CN107326091A - Molecular labeling Geo168 primers and the application of mountain system row Male cultivar identification are ground for Kiwi berry - Google Patents

Molecular labeling Geo168 primers and the application of mountain system row Male cultivar identification are ground for Kiwi berry Download PDF

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CN107326091A
CN107326091A CN201710740421.1A CN201710740421A CN107326091A CN 107326091 A CN107326091 A CN 107326091A CN 201710740421 A CN201710740421 A CN 201710740421A CN 107326091 A CN107326091 A CN 107326091A
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mountain
male
kiwi berry
geo168
mill
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CN107326091B (en
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钟彩虹
黄宏文
张琼
张鹏
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Wuhan Botanical Garden of CAS
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Abstract

The invention belongs to molecular biology and Biotechnology in Genetic Breeding field, it is more particularly to grind molecular labeling Geo168 primers and the application of mountain system row Male cultivar identification for Kiwi berry.The molecular labeling primer that the present invention is provided is Geo168 F:AAAATCTTACAAAAATATGCCGA and Geo168 R:TGATTTTCACGGGACTCTGC, using the primer, can accurately distinguish identification Kiwi berry mill mountain system row Male cultivar, can also distinguish other kinds in addition to mountain system row Male cultivar is ground, possess the universality of identification, be adapted to large-scale promotion.

