CN109055601B - Molecular marker primer for identifying kiwi fruit and kiwi fruit 2 variety and application - Google Patents

Molecular marker primer for identifying kiwi fruit and kiwi fruit 2 variety and application Download PDF

Info

Publication number
CN109055601B
CN109055601B CN201811157371.5A CN201811157371A CN109055601B CN 109055601 B CN109055601 B CN 109055601B CN 201811157371 A CN201811157371 A CN 201811157371A CN 109055601 B CN109055601 B CN 109055601B
Authority
CN
China
Prior art keywords
kiwi fruit
variety
primer
kiwi
geo11
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201811157371.5A
Other languages
Chinese (zh)
Other versions
CN109055601A (en
Inventor
钟彩虹
张琼
郑浩
张琦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuhan Botanical Garden of CAS
Original Assignee
Wuhan Botanical Garden of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuhan Botanical Garden of CAS filed Critical Wuhan Botanical Garden of CAS
Priority to CN201811157371.5A priority Critical patent/CN109055601B/en
Publication of CN109055601A publication Critical patent/CN109055601A/en
Application granted granted Critical
Publication of CN109055601B publication Critical patent/CN109055601B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12Q2600/13—Plant traits

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Botany (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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 2' variety and application thereof, wherein the primer comprises the following components in parts by weight: geo11-126-F: AAAAGGGGAACAAACCAAGG and Geo11-126-R: TGCCTCATTTAAACATCTCCAA. The primer provided by the invention is used for amplifying the DNA of the Chinese gooseberry No. 2, and only the Chinese gooseberry No. 2 can specifically amplify bands of 245bp, 251bp and 283 bp; according to the size and the number of different bands, the primers provided by the invention can also comprise the following components for different varieties: the kiwi fruit varieties such as 'Brulo', 'emerald' and 'noble and long', 'Huaguang No. 2', 'Haiword' and the like are distinguished.

