CN105384801A - Pea anti-powdery mildew er1 allele er1-7 and gene er1-7 linked-molecular marker - Google Patents

Abstract

The invention relates to a pea anti-powdery mildew er1 new allele er1-7 and a gene er1-7 linked-molecular marker, wherein the allele is positioned on an er1 gene locus of the VI<th> linkage group of a pea genetic map. A pea resource G0003967 is immune to China powdery mildew isolate EPYN and EPBJ. Through a genetical analysis of resistance, genetic linkage mapping and determining on a candidate gene PsMLO1cDNA sequence of er1, an anti-disease gene of G0003967 is authenticated, and it is found that compared with a wild type susceptible gene PsMLO1cDNA sequence, the 111<th>-120<th> base groups of an open reading frame of the PsMLO1cDNA sequence of the G0003967 are deleted, thus causing change of protein functions of PsMLO1. Such small fragment deletion mutation is a new PsMLO1 deviant form, and shows that the anti-powdery mildew gene contained in the G0003967 is a new er1 allele and is named as er1-7, thus providing a new gene resource for molecular breeding of powdery mildew resistance of pea resource.

Description

Pea mildew-resistance er1 allelotrope er1-7 and the molecule marker chain with gene er1-7

Technical field

The present invention relates to plant pathology, genetic breeding and biology field, specifically, relate to pea mildew-resistance er1 neomorph er1-7 and the molecule marker chain with gene er1-7.

Background technology

Pea (PisumsativumL.) is legume crop important in the world.The powdery mildew of pea caused by powdery mildew (ErysiphepisiD.C.) is global important disease, this disease generally occurs in temperate zone and subtropical zone, under the weather condition that particularly warm, night is cold by day, harm is serious, cause 25% ~ 50% production loss, the Foreign Banks' Entries loss of severe infections can reach more than 80% (Nisaretal., 2011; Fondevillaetal., 2012).

Control powdery mildew of pea is the most economical, effective and the method for environmental safety is plantation disease-resistant variety.At present, identify a large amount of mildew-resistance pea resources abroad, and in Resistance resource, identify the mildew-resistance gene of 3 independent inheritances, comprise 2 recessive gene er1 (Harlandetal., 1948) and er2 (Heringaetal., 1969) and 1 dominant gene Er3 (Fondevillaetal., 2007).The resistance mechanism of er1 gene suppresses pathogenic bacteria to the intrusion of epidermal cells of host, performance high resistance or immunity, resistance is not by the impact of envrionment conditions, there is wide spectrum, durable resistance, widespread use (Fondevillaetal., 2006) in the pea breeding of Europe, North America and Australia.The resistance mechanism of er2 is the development being limited powdery mildew in pathogenic bacteria invasion by the death of host cell, only on blade, show resistance, and resistance is by the impact of the many factors such as temperature, leaf age, show as susceptible as a rule, which has limited its application (Fondevillaetal., 2006) in breeding.Er3 finds recently in pea wild species P.fulvum, and its using value needs to study (Fondevillaetal., 2007) further.

So far, except identifying pea mildew-resistance gene er2 and identify Er3 in pea resource JI2480 in pea wild species P.fulvum, the research of a large amount of Resistance resource screening and genetic analysis aspect shows, the resistance of the mildew-resistance pea resource of many different geographic origin controls (Tiwarietal., 1997a by er1; Ghafooretal., 2012; Liuetal., 2003; Vaidetal., 1997).Therefore, in producing at present, the resistance of the mildew-resistance pea resource of application controls by er1.Along with the exploitation of pea molecule marker and the structure of genetic map, disease-resistant gene er1 and er2 is positioned in the 6th linkage group (LGVI) (Timmermanetal. of pea genetic map respectively, 1994) the and 3rd linkage group (LG III) (Katochetal., 2010) on, and the position of gene Er3 on pea genetic map also uncertain (Fondevillaetal., 2007).

