEG C of extensions 2min, (5) 72 DEG C of extension 5min；Wherein step (2), (3), (4) recycle for 35 totally, and 4
DEG C save.
Preferably, annealing temperature is determined according to selected ISSR primer, specifically such as in ISSR-PCR amplification program
Under: 53 DEG C of UBC840,55 DEG C of UBC841,55 DEG C of UBC811,55 DEG C of UBC834,52 DEG C of UBC835,46 DEG C of UBC843,
53 DEG C of UBC844,52 DEG C of UBC845,46 DEG C of UBC873, UBC880 50.
24 water lily initial species and primary mutation DNA fingerprinting table such as Fig. 5 institute based on the building of UBC840 molecular labeling
Show.
24 water lily initial species and primary mutation DNA fingerprinting table such as Fig. 6 institute based on the building of UBC841 molecular labeling
Show.
The primer for obtaining water lily DNA fingerprinting is obtaining the application in water lily DNA fingerprinting.
The obtained water lily DNA fingerprinting of the construction method of the water lily DNA fingerprinting is to water lily germ plasm resource
Classification and the application in identification.
Beneficial effects of the present invention:
The primer special provided by the invention for obtaining water lily DNA fingerprinting, cooperates ISSR-PCR reaction system, is obtained
Water lily DNA fingerprinting polymorphism it is high, reproducible, convenient for identifying.The something lost that the present invention passes through development water lily germ plasm resource
Character analysis and DNA fingerprinting building are passed, is water lily taxonomic identification, crossbreeding, the excavation of functional gene and utilization, figure position
Clone etc. provides technical support, is conducive to make full use of water lily germ plasm resource and excavates, formulates out the excellent water lily novel species of character
Matter has a very important significance water lily breeding research and innovation.
Detailed description of the invention
Fig. 1 is the agarose gel electrophoresis figure of 46 parts of water lily germ plasm resource DNA；
Fig. 2 is amplification electrophoretogram of the UBC840 to 46 parts of Nymphaea germplasm genomic DNAs；
Fig. 3 is amplification electrophoretogram of the UBC841 to 46 parts of Nymphaea germplasm genomic DNAs；
Fig. 4 is genetic similarity dendrogram of 46 parts of water lily germ plasm resources based on ISSR；
Fig. 5 is the DNA fingerprinting of water lily initial species and primary mutation based on the building of UBC840 molecular labeling；
Fig. 6 is the DNA fingerprinting of water lily initial species and primary mutation based on the building of UBC841 molecular labeling.
In Fig. 1, M:DL2000bp DNA ladder marker；1-46: it indicates for trying water lily sample；
In Fig. 2, Fig. 3, M:DL2000bp DNA ladder marker；1-24,25-46: it indicates for trying Nymphaea sample.
Specific embodiment
It, below will be to preferred reality of the invention in order to keep the purpose of the present invention, technical scheme and beneficial effects clearer
It applies example to be described in detail, to facilitate the technical staff to understand.
Embodiment
The present embodiment is using 46 parts of water lily germplasm as research material, wherein contain initial species 21, primary mutation 3, gardening
Kind 22.For examination sample standard deviation plantation in flowers research institute, Guangxi Academy of Agricultural Sciences water lily Germplasm Resources.Pick complete, nothing
Pest and disease damage, heavy water spire 1-2 piece undeployed are immersed in the label bag equipped with pure water 4 DEG C of refrigerators and save, sample order and
Number is shown in Table 1.
1 46 parts of water lily germ plasm resources of table and number table
1. the extraction and detection of water lily DNA
DNA, which is extracted, uses TIANGEN plant genes group DNA extraction kit, the electrophoresis in 1% Ago-Gel
DNA mass is detected, sees Fig. 1.DNA purity and concentration are detected with Gene Spec detection of nucleic acids instrument.Finally sample concentration is diluted to
50ng/uL, -20 DEG C of preservations.
