CN102994498B - 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 - Google Patents
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 Download PDFInfo
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Abstract
The invention relates to a molecular marker for co-separating a soybean anti-phytophthora megasperma candidate gene RpsWD15-1 and an application of the molecular marker for co-separating the soybean anti-phytophthora megasperma candidate gene RpsWD15-1. A disease-resistant variety wan bean 15 is taken as a male parent, a susceptible variety Williams (rps) is taken as a female parent, a generation F1 is generated through hybridizing, a population F2:3 is generated through self-crossing to be used as a mapping population, and an acknowledged soybean simple sequence repeats (SSR) marker and the SSR marker which is newly developed according to a soybean genomic sequence are utilized to carry out genetic mapping and fine mapping on an anti-disease gene which contains the wan bean 15. The anti-disease gene RpsWD15-1 is positioned on a chromosome No.17 and is positioned between the makers of Sattwd15-24/25(0.5centimeter) and Sattwd15-47(0.8centimeter). Moreover, a molecular marker Sattwd15-28 for co-separating the gene RpsWD15-1is also obtained. The marker can be used for soybean anti-phytophthora megasperma marker-assisted selection.
Description
Technical field
The invention belongs to agricultural biotechnology engineering and disease-resistant crops genetic breeding field, specifically, relate to a kind of and Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from molecule marker and application thereof.
Background technology
By soybean phytophthora (Phytophthora sojae Ka μ fmann & Gerdemann) soybean phytophthora root rot causing is one of Major Diseases of harm Soybean production.In recent years, in Heilongjiang Province, soybean main producing region, harm becomes increasingly conspicuous this disease, and harm soybean area reaches 15 * 10
4hectare.This disease can work the mischief at any growing stage of soybean, general field underproduction 10-30%, and serious plot can reach 60-90%, even causes total crop failure, and Soybean production is caused to serious threat.At present, soybean phytophthora root rot has become the Common Diseases that threatens soybean in China, so very urgent to the control of this disease.
Facts have proved, cultivate and plantation disease-resistant variety is the safest, economy and effective measures.For this reason, many scholars are doing a large amount of research aspect screening resistance resource and localization of disease resistance genes.Research shows, soybean is divided into qualitative character resistance (microspecies resistance) and quantitative character resistance to the resistance of phytophthora root rot.Qualitative character resistance is controlled by dominant gene, has complete resistance.So far, on 9 sites of soybean gene group, 15 anti-soybean phytophthora root rot genes (Rps) have been identified abroad, i.e. Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3a, Rps3b, Rps3c, Rps4, Rps5, Rps6, Rps7, Rps8 and theRps gene in cv.Waseshiroge, lay respectively at soybean gene group the 3rd, 16,13,18,18,18, (MLG N, J, F, G on 3,13 and No. 3 karyomit(e)s, G, G, N, F and N) (Sugimoto etc., 2012).The history that soybean phytophthora root rot occurs in China is shorter, and breeding for disease resistance work is also at the early-stage.At present, China has only identified 4 disease-resistant gene RpsYB30, RpsYD25, RpsZS18, RpsSN10 lays respectively at soybean gene group the 19th, 3, (MLG L, N, D1b and F) (Fan Aiying etc. on 2 and No. 13 karyomit(e)s, 2009, Sun Shi etc., 2011, Yao Haiyan etc., 2010, Yu Anliang etc., 2010, Zhu Zhendong etc., 2007).
China's soybean phytophthora has group structure complexity, and wide accommodation and virulence change the features such as fast, easily produces new virulence type microspecies and overcomes varietal resistance.Be 10-15 work-ing life that it is generally acknowledged soybean phytophthora root rot gene, but the speed that is overcome of new disease-resistant gene accelerates, as identified in Rps8 after less than 3 years, just found to overcome the soybean phytophthora bacterial strain of the new virulence type of its resistance.In 15 disease-resistant genes that large quantity research shows to have identified (except the Rps gene in cv.Waseshiroge), except Rps1c and Rps1k, be not highly resistant to the soybean phytophthora population of China lesion abroad.Therefore, be necessary to excavate energetically and utilize new resistance resource and constantly carry out the cumulative of disease-resistant gene, cultivate the lasting soybean varieties of resistance.
