CN105462963B - With the Soybean Resistance epidemic disease gene RpsZS18 molecular labeling isolated and its application - Google Patents
With the Soybean Resistance epidemic disease gene RpsZS18 molecular labeling isolated and its application Download PDFInfo
- Publication number
- CN105462963B CN105462963B CN201410415801.4A CN201410415801A CN105462963B CN 105462963 B CN105462963 B CN 105462963B CN 201410415801 A CN201410415801 A CN 201410415801A CN 105462963 B CN105462963 B CN 105462963B
- Authority
- CN
- China
- Prior art keywords
- soybean
- gene
- rpszs18
- disease
- label
- 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.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides the molecular labeling ZCindel246000 and its application that a kind of and Soybean Resistance epidemic disease gene RpsZS18 is isolated, the label is located between the SSR marker ZCSSR33 and ZCSSR46 of No. 2 chromosomes of soybean, and is 0cM with the genetic distance of gene RpsZS18.The present invention is male parent with disease-resistant variety precocity 18, and susceptible variety Williams is female parent, generates F by hybridizing and being selfed3Group develops new SSR marker with the soybean ssr marker announced in the section and using soybean genomic sequence according to first positioning result as mapping population, carries out finely positioning to the disease-resistant gene that precocity 18 contains.Disease-resistant gene RpsZS18 is positioned between the ZCSSR33 (0.7cM) of No. 2 chromosome and ZCSSR46 (2.8cM), and is isolated with SSR marker Satt172.According to the gene annotation in new definition section, predict RpsZS18 candidate gene, according to the candidate gene site sequence difference of anti-sense parent's prediction, it develops Indel and marks ZCindel24600, it is found to isolate label after the label is carried out group's verifying, and Indel label can be used in the antilemic assistant breeding of soybean.
Description
Technical field
The invention belongs to agricultural biotechnology engineerings and disease-resistant crops genetic breeding field, specifically, being related to one kind
With the Soybean Resistance epidemic disease gene RpsZS18 molecular labeling isolated and its application.
Background technique
The soybean blight as caused by soybean phytophthora (Phytophthora sojae Kaufmann&Gerdemann) is soybean
One of production destructive disease.The disease occurs in provinces such as Heilongjiang Province, China, Anhui, Fujian, Jilin, Xinjiang.Greatly
Beans phytophthora can infect soybean in soybean all breeding times, cause root-rot, stem rot, plant to be downgraded, is withered and dead, general field
Block underproduction 10-30%, serious plot is up to 60-90%, or even causes to have no harvest.At present the whole world every year because of phytophthora root rot of soybean caused by
Economic loss be about 1,000,000,000 dollars (Tyler, 2007).
Prevention and treatment soybean blight economy, effective and Environmental security method the most are plantation disease-resistant varieties.Currently, foreign countries are
17 resistant genes are identified on 8 locus in soybean genome.Respectively Rps1 (Bernard et al.,
1957)、Rps2(Kilen et al.,1974)、Rps3 (Mueller et al.,1978)、Rps4(Athow et al.,
1980), Rps5 (Buzzell and Anderson., 1981), Rps6 (Athow and Laviolette., 1982), Rps7
(Anderson and Buzzell., 1992), Rps8 (Gordon et al., 2006；Sandhu et al., 2005), soybean product
Kind of Waseshiroge Rps gene (Sugimoto et al., 2011), RpsUN1 and RpsUN2 (Lin et al.,
2013).Shared on the site Rps1 5 allele (Rps1a, 1b, 1c, 1d, 1k) (Bernard et al., 1957；
Buzzell and Anderson, 1992；Mueller et al., 1978), 3 allele are shared on the site Rps3
(Rps3a, 3b and 3c) (Mueller et al., 1978；Ploper et al.,1985).Rps1,Rps7,Waseshiroge
Rps gene and RpsUN1 be positioned on No. 3 chromosomes；Rps2 and RpsUN2 is positioned on No. 16 chromosome.
Rps3 and Rps8 is positioned on No. 13 chromosome；Rps4, Rps5 and Rps6 are positioned on No. 18 chromosome.
