CN101760513A - Chromosome mapping and molecule marking research on powdery mildew resisting gene of Elytrigia intermedium - Google Patents
Chromosome mapping and molecule marking research on powdery mildew resisting gene of Elytrigia intermedium Download PDFInfo
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Abstract
On the basis of obtaining a hybrid of Elytrigia intermedium and wheat and researching the cytogenetical characteristics, the trait characteristics, the microspore producing process and the like of hybrid offspring, the invention aims to carrying out chromosome mapping and primary molecule marking on an unknown powdery mildew resisting gene in the Elytrigia intermedium and makes early-stage preparations for the fine mapping location, the fine marking, the aggregation, the cloning and the like of the gene, and find a new germ plasm resource for the breeding for disease resistance work of wheat.
Description
Technical field
This research is the hybrid that has obtained middle couchgrass and wheat, and the cytogenetics characteristics of its filial generation, characteristic trait, sporule generating process etc. have been carried out on the basis of research, this unknown mildew-resistance gene in the middle couchgrass is carried out chromosomal localization and preliminary molecule marker, do preliminary preparation for further this gene being carried out work such as Fine Mapping, meticulous mark, polymerization, clone, and, belong to the Crop Genetics and Breeding field for the new germ plasm resource of wheat breeding for disease resistance work excavation.
Background technology
Wheat belongs to Gramineae (Poaceae) wheat family (Triticeae) Triticum.Wheat family is except that comprising important food crop such as wheat, barley, rye, also comprise herbage that some are good such as middle couchgrass, roegneria kamoji, lyme grass, wheatgrass, rely grass etc., other genus (table 1) in the kindred plant general reference wheat family of wheat except that Triticum.Plant surplus having 300 approximately in the wheat family, the basic number of chromosome is 7, contain 20 surplus kind of genome, ploidy not of the same race does not wait from diploid to the dodecaploid, wherein about 77% is perennial, all the other are annual (D.R.Deway, 1987; Dong Yuchen, 2001; He Ruifeng, 1993; Jin Shanbao, 1990).These sibling specieses all have the potentiality with wheat hybridizing, and the genetic resources that therefore is used for genetic improvement of wheat is very abundant.
Middle couchgrass [Elytigia intermedium (Host) Nevski, Thinopyrum intermedium (Host) Barkworth and Dewey or Agropyron intermedium (Host) Beauv.2n=6x=42] have another name called sky blue wheatgrass, be the perennial wild herb in the Gramineae wheat family (Triticeae).Middle couchgrass is three grades of GENE SOURCES of wheat, cold-resistant, drought-enduring, spend more manyly to fall in fact, anti-, high-quality, the yellow dwart of high anti-wheat, immune to wheat three rust, be the important anti-source of wheat.
Middle couchgrass originates in Eastern Europe, is an allohexaploid, chromosome number 2n=42.Natural distributed in Caucasia, the area, grassland, the southeast in the Central Asia, because of its adaptability is strong, now all there is distribution in a lot of countries in the world.Middle couchgrass is a kind of strong winter habit, long day, perennial herbaceous plant.In former producing region, the general 100-140cm of plant height, spike length 20-30cm, every fringe 20-30 small ear, every small ear 3-6 Xiao Hua.General early June blooms in Tai'an, Shandong, the general 30-50cm of spike length, and plant height Gao Zheke reaches more than the 150cm; Cross-pollination, setting percentage is low, mainly is based on vegetative propagation; Seed is tiny, thousand seed weight 4-6g.Its well developed root system has creeping underground stem, and regenerative power is strong.Winter resistance is strong, but under-40oC condition safe overwintering.Drought-enduring, can well grow in the area of annual rainfall 350mm.Salt tolerant alkali can be grown on the mild or moderate salty soils.Because its growth potential is strong, reproducibility is good, wide adaptability, and cauline leaf is in great numbers, and contains number of chemical compositions such as higher crude protein, robust fibre, crude fat, is a kind of good forage in pastoral area therefore.It is to disease immunity such as the leaf rust of wheat, bar rust, puccinia graminis, white powder in addition, contour anti-to smut, leaf blight and root rot, striped mosaic disease, and be the good resistance source of barley yellow dwarf, again can with wheat hybridizing, so it has become the valuable genetic resources that has important utility value in the genetic improvement of wheat.
