CN117965782A - Saline-alkali tolerant major QTL locus on brassica napus A04 chromosome, molecular marker and application - Google Patents
Saline-alkali tolerant major QTL locus on brassica napus A04 chromosome, molecular marker and application Download PDFInfo
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Abstract
The invention discloses a main effect QTL locus of a saline-alkali tolerant A04 chromosome of brassica napus, a related molecular marker and application thereof. The contribution rate of the QTL locus to the salt and alkali resistance of the cabbage type rape is 4.69%. The molecular marker is obtained through whole genome association analysis and comprises 11 nucleotide base insertion at 8857551bp, single nucleotide base mutation at 8857564bp, 1 nucleotide base insertion at 8857584bp, single nucleotide base mutation at 8857666bp and 3 nucleotide base insertion at 8857699bp on a rape reference genome A04 chromosome. The method can rapidly and accurately detect the haplotype of the main effect QTL locus with salt and alkali tolerance on the cabbage type rape A04 chromosome, and has important significance for accelerating the molecular breeding process of the cabbage type rape.
Description
Technical Field
The invention belongs to the field of molecular detection, in particular to salt and alkali resistant molecular detection of brassica napus, and particularly relates to development and application of a main effect QTL locus and a molecular marker of a salt and alkali resistant A04 chromosome of brassica napus.
Background
As the population worldwide continues to increase, the contradiction between shortage of grain and oil and the population continues to increase is increasingly prominent. The self-supporting rate of the vegetable oil in China is reduced year by year, and is currently less than 30 percent. Rape is the first large edible vegetable oil source in China, and accounts for about 55% of the yield of oil crops, and has important economic values of feeding, vegetable, sightseeing and the like. Therefore, the rape planting area and the yield are improved, the national edible oil safety can be ensured, and great social benefits are achieved.
Cabbage type rape is considered as a medium saline-alkali tolerant crop, short-term saline-alkali stress can cause the rape to suffer from high-permeability threat, and the phenomena of cell membrane damage, plant water loss, leaf wilting and the like are caused; the long-term saline-alkali stress produces ion toxicity to rape, causes unbalance of ion absorption and ion transport, causes nutrition unbalance and metabolic disturbance, and seriously inhibits the growth speed of new leaves and the advanced senescence and abscission of old leaves (ELPHICK ET AL 2010). Saline-alkali stress severely inhibits seed germination and seedling growth (REHMAN ET AL 2000,2000), severely limits normal growth and development of plants, and ultimately poses serious threat to crop yield and even death (Zhao Fugeng 2004). By breeding saline-alkali tolerant varieties, rape production is carried out on saline-alkali lands unsuitable for traditional agricultural production, and the method is an important way for expanding rape seeds.
Therefore, the identification of the important salt and alkali tolerance QTL in the brassica napus is significant for developing molecular markers for the QTL with high salt and alkali tolerance contribution rate of the brassica napus, can be used for cloning of salt and alkali tolerance genes and molecular marker assisted breeding, and has great significance for improving comprehensive utilization of a large amount of salt and alkali lands and improving self-sufficiency rate of vegetable oil.
Disclosure of Invention
The invention aims to develop a main effect QTL locus of the salt and alkali resistance of the brassica napus, wherein the QTL locus is positioned on a A04 chromosome of the brassica napus, has high contribution rate to the salt and alkali resistance of the brassica napus, plays a key role in the regulation and control of the salt and alkali resistance of the brassica napus, can be used as map cloning and molecular marker assisted breeding, and is suitable for large-scale popularization and application.
The developed salt and alkali resistant A04 chromosome major QTL locus of the brassica napus can be used for detecting the salt and alkali resistance of the brassica napus, predicting the salt and alkali resistance of the brassica napus, and has important significance for accelerating the molecular breeding process of the brassica napus.
The second purpose of the invention is to develop a molecular marker for salt and alkali resistance of the brassica napus, which can detect the salt and alkali resistance of the brassica napus, forecast the salt and alkali resistance of the brassica napus and has important significance for accelerating the molecular breeding process of the brassica napus.
