CN102220415B

CN102220415B - Markers of phytophthora sojae avirulence gene Avr5

Info

Publication number: CN102220415B
Application number: CN 201110094880
Authority: CN
Inventors: 董莎萌; 王源超; 张正光; 郑小波
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2011-04-15
Filing date: 2011-04-15
Publication date: 2013-01-23
Anticipated expiration: 2031-04-15
Also published as: CN102220415A

Abstract

The invention belongs to the field of biological technology, and relates to specific molecular markers of phytophthora sojae avirulence gene Avr5, which are CAPS-80316 and CAPS-80295. With a CAPS (Cleaved Amplified Polymorphic Sequence) molecular marker technology, two parent bacterial strains (P 6497, and P 7064) of phytophthora sojae and their 94 hybridized progeny colonies are processes through avirulence gene molecular marker selection and identification, and two molecular markers cosegregated with phytophthora sojae avirulence gene Avr5 are obtained. The invention can be applied in the research of the avirulence gene Avr5 distribution in natural colony, for revealing differentiation and variation characteristics of phytophthora sojae pathotype in fields, and assists in providing convenience for cloning the avirulence gene, and for finally identifying phytophthora sojae gene Rps5.

Description

The mark of soybean phytophthora nontoxic gene Avr5

Technical field

The present invention relates to the mark of soybean phytophthora nontoxic gene, be specifically related to closely two molecular marker gene CAPS-80316 and the CAPS-80295 of interlocking with soybean phytophthora nontoxic gene Avr5, and in the nontoxic gene labeling process used marking method.

Background technology

Soyabean phytophthora belongs to flagellum organic sphere oomycetes door downy mildew subclass, rotten mould order, pythiaceae, phytophthora (Tyler, 2007). and phytophthora includes more than 80 the mould kind of epidemic disease, and a variety of all is pathogenic bacteria important in the agriculture production.Different from the mould kind of most epidemic diseases, the host range of soyabean phytophthora is very narrow, mainly infects soybean, only can infect in addition the several plant of lupinus (Lupinus spp.).Soyabean phytophthora infects soybean, causes the sudden death of seedling and the root-rot of strain, is one of chief threat of Soybean production in the world wide.Cause direct economic loss up to tens00000000 dollars (Tyler, 2007) for every year worldwide Soybean production.This disease is in 1948 first in the discovery of the Indiana State of the U.S., then in succession this disease (Schmitthenner, 1985 have all been found in countries such as Australia, Canada, Brazil, Argentina, Japan, Italy, Singapore, Russia, Byelorussia, Ukraine, Kazakhstan, Hungary, Germany, Britain, France, Switzerland, New Zealand, Egypt, Nigeria, India; Wrather et al., 2001).China 1989 by Shen Chong Yao and Su Yanchun (1991) first northeastward the soybean main producing region be separated to this pathogenic bacteria, afterwards in Heilungkiang, Jilin, Beijing, Shandong, Anhui, Henan, Jiangsu, Zhejiang, the Inner Mongol, Hubei, Fujian, Xinjiang, Sichuan, Tianjin and Guizhou (Su Yanchun etc., 1993; Zhu Zhendong etc., 2003; Xu Jing waits quietly, and 2009; Tang Qinghua etc., 2009) etc. 15 provinces (urban district) also are in the news isolation identification in succession to soyabean phytophthora.Wherein comparatively serious in Heilungkiang and the generation of Fujian two provinces, other provinces sporadicly occur, and are still at present the important external Quarantine Objects of China.

In the long-term coevolution process of plant and oomycetes, oomycetes secretes a large amount of effector molecules (effectors) destroyed or delayed plant to the different sites of vegetable cell defense response (Birch et al., 2006 in order successfully to infect plant; Kamoun, 2006; Vleeshouwers et al., 2008).Studies show that in a large number " gene pairs gene " hypothesis that meets Flor of most of oomycetes disease, therefore, the same with a lot of plant pathogenetic bacterias and fungi, also exist a class effector molecule to be identified by the disease-resistant gene product of host plant (R protein) in the oomycetes, thereby cause strong plant immunization reaction and then the generation of prevention disease, and the gene of this class effector molecule of encoding is called as nontoxic gene (Avr gene).Interaction between the Avr protein of oomycetes secretion and the R protein of host plant is directly determining the generation of oomycetes disease, and therefore, the research that centers on the Avr gene is one of emphasis of oomycetes disease research always.In the past, the research of oomycetes Avr gene mainly concentrated in the evaluation and molecule marker of Avr gene, and recently, because being widely used of the new technologies such as the announcement of phytophthora genome sequence and particle gun, more than ten oomycetes Avr gene is cloned successively.At present, the oomycetes Avr gene that has been cloned into comprises coming the from the beginning ATR1 of Hyaloperonospora arabidopsis ^NdWsBAnd ATR13; From PiAvr3a, PiAvr4, PiAvrblb1 and the PiAvrblb2 of P.infestans, and from the PsAvr1b of P.sojae, PsAvr1a, PsAvr3a, PsAvr3c and PsAvr4/6 (Allen et al., 2004; Shan et al., 2004; Armstrong etal., 2005; Rehmany et al., 2005; Van Poppel et al., 2008; Vleeshouwers et al., 2008; Dong etal., 2009; Qutob et al., 2009; Sang-Keun et al., 2009; Dou et al., 2010).In addition the feature of these Avr genes on gene and genome structure, enter the transhipment mechanism of vegetable cell and the toxicity functional study that has also becomes study hotspot in Plant diseases.Start with from the clone of oomycetes nontoxic gene, explore origin and the evolution of nontoxic gene, clear and definite its function in the germ pathogenic course is understood the coevolution mechanism between nontoxic gene and the disease-resistant gene, for nearly step understanding with to prevent and treat such disease significant.

Forefathers find that soyabean phytophthora nontoxic gene Avr5 and Avr3a are positioned on the same linkage group, and genetic distance between the two is 4.5cM (May et al, 2002).Avr3a is cloned recently, and length of this genes encoding is the 112 amino acid whose outer albumen of secreting, and this provides clue for further seeking nontoxic gene Avr5.

Summary of the invention

Technical problem to be solved by this invention provides molecule marker, method and the primer thereof of a kind of easy, Rapid identification soybean phytophthora nontoxic gene Avr5, and screening obtains and the closely linked molecule marker of nontoxic gene Avr5.

Technical scheme provided by the invention is: a kind of molecule marker primer that detects nontoxic gene Avr5 in the soybean phytophthora bacterial strain, upstream primer nucleotide sequence are shown in SEQ ID NO.1, and the downstream primer nucleotide sequence is shown in SEQ ID NO.2.

The molecule marking method of nontoxic gene Avr5 in a kind of soybean phytophthora bacterial strain, utilize above-mentioned primer amplification soyabean phytophthora genomic dna to be measured, amplified production is carried out enzyme with the ScaI enzyme cut, if only obtain the fragment of 475bp, show that soyabean phytophthora genome to be measured contains the homozygote of Avr5; If obtain two kinds of fragments of 157bp and 318bp, show that soyabean phytophthora genome to be measured does not contain Avr5; If obtain 157bp, 318bp and three kinds of fragments of 475bp, show that soyabean phytophthora genome to be measured is the heterozygote that contains Avr5.

The invention provides the another kind of molecule marker primer that detects nontoxic gene Avr5 in the soybean phytophthora bacterial strain, the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4.

The invention provides the molecule marking method of nontoxic gene Avr5 in the another kind of soybean phytophthora bacterial strain, utilize above-mentioned primer amplification soyabean phytophthora genomic dna to be measured, amplified production is carried out enzyme with the EcoRI enzyme to be cut, if only obtain the fragment of 373bp, show that soyabean phytophthora genome to be measured contains the homozygote of Avr5; If obtain two kinds of fragments of 150bp and 217bp, show that soyabean phytophthora genome to be measured does not contain Avr5; If obtain 150bp, 217bp and three kinds of fragments of 373bp, show that soyabean phytophthora genome to be measured is the heterozygote that contains Avr5.

