CN101519692A - Molecular mark closely interlocked with green-keeping main effect QTL of corn leaf blade - Google Patents
Molecular mark closely interlocked with green-keeping main effect QTL of corn leaf blade Download PDFInfo
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- CN101519692A CN101519692A CN200910046639A CN200910046639A CN101519692A CN 101519692 A CN101519692 A CN 101519692A CN 200910046639 A CN200910046639 A CN 200910046639A CN 200910046639 A CN200910046639 A CN 200910046639A CN 101519692 A CN101519692 A CN 101519692A
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Abstract
The invention relate to a molecular mark closely interlocked with a green-keeping main effect QTL of a corn leaf blade. By constructing a corn molecular mark genetic map and quantitative character site analysis, the molecular mark closely interlocked with the green-keeping main effect QTL of the corn leaf blade is discovered. The molecular mark is applied to corn breeding and resource identification, and can predict the green-keeping performance of germplasm material leaf blades so as to achieve early prediction and screening of the corn leaf blade with the green-keeping performance of and improve the breeding efficiency.
Description
Technical field
The present invention relates to the corn breeding field, relate in particular to a kind of green-keeping capacity of maize molecule marker, relate to a kind of and maize leaf more specifically and protect the molecule marker of green property main effect QTL compact linkage and the application in corn breeding thereof.
Background technology
Corn (Zea mays L.) is important food and fodder crop in the world, and cultivated area is only second to wheat (Triticum aestivum L.) and paddy rice (Oryza sativa L.).Owing to the germ plasm resource scarcity, pacing up and down appears in Maize Production, makes slow progress in output, quality and resistance breeding in recent years, and breeding man is devoted to the excavation and the utilization of excellent genetic resources always, in the hope of making a breakthrough on corn germplasm innovation and breed breeding.
Aging is the final stage of growth and development of plants, and its characteristic feature is that chlorophyll degradation and photosynthetic capacity reduce, and aging will directly cause output to reduce and quality decline before the crop maturation.Protect green property and refer to protect the aging of green plant-growth anaphase blade or yellow and delay and keep green characteristic, be considered to desirable economical character (Theor Appl Genet, 1987,73,551-555 of crop; Ann Appl Biol, 1993,123,193-129; J Exp Bot., 2000,51,329-337; Theor Appl Genet, 2000,101,733-741; Haussmann et al, 2002,106,133-142).Research is thought, the corn variety that to protect green type corn variety be current most worthy, represented direction (In Genetic contribution to yield gains of five major crop plants (CSSA special publication 7) the .EDWRFehr.Madison:Crop Sciece Sciety ofAmerica. of corn variety evolution, 1984,15-47; Cereal Research Communications, 1998,26,161-167; Crop Science, 1991,31,47-248).
Therefore, it is to have important character the most permanent and that contact directly with output, feeding quality, resistance that maize leaf is protected green property, and high yield, many anti-, wide adaptabilities are the main directions of corn breeding, protect the important indicator that green proterties is the super breeding of current corn.Yet, protect the proterties that green proterties is a more complicated, be subjected to a plurality of Gene Handling, and exist mutually with environment and to do, make the kind of protecting green proterties with traditional method seed selection tool make slow progress.
The molecular mark technology that grew up in recent years, solving this problem effectively, by genetic map and quantitative trait locus (the Quantitative TratitLoci that makes up important anti-source, QTL) analyze, can find the molecule marker of protecting green property main effect QTL compact linkage (or be divided into from) with maize leaf.Use the molecule marker of protecting green property main effect QTL compact linkage with blade, can carry out the screening of generation morning in seedling stage, overcome environmental influence, eliminate the plant that protects green difference, save production cost, improve breeding and efficiency of selection germplasm materials.
