CN104141011B

CN104141011B - The method of a kind of assistant identification soybean mosaic disease resisting poison ospc gene and application thereof

Info

Publication number: CN104141011B
Application number: CN201410373427.6A
Authority: CN
Inventors: 韩英鹏; 赵雪; 李海燕; 李文滨
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2014-07-31
Filing date: 2014-07-31
Publication date: 2016-06-01
Anticipated expiration: 2034-07-31
Also published as: CN104141011A

Abstract

The present invention discloses method and the application thereof of a kind of assistant identification soybean mosaic disease resisting poison ospc gene, belongs to soybean heredity technical field. Method provided by the present invention is separation and Extraction DNA from soybean to be measured, pcr amplification is carried out by the primer of molecule marker SSR64 and SG1, recycling electrophoresis detection SSR64 primer extension product there is marker-free locus band and/or utilizes the genotype of the primer extension product of gene type analytical system analysis SG1, it is determined that whether soybean germplasm resource to be measured has mosaic disease resisting poison ospc gene. Utilize method provided by the present invention can realize the high efficiency selected of the resistant material to soybean Mosaic N1 poison strain, accelerate the seed selection of soybean SMV resistance new lines, set up novel soybean hybrid strain apolegamy system, the waste of man power and material can also be reduced, it is to increase breeding efficiency and accuracy simultaneously.

Description

The method of a kind of assistant identification soybean mosaic disease resisting poison ospc gene and application thereof

Technical field

The present invention relates to the method for a kind of assistant identification soybean mosaic disease resisting poison ospc gene and application thereof, belong to soybean heredity technical field.

Background technology

Soybean (Glycinemax) originates in China, is important cash crop. Soybean mosaic virus (soybeanmosaicvirus, SMV) disease is one of main disease of harm Soybean production, seriously affects soybean yields and quality. Cultivating disease-resistant variety is control the popular most effectual way of SMV at present. SMVN1 strain is one of the advantage virus strain in northeast soybean producing region, utilizes molecular marking technique location and the molecule marker of N1 strain resistance related gene and close linkage to be of value to the breeding practice of molecule assisted Selection resistant soybean kind.

Chinese scholars had reported the research of this respect. External aspect, Yu etc. (1994) identify the single dominant gene Rsv1 of SMV resistance, it is positioned on soybean molecule linkage group F, obtain 1 SSR marker HSP176 and 2 RFLP and mark pA186 and pK644a, all with Rsv site close linkage, its genetic distance is respectively 0.5cM, 1.5cM and 2.1cM. Rsv1 is positioned on the disease-resistant gene bunch of soybean molecule linkage group F by Imsande (1998). It is chain with Rsv1 that Hates etc. (2000) utilize AFLP label screening to go out R11 (518bp), R12 (171bp), R13 (261bp) and R14 (312bp) 4 mark, and genetic distance is all within 3.5cM. Jeong etc. (2002) find the dominant SMV resistance gene Rsv3 of the list independent with Rsv1 phase in Columbia, are located on linkage group B2. Rsv4 is positioned on linkage group D1b+W by Hayes etc. (2000), obtains linked marker Satt542 (4.7cM) and Satt558 (7.8cM). Domestic aspect, it is chain that Zheng Cuiming (2000) filters out 1 RAPD mark OPN980/1070 and N3 resistant gene relevant with resistant gene by BSA method, is 2.1cM with the genetic distance of disease-resistant gene. Liu Lijun etc. (2002) utilize the F2 colony of black agriculture 39 �� close rich 25, filter out codominance RAPD and mark OPN1400/1300, are 8.2cM with the genetic distance of black agriculture 39 disease-resistant gene. Wang Yongjun etc. (2004) utilize RIL colony to identify 5 SMV strains (Sa, Sc8, Sc9, N1 and N3), it has been found that 5 localization of disease resistance genes are on linkage group D1b+W. In theory, the molecule marker being less than 5cM with the genetic distance of disease-resistant gene just has the using value of molecule assisted Selection, and mark-gene genetic that conventional result of study obtains is distant, and range of application is less. Therefore, reduce the genetic distance in disease-resistant site, find the molecule marker with disease-resistant site close linkage and the effect tool improving molecule assisted Selection is of great significance.

