CN105925722B

CN105925722B - QTL related to soybean protein content, method for obtaining molecular marker, molecular marker and application

Info

Publication number: CN105925722B
Application number: CN201610541719.5A
Authority: CN
Inventors: 齐照明; 陈庆山; 辛大伟; 蒋洪蔚; 武小霞; 胡振帮; 杜翔宇; 齐慧冬; 李伟
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2016-07-11
Filing date: 2016-07-11
Publication date: 2020-02-14
Anticipated expiration: 2036-07-11
Also published as: CN105925722A

Abstract

The invention provides two QTLs related to soybean protein content, a molecular marker obtaining method and application thereof. QTL are respectively positioned on linkage groups A2 and N and respectively positioned by molecular markers Satt409 and Satt584, wherein the 5 '-3' primer of the molecular marker Satt409 is shown as SEQ ID NO.3, and the 3 '-5' primer is shown as SEQ ID NO. 4; the 5 '-3' primer of the molecular marker Satt584 is shown as SEQ ID NO.5, and the 3 '-5' primer is shown as SEQ ID NO. 6. And respectively taking the corresponding soybean individual genome DNA as a template, carrying out PCR amplification on the template by using the 5 '-3' primer and the 3 '-5' primer, and carrying out polyacrylamide gel electrophoresis separation on a product obtained by the PCR amplification to obtain a target molecular marker. The obtained molecular marker can be used for soybean molecular breeding.

Description

QTL related to soybean protein content, method for obtaining molecular marker, molecular marker and application

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a QTL related to soybean protein content, a molecular marker and an obtaining method and application of the QTL and the molecular marker.

Background

Soybeans are important economic crops in China and all over the world, because the soybeans are one of the main sources of plant protein required in daily life of people, and are also important feed crops in animal husbandry and breeding industries. Therefore, the protein content in the soybean kernels becomes one of the important indexes for measuring the soybean variety. However, the high-protein soybean variety mainly produced in China at present cannot meet the daily production and living needs of people. Therefore, the improvement of the protein content of the existing soybean variety by using the modern molecular breeding technology becomes a main task of contemporary breeding researchers.

The soybean protein content belongs to the quantitative trait inheritance controlled by multiple genes, and QTL analysis and positioning are carried out on the trait, so that SSR sites with use values can be discovered to cultivate new varieties with high quality and high yield. A large amount of soybean protein QTL is analyzed at home and abroad, but efficient loci are not effectively determined, and the real role of the loci is not determined, so that the method cannot be directly applied to actual breeding and important loci are lost.

In recent years, molecular marker assisted breeding has been developed, but has some disadvantages. Firstly, soybean protein is inherited from a quantitative trait controlled by multiple genes, the inheritance mode is controlled by multiple genes, and because a single locus has little influence on the trait, a plurality of genes are required to control the trait, and environmental factors have great influence on phenotype, the effect of the single locus is difficult to directly evaluate, so that the soybean protein cannot be applied to actual breeding. The initial step of understanding quantitative trait inheritance is to consider the micro-effective polygenes as a whole for analysis by means of biometrical methods. Subsequently, a micro-effective polygene hypothesis appeared, and it was considered that the quantitative inheritance is controlled by a plurality of micro-effective genes. Recent research efforts have confirmed that this hypothesis is deficient. Quantitative trait inheritance has the common regulation of a main effective gene and a plurality of micro-effective genes, and the interaction of the genes and the environment has great influence on the phenotype. It can be said that the quantitative trait is expressed under the influence of the major site, the minor site and the environmental factor. Therefore, it is very important to determine the effect value of each site on the protein content from the gene level.