For excavating new soybean resistance resource, Most scholars is carried out localization of disease resistance genes and molecular marker assisted selection breeding by SSR molecule marker (http://soybase.org).Such mark has the advantages such as high information quantity, reliability be strong, easy and quick, is widely used.In addition utilize the soybean gene group sequence (http://www.Phytozome.net) of having announced to develop corresponding molecule marker, can carry out Fine Mapping to gene, obtain the closely linked molecule marker of gene.
The soybean varieties Wan Dou15Shi Anhui Province Pan Cunhu farm institute of agricultural sciences, with what cover celebrating 13 systematic breeding forms after radiation, authorized by Anhui Province's quality in 1996.This kind has high resistance (Li Jun mountain 2007) to Viruses Infecting Soybean Plant disease and oidium.Early-stage Study result shows, this kind all has stronger resistance (Zhu Zhendong etc., 2001) to two of different virulence type soybean phytophthora bacterial strain PsMC1 and PsMC2.Chen Xiaoling etc. (2008) and summer yataghan etc. (2011) report that beans 15 pairs of soybean phytophthora bacterial strains in Anhui have resistance of wide spectrum again, infer that it may contain new disease-resistant gene.
Summary of the invention
The object of this invention is to provide a kind of and Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from molecule marker and application thereof.
In order to realize the object of the invention, a kind of and Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 of the present invention be divided into from molecule marker, wherein, described candidate gene RpsWD15-1 is positioned on No. 17 karyomit(e) of soybean, and between mark Sattwd15-24/25 and Sattwd15-47, be respectively 0.5cM and 0.8cM with the genetic distance of these two marks.
Described molecule marker is Sattwd15-28, containing repeating motif (AT)
23, and for the PCR primer pair of amplifier molecule mark Sattwd15-28 be:
Upstream primer F:5 '-GCTTCCTATCACTCTTTGCTG-3 ' and
Downstream primer R:5 '-TTAGGCTAATGATGCTG-3 '.
The genetic distance of molecule marker Sattwd15-28 and candidate gene RpsWD15-1 is 0cM.
The annealing temperature that pcr amplification is used is preferably 48 ℃, and amplified production size is 123bp.
Wherein, the nucleotide sequence of candidate gene RpsWD15-1 is as shown in SEQ ID NO.1.
The present invention be also provided for detecting with Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from the PCR primer pair of molecule marker Sattwd15-28, comprising:
Upstream primer F:5 '-GCTTCCTATCACTCTTTGCTG-3 ' and
Downstream primer R:5 '-TTAGGCTAATGATGCTG-3 '.
The present invention also provide with Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from the application of molecule marker in identifying resistant to phytophthora root rot soybean varieties, it comprises step: the genomic dna that 1) extracts soybean to be measured; 2) take the genomic dna of soybean to be measured is template, utilizes primers F and R described in claim 4, carries out pcr amplification reaction; 3) detect pcr amplification product, if can amplify the product of 123bp, be judged to be resistant to phytophthora root rot soybean varieties.
Wherein, the amplification system that PCR reaction is used is counted with 20 μ l: Soybean genomic DNA template concentrations 2ng/ μ L, 10 * PCR damping fluid, 2.5 μ l, four kinds of dNTP concentration are respectively 20 μ mol/L, upstream and downstream primer concentration is respectively 0.2 μ mol/L, and Taq archaeal dna polymerase 0.5U, uses ddH
2o complements to 20 μ l.
The condition that PCR reaction is used is: 95 ℃ 3 minutes; 94 ℃ 50 seconds, 48 ℃ 50 seconds, 72 ℃ 50 seconds, 35 circulations; 72 ℃ 10 minutes.
The present invention also provides the test kit for detection of resistant to phytophthora root rot soybean that contains above-mentioned primer pair.Preferred described test kit also comprises dNTPs, Taq archaeal dna polymerase, Mg
2+, one or more in PCR reaction buffer, standard positive template etc.
The present invention also provide above-mentioned and Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from the application of molecule marker in plant molecular marker assistant breeding.
The present invention further provides above-mentioned and Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from the application of molecule marker in Soybean Resistance phytophthora root rot molecular breeding.
