CN101942512B - Development and application of molecule marker for corn with considerable number of kernels and excellent allele function in low-nitrogen adverse environment - Google Patents
Development and application of molecule marker for corn with considerable number of kernels and excellent allele function in low-nitrogen adverse environment Download PDFInfo
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Abstract
The invention discloses development and application of a molecule marker for corn with considerable numbers of kernels and an excellent allele function in a low-nitrogen adverse environment. The molecular marker provided by the invention is DNA shown by a sequence 4 in a sequence list. Based on the marker, the invention provides a primer pair consists of the DNA shown by a sequence 2 in the sequence list and the DNA shown by a sequence 3 in the sequence list. The molecular marker and the primer pair can be used for assisting in identification of the corn with different single-strain kernel number properties in the low-nitrogen environment and further used for corn breeding. The method of the invention can be used for screening in an early seed or seedling stage, has a great instruction significance for breeding high-yield, stable-yield and stress-tolerance corn varieties, can acceleration breeding speed and reduce breeding cost, has the advantages of simple operation, low cost and short period, is suitable for popularization and application, is a quick selection method for the identification of the corn variety and has a very high development value.
Description
Technical field
The present invention relates to excellent allelotrope functional molecular marker development of high seed number and application under the low nitrogen adverse circumstance of corn.
Background technology
Corn is important food, feed and insutrial crop, in the national economic development, occupies critical role.China since two thousand seven, corn has been the first crop on the sown area.Nitrogenous fertilizer is the important limiting factor of corn yield.Yet the amplitude that amount of application of nitrogen fertilizer rises is far longer than the amplitude of corn yield increasing, and China's Maize Production is strong to the dependence of nitrogenous fertilizer, and the nitrogenous fertilizer utilising efficiency is low, and cereal crop nitrogen use efficiencies such as corn on average only are about 33%.A large amount of applied nitrogens also cause pollution of area source such as groundwater azotate content overproof, surface water body eutrophication.Therefore, basis and applied research that the corn nitrogen use efficiency is relevant with anti-low nitrogen are the great demands in China current agri-scientific research field, have crucial meaning.Current, the main path of corn variety raising the output is further to increase thickness of sowing, and along with the rising of density, the competition to the environment nutrient between the milpa will further aggravate.Therefore, nitrogen efficient with the seed selection of anti-low nitrogen corn variety be the important directions of breed of variety and genetic improvement in the following commercial corn breeding.
Often receive reasons such as polygenic locus control and environmental influence owing to complicated economical characters such as the efficient utilizations of nitrogen, conventional screening is difficult to receive ideal effect.Glutamine synthetase is cereal crop nitrogen use efficiency such as corn relevant nitrogen assimilation, activation, transhipment and the metabolism maincenter enzyme that assimilates approach again; Being the key gene of nitrogen assimilation and nitrogen use efficiency, also is the important point of penetration of understanding cereal crop nitrogen use efficiencies such as corn.Research also shows, therefore the height of kernal number, improves mealie portion kernal number under the low nitrogen adverse circumstance to the having the greatest impact of output under the low nitrogen situation, is the important directions of anti-low nitrogen breed improvement and selection.
In the breeding material seed selection process, hybridize, backcross or selfing process in all are artificial pollinations, pollination quality has directly determined kernal number and fecundity, the kernal number of treating the seed selection material is not the embodiment of genetic effect.Simultaneously, kernal number also receives the complex environment effects.Therefore, in the material chosen process, the conventional breeding method can't be counted proterties to corn kernel and selected.Also be usually said, character pair is selected can not respond in the breeding process.In addition, the performance that the output of breeding material is handled low nitrogen under the low nitrogen adverse circumstance of research need be set up barren test conditions.Developing the technology of selecting according to genotype under a kind of usual terms can effectively solve above-mentioned two problems, has important development and utilization and is worth.
Summary of the invention
The purpose of this invention is to provide excellent allelotrope functional molecular marker development of high seed number and application under the low nitrogen adverse circumstance of corn; The mark of a kind of 12bp of concrete design, based on the primer of this mark to and in the auxiliary application of differentiating in the corn that under low nitrogen environment, has different individual plant seed number proterties.
