CN108796113B - Molecular labeling primer and application for the red cultivar identification in Kiwi berry east - Google Patents
Molecular labeling primer and application for the red cultivar identification in Kiwi berry east Download PDFInfo
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- CN108796113B CN108796113B CN201810627233.2A CN201810627233A CN108796113B CN 108796113 B CN108796113 B CN 108796113B CN 201810627233 A CN201810627233 A CN 201810627233A CN 108796113 B CN108796113 B CN 108796113B
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Abstract
The invention belongs to molecular biology and Biotechnology in Genetic Breeding fields, it is more particularly to the molecular labeling primer and application for Kiwi berry ' east is red ' cultivar identification, the primer are as follows: Geo19-159-F:TGATTCGTTTTGACACAATGG and Geo19-159-R:TGCAGGCTATGGACTGAGTG.It is expanded using the red DNA in primer pair east provided by the invention, only the eastern red band for amplifying 280bp and 282bp that can be specific；, can also be right according to the size and number of different bands: Chu Hong, GT815, H-15, Hua Guang No. 2 numbers, Hua You, Hayward, Longshan is red, it is all over the sky it is red, mill mountain is No. 5 male, Qin Mei, QS-5, logical mountain 5, sub- No. 1 Kiwi berry of WH-1 or tall building distinguish.
Description
Technical field
The invention belongs to molecular biology and Biotechnology in Genetic Breeding field, it is more particularly to for Kiwi berry ' east is red ' kind
The molecular labeling primer and application of identification.
Background technique
China is the source area of actinidia and the original centre of origin of Kiwi berry genetic resources.In recent years, China
Kiwi berry industry expands steadily, and by 2017, national cultivated area 2,900,000 tons of yield, occupied first place in the world up to 2,400,000 mu.
In kind pattern, China is studied based on native country Kiwi berry genetic resources, and it is new to have selected the Kiwi berrys such as ' Jin Yan ', ' east is red '
Kind, cultivation extensively, revolutionizes the single pattern of world's Kiwi berry industry only ' Hayward ' kind in the world, pushes
The diversification of Kiwi berry market diversification and consumption.
With the increasingly release of Kiwi berry new varieties, the identification of new varieties is a great problem urgently to be resolved.Currently, to Mi
The identification of monkey peach patent kind only passes through the traditional means such as tree vigo(u)r, leaf morphology, florescence, floral shape, fruit appearance, inevitably by
The influence of environment and other human factors.To the uncertainty of Kiwifruit Cultivars, the healthy and sustainable development of industry, and seedling are limited
The wooden kind is impure or even wrong orchard worker's economic benefit will be caused to be badly damaged.
Molecular marking technique can quickly, it is accurate, efficiently kind is identified, to guaranteeing the correct of kind in agricultural production
Property has great importance.The suitable molecular labeling of selection and its mating detection technique, establish it is a set of easy, economical, accurate and
Efficient cultivar identification and fingerprint pattern technology system, be hit fake and forged seedling, protection plant genetics and breeding worker and
The legitimate rights and interests of peasant, specification China agricultural, commodities market there is an urgent need to.
The variation that molecular marking technique is generated in the genome by detecting bion, to carry out area to Different Individual
Point, it is the ideal genetic marker of one kind of development after morphological markers and biochemical marker.It is identified in plant germplasm resource
And in breeding, reach its maturity to the application of molecular labeling.SSR molecular marker quantity is abundant and is uniformly distributed in gene at random
Group is in codominance, and allelic variation is high, and easy to operate and stability is good, has been applied to the cultivar identifications sides such as soybean, apple and grape
Face.In Kiwi berry, it has been multiple excellent Kiwi berry product that molecular marking technique is applied to the identification of Kiwi berry male series of products
Kind is accurate to provide pollination staminiferous plant, promotes the breeding efficiency of Kiwi berry.