Particularly, of the present invention and Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from molecule marker and candidate gene RpsWD15-1 obtain by the following method:
Utilize the derivative F of Williams * Anhui beans 15 cross combinations
2:3the hereditary basis of 15 pairs of soybean phytophthora root rot resistances of Anhui beans, as mapping population, is studied by colony; Utilize the soybean ssr marker of having announced, the molecule marker that screening is chain with disease-resistant gene; Utilize soybean gene group sequence information, develop new SSR mark, this gene is carried out to Fine Mapping, obtain simultaneously with this gene be divided into from molecule marker.Thereby for Chinese People's Anti-Japanese Military and Political College's beans resistant to phytophthora root rot breeding provides assisted Selection, improve breeding efficiency, accelerate resistant to phytophthora root rot breeding work process.
Concrete technical scheme:
(1) the genetics of resistance analysis of Anhui beans 15
Using disease-resistant variety Anhui beans 15 as male parent, susceptible variety Williams(rps) as maternal, hybridization produces F
1generation, F
1for selfing, produce F
2generation, F
2102 F that produce for selfing
2:3family genetics of resistance is analyzed, disease-resistant gene is identified and mapping population.Adopt hypocotyl wound inoculation method, each Parentage determination 20-30 plant carries out genetics of resistance analysis to the reaction of soybean phytophthora bacterial strain PsMC1.After inoculation, at 25 ℃, humidity is under 80-100% condition, and then moisturizing 48h proceeds to hot-house culture, carries out state of an illness investigation after 6d.With reference to (2006) method evaluation standards such as Gordon, investigation F
2:3the separation ratio of anti-, separated, the susceptible family of family, the genetics of resistance rule of 15 pairs of phytophthora root rots of research Anhui beans.
(2) the extraction He Kanggan pond of gene DNA builds
Adopt CTAB method to extract 2 parents and 102 F
2:3the genomic dna of family.Each F
2:3family is got respectively 20 single-strain blade samples, and balanced mix is extracted DNA.According to (1991) such as Michelmore, get 10 disease-resistant, susceptible familys and build the Kang,Gan pond for separating of colony's fractional analysis (Bulk Segregating Analysis, BSA).
(3) utilize the disease-resistant gene of Anhui, SSR mark location beans 15
Soybean ssr marker sequence derives from http://soybase.org.Choosing at random SSR is marked at and between Anhui beans 15 and Williams, between ，Kang Gan pond, carries out pcr amplification and polymorphism screening.PCR reaction system is 20 μ l, contains 10 * PCR damping fluid, 2.5 μ l, four kinds of each 20 μ mol/L of dNTPs, and TaqDNA polysaccharase 0.5U, upstream and downstream primer is respectively 0.2 μ mol/L, and template DNA concentration is 2ng/ μ L.
PCR reacts amplification program, 95 ℃ of denaturations 3 minutes, and 94 ℃ of sex change 50 seconds, 47-51 ℃ of annealing 50 seconds, 72 ℃ are extended 50 seconds, from sex change, to extending, establish altogether 35 circulations, and last 72 ℃ are extended 10 minutes, 4 ℃ of preservations.Amplified production carries out electrophoresis and analysis in 8-12% polyacrylamide gel.
(4) Fine Mapping of disease-resistant gene
For this gene more effectively and is more accurately applied in molecular marker assisted selection breeding work, utilize the soybean gene group sequence information of having announced, develop new SSR mark, this gene is carried out to Fine Mapping.The SSR mark of having announced in utilizing soybase database is by the basis of disease-resistant gene Primary Location, first in Phytozome database (http://www.phytozome.net/soybean) download and the Liang Ge mark zone of this gene linkage between sequence, then use SSR Hunter 1.3(http: //en.bio-soft.net/dna/SSRHunter) find SSR site, finally use software Primer Premier 5.0(Premier Biosoft International, Palo Alto, CA) design SSR primer.PCR reaction system, amplification program and pcr amplification product detection method are with reference to the description in (3).
To thering is the SSR primer of polymorphism, carry out F
2:3colony's checking.At F
2:3the Zhong，Yu of the colony Anhui identical SSR primer of beans 15 genotype is designated as AA, and the SSR primer identical with Williams genotype is designated as BB, and heterozygous genes type is designated as AB.
(5) data analysis
To F
2:3population resistance separation and SSR are marked at F
2:3clastotype in family is carried out χ
2check.With Joinmap 4.0(Van Ooijen 2006) linkage relationship of software analysis SSR mark and disease-resistant gene.