The invention provides and auxiliary differentiate that the primer that under low nitrogen environment, has different individual plant seed number proterties corns is right, is that the primer of DNA composition shown in the sequence 3 of DNA shown in the sequence 2 of sequence table and sequence table is right.
Said primer is to can be used for the auxiliary corn that under low nitrogen environment, has different individual plant seed number proterties of differentiating.
The present invention also protects a kind of auxiliary discriminating under low nitrogen environment, to have the method for different individual plant seed number proterties corns, comprises the steps: that the genomic dna with corn to be measured is a template, with said primer to carrying out pcr amplification; If pcr amplification product is a kind of, and size is 329bp, and corn to be measured is homozygous for inserting; If pcr amplification product is a kind of, and size is 317bp, and corn to be measured is homozygous for disappearance; Lack homozygous (excellent genes type) corn and hanging down the individual plant seed number under the nitrogen environment in inserting homozygous (non-excellent genes type) corn in the individual plant kernal number height of eye under the low nitrogen environment.If pcr amplification product is two kinds, size is respectively 329bp and 317bp, and corn to be measured is an insertion/disappearance heterozygous, in the generation, separates obtaining lacking homozygous (excellent genes type) thereafter.
The present invention also protects a kind of corn breeding method, is that the homozygous corn of disappearance of selecting said method to identify carries out breeding.
The present invention also protects another kind of corn breeding method, comprises the steps: donor parents and receptor parent hybridization are obtained F1 generation, and F1 for selfing, is obtained F2 generation; Is template with F2 for the genomic dna of corn, with said primer to carrying out pcr amplification; Pcr amplification product is that a kind of and big or small F2 for 317bp is the corn that seed selection obtains for corn; The disappearance homozygous corn of said donor parents for adopting said method to identify, the insertion homozygous corn of said receptor parent for adopting said method to identify.
Said donor parents specifically can be corn variety CA156, and said receptor parent specifically can be corn variety military 314.
Pcr amplification product can adopt polyacrylamide gel electrophoresis argentation or sepharose electricity arteries and veins ethidium bromide (EB) to detect.
Said primer is to can be used for preparing the auxiliary test kit that under low nitrogen environment, has different individual plant seed number proterties corns of differentiating.
The present invention also protects the DNA shown in the sequence 4 of sequence table, i.e. the disappearance of 12bp zone, and this zone can be used as molecule marker, is used for auxiliary the discriminating under low nitrogen environment and has the corn of different individual plant seed number proterties, and then be used for corn breeding.
The above low nitrogen environment refers to that all total nitrogen content is lower than the soil of 0.4% (quality percentage composition).
The barren stable yields kind of anti-nitrogen is a developing direction, and high kernal number is important selection proterties under the low nitrogen adverse circumstance of selection.Method of the present invention can be used for selecting to hang down the corn variety that has high kernal number proterties under the nitrogen adverse circumstance, uses it for breeding.Method of the present invention is seed (get the part endosperm and do not influence seed vitality after planting) or the screening of seedling stage in early days, and acceptor can be known corn breeding parent commonly used, also can be unknown material; Like germ plasm resources such as self-mating system, cross-fertilize seed, local variety, the open colonies of pollinating, obtain to contain the individuality of target gene type, cultivation high yield, stable yields, degeneration-resistant corn variety are had important directive function; Be selection, accelerate breeding speed, reduce the breeding cost to gene; And have simple to operate; Advantage with low cost, that the cycle is short is suitable for applying; For the evaluation of corn germplasm provides a kind of system of selection efficiently, have very high exploitation and be worth.
Description of drawings
Fig. 1 is the path analysis of the effect between each character pair yield traits and the proterties under 2 years low nitrogen environments; Chlorophyll content: decide relative chlorophyll content value of typhon mouth phase with the SPAD instrumentation; Living weight: with relative living weight NDVI of the typhon mouth phase value of GreenSeeker mensuration; *: the p=0.01 level is remarkable; Ns: not remarkable.