Utilize the soyabean phytophthora genome database finished order-checking and to have announced ( Http:// genome.jgi-psf.org/Annotator/servlet/jgi.annotation.Annotation? pDb=Physo1_1), determine that the sequence location of known non-toxic Gene A vr3a in genome database is Scaffold_80:300039-300374, the genetic distance between Avr5 and the Avr3a is 4.5cM.

Utilize inquiry and the download function of soyabean phytophthora genome database, download the interior nucleotide sequence of 40kb genome area of Scaffold_80:280000-320000, find intergenic region (Intergenic Region) by its website, and for go forward side by side performing PCR amplification of the primer that these zone design amplification lengths are 1000bp, by the genomic dna difference purifying recovery product of pcr amplification from two parent strains, send company's order-checking.

By the result of returning that checks order, utilize the alternative sudden change of the Seqman program looks sequence among the Lasergene, perhaps insertion/deletion mutational site, seek again the restriction enzyme site of difference by SeqBuilder software, then the PCR product of previous correspondence is carried out enzyme and cut, thereby electrophoresis is determined differential fragment and obtained high-quality CAPS molecule marker.

Utilize simultaneously the soybean phytophthora bacterial strain hybridization generation F1 contain respectively Avr5 and not contain the Avr5 gene for heterozygote, selfing produces 94 F2 generations again, with these F2 for inoculation to the L85-3059 soybean of (containing the Rps5 disease-resistant gene), determine the toxicity of these bacterial strains, utilize simultaneously the CAPS molecule marker obtained at F2 for the enterprising row labels of different loci.

Comprehensive Pathogenicity and CAPS mark result compare for phenotype and genotype F2, the phenotype of finding CAPS-80316 and two molecule markers of CAPS-80295 and Avr5 is on all four at 94 F2 in generation, thereby determines that these two molecule markers and this nontoxic gene Avr5 closely interlock.

Compare with existing molecular marking technique, its advantage and positively effect show:

The present invention not only can study the distribution situation of nontoxic gene Avr5 in natural population, discloses soyabean phytophthora in the characteristics of field pathotype dissociation; The inside and outside difference of soyabean phytophthora on this site of comparator, the mould colony of clarification Chinese soybean epidemic disease originates from abroad; Can study origin, the evolution of nontoxic gene Avr5 and common chain Avr3a nontoxic gene, the process of variation; Two marks that the present invention determines can be for clone's nontoxic gene Avr5 provides good clue, also the final Soybean Resistance phytophthora gene Rps5 that identifies provides science to help, and helps scientist to carry out the seed selection of soybean disease-resistant variety.

Description of drawings

The restriction enzyme mapping of Fig. 1: CAPS-80316 and CAPS-80295 molecule marker.

The comparison of Fig. 2: CAPS-80295 nucleotide sequence in R2 (P6497) bacterial strain and R7 (P7064) bacterial strain.

The comparison of Fig. 3: CAPS-80316 nucleotide sequence in R2 (P6497) bacterial strain and R7 (P7064) bacterial strain.

Embodiment

Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does the example explanation.

Embodiment 1

1, strains tested

Soybean phytophthora genome sequencing bacterial strain R2 (P6497) and R7 (P7064) are Mark Gijzen researcher (south Crop protection institute of Ministry of Agriculture Canada) present, and planting disease by Agricultural University Of Nanjing is that preserve in the molecular biology of fungi laboratory.These two bacterial strains are disclosed in Qutob D, Tedman-Jones J, Dong S, Kuflu K, Pham H, et al. (2009) Copy number variation and transcriptional polymorphisms of Phytophthora sojaeRXLR effector genes Avrla and Avr3a.PLoS One 4:e5066.Bacterial strain is stored in the 10%V8 solid medium, and storage temperature is 14 ℃.

