CN109295248A - For detecting primer, kit, detection method and the application of the molecular labeling of control corn stem intensity main effect QTL linkage - Google Patents
For detecting primer, kit, detection method and the application of the molecular labeling of control corn stem intensity main effect QTL linkage Download PDFInfo
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Abstract
The present invention relates to primer, kit, detection method and the applications of the molecular labeling for detecting control corn stem intensity main effect QTL linkage, belong to maize genetic breeding and technical field of molecular biology.The present invention is by including the recombinant inbred lines of 241 familys to 58 × D863F of Zheng building and carrying out genetic linkage maps analysis, detect the QTL of 4 control corn stem intensity, wherein identify straw stiffness main effect QTL qRPR1.07 one high jointly on the 1st position chromosome bin1.07 of Hainan Le Dong and Henan Yuanyang Demonstration Base corn, between 2 SSR markers bnlg1556 and umc2232, nearly 20% hereditary variation can be explained.The molecular labeling of above-mentioned control corn stem intensity main effect QTL linkage can grow the corn material for orienting, accurately filtering out high straw stiffness early period, save breeding cost and time, be used for corn breeding resistant to lodging.
Description
Technical field
The present invention relates to for detecting control corn stem intensity main effect QTL linkage the primer of molecular labeling, kit,
Detection method and application belong to maize genetic breeding and technical field of molecular biology.
Background technique
Corn is one of most important cereal crops in China, in recent years along with the raising of maize production large-scale degree,
The requirement that corn variety adapts to entire mechanization is also more more and more urgent.Currently, it is the development of corn entire mechanization that machine receipts level is low
One " bottleneck " encountered in the process, and corn stem lodging be restrict China's corn harvest mechanization development principal element it
One；Being increasing with global extreme climate occurrence frequency simultaneously, the stable yields lodging resistance of corn variety require also higher and higher.
Research shows that the whole world is every year because up to 5%~20%, lodging not only seriously affects corn and produces for corn yield loss caused by lodging
It measures and restricts the raising of corn harvest mechanization technology.
Corn lodging is a complicated process, according to the difference of corn lodging feature be classified as root fall, stem fall and stem
Roll over three types；Research shows that stalk puncture strength has the correlation of height with field lodging rate, and exist significantly with lodging
Negative correlation.(the lodging genetic research crop of the corn stem puncture-resistant intensity such as Feng Guang, Liu Zhifang, Li Yanyan such as Feng Guang
Journal, 2009,35 (11): 2133-2138.), (Feng Guang, Jing Xiqiang, Li Yanyan wait the phase of corn stem character with lodging
It closes with path analysis North China Agricultural Journal, 2010a, 25:72-74.) by carrying out stalk puncture to corn overground part third stipes
Strength detection analyzes its inheritance, and the heredity of discovery corn stem puncture strength meets key-gene and add gene mould
Type is a quantitative character by controlled by multiple genes.(Hu HX, Meng YJ, Wang HW, the et al.Identifying such as Hu
quantitative trait loci and determining closely related stalk traits for rind
penetrometer resistance in a high-oil maize population.Theoretical and
Applied Genetics, 2012,124:1439-1447.) recombinant inbred lines established using B73 × Ce03005 are total
Detect 9 QTLs relevant to stalk puncture strength, single-action answers the contribution rate of QTL between 1.15%~12.43%.Meng
Deng (Meng Y, Li J, Liu J, et al.Ploidy effect and genetic architecture
exploration of stalk traits using DH and its corresponding haploid
2016,16 (1): populations in maize.Bmc Plant Biology 50.) utilizes Zheng58 × Chang7-2
The group of DH system of initiative and a monoploid group, detect 2 and stalk puncture strength phase on the 1st and the 5th chromosome
The QTL of pass.In terms of corn stem intensity QTL positioning, the result of different researcher's positioning is not the same.
Therefore, breeding and to plant high straw stiffness kind be to reduce corn lodging and raising machine produces effects the effective way of rate.
Researcher carried out QTL positioning to corn stem intensity using different genetic group in recent years, detected under various circumstances
The QTL arrived is not the same, and positioning section is larger, limits its utilization efficiency in breeding.
Summary of the invention
To solve the above-mentioned problems, the purpose of the present invention is to provide one kind for detecting control corn stem intensity main effect
The primer of QTL chain molecular labeling, the primer can detecte the straw stiffness of corn strain.