So far, China has identified 9 Soybean Resistance epidemic disease genes, i.e. RpsYB30, RpsYD25, RpsZS18,
RpsSN10, Rps9, RpsSu, RpsYD29, Rps10 and RpsJS.Disease-resistant gene RpsYB30 has been positioned in soybean No. 19 dye
Colour solid (Zhu Zhendong, 2007).RpsYD25 is by positioning No. 3 chromosome of soybean (model love grain husk etc., 2009)；RpsZS18 is positioned
On No. 2 chromosome of soybean (Yao Haiyan, 2010)；RpsSN10 be located on No. 13 chromosome of soybean (Yu Anliang,
2010)；Rps9 is positioned on No. 3 chromosome of soybean (Wu et al., 2011).RpsSu has been positioned in soybean No. 10
On chromosome (force dawn tinkling of pieces of jade etc., 2011).RpsYD29 is positioned in No. 3 chromosome (Zhang et al., 2013a).Rps10
It is positioned on No. 17 chromosome of soybean (Zhang et al., 2013b).The RpsJS of latest find is located in No. 18 dyeing
On body (Sun et al., 2013).
Soybean phytophthora group structure is complicated, is easy to produce new virulence type.It is generally acknowledged that the use of Soybean Resistance epidemic disease gene
Service life is 10-15, but the speed that new disease-resistant gene is eliminated is being accelerated.It is new it is therefore desirable to excavate and utilize energetically
Resistance resource and constantly carry out the cumulative of disease-resistant gene, cultivate the lasting soybean varieties of resistance.
As molecular markers development technology continues to develop, different types of molecular labeling is constantly applied to the excavation of gene
In mapping, finer positioning is realized.The features such as SSR marker since its quantity is big, polymorphism is high, easy to operate, is current
A kind of most commonly used molecular labeling of application range.But with the development of sequencing technologies, the polymorphisms such as SNP and Indel are stronger
Molecular labeling is developed and is applied in the positioning of gene.The completion of soybean genome sequencing is the fine fixed of soybean gene
The exploitation of position, clone and functional label is provided convenience.By the sequence information of soybean genome, design and using SSR and
SNP marker has been widely applied to the positioning of soybean gene, the exploitation of functional label.
Soybean varieties precocity 18 is
Female parent, 7821 (Shade-enduring black soybeans × mutagenesis 31) are that male parent progress composite-crossing breeding forms；Precocious No. 18 highly resistance mosaic virus,
Graywall and purple spot.It is resistant to lodging, cracking resistance pod.The precocious 18 pairs of soybean phytophthoras of Zhu Zhendong etc. (2006) discovery have resistance of wide spectrum.
Yao Haiyan etc. (2010) identifies an anti-epidemic disease gene RpsZS18 in precocity 18, and the assignment of genes gene mapping is dyed at soybean No. 2
Between body (LG D1b) SSR marker Sat_069 and Sat_183.
Summary of the invention
The object of the present invention is to provide one with the Soybean Resistance epidemic disease gene RpsZS18 molecular labeling isolated and its answer
With.
In order to achieve the object of the present invention, the of the invention one molecule mark isolated with Soybean Resistance epidemic disease gene RpsZS18
Remember that ZCindel246000, the molecular labeling are located at the SSR marker ZCSSR33 (0.7cM) and ZCSSR46 of No. 2 chromosomes of soybean
Primer pair between (2.8cM), for specific PCR amplifier molecule label ZCindel246000 are as follows:
Upstream primer F:5 '-CAGATAGAGTTTTCCTACACAGG-3 ' and
Downstream primer R:5 '-AGTAGAAGCAAAGAGGGACT-3 '.
The genetic distance of molecular labeling ZCindel266000 and gene RpsZS18 is 0cM.
The annealing temperature that PCR amplification uses is 58.4 DEG C, and amplified production size is 279bp (sequence 1).
The present invention also provides the molecular labeling ZCindel266000 to identify the application in anti-epidemic disease soybean varieties, packet
Include following steps:
1) genomic DNA of soybean to be measured is extracted；
2) using the genomic DNA of soybean to be measured as template, using above-mentioned primers F and R, pcr amplification reaction is carried out；
3) pcr amplification product is detected, if it is possible to amplify the product of 279bp, then determine that soybean to be measured is anti-epidemic disease product
Kind.
Wherein, the amplification system that pcr amplification reaction uses is calculated as with 10 μ l: Soybean genomic DNA template concentrations 2ng/ μ
L, 2 × PCR MasterMix (Tiangen) 4 μ l, primers F and each 0.2 μm of ol/L of R, use ddH2O complements to 10 μ l.