Recently, Humphry etc. (2011) and Pavan etc. (2011) study discovery, and pea mildew-resistance gene er1 produces by with barley sense powdery mildew gene (MLO) sequence homology PsMLO afunction.Under field conditions (factors), pea PsMLO homologous sequence generation base deletion, insertion, replacement etc. cause different er1 allelotrope to produce, as Mexique4, Stratagem, JI210 and JI1951 cause different er1 allelotrope to produce because the position of sudden change is different from mode, i.e. er1-1, er1-2, er1-3 and er1-4 (Humphryetal., 2011).Another allelotrope of er1 er1-5 (Pereiraetal., 2010 are obtained by chemical mutagen process pea sense Powdery Mildew susceptible variety; Humphryetal., 2011).Recently, Sunetal (2015b) identifies er1 neomorph er1-6 in Chinese Local Varieties of Pea.

China is less to pea Resistence research, and current research mainly concentrates on the Screening germplasm of pea mildew-resistance, has identified some mildew-resistance resources.Recently, Wang Zhongyi etc. (2013) and pair Haining etc. (2014), under control culture condition, have filtered out many immune resources.Disease-resistant er1 allelotrope er1-1, er1-2, er1-6 (Sunetal., 2015a, 2015b is identified in these pea Resistant germplasm; Wang Zhongyi etc., 2015).

Summary of the invention

The object of this invention is to provide pea mildew-resistance er1 neomorph er1-7 and proteins encoded thereof.

Another object of the present invention is to provide the molecule marker chain with pea mildew-resistance allelotrope er1-7.

In order to realize the object of the invention, er1-7 albumen provided by the invention, its aminoacid sequence is as shown in SeqIDNo.1, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function.

The present invention also provides the pea mildew-resistance er1 allelotrope er1-7 of encoding said proteins, and its cDNA sequence is as shown in SeqIDNo.2.

The present invention also provides the molecule marker chain with pea mildew-resistance allelotrope er1-7.Described molecule marker comprises ScOPE16-1600, ScOPD-650, c5DNAme, PSAD60 and PSMPSA5.

Wherein, for the primer sequence of pcr amplification molecule marker ScOPE16-1600 as shown in SeqIDNo.3 and SeqIDNo.4;

For the primer sequence of pcr amplification molecule marker ScOPD-650 as shown in SeqIDNo.5 and SeqIDNo.6;

For the primer sequence of pcr amplification molecule marker c5DNAme as shown in SeqIDNo.7 and SeqIDNo.8;

For the primer sequence of pcr amplification molecule marker PSAD60 as shown in SeqIDNo.9 and SeqIDNo.10;

For the primer sequence of pcr amplification molecule marker PSMPSA5 as shown in SeqIDNo.11 and SeqIDNo.12.

PCR reaction system, by 10 μ l, comprising: 2 × TaqPCRMasterMix4 μ l, upstream and downstream primer 0.2 μm of ol/L, template DNA 20ng/ μ l, ddH ₂o complements to 10 μ l.

PCR response procedures: 95 DEG C of denaturations 5 minutes; 94 DEG C 30 seconds, 51 DEG C ~ 67 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations; 72 DEG C 10 minutes, 4 DEG C of preservations.

The annealing temperature of described molecule marker ScOPE16-1600, ScOPD-650, c5DNAme, PSAD60 and PSMPSA5 is respectively 67 DEG C, 65 DEG C, 58 DEG C, 57 DEG C, 51 DEG C.

The present invention further provides the application of described allelotrope er1-7 in the molecular breeding of pea resource mildew-resistance.

Pea resource G0003967 all shows immunity to Chinese powdery mildew isolate EPYN and EPBJ.In order to the disease-resistant er1 allelotrope of clear and definite pea mildew-resistance resource G0003967, cultivate excellent resistant variety, to reach the object effectively utilizing G0003967 disease-resistant gene, the present invention passes through resistant analysis, the candidate gene PsMLO1cDNA sequence of genetic linkage mapping and er1 measures, the disease-resistant gene of G0003967 is identified, compared with wild-type susceptible gene PsMLO1cDNA sequence, find PsMLO1cDNA sequence open reading frame 111st ~ 120 base deletions of G0003967, thus cause the change of PsMLO1 protein function.This small segment deletion mutantion is a kind of new PsMLO1 variant form, show that the gene of the mildew-resistance contained by G0003967 is a new er1 allelotrope, called after er1-7, this equipotential gene position is on the er1 locus of pea genetic map VI linkage group.