Summary of the invention
The purpose of this research is the hybrid that has obtained middle couchgrass and wheat, and the cytogenetics characteristics of its filial generation, characteristic trait, sporule generating process etc. have been carried out on the basis of research, this unknown mildew-resistance gene in the middle couchgrass is carried out chromosomal localization and preliminary molecule marker, do preliminary preparation for further this gene being carried out work such as Fine Mapping, meticulous mark, polymerization, clone, and be the new germ plasm resource of wheat breeding for disease resistance work excavation.
The chromosomal localization and the molecule marking research of middle couchgrass mildew-resistance gene:
1. materials and methods
1.1 material
The middle couchgrass of this research and utilization is former draws the northwest plant research institute from the Chinese Academy of Sciences, and Mr. Li Zhensheng provides by; Long fringe couchgrass and false roegneria kamoji are drawn the product money institute from the Chinese Academy of Agricultural Sciences, the complete binary alien addition line of the long fringe couchgrass of China spring one diploid 1E, 2E, 3E, 4E, 5E, 6E, 7E is provided by the Chinese Academy of Agricultural Sciences's product money, octoploid of wheat-wheatgrass mountain farming TE263 and binary alien addition line mountain farming Line15 are couchgrass and tobacco grower's 15 hybridization in the middle of this laboratory utilizes, backcross, respectively from BC
1F
6And BC
3F
5In select; Wheat breed tobacco grower 15, China spring and Huixian are red is preserved by national wheat improvement branch center, center (Tai'an); No. 15 bacterial classifications of Powdery Mildew are provided by the Chinese Academy of Agricultural Sciences Institute of Plant Protection, and preserve in this laboratory.
1.2 powder mildew resistance inoculation and evaluation
With the antagonism of the dominant races NO.15 microspecies of current wheat powdery mildew, sense parent, all F
2Individual plant carries out twice inoculation, once in tri-leaf period, is once blooming the post-grouting phase (CIMMTY), under the condition that susceptible contrast is fully fallen ill, investigation is anti-, the sense separation case, and every duplication in 2-3 days once, sick level identifies that the response type grade scale by propositions such as Sheng Baoqin carries out.
1.3 the extraction of genomic dna
The phenol-chloroform extraction method of Devos (1992) is adopted in the extraction of DNA, and change is arranged slightly, is suitable for the 1.5ml centrifuge tube and extracts.
(1) get fresh blade 0.2-0.3 gram, grind into powder rapidly under liquid nitrogen freezing is sub-packed in the 1.5ml centrifuge tube, adds " S " damping fluid 600 μ l mixings of preheating, 65 ℃ water-bath 1-2 hour, in the water-bath process jog for several times, abundant mixing.
(2) add isopyknic phenol-chloroform (V/V=1: 1), (do not stop to shake gently 10-15 minute) centrifugal 10 minutes of 12000rpm behind the mixing; Supernatant liquor is moved into the isopyknic chloroform of adding in another centrifuge tube, and 12000rpm is centrifugal 10 minutes behind the mixing.
(3) supernatant liquor is moved in another centrifuge tube, add the Virahol of 0.6 volume, mixing left standstill 10 minutes gently, ticked DNA precipitation and embathed drying at room temperature in centrifuge tube 2-3 time with 75% ethanol.
(4) dried DNA is dissolved in 500 μ l, 1 * TE (pH7.0), adds 3-5 μ l RNAase (10mg/ml) respectively, 37 ℃ of water bath heat preservations 1 hour.
(5) add isopyknic phenol-chloroform, 12000rpm is centrifugal 10 minutes behind the mixing, and supernatant liquor moves in another centrifuge tube, adds isopyknic chloroform, and 12000rpm is centrifugal 10 minutes behind the mixing.
