Molecular marker related to soybean aging tolerance, amplification primer thereof and application

Abstract

The present application relates to the field of soybean molecular breeding, and particularly relates to a molecular marker related to soybean aging resistance, an amplification primer thereof and application. The present application constructs a genetic population by taking cultivated soybean and wild soybean as parents, locates a QTL related to soybean aging resistance, which is located on chromosome 15 of soybean, and the marker interval is only 0.42 Mb, and screens a molecular marker SSR-15-327 related to soybean aging resistance, which can be used for molecular marker assisted selection breeding and mining of related functional genes. Meanwhile, wild resources can be used to broaden the genetic basis of cultivated soybean.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of soybean molecular breeding, and particularly relates to a molecular marker related to soybean aging tolerance, an amplification primer thereof and application. BACKGROUND

[0002] Soybean seed aging refers to an irreversible change of seed survival ability and germination ability decline or even loss caused by prolonging of seed storage time. The seed aging is related to seed and seedling growth, yield and quality, and seriously affects the preservation, utilization and development of germplasm resources, so it is of great significance to carry out breeding research on soybean aging tolerance. With the development of molecular genetics, molecular marker assisted selection breeding provides a new way for people to speed up the breeding process. Marker assisted selection breeding refers to indirect selection of target traits through molecular markers linked to functional genes during breeding selection. Using molecular markers to locate quantitative trait loci (QTL) is an important means of marker assisted selection breeding. At present, domestic and foreign researchers have reported some QTLs related to soybean aging tolerance. Most of the QTLs are obtained in primary mapping populations, and the positioning interval is large. Moreover, the parents of the population are mostly cultivated species, and the genetic diversity is relatively single in long-term domestication and selection of human beings. Wild resources have rich genetic variation and many excellent traits. How to fully and reasonably use wild resources to mine molecular markers related to soybean aging tolerance is a technical problem to be solved by the present application. SUMMARY

[0003] To solve the above technical problems, the present application provides a molecular marker related to soybean aging tolerance, an amplification primer thereof and application.

[0004] To achieve the above purpose, the present application adopts the following technical solutions:

[0005] In a first aspect of the present application, a molecular marker related to soybean aging tolerance is provided, and the sequence is shown as SEQ ID NO. 1.

[0006] In a second aspect of the present application, a primer for amplifying the molecular marker is provided, and the sequence is shown as SEQ ID NO. 2-3.

[0007] In a third aspect of the present application, the application of the primer in screening soybean lines with aging tolerance traits is provided.

[0008] In a fourth aspect of the present application, the application of the primer in soybean molecular breeding, breeding of transgenic soybean or improvement of soybean germplasm resources is provided, and the improvement refers to improving the aging tolerance of soybean.

[0009] In a fifth aspect, the application provides a method for identifying a soybean aging tolerance trait using the primer, comprising the following steps:

[0010] S1, extracting genomic DNA of reference soybeans and soybean materials to be identified; the reference soybeans are Jinnong 26, Jihel No. 4, Guoyu 100-4, Huangjin Yuan, Huili Zi, Miao Yi Ling, Jilin No. 3, Mushi No. 2, Jiu Nong 36, Xiaoli Mousi Dou, Qianjiahuang, Baikao, Hailong Zhui Dou, Hulin Laotai Chou, Shuangliao Chadou, Qunxuan No. 1 or Nongan Pingding 4;

[0011] S2, performing PCR amplification on the genomic DNA of the reference soybeans and the soybean materials to be identified using the amplification primer;

[0012] S3, performing electrophoresis analysis on the amplification products to identify the electrophoretic band type of the amplification products of the soybean materials;

[0013] S4, if the electrophoretic band type of the soybean materials to be identified is consistent with the electrophoretic band type of the reference soybeans, the soybean materials to be identified are identified as excellent aging-resistant materials.

[0014] The application has the following beneficial effects:

[0015] Germination potential refers to the percentage of the number of germinated seeds to the number of seeds of the sample to be tested when the number of germinated seeds reaches the highest peak in the germination process. High seed germination potential indicates strong seed vitality, uniform germination and consistent emergence. In the application, the germination potential of soybean seeds after aging treatment in an artificial aging box is used as an index for evaluating the aging resistance of soybean seeds. Heilongjiang main cultivar Suinong 14 is used as a recurrent parent and wild soybean variety ZYD00006 is used as a donor parent. After hybridization, multiple backcrossing and selfing, a population of whole genome introgression lines (CSSLs) is constructed. Each line contains only a few wild soybean introgression fragments, which can narrow the QTL interval, improve the positioning accuracy and precision, and develop molecular markers in the interval. This is very necessary for molecular marker-assisted selection breeding and functional gene research of soybean aging resistance, and also has important significance for enriching the genetic diversity of soybean cultivars.