Fig. 2 is F among the embodiment 5
2Evaluation electrophorogram for plant; Size: molecular weight marker; I: insertion (Insertion) genotype of isozygotying; D: homozygous deletion (Deletion) genotype; C: the codominance of insertion/disappearance heterozygosis (Co-dominant) genotype.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
180 corn inbred lines (seeing table 1) that are used for excellent allelotrope evaluation among the embodiment 1 are the parental inbred line of the current frequent application of China's corn breeding; The public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences.The genetic background analytical results of each self-mating system is referring to (Chuanxiao Xie, Shihuang Zhang, Minshun Li; Xinhai Li; Zhuanfang Hao, Li Bai, Degui Zhang; Yehong Liang. (2007) Inferring genome ancestry and estimating molecular relatedness among 187 Chinese maize inbredlines.J.Genet.& Genomics 34 (8): 738-748), and as the foundation of main hereditary monoid.
Table 1 correlation analysis natural population's self-mating system and pedigree source thereof
The genetic analysis of embodiment 1, corn kernel number
One, field test design and species test
180 corn inbred lines adopt α-lattice design, facility nitrogenous fertilizer and not 2 processing of nitrogen fertilizer application, and three repetitions are established in each processing.The long 3.6m of cell row, line-spacing 0.6m, spacing in the rows 0.2m.Each executed 60kg/hm respectively when nitrogen fertilizer application was handled (NP) sowing
2P
2O
5And 45kg/hm
2K
2O is as base fertilizer, and 3 leaf phases were executed 69kg/hm
2Purity nitrogen, the typhon mouth phase imposes 172.5kg/hm
2Purity nitrogen; Nitrogen fertilizer application (NDP) is handled, and the amount of application of phosphorus potash fertilizer is with to execute the nitrogen district identical, and do not refertilize other periods.Ripe back is that unit gathers in the crops with the sub-district, and each 2 strain of wardrobe end of line are gone in every sub-district, and to avoid the influence of edge effect, continuous 10 strains in the middle of the picking bring bigger above and below ground space advantage to avoid the place of being short of seedling to leave a blank to adjacent plant.The air-dry indoor conventional species test in back, the main individual plant kernal number that adopts during this patent is analyzed.The result sees table 2.
Each proterties associating variance analysis F value table of table 2
Annotate: * representes respectively that with material significance level is 0.05 and 0.01.
Conclusion: the corn kernel number mainly receives Genetic Control, and significantly affected by environment; Low nitrogen is handled the main source of the kernal number variation that is corn, and is huge to the influence of corn kernel number.
Two, the relation of kernal number and other Main Agronomic Characters
Investigate under the low nitrogen adverse circumstance of 180 self-mating systems and kernal number (EKN) and chlorophyll content (SPAD1: typhon mouth phase under the normal confession nitrogen condition; Flowering period), the dependency between typhon mouth phase homogenization vegetation index (NDVI), plant height (PH), male and female bloom interval (ASI), nitrogen utilizes agronomy efficient (AE), seed nitrogen content (GN), solid strain are counted percentage (ESP), bald point (BT) and 100-grain weight (HKW) etc. maybe the be relevant Main Agronomic Characters SPAD2:, 2 results saw table 3 in 2 years.
Genetic correlation (going up the diagonal lines district) phenotypic correlation property (following diagonal lines district) result under low nitrogen of table 3 and the normal nitrogen condition
Conclusion: the nitrogen stress condition supplies under the nitrogen condition with normal; Kernal number (EKN) all has higher genetic correlation and phenotypic correlation property with the agronomy utilising efficiency (AE) of output (GY) and nitrogen; The phenotype that shows kernal number character pair yield traits and nitrogen use efficiency has contribution, and more important dependency is arranged in the heredity.In many investigation proterties, kernal number is extremely important with output and nitrogen use efficiency under the normal nitrogen condition to low nitrogen.
The path analysis of the effect under 2 years low nitrogen environments of 180 self-mating systems between each character pair yield traits and the proterties is seen Fig. 1.Single arrow: path coefficient (path coefficients); Double-headed arrow: relation conefficient (correlation coefficients); Output, grain yield; Nitrogen agronomy efficient, agronomic efficiency (kg kg
-1N); Kernal number, ear kernel number; 100-grain weight (g), hundred kernels weight; N%: seed nitrogen content, N content in dry grains; Living weight: normalized differential vegetation index, Normalized difference vegetation index; SPAD: the green relative content SPAD of typhon mouth phase leaf evaluation; The remarkable p=0.01 level of *; Ns: not remarkable, the p=0.05 level.