The reaction condition that pcr amplification reaction uses are as follows: 95 DEG C 3 minutes；94 DEG C 45 seconds, 58.4 DEG C 45 seconds, 72 DEG C 45 seconds,
Totally 35 circulations；72 DEG C 10 minutes.
The present invention also provides the kit for detecting anti-epidemic disease soybean for PCR, containing above-mentioned for spy in the kit
The primer pair of specific PCR amplification molecular labeling ZCindel246000.Preferably, the kit further includes dNTPs, Taq DNA
Polymerase, Mg2+, PCR reaction buffer, one of standard positive template etc. or a variety of.
The present invention further provides the molecular labeling ZCindel266000 answering in Soybean Resistance epidemic disease molecular breeding
With.
Specifically, the molecular labeling of the invention isolated with Soybean Resistance epidemic disease gene RpsZS18 obtains by the following method
:
It is mapping population using F3 derived from Williams and precocious 18 cross combinations, according to anti-epidemic disease gene RpsZS18
First positioning result, new SSR marker is designed and developed in the positioning section and utilizes the SSR announced in positioning section
Label screens on parent and anti-, sense pond all with the molecular labeling of polymorphism, the SSR marker with polymorphism is carried out group
Experience card constructs new linkage map, realizes finely positioning.In new positioning section interior prediction candidate gene, to precocious 18 Hes
The candidate gene site of Williams is sequenced, exploitation Indel label, and carries out group's verifying, is obtained and gene RpsZS18
The label isolated.To provide assisted Selection for soybean blight-resistant breeding, breeding efficiency is improved, accelerates blight-resistant breeding work
Process.
Specific embodiment:
(1) resistant analysis of precocity 18
It is male parent with disease-resistant variety precocity 18, susceptible variety Williams (rps) is as maternal, hybridization generation F1Generation, F1Generation
Selfing generates F2Generation, F275 F that generation selfing generates3Resistant analysis, disease-resistant gene identification and the mapping population of family.It adopts
Resisted with the reaction of hypocotyl wound inoculation method, 20-25 plant pair soybean phytophthora bacterial strain PsUSAR2 of each Parentage determination
Property genetic analysis.At 24 DEG C after inoculation, under conditions of humidity is 100%, moisturizing 48 hours, it is then passed to hot-house culture, after 4 days
Carry out Disease investigation.Referring to (2006) method evaluation criterions such as Gordon, F is investigated3Disease-resistant, the separation, susceptible family of family
Segregation ratio, the genetics of resistance rule of the precocious 18 pairs of soybean blights of research.
(2) building of the extraction of gene DNA and anti-sense pond
The genomic DNA of 2 parents and 75 F3 familys is extracted using CTAB method.Each F3Family takes 20 single plants respectively
Leaf sample, mixed in equal amounts extract DNA.10 disease-resistant, susceptible family buildings are taken to use according to Michelmore etc. (1991) method
In disease-resistant, the susceptible pond of segregating population fractional analysis (Bulk Segregation Analysis, BSA).
(3) disease-resistant gene of precocity 18 is positioned using SSR marker
According to Yao Haiyan (2010) positioning result, SSR marker Sat_069 and Sat_ are downloaded in http://soybase.org
Gene order between 183.SSR marker is designed and developed according to gene order and is marked using the SSR announced in the section
Note carries out PCR amplification and polymorphism screening between 18 and Williams of precocity, between disease-resistant, susceptible pond.In parent and it will resist
SSR marker between sick, susceptible pond with polymorphism carries out group's verifying.To F3Population resistance separation and SSR marker are in F3Family
Clastotype in system carries out X2It examines.With the linkage relationship of 3.0 software of mapmaker analysis SSR marker and disease-resistant gene.Make
With MapDraw software building genetic linkage maps, new positioning section is obtained.PCR reaction system is 10 μ l, contains 2 × Taq
4 μ l of PCR MasterMix (Tiangen), each 0.2 μm of ol/L of upstream and downstream primer, template DNA concentration are 2ng/ μ l, use ddH2O
Complement to 10 μ l.
PCR react amplification program: 95 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 45 seconds, and 55 DEG C are annealed 45 seconds, and 72 DEG C extend 45
Second, totally 35 recycle；72 DEG C 10 minutes, 10 DEG C preservation.Amplified production carried out in 8-12% polyacrylamide gel electrophoresis and
Analysis.