Summary of the invention
One object of the present invention is to provide a kind of and maize leaf to protect the molecule marker of green property main effect QTL compact linkage.Find by qtl analysis, on the linkage group 2 respectively between mark umc1845 and umc1026, and respectively exist one to protect green property QTL site between umc1026 and the umc1541.Further analysis revealed umc1026 and corn master are imitated blade to protect green property QTL linked, can be used in the germplasm materials blade and protect the prediction of green property.
Another object of the present invention is to provide a kind of molecule marker, is applied to the green-keeping capacity of maize evaluation.This molecule marker and maize leaf are protected green property close linkage, and promptly having or not of this molecule marker is directly connected to the power that this plant protects green property.
Another purpose of the present invention is to provide a kind of molecule marker, is applied to identify in the breeding and the resource of corn.This molecule marker and maize leaf are protected green property close linkage, and promptly having or not of this molecule marker is directly connected to the power that this plant protects green property.In the middle of early prediction and screening to the green property of the guarantor of maize leaf, this molecule marker can overcome environmental influence, to realizing generation screening early the seedling stage of germplasm materials, eliminates the plant that protects green difference, improves breeding and efficiency of selection.
A kind of and maize leaf is protected the molecule marker of green property main effect QTL compact linkage, with 5 '-TCGTCGTCTCCAATCATACGTG-3 ' (SEQ ID No:1) and 5 '-GCTACACGATACCATGGCGTTT-3 ' (SEQ ID No:2) is that primer is right, the total DNA of corn that extracts is that template is carried out the polymerase chain reaction (polymerase chain reaction, PCR) length that obtains after the amplification is the dna fragmentation of 230bp.Its step is as follows:
1. the extraction of the total DNA of corn:
Be mixed for the extraction of total DNA after 2 young leaflet tablets of clip (arteries and veins in the removal) shred when corn 6～7 leaves.Adopt the CTAB method to extract total DNA, step is referring to document Nucleic Acids Res, and 1980,8,4321 and Genetics, 1994,138,1251-1274.
2.PCR amplification,
3. denaturing polyacrylamide gel electrophoresis,
4. silver dyes program.
A kind of acquisition is as follows with the molecular marker method that maize leaf is protected green property main effect QTL compact linkage:
(1) green-keeping capacity of maize F
2Segregating population and F
2:3The structure of macroscopical identification colony and protect the evaluation of green property
Select for use blade to protect the strong corn inbred line of green property neat 319 and be parent 1 (P
1) the corn inbred line Mo17 relatively poor with protecting green property be parent 2 (P
2) hybridize, obtain first-filial generation hybrid (F
1).By first generation of hybrid F
1Selfing obtains second filial generation (F
2) segregating population, F
2The pollination of segregating population individual plant selfing obtains hybrid three generations family (F
2:3) the macroscopical identification family.
Summer, (36 ° of 16 ' N, 117 ° of 9 ' E, height above sea level 128m) planted P in Shandong Agricultural University corn garden then
1, P
2, F
1And F
2, F wherein
2500 strains are planted by colony, picked at random 350 strains.Clip P when plant strain growth to 6～7 leaves
1, P
2, F
1With 350 F
22 young leaflet tablets of individual plant extract total DNA.Extract 350 F of total DNA
2Individual plant bagging in flowering period individual plant selfing, preparation F
2:3Family.The concrete grammar that extracts total DNA is the CTAB method, and step is referring to document Nucleic AcidsRes, 1980,8,4321 and Genetics, 1994,138,1251-1274.
Summer next year is (36 ° of 16 ' N, 117 ° of 9 ' E, height above sea level 128m) plantation P in Shandong Agricultural University corn garden
1, P
2With 166 F that the q.s seed is arranged
2:3Family.Plant and adopt randomized block design, be specially the single file district, 0.70 meter of line-spacing, long 4 meters of row, every row 15 strains, district's group is arranged at random, 3 repetitions.The field management routine operation guarantees respectively to handle the consistence of cultivation condition.Protect green property in flowering period and ripening stage evaluation blade.