Description

Molecular marker primer for identifying kiwi fruit and kiwi fruit 2 variety 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 type 2 varieties and application of the molecular marker primer.
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 No. 2 is short and cylindrical, the average fruit weight is 6-10 g, and the maximum fruit weight is 10.2 g; the peel and the pulp are green, the surface of the fruit is smooth and edible, and the soft and ripe fruit contains 24-27% of soluble solid, 11% of total sugar, 1.1% of total acid and 30m g/100g of Vc. Application for new plant variety protection in Ministry of agriculture in 2015. The development of the molecular identification marker applicable to the kiwi fruit variety, namely, the kiwi fruit variety 2, is beneficial to definitely defining patent varieties on the molecular level and is beneficial to identification and variety protection of the kiwi fruit variety.
Disclosure of Invention
The invention aims to provide a molecular marker primer for identifying kiwi fruit variety 2, which is named as Geo11-126 by utilizing a molecular marker technology, and primers designed aiming at the molecular marker primer are Geo11-126-F: AAAAG GGGAACAAACCAAGG and Geo11-126-R: TGCCTCATTTAAACATCTCCAA.
The invention also aims to provide application of the molecular marker primer for identifying the variety of kiwi fruit, namely kiwi fruit variety 2, 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 2' variety: 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. 2' variety by using a designed and synthesized primer, a molecular marker Geo11-126 is obtained by screening, and the designed primer is Geo11-126-F: AAAAGGGGAACAAACCAAGG and Geo11-126-R: TGCCT CATTTAAACATCTCCAA.
The application of the molecular marker primer for identifying the kiwi fruit ' kiwi fruit 2 ' variety in kiwi fruit breeding comprises the following steps of utilizing the primer to carry out kiwi fruit breeding, or utilizing the primer to identify the kiwi fruit variety, particularly the kiwi fruit 2 ' variety, or according to the sizes and the number of different strips, carrying out the following steps of: the kiwi fruits are classified as 'bruclo', 'jade', 'noble long', 'huaguang No. 2', 'seaford', 'jinkui', 'jinnong', 'gold peach', 'gold gorgeous', 'full sky red', 'MBR', 'milan No. 1', 'Moshan No. 4', 'Qingyuan Qin cui', 'Qimei', 'Tongshan No. 5', 'WH-1', 'Wuzhu No. 3', 'Xiangma No. 6', 'Xiyai No. 1', 'Xuxiang', 'Jianghuang No. one' and 'Jiangshoujiao' kiwi fruits.
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 2.
3. The molecular marker primer provided by the invention has high specificity and wide application range, can identify the variety of 'kiwi fruit No. 2' from more than 40 kiwi fruits of different varieties, utilizes the molecular marker Geo11-126 primer to amplify DNA, and can specifically amplify the bands of 245bp, 251bp and 283bp only for the kiwi fruit No. 2.
4. In addition to identifying the 'kiwi fruit No. 2' variety according to the specific bands, different varieties can also be identified according to the size and number of the bands: the kiwi fruits are classified as 'bruclo', 'jade', 'noble long', 'huaguang No. 2', 'seaford', 'jinkui', 'jinnong', 'gold peach', 'gold gorgeous', 'full sky red', 'MBR', 'milan No. 1', 'Moshan No. 4', 'Qingyuan Qin cui', 'Qimei', 'Tongshan No. 5', 'WH-1', 'Wuzhu No. 3', 'Xiangma No. 6', 'Xiyai No. 1', 'Xuxiang', 'Jianghuang No. one' and 'Jiangshoujiao' kiwi fruits.
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 2' variety:
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. 2' variety by using a designed and synthesized primer, a molecular marker Geo11-126 is obtained by screening, and the designed primer is Geo11-126-F: AAAAGGGGAACAAACCAAGG and Geo11-126-R: TGCCTCATTTAAACATCTCCAA.
Example 2:
the application of the molecular marker primer for identifying the kiwi fruit variety' kiwi fruit 2 # 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
Figure BDA0001819211440000031
Figure BDA0001819211440000041
The primer F and the primer R are as follows: geo11-126-F: AAAAGGGGAACAAACCAAGG and Geo11-126-R: TGCCTCATTTAAACATCTCCAA.
PCR reaction conditions
Figure BDA0001819211440000042
(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 Geo11-126 is utilized to amplify the kiwi fruit variety, only the kiwi fruit No. 2 can specifically amplify the bands of 245bp, 251bp and 283bp, and simultaneously, different varieties comprise the following bands according to the sizes and the numbers of different bands: the kiwi fruits are classified as 'bruclo', 'jade', 'noble long', 'huaguang No. 2', 'seaford', 'jinkui', 'jinnong', 'gold peach', 'gold gorgeous', 'full sky red', 'MBR', 'milan No. 1', 'Moshan No. 4', 'Qingyuan Qin cui', 'Qimei', 'Tongshan No. 5', 'WH-1', 'Wuzhu No. 3', 'Xiangma No. 6', 'Xiyai No. 1', 'Xuxiang', 'Jianghuang No. one' and 'Jiangshoujiao' kiwi fruits. Specific amplification results are shown in table 1.
TABLE 1 names of different varieties of Kiwi fruits and PCR amplification results of Geo11-126 primers
Figure BDA0001819211440000051
Figure BDA0001819211440000061
Sequence listing
<110> Wuhan plant garden of Chinese academy of sciences
<120> molecular marker primer for identifying kiwi fruit and kiwi fruit No. 2 variety and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaaaggggaa caaaccaagg 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaaaggggaa caaaccaagg 20

Claims (4)

1. A molecular marker primer for identifying kiwi fruit and kiwi fruit 2 varieties is as follows: geo11-126-F: AAAAGGGGAACAAACCAAGG and Geo11-126-R: TGCCTCATTTAAACATCTCCAA.
2. The application of the primer of claim 1 in identification of kiwi fruit and kiwi fruit variety No. 2.
3. The primer of claim 1, wherein the primer is used for identifying varieties of kiwi fruits such as 'Brulo', 'emerald', 'noble', 'Huaguang No. 2', 'Haiword', 'Jinkui', 'Jinnong', 'gold peach', 'Jinyan', 'Mantianhong', 'Miliang No. 1', 'Moshan No. 4', 'Qingyuan Qiun', 'Qimei', 'Tongshan No. 5', 'Wuzhu No. 3', 'Xiangma No. 6', 'Asia No. 1', 'Xixiang', 'Yuhuang No. one' or 'Jiangshan' kiwi fruits.
4. A kit for identifying kiwi fruit and kiwi fruit No. 2 varieties comprises primers: geo11-126-F: AAAAGGGGAACAAACCAAGG and Geo11-126-R: TGCCTCATTTAAACATCTCCAA.
CN201811157371.5A 2018-09-30 2018-09-30 Molecular marker primer for identifying kiwi fruit and kiwi fruit 2 variety and application Active CN109055601B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811157371.5A CN109055601B (en) 2018-09-30 2018-09-30 Molecular marker primer for identifying kiwi fruit and kiwi fruit 2 variety and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811157371.5A CN109055601B (en) 2018-09-30 2018-09-30 Molecular marker primer for identifying kiwi fruit and kiwi fruit 2 variety and application