(4) prediction of candidate gene and the discovery of label is isolated
The information for annotating gene in new positioning section is searched in http://soybase.org, finds gene
Glyma.02g246000 is the Ser/Thr protein kinase type with typical disease-resistant gene structure.By 18 and Williams of precocity
The corresponding site middle gene Glyma.02g246000 is sequenced.The site gene Glyma.02g246000 is compared in anti-, sense parent
Sequencing result in this develops Indel by difference site and marks ZCindel246000.Progress group is marked to test Indel
Card finds it to isolate label.Glyma.02g246000 is possible for controlling the candidate gene model of precocious 18 resistances.
Soybean blight is one of Soybean production process destructive disease.The present invention utilizes SSR and Indel molecule mark
Note method carries out finely positioning to Soybean Resistance epidemic disease gene RpsZS18, and finds that it isolates the molecule mark with close linkage
Note.The disease-resistant gene has resistance of wide spectrum to soybean phytophthora, and the generation of China's soybean blight can be effectively controlled, mitigate the disease and make
At production loss.Using the Indel molecular labeling ZCIndel246000 isolated with the gene, it can effectively be applied to molecule
In marker-assisted breeding, the disadvantage that the period needed for conventional breeding methods is long is overcome；Based on PCR technology, indoors to soybean resource
Carry out Analysis of Resistance；It lays the foundation simultaneously to clone the gene, this has the molecular genetics mechanism for further appreciating that the gene
It is significant, it practices in Soybean production, there is important value in breeding work and disease-resistant theoretical research.
The present invention has the advantages that
(1) present invention has for the first time carried out the Soybean Resistance epidemic disease gene RpsZS18 found on No. 2 chromosomes finely fixed
Position.Gene pairs China soybean phytophthora has resistance of wide spectrum, and the generation of China's soybean blight can be effectively controlled, mitigate the disease and make
At production loss.
(2) the molecular labeling ZCindel246000 provided by the invention isolated with anti-epidemic disease gene RpsZS18 is
The molecular labeling obtained in precocity 18 and its filial generation family of resistance.Can be used for the identification of anti-soybean blight resource and
The molecular mark of anti-soybean blight offspring.
(3) the molecular labeling ZCindel246000 provided by the invention isolated with anti-epidemic disease gene RpsZS18 is
The clone of the gene and sequencing lay the foundation, thus the structure feature of the clearly sequence information.In addition, according to the molecule isolated
Label and soybean genomic sequence information, carry out the haplotype analysis of the gene, are expected to clone and obtain new disease-resistant gene.
Detailed description of the invention
Fig. 1 is 1 mid-early maturity 18 (left side 3) of the embodiment of the present invention and Williams (right side 3) inoculation soybean phytophthora bacterial strain PsUSAR2
4 days Disease investigation results afterwards.
Fig. 2 is Soybean Resistance epidemic disease gene RpsZS18 genetic linkage maps in the embodiment of the present invention 1.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.
The candidate gene of 1 Soybean Resistance epidemic disease gene RpsZS18 of embodiment and its molecular labeling isolated
The acquisition of ZCindel246000
1, the resistant analysis of precocity 18
It is male parent with disease-resistant variety precocity 18, susceptible variety Williams (rps) is as maternal, hybridization generation F1Generation, F1Generation
Selfing generates F2Generation, F275 F that generation selfing generates3Resistant analysis, disease-resistant gene identification and the mapping population of family.It adopts
With hypocotyl wound inoculation method, the reaction of 20-25 plant pair soybean phytophthora bacterial strain PsUSAR2 of each Parentage determination is carried out
Resistant analysis.At 24 DEG C after inoculation, under conditions of humidity is 100%, moisturizing 48 hours, then incoming hot-house culture, 4 days
Carry out Disease investigation (Fig. 1) afterwards.Referring to (2006) method evaluation criterions such as Gordon, it is specified that the plant death rate is less than 20%
Family is pure and mild disease-resistant family, and it is pure and mild susceptible family that the death rate, which is greater than 80%, and the family that the death rate is 21-79% is anti-
Property separation family, investigate F3The anti-of family, separation, susceptible family segregation ratio, the resistance of the precocious 18 pairs of soybean blights of research loses
Pass rule.
