CN105907755A - Molecular marker closely linked with resistance of hot peppers to cucumber mosaic virus and application of molecular marker - Google Patents

Molecular marker closely linked with resistance of hot peppers to cucumber mosaic virus and application of molecular marker Download PDF

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CN105907755A
CN105907755A CN201610405984.0A CN201610405984A CN105907755A CN 105907755 A CN105907755 A CN 105907755A CN 201610405984 A CN201610405984 A CN 201610405984A CN 105907755 A CN105907755 A CN 105907755A
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molecular marker
resistance
primer
fructus capsici
mosaic virus
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CN105907755B (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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    • 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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    • 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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    • 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 invention relates to the field of molecular markers, in particular to a molecular marker closely linked with resistance of red peppers to a cucumber mosaic virus. The molecular marker is located on chromosome 12 of a hot pepper, and the genetic distance between the locus and side wing SNP marked UN54228_1146 is 1 cM. By means of the molecular marker, CMV resistance of the hot peppers can be identified, and the breeding efficiency is improved. Compared with other previously discovered molecular markers, the genetic distance between SNP marked UN54228_1146 and the resistance locus is 1 cM, and the molecular marker has important significance in CMV resistance breeding of the hot peppers.

Description

Molecular marker closely linked with Fructus Capsici anti cucumber mosaic virus and application thereof
Technical field
The present invention relates to field of molecular marker, be specifically related to molecule mark closely linked with Fructus Capsici anti cucumber mosaic virus Note.
Background technology
Fructus Capsici is the most extensively planted, and is the second largest vegetable crop of China.But, along with pepper planting area Constantly increasing, the harm that Fructus Capsici is produced by disease is the most serious, wherein cucumber mosaic virus (Cucumber mosaic Virus, CMV) become one of China Fructus Capsici main virosis becoming product that causes harm.Fructus Capsici have a strong impact on after being caused harm by CMV quality and Yield, thus cultivate one of Fructus Capsici anti-CMV kind target becoming Fructus Capsici resistance breeding.But CMV anti-with Fructus Capsici is chain at present The report of labelling is less, there is presently no the molecular marker that can be used for Fructus Capsici anti cucumber mosaic virus assistant breeding.Thus probe into CMV relevant molecular marker anti-to Fructus Capsici is significant.
Summary of the invention
It is an object of the invention to provide a kind of and closely linked molecular marker of Fructus Capsici anti cucumber mosaic virus.
It is used for assisting anti cucumber mosaic virus molecular marker can be obtained by following steps according to the present invention:
The present invention is with 118 strains of RIL colony of pepper disease resistance kind Perennial and susceptible variety 83-58 and structure thereof System is test material, with the SNP marker developed based on transcript profile, uses KASPar genotyping technique to build genetic map, then Carrying out QTL location in conjunction with phenotypic data, result navigates to a CMV relevant QTL site anti-to Fructus Capsici on No. 12 chromosomes, This site is 1cM with the genetic distance of flank SNP marker UN54228_1146, can effectively be used for assistant breeding and work.
The KASPar primer for Fructus Capsici anti-CMV specificity screening of the present invention, its information is as follows.
It is an advantage of the invention that not by Disease Resistance Identification, be the DNA detection in seedling stage to pepper plant, it is possible to sentence Disconnected Fructus Capsici, for the resistance of CMV, can be widely applied to molecular mark.And this labelling can divide with KASPar gene Type technology carries out mass detection, can improve efficiency, shortens qualification time.
The molecular marker that the present invention provides achieves the qualification at CMV anti-to pepper plant in seedling stage, substantially reduces breeding Time, manpower and materials are saved.KASPar genotyping technique is used by the specific primer utilizing the present invention to provide, in addition Resistance sense parent Perennial, 83-58 compare, with resistance parent's Perennial homogenic type and the tool of heterozygous genotypes There is resistance, with Susceptible parent 83-58 has a homologous genes type, not there is resistance.Can CMV anti-to Fructus Capsici by this molecular marker Resistance is identified, improves breeding efficiency.Compare other molecular marker found before, the SNP marker of the present invention UN54228_1146 is 1cM with the genetic distance of resistance locus, significant in Fructus Capsici anti-CMV breeding.
Detailed description of the invention
Following example further illustrate present disclosure, but should not be construed as limitation of the present invention.Without departing substantially from In the case of present invention spirit and essence, the amendment that the inventive method, step or condition are made or replacement, belong to the present invention Scope.
The present invention is with pepper disease resistance kind Perennial and the F of susceptible variety 83-581And constructed RIL (F8) 118 strains of colony are test material, with three lives cigarette for virus expanding propagation material.
1, RIL population resistance is identified
Parents, 118 strains of F1, RIL colony, each plantation three repetition, each repetition plants 10.In Fructus Capsici kind Plant plantation of previous moon three lives cigarette, for the expanding propagation of CMV cause of disease.
Fresh tobacco disease leaf is weighed and saves backup, when tobacco seedling length to 5-6 sheet leaf, according to fresh sick leaf and 3mM pH= The PBS mass volume ratio 1:5 of 8, grinds to form homogenate in ice bath, and 3000rpm is centrifuged, and supernatant is inoculation liquid, puts In ice standby.To the tobacco seedling blade of 5,6 leaves, spread a little 600 mesh corundum long, use frictional inoculation method, finger Dipping a small amount of inoculation liquid, rub blade face gently, washes away the unnecessary juice in blade face with water after 30min；Inoculate once after 3 days.Inoculation The growth temperature of rear material controls at about 28 DEG C.After gathering inoculation, the tobacco leaf having obvious disease of 10d connects as Fructus Capsici Planting cause of disease, now the pathogenecity of cucumber mosaic virus is the strongest.The tobacco disease prophyll of collection is transferred to long to three, four leaves Pepper plant, inoculation method ibid, inoculates the 1st of pepper seedling the, 2 true leaves, with continuous sample adding apparatus every blade inoculation 20 μ L Inoculation liquid, 40 μ L inoculation liquid are inoculated in i.e. every strain.After inoculation, the growth temperature of material controls at about 28 DEG C.Reference after inoculation 25d Country's " eight or five " pepper disease resistance breeding project component grade standard (table 1) carries out Resistance Identification, and three averages repeated are as this material The material reaction to CMV resistance level.
Disease index (DI) computing formula: disease index (DI)=[∑ (morbidity strain number × disease progression at different levels)/(9 × investigation Strain number)] × 100.
The disease resistance criteria for classifying: immunity (I): DI=0；High anti-(HR): 0 < DI≤5；Disease-resistant (R): 5 < DI≤15；In anti- (MR): 15 < DI≤30；Susceptible (S): 30 < DI≤40；High sense (HS): DI > 40.
Table 1 country " eight or five " pepper disease resistance breeding project component grade standard
2, the structure of genetic linkage maps
2.1KASPar labelling
Every pair of KASPar molecular marker of the present invention contains 3 primer sequences: A1, A2, C.Wherein A1, A2 are only It is that the SNP site of end there are differences, at the 5 ' ends of A1, A2 respectively added with common joint sequence: 5 ' GAAGGTGACCAAGTTCATGCT3’、5’GAAGGTCGGAGTCAACGGATT3’.Carry in these two sections of sequences and Master Mix Having the complementary of fluorescence, can excite fluorescence, C after pairing is reverse complementary sequence.
Primer premixes
Table 2 KASPar primer premixes
The reading of fluorescent value is at Roche 480 II fluorescent quantitation instrument, according to 37 DEG C of 1min, Read fluorescence 1s Carry out, analyze according still further to Endpt Geno and read fluorescent value.RIL colony polymorphism is added up, with parent's Perennial phase Same Allele X-type is designated as " a ", and the Allele Y type identical with parent 83-58 is designated as banding pattern " b ", heterozygous sites (Both Alleles) be designated as " h ", fail identify be designated as "-".Joinmap4.0 software is utilized to carry out the structure of genetic linkage maps, right The initial data of RIL colony gene type carries out standardization processing, and is converted into the concrete form of software requirement；Again by obtained Data import software carry out computational analysis and build linkage map.Parameter is set to LOD >=3, and step-length is 0.5.Use Kosambi Function carries out the calculating of the genetic distance, and result cM (cM) represents.
3, the acquisition of anti cucumber mosaic virus SPN labelling
Based on the linkage map built, in conjunction with phenotypic evaluation data, MapQTL 4.0 software is utilized to carry out QTL location. Use Interval mapping method (IM, Interval Mapping), determine that QTL site exists in conjunction with Permutation Test inspection Time corresponding LOD value, and a QTL site will be made from the point of view of the highest for LOD value in linkage group, and analyze QTL additive effect and The contribution rate of phenotypic variation.Result navigates to three sites (table 3) on No. 12 chromosomes.
The essential information of table 3 Fructus Capsici anti cucumber mosaic virus QTL
Wherein labelling UN54228_1146 and main effect QTL site CMV12.3 close linkage.Can be used for Fructus Capsici anti-CMV molecule Marker-assisted breeding.