The present invention also provides a kind of for detecting the reagent of the molecular labeling of control corn stem intensity main effect QTL linkage
Box, the kit can detecte the straw stiffness of corn strain, and then judge that corn material to be measured is high straw stiffness corn plants
System or low straw stiffness corn strain.
The present invention also provides a kind of method for detecting corn strain straw stiffness, this method may determine that corn to be measured
Material is high straw stiffness corn strain or low straw stiffness corn strain, and then does standard for the breeding resistant to lodging of next step corn
It is standby.
Finally, the present invention provide a kind of above-mentioned primer, kit or detection method screening with identify that the high stalk of corn is strong
The application in terms of application and breeding in terms of degree germ plasm resource.
To achieve the goals above, the technical scheme adopted by the invention is that:
It is a kind of for detecting the primer of the molecular labeling of control corn stem intensity main effect QTL linkage, primer sequence is as follows:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
It can detecte the molecule mark of control corn stem intensity main effect QTL compact linkage by the molecular labeling primer
Note, the molecular labeling can improve the efficiency of breeding resistant to lodging in its straw stiffness of corn growth early prediction.
It is a kind of for detecting the kit of the molecular labeling of control corn stem intensity main effect QTL linkage, including following draw
Object:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
The primer that the kit is included can detecte the molecule mark of control corn stem intensity main effect QTL compact linkage
Note, the molecular labeling can improve the efficiency of breeding resistant to lodging in its straw stiffness of corn growth early prediction.
The above-mentioned kit for detecting the molecular labeling of control corn stem intensity main effect QTL linkage further includes Taq
Master Mix、buffer、dNTP、ddH2O.It can detect corn stem intensity, the germplasm money of screening high-quality using the kit
Source.
A method of for detecting corn strain straw stiffness, comprising the following steps: with corn gene group DNA to be measured
To expand template, PCR amplification is carried out with primer, PCR product obtains polymorphic molecular marker band after detecting, if PCR is produced
Object band is 189bp, then can determine whether that, containing high straw stiffness gene in corn material to be measured, phenotype is high straw stiffness corn
Strain；The sequence of the primer is as follows:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
The identification method is easy to operate, as a result accurately, can provide support for corn molecular mark resistant to lodging.
The reaction system of above-mentioned PCR amplification includes: 0.1 μ L of 2.5U/ μ L Taq enzyme, 10 × buffer, 1.6 μ L, DNA mould
2.0 μ L of plate, 10 μm of ol/L primers Fs 1, each 0.2 μ L of R1,10mmol/L dNTP 0.15 μ L and ddH2O 5.75μL.The PCR expands
Each reagent dosage of increasing system is moderate, the good connecting effect of enzyme under the reaction system.
The response procedures of above-mentioned PCR amplification include: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 50s, 60 DEG C of annealing 50s, 72 DEG C
Extend 50s, 35 circulations；Last 72 DEG C of extensions 5min, 4 DEG C of preservations.The product amplified under the response procedures without specificity more
It is good, no miscellaneous peak interference.
Primer for detecting the molecular labeling of control corn stem intensity main effect QTL linkage is high in screening and identification corn
Application in terms of straw stiffness germ plasm resource and/or the application in terms of breeding.It can screening rapidly and efficiently using the primer
With the germ plasm resource of the identification high straw stiffness of corn.
Kit for detecting the molecular labeling of control corn stem intensity main effect QTL linkage is screening and identification corn
Application in terms of high straw stiffness germ plasm resource and/or the application in terms of breeding.It can rapidly and efficiently using the kit
The germ plasm resource of screening and the identification high straw stiffness of corn, shortens breeding time.
For detecting the method for corn strain straw stiffness in terms of screening with the identification high straw stiffness germ plasm resource of corn
Application and/or the application in terms of breeding.It can be with the straw stiffness of early prediction corn material, rapidly and efficiently using this method
Screening with identification the high straw stiffness of corn germ plasm resource, shorten breeding time.
Detailed description of the invention
Fig. 1 is the stalk slice map of Zheng 58 in test example of the present invention；
Fig. 2 is the stalk slice map of D863F in test example of the present invention；
Fig. 3 is RIL population genetic linkage map in test example of the present invention；
Fig. 4 is using molecular labeling bnlg1556 in test example of the present invention in 58, D863F and 18 F of Zheng2Expansion in family
Increase banding pattern electrophoretogram；
Wherein, 1~18 expression F in Fig. 4218 single plants of family.
Specific embodiment
Specific embodiments of the present invention will be further explained with reference to the accompanying drawing.
