CN105462963A - Molecular marker coseparated from soybean epidemic disease resisting gene RpsZS18, and application thereof - Google Patents
Molecular marker coseparated from soybean epidemic disease resisting gene RpsZS18, and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker ZCindel246000 coseparated from a soybean epidemic disease resisting gene RpsZS18, and an application thereof. The marker is positioned between the SSR markers ZCSSR33 and ZCSSR46 of a soybean No.2 chromosome, and has a genetic distance of 0cM to the gene RpsZS18. A new SSR marker is exploited by using a soybean SSR marker in an interval and using a soybean genome sequence according to a preliminary positioning result with an F3 column generated through hybridizing and selfing disease resisting variety Zaoshu 18 used as a male parent and a susceptible variety Williams used as a female parent as a mapping colony, and a disease resisting gene contained in the Zaoshu 18 is finely positioned. The disease resisting gene RpsZS18 is positioned between the ZCSSR33 (0.7cM) and ZCSSR46 (2.8cM) of the No.2 chromosome, and is coseparated from an SSR marker Satt172. An RpsZS18 candidate gene is predicated according to gene note in the new positioning interval, the Indel marker ZCindel24600 is exploited according to the candidate gene site sequence difference predicated by resistant and susceptible parents, a colony verification result shows that the marker is a coseparated marker, and the Indel marker can be used in auxiliary breeding of soybean epidemic disease resistance.
Description
Technical field
The invention belongs to agricultural biotechnology engineering and disease-resistant crops genetic breeding field, specifically, relate to a kind of be divided into Soybean Resistance epidemic disease gene RpsZS18 from molecule marker and application.
Background technology
The soybean blight caused by soybean phytophthora (PhytophthorasojaeKaufmann & Gerdemann) is a kind of destructive disease in Soybean production.Province occurs this disease in Heilongjiang Province of China, Anhui, Fujian, Jilin, Xinjiang etc.Soybean phytophthora all can infect soybean soybean all breeding times, causes root-rot, stem rot, plant is downgraded, withered and dead, and general field underproduction 10-30%, serious plot can reach 60-90%, even causes total crop failure.The financial loss that the current whole world causes because of phytophthora root rot of soybean is every year approximately 1,000,000,000 dollars (Tyler, 2007).
Control soybean blight is the most economical, effective and the method for environmental safety is plantation disease-resistant variety.At present, 8 locus abroad in soybean gene group identify 17 resistant genes.Be respectively Rps1 (Bernardetal., 1957), Rps2 (Kilenetal., 1974), Rps3 (Muelleretal., 1978), Rps4 (Athowetal., 1980), Rps5 (Buzzell and Anderson., 1981), Rps6 (Athow and Laviolette., 1982), Rps7 (Anderson and Buzzell., 1992), Rps8 (Gordonetal., 2006; Sandhuetal., 2005), Rpsgene (Sugimotoetal., 2011), RpsUN1 and RpsUN2 (Linetal., 2013) of soybean varieties Waseshiroge.Rps1 site has 5 allelotrope (Rps1a, 1b, 1c, 1d, 1k) (Bernardetal., 1957; Buzzell and Anderson, 1992; Muelleretal., 1978), Rps3 site has 3 allelotrope (Rps3a, 3b and 3c) (Muelleretal., 1978; Ploperetal., 1985).Rpsgene and RpsUN1 of Rps1, Rps7, Waseshiroge is positioned on No. 3 karyomit(e)s; Rps2 and RpsUN2 is positioned on No. 16 karyomit(e).Rps3 and Rps8 is positioned on No. 13 karyomit(e); Rps4, Rps5 and Rps6 are positioned on No. 18 karyomit(e).
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 karyomit(e) (Zhu Zhendong, 2007).RpsYD25 is located soybean No. 3 karyomit(e) (Fan Aiying etc., 2009); RpsZS18 has been positioned on soybean No. 2 karyomit(e) (Yao Haiyan, 2010); RpsSN10 is located on soybean No. 13 karyomit(e) (Yu Anliang, 2010); Rps9 is positioned on soybean No. 3 karyomit(e) (Wuetal., 2011).RpsSu has been positioned on soybean No. 10 karyomit(e) (Wu Xiaoling etc., 2011).RpsYD29 is positioned in No. 3 karyomit(e) (Zhangetal., 2013a).Rps10 is positioned on soybean No. 17 karyomit(e) (Zhangetal., 2013b).The RpsJS of latest find is positioned at (Sunetal., 2013) on No. 18 karyomit(e)s.