In addition, the hierarchical evaluation is a statistical method by means of chi-square test and T test, and is used for analyzing the effect value of a group of influence factors on a certain effect. Is a statistical method for analyzing a certain character by comprehensive evaluation criteria. Until now, the use of stratified evaluations in the biological field is not common. At present, a plurality of soybean breeding scholars at home and abroad study the quantitative inheritance characteristics of soybeans. Most of the published articles related to MAS and QTL positioning are potential application values of researched markers and loci, the MAS and QTL positioning methods are rarely really applied to actual breeding, few people draw conclusions on effect values of quantitative genetic genes, and the MAS and QTL positioning methods are blank on the aspect of soybean kernel protein content. It is well known that the protein content of soybean kernel is quantitative trait inheritance, controlled by multiple genes. Among these genes, certain genes have a large influence on the trait, and some have a small influence. Before the specific influence of each gene on the protein content, a trait, was known, it was difficult to apply these directly to actual breeding. The adoption of the hierarchical evaluation is a relatively effective solution.

Disclosure of Invention

Aiming at the problems, the invention provides 5 QTLs closely related to the soybean protein content in soybeans by statistical analysis methods such as hierarchical evaluation and the like, and accurately verifies the effect value and the action mode of the QTLs related to the soybean protein content, 3 key QTLs are found by calculating the change rate to increase the protein content, and 2 key QTLs are found to reduce the soybean protein content. So that the method can be directly applied to actual molecular breeding.

Among the five QTLs provided by the invention, Satt683 belongs to a major QTL of the protein content of soybean kernels. Satt409, Satt180, Satt584 and Satt181 belong to the high-efficiency QTL.

The alleles Satt683-3, Satt409-2 and Satt584-5 contained in the soybean grain have positive correlation with the characteristic expression of soybean protein content, namely the corresponding alleles of the soybean grain have the effect of promoting the soybean protein content to increase, and the Satt181-2 and Satt180-5 have negative correlation with the characteristic expression of the soybean protein content, namely the corresponding alleles of the soybean grain have the effect of inhibiting the large protein content to increase.

The information for 3 QTLs associated with soybean protein traits is shown in table 1, where the alleles listed are alleles that are positively associated with a trait.

TABLE 13 QTLs associated with the traits of soybean proteins

The information for 2 QTLs associated with a soybean protein trait is shown in table 2, where the alleles listed are alleles that are negatively associated with the trait.

TABLE 22 QTLs associated with soybean protein traits

Can be directly amplified by using a primer to identify whether a variety to be detected contains the allele, and is used for molecular marker-assisted selection.

The invention provides a QTL related to the content of soybean protein: located on linkage group N, at genomic position 27.787cM on the public map, located by the molecular marker Satt 683; the 5 '-3' primer of the molecular marker Satt683 is shown as SEQ ID NO.1, and the 3 '-5' primer is shown as SEQ ID NO. 2.

The QTL comprised an allele Satt683-3 with an effect value of 12%, which allele was positively correlated with soy protein content.

The invention also provides a method for obtaining the molecular marker of the QTL related to the soybean protein content, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.1, and the 3 '-5' primer is shown as SEQ ID NO. 2; taking the soybean variety northern bean No.5 as a recurrent parent, hybridizing with a swan egg ZDD02114, and obtaining soybean individual genome DNA after twice hybridization and twice backcross as a template; the above template was PCR-amplified using the above 5 '-3' primer and 3 '-5' primer in a PCR amplification system of 10. mu.L including 0.8. mu.L of 1.5 ng/. mu.L DNA template, 0.5. mu. mol/L of 5 '-3' primer, 0.5. mu.L of 3 '-5' each, 200mmol/L dNTP 0.6. mu.L, 0.05. mu.L of 5U/. mu.L Taq enzyme, 10XBuffer 1. mu.L, and MgCl as the cation₂The final concentration is 1.5-2.0mmol/L, and the rest is complemented with ultrapure water; the PCR amplification procedure was pre-denaturation at 94 ℃ for 5 min(ii) a Denaturation at 94 ℃ for 40 seconds, annealing at 47 ℃ for 40 seconds, extension at 72 ℃ for 40 seconds, and circulation for 35 times; extension at 72 ℃ for 5 minutes; the reaction was terminated at 4 ℃. Subjecting the product obtained by the PCR amplification to a PCR product deformation program: 5 minutes at 94 ℃, and obtaining amplified fragments with the size of 450bp, 430bp or 410bp after electrophoretic separation, namely the target molecular marker.

