CN102234642B - Molecular marker of major dominant quantitative trait loci (QTL) for regulating and controlling maize leaf angle and method and application thereof - Google Patents
Molecular marker of major dominant quantitative trait loci (QTL) for regulating and controlling maize leaf angle and method and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker of major dominant quantitative trait loci (QTL) for regulating and controlling a maize leaf angle. The molecular marker consists of two pairs of primers, namely p-bnlg1484 and LA25. Through a simple sequence repeat (SSR) molecular marker and QTL analysis, a QTL for regulating and controlling an angle exists between a bnlg1484 marker and an LA5025 marker of the marker on a first chromosome, is positioned at the 1.02 locus of the first chromosome between the bnlg1484 marker and the LA25 marker, and has the contribution rate of 20.4 percent. Analysis shows that the presence or not of the marker is directly related to the size of the leaf angle of a plant, and the marker can be used for predicting the size of the leaf angle of a planted material.
Description
Technical field
The present invention relates to technical field of bioengineering, be specifically related to a kind of molecule marker and method and application that regulates and controls the main effect QTL compact linkage of leaf of Semen Maydis angle.
Background technology
Corn is the second largest farm crop of China.Corn is the high yield food crop, is again important diversified economy crop, and industrial chain is the longest in food crop, and value-rising is the highest.The Yield and quality of corn is related to fodder industry, foodstuffs industry, chemical engineering industry and energy industry.China recent years corn consumption continues to increase, and has formed take the industrial pattern chain of Maize Production as basis.Improve per unit area yield, increase and always produce, develop Maize Production, for socio-economic development, have important shoring of foundation effect, for realizing, by large agricultural country, move towards agricultural power, realize that agricultural produce synergy, increasing peasant income etc. all have important strategic importance.From development trend, tight slightly general layout will appear in domestic supply and demand of corn.Because China's cultivated land resource is very limited, the trend that cultivated land resource reduces is difficult to reverse, and increases the corn ultimate production and mainly relies on the raising unit yield.In decades, the individual plant productivity of american corn cross-fertilize seed does not have obviously to improve and the major cause that increases productivity is to have strengthened planting density and resistance in the past.So, be accompanied by cultivated area and reduce gradually, within the coming years, rely on and increase the major objective that planting density raising per unit area yield will remain the selection of breeding man.Plant Type in Maize determines planting density and affects canopy light intercepting and capturing rate, disease resistance and lodging resistance, so the plant type improvement is the important component part of modern breeding target, is also one of effective way that further improves corn yield yet to a great extent.Therefore, the genetic development of research Plant Type in Maize correlated character is to promoting the anti-close type breeding of new variety of corn to have important theory and practice meaning.
The leaf of Semen Maydis angle is an important factor that affects Plant Type in Maize.From the dense planting viewpoint, the milpa with relatively large leaf angle has affected light in maize canopy and has distributed, and has increased canopy to the sheltering from heat or light of lower leave, and then has reduced photosynthetic efficiency; And light distributes more rationally in the milpa maize canopy of leaf angle less, has reduced canopy to the sheltering from heat or light of lower leave, and then has improved effective intercepting quantity of light, has improved Productivity of planting group.Leaf angle and planting density are widely studied on the impact of output, and under the dense planting condition, the liguleless2 cross-fertilize seed that contains liguliss gene is compared average yield per mu high by 41.2% (Pendleton et al. 1968) with normal cross-fertilize seed; Cross-fertilize seed liguleless1 and liguleless2 that cross-fertilize seed B14 * Oh43 that the leaf angle is large and Oh43 * R177 and leaf angle are little plant under different density, result shows that liguleless2 is 75, under the planting density of 000 and 90,000 strains/hectare, output is significantly higher than corn hybrid seed B14 * Oh43 and Oh43 * R177 (Lambert and Johnson 1978).In addition, Childs, in America Corn Yield Contest in 2002, has created the high yield record of 1850 kg/acres with pioneer cross-fertilize seed 34N44, and planting density reaches 7224 strains/mu, and high planting density reaches 8237 strains/mu; Li Denghai was created the summer corn high yield record of 1290 kg/acres with cross-fertilize seed DH3719 in 2005, its density is near 7000 strains/mu.These statements of facts, Compact-type Corn Hybrids can increase to greatest extent planting density and then improve corn unit surface population yield.The major cause of corn yield raising in recent years is not the heterotic raising of individual plant, but plants by unit surface the raising that more strain number is colony's hybrid vigour and resistance.China's corn planting density is lower at present, and the maximum potential that further improves output just is to create the suitable plant type of leaf angle and improves planting density.