Soybean phytophthora root rot is a kind of serious plant disease that affects Soybean production.The present invention utilizes SSR molecule marking method, on No. 17 karyomit(e) of soybean, located first a new disease-resistant candidate gene RpsWD15-1 and obtained with this disease-resistant gene RpsWD15-1 be divided into from and closely linked molecule marker.This disease-resistant gene has resistance of wide spectrum, can effectively control the generation of China's soybean phytophthora root rot, alleviates the production loss that this disease causes.Utilize with this gene be divided into from SSR mark Sattwd15-28, can effectively be applied in molecular marker assisted selection breeding work, overcome long shortcoming of needed cycle of conventional breeding method; PCR-based technology, carries out Analysis of Resistance indoor to soybean resource; For this gene of clone lays the foundation, and it is carried out to structure and function analysis, this molecular genetics mechanism for further this gene of understanding is significant simultaneously, puts into practice, has very important value in breeding work and disease-resistant theoretical investigation at Soybean production.Its advantage is:
(1) the present invention has located a new anti-soybean phytophthora root rot candidate gene RpsWD15-1 first on No. 17 karyomit(e) of soybean.The soybean phytophthora bacterial strain PsMC1(virulence type that this gene Shi Dui China virulence is stronger: 1a, 1c, 1k, 2,3b, 3c, 4,5,6,7,8, ZS18) have in the soybean varieties Anhui beans 15 of complete resistance and obtain.，Gai gene pairs China soybean phytophthora has resistance of wide spectrum simultaneously, can effectively control the generation of China's soybean phytophthora root rot, alleviates the production loss that this disease causes.
(2) of the present invention and candidate gene RpsWD15-1 be divided into from molecule marker Sattwd15-28, the new mark obtaining in the stable filial generation family of Shi Anhui beans 15 and resistance thereof, can be for soybean phytophthora root rot cultivar identification and the Soybean Resistance molecular marker assisted selection breeding in generation after being ill.
(3) of the present invention and candidate gene RpsWD15-1 be divided into from molecule marker Sattwd15-28, for the Cloning and sequencing of this gene lays the foundation, thus clearly this sequence information and constitutional features.Meanwhile, according to be divided into from molecule marker and soybean gene group sequence information, carry out the haplotype analysis of this gene, be expected to be cloned into new disease-resistant gene.
Accompanying drawing explanation
Fig. 1 is the growing state of inoculating soybean phytophthora PsMC1 bacterial strain 6dHou， Anhui beans 15 and Williams in the embodiment of the present invention 1.
Fig. 2 is soybean phytophthora root rot candidate gene RpsWD15-1 and chain SSR mark genetic linkage map thereof in the embodiment of the present invention 1.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The candidate gene RpsWD15-1 that embodiment 1 Soybean Resistance phytophthora root rot is new and be divided into from the acquisition of molecule marker Sattwd15-28
The genetics of resistance analysis of 1.1 Anhui beans 15
Using disease-resistant variety Anhui beans 15 as male parent, susceptible variety Williams(rps) as maternal, preparing hybrid combination results F
1generation, F
1for selfing, produce F
2generation, F
2102 F that produce for selfing
2:3family is identified and mapping population as genetics of resistance analysis, disease-resistant gene.Adopt hypocotyl wound inoculation method, each Parentage determination 20-30 plant carries out genetics of resistance analysis to the reaction of bacterial strain PsMC1.After inoculation, at 25 ℃, humidity is under 80-100% condition, and then moisturizing 48h proceeds to hot-house culture, carries out state of an illness investigation after 6d.With reference to Gordon(2006) etc. method evaluation standard, to be less than 20% family be pure and mild disease-resistant family to plant mortality ratio, it is pure and mild susceptible family that mortality ratio is greater than 80% family, and the family that mortality ratio is 21-79% be resistance separated family, investigation F
2:3the separation ratio of anti-, separated, the susceptible family of family, the genetics of resistance rule (Fig. 1) of 15 pairs of phytophthora root rots of research Anhui beans.
102 derivative F of Williams * Anhui beans 15 cross combinations
2:3family shows the Resistance Identification result of bacterial strain PsMC1, and 31 familys are pure and mild disease-resistant, and 42 familys are heterozygous, and 29 familys are susceptible, through χ
2the separated 1:2:1 that meets expectation of check, the resistance that shows 15 couples of soybean phytophthora bacterial strain PsMC1 of Anhui beans is by a dominant Dominant gene, by this candidate gene called after RpsWD15.