' east red ' Kiwi berry single fruit weight is at 80-130 grams, and fruit is short cylindrical shape, the hairless green and brown color of pericarp, pulp succulence,
It is sour-sweet moderate.Total sugar content is up to 13.45 ﹪, and total acid only has 0.49 ﹪, 16.5 ﹪ of soluble solid, is rich in calcium, iron, potassium
Etc. several mineral materials and 17 kinds of amino acid.Exploitation is suitable for the Molecular Identification label of Kiwi berry ' east red ' kind, be conducive to from point
Patent kind is clearly defined in sub- level, carries out identification and kind protection conducive to Kiwifruit Cultivars.
Summary of the invention
It is an object of the invention to utilize molecular marking technique, a kind of point for Kiwi berry ' east is red ' cultivar identification is provided
Sub- labeled primer, the molecular labeling are named as Geo19-159, and the primer for its design is Geo19-159-F:
TGATTCGTTTTGACACAATGG and Geo19-159-R:TGCAGGCTATGGACTGAGTG.
It is another object of the present invention to provide the molecular labeling primers for being used for Kiwi berry ' east is red ' cultivar identification to exist
Application in Kiwi berry breeding.
In order to achieve the above object, the present invention takes following technical measures:
The acquisition of molecular labeling primer for Kiwi berry ' east red ' cultivar identification: applicant is from Kiwi berry genome sequence
Download ' Red Male ' Kiwi berry complete genome sequence in website (http://bioinfo.bti.cornell.edu/kiwi).Pass through SSR
Finder software developing SSR within the scope of full-length genome marks, and is annotated according to gene structure as a result, selecting gene loci design
SSR label primer.Using the primer of design synthesis, PCR amplification is carried out to Kiwi berry ' east is red ' kind, screening obtains molecular labeling
Geo19-159, the primer for its design is Geo19-159-F:TGATTCGTTTTGACACAATGG and Geo19-159-R:
TGCAGGCTATGGACTGAGTG。
Application of the molecular labeling primer in Kiwi berry breeding for Kiwi berry ' east is red ' cultivar identification, including the use of this
The primer of invention is used for Kiwi berry breeding, or utilizes primer pair Kiwifruit Cultivars of the invention, especially ' east red ' kind into
Row identification, only ' east red ' Kiwi berry can specificity expand 280bp and 282bp segment, or according to the big of different bands
Small and number includes: ' Chu Hong ', ' GT815 ', ' H-15 ', ' magnificent light 2 ', ' Hua You ', ' Hayward ', ' Longshan to different cultivars
Red ', ' is all over the sky red ', ' mill mountain is No. 5 male ', ' Qin Mei ', ' QS-5 ', ' logical mountain 5 ', ' WH-1 ' or ' tall building is No. 1 sub- ' Kiwi berry carry out
It distinguishes.
Compared with prior art, advantages of the present invention:
1. morphological markers simple, intuitive, but quantity is few, polymorphism is poor, vulnerable to environmental influence.Molecular labeling operation
Simplicity, it is detectable in each tissue and developmental stage of plant, not by season and environmental restrictions.Quantity is more, throughout entire
Genome.Polymorphism is high, and there are many allelic variations between kind.
2. primer of the invention can expand in kiwi series kind and obtain different size of DNA fragmentation.The molecule mark
Note amplification stability can be used to carry out the identification of Kiwi berry ' east is red ' kind.
3. molecular labeling primer specificity provided by the invention is high, applied widely, can be from the Mi of 40 multiple and different kinds
' east is red ' kind is identified in monkey peach, is expanded using molecular labeling Geo19-159 primer pair ' east is red ' DNA, it is only eastern
The red band for amplifying 280bp and 282bp that can be specific.
4. in addition to identifying ' east red ' kind according to specific band, can also according to stripe size and number to ' Chu Hong ',
' GT815 ', ' H-15 ', ' magnificent light 2 ', ' Hua You ', ' Hayward ', ' Longshan is red ', ' all over the sky red ', ' mill mountain is No. 5 male ', ' Qin
The logical mountain 5 of beauty ', ' QS-5 ', ' ', ' WH-1 ' or ' tall building is No. 1 sub- ' Kiwi berry distinguish.
Specific embodiment
Technical solution of the present invention is if not otherwise specified the conventional scheme of this field；The reagent or material,
If not otherwise specified, business or disclosed channel are derived from.