(6) get the sodium-acetate mixing that supernatant liquor adds the 3M of 1/10 volume, the cold dehydrated alcohol that adds 2 times of volumes, stand at low temperature 10 minutes (best-20～-40 ℃) behind the mixing is chosen the DNA precipitation, embathe 2-3 time with 75% ethanol, treat to add 50 μ l * TE (pH7.0) after the DNA drying.
1.4BSA (Bulked Segregate Analysis) divides the pond:
Press the segregating population cluster analysis method (BSA method) of (1991) propositions such as Michelmore, set up disease-resistant gene pond and susceptible gene pool, the primer that shows polymorphism between parents is further screened.
Disease-resistant pond and susceptible pond: with F
2The individual plant of colony extracts total genomic dna respectively, and 10 disease-resistant individual plants of picked at random are with its DNA balanced mix, and 10 susceptible individual plants of picked at random are set up disease-resistant pond and susceptible pond respectively with its DNA balanced mix.
1.5RAPD-PCR analyze
1.5.1 the dilution of primer source and primer
350 ten base random primers that are used for increasing, 230 (S1-S230) available from Shanghai bio-engineering corporation (Sangon), 120 (OPA1-20, OPE1-20, OPF1-20, OPH1-20, OPT1-20 and OPV1-20) are available from Beijing ancient cooking vessel state bio-engineering corporation.S series primer is the 0.2OD/ pipe, and every pipe adds the dilution of 200 μ l ultrapure waters.
1.5.2PCR reaction system
10×PCR?Buffer 2.5μl
Mg
2+(25mM) 2μl
dNTP(2.5mM) 1.5μl
TaqE(5U/μl) 0.25μl
Primer?pair(5μM) 1μl
DNA(30ng/μl) 2μl
ddH
2O 15.75μl
Cumulative volume (Total) 25 μ l
Attached 10 * PCR Buffer Tris (pH8.3) 100mM
KCl 500mM
MgCl
2 15mM
1.5.3PCR response procedures
PCR is reflected on the Thermal Cycler 9600PCR and carries out.Once increase in advance earlier: 94 ℃ 3 minutes, 36 ℃ 1 minute, 72 ℃ 2 minutes, carry out the amplification of 45 round-robin then, 94 ℃ 15 seconds, 36 ℃ 30 seconds, 72 ℃ of each circulations 1 minute, last 72 ℃ were extended 4 minutes.It is to be detected that amplification finishes back 4 ℃ of preservations.
1.5.4 the detection of amplified production
Add an amount of bromjophenol blue (product/bromjophenol blue 4V/1V) in the amplified production, mixing separates (electrophoretic voltage 5V/cm) with 1.4% agarose gel electrophoresis, and gel is observed with the photograph of Tanon Gis-2010 type gel imaging system after EB dyeing.
1.6 the preparation of agents useful for same
(1)1MTris-HCl(pH8.0)
800ml H
2Add 121.1gTris among the O,, be settled to 1L with HCl adjust pH to 8.0, sterilization, standby.
(2)0.5MEDTA(pH8.0)
800mlH
2Add 186.1gNa among the O
2EDTA-2H2O with NaOH adjust pH to 8.0, is settled to 1L, and sterilization is standby.
(3)3MNaAc(pH5.2)
600ml H
2Add 408.24gNaAc-3H among the O
2O after the dissolving, with Glacial acetic acid adjust pH to 5.2, is settled to 1L, and sterilization is standby.
(4)100×TE(pH8.0)
800ml H
2Add 121.1g Tris among the O, 37.23g Na
2EDTA-2H
2O with HCl adjust pH to 8.0, is settled to 1L, and sterilization is standby.
(5) preparation of RNA enzyme
Solid RNA enzyme is dissolved in 0.01MNaAc, and making enzyme concn is 10mg/ml, is heated to 100 ℃ and handles 15 to 20 minutes, slowly is cooled to room temperature, adds 0.1 volume 1M Tris-Hcl (pH7.4), is stored in-20 ℃ of refrigerators after the packing.
(6) preparation of EB
The 100mg ethidium bromide is dissolved in 10ml H
2Among the O, in brown bottle, keep in Dark Place.