Specifically, the molecular marker is closely linked with the main effect QTL site of the brassica napus A04 chromosome, and comprises INDEL_A04_8857551, SNP_A04_8857564, INDEL_A04_8857584, SNP_A04_8857666 and INDEL_A04_8857699, wherein,
The molecular marker INDEL_A04_8857551 is positioned at 8857551bp of a brassica napus A04 chromosome, the base at 8857551bp is ATATATTAAATC or A, and the mutation leads to polymorphism;
The molecular marker SNP_A04_8857564 is positioned at 8857564bp of the brassica napus A04 chromosome, the base at 8857564bp is T or G, and the mutation leads to polymorphism;
The molecular marker INDEL_A04_8857584 is positioned at 8857584bp of a brassica napus A04 chromosome, the base at 8857584bp is TA or T, and the mutation leads to polymorphism;
the molecular marker SNP_A04_8857666 is positioned at 8857666bp of a brassica napus A04 chromosome, the base at 8857666bp is G or A, and the mutation leads to polymorphism;
The molecular marker INDEL_A04_8857699 is positioned at 8857699bp of a brassica napus A04 chromosome, a base at 8857699bp of the A04 chromosome is TTAG or T, and the mutation leads to polymorphism.
The salt-tolerant alkalinity of brassica napus can be detected by using any one or a combination of the above molecular markers.
The invention further aims to provide a primer for molecular marking of a main effect QTL locus of a salt-tolerant A04 chromosome of the brassica napus, which can detect the salt-tolerant alkalinity of the brassica napus, forecast the salt-tolerant alkalinity of the brassica napus and has important significance for accelerating the molecular breeding process of the brassica napus.
Specifically, the primer can specifically amplify the above molecular marker and detect a polymorphism caused by mutation thereof, wherein,
The primer of the molecular marker INDEL_A04_8857551 is designed by taking DNA fragments of 400bp sequences before and after 8857551bp of the A04 chromosome of brassica napus as templates, wherein the DNA fragments are shown in SEQ ID NO:1 is shown in the specification;
The primer of the molecular marker SNP_A04_8857564 is designed by taking DNA fragments of 400bp sequences before and after 8857564bp of the A04 chromosome of brassica napus as templates, wherein the DNA fragments are shown in SEQ ID NO:2 is shown in the figure;
The primer of the molecular marker INDEL_A04_8857584 is designed by taking DNA fragments of 400bp sequences before and after 8857584bp of the A04 chromosome of brassica napus as templates, and the DNA fragments are shown as SEQ ID NO:3 is shown in the figure;
The primer of the molecular marker SNP_A04_8857666 is designed by taking DNA fragments of 400bp sequences before and after 8857666bp of the A04 chromosome of brassica napus as templates, wherein the DNA fragments are shown in SEQ ID NO:4 is shown in the figure;
The primer of the molecular marker INDEL_A04_8857699 is designed by taking DNA fragments of 400bp sequences before and after 8857699bp of the A04 chromosome of brassica napus as templates, and the DNA fragments are shown as SEQ ID NO: shown at 5.
The salt-tolerant alkalinity of brassica napus can be detected by using any one or a combination of the above molecular markers.
Further, the invention provides a specific molecular marker primer, which is:
PCR primer designed for molecular marker INDEL_A04_8857551 with sequence shown in SEQ ID NO: 6. 7, comprising an upstream primer and a downstream primer;
PCR primer designed for molecular marker SNP_A04_8857564 has sequence as shown in SEQ ID NO: 8. 9, 10, comprising an upstream universal primer and two downstream specific primers;
PCR primer designed for molecular marker INDEL_A04_8857584 with sequence shown in SEQ ID NO: 11. 12, comprising an upstream primer and a downstream primer;
PCR primer designed for molecular marker SNP_A04_8857666 has sequence as shown in SEQ ID NO: 13. 14, 15, comprising two upstream specific primers and one downstream universal primer;
PCR primer designed for molecular marker INDEL_A04_8857699 with sequence shown in SEQ ID NO: 16. 17, comprising an upstream primer and a downstream primer.