F derived from 18 cross combinations of 75 Williams and precocity3Resistance Identification result table of the family to bacterial strain PsUSAR2
Bright, pure and mild disease-resistant family is 18, and pure and mild susceptible family is 14, and heterozygous family is 43.Through X2Inspection meets desired
The segregation ratio of 1:2:1.
2, the extraction of Soybean genomic DNA and the building of anti-sense pond
2 parents and 75 F are extracted using CTAB method3The genomic DNA of family.Each F3Family takes 20 single plants respectively
Leaf sample, mixed in equal amounts are put into the centrifuge tube of 2.0ml.As for grind into powder in liquid nitrogen, 800 μ l are added into centrifuge tube
The CTAB Extraction buffer of 65 DEG C of preheatings, saves 30-50 minutes in 65 DEG C of water-baths after mixing；Centrifuge tube is taken out, room is cooled to
800 μ l phenol: chloroform: isoamyl alcohol (volume ratio 25:24:1) are added in temperature, extract 10 minutes, gently sway, mix well it,
12000rpm is centrifuged 10 minutes；It takes supernatant into another 1.5ml centrifuge tube, isometric chloroform: isoamyl alcohol (volume is added
Than 24:1), after mixing well 10 minutes, 12000 centrifugations 10 minutes；It takes supernatant into another 1.5ml centrifuge tube, is added 0.8
The isopropanol of times volume pre-cooling, slowly mixes, with -20 DEG C at place 30-60 minutes, make DNA with Precipitation；It is heavy to choose DNA
Shallow lake is transferred in 1.5ml centrifuge tube, with 75% ethanol wash 2 times, is finally dehydrated with dehydrated alcohol, drying, such as appropriate sterilizing of family
Ultrapure water.After DNA dissolution, detected with the integrality and ultraviolet spectrophotometry of 0.8% agarose gel electrophoresis detection DNA
The concentration of DNA sample.
Segregating population fractional analysis (Bulk is used for according to the methods of Michelmore etc. (1991) building
Segregating Analysis, BSA) anti-, sense pond.Respectively take the DNA of 10 disease-resistant familys of 1 μ l be mixed in one it is new from
In heart pipe, it is built into anti-pond.Pond construction method is felt referring to anti-pond construction method.
3, the disease-resistant gene of precocity 18 is positioned using SSR marker
According to Yao Haiyan (2009) positioning result, SSR marker Sat_069 and Sat_ are downloaded from http://soybase.org
Gene order between 183.176 pairs of SSR markers are designed and developed according to gene order and using having announced in the section
23 pairs of SSR markers resist between 18 and Williams of precocity, carry out PCR amplification and polymorphism screening between sense pond.It will be in parent
This SSR marker with polymorphism between anti-, sense pond carries out group's verifying.To F3Population resistance separation and SSR marker are in F3Family
Clastotype in system carries out X2It examines.With the linkage relationship of 3.0 software of mapmaker analysis SSR marker and disease-resistant gene.
Using MapDraw software building genetic linkage maps, new positioning section is obtained.PCR reaction system is 10 μ l, contains 2 × Taq
4 μ l of PCR MasterMix, each 0.2 μm of ol/L of upstream and downstream primer, template DNA concentration are 2ng/ μ l, use ddH2O supplies 10 μ
l。
PCR react amplification program: 95 DEG C initial denaturation 3 minutes；94 DEG C are denaturalized 45 seconds, and 55 DEG C are annealed 45 seconds, and 72 DEG C extend 45
Second, totally 35 recycle；72 DEG C 10 minutes, 10 DEG C preservation.Amplified production carried out in 8-12% polyacrylamide gel electrophoresis and
Analysis.
It is verified in newly-designed 176 couples of SSR by parental line selection and group.There are 13 pairs there is polymorphism in group；
There are 4 betweens there is polymorphism (table 1) group in the 23 pairs of primers searched in the genome.Wherein 16 labels are equal in group
The distribution of 1:2:1 is presented, 1 label ZCSSR8 is dominant marker, is distributed in 3:1.
Using 3.0 software of mapmaker analysis shows, this 17 pairs of polymorphism SSR primers and RpsZS18 chain (table 1).
Using MapDraw Software on Drawing genetic linkage maps, RpsZS18 is positioned between label ZCSSR33 and ZCSSR46, heredity
Distance respectively 0.7cM and 2.8cM, while being isolated with SSR marker Satt172, the genetic distance with RpsZS18 is 0cM
(Fig. 2).