Summary of the invention

For solving the problem, the present invention provides a kind of assistant identification soybean mosaic disease resisting poison method of ospc gene and application thereof, take technical scheme as follows:

It is an object of the present invention to provide the method for a kind of assistant identification soybean mosaic disease resisting poison ospc gene, it is characterized in that, separation and Extraction DNA from soybean to be measured, pcr amplification is carried out by the primer of molecule marker SSR64 and SG1, recycling electrophoresis detection SSR64 primer extension product there is marker-free locus band and/or utilizes the genotype of the primer extension product of gene type analytical system analysis SG1, it is determined that whether soybean germplasm resource to be measured has mosaic disease resisting poison ospc gene.

The step of described method is as follows:

1) DNA of soybean sample to be measured is extracted;

2) taking step 1) DNA that extracts as template, carry out pcr amplification by the primer of the primer of molecule marker SSR64 and molecule marker SG1;

3) electrophoresis detection step 2 is utilized) amplified production of the primer of gained SSR64 and/or utilize gene type analytical system to analyze the genotype of SG1 primer extension product, it is determined that whether soybean germplasm resource to be measured has marker gene locus band.

The primer of described molecule marker SSR64 is as shown in SEQIDNO.1-NO.2; The primer of described molecule marker SG1 is as shown in EQIDNO.3-NO.4.

Described pcr amplification, response procedures is 94 DEG C of denaturation 6min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, and 35 circulations, 72 DEG C of ends extend 5min.

Described marker gene site, numbering RsmvN1-2, is positioned at the F linkage group of soybean, specifically Chinese People's Anti-Japanese Military and Political College's Tofu pudding mosaic virus ospc gene be divided into from molecule marker SSR64 and SG1 between, and with molecule marker SSR64 and SG1 close linkage.

Described marker gene locus band, the product size of SRR64 primer amplification is the product size of 152bp, SG1 primer amplification is 80bp.

Described gene type analytical system is analyzed, and is by after centrifugal for SG1 primer PCR amplified production, the fluorescent signal of scanning samples, scanning starting temperature is 70 DEG C, end temp 96 DEG C, scanning terminates rear temperature and is down to 67 DEG C, judges whether scanning gained solubility curve morphs.

The concrete steps of described method are as follows:

1) DNA in soybean sample blade to be measured is extracted;

2) taking step 1) DNA that extracts is as template, pcr amplification is carried out respectively again by the primer of molecule marker SG1 shown in the primer of the SSR64 of molecule marker shown in SEQIDNO.1-NO.2 and SEQIDNO.3-NO.4, response procedures is 94 DEG C of denaturation 6min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations, and 72 DEG C of ends extend 5min;

3) polyacrylamide gel electrophoresis separation detection step 2 is utilized) amplified production of the molecule marker SSR64 primer of gained, whether detection is the band of 152bp containing size;

4) gene type analytical system analytical procedure 2 is utilized) genotype of the amplified production of the molecule marker SG1 primer of gained, by the amplified production of gained centrifugal 5min under 4000rpm, again the fluorescent signal of sample is scanned, scanning initial temperature is 70 DEG C, end temp is 96 DEG C, scanning is cooled to 67 DEG C after terminating, and judges whether scanning gained melting curve exists variation.

Described method is used for the genetic breeding of the poison qualification of ospc gene of soybean mosaic disease resisting, screening and soybean.

The useful effect of the present invention is as follows:

The present invention is by utilizing two to primer, by template of the genomic dna of single strain to be measured, two marker sites are carried out pcr amplification, the high efficiency selected of the resistant material to soybean Mosaic N1 poison strain can be realized, accelerate the seed selection of soybean SMV resistance new lines, set up novel soybean hybrid strain apolegamy system.

The present invention can avoid loaded down with trivial details phenotype inoculation qualification and environment on the impact of identification result, genomic dna can be extracted and pcr amplification reaches the object of Single-plant selection at cotyledon period, eliminate susceptible plant, offspring colony (the low generation is to advanced lines) efficiency of selection all can be reached 100%, reduce the waste of man power and material, it is to increase breeding efficiency and accuracy.

Accompanying drawing explanation

The gene locus RsmvN1-1 of Fig. 1 SMVN1 strain and the molecule marker of close linkage.

Fig. 2 population segment offspring is in the pcr amplification result in SSR64 site;

(10-26: system of the family numbering for sample).

Fig. 3 population segment offspring is at the melting curve of the pcr amplification product of SG1;

(in figure darker curve be carry east agriculture 93-046 allelotrope offspring's strain, arrow place from top to bottom sample representated by curve family system be numbered 10,11,12,13 and 17; Lighter curve represents carries offspring's strain of conrad allelotrope, and the system of family of arrow place sample representated by curve from top to bottom is numbered 14,15,16,18 and 19).

Embodiment

Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.

In following embodiment, unless stated otherwise, otherwise all operations is ordinary method.