Soybean phytophthora group structure is complicated, easily produces new virulence type.It is generally acknowledged that the work-ing life of Soybean Resistance epidemic disease gene is 10-15, but the speed that new disease-resistant gene is eliminated is accelerated.Therefore be necessary to excavate and utilize new Resistance resource energetically and constantly carry out the cumulative of disease-resistant gene, cultivating the soybean varieties that resistance is lasting.
Along with molecular markers development technology development, dissimilar molecule marker be constantly applied to gene excavation and mapping in, realize meticulousr location.SSR marker due to its quantity large, the features such as polymorphism is high, simple to operate are current range of application the most a kind of molecule marker.But along with the development of sequencing technologies, the molecule marker that the polymorphisms such as SNP and Indel are stronger is developed and is applied in the location of gene.The exploitation completed as the Fine Mapping of soybean gene, clone and functional label of soybean genome sequencing is provided convenience.By the sequence information of soybean gene group, SSR and SNP marker designed and utilized, the location of soybean gene, the exploitation of functional label have been widely applied to.
Soybean varieties precocity 18 is Institute of Genetics, Academia Sinica's nineteen eighty-twos is female parent with 7902 (Clarke 63 × mutagenesis 30), and 7821 (Shade-enduring black soybean × mutagenesis 31) form for male parent carries out composite hybridization seed selection; Precocious No. 18 high resistance mosaic virus, graywall and anaphylactoid purpuras.Resistant to lodging, cracking resistance pod.Zhu Zhendong etc. (2006) find that precocious 18 pairs of soybean phytophthoras have resistance of wide spectrum.Yao Haiyan etc. (2010) are a qualification anti-epidemic disease gene RpsZS18 in precocious 18, and by this assignment of genes gene mapping soybean No. 2 karyomit(e)s (LGD1b) between SSR marker Sat_069 and Sat_183.
Summary of the invention
The object of this invention is to provide one with Soybean Resistance epidemic disease gene RpsZS18 be divided into from molecule marker and application.
In order to realize the object of the invention, of the present invention one with Soybean Resistance epidemic disease gene RpsZS18 be divided into from molecule marker ZCindel246000, described molecule marker is positioned between soybean No. 2 chromosomal SSR marker ZCSSR33 (0.7cM) and ZCSSR46 (2.8cM), and the primer pair for specific PCR amplifier molecule mark ZCindel246000 is:
Upstream primer F:5 '-CAGATAGAGTTTTCCTACACAGG-3 ' and
Downstream primer R:5 '-AGTAGAAGCAAAGAGGGACT-3 '.
The genetic distance of molecule marker 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 application of described molecule marker ZCindel266000 in the anti-epidemic disease soybean varieties of qualification, comprises the following steps:
1) genomic dna of soybean to be measured is extracted;
2) with the genomic dna of soybean to be measured for template, utilize above-mentioned primers F and R, carry out pcr amplification reaction;
3) detect pcr amplification product, if the product of 279bp can be amplified, then judge that soybean to be measured is as anti-epidemic disease kind.
Wherein, the amplification system that pcr amplification reaction uses is counted with 10 μ l: Soybean genomic DNA template concentrations 2ng/ μ l, 2 × PCRMasterMix (Tiangen) 4 μ l, primers F and each 0.2 μm of ol/L of R, use ddH
2o complements to 10 μ l.
Pcr amplification reaction use reaction conditions be: 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 is also provided for the test kit that PCR detects anti-epidemic disease soybean, containing the above-mentioned primer pair for specific PCR amplifier molecule mark ZCindel246000 in described test kit.Preferably, described test kit also comprises dNTPs, Taq DNA polymerase, Mg
2+, PCR reaction buffer, one or more in standard positive template etc.
The present invention further provides the application of described molecule marker ZCindel266000 in Soybean Resistance epidemic disease molecular breeding.
