CN101760556A - Molecular marker technology for identifying red grains and white grains of wheat - Google Patents
Molecular marker technology for identifying red grains and white grains of wheat Download PDFInfo
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- CN101760556A CN101760556A CN 201010107867 CN201010107867A CN101760556A CN 101760556 A CN101760556 A CN 101760556A CN 201010107867 CN201010107867 CN 201010107867 CN 201010107867 A CN201010107867 A CN 201010107867A CN 101760556 A CN101760556 A CN 101760556A
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Abstract
The invention discloses a molecular marker technology for identifying red grains and white grains of wheat, which comprises the following steps: utilizing synthetic hexapliod wheat derived varieties of red grain wheat Chuanmai 42 and Sichuan cultivated white grain wheat Chuan W565 for hybridization, constructing an F2 population for genetic analysis of red grain genes, and harvesting 1015 F2 plants for identifying grain color and carrying out the genetic analysis and the molecular marking of the red grain genes of Chuanmai 42, wherein 241 white grain single plants in the F2 population are selected, and an F2-3 family is constructed for verifying the F2 result. The molecular marker technology utilizes an SSR modular marker for carrying out mapping on the red grain genes carried in the synthetic hexapliod wheat derived variety-Chuanmai 42, thereby being fast, accurate and effective to select the white grain variety, eliminating the red grain variety and accelerating the breeding process of the white grain wheat.
Description
Technical field
The present invention is a kind of SSR of utilization molecule marker, distinguishes the technology of red grain of wheat and white grain, belongs to the agricultural biotechnology engineering field.
Background technology
The wheat grain color is the important character that influences flour quality and output, and in the abrasive dust process, red grain wheat influences brightness (Flintham, 2000 of flour; Warner et al., 2000; Himi et al., 2002), each national peasant likes planting the white wheat kind in the world.The wheat grain color is respectively by wheat 3A, 3B, long-armed red grain gene (R-A1, R-B1, the R-D1) control of 3D karyomit(e); red grain gene all is a dominant inheritance; has additive effect; 3 recessive sites are pure and mild could to show white grain; and the performance of grain look affected by environment; this just makes with the naked eye selects white grain to have certain difficulty, and grain-by-grain seed selection is selected and can only be carried out after the seed results in vain, and the cycle is long.
The synthetic wheat is by tetraploid and diploid Triticum tauschii synthetic, and the excellent genes that is richly stored with also carries some unfavorable genes, and red graininess shape is exactly the important factor that restriction breeding man utilizes the synthetic wheat.Synthetic wheat derived varieties river wheat 42 high yields, stable yields, anti-bar become rusty, and have become the important gene source of improving yield of wheat, the breeding of anti-bar rust.Yet, because the influence of red graininess shape has limited the use of river wheat 42.Therefore, utilize select easily, method fast and effectively, making river wheat 42 is very significant by red grain to white transformation.The white grain gene of molecular marker assisted selection can be selected seedling stage, and not affected by environment, quicker, easier, more accurate.Up to the present, R-A1, R-B1, R-D1 gene have utilized common wheat to pass through RFLP, STS, EST and the mapping of SSR molecule marker (Flintham andHumphrey, 1993; Gale et al., 1995; Nalam et al., 2006; Kuraparthy et al., 2008, Sherman et al., 2008).
Summary of the invention
Make at the white wheat kind and with the naked eye to select difficulty big; and can only after the seed results, carry out; the problem that cycle is long; the object of the present invention is to provide a kind of molecular marking technique of differentiating red grain of wheat and white grain; utilize the SSR molecule marker; the red grain gene that synthetic wheat derived varieties river wheat 42 is carried is mapped, in the hope of for providing molecule marker fast and effectively as the seed selection of the white wheat kind of genetic resources with synthetic wheat and river wheat 42.