Embodiment 1:
The acquisition of molecular labeling primer for Kiwi berry ' east is red ' cultivar identification:
Applicant downloads from Kiwi berry genome sequence website (http://bioinfo.bti.cornell.edu/kiwi)
' Red Male ' Kiwi berry complete genome sequence.By SSR Finder software, developing SSR is marked within the scope of full-length genome, according to gene
Structure annotation is as a result, selection gene loci designs SSR label primer.Using the primer of design synthesis, to Kiwi berry ' east is red ' product
Kind carries out PCR amplification, and screening obtains molecular labeling Geo19-159, and the primer for its design is Geo19-159-F:
TGATTCGTTTTGACACAATGG and Geo19-159-R:TGCAGGCTATGGACTGAGTG.
Embodiment 2:
Application of the molecular labeling primer in cultivar identification for Kiwi berry ' east is red ' cultivar identification:
(1) DNA sample (the specific variety name such as table 1 of 40 multiple and different kind Kiwi berrys is extracted respectively according to CTAB method
It is shown).
(2) mixture that 990ul HIDI and 10ul ROX500 or LIZ500 are drawn using sample injector, it is anti-to be added to 96 holes
It answers in plate, every hole 10ul.
(3) 96 orifice plates are placed in plate centrifuge, centrifugation 500g stops.
(4) each sample DNA, each sample 50pg are separately added into the corresponding hole of 96 orifice plates.
(5) corresponding reagent is added to 96 orifice plates referring to following PCR reaction system, seals 96 orifice plates using sealing plate film, vibrates,
96 orifice plates are placed in plate centrifuge, centrifugation 500g stops.PCR reaction is carried out according to following procedure.
A.PCR reaction system
The preparation of 25 μ l systems
The primers F and primer R are as follows: Geo19-159-F:TGATTCGTTTTGACACAATGG and Geo19-159-R:
TGCAGGCTATGGACTGAGTG。
B.PCR reaction condition
(6) 96 orifice plates are placed in rapid cooling on mixture of ice and water immediately after EP (end of program), then 96 orifice plates is placed in plate
In centrifuge, centrifugation 2000g stops.
(7) amplified fragments are detected using 3730xl sequencing analysis instrument (American AB I company).
(8) SSR amplification data is analyzed using Genemapper software, using STR capillary electrophoresis detection amplified fragments peak,
Abscissa is clip size, and ordinate is fluorescence intensity level.
(9) Kiwifruit Cultivars are expanded using label Geo19-159, only ' east is red ' Kiwi berry expands to obtain 280bp and 282bp
Specific band, while according to the size and number of different bands, to different cultivars include: ' Chu Hong ', ' GT815 ', ' H-15 ',
' magnificent light 2 ', ' Hua You ', ' Hayward ', ' Longshan is red ', ' all over the sky red ', ' mill mountain is No. 5 male ', ' Qin Mei ', ' QS-5 ', ' logical mountain 5
Number ', ' WH-1 ' or ' tall building is No. 1 sub- ' Kiwi berry distinguishes.Specific amplification is as shown in table 1.