Output (GY) The results of path analysis shows: the kernal number under the low nitrogen situation is high to the output direct effect, is one of most important phenotype factor in the investigation project.Therefore, the kernal number of the low nitrogen situation of corn is the most relevant with corn yield.
The design that the discovery of embodiment 2, molecule marker and special primer are right
One, the discovery of molecule marker
Analyze the sequencing result of 180 self-mating system glutamine synthetase gene Gln1-3, find insertion/disappearance (Indels) site of a 12bp, be positioned at this upstream region of gene control region-274 to-285, sequence is following:
5’-CCGCGTCGAGGA-3’。
Gene with above-mentioned sequence is a wild-type, and the gene that lacks above-mentioned sequence is a mutant.
Two, the right design of special primer
According to the sequences Design special primer to as follows:
Forward primer: 5 '-ATGTAACTGACAGGTGGGCCT-3 ' (sequence 2 of sequence table);
Reverse primer: 5 '-TTGAGATTGAGGACGAAGGGA-3 ' (sequence 3 of sequence table).
With wild-type glutamine synthetase gene Gln1-3 is template, pcr amplification is obtained the dna fragmentation shown in the sequence 1 of sequence table with above-mentioned primer, this dna fragmentation be glutamine synthetase gene Gln1-3 upstream regulatory region-60 to-388,329bp altogether.With mutant glutamine synthetase gene Gln1-3 is template, with above-mentioned primer pcr amplification is obtained the sequence 1 of sequence table is lacked from the dna fragmentation shown in 5 ' terminal 104 to 115 Nucleotide, 317bp altogether.
Because difference in size is 12bp, detect for making things convenient for PCR, can select for use based on polyacrylamide gel electrophoresis to combine silver staining method to detect amplified fragments.Insert the band that homozygous sample electrophoresis shows a 329bp, lack the band that homozygous sample electrophoresis shows a 317bp, the heterozygous sample electrophoresis shows two bands of 329bp and 317bp.Therefore, the mark of this detection is a codominant marker, not only can distinguish the different gene type, can also distinguish not homoallelic heterozygous individual of this gene and homozygous individual.
The association analysis of corn kernel number under the insertion/disappearance of embodiment 3,12bp and the low nitrogen environment
One, under the low nitrogen adverse circumstance with normal condition under phenotypic evaluation
1, test place and soil nutrient content analysis
Carrying out crop science institute of Ya Cheng the Chinese Academy of Agricultural Sciences test base (Shui Nan village) in Hainan in the winter in 2007,2008, experimental field is sandy loam, for reaching low nitrogen adverse circumstance, exhausts through the nitrogen of 3 year 6 season of growth and handling.Treatment process is per season maize planting cross-fertilize seed, does not apply fertilizer, and complete stool removes after the corn maturation.