The acquisition of the gene locus RsmvN1-2 of embodiment 1 anti-soybean Mosaic (SMV) N1 strain

1. recombinate the structure of self-mating system and F2 segregating generation colony and the qualification of SMVN1 strain

Hybridize with Chinese soybean Cultivar east agriculture 93-046 (��) and rich 25 (��) of conjunction, obtain Hybrids F1; Obtain filial generation by F-1 hybrids self-pollination, pass method (SSD) by filial generation single and obtain 130 F2:6 generation restructuring self-mating systems; Utilize above-mentioned parents, obtain Hybrids F1 and continue selfing and obtain F2 segregating generation individuality totally 2000 seeds.

In 2007,2008,2009 respectively in the indoor inoculation qualification that 130 strains and parent are carried out N1 microspecies of independent anti-aphid net, repeat for three times. In 2013 in the indoor inoculation qualification that 2000 F2 generation single strains and parent are carried out N1 microspecies of independent anti-aphid net, inoculation method: gather the sick leaf in poison source and put into mortar, the phosphoric acid buffer (pH=7.0) (the sick leaf of 10ml/g) and the 600 object silicon carbide that add 0.01mol/L are a little, and disease leaf is ground to form homogenate shape. Inoculation liquid is dipped when fully being launched by Sheng Zhenye along vein frictional inoculation, immediately with tap water blade surface residue after inoculation with writing brush. Respectively at latter 20 days of inoculation and 30 days investigation incidences.

2. genetic map construction and localization of disease resistance genes

It is chosen at this SSR and SNP primer with amplification polymorphism of father and mother and carries out pcr amplification and gene type analysis taking the DNA that 130 families are as template, according to gene type situation between colony of the separation of SSR marker genotype in colony and SNP, the individuality of maternal type is designated as " A ", the individuality of male parent type is designated as " B ", assorted that close unclear with disappearance be designated as "-".

Involved experimental technique is as follows:

DNA extraction method adopts the SDS method improved, get fully grinding in 0.1g blade liquid nitrogen, powder proceeds in 2ml centrifuge tube, add 600 �� l Extraction buffer (0.1MTris-HClpH=8.0, 0.05MEDTApH=8.0, 0.5MNaCl, 1% mercaptoethanol) and 120 �� l10%SDS solution, 65 DEG C of water-bath 30min, add 800 �� l phenol-chloroform (V:V=1:1), mixed even, put upside down 15-20 time gently, the centrifugal 10min of 12000r/min, get supernatant to move to new 2ml centrifuge tube, add equal-volume chloroform-primary isoamyl alcohol (V:V=24:1), mixed even, put upside down 15-20 time gently, the centrifugal 10min of 12000r/min, get supernatant to move to new 2ml centrifuge tube, add the pre-cold isopropanol of equal-volume, put upside down mixed even gently, to white flocks occurs, leave standstill 10 minutes, the centrifugal 1min of 12000r/min, abandon supernatant, the �� l70% ethanol that adds 700 in precipitation blows washes DNA, after repeating to blow and washing 2 times, air-dry precipitation, add deionized water dissolving, for subsequent use.

By the SNP marker that the present invention relates to and SSR marker to the PCR amplification method of kind to be measured (being) DNA profiling: wherein the PCR reaction system of SSR marker is 20 �� l, comprise 2 �� l genomic dnas (100ng/ �� l), 1.5 �� lMgCl2 (25mM), 0.25 �� ldNTPmixtures (10mM), 2 �� l10 �� PCRbuffer, 3 �� l primers (2 ��Ms), 0.25 �� lTaqpolymerase (10units/ �� l) and 11 �� l deionized waters; The PCR system of SNP marker is 10 �� l, comprise 1 �� l genomic dna (100ng/ �� l), 1 �� lLCGreenplus (Idaho company), 0.75 �� lMgCl2 (25mM), 0.2 �� ldNTPmixtures (10mM), 1 �� l10 �� PCRbuffer, 2 �� l primers (2 ��Ms), 0.2 �� lTaqpolymerase (10units/ �� l) and 2.85 �� l deionized waters. PCR response procedures is 94 DEG C of denaturation 6min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, and 35 circulations, 72 DEG C of ends extend 5min. The PCR primer of SSR marker is separated with the polyacrylamide gel electrophoresis of 6%. The PCR primer of SNP marker adopts LightscannerHRM high resolving power melting curve abrupt climatic change/gene type analytical system to carry out analyzing (Idaho company). Detailed process is:

1) after pcr amplification reaction terminates, genotyping is carried out with LightScanner, the sample first drawing 10 �� L is in 96 orifice plates for detecting, cover the mineral oil of 15 �� L above, to prevent the volatilization of sample, afterwards by two 96 orifice plate trims, put into low speed centrifuge centrifugal, speed: 4000rpm, time: 5min.