1 different cultivars Kiwi berry title of table and Geo19-159 primer PCR amplification
| Variety name | Analyze result | Variety name | Analyze result |
| Bu Lunuo | 276bp | Stalwart honey | 288bp |
| River Mi 3 | 288bp | Longshan is red | 292bp |
| Chu Hong | 274bp、276bp | It is all over the sky red | 278bp、282bp |
| Emerald | 276bp | MBR | 288bp、290bp |
| East is red | 280bp、282bp | Summer millet | 288bp |
| Feng Yue | 288bp | Grind mountain 4 | 276bp、284bp |
| GT815 | 274bp、276bp、282bp | It is No. 5 male to grind mountain | 286bp、290bp |
| Your length | 276bp、282bp | Qingyuan Qiu Cui | 288bp |
| Osmanthus sea 4 | 284bp、288bp | Qin Mei | 278bp、286bp、288bp |
| H-15 | 278bp、280bp | QS-5 | 288bp、298bp |
| YX-H8 | 288bp | Logical mountain 5 | 278bp |
| Magnificent light 2 | 288bp、292bp | WH-1 | 286bp |
| Hua You | 276bp、288bp | Force plants No. 3 | 288bp |
| Hayward | 274bp | Hunan fiber crops No. 6 | 276bp |
| Jin Kui | 278bp、286bp | Tall building is No. 1 sub- | 276bp、282bp、284bp |
| Jin Mei | 274bp、280bp | It is new to see No. 2 | 284bp、288bp |
| Peasants who dig gold | 276bp、282bp | Select No. 1 in west | 278bp、286bp |
| Jin Tao | 274bp、280bp | Xu Xiang | 288bp |
| Jin Xia | 288bp | Henan emperor's No.1 | 276bp、284bp |
| Jin Yan | 274bp、280bp | It is early fresh | 288bp、290bp |
| Gold and jade | 288bp | Rivers and mountains is tender | 276bp |
| Jin Yuan | 274bp、280bp |
Sequence table
<110>Wuhan Botanical Garden, Chinese Acadmey of Sciences
<120>molecular labeling primer and application for the red cultivar identification in Kiwi berry east
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tgattcgttt tgacacaatg g 21
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tgcaggctat ggactgagtg 20
Claims (4)
1. for the molecular labeling primer of the red cultivar identification in Kiwi berry east, the primer are as follows: Geo19-159-F:
TGATTCGTTTTGACACAATGG and Geo19-159-R:TGCAGGCTATGGACTGAGTG.
2. application of the primer described in claim 1 in Kiwi berry breeding, the Kiwi berry be red east, Chu Hong, GT815,
H-15, Hua Guang No. 2 numbers, Hua You, Hayward, Longshan is red, it is all over the sky it is red, mill mountain is No. 5 male, Qin Mei, QS-5, logical mountain 5, WH-1 or tall building it is sub-
No. 1.
3. application of the primer described in claim 1 in Kiwifruit Cultivars identification, the Kiwi berry be the red, Chu Hong in east,
GT815, H-15, Hua Guang No. 2 numbers, Hua You, Hayward, Longshan is red, it is all over the sky it is red, mill mountain is No. 5 male, Qin Mei, QS-5, logical mountain 5, WH-1
Or tall building is No. 1 sub-.
4. a kind of kit for the red cultivar identification in Kiwi berry east, including primer: Geo19-159-F:
TGATTCGTTTTGACACAATGG and Geo19-159-R:TGCAGGCTATGGACTGAGTG.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107326081A (en) * | 2017-07-26 | 2017-11-07 | 中国农业科学院特产研究所 | A kind of authentication method of Germplasm Resources in Genera Actinidia and its application |
| CN107385071A (en) * | 2017-08-25 | 2017-11-24 | 中国科学院武汉植物园 | Molecular labeling primer and application for Kiwi berry mill mountain system row Male cultivar identification |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107326081A (en) * | 2017-07-26 | 2017-11-07 | 中国农业科学院特产研究所 | A kind of authentication method of Germplasm Resources in Genera Actinidia and its application |
| CN107385071A (en) * | 2017-08-25 | 2017-11-24 | 中国科学院武汉植物园 | Molecular labeling primer and application for Kiwi berry mill mountain system row Male cultivar identification |
Non-Patent Citations (3)
| Title |
|---|
| Development and Application of Transcriptome-Derived Microsatellites in Actinidia eriantha (Actinidiaceae);Rui Guo等;《Frontiers in Plant Science》;20170825;第8卷;Article 1383，第1-13页 * |
| 猕猴桃种间高密度遗传图谱的构建及果实性状QTLs定位;刘春燕;《中国博士学位论文全文数据库 农业科技辑》;20160915(第09期);D048-7，参见全文 * |
| 红心猕猴桃新品种‘东红’的选育;钟彩虹等;《果树学报》;20161231;第33卷(第12期);第1596-1599页，参见全文，尤其是摘要、第1597左栏第2段-第1599页左栏第6段 * |
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