[0016] So far, most of the QTL mapping studies on soybean aging tolerance-related traits are primary mapping, and the distance between markers and target traits is too large, and the accuracy of their positioning is poor. QTL random positioning is a diversified recombination process in different genetic backgrounds of different combinations, so the QTL positioned has hybrid combination specificity, the marker interval is too large, and it may be lost with the change of generation and genetic background, which is not conducive to application. It is difficult to carry out molecular assisted selection. The present application uses cultivated soybean and wild bean as parents to construct a genetic population, locates a QTL related to soybean aging tolerance, the marker interval is only 0.42 Mb, and screens a molecular marker SSR-15-327 related to soybean aging tolerance, which can be used for molecular marker assisted selection breeding and mining of related functional genes. At the same time, wild resources can be used to broaden the genetic basis of cultivated soybean. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The process diagram for mapping population construction.

[0018] Figure 2 The resequencing flow chart.

[0019] Figure 3 The maternal chromosome coverage depth distribution chart.

[0020] Figure 4 The SNP marker and Bin marker distribution density chart of each chromosome.

[0021] Figure 5 The germination potential of whole genome intro lines.

[0022] Figure 6 The polyacrylamide gel electrophoresis result. DETAILED DESCRIPTION

[0023] The present application will be described in detail below in conjunction with the drawings and specific examples, but should not be understood as limiting the present application. If not specially stated, the technical means used in the following examples are the conventional means familiar to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specially stated, can be obtained from commercial channels.

[0024] Example 1: Obtaining of QTL related to soybean aging tolerance

[0025] I. Construction of mapping population

[0026] The present application uses Suinong 14 as the recurrent parent and wild bean ZYD00006 as the donor parent, and constructs a whole genome intro line population through continuous backcrossing and selfing, and obtains 220 offspring lines in 2013-2016. The specific generations are shown in Table 1. Figure 1 .

[0027] II. Population genotype detection

[0028] Re-sequencing the population to detect genotypes (process as shown in Figure 2 , including the steps of:

[0029] 1) Extracting DNA of parents and offspring strains by CTAB method. After the sample is detected and qualified, the DNA is randomly broken by ultrasonic crushing method, and the DNA fragments are subjected to end repair, 3' end A addition, sequencing adapter addition, purification, and PCR amplification to complete the construction of sequencing library. After the library is qualified by quality inspection, sequencing is performed by Illumina HiSeqTM sequencing platform.

[0030] 2) Re-positioning the sequencing reads obtained by re-sequencing to the reference genome for subsequent variant analysis. The short sequences obtained by high-throughput sequencing are aligned to the reference genome by using BWA software. The positions of Clean Reads on the reference genome are located by alignment, and the sequencing depth, genome coverage, and other information of each sample are counted and analyzed for variant detection.

[0031] 3) For the results obtained by BWA alignment, use the Mark Duplicate tool of Picard to remove duplicates and shield the influence of PCR-duplication. Use GATK for InDel Realignment, i.e. local re-alignment of sites near the alignment results with insertions and deletions to correct alignment errors caused by insertions and deletions. Use GATK for Base Recalibration to correct the quality value of the base. Use GATK for variant calling, mainly including SNP and InDel. Strictly filter SNPs: filter out SNPs within 5bp if there are 2 SNPs, filter out SNPs within 5bp near InDel, and filter out two InDels with a distance less than 10bp. Finally, 580524 SNP tags are screened for use.

[0032] 4) Using the obtained 580524 SNPs, sliding on the chromosome with 17 SNPs as a window and 1 SNP as a step, when the number of SNPs with aa genotype in the sliding window is greater than 12, the genotype is aa, when the number of SNPs with bb genotype in the sliding window is greater than 14, the genotype is bb, and the other cases are ab for genotype filling and correction.

[0033] 5) After the marker filling and correction, the Bin division was carried out according to the recombination of the offspring. The samples were arranged in order according to the physical position of the chromosome, and when the typing transition appeared in any sample, it was considered that the recombination breakpoint appeared, and then the SNP between the recombination breakpoints was divided into Bin. After the bin screening, finally 3196 Bins were used as the markers for mapping for positioning. Figure 4

[0034] III. Phenotype data acquisition

[0035] Soybean aging method: After being treated in an artificial aging box at 45℃ and 95% humidity for 4 days, the seeds were balanced at room temperature for 3-5 days for germination experiment.

