CN101153318B - Molecule mark for assisting root-knot nematodes N resistant gene selection - Google Patents
Molecule mark for assisting root-knot nematodes N resistant gene selection Download PDFInfo
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- CN101153318B CN101153318B CN2006101135321A CN200610113532A CN101153318B CN 101153318 B CN101153318 B CN 101153318B CN 2006101135321 A CN2006101135321 A CN 2006101135321A CN 200610113532 A CN200610113532 A CN 200610113532A CN 101153318 B CN101153318 B CN 101153318B
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Abstract
The invention provides a molecular mark assisting selection of a root-knot nematode resistant N gene. The invention adopts a F2 population which containing the 'N' gene and is composed of inbred line Carolina Wonder and susceptible inbred line solanaceous phylum and artificial inoculated root-knob nematode, together with the BSA and AFLP technologies, to develop molecular marks and obtain a molecular mark E39/M41-527.The primers augmenting the E39/M41-527 are represented in the sequence table SEQ ID NO.1, and NO.2. According the analysis of AFLP, the molecular mark E39/M41-527 shows polymorphism in a resistant-susceptible pool; and by the analysis of a Joinmap3.0 mapping software, the genetic distance of the N gene is 6.258 cm. The invention provides a foundation for selection of root-knot nematode resistant N gene in peppers.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of molecule marker that assisting root-knot nematodes N resistant gene is selected that can be used for.
Background technology
China is the country of world's pepper planting area maximum, and cultivated area in 2002 has reached 1,300,000 hectares, about 2,819 ten thousand tons of output.Root knot nematode serious harm capsicum.The southern capsicum of root knot nematode main harm China produces, but to produce increase, the raising of multiple crop index, the continuous cropping continuous cropping phenomenon of area general along with protection ground, the north in recent years, makes the north regional rapid spread of nematodiasiss.All cause more than 90% in the crop loss that root knot nematode causes by Meloidogyne incognita (M.incognita), peanut root-knot nematode (M.arenaria), javanese root knot nematode (M.javanica) and four kinds the most common of northern root knot nematode (M.hapla), the heaviest with Meloidogyne incognita harm.The easy contaminate environment of capsicum root knot nematode control chemical prevention, physical control highly energy-consuming and DeGrain.Therefore cultivate a kind of effective means that disease-resistant variety is the control nematode.Utilize traditional breeding method general easy affected by environment, efficiency of selection is low.Exploitation and the chain molecule marker of disease-resistant gene, utilizing the molecular marker assisted selection breeding is the high-efficient breeding means that developed recently gets up.At present, on capsicum, have been found that and the gene of the nematicide named has 6 at least, gene n, Me1, Me2, Me3, Me4, Me5 is the control of dominance single-gene, and wherein Me3 and Me4 are chain, Me3 is to temperature-stable (Huang Sanwen, 2000, gardening journal, 27 (supplementary issues): 515-521).
People such as calendar year 2001 C.Djian-Caporalino utilize the DH colony of AFLP technology and material " YoloWonder " " PM687 " to finish the molecular genetic of Me3 and Me4 are located, be located on same the karyomit(e), differ 9.9cM, and developed one with closely linked AFLP mark, and between genetic distance 2.7 ± 1.7cM only.(Djian-CaporalinoC.，Pijarowski?L.，Fazari?A.，Samson?M.，Gaveau?L.，O’Byrne?C.，LefebvreV.，Caranta?C.，Palloix?A.，Abad?P..2001.High-resolution?geneticmapping?of?the?pepper(Capsicum?annuum?L.)resistance?loci?Me3andMe4?conferring?heat-stable?resistance?to?root-knotnematodes(Meloidogyne?spp.).Theor?Appl?Genet，103：592-600)
The molecular genetic location of gene n is not also reported, because the N gene discovery early, disease resistance is (near immunity) by force, so more meaningful to the Study on Molecular Marker and the utilization of this gene.
Summary of the invention
The object of the present invention is to provide a kind of molecule marker that assisting root-knot nematodes N resistant gene is selected that can be used for.