The extraction He Kanggan pond of 1.2 Soybean genomic DNAs builds
Adopt CTAB method to extract 2 parents and 102 F
2:3the genomic dna of family.Each F
2:3family is got respectively 20 single-strain blade samples, and balanced mix is put into the centrifuge tube of 2.0mL.Be placed in liquid nitrogen and be ground into powder, the CTAB Extraction buffer to adding 800 μ L65 ℃ preheatings in centrifuge tube is incubated 30-50 minute after mixing in 65 ℃ of water-baths, frequently shakes in order to avoid agglomerating; Take out centrifuge tube, be cooled to room temperature, add 800 μ L phenol: chloroform: primary isoamyl alcohol (25:24:1), extracting 10 minutes, during put upside down gently at set intervals, it is fully mixed, centrifugal 10 minutes of 12000rpm; Get supernatant liquor in another 1.5mL centrifuge tube, add equal-volume chloroform: primary isoamyl alcohol (24:1), fully mixes after 10 minutes centrifugal 10 minutes of 12000rpm; Get supernatant liquor in another 1.5mL centrifuge tube, add the Virahol of 0.8 times of volume precooling, slowly mix, at-20 ℃, place 30-60 minute, make DNA with Precipitation; Choose DNA precipitation and be transferred in 1.5mL centrifuge tube, with 75% alcohol washing 2 times, finally, with dehydrated alcohol dehydration, dry up, add the ultrapure water of appropriate sterilizing.After DNA dissolves, the agarose gel electrophoresis with 0.8% detects the integrity of DNA and the concentration that ultraviolet spectrophotometry detects DNA sample, and DNA concentration is adjusted to 40ng/ μ L.
According to methods such as (1991) such as Michelmore, build the Kang,Gan pond for separating of colony's fractional analysis (Bulk Segregating Analysis, BSA).The DNA that respectively gets 10 disease-resistant familys of 1 μ L is mixed in a new centrifuge tube, is built into anti-pond, and DNA concentration is adjusted to 40ng/ μ L.Sense pond construction process is with reference to anti-pond construction process.
1.3 utilize the disease-resistant gene of Anhui, SSR mark location beans 15
Soybean ssr marker sequence derives from http://soybase.org.Choosing at random SSR is marked at and between Anhui beans 15 and Williams, between Ji Kanggan pond, carries out pcr amplification and polymorphism screening.
PCR reaction system is 20 μ l, contains 10 * PCR damping fluid, 2.5 μ l, four kinds of each 20 μ mol/L of dNTPs, and TaqDNA polysaccharase 0.5U, upstream and downstream primer is respectively 0.2 μ mol/L, and template DNA concentration is 2ng/ μ L.
PCR reacts amplification program, 95 ℃ of denaturations 3 minutes, and 94 ℃ of sex change 50 seconds, 47-51 ℃ of annealing 50 seconds, 72 ℃ are extended 50 seconds, from sex change, to extending, establish altogether 35 circulations, and last 72 ℃ are extended 10 minutes, 4 ℃ of preservations.
PCR product detects, and according to the ratio of 1:5,6 * sample-loading buffer of 4 μ l is joined in PCR product, and after mixing, amplified production carries out electrophoresis and analysis in 8-12% polyacrylamide gel.
Choose at random the SSR mark on soybean, find to be marked between two parents and to there is polymorphism from 21 couples of SSR on No. 17 karyomit(e).These 21 marks are detected to polymorphism for Kang Gan pond, wherein only have 9 marks to produce polymorphism, they may be chain with disease-resistant gene.Further use 102 derivative F of Williams * Anhui beans 15 cross combinations
2:3family is carried out chain checking to these 9 marks, and result shows to derive from 9 mark Sat_222 on No. 17 karyomit(e), Sat_292, and Satt514, Satt461, Satt528, Satt574, Satt543, Satt615 and Satt301 and RpsWD15 are chain.These 9 are marked at and in colony, are all 1:2:1 and distribute, and are codominant marker.With Joinmap 4.0 software analysis, show, RpsWD15-1 gene and this 9 marks are chain, and between mark Satt543 and Satt615, are respectively 2.2cM and 7.2cM with the genetic distance of these two marks.
