CN108796114A - Molecular labeling primer for the gorgeous cultivar identification of Kiwi berry gold and application - Google Patents
Molecular labeling primer for the gorgeous cultivar identification of Kiwi berry gold and application Download PDFInfo
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- CN108796114A CN108796114A CN201810627265.2A CN201810627265A CN108796114A CN 108796114 A CN108796114 A CN 108796114A CN 201810627265 A CN201810627265 A CN 201810627265A CN 108796114 A CN108796114 A CN 108796114A
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Abstract
The invention belongs to molecular biology and Biotechnology in Genetic Breeding field, be more particularly to be for the molecular labeling primer and application, the primer of the gorgeous cultivar identification of Kiwi berry gold：Geo20-168-F:AAAATCTTACAAAAATATGCCGA and Geo20-168-R:TGATTTTCACGGGACTCTGC.It is expanded using the gorgeous DNA of primer pair provided by the invention gold, the only golden gorgeous band for amplifying 233bp that can be specific；According to the size and number of different bands, primer provided by the invention also can be right：' Chu Hong ', ' emerald ', ' Feng Yue ', ' your length ', ' magnificent light 2 ', ' Hua You ', ' Jin Kui ', ' Jin Mei ', ' Jin Tao ', ' gold and jade ', ' gold circle ', ' mill mountain 4 ', ' mill mountain is No. 5 male ', ' WH-1 ', ' new to see No. 2 ', ' Xu Xiang ' or ' Henan emperor's No.1 ' Kiwifruit Cultivars distinguish.
Description
Technical field
The invention belongs to molecular biology and Biotechnology in Genetic Breeding field, it is more particularly to be used for Kiwi berry ' Jin Yan ' kind
The molecular labeling primer of identification and application.
Background technology
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, up to 2,400,000 mu, 2,900,000 tons of yield occupied first place in the world national cultivated area.
In kind pattern, China is studied based on native country Kiwi berry genetic resources, and it is new to have selected a series of Kiwi berrys such as ' Jin Yan '
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 identifications of new varieties is a great problem urgently to be resolved hurrily.Currently, to Mi
The identification of monkey peach patent kind only by traditional means such as tree vigo(u)r, leaf morphology, florescence, floral shape, fruit appearances, 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 ensureing the correct of kind in agricultural production
Property has great importance.Select suitable molecular labeling and its mating detection technique, establish it is a set of easy, economical, accurate and
Efficient cultivar identification and fingerprint pattern technology system, be the fake and forged seedling of strike, 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 a kind of ideal genetic marker 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, molecular marking technique is applied to the identification of Kiwi berry male series of products, has been multiple excellent Kiwi berry product
Kind is accurate to provide pollination staminiferous plant, promotes the breeding efficiency of Kiwi berry.
' Jin Yan ' is first interspecific hybridization Huang meat new varieties cultivated for business, obtains within 2009 national kind power
(CNA20070118.5), pass through national variety certification (S-SV-AE-019-2010) within 2010.' Jin Yan ' fruit elongated cylindrical,
100~120 grams of Mean Fruit Weight, pulp yellow is fine and juicy, and savory sweet tea reaches as high as 20% containing soluble solid；Fruit goods
The frame phase is long, pole storage tolerance.' Jin Yan ' fruit composite merchandise protrudes, and has become Neihuang County of state meat Kiwi berry main breed at present.It opens
Hair is marked suitable for the Molecular Identification of Kiwi berry ' Jin Yan ' kind, is conducive to clearly define patent kind from molecular level, profit
In to Kiwifruit Cultivars carry out identification and kind protect.
Invention content
It is an object of the invention to utilize molecular marking technique, a kind of point for Kiwi berry ' Jin Yan ' cultivar identification is provided
Sub- labeled primer, the molecular labeling are named as Geo20-168, and the primer for its design is Geo20-168-F:
AAAATCTTACAAAAATATGCCGA and Geo20-168-R:TGATTTTCACGGGACTCTGC.