In order to achieve the above object, the present invention has adopted following technical scheme:
The present invention utilizes synthetic wheat derived varieties red grain wheat river wheat 42 and Sichuan cultivation white wheat river W565 hybridization, makes up F
2Colony is used for the genetic analysis of red grain gene.River wheat 42, river W565, F
1, F
2Single-strain planting is in the experimental plot, Chengdu.1015 F
2Plant is used to identify the genetic analysis of seed color and river wheat 42 red grain genes by results, wherein, and F
2241 white grain individual plants are selected in the colony, make up F
2-3Family is used to verify F
2The result.
(1) DNA extraction and SSR mark: get parents, F2 and F2-3 young leaflet tablet seedling stage, utilize the CTAB method to extract DNA; Select the long-armed hereditary difference of going up SSR primer screening parents river wheat 42 and river W565 of wheat 3D for use;
PCR reaction conditions, amplification system are as follows: the 15ul system:
1 * PCR damping fluid, template 1.5ul, magnesium ion concentration 1.5mM, dNTP concentration 0.2mM, primer concentration are 0.2uM, rTaq enzyme 1U adds aseptic deionized water or ddH2O water to 15ul.
The PCR program is:
1) 94 ℃ of pre-sex change 5min
2) touchdown PCR program totally 11 each 1 ℃ of reductions that circulate of circulation
94 ℃, 45 seconds
65 ℃, 45 seconds
72 ℃, 45 seconds
3) 30 circulations of conventional PCR program
94 ℃, 45 seconds
55 ℃, 45 seconds
72 ℃, 45 seconds
4) last amplification: 72 ℃, 10min
Electrophoresis detection: polyacrylamide concentration is 6%, and wherein, acrylamide: methylene diacrylamide=39: 1, the applied sample amount of amplified production is 5-8ul in each point sample hole.0.1% cma staining 10 minutes behind the electrophoresis 1h under the 400V constant-pressure conditions behind the prerunning 20min under the 220V constant-pressure conditions, 2% NaOH and 1.5% formaldehyde develop to band and occur, and take a picture at last and preserve;
(2) macroscopical identification: during the wheat fully matured, results F1 seed, F2 individual plant and F2-3 family, each strain system gets 30-40 grain seed and is placed on soaked overnight in the 5%NaOH solution, and naked eyes are identified the seed color, seed be dark red be red grain, for straw-colored be white grain;
(3) genetic distance calculates and the linkage map structure:
Utilize parents to have the primer scanning F of polymorphism
2White grain individual plant in the colony is in conjunction with the result who identifies the seed color, by chi square test estimation F
2The fitness of separating ratio between colony's observed value and desired value.The estimation of genetic distance is with the genetic distance (Lincoln etal, 1992) between Mapmaker3.0 calculating mark and the disease-resistant gene, with Kosambi function scaling system distance (Kosambi, 1944), the LOD value is 3.0, draws genetic map (Liu with Mapdraw, Meng, 2003).
(4) result
1) according to the seed color identification, river wheat 42 red grain gene R-D1 are dominant inheritance, 1015 F
2The red grain of 774 strains, the white grain of 241 strains are arranged in the plant, separate, show that the red grain gene that river wheat 42 is carried is controlled by single-gene than being 3: 1;
2) the SSR primer of 8 3D karyomit(e)s on long-armed is used to scan F
2241 recessive individual plants in the colony; according to the phenotype of 241 recessive individual plant SSR genotype and seed color, make up linkage map, 5 SSR primers and red grain gene R-D1 close linkage; the R-D1 gene is between Xgwm3 and two SSR marks of Xgwm314, and genetic distance is 2.1 respectively, 2.5CM.
The polymerase chain amplification reaction system that the present invention uses is conventional PCR system, General Instrument and reagent are all used in electrophoresis and dyeing, without any need for special instrument and special reagent, the PCR of the production of any company reaction instrument, electrophoresis chamber and electrophoresis apparatus, the reagent that any biological reagent company produces all can use and reach purpose of the present invention.