Publications (2)

Publication Number Publication Date
CN109055601A CN109055601A (en) 2018-12-21
CN109055601B true CN109055601B (en) 2021-06-11

Family

ID=64767176

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811157371.5A Active CN109055601B (en) 2018-09-30 2018-09-30 Molecular marker primer for identifying kiwi fruit and kiwi fruit 2 variety and application

Country Status (1)

Country Link
CN (1) CN109055601B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104313019A (en) * 2014-09-30 2015-01-28 中国科学院武汉植物园 SSR molecular marker A002 for identifying males and females of kiwi fruit hybrid populations
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104313019A (en) * 2014-09-30 2015-01-28 中国科学院武汉植物园 SSR molecular marker A002 for identifying males and females of kiwi fruit hybrid populations
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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Development and Application of Transcriptome-Derived Microsatellites in Actinidia eriantha (Actinidiaceae);Rui Guo等;《Front Plant Sci》;20170825;第8卷;Article1383 第1-13页 *
Development of microsatellite markers in Actinidia arguta (Actinidiaceae) based on the NCBI data platform;Yuping Man等;《Am J Bot》;20111031;第98卷(第11期);e310-e315 *
猕猴桃 EST-SSR 引物筛选及通用性分析;廖娇等;《果树学报》;20111110;第28卷(第6期);第1111-1116页 *

Also Published As

Publication number Publication date
CN109055601A (en) 2018-12-21

Similar Documents

Publication Publication Date Title
CN107385071B (en) Molecular marker primer for identifying male varieties of kiwi fruit Moshan series and application
CN107338318B (en) Molecular marker Geo101 primer for identifying male varieties of kiwi fruit Moshan series and application
CN107201404B (en) Molecular biological identification method for sex of asparagus hermaphrodite plants and application thereof
CN107326091B (en) Molecular marker Geo168 primer for identifying male varieties of kiwi fruit Moshan series and application
CN108359738B (en) Torreya sinensis EST-SSR primer and variety fingerprint construction method
CN108796111B (en) Molecular marker primer for identifying kiwi fruit and golden plum varieties and application
CN109055599B (en) Molecular marker primer for identifying Jinmei variety of kiwi fruit and application thereof
CN109055601B (en) Molecular marker primer for identifying kiwi fruit and kiwi fruit 2 variety and application
CN111349717A (en) SSR (simple sequence repeat) markers of sweet cherry stock resources and fingerprint spectrum database thereof
CN108085409B (en) Screening method of fir reference gene in different tissues and application of screening gene as reference gene
CN109055597B (en) Molecular marker primer for identifying kiwi fruit and kiwi fruit variety No. 1 and application
CN113151568B (en) SNP (Single nucleotide polymorphism) site with closely linked capsaicin content, CAPS (cleaved amplified polymorphic sequence) molecular marker of SNP site and application of CAPS molecular marker
CN109055600B (en) Molecular marker primer for identifying variety No. 2 of kiwi fruit Hongtianhong and application thereof
CN108796115B (en) Molecular marker primer for identifying kiwi fruit Jinyu variety and application thereof
KR20180075451A (en) Identification method the variety of Cherry Rootstock
CN112746124B (en) SSR primer and method for purity identification of cauliflower hybrid seeds
CN108796112B (en) Molecular marker primer for identifying kiwi fruit golden peach variety and application
CN109055602B (en) Molecular marker primer for identifying kiwi fruit-Dongfei variety and application thereof
CN108796116B (en) Molecular marker primer for identifying golden round variety of kiwi fruit and application thereof
CN108977575B (en) Molecular marker primer for identifying kiwi fruit RC197 variety and application
CN108998566B (en) Molecular marker primer for identifying super-red kiwi variety and application thereof
CN109207629B (en) SSR molecular marker BvRE049 for detecting granularity of beet seeds and application thereof
Guan et al. Identification of superior clones by RAPD technology in Xanthoceras sorbifolia Bge.
CN114634991B (en) InDel marker for identifying high-variety coconuts and application thereof
CN113981124B (en) Sakura SSR molecular marker primer and application thereof in identification of 42 sakura varieties

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant