CN107254535B - SNP molecular markers associated with maize salt tolerance and their applications - Google Patents

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 markers associated with maize salt tolerance and their applications

technical field

The invention relates to the technical field of crop molecular marker-assisted breeding, in particular to the main QTL of maize salt tolerance at maturity and its closely linked SNP molecular markers related to maize salt tolerance traits and its application.

Background technique

Corn is an important food, fodder and economic crop in the world, and it is also the largest crop with the widest planting area and the highest total output in my country. Corn production is closely related to the sustainable and stable development of China's grain. However, maize is a salt-sensitive plant that can tolerate soil salt concentrations ranging from 0.3-0.7%. Soil salinity will cause low germination rate, weak seedlings, withering and even death of corn, which will eventually lead to reduced yield and quality. There are 954.38 million hectares of saline-alkali land in the world, accounting for more than 7% of the world's total land area and more than 20% of the arable land. China has 99.133 million hectares of saline-alkali land, ranking third in the world's saline-alkali land area (Zhang Jianfeng et al., 2008, Soil and Water Conservation Research 15:74-78). With the sharp reduction of arable land, it is very urgent and necessary to develop and utilize salt-damaged land. At present, the utilization of salt-tolerant germplasm resources is an important means to solve this problem.

The use of traditional breeding methods to select salt-tolerant corn planting resources has problems such as low selection rate and long cycle. Molecular marker-assisted breeding is an effective means to solve this problem. The premise of using molecular markers to improve salt tolerance in maize is to obtain practical molecular markers closely linked with salt tolerance. Therefore, it is of great significance to carry out research on the mapping of salt-tolerant QTL and the development of molecular markers at the maturity stage of field maize, which is of great significance to accelerate the breeding of salt-tolerant maize planting resources and promote the sustainable development of maize production in my country.

QTL mapping is an accurate statistical method for detecting the genetic basis of quantitative traits such as salt tolerance. Previous studies have been conducted on the salt tolerance QTL of various crops, in order to identify salt tolerance control factors and apply them to molecular marker-assisted breeding. The predecessors have carried out mapping analysis of salt tolerance QTL correlation in crops such as soybean (Guan et al., 2014, The Crop Journal 2: 358-365), barley (Ahmadi-Ochtapeh et al., 2015, Biologia Plantarum 59: 283-290), and , under the guidance of QTL mapping results, further successfully identified rice (Ren et al., 2005, Nature Genetics 37: 1141-1146) and wheat (Munns et al., 2012, Nature Biotechnology 30: 360-364) salt tolerance genes and applied to Crop genetic improvement.

Compared with other crops, there are relatively few reports on the genetic and molecular mechanism of salt tolerance in maize. SNP markers were used to construct a high-density genetic linkage map, combined with plant height phenotype data to map the salt tolerance QTL in field maize at maturity and develop molecular markers. It is extremely necessary to provide practical and effective salt-tolerant linked markers for molecular marker-assisted breeding.

SUMMARY OF THE INVENTION

The first objective of the present invention is to provide major QTLs associated with maize salt tolerance traits.

The second object of the present invention is to provide a SNP molecular marker closely linked with the major QTL of maize salt tolerance, and a specific primer pair for amplifying the molecular marker.

The third object of the present invention is to provide the application of the above-mentioned SNP molecular marker.

The purpose of this invention is to realize through the following technical solutions:

Based on the above purpose, the applicant used 240 DH lines as materials, used SNP markers to construct a high-density genetic linkage map, and combined with the plant height phenotype data to map the salt tolerance QTL of field maize at maturity. Specifically, 240 DH lines obtained by induction and doubling with Xianyu 335 (female parent: PH6WC, salt-tolerant; male parent: PH4CV, salt-sensitive) as the basic material and their parental parents PH6WC and PH4CV were tested in Tongzhou District, Beijing The base (salt-damaged land) and the Changping District test base (normal soil) have been planted in spring for 3 consecutive years. Each year the trial used a randomized block design with two independent replicates. At the physiological maturity stage of maize material, use a plant height measuring instrument to measure from the first palea at the top of the ear to the ground level, and calculate the plant height at the mature stage.

Using hundreds of selected representative maize varieties, 56,110 SNP loci in the MaizeSNP50K chip of Illumina company in the United States were screened and evaluated according to the principles of locus duplication, signal intensity, deletion rate, polymorphism, and uniform distribution. 3072 core SNP sites were determined and customized into a SNP chip product maizeSNP3072 (Illumina, USA) (Tian et al., 2015, Molecular Breeding 35:136). DNA was extracted from the leaves of 240 DH lines with a plant DNA extraction kit from TianGen Company, and then DNA hybridization and chip scanning were performed according to the standard experimental procedures of ABI Company, and a high-density genetic linkage map was constructed using the Kosambi function module of JoinMap4 software.