It is another object of the present invention to provide the molecular labeling primers for being used for Kiwi berry ' Jin Yan ' 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 ' Jin Yan ' cultivar identification：Applicant is from Kiwi berry genome sequence
Website (http://bioinfo.bti.cornell.edu/kiwi) download ' Red Male ' Kiwi berry complete genome sequence.Pass through SSR
Finder softwares 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 ' Jin Yan ' kind, screening obtains molecular labeling
Geo20-168, the primer for its design are Geo20-168-F:AAAATCTTACAAAAATATGCCGA and Geo20-168-R:
TGATTTTCACGGGACTCTGC。
Application of the molecular labeling primer in Kiwi berry breeding for Kiwi berry ' Jin Yan ' cultivar identification, including the use of this
The primer of invention is used for Kiwi berry breeding, or is identified using the kind of the primer pair Kiwi berry of the present invention, especially reflects
Determine Kiwi berry ' Jin Yan ' kind, only ' Jin Yan ' Kiwi berry expands to obtain the specific band of 233bp；Or according to stripe size sum number
Mesh, to ' Chu Hong ', ' emerald ', ' Feng Yue ', ' your length ', ' magnificent light 2 ', ' Hua You ', ' Jin Kui ', ' Jin Mei ', ' Jin Tao ', ' gold
It is beautiful ', ' gold circle ', ' mill mountain 4 ', ' mill mountain is No. 5 male ', ' WH-1 ', ' new to see No. 2 ', ' Xu Xiang ' or ' Henan emperor's No.1 ' Kiwi berry into
Row is distinguished.
Compared with prior art, advantages of the present invention：
1. morphological markers simple, intuitive, but quantity is few, polymorphism is poor, easily by environmental influence.Molecular labeling operates
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. the primer of the present invention can expand in kiwi series kind obtains different size of DNA fragmentation.The molecule mark
Note amplification stability, can be used to carry out the identification of Kiwi berry ' Jin Yan ' 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
' Jin Yan ' kind is identified in monkey peach, is expanded using molecular labeling Geo20-168 primer pairs ' Jin Yan ' DNA, it can be special
The band of anisotropic acquisition 233bp.
4. primer provided by the invention can also be according to stripe size and number, to ' Chu Hong ', ' emerald ', ' Feng Yue ', ' expensive
It is long ', ' magnificent light 2 ', ' Hua You ', ' Jin Kui ', ' Jin Mei ', ' Jin Tao ', ' gold and jade ', ' gold circle ', ' mill mountain 4 ', ' grind mountain hero 5
Number ', ' WH-1 ', ' new to see No. 2 ', ' Xu Xiang ' or ' Henan emperor's No.1 ' Kiwi berry distinguishes.
Specific implementation mode
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 ' Jin Yan ' cultivar identification：
Applicant is from Kiwi berry genome sequence website (http://bioinfo.bti.cornell.edu/kiwi) it downloads
' Red Male ' Kiwi berry complete genome sequence.By SSR Finder softwares, developing SSR marks within the scope of full-length genome, according to gene
Structure annotates as a result, selection gene loci designs SSR label primer.Using the primer of design synthesis, to Kiwi berry ' Jin Yan ' product
Kind carries out PCR amplification, and screening obtains molecular labeling Geo20-168, and the primer for its design is Geo20-168-F:
AAAATCTTACAAAAATATGCCGA and Geo20-168-R:TGATTTTCACGGGACTCTGC.
Embodiment 2：
Application of the molecular labeling primer in cultivar identification for Kiwi berry ' Jin Yan ' 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 methods
It is shown).
(2) it uses sample injector to draw the mixture of 990ul HIDI and 10ul ROX500 or LIZ500, it is anti-to be added to 96 holes
It answers in plate, per 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 with reference to following PCR reaction systems, seals 96 orifice plates using sealing plate film, vibrates,
96 orifice plates are placed in plate centrifuge, centrifugation 500g stops.PCR reactions are carried out according to following procedure.
A.PCR reaction systems
The preparation of 25 μ l systems
The primers F and primer R be：Geo20-168-F:AAAATCTTACAAAAATATGCCGA and Geo20-168-
R:TGATTTTCACGGGACTCTGC。
B.PCR reaction conditions
(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 tablet
In centrifuge, centrifugation 2000g stops.
(7) 3730xl sequencing analysis instrument (American AB I companies) is used to detect amplified fragments.
(8) Genemapper softwares are used to analyze SSR amplification datas, using STR capillary electrophoresis detection amplified fragments peak,
Abscissa is clip size, and ordinate is fluorescence intensity level.
(9) using label Geo20-168 primer amplification Kiwi berry different cultivars DNA, only ' Jin Yan ' Kiwi berry expands to obtain
The specific band of 233bp, while including to different cultivars according to different bands：' Chu Hong ', ' emerald ', ' Feng Yue ', ' your length ',
' magnificent light 2 ', ' Hua You ', ' Jin Kui ', ' Jin Mei ', ' Jin Tao ', ' gold and jade ', ' gold circle ', ' mill mountain 4 ', ' mill mountain is No. 5 male ',
' WH-1 ', ' new see No. 2 ', ' Xu Xiang ' or ' Henan emperor's No.1 ' Kiwi berry distinguishes.Specific amplification is as shown in table 1.