Protect green property size and show, be i.e. greenery area relatively, relative greenery area=(ripening stage plant greenery area/flowering period maximum leaf area) * 100% (Cereal ResearchCommunications, 1998,26,161-167 with protecting green kilsyth basalt; Theor Appl Genet., 2002,106,133-142).
At F
2:3Each identifies in the middle of sub-district 5 individual plants of picked at random mark of listing family, ripening stage is investigated the greenery area of each individual plant of listing of different familys, it is ripening stage leaf area=length * wide * 0.75, blade with each sub-district each family of mean value calculation is protected green degree, be the ripening stage greenery area and flowering period greenery area ratio (Theor Appl Genet., 2002,106,133-142).
(2) molecular marker analysis, genetic map construction and qtl analysis
Be chosen at the combination of primers that has amplification polymorphism between two parents, to 166 F
2:3Family is protected green proterties and is analyzed.Separate F
2Total DNA of each individual plant maize leaf of colony, adopt little satellite (Simple sequence repeat, SSR) the molecule marker primer carries out pcr amplification, amplified production on the 6g/100ml polyacrylamide gel from after, obtain the molecule marker polymorphism data, the polymorphism data that obtains is adopted software MAP MAKER/EXP3.0 (Lincoln et al, WhiteheadInstitute Technical Report, Whitehead Institute, Cambridge, Massachusetts, USA, 1992), set LOD 〉=3.0, select the Kosambi function to make up the genetic linkage maps of S06.Complex inheritance spectrum data and guarantor's green property materials of identification (referring to step (1)), adopt WinCart QTL2.5 to carry out (Wang et al earlier, Department of Statistics, North CarolinaStateUniversity, Raleigh, NC.2007, http://www.statgen.ncsu.edu/qtlcart/WQTLCart.htm), analyze QTL, (CompositeInterval Mapping, CIM) mode is judged (the LOD threshold value setting is 2.5) to utilize composite interval mapping again.When LOD greater than 2.5 the time, illustrate that there is the chain site of QTL in this interval.
Through composite interval mapping (Composite Interval Mapping, CIM) analyze discovery at the umc1026 of linkage group 2 mark, i.e. 5 '-TCGTCGTCTCCAATCATACGTG-3 ' and 5 '-GCTACACGATACCATGGCGTTT-3 ' primer pair and neat 319 mainly imitate blade to protect green property QTL linked, and can be used for the germplasm materials blade and protect the prediction of green property.
The beneficial effect that technical solution of the present invention realizes:
The present invention is by SSR molecule marker and qtl analysis, on the linkage group 2 respectively between mark umc1845 and umc1026, and respectively exist one to protect green property QTL site between umc1026 and the umc1541.Further analysis revealed umc1026, promptly 5 '-TCGTCGTCTCCAATCATACGTG-3 ' and 5 '-GCTACACGATACCATGGCGTTT-3 ' primer is right, imitating blade with the corn master, to protect green property QTL linked, promptly having or not of this molecule marker is directly connected to the power that this plant protects green property, can be used in the germplasm materials blade and protects prediction of green property or evaluation.
, use gel electrophoresis to separate and obtain the band of length after the total DNA of corn is carried out pcr amplification with this primer as 230bp.This molecule marker and maize leaf are protected green property close linkage, and promptly having or not of this molecule marker is directly connected to the power that this plant protects green property.In the middle of the breeding and resource evaluation or early prediction and screening to the green property of the guarantor of maize leaf of corn, this molecule marker can overcome environmental influence, for screening, eliminates the plant that protects green difference morning, raising breeding and efficiency of selection to realizing the seedling stage of germplasm materials.Be applied in the middle of the green-keeping capacity of maize evaluation, can predict the green property of the guarantor of different germplasm materials blades.
