CN107254535B - SNP molecular marker related to salt tolerance of corn and application thereof - Google Patents

SNP molecular marker related to salt tolerance of corn and application thereof Download PDF

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CN107254535B
CN107254535B CN201710546524.4A CN201710546524A CN107254535B CN 107254535 B CN107254535 B CN 107254535B CN 201710546524 A CN201710546524 A CN 201710546524A CN 107254535 B CN107254535 B CN 107254535B
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CN107254535A (en
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赵久然
赵衍鑫
骆美洁
宋伟
邢锦丰
石子
张如养
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention provides SNP molecular markers related to salt tolerance of corn and application thereof, belonging to the technical field of crop molecular marker assisted breeding, wherein 240 DH lines are used as materials, a high-density genetic linkage map is constructed by utilizing the SNP markers, plant height phenotype data is combined, the salt tolerance QT L of the mature period of the field corn is positioned, the molecular markers closely linked with the SNP molecular markers are found to be related to the salt tolerance of the corn, the SNP molecular markers are PZE101094436, and the markers are obtained by amplifying primers with nucleotide sequences shown as SEQ ID NO. 1-3.

Description

SNP molecular marker related to salt tolerance of corn and application thereof
Technical Field
The invention relates to the technical field of crop molecular marker assisted breeding, in particular to a salt-tolerant main effect QT L in a mature period of corn, an SNP molecular marker closely linked with the same and related to salt-tolerant traits of the corn and application of the SNP molecular marker.
Background
Corn is a globally important grain and feed crop and an economic crop, and is the first crop with the widest planting area and the highest total yield in China. The corn production is closely related to the sustainable and stable development of Chinese grains. However, corn is a salt-sensitive plant that can tolerate soil salt concentrations in the range of 0.3-0.7%. The salt damage of the soil can cause low germination rate, weak seedlings, withering and even death of the corns, and finally, the yield is reduced and the quality is reduced. 9.5438 hundred million hectares of saline-alkali soil are in the world, which accounts for more than 7 percent of the total land area and more than 20 percent of arable land area. 9913.3 ten thousand hectares of saline-alkali soil exist in China, and the third place is occupied in the area ranking list of the world saline-alkali soil (Zhang Jianfeng et al, 2008, research on water and soil conservation 15: 74-78). With the rapid decrease of the arable land area, the development and the utilization of the land damaged by salt are urgent and necessary. At present, the utilization of salt-tolerant germplasm resources is an important means for solving the problem.
The molecular marker assisted breeding is an effective means for solving the problem, and the premise of improving the salt tolerance of the corn by utilizing the molecular marker is to obtain a practical molecular marker closely linked with the salt tolerance.
The QT L location is an accurate statistical method for detecting The genetic basis of quantitative traits such as salt tolerance, and The salt tolerance QT L of various crops has been studied in a location mode so as to identify The application of a salt tolerance control factor in molecular marker assisted breeding.
Compared with other crops, the research reports of the salt tolerance inheritance and molecular mechanism of the corn are relatively few, a high-density genetic linkage map is constructed by utilizing the SNP markers, positioning and molecular marker development are carried out on the salt tolerance QT L in the mature period of the corn in the field by combining plant height phenotype data, and practical and effective salt tolerance linkage markers can be provided for molecular marker assisted breeding.
Disclosure of Invention
The first purpose of the invention is to provide a main effect QT L related to the salt-tolerant trait of corn.
The second purpose of the invention is to provide an SNP molecular marker closely linked with the main effect QT L of the salt tolerance of the corn and a specific primer pair for amplifying the molecular marker.
The third purpose of the invention is to provide the application of the SNP molecular marker.