Summary of the invention
Purpose of the present invention provides a kind of molecule marker and method and application that regulates and controls the main effect QTL compact linkage of leaf of Semen Maydis angle.
Technical scheme of the present invention is: a kind of molecule marker that regulates and controls the main effect QTL of leaf of Semen Maydis angle, by p-bnlg1484 and two pairs of primers of LA25, formed, and the sequence of described primer p-bnlg1484 is:
F1：5＇-GTAAAAGACGACGACATTCCG-3＇
R1：5＇-GACGTGCACTCCGTTTAACA-3＇；
The sequence of described primer LA25 is:
F2：5＇-AAAGTCTCTCGTCCTCCAG-3＇
R2：5＇-GAACACTAACAAACATCGC-3＇。
The method of the molecule marker of the main effect QTL of described regulation and control leaf of Semen Maydis angle: take total corn DNA as template, carry out pcr amplification with primer pair p-bnlg1484 and LA25, obtain length through gel electrophoresis and be respectively 220 and 270 DNA fragmentation.
The molecule marker of the main effect QTL of described regulation and control leaf of Semen Maydis angle is in the early prediction of leaf of Semen Maydis angle and the application in screening.
The molecule marker of the main effect QTL of described regulation and control leaf of Semen Maydis angle is in the application of the seed selection of compact corn.
The invention has the beneficial effects as follows: the present invention is by SSR molecule marker and qtl analysis, at the QTL that has a regulation and control leaf corner dimension on the first chromosome between mark bnlg1484 and LA5025, it is positioned at 1.02 sites of the first chromosome, between mark bnlg1484 and label L A25, contribution rate is 20.4%.The analysis showed that having or not of this mark be directly connected to the size of Plant Leaf angle, can be used in the prediction of planting material leaf corner dimension.
After with this mark, total corn DNA being carried out to pcr amplification, use gel electrophoresis to separate to obtain length to be respectively 220 and 270 DNA fragmentation.Having or not of this mark is directly connected to this Plant Leaf corner dimension.Corn breeding and resource identify or early prediction and screening to the angles of corn plant leaves corner dimension in, this molecule marker can overcome environmental influence, and morning of planting material, for screening, is eliminated to the large plant of leaf angle, improves breeding and efficiency of selection.Be applied to, in the evaluation of leaf of Semen Maydis corner dimension, can predict different germplasm materials leaf corner dimensions.
The accompanying drawing explanation
Fig. 1 is that corn backcrosses is linkage group and QTL site plan thereof.
Embodiment
A kind of molecule marker that regulates and controls the main effect QTL of leaf of Semen Maydis angle, be comprised of p-bnlg1484 and two pairs of primers of LA25, and the sequence of described primer p-bnlg1484 is:
F1：5＇-GTAAAAGACGACGACATTCCG-3＇
R1：5＇-GACGTGCACTCCGTTTAACA-3＇；
The sequence of described primer LA25 is:
F2：5＇-AAAGTCTCTCGTCCTCCAG-3＇
R2：5＇-GAACACTAACAAACATCGC-3＇。
The method of the molecule marker of the main effect QTL of described regulation and control leaf of Semen Maydis angle: take total corn DNA as template, carry out pcr amplification with primer pair p-bnlg1484 and LA25, obtain length through gel electrophoresis and be respectively 220 and 270 DNA fragmentation.
The molecule marker of the main effect QTL of described regulation and control leaf of Semen Maydis angle is in the early prediction of leaf of Semen Maydis angle and the application in screening.