2) being scanned by the fluorescent signal of sample, the starting temperature arranging scanning is 70 DEG C, and end temp is 96 DEG C, keeps temperature to be 67 DEG C.

3) judging whether scanning gained melting curve morphs, wherein melting curve morphs (sample namely with solubility curve peak carries the positions such as disease-resistant), morphs as the position such as susceptible without melting curve.

Utilize Mapmaker/EXP3.0 mapping software, build molecular marker linkage maps. Application Group order carries out linkage analysis and grouping (LOD=5.0), linked marker less than 8 be optimized sequence with Compare order, more than 8 sort with Ripple order, error-detecting level is set to 1%, utilizes Kosambi function that recombination fraction is converted into genetic distance (cM). Chain accordingly to determine as anchor marker according to the SSR marker located by Song (2004) etc. Using the threshold that LOD >=2.0 exist as QTL, carry out QTL location with WinQTLcartographV2.0.

Result is as shown in Figure 1: the gene locus RsmvN1-1 (Fig. 1) of anti-soybean Mosaic (SMV) N1 strain, it is positioned the F linkage group of soybean, RsmvN1-1 is between SNP9 and SSR23, and the phenotypic variation can explained respectively is 48.64%.

The SSR primer that father and mother have amplification polymorphism between this it is chosen between SNP9 and SSR23 scope and in each 0.5Mbp genome range of upstream and downstream, extract F2 segregating generation to the susceptible single strain DNA of N1 strain, pcr amplification and gene type analysis is carried out taking it as template, according to the separation case of SSR marker genotype in colony, the individuality of maternal type is designated as " A ", the individuality of male parent type is designated as " B ", assorted that close unclear with disappearance be designated as "-". The experimental technique that application relates to is the same.

The genotype data that above-mentioned mark somatotype obtains is calculated as follows recombination fraction:

In formula, 1 B gene type is the genotype of disease-resistant donor east agriculture 93-046, and A genotype is the genotype that disease-resistant parent closes rich 25, and H is heterozygous genotypes number therebetween. Obtaining two with disease-resistant site recombination fraction according to above-mentioned formula is the molecule marker of 0, it is respectively SNP marker SG1 and SSR marker SSR64, this molecule marker be divided into disease-resistant gene from molecule marker, redefine SMVN1 strain disease-resistant site called after RsmvN1-2 (table 1) with this molecule marker.

F2 generation single strain that SMVN1 strain felt by table 1 distributes at molecule marker somatotype and the recombination fraction of RsmvN1-1 location proximate

The assisted Selection application of embodiment 2 gene locus RsmvN1-2 in hybridization advanced lines colony

After the gene locus RsmvN1-2 of anti-soybean Mosaic (SMV) N1 strain obtains, hybridize with Chinese soybean Cultivar east agriculture 93-046 (R) and conrad (S), utilize SSR64 and SG1 primer sequence (shown in SEQIDNO.1-NO.4) that 150 the F2:6 generation restructuring self-mating systems obtained are carried out pcr amplification, predict that these tested the anti-sense to two malicious strains being is reacted. Pcr amplification condition, electrophoresis and gene type assay condition are given condition identical with embodiment 1. 150 systems of family are carried out the anti-sense qualification of N1 virus strain simultaneously, compares the matching degree (see table 2, Fig. 2 and Fig. 3) of field test result and marker site pcr amplification assisting sifting result. Fig. 2 is the pcr amplification result of population segment offspring in SSR64 site, and Fig. 3 is the melting curve of population segment offspring at the pcr amplification product of SG1. As shown in Figures 2 and 3: the east allelotrope of agriculture 93-046 in SSR64 site is that the excellent of SMV resistance N1 strain waits position, the resistance man of N1 microspecies that to be field test with system of wherein consistent with disease-resistant parent's Dong Nong 93-046 band type family be is; Susceptible of system of consistent with Susceptible parent conrad band type family to be field test be N1 microspecies is. The result of result show tags assisting sifting and reality anti-sense qualification performance are very identical.