Soil is taken a sample before fertilising and is carried out nutrient content mensuration, gets 5 soil samples by five point samplings of routine, and the MV of getting each measured value is represented the soil nutrient content in this plot.Survey full nitrogen (Bremner with the Kai Shi distillation method; J.M.1996.Nitrogen-total.p.1085-112.In D.L.Sparks (ed) Methods of soil analysis.Part 3.Chemical methods.SSSA Book Ser.5.SSSA and ASA; Madison, WI.); Use HClO
4-H
2SO
4Disappear and boil-molybdenum antimony resistance colorimetric method survey content of tatal phosphorus (Olsen, S.R.and Sommers, L.E. (1982) Phosphorus.In A.L.Page; R.H.Miller; And D.R.Keeney (ed.): Methods of Soil Analysis (part2): Chemical and Microbiological Properties, 2nd end.ASA SSSA, Madision; WI, p.403-440); Survey available phosphorus (Olsen with sodium bicarbonate extraction-molybdenum antimony resistance colorimetric method (Olsen method); S.R.; C.V.Cole, F.S.Watanabe, and L.A.Dean.1954.Estimation of available phosphorus in soils by extraction with sodium bicarbonate.USDA Circ.939.U.S.Gov.Print.Office; Washington, DC.); Heat potassium dichromate oxidation-volumetry with oil bath and survey organic (GB 9834-88); Survey available potassium (Van Reeuwijk, L.P.1992.Procedures for soil analysis, 3rd ed.ISRIC, Wageningen, The Netherlands.) with ammonium acetate lixiviate-atomic method; Survey slowly available potassium (Van Reeuwijk, L.P.1992.Procedures for soil analysis, 3rd ed.ISRIC, Wageningen, The Netherlands.) with nitric acid extraction-atomic absorption spectrophotometry; Survey pH value (GB7859-57) with potential method (soil ratio is 2.5: 1).Nitrogen exhausts the plot and normally table 4 is seen in plot soil nutrient content and variance analysis.Low nitrogen plot (claim again nitrogen exhaust plot, NDP, Nitrogen depleted pool) and normal plot (NP, Applied nitrogen field) denitrogenate nutrient widely different outside, other nutrient remains basically stable.
The low nitrogen plot of table 4 and normal plot nutrient and contrast.
| Full nitrogen (g.kg 1) | Total phosphorus (g.kg -1) | Organic (g.kg -1) | Rapid available phosphorus (mg.kg -1) | Available potassium (mg.kg -1) | Total potassium K (mg.kg -1) | pH | |
| NDP | 0.49±0.04 | 0.52±0.04 | 8.93±0.88 | 30.04±6.44 | 179.13±26.83 | 432.79±70.84 | 6.31±0.09 |
| NP | 0.68±0.03 | 0.56±0.09 | 9.23±0.85 | 36.81±14.56 | 176.69±39.03 | 402.98±34.64 | 6.23±0.19 |
| t-Test | 11.45 | 1.45 | 1.18 | 0.97 | 0.13 | 0.73 | 0.74 |
| Pr＞t | 0 | 0.21 | 0.29 | 0.38 | 0.9 | 0.5 | 0.49 |
Carry out experiment in 2 years continuously: germplasm is in low nitrogen plot and normal plot respectively for 180 corn inbred lines, and each strain is each plot plantation 3 row, the long 3.6m of row, line-spacing 0.6m, spacing in the rows 0.2m, the individual plant kernal number of results back each self-mating system of statistics.
Two, the special primer of application implementation example 2 designs is to carrying out somatotype
Respectively 180 corn inbred lines are carried out gene type, step is following:
1, extracts gene DNA.
2, with the genomic dna be template, to carrying out pcr amplification, obtain pcr amplification product with the special primer of embodiment 2 design.
PCR reaction system (10 μ L) is seen table 5.
Table 5PCR reaction system
| Component | Final concentration | Add volume |
| ddH 2O | - | 6.02μL |
| 10 * Buffer (contains 20mmol/L Mg 2+) | 1× | 1μL |
| dNTP(25mmol/L?each) | Be 250 μ mol/L | 0.1μL |
| Primer (20 μ mol/L each) | 0.26μmol/L?each | 0.13μL |
| Taq enzyme (2U/ μ L) (ancient cooking vessel state company) | 0.5U | 0.25μL |
| DNA(10ng/μL) | 25ng | 2.5μL |
Add a cover 15 μ L MO (Sigma) on the reaction solution, to prevent moisture evaporation in the reaction process.
PCR response procedures (PTC-200PCR appearance; MJ Research, Waterson, MA): 94 ℃, 4 minutes, sex change in advance; 94 ℃ of sex change 45 seconds, 52 ℃ of annealing 30 seconds, 72 ℃ were extended 35 circulations 30 seconds; 72 ℃ are extended 8 fens kinds eventually; 4 ℃ of preservations.
3, pcr amplification product is carried out 4.5% polyacrylamide gel electrophoresis.