Wherein, when the INDEL primer is used for detection, the genomic DNA of the brassica napus is amplified by common PCR, the amplified product is subjected to electrophoresis detection, and the haplotype is determined according to the size of the product; in the detection using SNP primers, the KASP technology is used to amplify the genomic DNA of Brassica napus, and after the reaction, the fluorescence data is read and the haplotype is determined according to the fluorescence signal.
On the basis of the main effect QTL locus, the molecular marker and the primer provided by the invention, a series of derived inventions are also within the protection scope of the patent, and the derived inventions comprise but are not limited to kits and detection methods developed by utilizing the QTL, the molecular marker and the primer.
The invention further provides a method for detecting the salt-tolerant alkalinity of the brassica napus, which can detect the salt-tolerant alkalinity of the brassica napus, predicts the salt-tolerant alkalinity of the brassica napus and has important significance for accelerating the molecular breeding process of the brassica napus.
Specifically, the method takes cabbage type rape genomic DNA as a template, and uses the primer to detect molecular markers; the sequence of the 8857551bp of the A04 chromosome detected by the molecular marker INDEL_A04_8857551 is ATATATTAAATC, the sequence of the 8857564bp of the A04 chromosome detected by the molecular marker SNP_A04_8857564 is T, the sequence of the 8857584bp of the A04 chromosome detected by the molecular marker INDEL_A04_8857584 is TA, the sequence of the 8857666bp of the A04 chromosome detected by the molecular marker SNP_A04_8857666 is G, the sequence of the 8857699bp of the A04 chromosome detected by the molecular marker INDEL_A04_8857699 is TTAG, and the single or combined use of the molecular markers detects that the above base types determine that the brassica napus sample is homozygous saline-alkali resistant haplotype; the sequence of the base at 8857551bp on the A04 chromosome detected by the molecular marker INDEL_A04_8857551 is A, the sequence of the base at 8857564bp on the A04 chromosome detected by the molecular marker SNP_A04_8857564 is G, the sequence of the base at 8857584bp on the A04 chromosome detected by the molecular marker INDEL_A04_8857584 is T, the sequence of the base at 8857666bp on the A04 chromosome detected by the molecular marker SNP_A04_8857666 is A, the sequence of the base at 8857699bp on the A04 chromosome detected by the molecular marker INDEL_A04_8857699 is T, and the single or combined use of the molecular markers detects that the base types are all homozygous and saline-alkali intolerant haplotypes of the brassica napus sample; if the molecular markers are used singly or in combination and two base types are detected simultaneously, the brassica napus sample is judged to be heterozygous haplotype.
The beneficial effects of the invention are mainly as follows:
The method can detect the salt-tolerant alkalinity of the brassica napus, can predict the salt-tolerant alkalinity of the brassica napus, can effectively select the salt-tolerant alkalinity of the brassica napus, can be used for molecular marker assisted breeding of the brassica napus with salt-tolerant alkalinity, accelerates the progress of high-oil-content breeding of the brassica napus, is simple and quick to detect, has low cost, is not influenced by environment, and is suitable for large-scale popularization and application.
Drawings
Fig. 1: the invention discloses a schematic diagram of a distribution result of biomass saline-alkali tolerance coefficient of cabbage type rape after saline-alkali stress.
Fig. 2: according to the invention, the relative yield of the cabbage type rape subjected to saline-alkali stress is combined with genome variation to carry out Manhattan diagram of whole genome association analysis.
Fig. 3: in the invention, a schematic diagram of haplotype analysis is carried out by utilizing the molecular marker of the main effect QTL locus of the saline-alkali tolerant A04 chromosome of the brassica napus.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The technical means used in the examples are conventional technical means well known to those skilled in the art unless otherwise specified. The reagents used in the examples were commercially available unless otherwise specified.
Example 1 saline-alkaline stress treatment and phenotyping of related populations
(1) 5.7.2017, A seed material of 505 parts of cabbage type rape core (from agricultural university of Huazhong) was planted in the inner Mongolian autonomous region Bayan, wuyuan county (105 DEG 12 '-109 DEG 53' E,40 DEG 13 '-42 DEG 28' N). The experiment was divided into a control group (CK, soil salinity of 0.10% -0.25%, pH of 7.5-8.0) and a saline-alkali group (YJ, soil salinity of 0.35% -0.53%, pH of 8.0-8.5), and each treatment was set up in 2 replicates.