2.ISSR-PCR amplified reaction
Using the reaction system of 20ul, 2 × Es Taq Master Mix (containing dyestuff) 10uL is contained, for trying water lily mould
Plate DNA and ISSR primer each 1uL, the ddH of 8uL2O polishing.PCR amplification program are as follows: (1) 94 DEG C of initial denaturation 1min；(2)94℃
It is denaturalized 45s；(3) anneal 45s under different temperatures；(4) 72 DEG C of extension 2min；(5) 72 DEG C of extension 5min.Wherein step (2),
(3), (4) recycle for 35 totally, 4 DEG C of preservations.ISSR-PCR reaction product with 1.8% agarose gel electrophoresis, buffer be 1 ×
TAE。
The ISSR primer that 10 ISSR primers are designed and announced according to Canadian Columbia University used in the present embodiment
Sequence, by Shanghai, bioengineering Co., Ltd is synthesized.Annealing temperature is determined according to selected ISSR primer, is specifically shown in Table
2。
The amplification of 2 10 ISSR primers of table
*Note: R=(A, G), Y=(C, T)
*Note:R=(A, G), Y=(C, T)
As shown in table 2,10 ISSR primers coamplification in 46 parts of water lily germ plasm resources goes out 281 bands of a spectrum, wherein 281
All there is polymorphism, account for 100%.The clear band number that single primer amplification goes out between 23-35 item, at most draw by amplification number of sites
Object is UBC835, amplifies 35 sites, is at least UBC845, there is 23 sites, average polymorphic bands 28.1.It can be seen that
The efficiency that ISSR detects Nymphaea Genetic Diversity of Germplasm is very high, also indicates that Nymphaea germ plasm resource has on a molecular scale
There is genetic diversity extremely abundant.
Primer UBC840 is to the amplifications of 46 parts of water lily germplasm genomic DNAs shown in Fig. 2, primer UBC841 is to 46
The amplification of part water lily germplasm genomic DNA is as shown in Figure 3.
3. the ISSR clustering of water lily germ plasm resource
The mark information generated using 10 primers establishes the similarity factor matrix for sample sheet.Pass through software
NTYSYS-PC2.10e calculates genetic similarity GS (genetic similarity) to 46 parts of water lily germ plasm resources, according to GS
Value carries out clustering by non-weighting pairing arithmetic mean method (UPMGA), establishes genetic affinity dendrogram (see Fig. 4).
4. the DNA fingerprinting of water lily germ plasm resource constructs
Construct the primary germ plasm resource DNA fingerprinting of 24 portions of water lilys
The DNA fingerprinting of water lily initial species and primary mutation based on the building of UBC840 molecular labeling, is shown in Fig. 5.It is based on
The water lily initial species of UBC841 molecular labeling building and the DNA fingerprinting of primary mutation, are shown in Fig. 6.Wherein grey indicates the position
Point has band, i.e., is " 1 " in the 0-1 matrix of the site, colourless expression site is without band, i.e., the site is in 0-1 matrix
"0".Constructed finger-print can be used for the classification and identification of 24 parts of primary germ plasm resource of water lily.
Most water lily initial species and its mutation are closely similar before not blooming, are difficult to distinguish, such as blue star and white blue star,
Its blade profile, leaf color are all very much like, need Post flowering that can just distinguish.Small part is also difficult to distinguish under flowering conditions, such as Egyptian
White pond lily and its mutation pubescence tingia white pond lily, pattern are pure white, and zigzag blade is single to be difficult to distinguish from mode of appearance
It comes.The present invention passes through 10 ISSR primer pairs, the 24 water lily initial species filtered out and primary mutation constructs DNA fingerprinting,
Wherein primer UBC840 and UBC841 can individually identify 24 parts of primary germ plasm resources of water lily, other primers need multiple combinations
Effect identifies whole for trying primary germplasm water lily.The present invention is based on primer UBC840, UBC841 to construct 24 parts for trying water lily
The DNA fingerprinting of primary germplasm, the map can be used as the foundation of corresponding water lily primary germplasm identification and classification, while
The guarantee in molecular engineering level is provided for the taxonomic identification and intellectual property protection of new germ plasm and new varieties.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention rather than limits, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (8)
1. obtain water lily DNA fingerprinting primer, it is characterised in that: primer using UBC840, UBC841, UBC811,
One of UBC834, UBC835, UBC843, UBC844, UBC845, UBC873, UBC880 or a variety of.