4, the prediction of candidate gene and the discovery of label is isolated
It is analysed and compared in http://soybase.org database it is found that in label ZCSSR33 and ZCSSR46
Between have 16 prediction genetic models (table 2), 9 have functional annotation, wherein there is a genetic model
The albumen of the disease-resistant relevant Ser/Thr kinase domain of Glyma.02g246000 coded plant.By design overlapping primers to morning
The site Glyma.02g246000 and its upstream and downstream 1000bp sequence in ripe 18 and Williams carry out PCR amplification, to PCR
Amplified production is sequenced.The site for comparing Glyma.02g246000 in 18 and Williams of precocity finds that there are SNP multiple
Point and insertion and missing.Upstream and downstream primer development is designed according to the missing of one section of 13bp in the 3 ˊ-UTR region of its precocity 18
Indel marks ZCindel246000, the primer pair for specific PCR amplifier molecule label ZCindel246000 are as follows:
Upstream primer F:5 '-CAGATAGAGTTTTCCTACACAGG-3 ' (Seq ID No.1)
Downstream primer R:5 '-AGTAGAAGCAAAGAGGGACT-3 ' (Seq ID No.2)
Group's verifying is carried out with Indel label ZCindel246000, genetic linkage is calculated by 3.0 software of mapmaker
Distance constructs genetic linkage maps (Fig. 2) using MapDraw.The genetic distance of ZCindel246000 and RpsZS18 is 0cM,
Label ZCindel246000 is to isolate molecular labeling, and Glyma.02g246000 is possible for controlling the candidate of precocious 18 resistances
Genetic model.
Carry out group's verifying the following steps are included:
1) genomic DNA of soybean to be measured is extracted；
2) using the genomic DNA of soybean to be measured as template, using above-mentioned primers F and R, pcr amplification reaction is carried out；
3) pcr amplification product is detected, if it is possible to which the product (Seq ID No.3) for amplifying 279bp then determines to be measured big
Beans are anti-epidemic disease kind.
Wherein, the amplification system that pcr amplification reaction uses is calculated as with 10 μ l: Soybean genomic DNA template concentrations 2ng/ μ
L, 2 × PCR MasterMix (Tiangen) 4 μ l, primers F and each 0.2 μm of ol/L of R, use ddH2O complements to 10 μ l.
The reaction condition that pcr amplification reaction uses are as follows: 95 DEG C 3 minutes；94 DEG C 45 seconds, 58.4 DEG C 45 seconds, 72 DEG C 45 seconds,
Totally 35 circulations；72 DEG C 10 minutes.
The nucleotide of Soybean Resistance epidemic disease gene RpsZS18, candidate gene Glyma.02g246000 involved in the present invention
Sequence is respectively as shown in Seq ID No.4 and 5.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Bibliography
[1] Yao Haiyan, Wang Xiaoming, the SSR molecular marker of the precocious 18 resistant to phytophthora root rot genes of the soybean varieties such as military small phenanthrene
[J] plant genetic resources journal, 2010,11 (2): 213-217.
[2]MICHEMORE R W；PARAN I；KESSELI R V.Identification of markers linked
to disease resistance genes by bulk segregant analysis:a rapid method to
detect markers in specific genomic regions by using segregating populations
[J].Proceedings of the National Academy of Sciences of the United States of
America,1991,88:9823-9832.DOI:10.1073/pnas.88.21.9823.
[3]Gordon SG,St Martin SK,Dorrance AE(2006)Rps8 maps to a resistance
gene rich region on soybean molecular linkage group F.Crop Sci 46:168–173.
Claims (8)
1. the molecular labeling ZCindel246000 isolated with Soybean Resistance epidemic disease gene RpsZS18, which is characterized in that described point
Son label is located between the SSR marker ZCSSR33 and ZCSSR46 of No. 2 chromosomes of soybean, is used for specific PCR amplifier molecule mark
Remember the primer pair of ZCindel246000 are as follows:
Upstream primer F:5 '-CAGATAGAGTTTTCCTACACAGG-3 ' and
Downstream primer R:5 '-AGTAGAAGCAAAGAGGGACT-3 '；
Amplified production size is 279bp；
Wherein, the information of SSR marker ZCSSR33 and ZCSSR46 is as follows:
The start stop bit that SSR marker ZCSSR33 is located at No. 2 chromosomes of soybean is set to 43410490~43410642bp, for expanding
The primer for marking ZCSSR33 is upstream primer 5 '-CATTATTTCTTGTGGGT-3 ' A and downstream primer 5 '-
TGACAACATAGAACCAAA-3 ', amplified production size are 153bp；
The start stop bit that SSR marker ZCSSR46 is located at No. 2 chromosomes of soybean is set to 43522898~43523091bp-3 ', for expanding
The primer for increasing label ZCSSR46 is upstream primer 5 '-AAAGGGAGAAGCAAGTAAT-3 ' and downstream primer 5 '-
TCGCAAACAGTAAACACG-3 ', amplified production size are 194bp.