Utilization of the present invention contains the self-mating system Carolina Wonder (Fery of " N " gene, R.LDukes, P.D.Sr.Thies, J.A.1998. " Carolina Wonder ' and ' CharlestonBelle ': southern root knot nematode-resistant bell peppers.Hortscience; 33 (5): 900-902) the F2 colony of setting up with susceptible self-mating system eggplant door (available from vegetables gardening breeding development centre in Beijing); adopt the artificial inoculation root knot nematode; in conjunction with BSA (Bulked SegregatedAnalysis, discrete group cluster analysis method) and AFLP technological development molecule marker.The molecule marker that obtains is E39/M41-527.
The female parent of analyzing colony is the self-mating system Carolina Wonder of " N " gene; Male parent is susceptible self-mating system eggplant door; The two hybridization obtains F1 generation, and selfing obtains F2 generation again.
BSA (Bulked Segregated Analysis, discrete group cluster analysis method):
According to the disease resistance qualification result, with F
2Sick level index is high and minimum in the colony extreme anti-, sense plant respectively select 10 strains, and every strain is got 100ng DNA and mixed, and set up anti-pond and sense pond.DNA with anti-pond and sense pond is a masterplate, increases by the AFLP method, searches the differential fragment in two ponds, and the individual DNA with F2 colony is that masterplate carries out the AFLP amplification then, seeks the distribution of differential fragment in F2 colony.
Be to the F2 aflp analysis in generation below.
1, the enzyme of genomic dna is cut and is connected
Being connected of the double digestion of genomic dna and joint adopts a step to finish, and enzyme system is EcoRI/MseI, and reaction system is as shown in table 1, and 37 ℃ were reacted 8 hours down.
The EcoRI/MseI enzyme of table 1 capsicum genomic dna is cut and linked system
2, pre-amplification
Connection product with step 1 increases in advance.Reaction system is as shown in table 2.
The pre-amplification of table 2 reaction system
Primer sequence:
EOO：GAC?TGC?GTA?CCAATT?C
MOO：GAT?GAG?TCC?TGA?GTAA
Amplification program:
94 ℃ of 3min; 25 circulation (94 ℃ of 30s; 56 ℃ of 30s; 72 ℃ of 1min), 72 ℃ of 7min mend flat end, 4 ℃ of preservations.
Get 5ul pre-expansion volume increase thing and carry out the detection of 1.0% agarose gel electrophoresis.30 times of all the other pre-expansion volume increase thing dilutions ,-20 ℃ of preservations are standby.
3, selective amplification
Carry out selective amplification with after 30 times of the amplified production of step 2 dilutions, amplification system is as shown in table 3.
Table 3 selective amplification reaction system
Primer sequence:
EcoRI primer: 5 ' GAC TGC GTA CCAATT CAGA-3 '
MseI primer: 5 ' GAT GAG TCC TGA GTAAAGG-3 '
Amplification program:
Pre-94 ℃ of 5min of sex change, 12 circulation (94 ℃ of 30s; 65 ℃ (0.7 ℃/cycle) 30s; 72 ℃ of 1min), 25 circulation (94 ℃ of 30s; 56 ℃ of 30s; 72 ℃ of 1min), 72 ℃ of 5min mend flat end, 4 ℃ of preservations.
4, denaturing polyacrylamide gel electrophoresis
Sex change sample-loading buffer 6ul (98% methane amide, 10mM EDTA, 0.25% smell phenol indigo plant, 0.25% dimethylbenzene green grass or young crops) will be added, 95 ℃ of sex change 5min, cooling rapidly in ice bath then in the product of selective amplification.Adopt permanent power 70W electrophoresis 1h5min on 6% denaturing polyacrylamide gel, the application of sample amount is 5ul.
5, silver dyes detection
Found that there is significant difference in E39/M41-527 in these two groups.(as Fig. 1)
With aforesaid method all 320 individual plants are identified, and triplicate, the result is stable.There are 282 strains to use in the mark, several 230 strains of band strain are arranged, do not have several 52 strains of band strain, several 15 strains of exchange strain, several 26 strains of uncertain strain.Analyze through the Joinmap3.0 mapping software, genetic distance is 6.258cM.Mark E39/M41-527 and N gene close linkage are described, can be used as the molecule marker that assisting root-knot nematodes N resistant gene is selected with this its.