Particularly, of the present invention be divided into Soybean Resistance epidemic disease gene RpsZS18 from molecule marker obtain by the following method:
The F3 utilizing Williams and precocious 18 cross combinations to derive is mapping population, according to the first positioning result of anti-epidemic disease gene RpsZS18, in this positioning area, design and develop new SSR marker and utilize the SSR marker announced in positioning area, screen at parent and anti-, that sense Chi Shangdou has polymorphism molecule marker, the SSR marker with polymorphism is carried out colony's checking and build new linkage map, realize Fine Mapping.Predicting candidate gene in the new positioning area, checks order to the candidate gene site of precocious 18 and Williams, exploitation Indel mark, and carries out colony's checking, obtain to be divided into gene RpsZS18 from mark.Thus provide assisted Selection for Soybean Resistance epidemic disease resistant breeding, improve breeding efficiency, accelerate the blight-resistant breeding progress of work.
Specific embodiments:
(1) resistant analysis of precocious 18
With disease-resistant variety precocity 18 for male parent, susceptible variety Williams (rps) is as maternal, and hybridization produces F
1generation, F
1f is produced for selfing
2generation, F
2for 75 F that selfing produces
3resistant analysis, the disease-resistant gene of family are identified and mapping population.Adopt hypocotyl wound inoculation method, resistant analysis is carried out in the reaction of each Parentage determination 20-25 plant to soybean phytophthora bacterial strain PsUSAR2.At 24 DEG C after inoculation, humidity is under the condition of 100%, and moisturizing 48 hours, then imports hot-house culture into, carries out Disease investigation after 4 days.With reference to (2006) method evaluation standards such as Gordon, investigation F
3family disease-resistant, be separated, the segregation ratio of susceptible family, the genetics of resistance rule of the precocious 18 pairs of soybean blights of research.
(2) extraction of gene DNA and structure that is anti-, sense pond
CTAB method is adopted to extract the genomic dna of 2 parents and 75 F3 familys.Each F
3family gets 20 single-strain blade samples respectively, and balanced mix extracts DNA.Disease-resistant, the susceptible pond of 10 disease-resistant, susceptible familys structures for separating of colony's fractional analysis (BulkSegregationAnalysis, BSA) is got according to (1991) methods such as Michelmore.
(3) SSR marker is utilized to locate the disease-resistant gene of precocious 18
According to Yao Haiyan (2010) positioning result, download the gene order between SSR marker Sat_069 and Sat_183 at http://soybase.org.Design and develop SSR marker according to gene order and utilize the SSR marker announced in this interval, between precocious 18 and Williams, between disease-resistant, susceptible pond, carrying out pcr amplification and polymorphism screening.The SSR marker between parent and disease-resistant, susceptible pond with polymorphism is carried out colony's checking.To F
3population resistance separation and SSR marker are at F
3clastotype in family carries out X
2inspection.With the linkage relationship of mapmaker3.0 software analysis SSR marker and disease-resistant gene.Use MapDraw software building genetic linkage maps, obtain between new positioning area.PCR reaction system is 10 μ l, and containing 2 × TaqPCRMasterMix (Tiangen) 4 μ l, each 0.2 μm of ol/L of upstream and downstream primer, template DNA concentration is 2ng/ μ l, uses ddH
2o complements to 10 μ l.
PCR reacts amplification program: 95 DEG C of denaturations 3 minutes; 94 DEG C of sex change 45 seconds, 55 DEG C of annealing 45 seconds, 72 DEG C extend 45 seconds, totally 35 circulations; 72 DEG C 10 minutes, 10 DEG C of preservations.Amplified production carries out electrophoresis and analysis in 8-12% polyacrylamide gel.
(4) prediction of candidate gene and the discovery of common separation marking
In http://soybase.org, search the information of annotate genes between new positioning area, find that gene Glyma.02g246000 is the Ser/Thr protein kinase type with typical disease-resistant gene structure.Is checked order in site corresponding for gene Glyma.02g246000 in precocious 18 and Williams.Contrast gene Glyma.02g246000 site, at anti-, the sequencing result felt in parent, marks ZCindel246000 by difference site exploitation Indel.Indel mark is carried out colony's checking, finds that it is common separation marking.Glyma.02g246000 is probably for controlling the candidate gene model of precocious 18 resistances.
Soybean blight is a kind of destructive disease in Soybean production process.The present invention utilizes SSR and Indel molecule marking method, carries out Fine Mapping to Soybean Resistance epidemic disease gene RpsZS18, and find its be divided into from closely linked molecule marker.This disease-resistant gene has resistance of wide spectrum to soybean phytophthora, effectively can control the generation of China's soybean blight, alleviate the production loss that this disease causes.Utilize to be divided into this gene from Indel molecule marker ZCIndel246000, can effectively be applied in molecular mark, overcome the long shortcoming of cycle needed for conventional breeding methods; PCR-based technology, carries out Analysis of Resistance in indoor to soybean resource; Simultaneously for this gene of clone lays the foundation, this is significant to the molecular genetics mechanism understanding further this gene, puts into practice, has important value in breeding work and disease-resistant theoretical investigation at Soybean production.