(7)50×TAE(pH8.0)
800mlH
2Add 242.2g Tris among the O, 82.03g NaAc, 37.23g Na
2EDTA-2H
2O with Glacial acetic acid adjust pH to 8.0, is settled to 1L and preserves.Need be diluted to 1 * TAE during use.
(8)5×TBE
Tris27g, boric acid 13.75g, 0.5M EDTA 10ml is settled to 500ml, and is standby.
(9) preparation of damping fluid " S ":
100ml 1M Tris-HCl(pH8.0)
20ml 5N NaCl
100ml 0.5M EDTA(pH8.0)
100ml 20％ SDS
Be settled to 1000ml.
1.7 data analysis
Calculate recombination fraction (Mo Huidong, 1984) according to maximum likelihood method, and recombination fraction is converted into CentiMorgan (cM) by Kosambi (1944) coefficient.
The molecular marker analysis result of mildew-resistance gene
2. result and analysis
2.1RAPD analytical results
Extract mountain farming Line15, middle couchgrass and tobacco grower's 15 total genomic dna, carrying out RAPD analyzes, in 120 RAPD primers for examination, screen 59 discrepant primers between middle couchgrass and 15 liang of parents of tobacco grower altogether, wherein 1 special primer S170 (5 '-ACAACG CGAG-3 ') can stably amplify special band (Fig. 1 of 1 treaty 1200bp in mountain farming Line15 and middle couchgrass, shown in the arrow), and tobacco grower 15 lacks this band.Therefore S170-1200 can be used as the chromosomal special RAPD mark of the mountain additional middle couchgrass of agricultural Line15 institute, also further proves the genetic material that contains middle couchgrass among the mountain farming Line15 simultaneously on the dna molecular level.
2.2SSR analytical results
Be the chromosomal homology group's ownership of the preliminary additional middle couchgrass of clear and definite mountain farming Line15, it has been carried out ssr analysis, in 192 pairs of SSR primers for examination, screen 63 pairs of couchgrass and 15 discrepant primers of tobacco grower in the middle of the parent altogether, wherein primer Xgdm68-5D (P1:5 '-GCC TGA CCA CTC CCA TAA AA-3 '; P2:5 '-TCG GAAGGG GGA CTA TAC AA-3 ') in middle couchgrass and tobacco grower 15, all can stably amplify the special band about 1 treaty, 110～150bp, but the two clip size difference, mountain farming Line15 then can in stably amplify parents' special banding pattern (Fig. 2), so primer Xgdm68-5D can be used for identifying binary alien addition line mountain farming Line15.
3. discuss
Carry mildew-resistance gene in the dyeing of middle couchgrass E group
From the source of the powdery mildew resistance gene in wheat having found at present and named, still not from middle couchgrass.Utilize during common wheat and middle couchgrass hybridized seed selection! , in 2, in 3, in 4, in 5 (Sun Shancheng, 1981) and TAF46 partial amphidiploid and some alien addition lines such as (Larkin, 1996), but these materials are not all found the report of mildew-resistance.Couchgrass was to Powdery Mildew performance rabbit epidemic disease in the middle of we found through evaluation for many years, thereby infer that octoploid of wheat-wheatgrass or alien addition line that these have been bred may wherein may comprise the karyomit(e) that carries mildew-resistance gene not with all transferring in the genetic background of common wheat from the karyomit(e) of middle couchgrass.This research from the filial generation of susceptible variety tobacco grower 15 and middle couchgrass seed selection to the octoploid of wheat-wheatgrass and the binary alien addition line of Powdery Mildew performance immunity, and utilize them and then obtained some novel materials.The mildew-resistance octoploid of wheat-wheatgrass of seed selection and binary alien addition line, identify by the derivation that utilizes cover (21 a) Powdery Mildew toxicity physiological strain and 30 differential hosts to carry out disease-resistant gene and to show, its anti-spectrum is different with the mildew-resistance gene of having found, contains new mildew-resistance gene in the octoploid of wheat-wheatgrass of couchgrass and seed selection and the binary alien addition line in the middle of further specifying.Research by us imports the mildew-resistance gene of middle couchgrass in the genetic background of common wheat.