The purpose of the invention is realized by the following technical scheme:
based on the purposes, the applicant uses 240 DH lines as materials, utilizes SNP markers to construct a high-density genetic linkage map, combines plant height phenotype data, and positions salt-tolerant QT L in the mature period of corn in the field, wherein the DH lines and parent lines PH6WC and PH4CV are obtained by induced doubling of 240 parts of pioyu 335 (female parent: PH6WC, salt tolerance, male parent: PH4CV, salt sensitivity) as base materials, and are planted in continuous spring sowing in Tongzhou district test base (salt damage land) and Changsheng district test base (normal soil) of Beijing city for 3 years, each year test adopts random design, and independent repetition is carried out twice until the physiological mature period of the corn material, the plant height is counted from the first hull at the top of the topus to the ground level by using a plant height measuring instrument, and the plant height in the mature period is counted.
56110 SNP sites in a chip of MaizesNP50K from Illumina, USA are screened and evaluated by utilizing a plurality of selected hundreds of representative corn varieties according to the principles of site repetition, signal intensity, deletion rate, polymorphism, uniform distribution and the like, and 3072 core SNP sites are finally determined to be customized into an SNP chip product maizeSNP3072 (Illumina, USA) (Tian, et al, 2015, Molecular Breeding 35: 136). The DNA of 240 leaves of DH line was extracted by using the plant DNA extraction kit of TiangGen, followed by hybridization and chip scanning of DNA according to the standard experimental procedure of ABI, and high density genetic linkage map was constructed using Kosambi functional module of JoinMap4 software.
On the basis of high-density genetic linkage map construction, salt damage plant high phenotype data are combined, Windows QT L Cartogrer software is applied, a composite interval mapping method is adopted to position salt-tolerant main effect QT L in the mature period of field corn and develop tightly linked molecular markers, a main effect QT L is obtained, the molecular marker tightly linked with the main effect QT L is found to be related to the salt tolerance of the corn, the main effect QT L is named as qSPH1 and is positioned on the No.1 chromosome of the corn, and L OD is 22.4.
Further, the invention provides an SNP molecular marker related to the salt tolerance of corn, which is PZE101094436, the polymorphism of the SNP is G/A, and the SNP molecular marker is obtained by PCR amplification of a primer pair with a nucleotide sequence shown as SEQ ID NO. 1-3.
The invention provides application of the molecular marker in molecular assisted breeding of crops.
The invention provides application of the molecular marker in breeding crops with high salt tolerance.
The invention provides application of the molecular marker in screening salt-tolerant corn varieties.
The invention provides application of the molecular marker in predicting salt tolerance of corn.
The invention provides a specific primer pair for detecting SNP molecular markers related to salt tolerance of corn, which consists of 3 primers, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 1-3.
The invention provides application of the specific primer pair in improvement of corn germplasm resources.
The invention provides a method for identifying high-salt-tolerance corn, which comprises the following steps:
(1) extracting the genome DNA of the corn to be detected;
(2) taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification reaction by using a specific primer pair shown in SEQ ID NO. 1-3;
(3) when the primers shown in SEQ ID NO.2-3 are adopted, if the 19bp base of the amplification product is G, the salt tolerance of the corn to be detected is high, or when the 20bp base of the amplification product is A when the primers shown in SEQ ID NO.1 and 3 are adopted, the salt tolerance of the corn to be detected is low.
The invention has the beneficial effects that the maize salt-tolerant main effect QT L (shown in figure 1) and the SNP molecular marker tightly linked with the same are disclosed for the first time, and the molecular marker can be used for early prediction and screening of maize salt-tolerant characters and molecular assisted breeding to screen salt-tolerant germplasm resources.
Drawings
FIG. 1 shows the position of major QT L on chromosome 1 and L OD value by using plant height data of different year and three year average values, wherein SPH (2014/2015/2016) shows the plant height of the salt-damaged area in year (2014/2015/2016), and SPHmean shows the plant height of the salt-damaged area in three years.
