CN115725771A - Molecular marker related to soybean aging resistance, amplification primer and application thereof - Google Patents

Abstract

The invention relates to the field of soybean molecular breeding, in particular to a molecular marker related to soybean aging resistance, an amplification primer and application thereof. The method takes cultivated soybeans and wild beans as parents to construct a genetic population, positions 1 soybean aging-resistant related QTL (quantitative trait locus) on a No. 15 chromosome of the soybeans, has a marker interval of only 0.42Mb, screens 1 soybean aging-resistant related molecular marker SSR-15-327, and can be used for molecular marker-assisted selective breeding and mining related functional genes. Meanwhile, wild resources can be utilized, and the genetic basis of soybean cultivation is widened.

Description

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

Technical Field

The invention relates to the field of soybean molecular breeding, in particular to a molecular marker related to soybean aging resistance, an amplification primer and application thereof.

Background

The aging of soybean seeds refers to the irreversible change of reduced seed viability, reduced germination capacity and even loss of the germination capacity caused by the prolonged storage time of the seeds. The seed aging is not only related to the growth, development, yield and quality of seeds and seedlings, but also seriously influences the preservation, utilization and development of germplasm resources, so that the seed aging research has important significance on the breeding research of the soybean aging resistance traits. With the development of molecular genetics, molecular marker-assisted selective breeding provides a new way for people to accelerate the breeding process. Marker-assisted selective breeding refers to the indirect selection of a target trait by a molecular marker linked to a functional gene during breeding selection. The positioning of Quantitative Trait Loci (QTL) by using molecular markers is an important means for marker-assisted selective breeding. At present, researchers at home and abroad report some researches on soybean aging resistance related QTL, most QTL are obtained in a primary mapping population, and the positioning interval is large. In addition, most parents of the population are cultivars, and the genetic diversity of the cultivars is relatively single in long-term domestication and selection of human beings. The wild resources are rich in genetic variation and have a plurality of excellent properties, and the technical problem to be solved by the application is how to fully and reasonably utilize the wild resources and find the molecular marker related to the soybean aging resistance property.

Disclosure of Invention

In order to solve the technical problems, the invention provides a molecular marker related to soybean aging resistance, an amplification primer and application thereof.

In order to realize the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, molecular markers related to soybean aging resistance are provided, and the sequence is shown as SEQ ID NO. 1.

In a second aspect of the invention, primers for amplifying the molecular marker are provided, the sequences of which are shown in SEQ ID NO. 2-3.

In a third aspect of the invention, the application of the primer in screening the soybean line with the aging-resistant property is provided.

In a fourth aspect of the invention, the application of the primer in molecular breeding of soybean, cultivation of transgenic soybean or improvement of soybean germplasm resources is provided, wherein the improvement refers to improvement of the aging resistance of soybean.

In a fifth aspect of the invention, a method for identifying soybean aging resistance traits by using the primers is provided, which comprises the following steps:

s1, extracting reference soybean and soybean material genome DNA to be identified; the reference soybean is Jinong 26, jihei 4, guyun 100-4, gold element, fructus Piperis Longi, straw rain cape, jilin 3, japanese oyster 2, jinong 36, small grain fodder beans, jack yellow, white shells, syngnathus caucasianus, hulin Laotai, double Liao tea beans, group selection No.1 or nong' an Pingtui IV;

s2, carrying out PCR amplification on the reference soybean and the soybean material genome DNA to be identified by using an amplification primer;

s3, carrying out electrophoretic analysis on the amplification product, and identifying the electrophoretic band type of the soybean material amplification product;

and S4, if the electrophoresis band type of the soybean material to be identified is consistent with that of the reference soybean, identifying the soybean material as the excellent anti-aging material.

The invention has the following beneficial effects:

the germination vigor is the percentage of the number of the germinated seeds in the number of seeds of the variety to be tested when the number of the germinated seeds reaches the highest peak in the germination process, and the germination vigor of the seeds is high, which indicates that the seeds have strong vitality, are germinated in order and have consistent seedlings. According to the method, the soybean seeds are aged by using the artificial aging box, and the germination potential of the aged soybean seeds is used as an index for evaluating the aging resistance of the soybean seeds. And constructing a genome wide introductions line (CSSLs) population by crossing and backcrossing for multiple generations with the Sulnong 14 as a recurrent parent and the wild bean ZYD00006 as a donor parent. Each plant row only contains a few wild bean introduced fragments, so that the QTL interval can be shortened, the positioning precision and accuracy are improved, and molecular markers are developed in the interval, which is necessary for the auxiliary selection breeding and functional gene research of the aging-resistant molecular markers of the soybeans and has important significance for enriching the genetic diversity of soybean cultivars.