The invention also provides the molecular marker of the QTL related to the soybean protein content, which comprises the following components in percentage by weight: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.1, and the 3 '-5' primer is shown as SEQ ID NO. 2.

The invention also provides a method for soybean molecular breeding by using the molecular marker, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.1, and the 3 '-5' primer is shown as SEQ ID NO. 2; the 5 '-3' primer and the 3 '-5' primer are used for carrying out PCR amplification on the genome DNA of the soybean variety to be detected, the PCR amplification system is 10 mu L, the PCR amplification system comprises 0.8 mu L of DNA template with 1.5 ng/mu L, 0.5 mu mol/L of 5 '-3' primer, 0.5 mu L of 3 '-5' primer, 0.6 mu L of dNTP with 200mmol/L, 0.05 mu L of Taq enzyme with 5U/mu L and 10XBuffer1 mu L, and MgCl is adopted as cation₂The final concentration is 1.5-2.0mmol/L, and the rest is complemented with ultrapure water; the PCR amplification procedure is pre-denaturation at 94 ℃ for 5 minutes; denaturation at 94 ℃ for 40 seconds, annealing at 47 ℃ for 40 seconds, extension at 72 ℃ for 40 seconds, and circulation for 35 times; extension at 72 ℃ for 5 minutes; the reaction was terminated at 4 ℃. Subjecting the amplification product to a PCR product deformation program: and after electrophoretic separation at 94 ℃ for 5 minutes, if an amplified fragment with the size of 450bp, 430bp or 410bp exists in the amplified product, the soybean variety to be detected has QTLSatt683 related to the content of the soybean protein, if the amplified fragment with the size of 410bp exists in the amplified product, the soybean variety to be detected is a variety with allele Satt683-3 which is used for increasing the content of the soybean protein and is in the QTLSatt683 related to the content of the soybean protein, and if not, the variety to be detected does not have the QTL or the allele.

(II) the invention provides another QTL related to the soybean protein content: located on linkage group A2, having a genomic position of 145.565cM on the public map, located by the molecular marker Satt409, the 5 '-3' primer of the molecular marker Satt409 being shown as SEQ ID No.3 and the 3 '-5' primer as SEQ ID No. 4.

The QTL comprised an allele Satt409-2 with an effect value of 10%, which allele was positively correlated with soybean protein content.

The invention also provides a method for obtaining the molecular marker of the QTL related to the soybean protein content, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.3, and the 3 '-5' primer is shown as SEQ ID NO. 4; taking the soybean variety northern bean No.5 as a recurrent parent, hybridizing with a swan egg ZDD02114 or yellow, and obtaining soybean individual genome DNA after twice hybridization and twice backcross as a template; performing PCR amplification on the template by using the 5 '-3' primer and the 3 '-5' primer, wherein the PCR amplification system and the procedure are the same as those of the first step; and (3) carrying out electrophoretic separation on the product obtained by the PCR amplification to obtain amplified fragments with the size of 360bp, 330bp or 300bp, namely the target molecular marker.

The invention also provides the molecular marker of the QTL related to the soybean protein content, which comprises the following components in percentage by weight: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.3, and the 3 '-5' primer is shown as SEQ ID NO. 4.

The invention also provides a method for soybean molecular breeding by using the molecular marker, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.3, and the 3 '-5' primer is shown as SEQ ID NO. 4; the 5 '-3' primer and the 3 '-5' primer are used for carrying out PCR amplification on the genome DNA of the soybean variety to be detected, and the PCR amplification system and the procedure are the same; after the amplification products are subjected to electrophoretic separation, if amplification fragments with the sizes of 360bp, 330bp or 300bp exist in the amplification products, the soybean variety to be detected has QTLSatt409 related to the content of the soybean protein, if the amplification fragments with the sizes of 330bp exist in the amplification products, the soybean variety to be detected is a variety with allele Satt409-2 which is related to the content of the soybean protein and is used for increasing the content of the soybean protein in the QTLSatt409, and if not, the variety to be detected does not have the QTL or the allele.