(2) 4 Rows of cabbage type rape core germplasm materials are planted in each part, and the row spacing is 20cm. And thinning the seedlings in the 3-5 leaf period, wherein 8 plants are reserved in each row, and the plant spacing is about 20cm.
(3) 225Kg/hm 2 of compound fertilizer (N: P 2O5：K2 O=25:10:16) is applied as a base fertilizer during sowing, and 150kg/hm 2 of compound fertilizer (N: P 2O5：K2 O=25:10:16) is applied for 7 months and 1 day in 2017.
(4) And respectively carrying out sprinkling irrigation and watering on 5 months and 7 days, 5 months and 17 days and 7 months and 1 day of 2017.
(5) The cabbage type rape of the control group (CK) and the saline-alkali group (YJ) are respectively harvested according to different germplasm materials on the 9 th 2017 month 9 days (mature period). After the germplasm material is naturally dried, the dry weight (i.e. biomass) of the overground parts in the maturation period of various germplasm materials is examined.
(6) The saline-alkali resistance coefficient (saline-alkali resistance coefficient=saline-alkali group biomass/control group biomass) was calculated from the dry weight (i.e., biomass) of the aerial parts of the control group (CK) and the saline-alkali group (YJ), and the results are shown in table 1.
Table 1. 505 parts saline-alkali resistance coefficient value of cabbage type rape
Example 2 Whole genome correlation analysis
(1) The laboratory performs genomic re-sequencing on the collected correlated analysis population of 505 brassica napus compositions (Tang et al 2021), and obtains quality sequencing data by BWA (v 0.75) software (Heng et al 2009).
(2) The Darmor-bzh v 4.1.1 genome (http:// www.Genoscope.cns.fr/brassicanapus/data /) is used as a reference genome of the experiment, mutation analysis is carried out on the re-sequencing result of 505 cabbage type rape genomes, and 7,862,482 high-quality SNP and InDel markers are screened.
(3) In the whole genome association analysis, GEMMA software models are used, and a method of a mixed analysis model is adopted. By utilizing 7,862,482 high-quality SNP and InDel markers and combining a phenotype value (namely, saline-alkali tolerance coefficient), and taking 1.0e-06 as a significance threshold, the QTL locus related to saline-alkali tolerance is identified in the whole genome range.
(4) A main effect QTL locus with salt and alkali resistance is identified on the A04 chromosome of the cabbage type rape and is positioned between 8707583bp and 9007584bp of the A04 chromosome.
Example 3 candidate QTL site analysis and molecular marker development
(1) Based on the lead SNP (8857584 bp on A04 chromosome) in the main effect QTL locus with salt and alkali tolerance on A04 chromosome, the 150kb range at the upstream and downstream is defined as the QTL interval.
(2) Within this QTL interval, linkage Disequilibrium (LD) analysis was performed for each SNP and InDel marker.
(3) And calculating the contribution rate (PVE) of each SNP and InDel marker in the QTL interval to the saline-alkali tolerance.
(4) And screening candidate molecular markers in the QTL interval through LD values (R 2 > 0.98) between the SNP and the InDel markers and contribution rates (PVE > 0.4) of the SNP and the InDel markers to the saline-alkali tolerance, and identifying 16 SNP and 4 InDel molecular markers.