1 different cultivars Kiwi berry title of table and Geo20-168 primer PCR amplifications
| Variety name | Analysis result | Variety name | Analysis result |
| Bu Lunuo | 249bp | Stalwart honey | 249bp |
| River Mi 3 | 247bp、251bp | Longshan is red | 243bp |
| Chu Hong | 241bp、251bp | It is all over the sky red | 251bp |
| Emerald | 223bp | MBR | 249bp |
| East is red | 247bp、251bp | Summer millet | 249bp |
| Feng Yue | 247bp | Grind mountain 4 | 251bp、257bp |
| GT815 | 251bp | It is No. 5 male to grind mountain | 241bp、253bp |
| Your length | 263bp | Qingyuan Qiu Cui | 251bp |
| Osmanthus sea 4 | 253bp | Qin Mei | 245bp、249bp |
| H-15 | 241bp、251bp | QS-5 | 247bp、249bp |
| YX-H8 | 249bp | Logical mountain 5 | 243bp |
| Magnificent light 2 | 255bp | WH-1 | 253bp、257bp |
| Hua You | 257bp | Force plants No. 3 | 253bp |
| Hayward | 237bp、249bp | Hunan fiber crops No. 6 | 237bp、249bp |
| Jin Kui | 251bp、255bp | Tall building is No. 1 sub- | 247bp、249bp |
| Jin Mei | 241bp | It is new to see No. 2 | 229bp、243bp |
| Peasants who dig gold | 241bp、251bp | Select No. 1 in west | 245bp、249bp |
| Jin Tao | 221bp、243bp | Xu Xiang | 249bp、253bp、259bp |
| Jin Xia | 253bp | Henan emperor's No.1 | 235bp、241bp、255bp |
| Jin Yan | 233bp、241bp | It is early fresh | 253bp |
| Gold and jade | 257bp | Rivers and mountains is tender | 237bp、249bp |
| Jin Yuan | 235bp、241bp、257bp |
Sequence table
<110>Wuhan Botanical Garden, Chinese Acadmey of Sciences
<120>Molecular labeling primer for the gorgeous cultivar identification of Kiwi berry gold and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
aaaatcttac aaaaatatgc cga 23
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tgattttcac gggactctgc 20
Claims (6)
1. for the molecular labeling primer of the gorgeous cultivar identification of Kiwi berry gold, the primer is：Geo20-168-F:
AAAATCTTACAAAAATATGCCGA and Geo20-168-R:TGATTTTCACGGGACTCTGC.
2. application of the primer described in claim 1 in Kiwi berry breeding.
3. application of the primer described in claim 1 in Kiwifruit Cultivars identification.
4. application of the primer described in claim 1 in the gorgeous cultivar identification of Kiwi berry gold.
5. primer described in claim 1 is in ' Chu Hong ', ' emerald ', ' Feng Yue ', ' your length ', ' magnificent light 2 ', ' Hua You ', ' gold
Chief ', ' Jin Mei ', ' Jin Tao ', ' gold and jade ', ' gold circle ', ' mill mountain 4 ', ' mill mountain is No. 5 male ', ' WH-1 ', ' new sight 2 ', ' Xu Xiang '
Or ' the application in the identification of Henan emperor's No.1 ' Kiwifruit Cultivars.
6. a kind of kit for the gorgeous cultivar identification of Kiwi berry gold, including primer：Geo20-168-F:
AAAATCTTACAAAAATATGCCGA and Geo20-168-R:TGATTTTCACGGGACTCTGC.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101183987B1 (en) * | 2009-04-09 | 2012-09-27 | 대한민국 | SSR primer derived from Actinidia arguta and use thereof |
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-
2018
- 2018-06-19 CN CN201810627265.2A patent/CN108796114A/en active Pending
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|---|---|---|---|---|
| KR101183987B1 (en) * | 2009-04-09 | 2012-09-27 | 대한민국 | SSR primer derived from Actinidia arguta and use thereof |
| CN106498075A (en) * | 2016-11-25 | 2017-03-15 | 中国农业科学院郑州果树研究所 | Fructus actinidiae chinensiss InDel molecular markers and its screening technique and application |
| CN107326091A (en) * | 2017-08-25 | 2017-11-07 | 中国科学院武汉植物园 | Molecular labeling Geo168 primers and the application of mountain system row Male cultivar identification are ground for Kiwi berry |
| CN107338318A (en) * | 2017-08-25 | 2017-11-10 | 中国科学院武汉植物园 | Molecular labeling Geo101 primers and application for Kiwi berry mill mountain system row Male cultivar identification |
| 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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| 刘娟等: ""猕猴桃属16 个雄性材料遗传多样性的ISSR分析"", 《植物遗传资源学报》 * |
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