The present invention filters out two SSR primer Xgwm3 and Xgwm314, is positioned at the both sides of red grain gene R-D1, and genetic distance is 2.1 respectively, 2.5CM.Without any need for special instrument and reagent, test is convenient, low price in the process of the test of the present invention.With these two red grain genes of primer assisted Selection, thereby the white grain of quicker, accurate and effective selection kind, eliminate red grain kind, accelerated the white wheat breeding process.
Description of drawings
Fig. 1 is the technology of the present invention route map
Fig. 2 is SSR primer Xgwm3, Xgwm314, and Xwmc552, it is the electrophorogram of synthetic wheat Syn769 polymorphism difference that Xbarc270 detects river wheat 42, river W565 and river wheat 42 parents.
Among Fig. 2: 1 for river wheat 42 parents are synthetic wheat Syn769, and 2 is that river wheat 42,3 is river W565.
Fig. 3 is the electrophorogram of SSR primer Xgwm3 scanning river wheat 42/ river W565 F2 colony.
Fig. 4 is the electrophorogram of SSR primer Xgwm314 scanning river wheat 42/ river W565F2 colony.
Fig. 5 is the linkage map of river wheat 42 red grain gene R-D1.The right of figure is the primer title, the left side of figure be genetic distance (Kosambi, CM).
Embodiment
(1) test materials
Synthetic wheat derived varieties red grain wheat river wheat 42 and Sichuan cultivation white wheat river W565, and river wheat 42/ river W565F2 colony (1015) and F2-3 family.River wheat 42, river W565, F1, F2 colony and F2-3 family are planted in the experimental plot, Chengdu.
(2) macroscopical identification
During the wheat fully matured, results F1 seed, F2 individual plant and F2-3 family, each strain system gets 30-40 grain seed and is placed on soaked overnight in the 5%NaOH solution, naked eyes are identified the seed color, seed be dark red be red grain, for straw-colored be white grain.
(3) DNA extraction and SSR mark
Get parents, F2 and F2-3 young leaflet tablet seedling stage, utilize the CTAB method to extract DNA.Select the long-armed hereditary difference of going up SSR primer screening parents river wheat 42 and river W565 of wheat 3D karyomit(e) for use.
PCR reaction conditions, amplification system following (15ul system):
1 * PCR damping fluid, template 1.5ul, magnesium ion concentration 1.5mM, dNTP concentration 0.2mM, primer concentration are 0.2uM, rTaq enzyme 1U adds aseptic deionized water or ddH2O water to 15ul.
The PCR program is:
1) 94 ℃ of pre-sex change 5min
2) touchdown PCR program totally 11 each 1 ℃ of reductions that circulate of circulation
94 ℃, 45 seconds
65 ℃, 45 seconds
72 ℃, 45 seconds
3) 30 circulations of conventional PCR program
94 ℃, 45 seconds
55 ℃, 45 seconds
72 ℃, 45 seconds
4) final step amplification: 72 ℃, 10min
Electrophoresis detection: polyacrylamide concentration is that 6% (acrylamide: methylene diacrylamide=39: 1), the applied sample amount of amplified production is 5-8ul in each point sample hole.0.1% cma staining 10 minutes behind the electrophoresis 1h under the 400V constant-pressure conditions behind the prerunning 20min under the 220V constant-pressure conditions, 2% NaOH and 1.5% formaldehyde develop to band and occur, and take a picture at last and preserve.
(4) genetic distance calculates and the linkage map structure
Utilize parents to have white grain individual plant in the primer scanning F2 colony of polymorphism,, estimate the fitness of separating ratio between F2 colony observed value and desired value by chi square test in conjunction with the result who identifies the seed color.With the genetic distance (Lincoln et al, 1992) between Mapmaker3.0 calculating mark and the disease-resistant gene, apart from (Kosambi, 1944), the LOD value is 3.0, draws genetic map (Liu, Meng, 2003) with Mapdraw with Kosambi function scaling system.