The present invention, by the er1 candidate gene PsMLO1cDNA sequential analysis to pea mildew-resistance resource G0003967, has found new resistance er1 allelotrope er1-7.Determine the cDNA sequence of neomorph er1-7.The first identified of the present invention neomorph er1-7 of er1, significant to pea mildew-resistance breeding work, the generation of powdery mildew of pea effectively can be controlled by breeding technique, alleviate the financial loss that this disease causes, and provide new genetic resources for the molecular breeding of pea resource mildew-resistance.

Accompanying drawing explanation

Fig. 1 is Resistant germplasm G0003967 (left side) and the phenotype of No. 6, susceptible variety dam pea (right side) inoculation pea powdery mildew isolate after EPYN10 days in the embodiment of the present invention 1.

Fig. 2 is the pea mildew-resistance er1 allelotrope er1-7 genetic linkage maps drawn in the embodiment of the present invention 2.

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 pea mildew-resistance kind G0003967 related in following examples, No. 6, pea sense Powdery Mildew kind dam pea all come from China national crop germplasm storehouse.

The resistant analysis of embodiment 1 pea mildew-resistance resource G0003967

One, vegetable material and inoculum

Pea mildew-resistance kind G0003967; No. 6, pea sense Powdery Mildew kind dam pea.Powdery mildew (Erysiphepisi) Chinese isolate EPYN.

Two, the resistant analysis of G0003967

With disease-resistant variety G0003967 for male parent, No. 6, susceptible variety dam pea (Er1) is as maternal, and hybridization produces F ₁generation, F ₁f is produced for selfing ₂generation, F ₂102 F are produced for selfing ₃family, carries out resistant analysis, disease-resistant gene qualification to above-mentioned colony, and with it for mapping population.F ₁and F ₂colony adopts excised leaf conidium to shake off method and inoculates, and when pea seedlings the 3rd or the 4th stipes mounted blade, takes blade to inoculate, F ₃colony adopts plant inoculation method, F ₃each family of colony selects 24 planting seeds, and qualification plant is to the anti-sense reaction of pea powdery mildew isolate EPYN.After inoculation, be positioned over 18 ~ 22 DEG C, cultivate under 12h illumination.After 10 days, adopt the severity Scaling standard of 0 ~ 4 grade, with reference to (2015) method investigation incidences such as Wang Zhongyi.Evaluation of resistance is disease-resistant (R) with 0 ~ 2 grade, and 3 ~ 4 grades is susceptible (S).Investigation F ₂and F ₃family anti-, be separated, the segregation ratio of susceptible family, carry out resistant analysis and study the genetics of resistance rule of G0003967 to powdery mildew of pea.

The survey showed that, and the plant of No. 6, Susceptible parent dam pea is covered by a large amount of powdery mildew conidium, and rank is 4 grades, shows as susceptible (Fig. 1 is right).And any susceptible symptom does not appear in disease-resistant parent G0003967, rank is 0 grade, shows as immunity (Fig. 1 is left).4 F that No. 6, dam pea is derivative with G0003967 cross combination ₁plant is all disease plant, 102 F that No. 6, dam pea is derivative with G0003967 cross combination ₂in individual plant, have 78 susceptible strains, 24 Resistant variants, through X ²inspection meets the segregation ratio of the 3:1 (S:R) of expectation.F ₃the Resistance Identification result of family to bacterial strain EPYN shows, pure and mild disease-resistant family is 24, and pure and mild susceptible family is 31, and heterozygous family is 47.Through X ²inspection meets the segregation ratio of the 1:2:1 (R:Rs:S) of expectation.

Three, the extraction of pea genomic dna

CTAB method is adopted to extract 2 parents and 102 F ₂the genomic dna of plant.By sample as using proof press grind into powder in liquid nitrogen, in centrifuge tube, adding 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 (25:24:1, volume ratio), 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 (24:1), fully mixing is after 10 minutes, 12000 centrifugal 10 minutes; Get supernatant liquor to move 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, the agarose gel electrophoresis with 1% detects the integrity of DNA and the concentration of ultraviolet spectrophotometry detection DNA sample.