Wherein, the homozygote of Avr5 is contained in R2 bacterial strain system, and the homozygote of Avr5 is not contained in R7 bacterial strain system.

2, design CAPS mark

Utilize inquiry and the download function of soyabean phytophthora genome database, download the interior nucleotide sequence of 40kb genome area of Avr3a region Scaffold_80:280000-320000, find intergenic region (Intergenic Region) by its website, and for the primer of the 1000bp product performing PCR amplification of going forward side by side of increasing of these zone design, by the genomic dna difference purifying recovery product of pcr amplification from two parent strains, order-checking.Pass through sequencing result, utilize the alternative sudden change of the Seqman program looks sequence among the Lasergene, perhaps insertion/deletion mutational site, seek again the restriction enzyme site of difference by SeqBuilder software, then the PCR product of previous correspondence is carried out enzyme and cut, thereby electrophoresis is determined differential fragment and obtained high-quality CAPS molecule marker as shown in table 1 and primer.Its upstream primer nucleotide sequence of the labeled primer of CAPS-80316 is shown in SEQ ID NO.1, and the downstream primer nucleotide sequence is shown in SEQ ID NO.2.Its upstream primer nucleotide sequence of the primer of CAPS-80295 mark is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4.CAPS-80295 is marked at nucleotide sequence in R2 (P6497) bacterial strain shown in SEQID NO.5, and the nucleotide sequence in R7 (P7064) bacterial strain is shown in SEQ ID NO.6; CAPS-80316 is marked at nucleotide sequence in R2 (P6497) bacterial strain shown in SEQ ID NO.7, and the nucleotide sequence in R7 (P7064) bacterial strain is shown in SEQ ID NO.8.

Table 1

3, for the preparation that tries soybean seedling

Differential host's soybean planting is in the plastic flowerpot that vermiculite (d=2-4mm) is housed (d=10cm), 12 seeds of every basin kind, putting into 24-28 ℃, the greenhouse of 14h illumination (strong illumination)/10h dark grows, two true leaves grow but not expansion after 7 days, and thinning makes 10 seedling of every basin and fully waters for inoculation.Used soybean differential host L85-3059 is provided by south Crop protection institute of Ministry of Agriculture Canada Mark Gijzen laboratory, and susceptible check variety is Williams.

4, the mensuration of soybean phytophthora bacteria pathogenic

Adopt Hypocotyls inoculation technique (Qutob et al, 2009), measure the soybean phytophthora bacterial strain to differential host's toxicity.The soybean phytophthora bacterial strain on the LBA flat board dark 25 ℃ cultivated 7 days, be cut into afterwards the fritter of 2mm * 3mm as inoculum; The approximately long wound of 0.5cm is drawn at 1cm place under the cotyledon of soybean seedling downwards with scalper, is attached to the wound in then the inoculum mycelia being faced.Then moisturizing 12h after the inoculation puts into 24-28 ℃, the greenhouse of 14h illumination (strong illumination)/10h dark, 5 days " Invest, Then Investigate " results.Each differential host inoculates 10 strains, and with the criteria for classification of plant mortality ratio as anti-sense, plant mortality ratio≤30% is designated as disease-resistant (A); Plant mortality ratio 〉=70% is designated as susceptible (V); The plant mortality ratio is designated as intermediate type (I) (Ryley et al., 1998) between 30%-70%.Pathogenicity test results repeats 3 times.

