CN109055597B - Molecular marker primer for identifying kiwi fruit and kiwi fruit variety No. 1 and application - Google Patents

Molecular marker primer for identifying kiwi fruit and kiwi fruit variety No. 1 and application Download PDF

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CN109055597B
CN109055597B CN201811133105.9A CN201811133105A CN109055597B CN 109055597 B CN109055597 B CN 109055597B CN 201811133105 A CN201811133105 A CN 201811133105A CN 109055597 B CN109055597 B CN 109055597B
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kiwi fruit
variety
primer
kiwi
molecular marker
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CN109055597A (en
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张琼
钟彩虹
郑浩
刘小莉
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Wuhan Botanical Garden of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a molecular marker primer for identifying kiwi fruit' kiwi fruit 1 # variety and an application thereof, wherein the primer comprises the following components: geo29-201-F: GGAAAGGTCGAAACCATAAGA and Geo29-201-R: TTTTTCAGAAATCCAAAAGAAAAA. The primer provided by the invention is used for amplifying the Chinese gooseberry No. 1 DNA, and only the Chinese gooseberry No. 1 can specifically amplify a 302bp strip; depending on the size and number of different strips, it is also possible to pair: the kiwi fruits of 'Miliang No. 1' and 'Jiangshan jiao' are distinguished.