Until now, most of QTL positioning researches on soybean aging-resistant related traits are primary positioning, and the positioning accuracy of the markers is poor due to overlarge distances between the markers and the target traits. Random QTL positioning is a diversified recombination process under different genetic backgrounds of different combinations, so that the positioned QTL has the specificity of hybridization combination, has overlarge marker interval, can be lost along with the change of generations and genetic backgrounds, and is not beneficial to application. It is difficult to perform molecular assisted selection. The method takes cultivated soybeans and wild beans as parents to construct a genetic population, positions 1 QTL (quantitative trait loci) related to soybean aging resistance, has a marker interval of only 0.42Mb, screens 1 molecular marker SSR-15-327 related to soybean aging resistance, and can be used for molecular marker-assisted selective breeding and mining related functional genes. Meanwhile, wild resources can be utilized, and the genetic basis of soybean cultivation is widened.

Drawings

FIG. 1 is a drawing of the construction process of mapping population.

FIG. 2 is a flow chart of the re-sequencing.

FIG. 3 is a maternal chromosome coverage depth profile.

FIG. 4 is a distribution density chart of SNP markers and Bin markers of each chromosome.

FIG. 5 shows the germination vigor of the whole genome introduced line.

FIG. 6 shows the result of polyacrylamide gel electrophoresis.

Detailed Description

The invention is described in detail below with reference to the drawings and specific examples, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: acquisition of soybean aging-resistant-related QTL

1. Construction of mapping populations

The invention uses the seinquine 14 as a recurrent parent and the wild bean ZYD00006 as a donor parent, constructs a whole genome introgression line group through continuous backcross and selfing, and obtains 220 progeny strains in 2013-2016. See FIG. 1 for a specific generation.

2. Group genotype detection

The population is subjected to re-sequencing to detect the genotype (the flow is shown in figure 2), and the steps comprise:

1) And (3) extracting DNA of parent and progeny strains by using a CTAB method. And after the sample is detected to be qualified, randomly breaking the DNA by using an ultrasonic crushing method, repairing the tail end of a DNA fragment, adding A to the 3' end, adding a sequencing joint, purifying and performing PCR amplification to complete the construction of a sequencing library. The library is qualified by quality inspection and then sequenced by an Illumina HiSeqTM sequencing platform.

2) And (4) repositioning sequencing reads obtained by re-sequencing on a reference genome for subsequent variation analysis. And (3) comparing the short sequence obtained by the second generation high-throughput sequencing with a reference genome by using BWA software. And (3) positioning the positions of the Clean Reads on the reference genome by comparison, counting information such as sequencing depth, genome coverage and the like of each sample, and detecting variation.

3) For the results obtained from BWA alignment, duplicate was removed using Picard's Mark Duplicate tool, masking the effect of PCR-duplication. And (3) performing InDel reaction by using the GATK, namely performing local re-alignment on sites near the alignment result with the insertion deletion, and correcting the error of the alignment result caused by the insertion deletion. The Base quality values were corrected by Base Recalibration using GATK. Mutation detection (variant calling) was performed using GATK, mainly including SNP and InDel. Stringent filtration of SNPs: SNP cluster filtration (filtration if 2 SNPs exist within 5 bp), and SNP filtration near InDel (filtration of SNPs within 5bp near InDel); and adjacent INDEL filtering (two INDEL distances less than 10bp filtered out). The number of available SNP tags for the final screening was 580524.

4) And carrying out sliding scanning on the chromosome by using the obtained 580524 SNPs with 17 SNPs as a window and 1 SNP as a step length, typing as aa when the SNP in the sliding window is typed as aa is more than 12, typing as bb when the SNP in the sliding window is typed as bb is more than 14, and carrying out genotype filling and correction on ab in other cases.

5) And after the mark filling and correction are completed, performing Bin division according to the recombination condition of the filial generation. The samples were arranged in order according to the physical location of the chromosome, and when any sample showed a typing transition, it was considered that a recombination breakpoint occurred, then the SNPs between recombination breakpoints were classified into bins, and then Bin screening was performed, and finally 3196 bins were used as markers for mapping to locate (FIG. 4).

3. Phenotypic data acquisition

The soybean aging method comprises the following steps: the germination experiment was carried out in an artificial aging oven at 45 ℃ and 95% humidity for 4 days, and then the germination experiment was carried out at room temperature for 3-5 days.