The self-mating system source of plant of the present invention is as follows:
Described " neat 319 " are that the inbreeding of more generation seed selection forms from U.S. Pioneer Electronic Corp. corn hybrid seed 78599, the corn inbred line that the green property of guarantor is strong, combining ability is high, lodging resistance is strong and comprehensive proterties is good of Shandong Province Academy of Agricultural Sciences 20th century seed selection nineties.This used self-mating system comes from corn institute of Shandong Province Academy of Agricultural Sciences in the middle of the present invention.
Described " Mo17 " after to be the seventies in 20th century from the U.S. introduce once by China's widespread use, protect the relatively poor corn inbred line of green property, be the second cycle line of from 187-2 * 103 filial generations, selecting.This used self-mating system comes from Crop Breeding Cultivation Inst., Chinese Agriculture Academy in the middle of the present invention.
Described " 8112 " are that isolation and selection forms from the american goods corn.This used self-mating system comes from Laizhou City, Shandong Province institute of agricultural sciences in the middle of the present invention.
Described " yellow morning 4 " is that " Siping City, pool head " self-mating system breeding Tanaka selects.This used self-mating system comes from Crop Breeding Cultivation Inst., Chinese Agriculture Academy and Crop Inst., Beijing Agriculture and Forest Sciences Academy in the middle of the present invention.
Described " tucking in 502 " is that seed selection forms from 340 * yellow 4 cross materialses early.This used self-mating system comes from west, Laizhou City, Shandong Province by seeds company in the middle of the present invention.
Described " dust former 311 " is the second cycle line of selecting from Egyptian 205 * former military 02 filial generation.This used self-mating system comes from Beijing Agricultural College in the middle of the present invention.
Described " lucky 853 " is that seed selection forms from (yellow early 4 * from 330) * yellow 4 cross materialses early.This used self-mating system comes from Jilin Academy of Agricultural Science corn institute in the middle of the present invention.
Described " tucking in 488 " is the second cycle line of selecting from 8112 * 5003 filial generations.This used self-mating system comes from Laizhou City, Shandong Province corn institute in the middle of the present invention.
Described " Shen 137 " is that seed selection forms from U.S. germplasm 6JK1118.This used self-mating system comes from Shenyang Academy of Agricultural Sciences in the middle of the present invention.
Described " tucking in 478 " is the second cycle line of selecting from 8112 * 5003 filial generations.This used self-mating system comes from Laizhou City, Shandong Province corn institute in the middle of the present invention.
Described " 3195 " are the second cycle lines of selecting from red 340 * trip nine wide filial generations.This used self-mating system comes from kind industry company limited of Shenyang Academy of Agricultural Sciences in the middle of the present invention.
Described " prosperous 7-2 " is the second cycle line that seed selection becomes in No. 7, prosperous list (V59 * Huang early 4), imports subtropics corn germplasm S901 (SWANI selects system) again.This used self-mating system comes from kind industry company limited of the high section in Henan Province in the middle of the present invention.
Described " 178 " are that the inbreeding of more generation seed selection forms from U.S. Pioneer Electronic Corp. corn hybrid seed 78599.This used self-mating system comes from China Agricultural University in the middle of the present invention.
Described " BM " is the second cycle line of selecting from 230B * Mo17 filial generation.This used self-mating system comes from Tangshan City research of agricultural science institute in the middle of the present invention.
Described " neat 205 " are the second cycle lines of selecting from S3 * Po70 filial generation.This used self-mating system comes from Crop Breeding Cultivation Inst., Chinese Agriculture Academy in the middle of the present invention.
Described " E28 " is to be anti-source and trip's 9 wide hybridization with A619Ht, and with trip 9 wide third backcross generations, selfing two generations seed selection forms continuously.This used self-mating system comes from the academy of agricultural sciences, Dandong City in the middle of the present invention.
Described " military 314 " are that seed selection forms from Huang 4 * (military 302D * yellow explosion) cross materialses morning.This used self-mating system comes from Shaanxi high-efficiency agriculture institute in the middle of the present invention.