The invention has the advantages that:
(1) the present invention has carried out Fine Mapping to the Soybean Resistance epidemic disease gene RpsZS18 that No. 2 karyomit(e)s find first.This gene pairs China soybean phytophthora has resistance of wide spectrum, effectively can control the generation of China's soybean blight, alleviate the production loss that this disease causes.
(2) provided by the invention and anti-epidemic disease gene RpsZS18 be divided into from molecule marker ZCindel246000, be precocious 18 and resistance filial generation family in the molecule marker that obtains.Can be used for the molecular mark of the qualification of anti-soybean blight resource and anti-soybean blight offspring.
(3) provided by the invention and anti-epidemic disease gene RpsZS18 be divided into from molecule marker ZCindel246000, for the Cloning and sequencing of this gene lays the foundation, thus the constitutional features of clear and definite this sequence information.In addition, according to be divided into from molecule marker and soybean genomic sequence information, carry out the haplotype analysis of this gene, be expected to clone and obtain new disease-resistant gene.
Accompanying drawing explanation
Fig. 1 is that the embodiment of the present invention 1 mid-early maturity 18 (left 3) and Williams (right 3) inoculate 4 days Disease investigation results after soybean phytophthora bacterial strain PsUSAR2.
Fig. 2 is Soybean Resistance epidemic disease gene RpsZS18 genetic linkage maps in the embodiment of the present invention 1.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
The candidate gene of embodiment 1 Soybean Resistance epidemic disease gene RpsZS18 and be divided into from the acquisition of molecule marker ZCindel246000
1, the resistant analysis of precocious 18
With disease-resistant variety precocity 18 for male parent, susceptible variety Williams (rps) is as maternal, and hybridization produces F
1generation, F
1f is produced for selfing
2generation, F
2for 75 F that selfing produces
3resistant analysis, the disease-resistant gene of family are identified and mapping population.Adopt hypocotyl wound inoculation method, resistant analysis is carried out in the reaction of each Parentage determination 20-25 plant to soybean phytophthora bacterial strain PsUSAR2.At 24 DEG C after inoculation, humidity is under the condition of 100%, and moisturizing 48 hours, then imports hot-house culture into, carries out Disease investigation (Fig. 1) after 4 days.With reference to (2006) the method evaluation standard such as Gordon, the family that regulation plant mortality ratio is less than 20% is pure and mild disease-resistant family, mortality ratio be greater than 80% be pure and mild susceptible family, and the family that mortality ratio is 21-79% is Resistant segregation family, investigation F
3family anti-, be separated, the segregation ratio of susceptible family, the genetics of resistance rule of the precocious 18 pairs of soybean blights of research.
The F that 75 Williams and precocious 18 cross combinations derive
3the Resistance Identification result of family to bacterial strain PsUSAR2 shows, pure and mild disease-resistant family is 18, and pure and mild susceptible family is 14, and heterozygous family is 43.Through X
2inspection meets the segregation ratio of the 1:2:1 of expectation.
2, the extraction of Soybean genomic DNA and anti-sense pond build
CTAB method is adopted to extract 2 parents and 75 F
3the genomic dna of family.Each F
3family gets 20 single-strain blade samples respectively, and balanced mix puts into the centrifuge tube of 2.0ml.As for grind into powder in liquid nitrogen, in centrifuge tube, add the CTAB Extraction buffer of 800 μ l65 DEG C preheatings, after mixing, in 65 DEG C of water-baths, preserve 30-50 minute; Take out centrifuge tube, be cooled to room temperature, add 800 μ l phenol: chloroform: primary isoamyl alcohol (volume ratio 25:24:1), extract 10 minutes, sway gently, make it fully mix, centrifugal 10 minutes of 12000rpm; Get supernatant liquor in another 1.5ml centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (volume ratio 24:1), fully mixing is after 10 minutes, 12000 centrifugal 10 minutes; Get supernatant liquor in another 1.5ml centrifuge tube, add the Virahol of 0.8 times of volume precooling, slowly mix, with-20 DEG C at place 30-60 minute, make DNA with Precipitation; Choosing DNA precipitation is transferred in 1.5ml centrifuge tube, and with 75% ethanol wash 2 times, finally dewater with dehydrated alcohol, dry up, family is as the ultrapure water of appropriate sterilizing.After DNA dissolves, detect the integrity of DNA and the concentration of ultraviolet spectrophotometry detection DNA sample with 0.8% agarose gel electrophoresis.