The Fine Mapping of 1.4 disease-resistant genes
For this gene more effectively and is more accurately applied in molecular marker assisted selection breeding work, utilize the soybean gene group sequence of having announced to develop new SSR mark, this gene is carried out to Fine Mapping.In Phytozome database, (http://www.phytozome.net/soybean) compare of analysis is known, and between Satt543 and Satt615, physical distance is 4.29Mb and downloads this sector sequence; With SSR Hunter 1.3 (http://en.bio-soft.net/dna/SSRHunter), search out altogether 671 GeSSR sites, being wherein greater than 10 SSR sites of repeating motif has 414; With software Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA), on this 414 GeSSR site, design primer, 414 SSR marks of middle acquisition.PCR reaction system, amplification program and detection method are with reference to the description in (3).
Adopt BSA method, obtain altogether the SSR mark of 17 polymorphisms, may be chain with disease-resistant gene.Further use 102 derivative F of Williams * Anhui beans 15 cross combinations
2:3family is carried out chain checking (table 1) to these 17 marks.Result shows, these 17 are marked at and in colony, are all 1:2:1 and distribute, and are codominant marker.
Table 1 is according to soybean gene group sequences Design and the Molecular Marker Information chain with candidate gene RpsWD15-1
With Joinmap 4.0 software analysis, show, RpsWD15 gene and this 26 marks are chain, and between mark Sattwd15-24/25 and Sattwd15-47, are respectively 0.5cM and 0.8cM with the genetic distance of these two marks.Meanwhile, also obtain with RpsWD15-1 candidate gene be divided into from molecule marker Sattwd15-28, the genetic distance of mark Sattwd15-28 and candidate gene RpsWD15-1 is 0cM(Fig. 2).
Known in the comparison of Phytozome database inner analysis, the genetic model (table 2) that has 8 predictions between mark Sattwd15-24/25 and Sattwd15-47, the albumen that wherein has the serine/threonine structure that two genetic model Glyma17g28950.1 are relevant to plant disease-resistant with Glyma17g28970.1 coding, and in the present invention, with disease-resistant gene be divided into from molecule marker Sattwd15-28 be the primer according to genetic model Glyma17g28950.1 design.Therefore Glyma17g28950.1 may be the candidate gene of Anhui beans 15, i.e. called after RpsWD15-1.
On No. 17 karyomit(e) of table 2 soybean, the gene between mark Sattwd15-24/25 and Sattwd15-47
According to 5 of genetic model Glyma17g28950.1 '-UTR and 3 '-UTR design primer (upstream primer: 5 '-CTCGTCAACCAATAACCCAT-3 '; Downstream primer: 5 '-CAAAATCCCAATAACCAAA-3 ') ，Yi Anhui beans 15 genomes are that template is carried out pcr amplification.The TIANgel MidiPurification Kit of TIANGEN company for the object fragment obtaining, reference reagent box specification sheets reclaims object fragment.
The object fragment that recovery obtains adopts the pMD18-T test kit (TaKaRa Code:D101A) of TaKaRa company, and reference reagent box specification sheets is connected object fragment and transforms with pMD18-T carrier, and concrete operation step is as follows:
(1) in Eppendorf tube, prepare 5 μ lDNA solution, comprise the pMD18-T carrier of 1 μ l, the Insert Fragment of 3 μ l, the ddH of 1 μ l
2o.
(2) add the Solution 1 of 5 μ l.
(3) 16 ℃ of reactions, after 10 hours, connect product by 10 μ l and join in the JM109 competence intestinal bacteria of 100 μ l, place 30 minutes in ice.
(4) 42 ℃ are heated 45 seconds, then in ice, place 1 minute.
(5) add the LB substratum of 600 μ l, 37 ℃ of concussions are cultivated 45 minutes.
(6) on the LB solid medium that contains X-gel, IPTG and Amp, cultivate, form single bacterium colony.
(7) the single white colony of picking, and after concussion is cultivated in containing the liquid nutrient medium of Amp, by PCR method, confirm the size of Insert Fragment in carrier.Sequencing result shows, the nucleotide sequence of Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 is as shown in SEQ ID NO.1, and CDS sequence is as shown in SEQ ID NO.2, and the aminoacid sequence of its coding is as shown in SEQ ID NO.3.
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, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
- With Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from molecule marker Sattwd15-28, it is characterized in that, described candidate gene RpsWD15-1 is positioned on No. 17 karyomit(e) of soybean, its nucleotide sequence is as shown in SEQ ID NO.1;Specific PCR primer pair for amplifier molecule mark Sattwd15-28 is:Upstream primer F:5 '-GCTTCCTATCACTCTTTGCTG-3 ' andDownstream primer R:5 '-TTAGGCTAATGATGCTG-3 '.