In 10 μ LPCR amplified productions, add 5 μ L3 * Loading Buffer, behind the mixing,, put immediately, obtain sample to more than the cooled on ice 10min at 95 ℃ of sex change 5min.Each well is clicked and entered 4 μ L samples (molecular weight standard is pBR322DNA/MspI), the about 40-60min of electrophoresis under the permanent power of 70W.
Electrophoresis carries out silver and dyes colour developing after finishing.
Staining fluid is by 1g AgNO
3Form dyeing 10-15min with 1.5mL 37% (volumn concentration) formalin.
Developing solution is by the 30g anhydrous Na
2CO
3, 200 μ L 1%Na
2S
2O
3The aqueous solution and 1.5mL 37% (volumn concentration) formalin are formed, to being with line to occur.
Stop bath is 10% (volumn concentration) aqueous acetic acid, photographic fixing 2min.
Three, the genotype of each sample and individual plant kernal number
Obtained to supply the group structure data (being the Q data) of examination material according to the method for Pritchard etc. 2000, combined general linear model (GLM) and the cognation between compounded linear model (MLM) Model Calculation proterties and the mark under many environment (e) condition with paired family index (k) data in the group structure of delivering on " Nature Genetics " (Q) of utilizing respectively according to Yu etc. 2005.Use TASSEL2.0 software (Cornell Univ USA's exploitation) to carry out correlation analysis based on candidate gene.The group structure data (Q) of confession examination material and sibship aiding data etc. have been published data (Chuanxiao Xie; Marilyn Warburton; Mingshun Li; Xinhai Li; Muji Xiao, Zhuanfang Hao, Qi Zhao and Shihuang Zhang. (2008) An analysis ofpopulation structure and linkage disequilibrium using multilocus data in 187maize inbred lines.Mol.Breeding 21 (4): 407-418).The genotype data of target gene: (Gln1-3 180lines alignment rusults.phy) (annotate: presents needs software such as PHYLIP to open, and this full length gene sequence data of all material is arranged, and it is interval not only to contain this marker development).The phenotypic evaluation data: individual plant kernal number correlation analysis result sees table 6 under glutamine synthetase gene Gln1-3 gene and the low nitrogen adverse circumstance.
Kernal number correlation analysis result under table 6 glutamine synthetase gene Gln1-3 gene and the low nitrogen adverse circumstance
| The site | The position | Type | Sequence 5 '-3 ' | p?Marker | Unit point is explained phenotype (%) |
| -285 | 5 ' control region | Indels | CCGCGTCGAGGA | 0.003 | 16.85 |
| 2581 | The 9th exon | SNP | C/G | 0.852 | 3.86 |
| 396 | The 1st intron | Indels | CCTCTTGTTT | 0.218 | 3.52 |
| 3265 | 3 ' control region | Indels | GGCCG | 0.060 | 2.53 |
| -111 | 5 ' control region | SNP | C/G | 0.005 | 2.36 |
| 1422 | The 4th intron | SNP | T/C | 0.000 | 2.13 |
| 2934 | The 10th exon | SNP | C/G | 0.404 | 1.17 |
It is strong to be positioned under insertion/disappearance (Indels) site and the low nitrogen adverse circumstance of a 12bp of this gene-285 upstream regulatory region individual plant kernal number cognation, and the explainable phenotypic variation of unit point has very high marker development and utility value up to 16.85% (p＜0.05).
Individual plant kernal number cognation is strong under insertion/disappearance (Indels) site of this 12bp and the low nitrogen adverse circumstance; Wherein the allelotype of disappearance is excellent allelotype (absence type individual plant kernal number is higher than insert type individual plant kernal number); The explainable phenotypic variation of unit point has very high marker development and utility value up to 16.85% (p＜0.05).
The association analysis of corn individual plant kernal number under the insertion/disappearance of embodiment 4,12bp and the low nitrogen environment
127 corn inbred lines: the public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences; Reference: Chuanxiao Xie; Shihuang Zhang, Minshun Li, Xinhai Li; Zhuanfang Hao; Li Bai, Degui Zhang, Yehong Liang. (2007) Inferring genome ancestry and estimatingmolecular relatedness among 187 Chinese maize inbred lines.J.Genet.&Genomics 34 (8): 738-748.