4. conclusion
Utilize 7 the long fringe couchgrass of China spring-diploid binary alien addition lines of cover (1E-7E) to hybridize respectively and obtain F with mildew-resistance binary alien addition line mountain farming Line15
1In generation, is to F
1Seville orange flower powder parent cell I meiosis metaphase chromosome configuration carries out statistic analysis result and shows, only at the F of 1E * mountain farming Line15
1Seville orange flower powder parent cell I meiosis metaphase observes the chromosome configuration of 2n=22II, infers that tentatively the middle couchgrass karyomit(e) among the mountain farming Line15 may belong to E
eThe homology group of first part in the genome.
Description of drawings
The RAPD result of Fig. 1 primer S170 (A, middle couchgrass, B, tobacco grower 15, M, Maker, 1-6, the different individual plants of mountain farming line15, arrow is depicted as special band)
The SSR result of Fig. 2 primer Xgdm68-5D (A, middle couchgrass, B, tobacco grower 15, M, Maker, 1-6, the different individual plants of mountain farming line15)
Embodiment
1.1 material
The middle couchgrass of this research and utilization is former draws the northwest plant research institute from the Chinese Academy of Sciences, and Mr. Li Zhensheng provides by; Long fringe couchgrass and false roegneria kamoji are drawn the product money institute from the Chinese Academy of Agricultural Sciences, the complete binary alien addition line of the long fringe couchgrass of China spring-diploid 1E, 2E, 3E, 4E, 5E, 6E, 7E is provided by the Chinese Academy of Agricultural Sciences's product money, octoploid of wheat-wheatgrass mountain farming TE263 and binary alien addition line mountain farming Line15 are couchgrass and tobacco grower's 15 hybridization in the middle of this laboratory utilizes, backcross, respectively from BC
1F
6And BC
3F
5In select; Wheat breed tobacco grower 15, China spring and Huixian are red is preserved by national wheat improvement branch center, center (Tai'an); No. 15 bacterial classifications of Powdery Mildew are provided by the Chinese Academy of Agricultural Sciences Institute of Plant Protection, and preserve in this laboratory.
1.2 powder mildew resistance inoculation and evaluation
With the antagonism of the dominant races NO.15 microspecies of current wheat powdery mildew, sense parent, all F
2Individual plant carries out twice inoculation, once in tri-leaf period, is once blooming the post-grouting phase (CIMMTY), under the condition that susceptible contrast is fully fallen ill, investigation is anti-, the sense separation case, and every duplication in 2-3 days once, sick level identifies that the response type grade scale by propositions such as Sheng Baoqin carries out.
1.3 the extraction of genomic dna
The phenol-chloroform extraction method of Devos (1992) is adopted in the extraction of DNA, and change is arranged slightly, is suitable for the 1.5ml centrifuge tube and extracts.
(1) get fresh blade 0.2-0.3 gram, grind into powder rapidly under liquid nitrogen freezing is sub-packed in the 1.5ml centrifuge tube, adds " S " damping fluid 600 μ l mixings of preheating, 65 ℃ water-bath 1-2 hour, in the water-bath process jog for several times, abundant mixing.
(2) add isopyknic phenol-chloroform (V/V=1: 1), (do not stop to shake gently 10-15 minute) centrifugal 10 minutes of 12000rpm behind the mixing; Supernatant liquor is moved into the isopyknic chloroform of adding in another centrifuge tube, and 12000rpm is centrifugal 10 minutes behind the mixing.
(3) supernatant liquor is moved in another centrifuge tube, add the Virahol of 0.6 volume, mixing left standstill 10 minutes gently, ticked DNA precipitation and embathed drying at room temperature in centrifuge tube 2-3 time with 75% ethanol.
(4) dried DNA is dissolved in 500 μ l, 1 * TE (pH7.0), adds 3-5 μ l RNAase (10mg/ml) respectively, 37 ℃ of water bath heat preservations 1 hour.
(5) add isopyknic phenol-chloroform, 12000rpm is centrifugal 10 minutes behind the mixing, and supernatant liquor moves in another centrifuge tube, adds isopyknic chloroform, and 12000rpm is centrifugal 10 minutes behind the mixing.