Four, the disease-resistant gene linked marker of G0003967 is analyzed and location

Known and that er1 is chain 10 molecule markers are utilized to carry out pcr amplification between parent G0003967 and No. 6, dam pea and polymorphism screens (table 1).PCR reaction system is 10 μ l, comprising: 2 × TaqPCRMasterMix4 μ l, upstream and downstream primer 0.2 μm of ol/L, template DNA 20ng/ μ l, ddH ₂o complements to 10 μ l.PCR response procedures: 95 DEG C of denaturations 5 minutes; 94 DEG C 30 seconds, 51 DEG C ~ 67 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations; 72 DEG C 10 minutes, 4 DEG C of preservations.Amplified production 8% Polyacrylamide Gel Electrophoresis.

By parental line selection, in 10 linked markers, there are 5 to be marked between parent there is polymorphism, the molecule marker between parent with polymorphism is carried out F ₂colony's checking (Fig. 2).Molecule marker is at F ₂clastotype in colony carries out X ²inspection.Wherein, 2 SCAR mark ScOPE16-1600 and ScOPD-650 are dominant marker, and it all presents the distribution of 3:1 in colony; 1 genetic marker c5DNAme and 2 SSR marker PSAD60 and PSMPSA5 is codominant marker, and it all presents 1:2:1 distribution in colony.

With the linkage relationship of mapmaker3.0 software analysis molecule marker and disease-resistant gene.Use MapDraw software building genetic linkage maps, the interval of location disease-resistant gene.Use mapmaker3.0 software linkage analysis to show, these 5 pairs of polymorphism primers and disease-resistant gene er1-7 all chain.Use MapDraw Software on Drawing genetic linkage maps, er1-7 is positioned between genetic marker c5DNAme and SCAR mark ScOPE16-1600, and genetic distance is respectively 4.2cM and 8.3cM (Fig. 2).

The molecule marker primer sequence information that table 1. is chain with er1

The er1 candidate gene PsMLO1cDNA Sequence Identification of embodiment 2 Resistance resource

The plant total serum IgE of pea resource G0003967 and No. 6, susceptible check variety dam pea is extracted with RNAprep plant total RNA extraction reagent box (centrifugal column type, biochemical purchased from sky root).Concrete operation method is as follows:

1) get about 100mg pea young leaflet tablet rapid grind into powder in liquid nitrogen, add 450 μ LRL (adding beta-mercaptoethanol to final concentration before use is 1%), vortex concuss mixes;

2) all solution is transferred to (Filter column CS is placed in collection tube) on Filter column CS, the centrifugal 5min of 12000rpm, the supernatant in careful absorption collection tube is in the centrifuge tube of RNase-free;

3) in centrifuge tube, slowly add the dehydrated alcohol of 0.5 times of volume, mixing, the solution obtained is proceeded in adsorption column CR3 together with precipitation, leave standstill the centrifugal 1min of 2min, 12000rpm, outwell the waste liquid in collection tube;

4) in adsorption column CR3, add 500 μ L protein liquid removal RW1, leave standstill the centrifugal 1min of 2min, 12000rpm, outwell the waste liquid in collection tube;

5) preparation of DNase I working fluid: get 10 μ LDNase I storage liquid and put into new RNase-free centrifuge tube, add 70 μ LRDD solution, softly mix;

6) add DNase I working fluid of 80 μ L to adsorption column CR3 central authorities, room temperature places 20min;

7) in adsorption column CR3, add 350 μ L protein liquid removal RW1, leave standstill the centrifugal 1min of 2min, 12000rpm, outwell the waste liquid in collection tube;

8) in adsorption column CR3, add 500 μ L rinsing liquids RW (adding dehydrated alcohol before use), room temperature leaves standstill 2min, and the centrifugal 1min of 12000rpm, outwells the waste liquid in collection tube;

9) repeating step 8);

10) the centrifugal 2min of 12000rpm, outwells waste liquid, adsorption column CR3 is placed in room temperature and places several minutes, thoroughly dries residual rinsing liquid;

11) adsorption column CR3 is put into a new RNase-free centrifuge tube, the unsettled dropping 50 in the mid-way to adsorption film μ LRNase-freeddH ₂o, room temperature places the centrifugal 2min of 10min, 12000rpm, obtains RNA solution;

12) RNA integrity detection: the gel electrophoresis of plain agar sugar, deposition condition: gum concentration 1.2%; 1 × TBE electrophoretic buffer; 150V; 15min.