2. the construction method of water lily DNA fingerprinting, it is characterised in that: specifically includes the following steps:
1) water lily DNA is extracted；
2) it ISSR-PCR amplified reaction and electrophoresis detection: is expanded using ISSR primer pair water lily DNA, obtained ISSR-
PCR reaction product is detected with 1.8% agarose gel electrophoresis；The ISSR primer is slept using acquisition described in claim 1
The primer of lotus DNA fingerprinting；
3) DNA fingerprinting constructs: establishing finger-print according to the stripe information that every primer is presented: with water lily germ plasm resource volume
Number it is file, is row with the site number of every bands of a spectrum；Wherein the site has amplified band to be expressed as grey, i.e. the site
It is " 1 " in 0-1 matrix, it is colourless which does not have amplified band to be expressed as, i.e., the site is " 0 " in 0-1 matrix, obtains
The DNA fingerprinting table of different water lily kinds.
3. the construction method of water lily DNA fingerprinting according to claim 2, it is characterised in that: water lily DNA, which is extracted, to be used
Completely, no disease and pests harm, heavy water spire undeployed；Water lily DNA sample concentration is 50 ng/uL.
4. the construction method of water lily DNA fingerprinting according to claim 2, it is characterised in that: ISSR-PCR amplification is anti-
Answer system are as follows: using the reaction system of 20ul, including 2 × Es Taq Master Mix(contains dyestuff) 10 uL, water lily DNA and
ISSR primer each 1 uL, the ddH of 8 uL2O polishing.
5. according to the construction method of the described in any item water lily DNA fingerprintings of claim 2-4, it is characterised in that: ISSR-
PCR amplification program are as follows: (1) 94 DEG C of initial denaturation 1 min, (2) 94 DEG C of denaturation 45s, 46-55 DEG C of (3) annealing 45s, (4) 72 DEG C
Extend 2 min, (5) 72 DEG C of 5 min of extension；Wherein step (2), (3), (4) recycle for 35 totally, 4 DEG C of preservations.
6. according to the construction method of the described in any item water lily DNA fingerprintings of claim 5, it is characterised in that: ISSR-PCR
In amplification program, annealing temperature is determined according to selected ISSR primer, specific as follows: 53 DEG C of UBC840, UBC841 55
DEG C, 55 DEG C of UBC811,55 DEG C of UBC834,52 DEG C of UBC835,46 DEG C of UBC843,53 DEG C of UBC844,52 DEG C of UBC845,
46 DEG C of UBC873, UBC880 50.
7. the primer according to claim 1 for obtaining water lily DNA fingerprinting is obtaining answering in water lily DNA fingerprinting
With.
8. according to the obtained water lily DNA fingerprint of construction method of the described in any item water lily DNA fingerprintings of claim 2-6
Map is classified and the application in identification to water lily germ plasm resource.
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| CN113832254A (en) * | 2021-11-12 | 2021-12-24 | 广西壮族自治区农业科学院 | SSR primer pair of water lily, synthetic method and application |
| CN113832254B (en) * | 2021-11-12 | 2023-11-14 | 广西壮族自治区农业科学院 | SSR primer pair of water lily, and synthetic method and application thereof |
| CN114766348A (en) * | 2022-05-17 | 2022-07-22 | 中国科学院昆明植物研究所 | Method for identifying two parents of water lily hybrid based on ITS sequence and matK sequence |
| CN116287380A (en) * | 2023-02-01 | 2023-06-23 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | SRAP molecular marker primer group for identifying water lily varieties or detecting genetic diversity of water lily germplasm resources and application thereof |
| CN116287380B (en) * | 2023-02-01 | 2024-04-26 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | SRAP molecular marker primer group for identifying water lily varieties or detecting genetic diversity of water lily germplasm resources and application thereof |
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