The molecule marker of the main effect QTL of described regulation and control leaf of Semen Maydis angle is in the application of the seed selection of compact corn.
Fig. 1 is that corn backcrosses is linkage group and QTL site plan thereof, and wherein, the figure left side is marking path (unit: centimorgan); Right side is the mark title.
| QTL | Karyomit(e) | The LOD value | Between mark zone | Contribution rate (%) |
| qLA1 | 1 | 8.6 | LA5025 | 38.6 |
A kind of detailed step of method of the molecule marker that obtains leaf of Semen Maydis angle main effect QTL is as follows:
(1) leaf of Semen Maydis included angle B C3F2 and the structure of BC3F2:3 family macroscopical identification colony and the tolerance of leaf angle
Select the corn inbred line Henan 82 that the leaf angle is little, plant type is compact to be parent 1(P1) with the corn inbred line Shen 137 that the leaf angle is large, plant type is loose, be parent 2(P2) hybridize, obtain first-filial generation hybrid (F1). by first generation of hybrid F1 and Shen 137 continuous hybrid 3 times, obtain third backcross generation (BC3F1).The BC3F1 selfing obtains the BC3F2 segregating population, and the pollination of BC3F2 segregating population individual plant selfing obtains the macroscopical identification family of hybrid three generations's family (BC3F2:3).
Spring is at Agricultural University Of He'nan scientific and educational park plantation P1, P2, F1 and BC3F2 then.Wherein, BC3F2 colony plantation 300 strains.2 young leaflet tablets getting P1, P2, F1 and 300 strain BC3F2 individual plants when plant strain growth during to 5-7 sheet leaf extract total DNA.Extract 300 strain BC3F2 individual plant bagging in the flowering period individual plant selfing of total DNA, preparation BC3F2 family.Extract the SDS method of the concrete grammar of total DNA for improvement.
At Sanya, Hainan, plant P1, P2 and 255 BC3F2:3 familys that enough grain weights are arranged winter then.Plant and adopt randomized block design, be specially the single file district, 0.70 meter of line-spacing, row length is 4 meters, every row 15 strains, random district group is arranged, 3 repetitions.The field management routine operation guarantees respectively to process the consistence of cultivation condition.10 days investigation leaf angles after blooming.
The leaf angle is stem and the angle that will investigate the vein of the blade outside of belly.In each BC3F2:3 family, from centre, get at random the vaned leaf angle on 5 continuous individual plant tolerance fringe tops, with the mean value of all tolerance leaf angles of every strain, represent the individual plant leaf angle of investigation, then with the mean value of all tolerance individual plants of each residential quarter, represent the leaf angle value of each family.
The length of carrying out obtaining after pcr amplification as template take the total corn DNA that extracts is respectively 220 and 270 DNA fragmentation, and its step is as follows:
(1) extraction of total corn DNA:
After when corn 5-10 sheet leaf, getting 1 tender appropriate blade of children and grinding with liquid nitrogen for the extraction of total DNA.Adopt the SDS method of improvement to extract total DNA.Concrete steps are as follows:
1. get maize leaf grinding powder in liquid nitrogen that 5g is fresh, in the centrifuge tube of the 50ml that packs into;
2. in centrifuge tube, add the SDS extracting solution 15ml that is preheated to 65 ℃, then add 30 μ l beta-mercaptoethanols to mix, 65 ℃ of water-bath 40 ~ 60min in water-bath;
3. take out and add 5M KAC 5mL, ice-water bath 10min;
4. add the 15ml chloroform
:Primary isoamyl alcohol (24
:1) fully mix, slowly shake to being blackish green, standing 10min under room temperature, 4 ℃, centrifugal 15 min of 12000r/min;
5.; Get supernatant liquor, add the Virahol of about 15ml precooling (20 ℃), rotation mixes, and is placed in 4 ℃ of refrigerators, and DNA is cotton-shaped and separates out, and with glass needle, the DNA hook is gone out, and is placed in the 7ml centrifuge tube, adds 10nM MNH
4In 76% ethanol of AC, soak 2h;
6. outwell alcohol, by the dry 2h of DNA, add appropriate TE dissolving DNA, DNA adds 40-50 μ l RNase(10mg/ml after dissolving), 37 ℃ of insulation 1h, add equal-volume phenol
:Chloroform
:Primary isoamyl alcohol (25
:24
:1) mixed solution, shake 10min gently, the centrifugal 10min of 8000r/min;
7. get supernatant liquor in another 7ml centrifuge tube, add the equal-volume chloroform
:Primary isoamyl alcohol (24
:1), shake gently 10min, the centrifugal 10min of 8000r/min;
8. get supernatant liquor in the 50ml centrifuge tube, add (precooling is-20 ℃) Virahol of 200% volume, be placed in 4 ℃ of refrigerator 10min, DNA slowly separates out;
9. hook goes out in DNA to 1.5ml centrifuge tube, adds appropriate 76% alcohol immersion 30min, the centrifugal 2min of 8000r/min, Air drying 6 ~ 12h;
10. add 300-500 μ l TE to dissolve, fully dissolving DNA, be placed in-20 ℃ of refrigerators and preserve.