Description

For Kiwi berry grind the identification of mountain system row Male cultivars molecular labeling Geo168 primers and Using
Technical field
The invention belongs to molecular biology and Biotechnology in Genetic Breeding field, it is more particularly to be used for Kiwi berry mill mountain system row hero The molecular labeling Geo168 primers of property cultivar identification and application.
Background technology
In recent years, China's Kiwi berry industry is expanded steadily, and by 2016, national cultivated area was up to 2,200,000 mu, yield 260 Ten thousand tons, the output value is more than 20,000,000,000 yuan.Actinidia pollen abortion in dioecism type, i.e. female flower, and ovary development in male flower Incomplete and ovule abortion.Therefore, in Kiwi berry breeding and the cultivation of business orchard, the pollen of excellent staminiferous plant need to be generally selected, Cross-pollination is carried out, could realize that Kiwi berry is solid.
In recent years, the serial Kiwifruit Cultivars of ' mill mountain ' male of Wuhan Botanical Garden, Chinese Acadmey of Sciences seed selection (' grind mountain hero 1 Number ', ' mill mountain is male No. 2 ', ' mill mountain is male No. 3 ', ' mill mountain 4 ' and ' mill mountain is male No. 5 '), with florescence covering is wide, flower amount is big, spend The features such as powder germination rate is high, it has also become the supporting pollination staminiferous plant of multiple excellent Kiwifruit Cultivars.Such as ' mill mountain is male No. 2 ' Kiwi berry Chinese gooseberry pollination that can not only be to early florescence and middle florescence, and can promote the serial kiwifruit fruit quality improving of red heart, Increase endocarp red accumulation.At present, the identification to ' mill mountain ' series of products only passes through tree vigo(u)r, leaf morphology, florescence and flower-shape The traditional means such as state, are influenceed by environment and other human factors unavoidably.The uncertainty identified ' mill mountain ' series of products, Cause bigger difficulty to Kiwi berry breeding work, and mix or mistake Male cultivar also inherently the production of influence fruit and Fruit quality.
The application of molecular marking technique provides objective and accurate means for cultivar identification.Molecular marking technique passes through inspection The variation that bion produces on genome is surveyed, is after morphological markers and biochemical marker so as to be made a distinction to Different Individual Afterwards, a kind of ideal genetic marker of development.In plant germplasm resource identification and breeding, the application to molecular labeling Reach its maturity.RAPD molecular labelings polymorphic frequency is high, high specificity, is widely used in the plant of the grass family such as wheat, paddy rice and corn The Germplasm Identification of thing.SSR molecular marker allelic variation is high and stability is good, is reflected applied to resources such as soybean, apple and grapes Fixed research.SNP marker polymorphism is high and distribution is wide, is appropriate for cultivar identification and sort research.In Kiwi berry, open Send out SNP marker and marker assisted selection is carried out to Main Agronomic Characters such as kiwi sugar, acid and fruit sizes.However, right The molecular markers for identification research of Kiwi berry male series of products is not yet carried out.
Exploitation is marked suitable for the Molecular Identification of male rhesus macaque Peach cultivars, can targetedly match parent sire of hybrid pigs, favorably In improving the breeding efficiency to Kiwi berry, Kiwi berry industrial economy benefit is lifted.
The content of the invention
It is an object of the invention to utilize molecular marking technique to be used for Kiwi berry mill mountain system row Male cultivar there is provided one kind to reflect Fixed molecular labeling Geo168 primers, described molecular labeling is named as Geo168, and the primer for its design is Geo168-F: AAAATCTTACAAAAATATGCCGA and Geo168-R:TGATTTTCACGGGACTCTGC.
It is another object of the present invention to provide be used for the molecular labeling that Kiwi berry grinds the identification of mountain system row Male cultivar Application of the Geo168 primers in Kiwi berry breeding.
In order to achieve the above object, the present invention takes following technical measures：
The acquisition of the molecular labeling Geo168 primers of mountain system row Male cultivar identification is ground for Kiwi berry：Applicant is from macaque Peach genome sequence website (http://bioinfo.bti.cornell.edu/kiwi) download ' Red Male ' Kiwi berry full genome sequence Row.Different repeat units SSR sites are excavated in the range of full-length genome, gene structure annotation result is compared, selects in gene regions Between SSR sites design amplimer.Using the primer of design synthesis, performing PCR amplification is entered to Kiwi berry ' mill mountain ' series of products, Screening obtains molecular labeling Geo168, and the primer for its design is Geo168-F:AAAATCTTACAAAAATATGCCGA and Geo168-R:TGATTTTCACGGGACTCTGC。
The molecular labeling Geo168 primers of mountain system row Male cultivar identification answering in Kiwi berry breeding is ground for Kiwi berry With being identified that the kind of the Kiwi berry includes but is not limited to using the kind of primer pair Kiwi berry of the present invention：' mill mountain Male No. 1 ', ' mill mountain is male No. 2 ', ' mill mountain is male No. 3 ', ' mill mountain 4 ', ' mill mountain is male No. 5 ', ' Chu Hong ', ' magnificent light 2 ', ' select 1 in west Number ' or ' Hua You ' Kiwi berry.
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 is operated Simplicity, in each tissue and developmental stage of plant, can detect, is not limited by season and environment.Quantity is more, throughout whole Genome.Polymorphism is high, there are many allelic variations between kind.