[0036] Germination experiment: The germination experiment was carried out according to the germination conditions in the International Seed Testing Rules with slight modification. A layer of filter paper was placed in a culture dish as a germination bed, 40 soybean seeds were placed in each culture dish, water was added until a thin water film was formed on the paper, and then a layer of moist filter paper was covered on the seeds. 3 times. Germination in a constant temperature incubator at 20℃ in the dark. The number of seeds germinated was recorded daily, and the germination potential (on the 4th day of culture) was calculated as shown in Table 2. Figure 5

[0037] IV. QTL analysis of soybean aging resistance

[0038] ICIM (complete interval mapping) method in ICIMapping 4.1 software was used for QTL positioning of population aging resistance, ICIM-ADD module was selected for analysis, and CSL template was imported for line population. The LOD value was set to be ≥2.5, and QTL analysis of soybean aging resistance was carried out. A QTL related to soybean aging resistance was found on chromosome 15, with a physical position of 6999904bp-7413169bp, as shown in Table 1.

[0039] Table 1. QTL interval related to soybean aging resistance

[0040]

[0041] Example 2: Molecular markers related to soybean aging resistance, amplification primers thereof and application

[0042] A molecular marker related to soybean aging resistance was identified in the interval using 60 soybean existing varieties, and a molecular marker related to aging resistance was identified, which was located at 7213940bp-7213966bp on soybean chromosome 15, named SSR-15-327. The nucleotide sequences are as follows:

[0043] SSR-15-327 (SEQ ID NO. 1): AATAATAATAATAATAATAATAATAAT. ​​

[0044] The amplification primers are respectively:

[0045] SSR-15-0327F (SEQ ID NO. 2): 5'-TGGTCCAACTAGCGATAGGG-3';

[0046] SSR-15-0327R (SEQ ID NO. 3): 5'-GCCGTTGTTATTGGACTTGG-3'.

[0047] The method for identifying the soybean aging resistance trait comprises the following steps:

[0048] S1, extracting soybean leaf genomic DNA by CTAB method;

[0049] S2, performing PCR amplification on the genomic DNA of the soybean material to be identified by using the amplification primers; the amplification system and procedure are shown in Table 2 and Table 3;

[0050] S3, performing electrophoresis analysis on the amplification products to identify the electrophoretic band type of the amplification products of the soybean material.

[0051] S4, if the electrophoretic band type of the material to be identified is consistent with the identified soybean aging resistance advantage band type (band type 1) in the present application, it is a soybean aging resistance excellent material. Figure 1

[0052] Table 2 PCR reaction system

[0053]

[0054] Table 3 PCR reaction procedure

[0055]

[0056] Results: The phenotype and genotype statistics of the existing varieties used for identifying the soybean aging resistance related molecular markers are shown in Table 4, the polyacrylamide gel electrophoresis results are shown in Figure 6 Table 5 shows the multiple comparison results of the germination potential of soybean varieties with different genotypes.

[0057] Table 4 Phenotype and genotype statistics of existing varieties used for identifying soybean aging resistance related molecular markers

[0058]

[0059]

[0060] Table 5 Multiple comparison results of germination potential of soybean varieties with different genotypes

[0061]

[0062] ​* represents a 0.05 level of significance.

[0063] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, it is intended that the appended claims include all such modifications and variations as fall within the scope of the present application.

[0064] It is apparent that those skilled in the art can make modifications and variations to the application without departing from the spirit and scope of the application. Accordingly, it is intended that the application embrace all such modifications and variations as fall within the scope of the claims and their equivalents.

Claims

1. A method of identifying soybean aging tolerance traits, comprising, Comprising the following steps: S1, extracting reference soybean and soybean material to be identified genomic DNA; the reference soybean is Jinnong 26, Jihel No. 4, Guoyu 100-4, Huangjin Yuan, gray pod, Mao Yi Ling, Jilin No. 3, Mushi No. 2, nine farmers 36, small grain Moushi bean, Qianjiahuang, white shell, Hailong pig kidney bean, Hulin Laotai Chao, double Liaochao bean, group selection No. 1 or Nongan Pingding four; S2, using the sequence as shown in SEQ ID NO. 2-3 amplification primer pair to amplify the genomic DNA of reference soybean and soybean material to be identified by PCR; S3, electrophoresis analysis of the amplification product, identification of soybean material amplification product electrophoresis band type; S4, if the electrophoresis band type of the soybean material to be identified is consistent with the electrophoresis band type of the reference soybean, it is identified as an excellent material resistant to aging.

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