(2) pcr amplification
Described PCR cumulative volume is 15 μ l, and reaction system is:
10 * buffer(contains Mgcl
2) μ l l.5
0.5mmol/l dNTP 0.3μl
Taq enzyme (1U) 0.1 μ l
20ng/μ l primer 1.5 μ l
ddH
2O 8.6μl
20ng/ μ l template DNA 3.0 μ l
After each reactive component is mixed, add 20 μ l mineral oil to cover, on PTC-200 type PCR instrument, increase.
The PCR response procedures:
Step1： 95℃2min
Step2： 95℃ 1min
Step3： 65℃ 1min；-1℃/cycle
Step4： 72℃ 1.5min
Step5 goto step 2,7 times
Step6 95℃ 1min
Step7 58℃1min
Step8 72℃1.5min
Step9 goto step 6,28 times
Step10 72℃5min
Step11:4 ℃ of preservation
The detection of amplified production
1. DNA amplification sex change
In amplified production, add 6 μ l loading buffer, 95 ℃ of sex change 6min.Be placed in immediately mixture of ice and water standby.
Loading buffer:98% methane amide 49ml (100%), 10mM EDTA (PH=8.0) 1ml (0.5M PH=8.0), 0.25% bromjophenol blue 0.125g, the blue or green 0.125g of 0.25% dimethylbenzene.
2. electrophoresis is prepared
The preparation of sheet glass: sheet glass scrub repeatedly, clean, dry with alcohol with dish detergent.After notch board being coated to 2% Repel Silane in stink cupboard, then with alcohol clean, drying, another piece flat board is coated to 0.5% Bingding Silane.In operating process, prevent that two sheet glass from polluting mutually, after finish-drying, carry out again sheet glass assembling, encapsulating.
The assembling of sheet glass: large plate is kept flat, and platelet presses on large plate and (scribbles the two sides of silane inwards), and centre separates with press strip, puts the large clamp of rear use well and fixes.
The preparation of polyacrylamide gel: 6%PA glue 50ml, 10% ammonium persulphate 250 μ l, TEMED 50 μ l.Shake up rear encapsulating.
Encapsulating: the polyacrylamide gel that will configure is along sheet glass groove limit tapped, limit fill with gently into, must be even, prevent bubble.Treat that solation is moving to bottom, at the encapsulating mouth, insert comb, and notice preventing that the comb bottom from producing bubble.With a strong large clip, comb is clamped, it is fixed, to avoid producing glued membrane, affect point sample.If spill, should in time add polyacrylamide solution.Put room temperature and allow its polymerization 30min at least.
3. the electrophoresis of amplified production
After gelling is solid, take out comb, wash the top gel off and notice that especially seam crossing will clean surely.First groove (negative electrode) electrode buffer of packing under electrophoresis chamber, be contained in the gel slab of polymerization in electrophoresis chamber, in upper groove, injects the electrode buffer of 1 * TBE (diluting with 10 * TBE).