Described " B73 " is U.S.'s germplasm, and seed selection forms from BSSS.This used self-mating system comes from China Agricultural University in the middle of the present invention.
Described " H21 " is the second cycle line of early selecting 4 * H84 filial generation from Huang.This used self-mating system comes from Laiyang Agricultural College in the middle of the present invention.
Described " lucky 846 " are the second cycle lines of selecting from lucky 63 * Mo17 filial generation.This used self-mating system comes from Jilin Academy of Agricultural Science corn institute in the middle of the present invention.
Described " literary composition yellow 31413 " is the second cycle line of selecting from blue or green 1331 filial generations of Huang 4 * literary composition morning.This used self-mating system comes from the Agricultural Science Institute, Yantai of Shandong Province in the middle of the present invention.
Described " Zheng 32 " are to select excellently from U.S.'s seed of single cross 3382 through individual plant, and continuously selfing is bred.This used self-mating system comes from Henan Academy of Agricultural Sciences in the middle of the present invention.
Description of drawings
Fig. 1 is neat 319 corn inbred line linkage groups 2 and QTL site plan thereof, and wherein, the figure left side is a marking path (unit: centimorgan); The right side is the mark title.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with accompanying drawing, embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.
If the used reagent of the present invention is unexplained reference, all available from Sigma-aldrich (company of Sigma-Aldrich).
The present invention relates to molecular biology experiment, as not dated especially, all with reference to oneself " molecular cloning " book (J. Sa nurse Brooker, E.F. be Ritchie, T. Manny A Disi work not, Science Press, 1994).This book and follow-up publication version thereof are those skilled in the art's the most frequently used reference books with directiveness when carrying out the experimental implementation relevant with molecular biology.
Determining of embodiment 1 molecule marker
(1) green-keeping capacity of maize F
2Segregating population and F
2:3The structure of macroscopical identification colony and protect the evaluation of green property
Select for use blade to protect the strong corn inbred line of green property neat 319 and be parent 1 (P
1) the corn inbred line Mo17 relatively poor with protecting green property be parent 2 (P
2) hybridize, obtain first-filial generation hybrid (F
1).By first generation of hybrid F
1Selfing obtains second filial generation (F
2) segregating population, F
2The pollination of segregating population individual plant selfing obtains hybrid three generations family (F
2:3) the macroscopical identification family.
Summer, (36 ° of 16 ' N, 117 ° of 9 ' E, height above sea level 128m) planted P in Shandong Agricultural University corn garden then
1, P
2, F
1And F
2, F wherein
2500 strains are planted by colony, picked at random 350 strains.When plant strain growth to 6～7 leaves, clip P
1, P
2, F
1With 350 F
22 young leaflet tablets of individual plant extract total DNA.Extract 350 F of total DNA
2Individual plant bagging in flowering period individual plant selfing, preparation F
2:3Family.The concrete grammar that extracts total DNA is the CTAB method, and step is referring to document Nucleic AcidsRes, 1980,8,4321 and Genetics, 1994,138,1251-1274.
Summer next year is (36 ° of 16 ' N, 117 ° of 9 ' E, height above sea level 128m) plantation P in Shandong Agricultural University corn garden
1, P
2With 166 F that the q.s seed is arranged
2:3Family.Plant and adopt randomized block design, be specially the single file district, 0.70 meter of line-spacing, long 4 meters of row, every row 15 strains, district's group is arranged at random, 3 repetitions.The field management routine operation guarantees respectively to handle the consistence of cultivation condition.Protect green property in flowering period and ripening stage evaluation blade.
Protect green property size and show, be i.e. greenery area relatively, relative greenery area=(ripening stage plant greenery area/flowering period maximum leaf area) * 100% (Cereal ResearchCommunications, 1998,26,161-167 with protecting green kilsyth basalt; Theor Appl Genet., 2002,106,133-142).