(5) From the molecular markers screened in the previous step of analysis, molecular markers INDEL_A04_8857551, SNP_A04_8857564, INDEL_A04_8857584, SNP_A04_8857666 and INDEL_A04_8857699 which are close to the physical position of the lead SNP in the QTL interval are selected for haplotype analysis of the QTL locus. The molecular markers INDEL_A04_8857551, SNP_A04_8857564, INDEL_A04_8857584, SNP_A04_8857666 and INDEL_A04_8857699 are used singly or in combination for detecting the base types, and when the sequences of detected bases are ATATATTAAATC, T, TA, G, TTAG, the brassica napus sample is a homozygous saline-alkali tolerant haplotype (haplotype: hap.A); the molecular markers INDEL_A04_8857551, SNP_A04_8857564, INDEL_A04_8857584, SNP_A04_8857666 and INDEL_A04_8857699 are singly or in combination used for detecting the base types, and when the detected base sequences are A, G, T, A, T, the brassica napus sample is homozygous saline-alkali intolerant haplotype (haplotype: hap.B). There was a significant difference in saline-alkali tolerance coefficient between the haplotypes identified as rap.a and rap.b (p=0.00062).
(6) Reference is made to SEQ ID NO:1 to SEQ ID NO:5, designing a primer for the molecular marker. The primer pair for detecting the molecular marker INDEL_A04_8857551 is an upstream primer INDEL-1F (5'-TTTAAAATGGGATAACTTCTGC-3') and a downstream primer INDEL-1R (5'-ATCAAACCAGGATGAATCTC-3'); the primer pair for detecting the molecular marker SNP_A04_8857564 is an upstream universal primer C-SNP1-F (5'-TGGGAAACTGGAATCACCT-3') and downstream specific primers X-SNP1-R (5'-GGATACATAAAGTGGTGGAGA-3') and Y-SNP1-R (5'-GGATACATAAAGTGGTGGAGC-3'); the primer pair for detecting the molecular marker INDEL_A04_8857584 is an upstream primer INDEL-3F (5'-ATGGGATAACTTCTGCATT-3') and a downstream primer INDEL-3R (5'-CAATCAAACCAGGATGAATCTC-3'); the primer pair for detecting the molecular marker SNP_A04_8857666 is a specific upstream primer X-SNP2-F (5'-ATTCCCAAACTGGGAAACTG-3'), a specific Y-SNP2-F (5'-ATTCCCAAACTGGGAAACTA-3') and a specific downstream universal primer C-SNP2-R (5'-GGAGAATCATCAGGAAGTTG-3'); the primer pair for detecting the molecular marker INDEL_A04_8857699 is an upstream primer INDEL-3F (5'-CAAATCAAGCTTCTCACAAAGA-3') and a downstream primer INDEL-3R (5'-TGAATAAACCTCATAGGGGAG-3').
(7) The molecular markers SNP_A04_8857564 and SNP_A04_8857666 are detected by using the KASP technology, and the KASP reaction is carried out on a LGC SNPLINE genotyping platform. The reaction system is as follows: mu.L of 2 XFLU-ARMS 2 XPCR mix VD (Guangzhou Gude), 0.2. Mu.L of typing primer X (10. Mu.M), 0.2. Mu.L of typing primer Y (10. Mu.M), 0.4. Mu.L of universal primer C (10. Mu.M), 4.2. Mu.L of template (rape leaf genomic DNA sample). The PCR amplification conditions were: pre-denaturation was carried out at 95℃for 1 min; the annealing temperature is reduced by 0.8 ℃ in each cycle for 5 seconds at 95 ℃ and 15 seconds at 63.4 ℃, and drop PCR is carried out in 9 cycles; amplifying at 95 ℃ for 5 seconds and at 57.5 ℃ for 15 seconds in 35 cycles; fluorescence scanning was performed at 16℃for 20 seconds for a total of 2 cycles. After the reaction is finished, fluorescent data are read, and if only a fluorescent signal corresponding to the primer X is detected, the detection site is a homozygous saline-alkali tolerant haplotype; if only the fluorescent signal corresponding to the primer Y is detected, the detection site is a homozygous saline-alkali intolerant haplotype; if two fluorescent signals are detected simultaneously, the detection site is a heterozygous haplotype.