One, under the low nitrogen adverse circumstance with normal condition under phenotypic evaluation
Phenotypic evaluation time and place are respectively: Beijing (sowing in spring in 2007), Gongzhuling (spring sowing of Gongzhuling, Jilin in 2007), Sanya (winter sowing in 2007, winter sowing in 2008).
Facility nitrogenous fertilizer and not 2 processing of nitrogen fertilizer application, three repetitions are established in each processing.The long 3.6m of cell row, line-spacing 0.6m, spacing in the rows 0.2m.Nitrogen fertilizer application is handled: execute 60kg/hm during sowing
2P
2O
5And 45kg/hm
2K
2O is as base fertilizer, and 3 leaf phases were executed 69kg/hm
2Purity nitrogen, the typhon mouth phase imposes 172.5kg/hm
2Purity nitrogen; Nitrogen fertilizer application is not handled: execute 60kg/hm during sowing
2P
2O
5And 45kg/hm
2K
2O is as base fertilizer, and the whole experimental phase is nitrogen fertilizer application not.Ripe back is that unit gathers in the crops with the sub-district, and each 2 strain of wardrobe end of line are gone in every sub-district, and to avoid the influence of edge effect, continuous 10 strains in the middle of the picking bring bigger above and below ground space advantage to avoid the place of being short of seedling to leave a blank to adjacent plant.The air-dry indoor conventional species test in back, the main individual plant kernal number mean number that adopts in this analysis.The result sees table 7.Investigate individual plant kernal number under low nitrogen (LN) and normal nitrogen (HN) situation, calculate the coefficient and the index of average individual plant kernal number under low nitrogen and the normal nitrogen situation.
Coefficient=LN individual plant kernal number/HN individual plant kernal number * 100%;
Index=(HN individual plant kernal number-LN individual plant kernal number)/HN individual plant kernal number * 100%.
The result sees table 7.
Two, the special primer of application implementation example 2 designs is to carrying out somatotype
Respectively 127 corn inbred lines are carried out gene type, step is with the step 2 of embodiment 3.
The band of a 329bp of electrophoresis showed for inserting homozygous sample, show a 317bp band be the homozygous sample of disappearance.
The somatotype result sees table 7.
Table 7 donor and the individual plant kernal number phenotype of recommending acceptor target label genotype and 6 environment in 2 years 3 places
Have the missing gene type corn inbred line CA156, close 344 or neat 205 types individual plant kernal number difference under normal nitrogen situation under the low nitrogen little, coefficient is high, index is low, can be used as donor parents during breeding.124 genotypic corn inbred lines of insertion individual plant kernal number difference under normal nitrogen situation under the low nitrogen is big, and coefficient is low, and index is high, can be used as receptor parent during breeding.Breeding method can have following several kinds: 1) backcross improvement: through acceptor self-mating system in the method improvement table of backcrossing 7; With donor self-mating system in the table 6 is recurrent parent; Utilize special primer of the present invention right in the process of hybridizing and backcrossing, high corn kernel is counted the excellent allelotrope of glutamine synthetase Gln1-3 gene under the low nitrogen adverse circumstance of selection; 2) pedigree method choosing system: through the method for hybridization, select self-mating system CA156, close 344 or neat 205 with table 6 in donor hybridization between selfed lines or three hand over the back to select to contain the excellent allelotrope of glutamine synthetase Gln1-3 gene to carry out pedigree method choosing system; 3) improvement of other unknown material: through the method for hybridizing or backcrossing; With corn inbred line CA156, close 344 or neat 205 for excellent allelotrope donor; In hybridization or process such as backcross; Select in other unknown material of improvement, screening contains under the low nitrogen adverse circumstance high corn kernel counts excellent allelotrope breeding material of glutamine synthetase Gln1-3 gene or intermediate materials.
Embodiment 5, special primer are to the application in breeding
CA156 is a donor with disappearance (D) excellent genes type, and inserting (I) genotype military 314 is acceptor, obtains F
1Generation; With F
1For selfing, obtain 32 strain F
2For colony.To F
2For colony wherein 32 strain individualities extract genomic dna respectively, to carrying out PCR, through the electrophoresis detection pcr amplification product, method is with the step 2 of embodiment 3 with the special primer of embodiment 2 design.