2. molecular labeling according to claim 1, which is characterized in that carrying out the annealing temperature that PCR amplification uses is 58.4
℃。
3. molecular labeling as claimed in claim 1 or 2 is identifying the application in anti-epidemic disease soybean varieties, which is characterized in that including with
Lower step:
1) genomic DNA of soybean to be measured is extracted；
2) using the genomic DNA of soybean to be measured as template, using as claimed in claim 1 or 22 primers Fs and R, it is anti-to carry out PCR amplification
It answers；
3) pcr amplification product is detected, if it is possible to amplify the product of 279bp, then determine that soybean to be measured is anti-epidemic disease kind.
4. application according to claim 3, which is characterized in that the amplification system that pcr amplification reaction uses in step 2) with
10 μ l are calculated as: 4 μ l of Soybean genomic DNA template concentrations 2ng/ μ l, 2 × PCR MasterMix, primers F and each 0.2 μm of ol/ of R
L uses ddH2O complements to 10 μ l.
5. application according to claim 3, which is characterized in that the reaction condition that pcr amplification reaction uses in step 2) are as follows:
95 DEG C 3 minutes；94 DEG C 45 seconds, 58.4 DEG C 45 seconds, 72 DEG C 45 seconds, totally 35 circulation；72 DEG C 10 minutes.
6. detecting the kit of anti-epidemic disease soybean for PCR, which is characterized in that contain claims 1 or 2 institute in the kit
The primer pair for specific PCR amplifier molecule label ZCindel246000 stated；Wherein, it is used for specific PCR amplifier molecule
Mark the primer pair of ZCindel246000 are as follows:
Upstream primer F:5 '-CAGATAGAGTTTTCCTACACAGG-3 ' and
Downstream primer R:5 '-AGTAGAAGCAAAGAGGGACT-3 '.
7. kit according to claim 6, which is characterized in that the kit further includes dNTPs, Taq DNA polymerization
Enzyme, Mg2+, PCR reaction buffer and standard positive template.
8. application of the molecular labeling as claimed in claim 1 or 2 in Soybean Resistance epidemic disease molecular breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410415801.4A CN105462963B (en) | 2014-08-21 | 2014-08-21 | With the Soybean Resistance epidemic disease gene RpsZS18 molecular labeling isolated and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410415801.4A CN105462963B (en) | 2014-08-21 | 2014-08-21 | With the Soybean Resistance epidemic disease gene RpsZS18 molecular labeling isolated and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105462963A CN105462963A (en) | 2016-04-06 |
| CN105462963B true CN105462963B (en) | 2019-04-16 |
Family
ID=55601124
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410415801.4A Expired - Fee Related CN105462963B (en) | 2014-08-21 | 2014-08-21 | With the Soybean Resistance epidemic disease gene RpsZS18 molecular labeling isolated and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105462963B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110273022B (en) * | 2019-08-01 | 2023-07-18 | 安阳市农业科学院 | Molecular marker, primer, detection method and application for detecting soybean phytophthora root rot resistance gene |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003094601A1 (en) * | 2002-05-10 | 2003-11-20 | The Ohio State University Research Foundation | Identification of soybeans having resistance to phytophthora sojae |
| CN101942520A (en) * | 2010-10-14 | 2011-01-12 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN102994498A (en) * | 2012-11-23 | 2013-03-27 | 中国农业科学院作物科学研究所 | Molecular marker for co-separating soybean anti-phytophthora megasperma candidate gene RpsWD15-1 and application of molecular marker for co-separating soybean anti-phytophthora megasperma candidate gene RpsWD15-1 |
-
2014
- 2014-08-21 CN CN201410415801.4A patent/CN105462963B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003094601A1 (en) * | 2002-05-10 | 2003-11-20 | The Ohio State University Research Foundation | Identification of soybeans having resistance to phytophthora sojae |