FIG. 2 result of SNP site genotype analysis of 13 corn samples by PZE101094436SNP marker the KASP method was performed to detect SNP gene sites according to the standard experimental procedure of L analysis of the Goverment Chemist (L GC) company, and the data was analyzed and derived by Kraken software.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The 240 corn DH lines and the corn germplasm resources such as parent materials and the like used in the embodiment of the invention are from the corn germplasm resource library of the corn research center of the agriculture and forestry academy of sciences of Beijing.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 maize salt tolerance-related major QT L location
1. Analysis of soil composition
Sampling was performed according to 5-point sampling method (Zhai et al 2016, Advance Journal of Food Science)&Technology11:88-94), composition analysis of soil at 5 representative locations in Tongzhou and Changping land, Total salt concentration was determined by the sludge drying method (W ü st et al, 2000, Journal of Archaeological Science 27:1161-+The concentration was measured. After measuring the soil components at 0-20cm and 20-40cm depths, statistical analysis of the measured data was performed using the t-test. As a result, it was found that the total salt concentration and Na of Tongzhou soil were 0 to 20cm and 20 to 40cm deep+The concentration is obviously higher than that of Changping control soil. While the pH value was not significantly different in the soil at the two locations. The pH value of the Tongzhou soil is normal, and the soil is mainly subjected to salt damage.
2. Determination of plant height in mature period of corn
240 maize DH lines and their parental material were grown in the Beijing Tong salt plots (TZ, N39 ° 41 ' 49.70 ", E116 ° 40 ' 50.75 ') and Changping Normal (CP, N40 ° 10 ' 50.38", E116 ° 27 ' 15.40 "), respectively, in 2014, 2015 and 2016. The experiment was conducted in a randomized block design with two independent replicates per year. For each replicate, one row was planted per DH maize line, and 20 plants were planted per seed. Each row is 5m long and the row spacing is 60 cm. At the mature stage of maize, the DH line and parental plant height are determined. Analysis of measurement data shows that the inhibition degree of the DH line female parent plant height under salt stress is obviously lower than that of the DH line male parent, the salt damage plant height and the heritability of the normal plant height are respectively 74.7 percent and 86.4 percent; and the correlation between the plant height of the salt-damaged field and the plant height of the normal field is low, and the correlation coefficient is 0.397.
3. Genetic linkage map construction and QT L location
By utilizing a plurality of selected hundreds of representative corn varieties, 56110 SNP loci in a maize NP50K chip (products of Illumina company in America) are screened and evaluated according to the principles of locus repetition, signal intensity, deletion rate, polymorphism, uniform distribution and the like, and 3072 core SNP loci are finally determined to be customized into a SNP chip product, namely, maize SNP 3072. DNA was extracted from 240 leaves of DH line by using a plant DNA extraction kit of TianGen, and then DNA hybridization and chip scanning were performed according to the standard experimental procedure of ABI, so that a genetic linkage map was constructed using Kosambi function module of JoinMap4 software. A genetic linkage map constructed using 1317 SNPs with polymorphisms in the parents of the DH line covers a total distance of 1462.05cM of 10 chromosomes in the maize genome, and the spacing between SNPs is about 1.11 cM.
The salt tolerance QT L was then located using a complex interval mapping method based on phenotypic and genotypic data using 3-year salt damage plant height as phenotypic data, located on chromosome 1, at a major QT L, designated qSPH1 (L OD 22.4), capable of accounting for 31.24% of phenotypic variation, located between the PZE101094436 and PZE101150513SNP markers.
QT L located based on normal plant height is distributed on chromosomes 4, 5, 8 and 9, and is completely different from the position of the main effect QT L of qSPH1, which indicates that the qSPH1 controls the salt tolerance of corn and is not related to the plant height.
Example 2 development and application of SNP marker closely linked with salt-tolerant major QT L
Through genetic localization, the salt tolerance main effect QT L qSPH1 is located between molecular markers PZE101094436 and PZE101150513, so that PZE101094436 is a molecular marker closely linked with the molecular marker.