Prerunning: firm power 40W, the about 30min of prerunning.
With suction pipe, remove urea and the bubble that precipitates on the glue face, insert comb.
Electrophoresis: add amplification sample 4 ~ 6 μ l through sex change with liquid-transfering gun, connect with the mains, electrophoresis.Parameter is firm power 50W, and electrophoresis to tetrabromophenol sulfonphthalein arrives the electrophoresis chamber bottom, cuts off the electricity supply.(degree distinguished depending on SSR amplified production molecular size range and difference banding pattern is adjusted electrophoresis time).
Electrophoresis is put the upper strata electrophoretic buffer after finishing, and unloads lower glass plate, takes off comb, carefully separates two sheet glass, and sex change glue is close on the sheet glass that scribbles Binding Silane.
4. the silver of amplified production dyes detection
Decolouring and fixing: after electrophoresis, gel is put into to stationary liquid (900ml distilled water+100ml ethanol+5ml glacial acetic acid), shaken gently 15 ~ 20min colourless to indicator.
Washing: with 1000ml distillation washing 3min.
Silver dyes: put into staining fluid (2gAgNO
3+ 1000ml distilled water), in, shake gently about 30min.
Rinsing: the gel after dyeing is put into distilled water 2 ~ 3s, takes out rapidly and hold up control water.
Develop: offset plate is put into to developing solution (1000ml distilled water+15gNaOH+5ml formaldehyde) and shake to the demonstration of DNA band.
Stop developing: offset plate is taken out and puts into stop buffer (0.75% Na
2CO
3Solution), in, stop developing.
Washing: distilled water immersion is washed gel 10min, pulls airing out, and at room temperature seasoning is preserved.
Preserve: the statistics banding pattern, take a picture or scanning.
Utilize the method dyeing, the background of glue is brown, and generally glue first occurs brownly, and then the banding pattern of DNA becomes red-brown to occur gradually.There is not the excessive phenomenon that occurs that background is dark of dyeing in the method.
Attached: the preparation of 6%PA glue:
Acrilamide 57g, Bis-acrilamide 3g, 10 * TBE 50ml, urea 420g.After stirring and dissolving, adding distil water is to 1000ml, and it is standby that filtration is placed on room temperature
The preparation of 10 * TBE:
Tris 108g, boric acid (Boric Acid) 55g, Na
2EDTA.2H
2O 7.44g.
2% Repel Silane：
490ml chloroform (analytical pure), add 10ml Repel Silane, room temperature preservation after mixing.
0.5% Binding Silane：
The ethanol of 3ml 95% adds 15 μ l glacial acetic acids and 15ul Binding Silane.
10% ammonium persulphate (Ammonium Persu Lphate):
Ammonium persulphate 2g, ultrapure water 18ml, after dissolving, be sub-packed in the centrifuge tube of 1.5ml-20 ℃ of preservations.
5. genotype record
F
2:3On each site of family colony, mainly contain 3 kinds of banding patterns: the banding pattern, the banding pattern in male parent Shen 137, the heterozygous that derive from maternal Henan 82.
(2) genetic map construction and qtl analysis
Be chosen at the mark that has polymorphism between two parents, 255 BC3F2:3 family leaf angle proterties are analyzed, separate total DNA of each individual plant maize leaf of BC3F2 colony, adopt micro-satellite (simple sequence repeat, SSR) molecule marker primer to carry out pcr amplification, amplified production is after separating on the 6g/100ml polyacrylamide gel, obtain the molecule marker polymorphism data, first carry out two point analysiss, with " GROUP " instruction, infer possible linkage group, the LOD value is greater than 3.0.LOD=3.0 is that two of judgements mark whether chain important thresholding.With reference to part telltale mark sequence, utilize and multiple spot analysis to build the skeleton construction of linkage group at 3, then use " COMPARE ", " TRY " and " RIPPLE " instruction to determine putting in order of each linkage group mark.By multiple spot, analyze the recombination value between two adjacent marks, utilize the Kosambi function to convert recombination value to map unit (cM), with " Mapdraw ", build linkage map.