At F
2:3Each identifies in the middle of sub-district 5 individual plants of picked at random mark of listing family, ripening stage is investigated the greenery area of each individual plant of listing of different familys, it is ripening stage leaf area=length * wide * 0.75, blade with each sub-district each family of mean value calculation is protected green degree, be the ripening stage greenery area and flowering period greenery area ratio (Theor Appl Genet., 2002,106,133-142).
(2) molecular marker analysis, genetic map construction and qtl analysis
Be chosen at the combination of primers that has amplification polymorphism between two parents, to 166 F
2:3Family is protected green proterties and is analyzed.Separate F
2Total DNA of each individual plant maize leaf of colony, adopt little satellite (Simple sequence repeat, SSR) the molecule marker primer carries out pcr amplification, amplified production on the 6g/100ml polyacrylamide gel from after, obtain the molecule marker polymorphism data, the polymorphism data that obtains is adopted software MAP MAKER/EXP3.0 (Lincoln et al, WhiteheadInstitute Technical Report, Whitehead Institute, Cambridge, Massachusetts, USA, 1992), set LOD 〉=3.0, select the Kosambi function to make up the genetic linkage maps of S06.Complex inheritance spectrum data and guarantor's green property materials of identification (referring to step (1)), adopt WinCart QTL2.5 to carry out (Wang et al earlier, Department of Statistics, North CarolinaState University, Raleigh, NC.2007, http://www.statgen.ncsu.edu/qtlcart/WQTLCart.htm), analyze QTL, (CompositeInterval Mapping, CIM) mode is judged (the LOD threshold value setting is 2.5) to utilize composite interval mapping again.When LOD greater than 2.5 the time, illustrate that there is the chain site of QTL in this interval.
Through composite interval mapping (Composite Interval Mapping, CIM) analyze discovery on linkage group 2, respectively between mark umc1845 and umc1026, and respectively exist one to protect green property QTL site between umc1026 and the umc1541, its corresponding LOD value is respectively 4.21 and 3.02, explain that respectively heritable variation is 10.67% and 11.11% (see Table 1 and Fig. 1), it closes on mark and is all umc1026, promptly 5 '-TCGTCGTCTCCAATCATACGTG-3 ' and 5 '-GCTACACG ATACCATGGCGTTT-3 ' primer is right, the result shows umc1026 and neat 319 main imitate blade to protect green property QTL linked, and can be used for the germplasm materials blade and protect the prediction of green property.
The molecule marker umc1026 mark of protecting green property main effect QTL linkage with neat 319 blades of the green self-mating system of guarantor is with the neat 319/Mo17 second filial generation of corn (F
2) the blade DNA isolation, adopt 5 '-TCGTCGTCTCCAATCATACGTG-3 ' and 5 '-GCTACACGATACCATGGCGTTT-3 '; Carry out pcr amplification, amplified production upward obtains after the electrophoretic separation at 1.5% sepharose (SCAR, STS) and 6g/100ml polyacrylamide gel (SRAP, SSR), and size is 230bp.
The neat 319/Mo17 green-keeping capacity of maize main effect QTL of table 1 is analyzed
The pcr amplification of embodiment 2 molecule markers
1. the extraction of the total DNA of corn:
Be mixed for the extraction of total DNA after 2 young leaflet tablets of clip (arteries and veins in the removal) shred when corn 6～7 leaves.With fresh blade (about 5g) quick-frozen in liquid nitrogen, quick grind into powder in mortar, change in the 50ml centrifuge tube, the CTAB extraction buffer (83.5mmol/L Tris-7.5,1.175mol/L NaCl, the 16.7mmol/L EDTA-8.0 that add 65 ℃ of preheatings, 1.67%CTAB, the 15ml of 1% β-ME), the mixing that fully vibrates is incubated 1.5～2h (swaying once every 10min) in 65 ℃ of thermostat containers.Take out, to be cooledly add isopyknic chloroform to room temperature: primary isoamyl alcohol (24: 1) shakes up 5～10min gently.Centrifugal 10min under the room temperature (8,000r/min), get supernatant liquor and go to the 50ml centrifuge tube, add the Virahol of 2/3 volume, shake up gently, leave standstill for some time under the room temperature after, obtain DNA precipitation.With DNA with twice of 70% washing with alcohol.