Selecting 13 laboratories to identify salt-tolerant maize inbred lines, wherein 6 salt-tolerant (Jing 725, PH6WC, Jing 724, 91227, A9241 and Jing 464) and 7 salt-tolerant (PH4CV, DH382, D9B, D9H, Jing 4055, B547 and MC01) (L uo and the like, 2017 and Maydica 62:11) are selected, analyzing genotypes of the above materials by utilizing a PZE101094436SNP marker and combining with a KASP technology, adopting 3 specific primers designed by the invention, nucleotide sequences of the specific primers are respectively shown as SEQ ID NO.1-3, adopting a KASP method to detect SNP gene sites according to a standard experiment step of L analysis of the Government Chemist (L GC) company, and adopting primers shown as SEQ ID NO.2-3, if a 19bp base of an amplification product is G, the maize to be detected has high salt-tolerant capability, and adopting SEQ ID NO.1 and 20bp base of a low-tolerant amplification product.
The main test steps are (1) after DNA extraction with the plant DNA extraction kit from TiangGen, diluting the DNA to 50 ng/. mu.l with a Replikator well plate duplicator from L GC, transferring 1.5. mu.l of DNA from each sample to 384 plates (black 384 well plates) (2) adding primers (SEQ ID No.1-3), KASP 2 × Master Mix, DNase/RNase-Free deioned Water from L GC to 384 plates (3) adding the 384 plates with the mixed solution to 384 plates with a Merodian microdispenser from L GC, (4) amplifying the target DNA fragments with a Kube heat sealer from L GC, using a Hydrocycle Water bath from L GC, the amplification system is 94 ℃ 15min, 94 ℃ 20s 61-55 ℃ 1min (10 cycles for each cycle), 94 ℃ 20s 55 ℃ 1min, 82 ℃ for 26 ℃ GC, and the amplification system is 80 ℃ for 26 ℃ samples from Astar GC 82plusThe SNP scanner was scanned and the data was finally analyzed using Kraken software and the results derived as shown in figure 2.
The result shows that the PZE101094436 marker can distinguish salt-tolerant inbred lines from salt-tolerant inbred lines, and the salt-tolerant identification is consistent with the SNP result.
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> agriculture and forestry academy of sciences of Beijing City
<120> SNP molecular marker related to salt tolerance of corn and application thereof
<130>KHP171113530.2
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Claims (5)

1. The application of the SNP molecular marker related to the salt tolerance of the corn in breeding the corn with high salt tolerance is characterized in that the SNP molecular marker is PZE101094436, and when the primer shown in SEQ ID NO.2-3 is adopted for amplification, the 19bp base polymorphism of an amplification product is G/A.
2. The application of a specific primer group in screening salt-tolerant corn varieties is characterized in that the specific primer group comprises three primers, and nucleotide sequences of the three primers are respectively shown in SEQ ID NO. 1-3.
3. The application of the SNP molecular marker related to the salt tolerance of the corn in predicting the salt tolerance of the corn is disclosed, wherein the SNP molecular marker is PZE101094436, and when the primer shown in SEQ ID NO.2-3 is adopted for amplification, the 19bp base polymorphism of an amplification product is G/A.
4. The application of the specific primer group in predicting the salt tolerance of the corn is characterized in that the specific primer group consists of three primers, and the nucleotide sequences of the primers are respectively shown in SEQ ID NO. 1-3.
5. A method for identifying corn with high salt tolerance is characterized by comprising the following steps:
(1) extracting the genome DNA of the corn to be detected;
(2) taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification reaction by using a specific primer group; the specific primer group comprises three primers, and the nucleotide sequences of the three primers are respectively shown in SEQ ID NO. 1-3;
(3) when the primers shown in SEQ ID NO.2-3 are adopted, if the 19bp base of the amplification product is G, the salt tolerance of the corn to be detected is high; when the primers shown in SEQ ID NO.1 and SEQ ID NO. 3 are adopted, the 20bp base of the amplification product is A, and the salt tolerance of the corn to be detected is low.
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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
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