5, the extraction of genomic dna

Adopt CTAB (Cetyltrimethylammonium bromide) method to extract soybean phytophthora genomic dna (Doyle﹠amp; Doyle, 1987), concrete operations are as follows:

(1) gets hypha powder after about 50mg lyophilize in 1.5ml EP pipe, add 900 μ l 2%CTAB (cetyl trimethylammonium bromide) extracting solution (2%CTAB; 100mmol/L Tris-HCl, PH 8.0; 20mmol/L EDTA, PH8.0; 1.4mol/L abundant mixing on eddy mixer NaCl) and behind the 90 μ l 10%SDS (Sodium dodecylbenzene sulfonate);

(2) 55-60 ℃ of water-bath 1h, every 15min vibration mixing is once;

(3) the centrifugal 10min of 12000rpm/min gets supernatant liquor, add isopyknic phenol chloroform primary isoamyl alcohol (25: 24: 1) carry 1 time;

(4) the centrifugal 10min of 12000rpm/min draws supernatant liquor, adds equal-volume chloroform extracting 1 time;

(5) the centrifugal 10min of 12000rpm/min draws supernatant liquor, the ice dehydrated alcohol that adds the 3MNaAC solution of 0.1 * volume and the 2 * volume precipitation genomic dna that spends the night;

(6) with 70% washing with alcohol twice of precooling, then put 37 ℃ of lower dryings;

(7) dried DNA leaves standstill the 1h degradation of rna with 40 μ l, 1 * TE (10mmol/L Tris-HCL, 0.1mmol/L EDTA, PH8.0) dissolving rear (containing 50 μ g/ml RNase among the TE) in 37 ℃;

(8) get 2 μ l DNA samples electrophoresis on 1% Agarose glue, UV spectrophotometer measuring DNA concentration and purity, and be diluted to approximately behind the 25ng/ μ l in-20 ℃ of refrigerators for subsequent use.

6, pcr amplification and enzyme are cut detection

Employing contains molecule marker that the two pairs of CAPS molecule marker primer amplifications of CAPS mark CAPS-80316 and CAPS-80295 obtain and carry out screening with nontoxic gene Avr5 linked marker in two parent.The PCR reaction system of CAPS mark is: reaction cumulative volume 25ul, 2.5ul PCR Buffer wherein, 0.5ul Mg2+, two primers (each 0.25ul), rTaq enzyme (TAKARRA) 0.1ul, dNTP 0.4ul, genomic templates 0.1-0.5ng, ddH2O are that the concrete response procedures of 20ul. is: 98 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 40s, 35 circulations of increasing.Get 5ul PCR product, add restriction endonuclease 0.2ul, enzyme is cut Buffer 1ul, and ddH2O is 3.8ul, and 37 ℃ were reacted 2 hours, and got product 5ul and be splined on 0.8% agarose gel electrophoresis analysis.

Obtain two CAPS molecule marker: CAPS-80316 and CAPS-80295 by screening.Take the phytophthora genomic dna as template and the pair of primers amplification of CAPS-80316 be 475bp, cut the R2 product with the ScaI enzyme and still obtain the 475bp fragment, enzyme is cut two kinds of fragments that the R7 product obtains 157bp and 318bp, and enzyme is cut the heterozygote product and obtained 157bp, 318bp and three kinds of fragments of 475bp (seeing Fig. 1).Take the phytophthora genomic dna as template and the pair of primers amplification of CAPS-80295 be 373bp, cut the R2 product with the EcoRI enzyme and still obtain the 373bp fragment, enzyme is cut two kinds of fragments that the R7 product obtains 150bp and 217bp, and enzyme is cut the heterozygote product and obtained 150bp, 217bp and three kinds of fragments of 373bp (seeing Fig. 1).

The electrophoretogram of two molecule marker CAPS-80316 of above presentation of results and CAPS-80295 and the phenotype whether soyabean phytophthora carries Avr5, fully coupling.Illustrate the same with R2 is the homozygote that contains Avr5, and illustrate the same with R7 is the homozygote that does not contain Avr5, and illustrate the same with F1 is the heterozygote that contains Avr5.

Embodiment 2

Be the further above-mentioned CAPS mark of checking, F1 and the F2 offspring of the sexual mating that derives from R2 (P6497) and R7 (P7064) detected.