Corn material Zheng 58 of the invention is from Henan Academy of Agricultural Sciences, and D863F is from the Chinese Academy of Agricultural Sciences.
For detecting the embodiment 1 of the primer of the molecular labeling of control corn stem intensity main effect QTL linkage
The primer of the molecular labeling of the detection control corn stem intensity main effect QTL linkage of the present embodiment, primer sequence is such as
Under:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
For detecting the embodiment 1 of the kit of the molecular labeling of control corn stem intensity main effect QTL linkage
The kit of the molecular labeling for detecting control corn stem intensity main effect QTL linkage of the present embodiment, including
Primer, Taq Master Mix, buffer, dNTP, ddH2The sequence of O, the primer are as follows:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
For detecting the embodiment 1 of the method for corn strain straw stiffness
The method that the present embodiment is used to detect corn strain straw stiffness, comprising the following steps:
(1) extraction of corn gene group DNA
In Maize at Seedling Stage, young green corn blade is chosen, liquid nitrogen grinding extracts DNA using CTAB method, and DNA sample is -20
It is saved backup at DEG C.
(2) PCR amplification
The corn gene group DNA extracted using step (1) carries out PCR as template, using the primer of molecular labeling bnlg1556
Amplification, the sequence of the primer are as follows:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
PCR reaction system is 10 μ L, comprising: 0.1 μ L of 2.5U/ μ L Taq enzyme, 10 × buffer, 1.6 μ L, DNA template
2.0 μ L, 10 μm of ol/L primers Fs 1, each 0.2 μ L of R1,10mmol/L dNTP 0.15 μ L and ddH2O 5.75μL。
Pcr amplification reaction program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 50s, 60 DEG C of annealing 50s, 72 DEG C of extension 50s,
35 circulations；Last 72 DEG C of extensions 5min, 4 DEG C of preservations.
(3) data are analyzed
Amplified production carries out capillary fluorescence electrophoresis using ABI 3500XL Genetic Analyser.Using 384 hole electrophoresis plates, often
Hole is separately added into 7 μ L formamides, 0.04 μ L liz500 molecular weight internal standard (Applied Biosystems, USA) and 2 μ l PCR
Product after 95 DEG C of denaturation 3min, carries out capillary fluorescence electrophoresis using ABI 3500XL Genetic Analyser, in Genographer
Analytical electrophoresis strip data on 2.1 softwares.
(4) electrophoresis result determines
The method of result is determined according to electrophoretic band are as follows: if PCR product band is 189bp, can determine whether corn to be measured
Contain high straw stiffness gene in material, phenotype is high straw stiffness corn strain.
The embodiment 1 of the application of the primer of the molecular labeling of detection control corn stem intensity main effect QTL linkage
The application of the primer of the molecular labeling of the control corn stem intensity main effect QTL linkage of the present embodiment is specially to sieve
Application in terms of choosing and the identification high straw stiffness germ plasm resource of corn and/or the application in terms of breeding, with corn gene to be measured
Group DNA is template, controls this molecule mark of the molecular labeling primer sequence of corn stem intensity main effect QTL linkage using of the invention
Remember that primer carries out PCR amplification, which can indicate straw stiffness gene in corn strain, and then can be in growth early period
Orientation, the corn material for accurately filtering out high straw stiffness.
The embodiment 1 of the application of the kit of the molecular labeling of detection control corn stem intensity main effect QTL linkage
The application of the kit of the molecular labeling of the control corn stem intensity main effect QTL linkage of the present embodiment specially exists
Application in terms of screening and the identification high straw stiffness germ plasm resource of corn and/or the application in terms of breeding.With corn-based to be measured
Because group DNA is template, using molecular labeling provided by the invention as primer, PCR amplification is carried out using kit provided by the invention,
Amplified production is detected through capillary fluorescence electrophoresis, straw stiffness gene in corn plants system can be indicated, judge that corn stem to be measured is strong
The size of degree is prepared for later period genetic breeding.
Detect the embodiment 1 of the application of the method for corn strain straw stiffness
The present embodiment carries out PCR amplification band by primer and filters out using the method for detection corn strain straw stiffness
Breeding is carried out with the corn strain of the high straw stiffness of the consistent genotype of parent D863F, cultivates high straw stiffness resistant to lodging
Corn strain, this method application can save breeding cost and time.