Anti-, the sense pond for separating of colony's fractional analysis (BulkSegregatingAnalysis, BSA) is built according to methods such as (1991) such as Michelmore.The DNA respectively getting 10 disease-resistant familys of 1 μ l is mixed in a new centrifuge tube, is built into anti-pond.Sense pond construction process is with reference to anti-pond construction process.
3, SSR marker is utilized to locate the disease-resistant gene of precocious 18
According to Yao Haiyan (2009) positioning result, download the gene order between SSR marker Sat_069 and Sat_183 from http://soybase.org.Design and develop 176 pairs of SSR marker according to gene order and utilize the 23 pairs of SSR marker announced in this interval, between precocious 18 and Williams, resisting, feeling between pond and carry out pcr amplification and polymorphism screening.The SSR marker between parent and anti-, sense pond with polymorphism is carried out colony's checking.To F
3population resistance separation and SSR marker are at F
3clastotype in family carries out X
2inspection.With the linkage relationship of mapmaker3.0 software analysis SSR marker and disease-resistant gene.Use MapDraw software building genetic linkage maps, obtain between new positioning area.PCR reaction system is 10 μ l, and containing 2 × TaqPCRMasterMix4 μ l, each 0.2 μm of ol/L of upstream and downstream primer, template DNA concentration is 2ng/ μ l, uses ddH
2o supplies 10 μ l.
PCR reacts amplification program: 95 DEG C of denaturations 3 minutes; 94 DEG C of sex change 45 seconds, 55 DEG C of annealing 45 seconds, 72 DEG C extend 45 seconds, totally 35 circulations; 72 DEG C 10 minutes, 10 DEG C of preservations.Amplified production carries out electrophoresis and analysis in 8-12% polyacrylamide gel.
By parental line selection and colony's checking in newly-designed 176 couples of SSR.13 are had to have polymorphism in colony; Genome has 4 to having polymorphism (table 1) between colony in the 23 pairs of primers searched.Wherein 16 are marked at the distribution all presenting 1:2:1 in colony, and 1 mark ZCSSR8 is dominant marker, distributes in 3:1.
Mapmaker3.0 software analysis is used to show, these 17 pairs of polymorphism SSR primers and RpsZS18 all chain (table 1).Use MapDraw Software on Drawing genetic linkage maps, RpsZS18 is positioned between mark ZCSSR33 and ZCSSR46, genetic distance is respectively 0.7cM and 2.8cM, be divided into SSR marker Satt172 simultaneously from, be 0cM (Fig. 2) with the genetic distance of RpsZS18.
4, the prediction of candidate gene and the discovery of common separation marking
Analyses and comparison are carried out known in http://soybase.org database, the genetic model (table 2) having 16 to predict between mark ZCSSR33 and ZCSSR46,9 have functional annotation, wherein have the albumen of a disease-resistant relevant Ser/Thr kinase domain of genetic model Glyma.02g246000 coded plant.By design overlapping primers, pcr amplification is carried out to the Glyma.02g246000 site in precocious 18 and Williams and upstream and downstream 1000bp sequence thereof, pcr amplification product is checked order.In comparison precocious 18 and Williams, the site of Glyma.02g246000 finds to exist multiple SNP site and insertion and disappearance.Disappearance design upstream and downstream primer development Indel according to one section of 13bp in the 3 ˊ-UTR region of its precocity 18 marks ZCindel246000, and the primer pair for specific PCR amplifier molecule mark ZCindel246000 is:
Upstream primer F:5 '-CAGATAGAGTTTTCCTACACAGG-3 ' (SeqIDNo.1)
Downstream primer R:5 '-AGTAGAAGCAAAGAGGGACT-3 ' (SeqIDNo.2)
Mark ZCindel246000 with Indel and carry out colony's checking, by mapmaker3.0 computed in software genetic linkage distance, use MapDraw to build genetic linkage maps (Fig. 2).The genetic distance of ZCindel246000 and RpsZS18 is 0cM, and mark ZCindel246000 is for being total to isolated molecule mark, and Glyma.02g246000 is probably for controlling the candidate gene model of precocious 18 resistances.