- 2. molecule marker according to claim 1, is characterized in that, the annealing temperature that pcr amplification is used is 48 ℃, and amplified production size is 123bp.
- For detection of with Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from the specific PCR primer pair of molecule marker, it is characterized in that, described candidate gene RpsWD15-1 is positioned on No. 17 karyomit(e) of soybean, and its nucleotide sequence is as shown in SEQ ID NO.1; Described primer pair comprises:Upstream primer F:5 '-GCTTCCTATCACTCTTTGCTG-3 ' andDownstream primer R:5 '-TTAGGCTAATGATGCTG-3 '.
- Described in claim 1 with Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from the application of molecule marker Sattwd15-28 in identifying resistant to phytophthora root rot soybean varieties, it comprises step:1) extract the genomic dna of soybean to be measured;2) take the genomic dna of soybean to be measured is template, utilizes primers F and R described in claim 1, carries out pcr amplification reaction;3) detect pcr amplification product, if can amplify the product of 123bp, be judged to be resistant to phytophthora root rot soybean varieties.
- 5. application according to claim 4, it is characterized in that, step 2) amplification system that in, PCR reaction is used is counted with 20 μ l: Soybean genomic DNA template concentrations 2ng/ μ L, 10 * PCR damping fluid, 2.5 μ l, four kinds of dNTP concentration are respectively 20 μ mol/L, upstream and downstream primer concentration is respectively 0.2 μ mol/L, and Taq archaeal dna polymerase 0.5U, uses ddH 2o complements to 20 μ l.
- 6. application according to claim 4, is characterized in that step 2) in the condition used of PCR reaction be: 95 ℃ 3 minutes; 94 ℃ 50 seconds, 48 ℃ 50 seconds, 72 ℃ 50 seconds, 35 circulations; 72 ℃ 10 minutes.
- 7. the test kit for detection of resistant to phytophthora root rot soybean that contains primer pair described in claim 3.
- 8. test kit according to claim 7, is characterized in that, described test kit also comprises dNTPs, Taq archaeal dna polymerase, Mg 2+, one or more in PCR reaction buffer, standard positive template.
- Described in claim 1 with Soybean Resistance phytophthora root rot candidate gene RpsWD15-1 be divided into from the application of molecule marker Sattwd15-28 in Soybean Resistance phytophthora root rot molecular breeding.
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| CN201210509161.4A CN102994498B (en) | 2012-11-23 | 2012-11-23 | 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 |
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| CN201210509161.4A CN102994498B (en) | 2012-11-23 | 2012-11-23 | 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 |
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| 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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| CN101677516A (en) * | 2007-04-20 | 2010-03-24 | 孟山都技术公司 | Methods and compositions for selecting soybean plants resistant to phytophthora root rot |
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Non-Patent Citations (8)
| Title |
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| Molecular Marker Analysis of Soybean Plant Introductions with Resistance to Phytophthora sojae;S.G.Gordon et al.;《Genetics and Resistance》;20070131;第97卷(第1期);113-118 * |
| S.G.Gordon et al..Molecular Marker Analysis of Soybean Plant Introductions with Resistance to Phytophthora sojae.《Genetics and Resistance》.2007,第97卷(第1期),113-118. |
| 分子标记技术在疫霉菌上的应用;郑金龙 陈鸿 易克贤;《安徽农学通报》;20080831;第14卷(第15期);63-66 * |
| 夏长剑.大豆抗疫霉根腐病基因分析及大豆品种皖豆15抗疫霉根腐病基因作图.《中国优秀硕士学位论文全文数据库农业科技辑》.2011,D046-45. |
| 大豆抗疫霉根腐病基因分析及大豆品种皖豆15抗疫霉根腐病基因作图;夏长剑;《中国优秀硕士学位论文全文数据库农业科技辑》;20111019;D046-45 * |
| 大豆抗疫霉根腐病基因鉴定及分子标记;霍云龙;《中国优秀硕士学位论文全文数据库农业科技辑》;20060316;D046-30 * |
| 郑金龙 陈鸿 易克贤.分子标记技术在疫霉菌上的应用.《安徽农学通报》.2008,第14卷(第15期),63-66. |
| 霍云龙.大豆抗疫霉根腐病基因鉴定及分子标记.《中国优秀硕士学位论文全文数据库农业科技辑》.2006,D046-30. |
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