Description

Molecular marker primer for identifying kiwi fruit and kiwi fruit variety No. 1 and application
Technical Field
The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a molecular marker primer for identifying kiwi fruit and kiwi fruit jujube No. 1 varieties and application thereof.
Background
China is the origin of the plant of the genus Actinidia and the origin of the genetic resource of Actinidia chinensis. In recent years, the kiwi fruit industry in China is steadily expanded, and the cultivation area reaches 240 million mu in the whole country and the yield reaches 290 million tons as long as 2017, which all live at the first place in the world. On the aspect of variety pattern, China is based on the research of local kiwi fruit genetic resources, breeds new kiwi fruit varieties such as 'Jinyan', 'Donghong', and the like, is widely cultivated in the global range, thoroughly changes the single pattern of the world kiwi fruit industry Wei 'Haowder' variety, and promotes the diversification of kiwi fruit market and consumption.
With the increasing introduction of new kiwi fruit varieties, the identification of new varieties is a great problem to be solved urgently. At present, the patent varieties of the kiwi fruits are only identified by traditional means such as tree vigor, leaf shape, flowering phase, flower shape, fruit appearance and the like, and are difficult to avoid the influence of environment and other human factors. The uncertainty of the kiwi fruit variety limits the healthy and continuous development of the industry, and the economic benefit of the fruit grower is seriously damaged due to impure seedling varieties and even mistakes.
The molecular marker technology can quickly, accurately and efficiently identify the variety, and has important significance for ensuring the correctness of the variety in agricultural production. The method selects proper molecular markers and matched detection technology thereof, establishes a set of simple, convenient, economic, accurate and efficient variety identification and fingerprint spectrum technical system, is a method for striking the legal rights and interests of counterfeit seedlings and protecting plant genetic breeding workers and farmers, and standardizes the urgent needs of the agricultural market in China.
The molecular marker technology distinguishes different individuals by detecting variation of biological individuals on genomes, and is an ideal genetic marker developed after morphological markers and biochemical markers. The application of molecular markers in plant germplasm resource identification and breeding is becoming more mature. The SSR molecular markers are abundant in quantity, randomly and uniformly distributed in a genome, co-dominant, high in allelic variation, simple and convenient to operate and good in stability, and have been applied to the identification of varieties such as soybeans, apples and grapes. In the kiwi fruit, the molecular marker technology is applied to identification of male series varieties of kiwi fruit, so that a pollination male plant is accurately provided for a plurality of excellent kiwi fruit varieties, and the breeding efficiency of kiwi fruit is improved.
The kiwi fruit of the kiwi fruit No. 1 is cylindrical, the shape of the kiwi fruit is beautiful, the peel is green, smooth and edible, the kiwi fruit is larger in size, the average fruit weight is 10-15g, and the maximum fruit weight is 21 g. The pulp is green, and the soft ripe fruit contains 16-20% of soluble solid, 9.7% of total sugar, 1.0% of total acid and Vc208mg/100 g. New plant variety protection of Ministry of agriculture was applied in 2013. The development of the molecular identification marker applicable to the kiwi fruit 'kiwi fruit No. 1' variety is beneficial to definitely defining patent varieties on the molecular level and carrying out identification and variety protection on the kiwi fruit variety.
Disclosure of Invention
The invention aims to provide a molecular marker primer for identifying kiwi fruit variety 1, which is named as Geo29-201 by utilizing a molecular marker technology, and primers designed aiming at the molecular marker are Geo29-201-F: GGAAAGGTCGAAACCATAAGA and Geo29-201-R: TTTTTCAGAAATCCAAAAGAAAAA.
The invention also aims to provide application of the molecular marker primer for identifying the variety of kiwi fruit, namely kiwi fruit variety No. 1, in kiwi fruit breeding.
In order to achieve the purpose, the invention adopts the following technical measures:
obtaining a molecular marker primer for identifying kiwi fruit 'kiwi fruit No. 1' varieties: applicants downloaded the ` Hongyang ` kiwi whole gene sequence from the Actinidia chinensis genomic sequence website (http:// bioinfo. bti. corn. edu/kiwi). SSR markers are developed in the whole genome range through SSR Finder software, and gene loci are selected to design SSR marker primers according to gene structure annotation results. PCR amplification is carried out on the kiwi fruit 'kiwi fruit No. 1' variety by utilizing a designed and synthesized primer, a molecular marker Geo29-201 is obtained by screening, and the designed primer is Geo29-201-F: GGAAAGGTCGAAACCATAAGA and Geo29-201-R: TTTTTCAGAAATCCAAAAGAAAAA.
The application of the molecular marker primer for identifying the kiwi fruit ' kiwi fruit 1 ' variety in kiwi fruit breeding comprises the steps of utilizing the primer to breed kiwi fruits, or utilizing the primer to identify the kiwi fruit variety, particularly the kiwi fruit 1 ' variety, or distinguishing the kiwi fruit ' Miliang No. 1 ' and the kiwi fruit ' Jiangshou ' according to the sizes and the number of different strips.
Compared with the prior art, the invention has the advantages that:
1. morphological markers are simple and intuitive, but are small in number, poor in polymorphism, and susceptible to environmental conditions. The molecular marker is simple and convenient to operate, can be detected in each tissue and development period of the plant body, and is not limited by seasons and environments. In large numbers, throughout the genome. The polymorphism is high, and a plurality of allelic variation exists among varieties.
2. The primer of the invention can be used for amplifying in kiwi fruit series varieties to obtain DNA fragments with different sizes. The molecular marker has amplification stability, and can be used for identifying kiwi fruit variety, namely, kiwi fruit variety No. 1.
3. The molecular marker primer provided by the invention has high specificity and wide application range, can identify the variety of 'kiwi fruit No. 1' from more than 40 kiwi fruits of different varieties, utilizes the molecular marker Geo29-201 primer to amplify DNA, and can specifically amplify a 302bp strip only from kiwi fruit No. 1.
4. Besides identifying the variety of the 'kiwi fruit No. 1' according to specific bands, the 'Miliang No. 1' and 'Jiangshan jiao' kiwi fruits can be distinguished according to the sizes and the number of the bands.
Detailed Description
The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, unless otherwise specified, are commercially or publicly available.
Example 1:
obtaining a molecular marker primer for identifying kiwi fruit 'kiwi fruit No. 1' varieties:
applicants downloaded the ` Hongyang ` kiwi whole gene sequence from the Actinidia chinensis genomic sequence website (http:// bioinfo. bti. corn. edu/kiwi). SSR markers are developed in the whole genome range through SSR Finder software, and gene loci are selected to design SSR marker primers according to gene structure annotation results. PCR amplification is carried out on the kiwi fruit 'kiwi fruit No. 1' variety by utilizing a designed and synthesized primer, a molecular marker Geo29-201 is obtained by screening, and the designed primer is Geo29-201-F: GGAAAGGTCGAAACCATAAGA and Geo29-201-R: TTTTTCAGAAATCCAAAAGAAAAA.
Example 2:
the application of the molecular marker primer for identifying the kiwi fruit 'kiwi fruit No. 1' variety in variety identification:
(1) DNA samples of more than 40 different varieties of kiwi fruits are respectively extracted according to a CTAB method (the specific variety names are shown in Table 1).
(2) A mixture of 990ul HIDI and 10ul ROX500 or LIZ500 was pipetted using a sample applicator and added to a 96 well reaction plate at 10ul per well.
(3) The 96-well plate was placed in a plate centrifuge and centrifuged at 500g immediately.
(4) The sample DNA was added to each well of a 96-well plate at 50pg per sample.
(5) The corresponding reagents were added to a 96-well plate according to the following PCR reaction system, the 96-well plate was sealed with a sealing membrane, shaken, and the 96-well plate was placed in a plate centrifuge and centrifuged at 500 g. The PCR reaction was carried out according to the following procedure.
PCR reaction System
Preparation of 25. mu.l System
Stencil (ul) 1
Primer F (ul) 0.5
Primer R (ul) 0.5
dNTP 10mM(μl) 0.5
Taq Buffer(μl) 2.5
25mM MgCl2(μl) 2.0
Taq enzyme (. mu.l) 5U/. mu.l 0.2
Water (ul) 17.8
The primer F and the primer R are as follows: geo29-201-F: GGAAAGGTCGAAACCATAAGA and Geo29-201-R: TTTTTCAGAAATCCAAAAGAAAAA.
PCR reaction conditions
Figure BDA0001814071550000041
(6) Immediately after the program is finished, the 96-well plate is placed on an ice-water mixture for quick cooling, and then the 96-well plate is placed in a plate centrifuge, and the centrifugation is stopped at 2000 g.
(7) The amplified fragments were detected using a 3730xl sequencing analyzer (ABI, USA).
(8) Analyzing SSR amplification data by using Genemapper software, and detecting amplified fragment peaks by using STR capillary electrophoresis, wherein the abscissa is the size of the fragment and the ordinate is the fluorescence intensity value.
(9) The marker Geo29-201 is used for amplifying kiwi fruit varieties, only kiwi fruit No. 1 can specifically amplify 302bp bands, and meanwhile, kiwi fruits No. 1 ' Miliang and ' Jiangshan jiao ' are distinguished according to the sizes and the number of different bands. Specific amplification results are shown in table 1.
TABLE 1 names of different varieties of Kiwi fruits and Geo29-201 primer PCR amplification results
Figure BDA0001814071550000042
Figure BDA0001814071550000051
Sequence listing
<110> Wuhan plant garden of Chinese academy of sciences
<120> molecular marker primer for identifying kiwi fruit and kiwi fruit jujube No. 1 variety and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggaaaggtcg aaaccataag a 21
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tttttcagaa atccaaaaga aaaa 24