On the basis of the construction of high-density genetic linkage map, combined with the phenotype data of plant height in salt-damaged land, the WindowsQTL Cartographer software was used to map the major QTLs for salt tolerance at maturity in field maize and develop closely linked molecules by using the composite interval mapping method. mark. A major QTL was obtained, and a molecular marker closely linked to it was found to be associated with maize salt tolerance. The major QTL, named qSPH1, is located on maize chromosome 1 with an LOD of 22.4.

Further, the present invention provides a SNP molecular marker related to maize salt tolerance traits, which is PZE101094436, the polymorphism of this SNP is G/A, and the nucleotide sequence is as shown in SEQ ID NO.1-3 primers obtained for PCR amplification.

The present invention provides the application of the above molecular markers in molecular-assisted breeding of crops.

The present invention provides the application of the above molecular marker in breeding crops with high salt tolerance.

The present invention provides the application of the above molecular markers in screening salt-tolerant corn varieties.

The present invention provides the application of the above molecular markers in predicting the salt tolerance of maize.

The present invention provides a specific primer pair for detecting SNP molecular markers related to maize salt tolerance, which consists of three primers, and the nucleotide sequences thereof are respectively as SEQ ID NO. 1-3.

The present invention provides the application of the above-mentioned specific primer pair in the improvement of maize germplasm resources.

The invention provides a method for identifying high salt tolerance corn, comprising the following steps:

(1) extracting the genomic DNA of the maize to be tested;

(2) using the DNA extracted in step (1) as a template, using the specific primer pairs shown in SEQ ID NO.1-3 to carry out PCR amplification reaction;

(3) When the primers shown in SEQ ID NO.2-3 are used, if the base of the 19 bp of the amplified product is G, the salt tolerance of the corn to be tested is high, or when the primers shown in SEQ ID NO.1 and 3 are used When the base of the 20 bp of the amplified product is A in the primer, the salt tolerance of the corn to be tested is low.

The beneficial effects of the present invention are: for the first time, the major QTL (as shown in Fig. 1 ) and its closely linked SNP molecular markers for salt tolerance in maize are disclosed, and the molecular markers can be used for early prediction and screening of salt tolerance traits in maize, as well as for carrying out Molecular-assisted breeding to screen for salt-tolerant germplasm resources.

Description of drawings

Figure 1. The location and LOD value of the major QTL on chromosome 1 obtained by using the plant height data of different years and three-year averages. In the figure, SPH(2014/2015/2016): (2014/2015/2016) plant height in salt-damaged land; SPHmean: three-year average of plant height in salt-damaged land.

Fig. 2 The results of genotype analysis of SNP loci of 13 maize samples by PZE101094436 SNP marker. The KASP method was used to detect SNP loci according to the standard experimental procedures of the Laboratory of the Government Chemist (LGC) company, and the data were analyzed and exported by Kraken software. Top left: the base of the SNP site is G, bottom right: the base of the SNP site is A, bottom left: negative control. Two replicates were performed for each sample.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention.

The 240 maize DH lines and their parental materials and other maize germplasm resources used in the examples of the present invention were all from the maize germplasm resource bank of the Maize Research Center of Beijing Academy of Agriculture and Forestry.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 Mapping of major QTLs related to salt tolerance in maize

1. Soil composition analysis

According to the 5-point sampling method (Zhai et al. 2016, Advance Journal of Food Science & Technology 11:88-94), soil composition analysis was carried out in 5 representative locations in Tongzhou and Changping. Total salt concentration was determined using the residue drying method (Wüst et al., 2000, Journal of Archaeological Science 27: 1161-1172). Soil pH was measured using a pH meter from Hach. The Na ⁺ concentration in the soil was measured using a flame emission spectrometer from PerkinElmer. After measuring soil composition at 0-20cm and 20-40cm depths, statistical analysis of the measured data was carried out using t-test. It was found that the total salt concentration and Na ⁺ concentration of Tongzhou soil at depths of 0-20cm and 20-40cm were significantly higher than those of Changping control soil. However, there was no significant difference in pH between the soils at the two locations. It shows that the soil pH value in Tongzhou is normal, and the soil mainly suffers from salt damage.