The result sees Fig. 2.Obtain 6 strain homozygous deletion (Deletion) genotype individual (D), codominance (Co-dominant) the genotype individuality (C) of 12 strain insertions/disappearance heterozygosis, 14 strains insertion (Insertion) the genotype individuality (I) that isozygotys.Wherein, above-mentioned 6 strain homozygous deletion individualities can be used as the good homozygous individual of this genotype and directly get into Breeding Application, and the individual offspring of the codominance of 12 strain insertion/disappearance heterozygosis can use present method detection homozygous deletion individuality Breeding Application in addition again in the offspring.
F2 is planted the low nitrogen plot in embodiment 3 for colony, the average individual plant kernal number of results back each genotype colony of statistics.Genotype is that the average individual plant kernal number of the colony of I is 125.2, and genotype is that the average individual plant kernal number of the colony of D is 324.5, and genotype is that the average individual plant kernal number of the colony of C is 308.4.
Claims (9)
1. auxiliary differentiating that the primer that under low nitrogen environment, has different individual plant seed number proterties corns is right, is that the primer of DNA composition shown in the sequence 3 of DNA shown in the sequence 2 of sequence table and sequence table is right.
2. the said primer of claim 1 is to having the application in the different individual plant seed number proterties corns auxiliary the discriminating under low nitrogen environment.
3. the auxiliary method that under low nitrogen environment, has different individual plant seed number proterties corns of differentiating comprises the steps: that the genomic dna with corn to be measured is a template, with the said primer of claim 1 to carrying out pcr amplification; If pcr amplification product is a kind of, and size is 329bp, and corn to be measured is homozygous for inserting; If pcr amplification product is a kind of, and size is 317bp, and corn to be measured is homozygous for disappearance; Lack homozygous corn and hanging down the individual plant seed number under the nitrogen environment in inserting homozygous corn in the individual plant kernal number height of eye under the low nitrogen environment.
4. a corn breeding method comprises the steps: donor parents and receptor parent hybridization are obtained F1 generation, and F1 for selfing, is obtained F2 generation; Is template with F2 for the genomic dna of corn, with the said primer of claim 1 to carrying out pcr amplification; Pcr amplification product is that a kind of and big or small F2 for 317bp is the corn that seed selection obtains for corn; The disappearance homozygous corn of said donor parents for adopting the said method of claim 3 to identify, the insertion homozygous corn of said receptor parent for adopting the said method of claim 3 to identify.
5. method as claimed in claim 4 is characterized in that: said donor parents is corn variety CA156, and said receptor parent is a corn variety military 314.
6. the said primer of claim 1 is to the application in the test kit that under low nitrogen environment, has different individual plant seed number proterties corns in the auxiliary discriminating of preparation.
7. the DNA shown in the sequence 4 of sequence table.
8. the said DNA of claim 7 has the application in the different individual plant seed number proterties corns auxiliary the discriminating under low nitrogen environment.
9. the application of the said DNA of claim 7 in corn breeding.
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| CN110189793B (en) * | 2019-06-04 | 2022-10-25 | 河南农业大学 | Hyperspectrum-based wheat nitrogen fertilizer physiological utilization rate estimation model construction and wheat variety classification with different nitrogen efficiencies |
| CN115820672A (en) * | 2022-12-26 | 2023-03-21 | 中国农业大学 | Corn nitrogen utilization related gene ZmNCRG2 and application thereof |
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Non-Patent Citations (3)
| Title |
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| Ming Lu等.Mapping of quantitative trait loci for kernel row number in maize.《Molecular Breeding》.2010,第28卷(第2期),143-152. * |
| 吴永升.玉米谷氨酰胺合成酶基因Gln1-3、Gln1-4氮利用效率关联性分析.《中国博士学位论文全文数据库农业科技辑》.2009,第2009年卷(第10期),D047-31. * |
| 吴永升等.玉米Gln1-3 gDNA序列分离、基因结构、保守功能域与等位变异分析.《作物学报》.2008,第34卷(第7期),1114-1120. * |
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