(5) result
1, according to the seed color identification, river wheat 42 red grain gene R-D1 are dominant inheritance, 1015 F
2The red grain of 774 strains, the white grain of 241 strains are arranged in the plant, separate, show that the red grain gene that river wheat 42 is carried is controlled by single-gene than being 3: 1.
2, the SSR primer of 8 3D on long-armed is used to scan F
2241 recessive individual plants (white grain) according to the phenotype of 241 recessive individual plant SSR genotype (as table 1) and seed color, make up linkage map in the colony.5 SSR primers and red grain gene R-D1 close linkage, R-D1 gene are between Xgwm3 and two SSR marks of Xgwm314, and genetic distance is 2.1 respectively, 2.5CM (as shown in Figure 5).
Table 1. is with the red grain gene R-D1 linkage analysis result of 5 SSR marks to 241 recessive individual plants of F2 colony
Annotate: A. river wheat 42 pure and mild genotype; B. the pure and mild genotype of river W565; H. heterozygosis type; C. variation type.
3, river wheat 42 red grain genes derive from synthetic wheat diploid parents Triticum tauschii.
Claims (1)
1. a molecular marking technique of differentiating river wheat 42 red grains and white grain utilizes synthetic wheat derived varieties red grain wheat river wheat 42 and Sichuan cultivation white wheat river W565 hybridization, makes up F
2Colony is used for the genetic analysis of red grain gene, 1015 F
2Plant is used to identify the genetic analysis and the molecule marker of seed color and river wheat 42 red grain genes by results, wherein, and F
2241 white grain individual plants are selected in the colony, make up F
2-3Family is used to verify F
2The result; It is characterized in that: these molecular marking technique concrete steps are as follows:
(1) DNA extraction and SSR mark: get parents, F2 and F2-3 young leaflet tablet seedling stage, utilize the CTAB method to extract DNA; Select the long-armed hereditary difference of going up SSR primer screening parents river wheat 42 and river W565 of wheat 3D karyomit(e) for use;
PCR reaction conditions, amplification system are as follows: the 15ul system:
1 * PCR damping fluid, template 1.5ul, magnesium ion concentration 1.5mM, dNTP concentration 0.2mM, primer concentration are 0.2uM, rTaq enzyme 1U adds aseptic deionized water or ddH2O water to 15ul;
The PCR program is:
1) 94 ℃ of pre-sex change 5min
2) touchdown PCR program totally 11 each 1 ℃ of reductions that circulate of circulation
94 ℃, 45 seconds
65 ℃, 45 seconds
72 ℃, 45 seconds
3) 30 circulations of conventional PCR program
94 ℃, 45 seconds
55 ℃, 45 seconds
72 ℃, 45 seconds
4) final step amplification: 72 ℃, 10min
Electrophoresis detection: polyacrylamide concentration is 6%, wherein, acrylamide: methylene diacrylamide=39: 1, the applied sample amount of amplified production is 5-8ul in each point sample hole, 0.1% cma staining 10 minutes behind the electrophoresis 1h under the 400V constant-pressure conditions behind the prerunning 20min under the 220V constant-pressure conditions, 2% NaOH and 1.5% formaldehyde develop to the band appearance, take a picture at last and preserve;
(2) macroscopical identification: during the wheat fully matured, results F
1Seed, F
2Individual plant and F
2-3The family seed, each strain system gets 30-40 grain seed and is placed on soaked overnight in the 5%NaOH solution, naked eyes are identified the seed color, seed be dark red be red grain, for straw-colored be white grain;
(3) genetic distance calculates and the linkage map structure: utilize parents to have the primer scanning F of polymorphism
2White grain individual plant in the colony is in conjunction with the result who identifies the seed color, by chi square test estimation F
2The fitness of separating ratio between colony's observed value and desired value is calculated genetic distance between mark and the disease-resistant gene with Mapmaker3.0, and with Kosambi function scaling system distance, the LOD value is 3.0, draws genetic map with Mapdraw;
(4) result
1) according to the seed color identification, river wheat 42 red grain gene R-D1 are dominant inheritance, 1015 F
2The red grain of 774 strains, the white grain of 241 strains are arranged in the plant, separate, show that the red grain gene that river wheat 42 is carried is controlled by single-gene than being 3: 1;
2) the SSR primer of 8 3D karyomit(e)s on long-armed is used to scan F
2241 recessive individual plants in the colony; according to the phenotype of 241 recessive individual plant SSR genotype and seed color, make up linkage map, 5 SSR primers and red grain gene R-D1 close linkage; the R-D1 gene is between Xgwm3 and two SSR marks of Xgwm314, and genetic distance is 2.1 respectively, 2.5CM.