(8) The molecular markers indel_a04_8857551, indel_a04_8857584 and indel_a04_8857699 were detected by ordinary PCR. The reaction system is as follows: 5. Mu.L of 2 XTaq Master Mix (Norflu), 0.5. Mu.L of upstream primer (10. Mu.M), 0.5. Mu.L of downstream primer (10. Mu.M), 4. Mu.L of template (rape leaf genomic DNA sample). The PCR amplification conditions were: 3 minutes at 95 ℃;95 ℃ for 15 seconds, 58 ℃ for 15 seconds, 72 ℃ for 20 seconds, and 35 cycles in total; and at 72℃for 5 minutes. Detecting by molecular marker INDEL_A04_8857551 electrophoresis, if the size of the PCR product is 133bp, the detection site is homozygous saline-alkali tolerant haplotype; if the size of the PCR product is 121bp, the detection site is a homozygous saline-alkali intolerant haplotype; if PCR products of 121bp and 133bp are detected simultaneously, the detection site is a heterozygous haplotype. Detecting by molecular marker INDEL_A04_8857584 electrophoresis, if the size of the PCR product is 129bp, the detection site is a homozygous saline-alkali tolerant haplotype; if the size of the PCR product is 117bp, the detection site is a homozygous saline-alkali intolerant haplotype; if PCR products of 117bp and 129bp are detected simultaneously, the detection site is a heterozygous haplotype. Detecting by molecular marker INDEL_A04_8857699 electrophoresis, if the size of the PCR product is 148bp, the detection site is homozygous saline-alkali tolerant haplotype; if the size of the PCR product is 145bp, the detection site is a homozygous saline-alkali intolerant haplotype; if PCR products of 145bp and 148bp are detected simultaneously, the detection site is a heterozygous haplotype.
Finally, the above examples are preferred embodiments of the present invention, and the present invention is not limited to the specific details in the above examples. Any modification, equivalent replacement, improvement, etc. made under the functional and structural principles of the present invention should be included in the protection scope of the present invention.
Description of the sequence Listing:
The sequence SEQ ID NO:1 is the genomic sequence of the 400bp sequences (total 801 bp) before and after the molecular marker INDEL_A04_ 8857551.
The sequence SEQ ID NO:2 is the genome sequence of 400bp sequences (total 801 bp) before and after the molecular marker SNP_A04_ 8857564.
The sequence SEQ ID NO:3 is the genomic sequence of 400bp (total 801 bp) before and after the molecular marker INDEL_A04_ 8857584.
The sequence SEQ ID NO:4 is the genome sequence of 400bp sequences (total 801 bp) before and after the molecular marker SNP_A04_ 8857666.
The sequence SEQ ID NO:5 is the genomic sequence of the 400bp sequences (total 801 bp) before and after the molecular marker INDEL_A04_ 8857699.
The sequence SEQ ID NO: 6. 7 is a PCR primer designed for the molecular marker INDEL_A04_8857551, including an upstream primer and a downstream primer.
The sequence SEQ ID NO: 8. 9, 10 are PCR primers designed for the molecular marker SNP_A04_8857564, comprising an upstream universal primer and two downstream specific primers.
The sequence SEQ ID NO: 11. 12 are PCR primers designed for the molecular marker INDEL_A04_8857584, including an upstream primer and a downstream primer.
The sequence SEQ ID NO: 13. 14, 15 are PCR primers designed for the molecular marker snp_a04_8857666, comprising two upstream specific primers and one downstream universal primer.
The sequence SEQ ID NO: 16. 17 are PCR primers designed for the molecular marker INDEL_A04_8857699, including an upstream primer and a downstream primer.
Claims (6)
1. A main effect QTL locus for salt and alkali resistance of cabbage type rape is characterized in that: the major QTL locus is located between the 8707583 ～ 9007584 th bases of the A04 chromosome of the brassica napus.
2. A cabbage type rape salt and alkali resistant molecular marker is characterized in that: the molecular marker is closely linked to the major QTL site of claim 1, including indel_a04_8857551, snp_a04_8857564, indel_a04_8857584, snp_a04_8857666, indel_a04_8857699, wherein,
The molecular marker INDEL_A04_8857551 is positioned at 8857551bp of a brassica napus A04 chromosome, the base at 8857551bp is ATATATTAAATC or A, and the mutation leads to polymorphism;
The molecular marker SNP_A04_8857564 is positioned at 8857564bp of the brassica napus A04 chromosome, the base at 8857564bp is T or G, and the mutation leads to polymorphism;
The molecular marker INDEL_A04_8857584 is positioned at 8857584bp of a brassica napus A04 chromosome, the base at 8857584bp is TA or T, and the mutation leads to polymorphism;
the molecular marker SNP_A04_8857666 is positioned at 8857666bp of a brassica napus A04 chromosome, the base at 8857666bp is G or A, and the mutation leads to polymorphism;
The molecular marker INDEL_A04_8857699 is positioned at 8857699bp of the brassica napus A04 chromosome, a base at 8857699bp of the A04 chromosome is TTAG or T, and the mutation leads to polymorphism.