1, soybean phytophthora F1 offspring's generation

The F1 offspring derives from the sexual mating of R2 (P6497) and R7 (P7064): the fritter of respectively two kinds of mycelia being cut 5mm is placed on the agar plate that same contains 2.5% (v/v) V-8 and 10 μ g/ml β-sitosterol, at least 30 days picking list oospore of 25 ℃ of dark growths, extract single oospore genomic dna, be confirmed to be heterozygote with the CAPS mark.Heterozygote F1 is placed on the agar plate that contains 2.5% (v/v) V-8 and 10 μ g/ml β-sitosterol for the fritter that mycelia cuts 5mm.

2, the acquisition of soyabean phytophthora F2 and Pathogenicity

F1 is produced F2 generation for selfing, 94 F2 offsprings of random choose bacterial strain and in order 1-94 arrange numbering, 94 F2 offspring inoculation can both be caused a disease to the Williams soybean varieties that does not contain any disease-resistant gene, illustrate that offspring's virulence is not affected.Being inoculated into upper discovery of the soybean varieties L85-3059 that contains disease-resistant gene Rps5 has 69 can not cause a disease, and 25 can cause a disease, and further specifying Avr5 is the aobvious Gene Handling of a list.

3, the phenotype of CAPS-80316 and CAPS-80295 mark and Avr5 matches among the F2

Extracted respectively the genomic dna of 1-94 F2 offspring bacterial strain.Take R2 and R7 parent strain and F1 for heterozygote as contrast, genotype (CAPS-80316 and CAPS-80295 labeling pattern) and the phenotype (in the toxicity that contains on the soybean L85-3059 of resistant gene Rps5) of each F2 offspring bacterial strain are compared, find that 69 F2 that can not cause a disease all are shown as R2 or H (F1 heterozygote) type for CAPS-80316 and two marks of CAPS-8029 of bacterial strain, 25 F2 that can cause a disease all are shown as the R7 type for CAPS-80316 and two marks of CAPS-8029 of bacterial strain, and above data declaration CAPS-80316 and CAPS-80295 mark and nontoxic gene Avr5 are closely linked.

Claims

1. molecule marker primer that detects nontoxic gene Avr5 in the soybean phytophthora bacterial strain, it is characterized in that: the upstream primer nucleotide sequence is shown in SEQ ID NO.1, and the downstream primer nucleotide sequence is shown in SEQ ID NO.2.

2. molecule marker primer that detects nontoxic gene Avr5 in the soybean phytophthora bacterial strain, it is characterized in that: the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4.

3. identify the genotypic molecule marking method of nontoxic gene Avr5 in the soybean phytophthora bacterial strain for one kind, it is characterized in that: utilize primer amplification claimed in claim 1 soyabean phytophthora genomic dna to be measured, amplified production is carried out enzyme with the ScaI enzyme to be cut, if only obtain the fragment of 475bp, show that soyabean phytophthora genome to be measured contains the homozygote of Avr5; If obtain two kinds of fragments of 157bp and 318bp, show that soyabean phytophthora genome to be measured does not contain Avr5; If obtain 157bp, 318bp and three kinds of fragments of 475bp, show that soyabean phytophthora genome to be measured is the heterozygote that contains Avr5.

4. identify the genotypic molecule marking method of nontoxic gene Avr5 in the soybean phytophthora bacterial strain for one kind, it is characterized in that: utilize primer amplification claimed in claim 2 soyabean phytophthora genomic dna to be measured, amplified production is carried out enzyme with the EcoRI enzyme to be cut, if only obtain the fragment of 373bp, show that soyabean phytophthora genome to be measured contains the homozygote of Avr5; If obtain two kinds of fragments of 150bp and 217bp, show that soyabean phytophthora genome to be measured does not contain Avr5; If obtain 150bp, 217bp and three kinds of fragments of 373bp, show that soyabean phytophthora genome to be measured is the heterozygote that contains Avr5.