2. the primer of the present invention amplification can obtain different size of DNA pieces in the male series of products of Kiwi berry ' mill mountain ' Section.The molecular labeling expands stability, can be used for the identification for carrying out the male series of products of Kiwi berry ' mill mountain '.
3. the primer that the present invention is provided can distinguish other kinds in addition to ' mill mountain ' serial Male cultivar, possesses identification Universality, is adapted to large-scale promotion.
Brief description of the drawings
Fig. 1 is that Capillary Electrophoresis is expanded in the male series of products of Kiwi berry ' mill mountain ' using molecular labeling Geo168 primers Obtain the peak figure of different DNA fragmentations.
Embodiment
Technical scheme of the present invention, is the conventional scheme of this area if not otherwise specified；The reagent or material, If not otherwise specified, business or disclosed channel are derived from.
Embodiment 1：
The acquisition of the molecular labeling Geo168 primers of mountain system row Male cultivar identification is ground for Kiwi berry：
Applicant is from Kiwi berry genome sequence website (http://bioinfo.bti.cornell.edu/kiwi) download ' Red Male ' Kiwi berry complete genome sequence.Different repeat units SSR sites are excavated in the range of full-length genome, gene structure note is compared Result is released, selects to design amplimer in the SSR sites of gene interval.Using the primer of design synthesis, to Kiwi berry ' mill mountain ' Series of products enters performing PCR amplification, and screening obtains molecular labeling Geo168, and the primer for its design is Geo168-F: AAAATCTTACAAAAATATGCCGA and Geo168-R:TGATTTTCACGGGACTCTGC.
Embodiment 2：
The molecular labeling Geo168 primers for grinding the identification of mountain system row Male cultivar for Kiwi berry are differentiating Kiwi berry mill mountain product Application in kind：
(1) extract respectively according to CTAB methods ' mill mountain is male No. 1 ', ' mill mountain is male No. 2 ', ' mill mountain is male No. 3 ', ' mill mountain 4 ' and The DNA sample of ' mill mountain is male No. 5 ' Kiwi berry.
(2) 990ul HIDI and 10ul ROX500 or LIZ500 mixture are drawn using sample injector, 96 holes are added to anti- Answer in plate, per hole 10ul.
(3) 96 orifice plates are placed in plate centrifuge, centrifugation 500g is to stop.
(4) the male serial sample DNA in 5 mill mountains, 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, seal 96 orifice plates using shrouding film, vibrate, 96 orifice plates are placed in plate centrifuge, centrifugation 500g is to stop.Enter performing PCR reaction according to following procedure.
A.PCR reaction systems
The preparation of 25 μ l systems
Template (μ l) 1
Primers F (μ l) 0.5
Primer R (μ l) 0.5
dNTP 10mM(μl) 0.5
Taq Buffer(μl) 2.5
25mM MgCl2(μl) 2.0
Taq enzyme (μ l) 5U/ μ l 0.2
Water (μ l) 17.8
Described primers F and primer R be：Geo168-F:AAAATCTTACAAAAATATGCCGA and Geo168-R: TGATTT TCACGGGACTCTGC。
B.PCR reaction conditions
(6) 96 orifice plates are placed on mixture of ice and water immediately after EP (end of program) and rapidly cooled down, then 96 orifice plates are placed in flat board In centrifuge, centrifugation 2000g is to stop.
(7) using 3730xl sequencing analysis instrument (American AB I companies) detection amplified fragments.
(8) Genemapper software analysis SSR amplification datas are used, using STR capillary electrophoresis detection amplified fragments peak, Abscissa is clip size, and ordinate is fluorescence intensity level.
(9) using Geo168 amplification mill mountain series of products is marked, male No. 1 mill mountain, mill mountain hero 2, mill can specific be distinguished Mountain is male No. 3, grind mountain 4 and mill mountain No. 5 (Fig. 1) of hero.As a result show, ' mill mountain is male No. 1 ' amplification obtains 2 bands, respectively 225bp And 247bp；' mill mountain is male No. 2 ' amplification obtains 2 bands, respectively 241bp and 249bp；' mill mountain is male No. 3 ' amplification obtains 1 band, For 261bp；' mill mountain 4 ' amplification obtains 2 bands, respectively 251bp and 257bp；' mill mountain is male No. 5 ' amplification obtains 2 bands, point Other 241bp and 253bp.
Embodiment 3：
Molecular labeling Geo168 primers answering in Kiwifruit Cultivars are differentiated for Kiwi berry male mill mountain cultivar identification With：
Extract ' mill mountain male No. 1 ', ' mill mountain is male No. 2 ', ' mill mountain is male No. 3 ', ' mill mountain 4 ', ' mill mountain is male No. 5 ', ' Chu Hong ', The DNA sample of ' magnificent light 2 ', ' selecting No. 1 in west ' or ' Hua You ' Kiwi berry, using the method for embodiment 2, utilizes Geo168-F: AAAATCTTACAAAAATATGCCGA and Geo168-R:TGATTTTCACGGGACTCTGC.Molecular labeling is carried out to above-mentioned kind Identification.
Using marking Geo168 amplification mill mountain series of products, can specificity distinguish ' mill mountain male No. 1 ', ' mill mountain is male No. 2 ', ' mill mountain is male No. 3 ', ' mill mountain 4 ', ' mill mountain is male No. 5 ', ' Chu Hong ', ' magnificent light 2 ', ' selecting No. 1 in west ' or ' Hua You ' Kiwi berry.Knot Fruit shows that ' mill mountain is male No. 1 ' amplification obtains 2 bands, respectively 225bp and 247bp；' mill mountain is male No. 2 ' amplification obtains 2 bands, Respectively 241bp and 249bp；' mill mountain is male No. 3 ' amplification obtains 1 band, is 261bp；' mill mountain 4 ' amplification obtains 2 bands, point Wei not 251bp and 257bp；' mill mountain is male No. 5 ' amplification obtains 2 bands, respectively 241bp and 253bp；' Chu Hong ' amplification obtains 2 Band, respectively 241bp and 251bp；' magnificent light 2 ' amplification obtains 1 band, is 255bp；' selecting No. 1 in west ' amplification obtains 2 bands, Respectively 245bp and 249bp；' Hua You ' amplification obtains 1 band, is 257bp.
Sequence table
<110>Wuhan Botanical Garden, Chinese Acadmey of Sciences
<120>Molecular labeling Geo168 primers and the application of mountain system row Male cultivar identification are ground for Kiwi berry
<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 (5)