The invention provides the molecule marker E39/M41-527 of N gene, and the method for aflp analysis, for the anti-root nematode of capsicum N gene provides the selection foundation.
Description of drawings
Fig. 1 is the right detected result of the silver of aflp analysis, and R1-R10 is 10 individual plants in disease-resistant pond among the figure; S1-S10 is 10 individual plants in susceptible pond; M: molecular weight marker; The arrow indicating positions is a mark, and disease-resistant individual plant all contains this band (not showing among 2 figure), and susceptible individual plant does not contain this band.
Fig. 2 is the distribution of AFLP mark (E39m41-527) in F2 colony, and wherein 1～17 is F2 colony individual plant, M: molecular weight standard (PBR322/MspI);
Fig. 3 is a Joinmap3.0 mapping software analytical results.
Embodiment
The female parent of analyzing colony is the self-mating system Carolina Wonder of " N " gene; Male parent is susceptible self-mating system eggplant door; The two hybridization obtains F1 generation, and selfing obtains F2 generation again.
According to the disease resistance qualification result, with F
2Sick level index is high and minimum in the colony extreme anti-, sense plant respectively select 10 strains, and every strain is got 100ng DNA and mixed, and set up anti-pond and sense pond.DNA with anti-pond and sense pond is a masterplate, increases by the AFLP method, searches the differential fragment in two ponds, and the individual DNA with F2 colony is that masterplate carries out the AFLP amplification then, seeks the distribution of differential fragment in F2 colony.
Table 4 has shown the situation that the disease resistance of F2 colony is identified.
Table 4F
2Colony's disease resistance is identified situation
Individual plant: 2, each 100ng of 3,4,5,6,7,8,9,10,12 DNA mixes and forms disease-resistant pond, and with individual plant: 29, each 100ng of 36,42,55,126,140,186,206,267,295 DNA mixes and forms susceptible pond.Be used for aflp analysis.
Aflp analysis
1, the preparation of material
Pepper seed is put into 65 degree water and was soaked 20 minutes, puts into 28 ℃ incubator vernalization after the taking-up.Sowing, the soil sterilization 170 of growing seedlings is spent 2 hours, seeding tray seedling culture, earthing 1cm keeps humidity and temperature, during seedling 2 leaves 1 core, extracts DNA.
2, the extraction of DNA
Extracting method adopts CTAB method extraction in a small amount.Step is as follows:
(1) gets the 1.5ml centrifuge tube and add fresh tender leaf a slice;
(2) grind to form broken foam behind the liquid nitrogen refrigerating, add 700ul2%CTAB (2ul BME0.4%) extracting solution;
(3) centrifuge tube is placed 65 ℃ of water-bath 1.0h, during shake once gently every 10min.Attention: the amount of sample can not be too many, otherwise owing to extract amount that damping fluid adds very little, lysis is insufficient;
(4) take out postcooling to room temperature, add 700ul chloroform/primary isoamyl alcohol (24:1) then and rotate gently and leave standstill 10min after shaking up;
(5) keep 4 ℃ of centrifugal 10min of following 10000rpm;
(6) slowly draw in the centrifuge tube that supernatant liquor joins the 1.5ml that has added the 500ul Virahol in advance with wide mouthful rifle head, mixing gently turns upside down;
(7) keep 4 ℃ of centrifugal 10min of following 10000rpm;
(8) slowly outwell whole liquid, add the ethanol rinsing DNA of 1ml70% at twice, room temperature is placed 5min, and the centrifugal 5min of 8000rpm abandons supernatant;
(9) air-dry under the room temperature, add 1 * TE100ul (1ul 10mg/ml RNase) dissolving DNA behind about 2～3h;
(10) insulation 1h removes RNA in 37 ℃ of water-baths;
(11) getting 2ul DNA, serves as that the concentration that 1.0% agarose gel electrophoresis detects DNA is carried out in contrast with λ DNA, and all dilution is 50ng/ul, and-20 ℃ of preservations are used for aflp analysis.