| CN101942520A (en) * | 2010-10-14 | 2011-01-12 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN102994498A (en) * | 2012-11-23 | 2013-03-27 | 中国农业科学院作物科学研究所 | Molecular marker for co-separating soybean anti-phytophthora megasperma candidate gene RpsWD15-1 and application of molecular marker for co-separating soybean anti-phytophthora megasperma candidate gene RpsWD15-1 |
Non-Patent Citations (3)
| Title |
|---|
| Genetic characterization and fine mapping of the novel Phytophthora resistance gene in a Chinese soybean cultivar;Zhang, Jiqing et al.;《THEORETICAL AND APPLIED GENETICS》;20130630;第126卷(第6期);第1555-1561页 * |
| Identification and Candidate Gene Analysis of a Novel Phytophthora Resistance Gene Rps10 in a Chinese Soybean Cultivar;Zhang, Jiqing et al.;《PLOS ONE》;20130725;第8卷(第7期);第e69799页 * |
| 大豆品种早熟18抗疫霉根腐病基因的SSR分子标记;姚海燕;《中国优秀硕士学位论文全文数据库 农业科技辑》;20100715(第7期);第D047-194页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105462963A (en) | 2016-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Alves-Santos et al. | A DNA-based procedure for in planta detection of Fusarium oxysporum f. sp. phaseoli | |
| RU2748688C2 (en) | Genetic locus associated with root and stem rot due to phytophthora in soy | |
| CN109628627A (en) | The SNP marker development and application of broad-spectrum rice-blast resistant gene of paddy rice Pigm | |
| Pattison et al. | Inheritance of Phytophthora root rot resistance in red raspberry determined by generation means and molecular linkage analysis | |
| CN114480709B (en) | Molecular marker for detecting wheat leaf rust resistance gene Lr47, detection method and application thereof | |
| CN110205396B (en) | SNP marker closely linked with irregular stripe character of cucumber fruit and application thereof | |
| CN116516049A (en) | Soybean protein content QTL locus qPRO_11_1, molecular marker and application thereof | |
| CN107385053A (en) | A kind of four primer molecule labeling methods identified rice and produce als gene mutation | |
| Vargas et al. | Polymorphisms in VvPel associate with variation in berry texture and bunch size in the grapevine | |
| CN107058519A (en) | Molecular labeling CAPs1516 and its application with Soybean Resistance epidemic disease gene close linkage | |
| CN108060162A (en) | SNP marker and its application with cucumber anti-epidemic disease character close linkage | |
| Cho et al. | Development of AFLP and CAPS markers linked to the scab resistance gene, Rvn2, in an inter-specific hybrid pear (Pyrus spp.) | |
| CN105462963B (en) | With the Soybean Resistance epidemic disease gene RpsZS18 molecular labeling isolated and its application | |
| El-Khishin et al. | AFLP fingerprinting of some Egyptian date palm (Phoenix dactylifera L.) cultivars | |
| US10590491B2 (en) | Molecular markers associated with Mal de Rio Cuarto Virus in maize | |
| CN111944920B (en) | InDel marker closely linked with melon epidemic disease resistance gene and application thereof | |
| EP2242850A1 (en) | Maize plants characterised by quantitative trait loci (qtl) | |
| Park et al. | Development of SNP markers for the identification of commercial Korean watermelon cultivars using Fluidigm genotyping analysis | |
| CN109722488B (en) | SCAR molecular marker linked with root knot nematode resistant gene Me3 and application thereof | |
| CA2804853A1 (en) | Soybean gene cluster regions associated with aphid resistance and methods of use | |
| CN113736866A (en) | SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof | |
| CN102994498B (en) | Molecular marker for co-separating soybean anti-phytophthora megasperma candidate gene RpsWD15-1 and application of molecular marker for co-separating soybean anti-phytophthora megasperma candidate gene RpsWD15-1 | |
| CN107130019B (en) | Molecular marker primer of rice local variety thin rice panicle blast resistance gene and application thereof | |
| CN104328114B (en) | Magnaporthe oryzae avirulence gene AvrPib specific molecular marker, and method and application thereof | |
| CN102965369B (en) | Molecule mark coseparated from candidate gene RpsWD15-2 for phytophthora root rot of soybean and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | 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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190416 Termination date: 20190821 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |