CN107254535A - The SNP marker related to salt tolerance of corn and its application - Google Patents
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Abstract
The invention provides the SNP marker related to salt tolerance of corn shape and its application, belong to crop molecular mark technical field.The present invention is using 240 DH systems as material, highdensity genetic linkage mapses are built using SNP marker, with reference to plant height phenotypic data, field corn maturity period salt tolerant QTL is positioned, it was found that related with salt tolerance of corn to the molecular labeling of its close linkage, the SNP marker is PZE101094436, and primer amplification of the mark as nucleotide sequence as shown in SEQ ID NO.1 3 is obtained.The molecular labeling can be used for the early prediction of salt tolerance of corn shape, screening, and for carrying out marker assisted selection, to screen Salinity tolerance germplasm resource.
Description
Technical field
The present invention relates to crop molecular mark technical field, more particularly to corn maturity period salt tolerant main effect
QTL and the SNP marker related with salt tolerance of corn shape to its close linkage and its application.
Background technology
Corn is important grain, feed and the economical crops in the whole world, is also China's cultivated area most wide, total output highest
The first big crop.Maize Production and the sustainable stable development of Grain in China are closely related.However, corn is salt density value plant,
Its endurable soil salt concentration range is 0.3-0.7%.Soil salt damage can cause that corn bud ratio is low, seedling is weak, it is withered even
Death, ultimately results in the underproduction and quality decline.There are 9.5438 hundred million hectares of salt-soda soils in the whole world, accounts for the total land area more than 7% in the world
And arable area more than 20%.There are 9913.3 ten thousand hectares of salt-soda soils in China, and the 3rd is accounted in world's saline alkali land area ranking list
Position (Zhang Jianfeng etc., 2008, research soil and water conservation 15:74-78).With the drastically reduction of arable area, development and utilization salt
Evil soil is very urgent and necessary.At present, the utilization of Salinity tolerance germplasm resource is the important means for solving this problem.
The seed selection for carrying out haloduric corn germplasm resource using traditional breeding method means has that selection rate is low, the cycle.
Molecular mark is the effective means for solving this problem.The premise that salt tolerance of corn is improved using molecular labeling is to obtain
Obtain the practical molecular labeling with salt tolerance close linkage.Therefore, the positioning of field corn maturity period salt tolerant QTL and molecular labeling are carried out
Developmental research, the seed selection to accelerating haloduric corn germplasm resource, promotes China's Maize Production sustainable development significant.
QTL positioning is a kind of statistical method for the hereditary basis for being accurately used for amount detection character such as salt tolerance.Forefathers
Position Research is carried out to the salt tolerant QTL of multiple kinds of crops, educated to identify that salt tolerant controlling elements are applied to molecular labeling auxiliary
Kind.Forefathers are to soybean (Guan etc., 2014, The Crop Journal 2:358-365), barley (Ahmadi-Ochtapeh
Deng 2015, Biologia Plantarum 59:283-290) etc. the salt tolerant QTL correlations of crop carry out positioning analysis, moreover,
Under the guidance of QTL positioning results, further successful identification to paddy rice (Ren etc., 2005, Nature Genetics 37:1141-
And wheat (Munns etc., 2012, Nature Biotechnology 30 1146):360-364) resistant gene of salt and it is applied to crop
In genetic improvement.
Other crops are compared, the heredity of corn salt tolerant and the research of molecular mechanism report are relatively fewer, are built using SNP marker
Highdensity genetic linkage mapses, are positioned and molecule mark with reference to plant height phenotypic data to field corn maturity period salt tolerant QTL
Note exploitation is extremely necessary, and effective salt tolerant linked marker can be provided for molecular mark.
The content of the invention
First purpose of the present invention is to provide the main effect QTL related to salt tolerance of corn shape.
Second object of the present invention is to provide the SNP marker with corn salt tolerant main effect QTL compact linkage, and amplification
The specific primer pair of the molecular labeling.
Third object of the present invention is to provide the application of above-mentioned SNP marker.
The purpose of the present invention is achieved through the following technical solutions：
Based on object above, applicant builds highdensity genetic linkage using 240 DH systems as material using SNP marker
Collection of illustrative plates, with reference to plant height phenotypic data, is positioned to field corn maturity period salt tolerant QTL.Specially 240 parts (female with first jade 335
This：PH6WC, salt tolerance；Male parent：PH4CV, salt density value) based on material through induction double obtain DH systems with and its Parent
PH6WC and PH4CV is in Tongzhou District, Beijing City proving ground (salt damage area) and Changping District proving ground (normal soil) continuous 3 year spring
Sowing planting.Annual experiment uses RANDOMIZED BLOCK DESIGN, carries out independence twice and repeats.To corn material physiological maturity, with strain
From the flower bran of day fringe top first, amount, to ground level, is calculated as maturity period plant height to height measuring instrument downwards.
Using selected hundreds of parts representative corn varieties, repeated according to site, signal intensity, miss rate, polymorphism,
The principles such as even distribution are screened to 56110 SNP sites in Illumina companies of U.S. MaizeSNP50K chips, assessed,
Finally determine that 3072 core SNP sites are customized to SNP chip product maizeSNP3072 (Illumina companies of the U.S.) (Tian
Deng 2015, Molecular Breeding 35:136).The blade of 240 DH systems is extracted with the DNA of plants of TianGen companies
Kit extracts DNA, carries out DNA hybridization and chip scanning according to the standard test step of ABI companies afterwards, uses
The Kosambi functional modules of JoinMap4 softwares build high density genetic linkage mapses.
On the basis of high density genetic linkage mapses structure, with reference to salt damage area plant height phenotypic data, using Windows
QTL Cartographer softwares, using composite interval mapping method to field corn maturity period salt tolerant main effect QTL carry out positioning and
Develop the molecular labeling of close linkage.A main effect QTL is obtained, molecular labeling and salt tolerance of corn with its close linkage is found
It is related.The main effect QTL is named as qSPH1, and on No. 1 chromosome of corn, LOD is 22.4.
Further, the invention provides the SNP marker related to salt tolerance of corn shape, it is
PZE101094436, the SNP polymorphism are G/A, are expanded as primer pair PCR of the nucleotide sequence as shown in SEQ ID NO.1-3
Increase and obtain.
The invention provides application of the above-mentioned molecular labeling in crop marker assisted selection.
The invention provides application of the above-mentioned molecular labeling in the high crop of seed selection salt resistance ability.
The invention provides application of the above-mentioned molecular labeling in screening haloduric corn kind.
The invention provides application of the above-mentioned molecular labeling in prediction corn salt resistance ability.
The invention provides the specific primer pair for detecting the SNP marker related to salt tolerance of corn, by 3
Primer is constituted, and its nucleotide sequence is respectively such as SEQ ID NO.1-3.
The invention provides above-mentioned specific primer to being improved in corn germ plasm resource in application.
The present invention provides a kind of method for identifying high salt resistance ability corn, comprises the following steps：
(1) genomic DNA of corn to be measured is extracted；
(2) DNA extracted using in step (1) is template, using the specific primer shown in SEQ ID NO.1-3 to carrying out
Pcr amplification reaction；
(3) when using primer shown in SEQ ID NO.2-3, if amplified production 19bp base is G, corn to be measured
Salt resistance ability is high, or when using SEQ ID NO.1, primer shown in 3 when amplified production 20bp base for A, then corn to be measured
Salt resistance ability is low.
The beneficial effects of the present invention are：First public corn salt tolerant main effect QTL (as shown in Figure 1) and with its close linkage
SNP marker, the molecular labeling can be used for the early prediction of salt tolerance of corn shape, screening, and auxiliary for carrying out molecule
Breeding is helped, to screen Salinity tolerance germplasm resource.