Claims (1)

1. The primer pairs GGAAAGGTCGAAACCATAAGA and TTTTTCAGAAATCCAAAAGAAAAA are applied to identification of kiwi fruit and kiwi fruit varieties No. 1, Miliang No. 1 and/or Jiangshan Jiaozuo kiwi fruit varieties.
CN201811133105.9A 2018-09-27 2018-09-27 Molecular marker primer for identifying kiwi fruit and kiwi fruit variety No. 1 and application Active CN109055597B (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104278097A (en) * 2014-09-30 2015-01-14 中国科学院武汉植物园 SSR molecular marker A003 for identifying sexes of kiwi fruit hybrid populations
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
CN106868201A (en) * 2017-04-27 2017-06-20 中国科学院武汉植物园 A kind of molecular labeling and application with kiwifruit fruit weight proterties QTL site close linkage
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
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104278097A (en) * 2014-09-30 2015-01-14 中国科学院武汉植物园 SSR molecular marker A003 for identifying sexes of kiwi fruit hybrid populations
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
CN106868201A (en) * 2017-04-27 2017-06-20 中国科学院武汉植物园 A kind of molecular labeling and application with kiwifruit fruit weight proterties QTL site close linkage
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
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 (2)

* Cited by examiner, † Cited by third party
Title
猕猴桃品种SSR分析的初步研究;郑轶琦等;《武汉植物学研究》;20031231;第444-448页 *
软枣猕猴桃新品种‘猕枣1 号;韩飞等;《园艺学报》;20161231;第2699–2700页 *

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