3, the optimization of aflp analysis and reaction system
The enzyme of A, genomic dna is cut and is connected
Being connected of the double digestion of genomic dna and joint adopts a step to finish, and enzyme system is EcoRI/MseI, and reaction system is as shown in table 5, and 37 ℃ were reacted 8 hours down.
The EcoRI/MseI enzyme of table 5 capsicum genomic dna is cut and linked system
B, pre-amplification
Connection product with step 1 increases in advance.Reaction system is as shown in table 6.
The pre-amplification of table 6 reaction system
Primer sequence:
EOO：GAC?TGC?GTA?CCAATT?C
MOO：GATGAGTCCTGA?GTA?A
Amplification program:
94 ℃ of 3min; 25 circulation (94 ℃ of 30s; 56 ℃ of 30s; 72 ℃ of 1min), 72 ℃ of 7min mend flat end, 4 ℃ of preservations.
Get 5ul pre-expansion volume increase thing and carry out the detection of 1.0% agarose gel electrophoresis.30 times of all the other pre-expansion volume increase thing dilutions ,-20 ℃ of preservations are standby.
C, selective amplification
Carry out selective amplification with after 30 times of the amplified production of step 2 dilutions, amplification system is as shown in table 7.
Table 7 selective amplification reaction system
Primer sequence:
EcoRI primer: 5 ' GAC TGC GTA CCAATT CAGA-3 '
MseI primer: 5 ' GAT GAG TCC TGA GTAAAGG-3 '
Amplification program:
Pre-94 ℃ of 5min of sex change, 12 circulation (94 ℃ of 30s; 65 ℃ (0.7 ℃/cycle) 30s; 72 ℃ of 1min), 25 circulation (94 ℃ of 30s; 56 ℃ of 30s; 72 ℃ of 1min), 72 ℃ of 5min mend flat end, 4 ℃ of preservations.
D, denaturing polyacrylamide gel electrophoresis
Sex change sample-loading buffer 6ul (98% methane amide, 10mMEDTA, 0.25% smell phenol indigo plant, 0.25% dimethylbenzene green grass or young crops) will be added, 95 ℃ of sex change 5min, cooling rapidly in ice bath then in the product of selective amplification.Adopt permanent power 70W electrophoresis 1h5min on 6% denaturing polyacrylamide gel, the application of sample amount is 5ul.
(1) cleaning of sheet glass
At first use tap water and dish detergent with long slab and short slab wash clean,, dry with distilled water flushing plate face.Again with dehydrated alcohol with sheet glass wiped clean again;
Attention: long slab and short slab should separate when washing plate, avoid mutual pollution.
(2) coated plate
The Repel solution for preparing promptly is coated on the short slab equably, the Binding liquid for preparing is coated on the long slab equably, place more than the 20min.In the coated plate process, should avoid two kinds of solution to pollute mutually.Binding is convenient to acrylamide gel and is fixed on the long slab, and Repel is easy to acrylamide gel to be separated with short slab.
(3) encapsulating
Long slab and short slab are assembled, transfer to level, check whether comb is suitable, and the optimum position of comb insertion.Preferably a hook is put on the next door, is used for hook and goes out the bubble that the encapsulating process produces.Get the polyacrylamide gel (determining the consumption of glue and suitable encapsulating mode according to the electrophoresis chamber of different model) of 60ml6%, 300ulAPS and 60ul TEMED be mixing gently, and one side pad of plate is played a Small angle with glue pouring into slowly.Behind the bottom, rapidly plate is placed level when glue, comb is inserted the appropriate location, and use clamp, in case point sample hourglass sample.Be used for electrophoresis more than the polymerization 2h;
(4) prerunning
Comb is carefully extracted, and the end that will insert comb with wash bottle cleans up, and wipes clean sheet glass then, after electrophoresis chamber is assembled, adds 1 * TBE in electrophoresis chamber, with paper electrode is dried, in case short circuit during electrophoresis.Prerunning 30min under permanent power 70W condition.(electrophoresis apparatus of different model adopts different firm powers);
(5) sex change
With 95 ℃ of sex change 5min of sample that choosing is expanded, cooling rapidly in ice bath then makes DNA keep the strand state;
(6) electrophoresis
After prerunning finishes, the bubble and the impurity piping and druming of glue face is clean, comb to be inserted gently, its degree of depth is for just entering glue face 1mm, point sample 5ul, permanent power 70W electrophoresis 1h5min; Attention: the employed best firm power difference of the electrophoresis chamber of different model, temperature remains on about 60 ℃ and is advisable during with electrophoresis, and the electrophoretic time can be split up into suitable by homogeneity range with each band.