2. Determination of plant height at mature stage of maize

240 maize DH lines and their parent materials were collected in Tongzhou salt-damaged land in Beijing (TZ, N39°41′49.70″, E116°40′50.75′) and Changping normal land (CP, N40°) in 2014, 2015 and 2016, respectively. 10′ 50.38″, E116° 27′ 15.40″) were planted. Each year the trial used a randomized block design with two independent replicates. For each replicate, each DH maize line was planted in one row with 20 plants per row. Every row is 5m long, and the row spacing is 60cm. In the maize maturity stage, the plant heights of the DH line and the parent parent are measured. Analysis of the measurement data shows that the degree of inhibition of the DH line female parent plant height by salt stress is significantly lower than that of the DH line male parent. The heritability of plant height in salt-damaged and normal fields was higher, 74.7% and 86.4%, respectively; and it was found that the correlation between plant height in salt-damaged and normal fields was low, with a correlation coefficient of 0.397.

3. Genetic linkage map construction and QTL mapping

Using selected hundreds of representative maize varieties, 56110 SNP loci in the MaizeSNP50K chip (product of Illumina, USA) were screened according to the principles of locus duplication, signal intensity, deletion rate, polymorphism, and uniform distribution. After evaluation, 3072 core SNP loci were finally determined to be customized into the SNP chip product maizeSNP3072. The leaves of 240 DH lines were extracted with the plant DNA extraction kit of TianGen Company, and then DNA hybridization and chip scanning were carried out according to the standard experimental procedures of ABI Company. , using the Kosambi function module of JoinMap4 software to construct a genetic linkage map. The genetic linkage map constructed by using 1317 SNPs with polymorphisms in DH line parents covers a total distance of 1462.05cM in 10 chromosomes in the maize genome, and the distance between SNPs is about 1.11cM.

Then, based on the phenotypic and genotypic data information, complex interval mapping was used to map salt tolerance QTLs. Using the phenotypic data of plant height in 3-year salt-damaged land, a major QTL located on chromosome 1, named qSPH1 (LOD 22.4), was able to explain 31.24% of the phenotypic variation, which was located at the PZE101094436 and PZE101150513 SNP markers between.

At the same time, it can be located in three different years, indicating that its effect is very stable and not affected by environmental factors. The QTLs located on the basis of normal plant height were distributed on chromosomes 4, 5, 8 and 9, which were completely different from qSPH1, the main effect QTL, indicating that qSPH1 controls maize salt tolerance, regardless of plant height.

Example 2 Development and application of SNP markers closely linked to major salt-tolerant QTLs

Through genetic mapping, the salt tolerance major QTL qSPH1 is located between the molecular markers PZE101094436 and PZE101150513, therefore, PZE101094436 is a molecular marker closely linked to it.

13 maize inbred lines with salt tolerance identified by laboratories were selected, of which 6 were salt-tolerant (Jing 725, PH6WC, Jing 724, 91227, A9241, and Jing 464), and 7 were salt-intolerant (PH4CV, DH382, D9B, D9H, Jing 4055, B547, MC01) (Luo et al., 2017, Maydica 62:11), using PZE101094436 SNP marker and combined with KASP technology to analyze the genotypes of the above materials, using the three specific primers designed in the present invention, its core The nucleotide sequences are shown in SEQ ID NO.1-3 respectively, and the detection of SNP loci by KASP method is carried out according to the standard experimental procedure of the Laboratory of the Government Chemist (LGC) company. When using primers shown in SEQ ID NO.2-3 If the base of the 19th bp of the amplified product is G, the salt tolerance of the corn to be tested is high; when the primers shown in SEQ ID NO. 1 and 3 are used, the base of the 20 bp of the amplified product is A, then the tested Corn has low salt tolerance.

The main test steps are as follows: (1) After extracting DNA with TianGen's plant DNA extraction kit, dilute the DNA to 50ng/μl with LGC's Replikator well plate replicator, and transfer 1.5μl of DNA from each sample to 384 plate (black 384-well plate). (2) Mix the primers (SEQ ID NO. 1-3), KASP 2×Master Mix, and DNase/RNase-Free Deionized Water in a certain proportion, and add them into the 384 plate with a Merodian microdispenser from LGC. (3) The 384 plate to which the mixed solution was added was sealed with a Kube heat sealer from LGC. (4) Amplify the target DNA fragments with the Hydrocycle water bath of LGC Company. The amplification system is: 94°C for 15min; 94°C for 20s 61-55°C for 1min (each cycle drops by 0.6°C) for a total of 10 cycles; 94°C for 20s 55 ℃ 1min 26 cycles. (5) Scan the amplified sample with the PHERAstar ^plus SNP scanner of LGC Company, and finally analyze the data with Kraken software and derive the results shown in Figure 2.

It was found that the PZE101094436 marker could distinguish salt-tolerant and salt-intolerant inbred lines, and the identification of salt tolerance was consistent with the SNP results.

Although the present invention has been described in detail above with general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art of. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

SEQUENCE LISTING

<110> Beijing Academy of Agriculture and Forestry

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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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