(6) get the sodium-acetate mixing that supernatant liquor adds the 3M of 1/10 volume, the cold dehydrated alcohol that adds 2 times of volumes, stand at low temperature 10 minutes (best-20～-40 ℃) behind the mixing is chosen the DNA precipitation, embathe 2-3 time with 75% ethanol, treat to add 50 μ l * TE (pH7.0) after the DNA drying.
1.4BSA (Bulked Segregate Analysis) divides the pond:
Press the segregating population cluster analysis method (BSA method) of (1991) propositions such as Michelmore, set up disease-resistant gene pond and susceptible gene pool, the primer that shows polymorphism between parents is further screened.
Disease-resistant pond and susceptible pond: with F
2The individual plant of colony extracts total genomic dna respectively, and 10 disease-resistant individual plants of picked at random are with its DNA balanced mix, and 10 susceptible individual plants of picked at random are set up disease-resistant pond and susceptible pond respectively with its DNA balanced mix.
1.5RAPD-PCR analyze
1.5.1 the dilution of primer source and primer
350 ten base random primers that are used for increasing, 230 (S1-S230) available from Shanghai bio-engineering corporation (Sangon), 120 (OPA1-20, OPE1-20, OPF1-20, OPH1-20, OPT1-20 and OPV1-20) are available from Beijing ancient cooking vessel state bio-engineering corporation.S series primer is the 0.2OD/ pipe, and every pipe adds the dilution of 200 μ l ultrapure waters.
1.5.2PCR reaction system
10×PCR?Buffer 2.5μl
Mg
2+(25mM) 2μl
dNTP(2.5mM) 1.5μl
TaqE(5U/μl) 0.25μl
Primer?pair(5μM)?1μl
DNA(30ng/μl) 2μl
ddH
2O 15.75μl
Cumulative volume (Total) 25 μ l
Attached 10 * PCR Buffer Tris (pH8.3) 100mM
KCl 500mM
MgCl
2 15mM
1.5.3PCR response procedures
PCR is reflected on the Thermal Cycler 9600PCR and carries out.Once increase in advance earlier: 94 ℃ 3 minutes, 36 ℃ 1 minute, 72 ℃ 2 minutes, carry out the amplification of 45 round-robin then, 94 ℃ 15 seconds, 36 ℃ 30 seconds, 72 ℃ of each circulations 1 minute, last 72 ℃ were extended 4 minutes.It is to be detected that amplification finishes back 4 ℃ of preservations.
1.5.4 the detection of amplified production
Add an amount of bromjophenol blue (product/bromjophenol blue 4V/1V) in the amplified production, mixing separates (electrophoretic voltage 5V/cm) with 1.4% agarose gel electrophoresis, and gel is observed with the photograph of Tanon Gis-2010 type gel imaging system after EB dyeing.
1.6 the preparation of agents useful for same
(1)1MTris-HCl(pH8.0)
800ml H
2Add 121.1gTris among the O,, be settled to 1L with HCl adjust pH to 8.0, sterilization, standby.
(2)0.5MEDTA(pH8.0)
800mlH
2Add 186.1gNa among the O
2EDTA-2H2O with NaOH adjust pH to 8.0, is settled to 1L, and sterilization is standby.
(3)3MNaAc(pH5.2)
600ml H
2Add 408.24gNaAc-3H among the O
2O after the dissolving, with Glacial acetic acid adjust pH to 5.2, is settled to 1L, and sterilization is standby.
(4)100×TE(pH8.0)
800ml H
2Add 121.1g Tris among the O, 37.23g Na
2EDTA-2H
2O with HCl adjust pH to 8.0, is settled to 1L, and sterilization is standby.
(5) preparation of RNA enzyme
Solid RNA enzyme is dissolved in 0.01MNaAc, and making enzyme concn is 10mg/ml, is heated to 100 ℃ and handles 15 to 20 minutes, slowly is cooled to room temperature, adds 0.1 volume 1M Tris-Hcl (pH7.4), is stored in-20 ℃ of refrigerators after the packing.