E, silver dye detection
(1) decolouring is with fixing: sheet glass is taken off from electrophoresis chamber, extract comb, and gently long slab is taken off, put into the glacial acetic acid solution of 2L10%, shake 20-30min gently, disappear to indicating dye and be advisable;
(2) rinsing: with the washing 2 times of vibrating gently of 2L distilled water, each 3min;
(3) dyeing: offset plate is put into the 2L staining fluid, and 30min gently vibrates;
(4) develop: take out in the staining fluid back rapidly in distilled water rinsing put into the developing solution of precooling once, whole process is no more than 10s as far as possible, fully concussion is clear until band;
(5) fixing: when band was no longer clear, it was glacial acetic acid solution that stationary liquid is put in the plate taking-up, stops 2min;
(6) rinsing: the glass plate is put into more than the distilled water rinsing 10min, and the plate rinsing time that needs to preserve should be longer, otherwise the easy flavescence of glue face;
(7) take out the back and dry naturally, the scanning or the preservation of taking a picture.
Found that there is significant difference in E39/M41-527 in these two groups.(as Fig. 1)
With aforesaid method all 320 individual plants are identified, and triplicate, the result is stable.There are 282 strains to use in the mark, several 230 strains of band strain are arranged, do not have several 52 strains of band strain, several 15 strains of exchange strain, several 26 strains of uncertain strain.(as Fig. 2) analyzes through the Joinmap3.0 mapping software, and genetic distance is 6.258cM.(as Fig. 3) illustrates mark E39/M41-527 and N gene close linkage, can be used as the molecule marker that assisting root-knot nematodes N resistant gene is selected with this its.
The application of embodiment 2 mark E39/M41-527
With above-mentioned aflp analysis method 282 individual plants that anti-thoughts are arranged are carried out the artificial inoculation evaluation and carry out the dna marker analysis simultaneously, and triplicate, the result is stable.Contain several 230 strains of strain of this mark, do not contain several 52 strains of strain of mark, be not inconsistent several 15 strains of strain, accuracy rate 94.6 with disease-resistant result.
Sequence table
＜110〉also academy of sciences's vegetable or flower institute of Chinese farming
＜120〉be used for the molecule marker that assisting root-knot nematodes N resistant gene is selected
<130>
<160>2
<170>PatentIn?version3.3
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<400>1
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<400>2
Claims (3)
1. the molecule marker that is used for the anti-root nematode of capsicum N gene assisted Selection, the right nucleotide sequence of the primer of this molecule marker that it is characterized in that increasing is shown in SEQ ID NO.1 and 2.
2. molecule marker as claimed in claim 1, the genetic distance that it is characterized in that this mark and N gene is 6.258cM.
3. the application of the described molecule marker of claim 1 in the capsicum seed selection.
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| CN109722488B (en) * | 2019-03-13 | 2022-07-15 | 中国农业科学院蔬菜花卉研究所 | SCAR molecular marker linked with root knot nematode resistant gene Me3 and application thereof |
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| WO1995035024A1 (en) * | 1994-06-17 | 1995-12-28 | The United States Of America, Represented By The Secretary, Department Of Agriculture | Plant virus resistance gene and methods |
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| WO1995035024A1 (en) * | 1994-06-17 | 1995-12-28 | The United States Of America, Represented By The Secretary, Department Of Agriculture | Plant virus resistance gene and methods |
Non-Patent Citations (2)
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| 分子标记技术及其在辣椒遗传育种上的应用.辣椒杂志 3.2004,(3),1-8. |
| 分子标记技术及其在辣椒遗传育种上的应用.辣椒杂志 3.2004,(3),1-8. * |
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