3. A molecularly imprinted primer according to claim 2, characterized in that: the primer can specifically amplify the molecular marker of claim 2 and detect polymorphism caused by mutation thereof, wherein,
The primer of the molecular marker INDEL_A04_8857551 is designed by taking DNA fragments of 400bp sequences before and after 8857551bp of the A04 chromosome of brassica napus as templates, wherein the DNA fragments are shown in SEQ ID NO:1 is shown in the specification;
The primer of the molecular marker SNP_A04_8857564 is designed by taking DNA fragments of 400bp sequences before and after 8857564bp of the A04 chromosome of brassica napus as templates, wherein the DNA fragments are shown in SEQ ID NO:2 is shown in the figure;
The primer of the molecular marker INDEL_A04_8857584 is designed by taking DNA fragments of 400bp sequences before and after 8857584bp of the A04 chromosome of brassica napus as templates, and the DNA fragments are shown as SEQ ID NO:3 is shown in the figure;
The primer of the molecular marker SNP_A04_8857666 is designed by taking DNA fragments of 400bp sequences before and after 8857666bp of the A04 chromosome of brassica napus as templates, wherein the DNA fragments are shown in SEQ ID NO:4 is shown in the figure;
The primer of the molecular marker INDEL_A04_8857699 is designed by taking DNA fragments of 400bp sequences before and after 8857699bp of the A04 chromosome of brassica napus as templates, and the DNA fragments are shown as SEQ ID NO: shown at 5.
4. Use of the major QTL locus of claim 1 or the molecular marker of claim 2 or the primer of claim 3 for detecting salt-tolerant alkalinity in brassica napus.
5. A kit for detecting salt-tolerant alkalinity of brassica napus is characterized in that: the kit contains the primer of claim 3.
6. A method for detecting salt-tolerant alkalinity of brassica napus, which is characterized in that the genomic DNA of brassica napus is used as a template, and the primer of claim 3 is used for detecting molecular markers; the sequence of the 8857551bp of the A04 chromosome detected by the molecular marker INDEL_A04_8857551 is ATATATTAAATC, the sequence of the 8857564bp of the A04 chromosome detected by the molecular marker SNP_A04_8857564 is T, the sequence of the 8857584bp of the A04 chromosome detected by the molecular marker INDEL_A04_8857584 is TA, the sequence of the 8857666bp of the A04 chromosome detected by the molecular marker SNP_A04_8857666 is G, the sequence of the 8857699bp of the A04 chromosome detected by the molecular marker INDEL_A04_8857699 is TTAG, and the single or combined use of the molecular markers detects that the above base types determine that the brassica napus sample is homozygous saline-alkali resistant haplotype; the sequence of the base at 8857551bp on the A04 chromosome detected by the molecular marker INDEL_A04_8857551 is A, the sequence of the base at 8857564bp on the A04 chromosome detected by the molecular marker SNP_A04_8857564 is G, the sequence of the base at 8857584bp on the A04 chromosome detected by the molecular marker INDEL_A04_8857584 is T, the sequence of the base at 8857666bp on the A04 chromosome detected by the molecular marker SNP_A04_8857666 is A, the sequence of the base at 8857699bp on the A04 chromosome detected by the molecular marker INDEL_A04_8857699 is T, and the single or combined use of the molecular markers detects that the base types are all homozygous and saline-alkali intolerant haplotypes of the brassica napus sample; if the molecular markers are used singly or in combination and two base types are detected simultaneously, the brassica napus sample is judged to be heterozygous haplotype.
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