Germination experiment: germination tests were performed according to germination conditions in International seed test protocols with minor modifications. A layer of filter paper is padded in each culture dish to serve as a germination bed, 40 soybean seeds are placed in each culture dish, water is added until a thin water film is seen on the paper, and a layer of wet filter paper is covered on the seeds. 3 replicates. Germinating in the incubator at a constant temperature of 20 ℃ in the dark. The number of seeds germinated was recorded daily and the germination vigor (day 4 of culture) was counted as shown in FIG. 5.

4. Soybean aging resistance QTL analysis

And (3) carrying out QTL positioning on aging resistance of the population by using an ICIM (complete interval mapping method) method in ICIMapping4.1 software, selecting an ICIM-ADD module for analysis, and adopting a CSL template for the introduction line population. And setting the LOD value to be more than or equal to 2.5, and carrying out QTL analysis on the soybean aging resistance. A soybean aging resistance related QTL is found on a chromosome 15, and the physical position is 6999904bp-7413169bp, as shown in Table 1.

TABLE 1 Soybean aging-resistant related QTL interval

Example 2: molecular marker related to soybean aging resistance, amplification primer and application thereof

60 parts of soybean existing varieties are utilized to identify molecular markers related to soybean aging resistance in an interval, and a molecular marker related to aging resistance is identified and located at 7213940bp-7213966bp of a soybean No. 15 chromosome and named as SSR-15-327. The nucleotide sequences are:

SSR-15-327(SEQ ID NO.1)：AATAATAATAATAATAATAATAATAAT。

the amplification primers are respectively:

SSR-15-0327F(SEQ ID NO.2)：5′-TGGTCCAACTAGCGATAGGG-3′；

SSR-15-0327R(SEQ ID NO.3)：5′-GCCGTTGTTATTGGACTTGG-3′。

the method for identifying the soybean aging-resistant character comprises the following steps:

s1, extracting soybean leaf genome DNA by a CTAB method;

s2, carrying out PCR amplification on the genomic DNA of the soybean material to be identified by using an amplification primer; the amplification system and procedure are shown in tables 2 and 3;

and S3, carrying out electrophoretic analysis on the amplification product, and identifying the electrophoretic band type of the amplification product of the soybean material.

And S4, if the electrophoresis band type of the material to be identified is consistent with the soybean aging resistant dominant band type (band type No.1 in figure 1) identified in the invention, the material is the soybean aging resistant excellent material.

TABLE 2 PCR reaction System

TABLE 3 PCR reaction procedure

As a result: statistics of phenotype and genotype of the existing varieties used for identifying the soybean aging-resistant related molecular markers are shown in table 4, a polyacrylamide gel electrophoresis result is shown in fig. 6, and a multiple comparison result of germination potentials of soybean varieties with different genotypes is shown in table 5.

TABLE 4 phenotype and genotype statistics for existing varieties used to identify molecular markers associated with soybean aging resistance

TABLE 5 multiple comparison results of germination potentials of soybean varieties of different genotypes

* Representing 0.05 level significance.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (5)

1. The molecular marker related to soybean aging resistance is characterized in that the sequence is shown in SEQ ID NO. 1.

2. The primer for amplifying the molecular marker of claim 1, wherein the sequence is shown in SEQ ID NO. 2-3.

3. Use of the primer of claim 2 for screening soybean lines having an aging-resistant trait.

4. The use of the primers of claim 2 in molecular breeding of soybean, breeding of transgenic soybean, or improvement of soybean germplasm resources, wherein the improvement is the improvement of the soybean aging resistance.

5. The method for identifying the soybean aging-resistant trait by using the primer of claim 2, comprising the following steps:

s1, extracting genome DNA of reference soybeans and soybean materials to be identified; the reference soybean is Jinong 26, jihei 4, guyun 100-4, gold element, fructus Piperis Longi, straw rain cape, jilin 3, japanese oyster 2, jinong 36, small grain fodder beans, jack yellow, white shells, syngnathus caucasianus, hulin Laotai, double Liao tea beans, group selection No.1 or nong' an Pingtui IV;

s2, carrying out PCR amplification on the reference soybean and the soybean material genome DNA to be identified by using an amplification primer;

s3, carrying out electrophoretic analysis on the amplification product, and identifying the electrophoretic band type of the soybean material amplification product;

and S4, if the electrophoresis band type of the soybean material to be identified is consistent with that of the reference soybean, identifying the soybean material as an excellent anti-aging material.

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