Test example
1, test material and field trial
Straw stiffness identification is carried out to 118 parts of corn inbred lines respectively within 2013 and 2014, as shown in table 1 below, in table 1
The straw stiffness value of each corn material is that the average value in 2 years is divided into 118 parts of materials according to corn stem average strength
High straw stiffness, middle straw stiffness and low straw stiffness three types.From table 1 it can be seen that self-mating system straw stiffness range exists
33.84~72.72N/mm2Between, filter out that comprehensive agronomy character is excellent from self-mating system, the low stem of straw stiffness significant difference
Stalk intensity corn material Zheng 58 and high straw stiffness corn material D863F hybridize as parent, and wherein Zheng 58 is from river
Southern Shanxi Academy of Agricultural Sciences, D863F is from the Chinese Academy of Agricultural Sciences.
Field trial carries out in Henan Province Yuanyang County academy of agricultural sciences of Henan Province agricultural science and technology experiment and demonstration base.2013 and
Random district's groups sowing is respectively adopted within 2014.Every part of expert evidence plantation row long 5m, line-spacing 0.6m, spacing in the rows 0.25m, 2 repetitions,
Normal field management.
1 118 parts of corn stem intensity tables of table
2, the building of inbreeding population
It is using Zheng 58 and D863F as the F of parents that corn, which recombinates inbreeding population RIL genetic map,1, utilize single seed heredity
Method is continuously selfed 6 generations progress informative population and is obtained.
3, genetic map construction and qtl analysis
The measurement for carrying out straw stiffness to Zheng parent 58 and D863F by using straw stiffness analyzer, by cross-sectional area
For 1.0mm2Gauge head be vertically pierced into the middle part of stalk overground part third internode, read and record test data.The results show that two
There are significant difference, the straw stiffness of D863F is 57.77N/mm to the straw stiffness of parent within 15 days after taking out hero2, Zheng 58 stalk
Intensity is 39.73N/mm2, the straw stiffness of D863F increases 18.04N/mm than Zheng 582.It is shown by micro- sem observation, phase
Than Zheng Yu 58, the stalk sclerderm tissue of D863F is thick, and vascular bundle number is more in unit area, and bundle sheath thickness is big, and lignifying is thin
Parietal cell mostly increases with bundle sheath thickness, and sclerderm tissue thickens (as shown in Figure 1 and Figure 2).
Include the recombinant inbred lines of 241 familys based on 58 × D863F of Zheng building, to recombinant inbred lines and its
Zheng parent 58 and D863F carry out parent's polymorphism mark screening, obtain 232 polymorphism SSR markers.It is soft using Jionmap4
Part, under the scene of LOD=2.5,1 genetic linkage maps comprising 215 SSR marker sites of building are chain comprising 10
Group, indicia distribution are relatively uniform.
Winter in 2016 and summer in 2017 plant RIL group, genotype combination phenotype in Hainan Le Dong and Henan Yuanyang respectively
Identification detects the QTL of 4 control corn stem intensity, the 1st chromosome of Le Dong and Henan Yuanyang two places corn in Hainan altogether
Straw stiffness main effect QTL qRPR1.07 one high is identified on the position bin1.07 jointly, is located at 2 SSR markers
Between bnlg1556 and umc2232, nearly 20% hereditary variation (as shown in Figure 3) can be explained.
The F derived from Zheng 58 and D863F6In RIL group, the strain RIL29 with 58 similar genetic background of Zheng is chosen, it should
Strain has imported the main effect QTL qRPR1.07 segment that corn stem intensity is controlled on the 1st article of chromosome of D863F, will
RIL29 first hybridizes to obtain F with Zheng 581Generation, F1In generation, is selfed the F for obtaining 186 single plants again2Segregating population.
4, assessment of the linked marker to filial generation allelic
From F218 single plants are randomly selected in 186 single plants of segregating population, using molecular labeling bnlg1556 to random
18 single plants chosen carry out PCR amplification, electrophoretic analysis its genotype, as a result as shown in figure 4, from fig. 4, it can be seen that 2,7,9,
11,12,14, No. 15 strain genotype are consistent with D863F, 1,3,4,6,8,13,16, No. 18 strain genotype and Zheng No. 58 one
It causes.By using straw stiffness analyzer to F2The measurement of straw stiffness is carried out for 186 strains in segregating population, as a result table
It is consistent with D863F that bright molecular labeling bnlg1556 filters out 42 pnca gene types in 186 single plants, straw stiffness average value
Reach 60.82N/mm2；45 pnca gene types and the consistent single plant of Zheng 58, straw stiffness average value reach 45.82N/mm2.Therefore
Molecular labeling bnlg1556 can grow the corn material for orienting, accurately filtering out high straw stiffness early period, save breeding
Cost and time are used for corn breeding resistant to lodging.
