CN105907755B - Molecular labeling and its application with capsicum anti cucumber mosaic virus close linkage - Google Patents

Molecular labeling and its application with capsicum anti cucumber mosaic virus close linkage Download PDF

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CN105907755B
CN105907755B CN201610405984.0A CN201610405984A CN105907755B CN 105907755 B CN105907755 B CN 105907755B CN 201610405984 A CN201610405984 A CN 201610405984A CN 105907755 B CN105907755 B CN 105907755B
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capsicum
mosaic virus
primer
cucumber mosaic
molecular labeling
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CN105907755A (en
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张宝玺
王立浩
张正海
曹亚从
王兴兴
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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    • 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
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    • 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
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    • 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

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Abstract

The present invention relates to field of molecular marker, and in particular to the molecular labeling with capsicum anti cucumber mosaic virus close linkage.The molecular labeling is located on No. 12 chromosomes of capsicum, and the genetic distance of the site and flank SNP marker UN54228_1146 are 1cM.By the molecular labeling can CMV resistance anti-to capsicum identify, improve breeding efficiency.Compared to the other molecular labelings found before, the genetic distance of SNP marker UN54228_1146 of the invention and resistance locus is 1cM, significant in the anti-CMV breeding of capsicum.

Description

Molecular labeling and its application with capsicum anti cucumber mosaic virus close linkage
Technical field
The present invention relates to field of molecular marker, and in particular to the molecule mark with capsicum anti cucumber mosaic virus close linkage Note.
Background technique
Capsicum is worldwide planted extensively, is the second largest vegetable crop in China.However, with pepper planting area Constantly increase, the harm that disease produces capsicum is also more serious, wherein cucumber mosaic virus (Cucumber mosaic Virus, CMV) cause harm China capsicum at production one of main virosis.Capsicum seriously affected after being caused harm by CMV quality and Yield, thus cultivate target one of of the anti-CMV kind of capsicum as capsicum resistance breeding.However it is chain with the anti-CMV of capsicum at present The report of label is less, and there is presently no the molecular labelings that can be used for capsicum anti cucumber mosaic virus assistant breeding.Thus probe into Molecular labeling relevant to the anti-CMV of capsicum is significant.
Summary of the invention
The purpose of the present invention is to provide a kind of and capsicum anti cucumber mosaic virus close linkage molecular labelings.
According to the present invention for assisting anti cucumber mosaic virus molecular labeling that can obtain by following steps:
The present invention is with pepper disease resistance kind Perennial and susceptible variety 83-58 and its 118 strains of RIL group of building System is test material, with the SNP marker developed based on transcript profile, constructs genetic map using KASPar genotyping technique, then QTL positioning is carried out in conjunction with phenotypic data, a QTL site relevant to the anti-CMV of capsicum is as a result navigated on No. 12 chromosomes, The genetic distance of the site and flank SNP marker UN54228_1146 are 1cM, can effectively be used to assistant breeding and work.
KASPar primer for the anti-CMV specificity screening of capsicum of the invention, information are as follows.
It is an advantage of the invention that only being detected in seedling stage to the DNA of pepper plant, so that it may sentence not by Disease Resistance Identification Capsicum break for the resistance of CMV, molecular mark can be widely applied to.And the label can use KASPar gene point Type technology carries out mass detection, and efficiency can be improved, and shortens qualification time.
Molecular labeling provided by the invention realizes the identification of CMV anti-to pepper plant in seedling stage, substantially reduces breeding Time has saved manpower and material resources.By using KASPar genotyping technique using specific primer provided by the invention, it is subject to Resistance sense parent Perennial, 83-58 are compared, with resistance parent's Perennial homogenic type and heterozygous genotypes tool It is resistant, do not have resistance with phase homogenic type with Susceptible parent 83-58.It can CMV anti-to capsicum by the molecular labeling Resistance is identified, breeding efficiency is improved.Compared to the other molecular labelings found before, SNP marker of the invention The genetic distance of UN54228_1146 and resistance locus is 1cM, significant in the anti-CMV breeding of capsicum.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to Range.
The present invention is with the F of both pepper disease resistance kind Perennial and susceptible variety 83-581And its constructed RIL (F8) 118 strains of group are test material, it is that virus expands numerous material with three lives cigarette.