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| CN 201010107867 CN101760556A (en) | 2010-02-10 | 2010-02-10 | Molecular marker technology for identifying red grains and white grains of wheat |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304507A (en) * | 2011-07-05 | 2012-01-04 | 华中农业大学 | Method for converting monomorphic SSR (Simple Sequence Repeat) marker into polymorphic marker |
| CN108315466A (en) * | 2018-04-02 | 2018-07-24 | 河南科技学院 | It is a kind of that for detecting in wheat, whether there is or not the primer special of yellow leaf gene, kit and its applications |
| CN108456745A (en) * | 2018-05-28 | 2018-08-28 | 安徽农业大学 | Grain Color in Wheat correlation CAPS labels and its detection method |
| CN116855504A (en) * | 2023-05-11 | 2023-10-10 | 四川农业大学 | Wheat red glume red stalk gene RgM G52 and KASP molecular marker and application thereof |
-
2010
- 2010-02-10 CN CN 201010107867 patent/CN101760556A/en active Pending
Non-Patent Citations (6)
| Title |
|---|
| 《Cereal Chem.》 19971031 Dowell Effect of NaOH on Visible Wavelength Spectra of Single Wheat Kernels and Color Classification Efficiency 617-620 1 第74卷, 第5期 2 * |
| 《Crop Science》 20080831 Sherman,et al. Microsatellite Markers for Kernel Color Genes in Wheat 1419-1424 第48卷, 2 * |
| 《Genome》 20050809 Himi,et al. Colour genes (R and Rc) for grain and coleoptile upregulate flavonoid biosynthesis genes in wheat 747-754 1 第48卷, 2 * |
| 《Seed Science Research》 20001231 Flintham,et al. Different genetic components control coat-imposed and embryo-imposed dormancy in wheat 43-50 1 第10卷, 2 * |
| 《Theor. Appl. Genet.》 20021231 Groos,et al. Study of the relationship between pre-harvest sprouting and grain color by quantitative trait loci analysis in a white×red grain bread-wheat cross 39-47 1 第104卷, 2 * |
| 《全国植物分子育种研讨会摘要集》 20090104 杨武云等 川麦42重要基因发掘及遗传转育 正文第8-10行 1 , 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304507A (en) * | 2011-07-05 | 2012-01-04 | 华中农业大学 | Method for converting monomorphic SSR (Simple Sequence Repeat) marker into polymorphic marker |
| CN108315466A (en) * | 2018-04-02 | 2018-07-24 | 河南科技学院 | It is a kind of that for detecting in wheat, whether there is or not the primer special of yellow leaf gene, kit and its applications |
| CN108315466B (en) * | 2018-04-02 | 2021-08-27 | 河南科技学院 | Special primer and kit for detecting existence of yellow leaf gene in wheat and application of special primer and kit |
| CN108456745A (en) * | 2018-05-28 | 2018-08-28 | 安徽农业大学 | Grain Color in Wheat correlation CAPS labels and its detection method |
| CN108456745B (en) * | 2018-05-28 | 2021-07-23 | 安徽农业大学 | Wheat grain color-related CAPS (cleaved amplified polymorphic sequence) marker and detection method thereof |
| CN116855504A (en) * | 2023-05-11 | 2023-10-10 | 四川农业大学 | Wheat red glume red stalk gene RgM G52 and KASP molecular marker and application thereof |
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Application publication date: 20100630 |