Sequence table
<110>Henan Academy of Agricultural Sciences's Crop Design center
<120>for detecting primer, the kit, detection method of the molecular labeling of control corn stem intensity main effect QTL linkage
And application
<160> 2
<170> SIPO Sequence Listing 1.0
<211> 20
<212> DNA
<213>upstream primer sequence F1
<400> 1
accgacctaa gctatgggct 20
<211> 20
<212> DNA
<213>downstream primer sequence R1
<400> 2
ccggttataa acacagccgt 20
Claims (9)
1. a kind of for detecting the primer of the molecular labeling of control corn stem intensity main effect QTL linkage, it is characterised in that: primer
Sequence is as follows:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
2. a kind of for detecting the kit of the molecular labeling of control corn stem intensity main effect QTL linkage, it is characterised in that: packet
Include following primer:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
3. according to claim 2 for detecting the reagent of the molecular labeling of control corn stem intensity main effect QTL linkage
Box, it is characterised in that: further include Taq Master Mix, buffer, dNTP, ddH2O。
4. a kind of method for detecting corn strain straw stiffness, it is characterised in that: the following steps are included: with corn-based to be measured
Because group DNA is amplification template, PCR amplification is carried out with primer, PCR product obtains polymorphic molecular marker band after detecting, such as
Fruit PCR product band is 189bp, then can determine whether that, containing high straw stiffness gene in corn material to be measured, phenotype is high stalk
Intensity corn strain；The sequence of the primer is as follows:
F1:5 '-ACCGACCTAAGCTATGGGCT-3 ' (SEQ ID NO.1)；
R1:5 '-CCGGTTATAAACACAGCCGT-3 ' (SEQ ID NO.2).
5. the method according to claim 4 for detecting corn strain straw stiffness, it is characterised in that: the PCR expands
The reaction system of increasing includes: that 0.1 μ L of 2.5U/ μ L Taq enzyme, 10 × buffer, 1.6 μ L, 2.0 μ L of DNA profiling, 10 μm of ol/L draw
Each 0.2 μ L of object F1, R1,10mmol/L dNTP 0.15 μ L and ddH2O5.75μL。
6. the method according to claim 5 for detecting corn strain straw stiffness, it is characterised in that: the PCR expands
The response procedures of increasing include: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 50s, 60 DEG C of annealing 50s, 72 DEG C of extension 50s, 35 recycle；
Last 72 DEG C of extensions 5min, 4 DEG C of preservations.
7. the primer as described in claim 1 for detecting the molecular labeling of control corn stem intensity main effect QTL linkage exists
Application in terms of screening and the identification high straw stiffness germ plasm resource of corn and/or the application in terms of breeding.
8. as claimed in claim 2 or claim 3 for detecting the reagent of the molecular labeling of control corn stem intensity main effect QTL linkage
Application of the box in terms of screening with the identification high straw stiffness germ plasm resource of corn and/or the application in terms of breeding.
9. the method for detecting corn strain straw stiffness as described in any one of claim 4~6 is in screening and identification
Application in terms of the high straw stiffness germ plasm resource of corn and/or the application in terms of breeding.
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Cited By (5)
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| CN110904266A (en) * | 2019-12-26 | 2020-03-24 | 北京市农林科学院 | Identification of maize stalk lodging resistance QTL and development and application of molecular marker |
| CN112795691A (en) * | 2021-03-24 | 2021-05-14 | 湖南农业大学 | Molecular marker linked with corn stem thickness and application thereof |
| CN113273460A (en) * | 2021-06-17 | 2021-08-20 | 宁波市农业科学研究院 | Lodging-resistant conventional japonica rice breeding method |
| 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 |
| CN115927704A (en) * | 2022-07-27 | 2023-04-07 | 湖南农业大学 | Molecular marker closely linked with length of corn stalk and application thereof |
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| 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 |
| CN112795691A (en) * | 2021-03-24 | 2021-05-14 | 湖南农业大学 | Molecular marker linked with corn stem thickness and application thereof |
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| CN113273460A (en) * | 2021-06-17 | 2021-08-20 | 宁波市农业科学研究院 | Lodging-resistant conventional japonica rice breeding method |
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