Brief description of the drawings
Fig. 1 using different year and the obtained main effect QTL of the plant height data of three annual means positioning its in No. 1 chromosome
On position and LOD value.SPH (2014/2015/2016) in figure:(2014/2015/2016) year salt damage area plant height；SPH
mean：The annual mean of salt damage area plant height three.
Fig. 2 PZE101094436SNP mark the SNP site genotyping result to 13 corn samples.According to
The standard test step of Laboratory of the Government Chemist (LGC) company carries out KASP methods detection SNP
Gene loci, data are with Kraken software analysis and export.The upper left corner：The base of SNP site is G, the lower right corner：SNP site
Base is A, the lower left corner：Negative control.Each sample is repeated twice.
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit and essence of the invention, the modifications or substitutions made to the inventive method, step or condition belong to the present invention
Scope.
The corn germ plasm resource such as 240 Maize DH Lines used and its Parent material is all from north in the embodiment of the present invention
Capital city agricultural and forest science institute corn research center corn germ plasm resource storehouse.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
The correlation main effect QTL positioning of the corn salt tolerant of embodiment 1
1st, soil constituent is analyzed
According to 5 point samplings (2016, the Advance Journal of Food such as Zhai Science＆Technology
11:88-94), the soil of 5 representative locations in Tongzhou and Changping ground is taken to carry out constituent analysis.Determined using residue oven drying method
Total salt concentration (W ü st etc., 2000, Journal of Archaeological Science 27:1161-1172).Using Hach
The pH meter of company determines soil pH value.Using the flame emission spectrometer of PerkinElmer companies to Na in soil+Concentration is carried out
Determine.After 0-20cm and 20-40cm deep soil compositions are determined, examined using t and statistical analysis is carried out to determination data.As a result
It was found that, the total salt concentration and Na of soil in 0-20cm and 20-40cm depth Tongzhou+Concentration conspicuousness compares soil higher than Changping.
And pH value does not have difference in two position soil.Show that Tongzhou soil pH value is normal, its soil is primarily present salt damage.
2nd, corn maturity period plant height is determined
240 Maize DH Lines and its Parent material are in 2014,2015 and 2016 respectively in Beijing Tongzhou salt damage area
(TZ, N39 ° 41 ' 49.70 ", E116 ° 40 ' 50.75 ') and Changping normally (CP, N40 ° 10 ' 50.38 ", E116 ° 27 '
15.40 ") planted.Annual experiment uses RANDOMIZED BLOCK DESIGN, carries out independence twice and repeats.For each repetition, each
A row plants in DH corns system, and 20 plants are planted per seeding.Per platoon leader 5m, row's spacing is 60cm.In the corn maturity period, DH systems and father are determined
Maternal plant height.Measurement data analysis is found, DH systems female parent plant height is substantially less than DH systems male parent, salt by the inhibition level of salt stress
Evil ground plant height, normally the genetic force of plant height is higher, and respectively 74.7% and 86.4%；And it was found that, salt damage area plant height with
Normally the correlation of plant height is relatively low, and coefficient correlation is 0.397.
3rd, genetic linkage mapses are built and QTL positioning
Using selected hundreds of parts representative corn varieties, repeated according to site, signal intensity, miss rate, polymorphism,
The principles such as even distribution are sieved to 56110 SNP sites in MaizeSNP50K chips (U.S.'s Illumina Products)
Choosing, assessment, finally determine that 3072 core SNP sites are customized to SNP chip product maizeSNP3072.The leaf of 240 DH systems
Piece extracts DNA with the plant DNA extraction kit of TianGen companies, is carried out afterwards according to the standard test step of ABI companies
DNA hybridization and chip scanning, make the structure that genetic linkage mapses are carried out using the Kosambi functional modules of JoinMap4 softwares
Build.Maize genome is covered using 1317 genetic linkage mapses in DH systems Parent constructed by the SNP with polymorphism
Spacing between the common 1462.05cM distances of 10 chromosomes, SNP is about 1.11cM.
Then according to phenotype and genotype data information, salt tolerant QTL is positioned using composite interval mapping method.Utilize
3 years salt damage area plant heights are as phenotypic data, and the main effect QTL navigated on No. 1 chromosome is named as qSPH1 (LOD
For that 22.4), can explain 31.24% phenotypic variation, it marks it positioned at PZE101094436 and PZE101150513SNP
Between.
Meanwhile, it can position under 3 different years and obtain, and show that its effect is highly stable, not by environmental factor
Influence.The QTL navigated to based on normally plant height is distributed in No. 4, No. 5, No. 8 and No. 9 chromosomes, with this main effect of qSPH1
QTL position is entirely different, shows that qSPH1 controls salt tolerance of corn, and unrelated with plant height.
Embodiment 2 and the SNP marker exploitation and application of salt tolerant main effect QTL compact linkage
By Genes location, salt tolerant main effect QTL qSPH1 is located at PZE101094436 and PZE101150513 molecular labelings
Between, so, PZE101094436 is the molecular labeling with its close linkage.
Choose 13 laboratory qualifications and cross the corn inbred line of salt tolerance, wherein 6 salt tolerants (capital 725, PH6WC, capital 724,
91227th, A9241, capital 464), 7 not salt tolerant (PH4CV, DH382, D9B, D9H, capital 4055, B547, MC01) (Luo etc.,
2017,Maydica 62:11), marked and the genotype of above material is entered using PZE101094436SNP with reference to KASP technologies
Row analysis, using 3 specific primers designing of the present invention, its nucleotide sequence is respectively as shown in SEQ ID NO.1-3, KASP
Method detects standard test of the SNP gene locis according to Laboratory of the Government Chemist (LGC) company
Step is carried out, and when using primer shown in SEQ ID NO.2-3, if amplified production 19bp base is G, corn to be measured is resistance to
Salt ability is high；When using SEQ ID NO.1, primer shown in 3 when amplified production 20bp base for A, then corn salt tolerant to be measured
Ability is low.
Its main experimental step is as follows：(1) extracted with the plant DNA extraction kit of TianGen companies after DNA, use LGC
DNA is diluted to 50ng/ μ l by the Replikator orifice plates duplicator of company, and 1.5 μ l DNA of each sample are transferred into 384
In sheet material (orifice plate of black 384).(2) by primer (SEQ ID NO.1-3), 2 × Master of KASP Mix, DNase/RNase-
Free Deionized Water are mixed by a certain percentage, are added with the micro knockouts of the Merodian of LGC companies in 384 plates.
(3) the Kube heat-sealing film instrument sealers of the 384 plates LGC companies of good mixed liquor are added.(4) with the Hydrocycle water-baths of LGC companies
Pot carries out target DNA fragments amplification, and amplification system is:94℃15min；(each circulation declines 94 DEG C of 20s 61-55 DEG C 1min
0.6 DEG C) totally 10 circulation；94 DEG C of 55 DEG C of 20s 1min 26 circulations.(5) by PHERA of the sample expanded with LGC companies
starplusSNP scanners sweep plate, finally with Kraken software analysis datas and export result as shown in Figure 2.
As a result find, PZE101094436 marks can be by salt tolerant and salt-tolerant inbred lines do not make a distinction, Tolerant salt and
SNP results are consistent.
Although having used general explanation, embodiment and experiment above, the present invention is described in detail,
But some modifications on the basis of the present invention, can be made to it or are improved, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed
Scope.
SEQUENCE LISTING
<110>Beijing City Agriculture and Forestry Institute
<120>The SNP marker related to salt tolerance of corn and its application
<130> KHP171113530.2
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
ggagccgtgg aagtgcgaga 20
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
gagccgtgga agtgcgagg 19
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
gccaagatgc tcgcaagtac cttat 25
Claims (10)
1. the SNP marker related to salt tolerance of corn shape, it is characterised in that it is PZE101094436, the SNP molecules
The polymorphism of mark is G/A, is expanded and obtained as primer PCR of the nucleotide sequence as shown in SEQ ID NO.1-3.