The gene locus RsmvN1-2 field test result of the anti-soybean Mosaic N1 strain of table 2 and accuracy

The application of embodiment 3 gene locus RsmvN1-2 in the single strain of anti-sense of F2 segregating generation assisting sifting

After the gene locus RsmvN1-2 of the N1 strain of anti-soybean Mosaic (SMV) obtains, utilize be divided into disease-resistant site from molecule marker SSR64 with SG1 to 60 eastern agriculture 93-046 for the mono-strain of F2 that one of parent is derivative carries out gene type (method described in analytical procedure and embodiment 1 is identical), predict that the anti-sense of the malicious strain of SMVN1 is reacted by these tested single strains. 60 mono-strains of F2 are carried out the anti-sense qualification of N1 microspecies simultaneously, compares field test result and the matching degree (see table 3) of mark assisting sifting result. The result of result show tags assisting sifting and reality anti-sense qualification performance are very identical.

Table 3 molecule marker is in the gene type result of the assisted Selection of F2 segregating generation 60 single strains

Illustrating: aR represents disease-resistant phenotype, S represents susceptible phenotype;

bRepresent the disease-resistant allelotrope (namely from the allelotrope of disease-resistant parent Dong Nong 93-046) of SG1 mark,Represent the susceptible allelotrope (namely from the allelotrope of Susceptible parent) of SG1 mark;

cRepresent the disease-resistant allelotrope (namely from the allelotrope of disease-resistant parent Dong Nong 93-046) of SSR64 mark,Represent the susceptible allelotrope (namely from the allelotrope of Susceptible parent) of SSR64 mark.

Although the present invention is with preferred embodiment openly as above; but it also is not used to limit the present invention; any person skilled in the art; not departing from spirit and scope of the invention; various changes and modification can be done; therefore, protection scope of the present invention should with being as the criterion that claim book defines.

Claims

1. the method for an assistant identification soybean mosaic disease resisting poison ospc gene, it is characterized in that, separation and Extraction DNA from soybean to be measured, pcr amplification is carried out by the primer of molecule marker SSR64 and SG1, recycling electrophoresis detection SSR64 primer extension product there is marker-free locus band and/or utilizes the genotype of the primer extension product of gene type analytical system analysis SG1, it is determined that whether soybean germplasm resource to be measured has mosaic disease resisting poison ospc gene; Described marker gene site Chinese People's Anti-Japanese Military and Political College's Tofu pudding mosaic virus ospc gene be divided into from molecule marker SSR64 and SG1 between, and with molecule marker SSR64 and SG1 close linkage.

2. method described in claim 1, it is characterised in that, step is as follows:

1) DNA of soybean sample to be measured is extracted;

3) electrophoresis detection step 2 is utilized) amplified production of the primer of gained SSR64 and/or utilize gene type analytical system to analyze the genotype of SG1 primer extension product, it is determined that whether soybean germplasm resource to be measured has marker gene locus band; Described marker gene site Chinese People's Anti-Japanese Military and Political College's Tofu pudding mosaic virus ospc gene be divided into from molecule marker SSR64 and SG1 between, and with molecule marker SSR64 and SG1 close linkage.

3. method described in claim 1 or 2, it is characterised in that, the primer of described molecule marker SSR64 is as shown in SEQIDNO.1-NO.2; The primer of described molecule marker SG1 is as shown in SEQIDNO.3-NO.4.

4. method described in claim 1 or 2, it is characterised in that, described pcr amplification, response procedures is 94 DEG C of denaturation 6min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, and 35 circulations, 72 DEG C of ends extend 5min.

5. method described in claim 1 or 2, it is characterised in that, described marker gene locus band, the product size of SSR64 primer amplification is the product size of 152bp, SG1 primer amplification is 80bp.

6. method described in claim 1 or 2, it is characterized in that, described gene type analytical system is analyzed, it is by after centrifugal for SG1 primer PCR amplified production, the fluorescent signal of scanning samples, scanning starting temperature is 70 DEG C, end temp 96 DEG C, scanning terminates rear temperature and is down to 67 DEG C, judges whether scanning gained solubility curve morphs.

7. method described in claim 1 or 2, it is characterised in that, concrete steps are as follows:

1) DNA in soybean sample blade to be measured is extracted;

4) Lightscanner gene type analytical system analytical procedure 2 is utilized) genotype of the amplified production of the molecule marker SG1 primer of gained, by the amplified production of gained centrifugal 5min under 4000rpm, again the fluorescent signal of sample is scanned, scanning initial temperature is 70 DEG C, end temp is 96 DEG C, scanning is cooled to 67 DEG C after terminating, and judges whether scanning gained melting curve exists variation.

8. the arbitrary described method of claim 1-7, for the genetic breeding of the poison qualification of ospc gene of soybean mosaic disease resisting, screening and soybean.