DNA is dried up, add fully dissolving of 2～3ml TE damping fluid (pH8.0) again, add 10～20 μ l RNAase A solution (10mg/ml), 37 ℃ of following water bath with thermostatic control 1～2h.Add isopyknic phenol-chloroform (1: 1), mixing 10min. gently, centrifugal 10min (4,000r/min).Get supernatant, add the 3mol/L NaAc (pH5.2) of 1/10 volume, add the precooling dehydrated alcohol of 2 times of volumes behind the mixing, and shake up gently, leave standstill for some time under the room temperature after, go out DNA with the glass stick hook and precipitate.With DNA with twice of 70% washing with alcohol.DNA is dried up, add 200～300 μ lTE-8.0 and fully dissolve.Measure DNA concentration with ultraviolet spectrometry range instrument, DNA concentration is transferred to 0.3 μ g/ μ l with TE damping fluid (pH8.0).4 ℃ of preservations are standby.
2.PCR amplification:
(1) reaction system of pcr amplification
(2) the hot program of pcr amplification reaction
Behind each reactive component mixing, on the PCR instrument, increase by above program.
(3) denaturing polyacrylamide gel electrophoresis
Instrument: the EPC3000 three permanent electrophoresis apparatuses that Liuyi Instruments Plant, Beijing produces
Glue type: 34 * 32 * 0.4mm
Gel: 6% polyacrylamide denaturant gel
Electrophoretic procedures:
Clean sheet glass: be stained with washing composition sheet glass scrub repeatedly with tap water, clean 2 times with distilled water, again with 95% alcohol scouring twice, drying.On long slab, be coated with the bonding silane of 1ml 0.5% (w/v), be coated with 1ml 2% (w/v) on another piece plate and separate silane.Prevent in the operating process that two blocks of glass from polluting mutually.
Treat to assemble electrophoresis chamber behind the sheet glass finish-drying, water level gauge detects.
The preparation of polyacrylamide gel
Encapsulating: use the 100ml syringe, irritate into coagulant liquid gently, prevent bubble via injection orifice.When treating that glue flow to above the hectograph, insert comb gently, it is condensed at least more than the 1h on top.
Prerunning: get 1500ml l * tbe buffer liquid, wherein 800ml adds anodal groove, and 700ml adds the negative pole groove, extracts comb.The permanent power prerunning of 80W 30min makes temperature reach 50 ℃.
Sex change: 10 μ l PCR samples add 3.5 μ l, 6 * Loading Buffer, behind the mixing, at 95 ℃ of sex change 5min, put into immediately more than the cooled on ice 10min.
Electrophoresis: with inhaling ball pressure-vaccum loading slot, remove bubble, insert the sample comb, each well adds 5 μ l samples.About the permanent power electrophoresis of 80W 1.0h.After electrophoresis finishes, carefully separate two sheet glass, gel can be close on the sheet glass that is coated with Binding Silance.
(4) silver dyes program
Fixing: as gel slab to be placed 2L acetate solution (10%), sway 20min gently.
Rinsing: sway rinsing 5min. with the 2L distilled water
Dyeing: (2g AgNO in the staining fluid of newly joining with 2L
3, 3ml 37% (v/v) formaldehyde), 30min gently vibrates.
Rinsing: with the rinsing of 2L distilled water, the time is no more than 10 seconds.