MRNA the 1st article of cDNA chain is synthesized with BioRT reverse transcription amplification (RT-PCR) test kit (two-step approach), with PsMLO1 Auele Specific Primer, pcr amplification (Pavanetal., 2013) is carried out to PsMLO1F/PsMLO1R (5 '-AAAATGGCTGAAGAGGGAGTT-3 '/5 '-TCCACAAATCAAGCTGCTACC-3 ').Pcr amplification product detects through 1.5%TBE agarose gel electrophoresis, adopts plain agar sugar gel DNA to reclaim test kit (centrifugal column type, biochemical purchased from sky root) and reclaims and purified pcr product.PCR primer after purifying is cloned in pZeroBack carrier or pMG-T carrier (biochemical purchased from sky root), Beijing Liuhe Huada Genomics Technology Co., Ltd is sent to check order, utilize that ClustalX2 software analysis is disease-resistant, PsMLO1cDNA sequence difference between susceptible variety, and compare (Humphryetal.2011) with known wild-type Foreign Banks' Entries PsMLO1cDNA sequence (GenBank:FJ463618).

Result shows, the PsMLO1cDNA sequence of No. 6, susceptible contrast dam pea is identical with wild-type PsMLO1 sequence (FJ463618), the PsMLO1cDNA of Resistant germplasm G0003967 is compared with wild-type PsMLO1cDNA sequence, there are 111st ~ 120 base deletions, thus cause the change of PsMLO1 protein function (SeqIDNo.1).This small segment disappearance does not also have relevant report at present, is a kind of new PsMLO1 mutant form (SeqIDNo.2), and this result shows containing new mildew-resistance allelotrope in G0003967, by its called after er1-7.This equipotential gene position is on the er1 locus of pea genetic map VI linkage group, and this disease-resistant gene has resistance of wide spectrum to powdery mildew of pea, effectively can control the generation of China's powdery mildew of pea, alleviate the production loss that this disease causes.This gene can effectively be applied in molecular breeding, to pea produce and breeding work significant.

The CDS sequence that the PsMLO1cDNA of pea mildew-resistance resource G0003967 is corresponding and encoding amino acid sequence thereof are shown in SeqIDNo.25.

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.

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Claims (6)

1.er1-7 albumen, is characterized in that, its aminoacid sequence is as shown in SeqIDNo.1, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function.

2. the pea mildew-resistance er1 allelotrope er1-7 of albumen described in coding claim 1.

3. allelotrope er1-7 as claimed in claim 2, it is characterized in that, its cDNA sequence is as shown in SeqIDNo.2.

4. the molecule marker chain with allelotrope er1-7 described in Claims 2 or 3, it is characterized in that, described molecule marker comprises ScOPE16-1600, ScOPD-650, c5DNAme, PSAD60 and PSMPSA5;

For the primer sequence of pcr amplification molecule marker ScOPE16-1600 as shown in SeqIDNo.3 and SeqIDNo.4;

For the primer sequence of pcr amplification molecule marker ScOPD-650 as shown in SeqIDNo.5 and SeqIDNo.6;

For the primer sequence of pcr amplification molecule marker c5DNAme as shown in SeqIDNo.7 and SeqIDNo.8;

For the primer sequence of pcr amplification molecule marker PSAD60 as shown in SeqIDNo.9 and SeqIDNo.10;

For the primer sequence of pcr amplification molecule marker PSMPSA5 as shown in SeqIDNo.11 and SeqIDNo.12.

5. molecule marker as claimed in claim 4, is characterized in that,

PCR reaction system, by 10 μ l, comprising: 2 × TaqPCRMasterMix4 μ l, upstream and downstream primer 0.2 μm of ol/L, template DNA 20ng/ μ l, ddH ₂o complements to 10 μ l;

PCR response procedures: 95 DEG C of denaturations 5 minutes; 94 DEG C 30 seconds, 51 DEG C ~ 67 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations; 72 DEG C 10 minutes, 4 DEG C of preservations.

6. molecule marker as claimed in claim 5, it is characterized in that, the annealing temperature of molecule marker ScOPE16-1600, ScOPD-650, c5DNAme, PSAD60 and PSMPSA5 is respectively 67 DEG C, 65 DEG C, 58 DEG C, 57 DEG C, 51 DEG C.