Claims (4)

1. with the closely linked molecular marker of Fructus Capsici anti cucumber mosaic virus, it is characterised in that described molecular marker is positioned at Fructus Capsici On No. 12 chromosomes, this site is 1cM with the genetic distance of flank SNP marker UN54228_1146.
2. for the KASPar primer of Fructus Capsici anti-CMV specificity screening, it is characterised in that the sequence of described primer is:
Front primer A1:TGGATTGGCAAACAACACGAATGACC；
Front primer A2:GCATCTTGAATGGAGATTTTCTTGACACGATG；
Rear primer C:GCATCTTGAATGGAGATTTTCTTGACACGATT.
3. the method identifying the anti-CMV of Fructus Capsici, it is characterised in that described method includes using following primer to carry out expanding Step:
The sequence of described primer is:
Front primer A1:TGGATTGGCAAACAACACGAATGACC；
Front primer A2:GCATCTTGAATGGAGATTTTCTTGACACGATG；
Rear primer C:GCATCTTGAATGGAGATTTTCTTGACACGATT.
4. the application of molecular marker closely linked with Fructus Capsici anti cucumber mosaic virus described in claim 1.
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Publication number Priority date Publication date Assignee Title
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
CN112216343A (en) * 2020-10-28 2021-01-12 遵义市农业科学研究院 Screening method of tomato spotted wilt virus resistant hot 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
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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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
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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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN112216343A (en) * 2020-10-28 2021-01-12 遵义市农业科学研究院 Screening method of tomato spotted wilt virus resistant hot pepper plants
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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