Carry out colony's checking to comprise the following steps:
1) genomic dna of soybean to be measured is extracted;
2) with the genomic dna of soybean to be measured for template, utilize above-mentioned primers F and R, carry out pcr amplification reaction;
3) detect pcr amplification product, if the product (SeqIDNo.3) of 279bp can be amplified, then judge that soybean to be measured is as anti-epidemic disease kind.
Wherein, the amplification system that pcr amplification reaction uses is counted with 10 μ l: Soybean genomic DNA template concentrations 2ng/ μ l, 2 × PCRMasterMix (Tiangen) 4 μ l, primers F and each 0.2 μm of ol/L of R, use ddH
2o complements to 10 μ l.
Pcr amplification reaction use reaction conditions be: 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 Soybean Resistance epidemic disease gene RpsZS18 related in the present invention, the nucleotide sequence of candidate gene Glyma.02g246000 are respectively as shown in SeqIDNo.4 and 5.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Reference
[1] Yao Haiyan, Wang Xiaoming, Wu little Fei etc. the SSR molecular marker [J] of the precocious 18 resistant to phytophthora root rot genes of soybean varieties. plant genetic resources journal, 2010,11 (2): 213-217.
[2]MICHEMORERW；PARANI；KESSELIRV.Identificationofmarkerslinkedtodiseaseresistancegenesbybulksegregantanalysis:arapidmethodtodetectmarkersinspecificgenomicregionsbyusingsegregatingpopulations[J].ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,1991,88:9823-9832.DOI:10.1073/pnas.88.21.9823.
[3]GordonSG,StMartinSK,DorranceAE(2006)Rps8mapstoaresistancegenerichregiononsoybeanmolecularlinkagegroupF.CropSci46:168–173.
Claims (8)
1. with Soybean Resistance epidemic disease gene RpsZS18 be divided into from molecule marker ZCindel246000, it is characterized in that, described molecule marker is between soybean No. 2 chromosomal SSR marker ZCSSR33 and ZCSSR46, and the primer pair for specific PCR amplifier molecule mark ZCindel246000 is:
Upstream primer F:5 '-CAGATAGAGTTTTCCTACACAGG-3 ' and
Downstream primer R:5 '-AGTAGAAGCAAAGAGGGACT-3 '.
2. molecule marker according to claim 1, is characterized in that, the annealing temperature of carrying out pcr amplification use is 58.4 DEG C, and amplified production size is 279bp.
3. the application of molecule marker described in claim 1 or 2 in the anti-epidemic disease soybean varieties of qualification, is characterized in that, comprise the following steps:
1) genomic dna of soybean to be measured is extracted;
2) with the genomic dna of soybean to be measured for template, utilize primers F described in claim 1 or 2 and R, carry out pcr amplification reaction;
3) detect pcr amplification product, if the product of 279bp can be amplified, then judge that soybean to be measured is as anti-epidemic disease kind.
4. application according to claim 3, it is characterized in that, step 2) in pcr amplification reaction use amplification system count with 10 μ l: Soybean genomic DNA template concentrations 2ng/ μ l, 2 × PCRMasterMix4 μ l, primers F and each 0.2 μm of ol/L of R, use ddH
2o complements to 10 μ l.
5. application according to claim 3, is characterized in that, step 2) in pcr amplification reaction use reaction conditions be: 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.
6. detect the test kit of anti-epidemic disease soybean for PCR, it is characterized in that, containing the primer pair for specific PCR amplifier molecule mark ZCindel246000 described in claim 1 or 2 in described test kit.
7. test kit according to claim 6, is characterized in that, described test kit also comprises dNTPs, Taq DNA polymerase, Mg
2+, PCR reaction buffer, one or more in standard positive template.
8. the application of molecule marker described in claim 1 or 2 in Soybean Resistance epidemic disease molecular breeding.
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| ZHANG, JIQING ET AL.: "Identification and Candidate Gene Analysis of a Novel Phytophthora Resistance Gene Rps10 in a Chinese Soybean Cultivar", 《PLOS ONE》 * |
| 姚海燕: "大豆品种早熟18抗疫霉根腐病基因的SSR分子标记", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110273022A (en) * | 2019-08-01 | 2019-09-24 | 安阳市农业科学院 | Detect molecular labeling, primer, detection method and the application of soybean resistant to phytophthora root rot gene |
| 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 |
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