1. a kind of Kiwi berry mill mountain system row Male cultivar identification molecular labeling primer, described primer：Geo168-F: AAAATCTTACAAAAATATGCCGA and Geo168-R:TGATTTTCACGGGACTCTGC.
2. application of the primer in Kiwi berry breeding described in claim 1.
3. application of the primer in Kiwifruit Cultivars identification described in claim 1.
4. application of the primer in Kiwi berry grinds the identification of mountain system row Male cultivar described in claim 1.
5. application according to claim 3, described Kiwifruit Cultivars include：Grind mountain hero 1, mill mountain is male No. 2, it is male to grind mountain No. 3, mill mountain 4, male No. 5 of mill mountain, Chu Hong, magnificent light 2, west select No. 1 or magnificent excellent Kiwi berry.
CN201710740421.1A 2017-08-25 2017-08-25 Molecular marker Geo168 primer for identifying male varieties of kiwi fruit Moshan series and application Active CN107326091B (en)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN108796114A (en) * 2018-06-19 2018-11-13 中国科学院武汉植物园 Molecular labeling primer for the gorgeous cultivar identification of Kiwi berry gold and application
CN108796115A (en) * 2018-06-19 2018-11-13 中国科学院武汉植物园 Molecular labeling primer for Kiwi berry gold and jade cultivar identification and application
CN109055597A (en) * 2018-09-27 2018-12-21 中国科学院武汉植物园 Molecular labeling primer and application for No. 1 cultivar identification of Kiwi berry Mi jujube
CN109055600A (en) * 2018-09-30 2018-12-21 中国科学院武汉植物园 Expire the molecular labeling primer and application of Tianhong No.2 cultivar identification for Kiwi berry

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108796114A (en) * 2018-06-19 2018-11-13 中国科学院武汉植物园 Molecular labeling primer for the gorgeous cultivar identification of Kiwi berry gold and application
CN108796115A (en) * 2018-06-19 2018-11-13 中国科学院武汉植物园 Molecular labeling primer for Kiwi berry gold and jade cultivar identification and application
CN108796115B (en) * 2018-06-19 2021-06-11 中国科学院武汉植物园 Molecular marker primer for identifying kiwi fruit Jinyu variety and application thereof
CN109055597A (en) * 2018-09-27 2018-12-21 中国科学院武汉植物园 Molecular labeling primer and application for No. 1 cultivar identification of Kiwi berry Mi jujube
CN109055597B (en) * 2018-09-27 2021-08-03 中国科学院武汉植物园 Molecular marker primer for identifying kiwi fruit and kiwi fruit variety No. 1 and application
CN109055600A (en) * 2018-09-30 2018-12-21 中国科学院武汉植物园 Expire the molecular labeling primer and application of Tianhong No.2 cultivar identification for Kiwi berry
CN109055600B (en) * 2018-09-30 2021-05-04 中国科学院武汉植物园 Molecular marker primer for identifying variety No. 2 of kiwi fruit Hongtianhong and application thereof

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