1, RIL population resistance is identified
Parents, F1, RIL group 118 strains, three repetitions of each plantation, it is each repeat plantation 10.In capsicum kind The previous plantation moon, the cigarette three lives is planted, the expansion for CMV cause of disease is numerous.
The weighing of fresh tobacco disease leaf is saved backup, when tobacco seedling length to 5-6 piece leaf, according to fresh sick leaf and 3mMpH=8 PBS buffer solution mass volume ratio 1:5, be ground into homogenate in ice bath, 3000rpm centrifugation, supernatant is inoculation liquid, is placed in It is spare in ice.A little 600 mesh diamond dust is spread on the long tobacco seedling blade to 5,6 leaves, using frictional inoculation method, finger-dipping A small amount of inoculation liquid is taken, gently rub blade face, washes away the extra juice in blade face with water after 30min；It is inoculated after 3 days primary.After inoculation The growth temperature of material is controlled at 28 DEG C or so.10d's has the tobacco leaf of obvious illness to be inoculated with as capsicum after acquisition inoculation Cause of disease, the pathogenecity of cucumber mosaic virus is most strong at this time.The tobacco disease prophyll of acquisition is transferred to the peppery of long to three, four leaves Pepper plant, inoculation method are same as above, and are inoculated with the 1st, 2 true leaf of pepper seedling, are connect with 20 μ L of every blade inoculation of continuous sample adding apparatus Kind liquid, i.e., every plant 40 μ L inoculation liquids of inoculation.The growth temperature of material is controlled at 28 DEG C or so after inoculation.It is inoculated with after 25d referring to state Family's " eight or five " pepper disease resistance breeding project group grade scale (table 1) carries out Resistance Identification, and three duplicate mean values are as the material Reaction to CMV resistance level.
Disease index (DI) calculation formula: disease index (DI)=[∑ (morbidity strain number × disease series at different levels)/(9 × investigation Strain number)] × 100.
The disease resistance criteria for classifying: immune (I): DI=0；Highly resistance (HR): 0 < DI≤5；Disease-resistant (R): 5 < DI≤15；In resist (MR): 15 < DI≤30；Susceptible (S): 30 < DI≤40；Height sense (HS): DI > 40.
National " eight or five " pepper disease resistance breeding project group grade scale of table 1
2, the building of genetic linkage maps
2.1KASPar label
Each pair of KASPar molecular labeling of the present invention contains 3 primer sequences: A1, A2, C.Wherein A1, A2 be only It is that the SNP site of end has differences, at the 5 ' ends of A1, A2 respectively added with common joint sequence: 5 ' GCATCTTGAATGGA GATTTTCTTGACACGATG 3',5'GCATCTTGAATGGAGATTTTCTTGACACGATT 3'.This two sections of sequences with Sequence in Master Mix with fluorescence is complementary, fluorescence can be excited after pairing, C is reverse complementary sequence.
Primer premix
2 KASPar primer of table premix
The reading of fluorescent value is in 480 II fluorescent quantitation instrument of Roche, according to 37 DEG C of 1min, Read fluorescence 1s It carries out, is analyzed according still further to Endpt Geno and read fluorescent value.RIL group polymorphism is counted, with parent's Perennial phase Same Allele X-type is denoted as " a ", and Allele Y type identical with parent 83-58 is denoted as banding pattern " b ", heterozygous sites (Both Alleles it) is denoted as " h ", fail identification is denoted as "-".The building of genetic linkage maps is carried out using Joinmap4.0 software, it is right The initial data of RIL group Genotyping carries out standardization processing, and is converted into the specific format of software requirement；It again will be acquired Data import software and calculate and analysis and construct linkage map.Parameter is set as LOD >=3, step-length 0.5.Use Kosambi Function carries out the calculating of genetic distance, is as a result indicated with cM (cM).
3, the acquisition of anti cucumber mosaic virus SPN label
Based on the linkage map constructed, in conjunction with phenotypic evaluation data, QTL positioning is carried out using 4.0 software of MapQTL. Using Interval mapping method (IM, Interval Mapping), is examined in conjunction with Permutation Test and determine that QTL site exists When corresponding LOD value, and regard the highest point of LOD value in linkage group as a QTL site, and analyze QTL additive effect and The contribution rate of phenotypic variation.As a result three sites (table 3) are navigated on No. 12 chromosomes.
The essential information of 3 capsicum anti cucumber mosaic virus QTL of table
Wherein label UN54228_1146 and main effect QTL site CMV12.3 close linkage.It can be used for the anti-CMV molecule of capsicum Marker-assisted breeding.
Sequence table
<110>Vegetable & Flower Inst., Chinese Academy of Agriculture Science
<120>with the molecular labeling of capsicum anti cucumber mosaic virus close linkage and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gcatcttgaa tggagatttt cttgacacga tg 32
<210> 2
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gcatcttgaa tggagatttt cttgacacga tt 32
<210> 3
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tggattggca aacaacacga atgacc 26