complex inheritance spectrum data and leaf angle appraising datum, utilize WinQTLcartV2.5 software composite interval mapping (Composite Interval Mapping, CIM) method is carried out QTL location and Effect Estimation, every 2cM carries out full genome scanning to each proterties, to determine each proterties QTL number and the position on karyomit(e) thereof, according to Churchill and Doerge(1994) method, permutation 1000 times, the significance level of setting QTL is 0.05(Chromosome-wise Type I error rate=0.05), LOD threshold value (being set as 2.5) according to permutation test, as LOD greater than 2.5 the time, illustrate that there is the chain site of QTL in this interval.Through CIM analyze to find 1.02 region memories on the first chromosome at a QTL between mark bnlg1484 and LA5025, this QTL reduces leaf angle allelotrope from the little parent Henan 82 of leaf angle, and can be used for the prediction of planting material leaf corner dimension.
The self-mating system source of plant of the present invention is as follows:
Described " Henan 82 " derives from Henan and combines C3 improvement colony's (Reid blood relationship) No. 5, from this colony, selects fine individual plant to form by the inbreeding of more generation seed selection.This self-mating system used in the present invention comes from Agricultural University Of He'nan.
Described " Shen 137 " derives from seed selection in U.S. germplasm 6JK1118 and forms.This self-mating system used in the present invention comes from Shenyang Academy of Agricultural Sciences.
Claims (3)
1. molecule marker that regulates and controls the main effect QTL of leaf of Semen Maydis angle, it is characterized in that: take total corn DNA as template, with primer pair p-bnlg1484 and LA5025, carry out pcr amplification, through gel electrophoresis, obtain length and be respectively 220 and 270 DNA fragmentation, at the QTL that has a regulation and control leaf corner dimension on the first chromosome between mark bnlg1484 and LA5025, it is positioned at 1.02 sites of the first chromosome, between mark bnlg1484 and label L A5025, the sequence of described mark p-bnlg1484 is:
F1：5′-GTAAAAGACGACGACATTCCG-3′
R1：5′-GACGTGCACTCCGTTTAACA-3′；
The sequence of described label L A5025 is:
F2：5′-AAAGTCTCTCGTCCTCCAG-3′
R2：5′-GAACACTAACAAACATCGC-3′。
2. the molecule marker of the main effect QTL of regulation and control leaf of Semen Maydis angle as claimed in claim 1 is in the early prediction of leaf of Semen Maydis angle and the application in screening.
3. the molecule marker of the main effect QTL of regulation and control leaf of Semen Maydis angle as claimed in claim 1 is in the application of the seed selection of compact corn.
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| CN104480121B (en) * | 2014-12-17 | 2017-06-16 | 河南农业大学 | Control ZmCLA1 genes and its method in the seed selection type of resistance to more seedlings corn and the application of maize leaves corner dimension |
| CN106701967B (en) * | 2017-01-22 | 2020-03-31 | 甘肃农业大学 | Molecular marker for regulating main effect QTL (quantitative trait locus) of corn leaf angle and application method thereof |
| CN106591325B (en) * | 2017-02-09 | 2019-06-21 | 河南农业大学 | A kind of method and its application using corn ZmDPS10-2 gene breeding early blossoming corn |
| CN107447022B (en) * | 2017-09-11 | 2020-09-18 | 河南省农业科学院粮食作物研究所 | SNP molecular marker for predicting corn heterosis and application thereof |
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| CN1372789A (en) * | 2002-04-02 | 2002-10-09 | 西北农林科技大学 | Maize inbred line germplasm material and hybrid breeding method thereof |
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Non-Patent Citations (2)
| Title |
|---|
| 赵文明, 等.玉米株型性状QTLs 作图群体分析.《玉米科学》.2008,第16卷(第1期),25-28. * |
| 赵文明.玉米株型相关性状QTL定位与分析.《中国优秀硕士学位论文全文数据库农业科技辑》.2009,(第04期),19-23以及摘要. * |
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