(6) preparation of EB
The 100mg ethidium bromide is dissolved in 10ml H
2Among the O, in brown bottle, keep in Dark Place.
(7)50×TAE(pH8.0)
800mlH
2Add 242.2g Tris among the O, 82.03g NaAc, 37.23g Na
2EDTA-2H
2O with Glacial acetic acid adjust pH to 8.0, is settled to 1L and preserves.Need be diluted to 1 * TAE during use.
(8)5×TBE
Tris27g, boric acid 13.75g, 0.5M EDTA 10ml is settled to 500ml, and is standby.
(9) preparation of damping fluid " S ":
100ml 1M Tris-HCl(pH8.0)
20ml 5N NaCl
100ml 0.5M EDTA(pH8.0)
100ml 20％ SDS
Be settled to 1000ml.
1.7 data analysis
Calculate recombination fraction (Mo Huidong, 1984) according to maximum likelihood method, and recombination fraction is converted into CentiMorgan (cM) by Kosambi (1944) coefficient.
Claims (2)
1. in 120 RAPD primers for examination, screen 59 discrepant primers between middle couchgrass and 15 liang of parents of tobacco grower altogether, wherein 1 special primer S170 (5 '-ACA ACG CGA G-3 ') can stably amplify the special band of 1 treaty 1200bp in mountain farming Line15 and middle couchgrass, and tobacco grower 15 lacks this band, therefore S170-1200 can be used as the chromosomal special RAPD mark of the mountain additional middle couchgrass of agricultural Line15 institute, also further proves the genetic material that contains middle couchgrass among the mountain farming Line15 simultaneously on the dna molecular level.
2. be the chromosomal homology group's ownership of the preliminary additional middle couchgrass of clear and definite mountain farming Line15, it has been carried out ssr analysis, in 192 pairs of SSR primers for examination, screen 63 pairs of couchgrass and 15 discrepant primers of tobacco grower in the middle of the parent altogether, wherein primer Xgdm68-5D (P1:5 '-GCC TGA CCA CTC CCA TAA AA-3 '; P2:5 '-TCGGAA GGG GGA CTA TAC AA-3 ') in middle couchgrass and tobacco grower 15, all can stably amplify the special band about 1 treaty, 110～150bp, but the two clip size difference, mountain farming Line15 then can in stably amplify parents' special banding pattern, so primer Xgdm68-5D can be used for identifying binary alien addition line mountain farming Line15.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810160298A CN101760513A (en) | 2008-11-19 | 2008-11-19 | Chromosome mapping and molecule marking research on powdery mildew resisting gene of Elytrigia intermedium |
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|---|---|---|---|
| CN200810160298A CN101760513A (en) | 2008-11-19 | 2008-11-19 | Chromosome mapping and molecule marking research on powdery mildew resisting gene of Elytrigia intermedium |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642916A (en) * | 2013-12-03 | 2014-03-19 | 四川农业大学 | Method for quickly and effectively identifying genome E<e> of elytrigia desv. |
| CN104774968A (en) * | 2015-05-08 | 2015-07-15 | 周口市农业科学院 | Method for identifying wheat variety of Zhoumai 22 |
| CN110643731A (en) * | 2019-10-30 | 2020-01-03 | 山西省农业科学院作物科学研究所 | Rapid detection of thinopyrum intermedium 6JSSpecific molecular marker of chromosome and application |
-
2008
- 2008-11-19 CN CN200810160298A patent/CN101760513A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642916A (en) * | 2013-12-03 | 2014-03-19 | 四川农业大学 | Method for quickly and effectively identifying genome E<e> of elytrigia desv. |
| CN103642916B (en) * | 2013-12-03 | 2015-03-25 | 四川农业大学 | Method for quickly and effectively identifying genome E<e> of elytrigia desv. |
| CN104774968A (en) * | 2015-05-08 | 2015-07-15 | 周口市农业科学院 | Method for identifying wheat variety of Zhoumai 22 |
| CN110643731A (en) * | 2019-10-30 | 2020-01-03 | 山西省农业科学院作物科学研究所 | Rapid detection of thinopyrum intermedium 6JSSpecific molecular marker of chromosome and application |
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