2. application of the molecular labeling in crop marker assisted selection described in claim 1.
3. application of the molecular labeling in the high crop of seed selection salt resistance ability described in claim 1.
4. application of the molecular labeling in screening haloduric corn kind described in claim 1.
5. application of the molecular labeling in prediction corn salt resistance ability described in claim 1.
6. the specific primer pair for detecting the SNP marker related to salt tolerance of corn, it is characterised in that drawn by three
Thing is constituted, and its nucleotide sequence is respectively as shown in SEQ ID NO.1-3.
7. the specific primer described in claim 6 to being improved in corn germ plasm resource in application.
8. a kind of method for identifying high salt resistance ability corn, it is characterised in that comprise the following steps：
(1) genomic DNA of corn to be measured is extracted；
(2) DNA extracted using in step (1) is template, using the specific primer described in claim 6 to entering performing PCR amplification
Reaction；
(3) when using primer shown in SEQ ID NO.2-3, if amplified production 19bp base is G, corn salt tolerant to be measured
Ability is high；When using SEQ ID NO.1, primer shown in 3 when amplified production 20bp base for A, then corn salt tolerant energy to be measured
Power is low.
9. the related main effect QTL of corn salt tolerant, it is qSPH1, and on No. 1 chromosome of corn, LOD is 22.4, can be solved
Release 31.24% phenotypic variation, its LOD peak is located at No. 1 chromosome 88.51cM position, and its confidential interval is No. 1 chromosomal inheritance
Apart from 77.61-98.18cM, physical distance is 92353978-194525458.
10. application of the main effect QTL in screening haloduric corn kind described in claim 9.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795951A (en) * | 2018-06-29 | 2018-11-13 | 北京市农林科学院 | A kind of corn resistant gene of salt and its molecular labeling and application |
| CN117737284A (en) * | 2023-12-11 | 2024-03-22 | 广东省农业科学院作物研究所 | Allele and molecular marker for identifying thin-skin sweet corn, identification method and application |
| CN117925901A (en) * | 2024-03-19 | 2024-04-26 | 东北农业大学 | Molecular marker developed based on gene ZmArg and related to salt and alkali tolerance of corn and application thereof |
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| CN103160502A (en) * | 2013-02-28 | 2013-06-19 | 南通新禾生物技术有限公司 | Single nucleotide polymorphism (SNP) molecular markers for corn germplasm salt-resistant quantitative trait loci (QTL) and application thereof |
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| CN103160502A (en) * | 2013-02-28 | 2013-06-19 | 南通新禾生物技术有限公司 | Single nucleotide polymorphism (SNP) molecular markers for corn germplasm salt-resistant quantitative trait loci (QTL) and application thereof |
| CN104532359A (en) * | 2014-12-10 | 2015-04-22 | 北京市农林科学院 | Core SNP sites combination maizeSNP384 for building of maize DNA fingerprint database and molecular identification of varieties |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795951A (en) * | 2018-06-29 | 2018-11-13 | 北京市农林科学院 | A kind of corn resistant gene of salt and its molecular labeling and application |
| CN117737284A (en) * | 2023-12-11 | 2024-03-22 | 广东省农业科学院作物研究所 | Allele and molecular marker for identifying thin-skin sweet corn, identification method and application |
| CN117925901A (en) * | 2024-03-19 | 2024-04-26 | 东北农业大学 | Molecular marker developed based on gene ZmArg and related to salt and alkali tolerance of corn and application thereof |
| CN117925901B (en) * | 2024-03-19 | 2024-05-31 | 东北农业大学 | Molecular marker developed based on gene ZmArg and related to salt and alkali tolerance of corn and application thereof |
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