Develop: (developing solution contains anhydrous Na in the 2L developing solution
2CO
360g, Na
2S
2O
3400 μ l, 37% formaldehyde 3ml, 5h preparation in advance places 4 ℃ of refrigerator precoolings) sway gently, until the band line occurring.
Photographic fixing: photographic fixing 5min in 2L 10% (v/v) acetum.
Rinsing: with 2L distilled water rinsing 5min.
Dried glue: dry more than the 24h naturally under the room temperature.
Embodiment 3 protects the linked checking of molecule marker in different germplasm materials of green property main effect QTL with neat 319 blades
What obtain protects the molecule marker umc1026 of green property main effect QTL compact linkage with maize leaf, has also carried out blade different germplasm materials plant and has protected the prediction of green property.
Separate earlier total DNA from their seedling leaf, the primer that utilizes mark umc1026 then is to carrying out pcr amplification to these total DNA, and determines whether to exist corresponding mark by passing judgment on collection of illustrative plates.When having corresponding amplified band, it is better to illustrate that this strain is protected green property, and it is general or relatively poor not exist then explanation to protect green property.Based on these judgment criterion the green property of the guarantor of different self-mating system plant is predicted then.The late growth stage blade of measuring tested sample is protected green property and is compared with predicting the outcome subsequently.The results are shown in Table 2, it is very identical with measured result to predict the outcome.
Table 2 is protected green property with umc1026 prediction germplasm materials blade
The 230bp amplified band of the underlined umc1026 of "+" expression
The 230bp amplified band of the unmarked umc1026 of "-" expression
Experimental result shows that the umc1026 molecule marker is not only applicable to the green property of the guarantor prediction of neat 319 self-mating systems and hybridization system, also is applicable to the green property of the guarantor prediction of other numerous corn strains.
Sequence table
＜110〉Academy of Agricultural Sciences, Shanghai City
＜120〉protect the molecule marker of green property main effect QTL compact linkage with maize leaf
<130>0911091
<160>1
<170>PatentIn?version?3.3
<210>SEQ?ID?No?1
<211>22
<212>DNA
＜213〉primer before the umc1026
<400>1
<210>SEQ?ID?No?2
<211>22
<212>DNA
＜213〉primer behind the umc1026
<400>1
Claims (5)
1. protect the molecule marker of green property main effect QTL compact linkage with maize leaf for one kind, it is made up of 5 '-TCGTCGTCTCCAATCATACGTG-3 ' and 5 '-GCTACACGATACCATGGCGTTT-3 ' primer.
2. according to claim 1 and maize leaf is protected the molecule marker of green property main effect QTL compact linkage, is after template is carried out pcr amplification with the total DNA of corn, obtains the dna fragmentation that length is 230bp through gel electrophoresis.
3. according to claim 1 and maize leaf is protected the molecule marker of green property main effect QTL compact linkage, the application in the green-keeping capacity of maize evaluation.
4. according to claim 1 and maize leaf is protected the molecule marker of green property main effect QTL compact linkage, in the breeding of corn and the application in the resource evaluation.
5. according to claim 1 and maize leaf are protected the molecule marker of green property main effect QTL compact linkage, to the application in the green property of the guarantor of maize leaf early prediction and the screening.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN112094941A (en) * | 2020-10-19 | 2020-12-18 | 中国科学院植物研究所 | Two main effect QTLs for regulating and controlling maize leaf senescence and molecular marker and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112094941A (en) * | 2020-10-19 | 2020-12-18 | 中国科学院植物研究所 | Two main effect QTLs for regulating and controlling maize leaf senescence and molecular marker and application thereof |
| CN114292942A (en) * | 2020-10-19 | 2022-04-08 | 中国科学院植物研究所 | Major QTL for regulating and controlling maize leaf senescence and molecular marker and application thereof |
| CN114292942B (en) * | 2020-10-19 | 2024-01-30 | 中国科学院植物研究所 | Main effect QTL for regulating and controlling corn leaf senescence, molecular marker and application thereof |
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