Claims (2)

1. point of the flank SNP marker UN54228_1146 as QTL site close linkage related to capsicum anti cucumber mosaic virus The application of son label, wherein the QTL site is on No. 12 chromosomes, the QTL site and the flank SNP marker The genetic distance of UN54228_1146 is 1cM, and the specific primer of the flank SNP marker UN54228_1146 includes:
Preceding primer A1:5'- GCATCTTGAATGGAGATTTTCTTGACACGATG-3'；
Preceding primer A2:5'- GCATCTTGAATGGAGATTTTCTTGACACGATT-3'；
Primer C:5'- TGGATTGGCAAACAACACGAATGACC-3' afterwards.
2. a kind of method for identifying the anti-CMV of capsicum, which is characterized in that the method includes using flank SNP marker The step of specific detection primer of UN54228_1146 is detected, the specific primer include:
Preceding primer A1:5'- GCATCTTGAATGGAGATTTTCTTGACACGATG-3'；
Preceding primer A2:5'- GCATCTTGAATGGAGATTTTCTTGACACGATT-3'；
Primer C:5'- TGGATTGGCAAACAACACGAATGACC-3' afterwards.
CN201610405984.0A 2016-06-10 2016-06-10 Molecular labeling and its application with capsicum anti cucumber mosaic virus close linkage Active CN105907755B (en)

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CN107151709A (en) * 2017-07-13 2017-09-12 中国农业科学院蔬菜花卉研究所 The SNP marker related to capsicum pungent and its application
CN111808984A (en) * 2020-08-24 2020-10-23 中国农业科学院蔬菜花卉研究所 SNP (Single nucleotide polymorphism) marker related to hot pepper cytoplasmic male sterility restoring gene, specific primer and application of specific primer
CN112216343B (en) * 2020-10-28 2023-08-15 遵义市农业科学研究院 Screening method of tomato spotted wilt virus resistant pepper plants

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CN105296475A (en) * 2015-11-06 2016-02-03 江苏省农业科学院 Molecular marker interlocked with pepper cucumber mosaic virus disease resistance gene qcmv-2-1 and application of molecular marker
CN105368927A (en) * 2015-09-16 2016-03-02 北京市农林科学院 Molecular marker genSSR3000 closely linked with pepper CMV resistance gene and acquisition and application
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CN105296475A (en) * 2015-11-06 2016-02-03 江苏省农业科学院 Molecular marker interlocked with pepper cucumber mosaic virus disease resistance gene qcmv-2-1 and application of molecular marker
CN107488732A (en) * 2017-10-09 2017-12-19 贵州省产品质量监督检验院 Detect triple fluorescent PCR primer group, probe groups, kit and the method for capsicum transgene component

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《辣椒抗CMV的遗传分析及相关QTL定位》;姚明华等;《中国优秀硕士学位论文全文数据库农业科技辑》;20140215(第2期);第30、69和90页 *
《辣椒根结线虫抗性基因Mel的精细定位》;王学瑛等;《中国优秀硕士学位论文全文数据库农业科技辑》;20160115(第1期);第16-17、58-68页 *

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