(III) the invention provides another QTL related to the soybean protein content: located on linkage group N, at the genomic position of the public map 29.37cM, located by the molecular marker Satt584, the 5 '-3' primer of the molecular marker Satt584 is shown as SEQ ID No.5 and the 3 '-5' primer is shown as SEQ ID No. 6.

The QTL contained the allele Satt584-5 with an effect value of 10%, which allele was positively correlated with the soy protein content.

The invention also provides a method for obtaining the molecular marker of the QTL related to the soybean protein content, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.5, and the 3 '-5' primer is shown as SEQ ID NO. 6; taking the soybean variety northern bean No.5 as a recurrent parent, hybridizing with a swan egg ZDD02114, and obtaining soybean individual genome DNA after twice hybridization and twice backcross as a template; performing PCR amplification on the template by using the 5 '-3' primer and the 3 '-5' primer, wherein the PCR amplification system and the procedure are the same as those of the first step; and (3) carrying out electrophoretic separation on the product obtained by the PCR amplification to obtain amplified fragments with the size of 400bp, 350bp or 300bp, namely the target molecular marker.

The invention also provides the molecular marker of the QTL related to the soybean protein content, which comprises the following components in percentage by weight: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.5, and the 3 '-5' primer is shown as SEQ ID NO. 6.

The invention also provides a method for soybean molecular breeding by using the molecular marker, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.5, and the 3 '-5' primer is shown as SEQ ID NO. 6; the 5 '-3' primer and the 3 '-5' primer are used for carrying out PCR amplification on the genome DNA of the soybean variety to be detected, and the PCR amplification system and the procedure are the same; after the amplification products are subjected to electrophoretic separation, if the amplification fragments with the sizes of 400bp, 350bp or 300bp exist in the amplification products, the soybean variety to be detected has QTLSatt584 related to the soybean protein content, if the amplification fragments with the sizes of 300bp exist in the amplification products, the soybean variety to be detected is a variety with allele Satt584-5 which is used for increasing the soybean protein content in the QTLSatt584 related to the soybean protein content, and otherwise, the variety to be detected does not have the QTL or the allele.

(IV) the invention provides another QTL related to the soybean protein content: located on linkage group C1, having a genomic position of 105.092cM on the public map, located by the molecular marker Satt181, the 5 '-3' primer of the molecular marker Satt181 being shown as SEQ ID No.7 and the 3 '-5' primer as SEQ ID No. 8.

The QTL contained the allele Satt181-2 with an effect value of 8%, which allele was negatively associated with soybean protein content.

The invention also provides a method for obtaining the molecular marker of the QTL related to the soybean protein content, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.7, and the 3 '-5' primer is shown as SEQ ID NO. 8; taking the soybean variety BeiDou No.5 as a recurrent parent, hybridizing with semen sojae atricolor or Aika 166, and obtaining soybean individual genome DNA after twice hybridization and twice backcross as a template; performing PCR amplification on the template by using the 5 '-3' primer and the 3 '-5' primer, wherein the PCR amplification system and the procedure are the same as those of the first step; and (3) carrying out electrophoretic separation on the product obtained by the PCR amplification to obtain amplified fragments with the size of 300bp, 280bp or 180bp, namely the target molecular marker.

The invention also provides the molecular marker of the QTL related to the soybean protein content, which comprises the following components in percentage by weight: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.7, and the 3 '-5' primer is shown as SEQ ID NO. 8.

The invention also provides a method for soybean molecular breeding by using the molecular marker, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.7, and the 3 '-5' primer is shown as SEQ ID NO. 8; the 5 '-3' primer and the 3 '-5' primer are used for carrying out PCR amplification on the genome DNA of the soybean variety to be detected, and the PCR amplification system and the procedure are the same; after the amplification products are subjected to electrophoretic separation, if amplification fragments with the sizes of 300bp, 280bp or 180bp exist in the amplification products, the soybean variety to be detected has QTLSatt181 related to the content of the soybean protein, if the amplification fragments with the sizes of 280bp exist in the amplification products, the soybean variety to be detected is a variety with allele Satt181-2 which reduces the content of the soybean protein in the QTLSatt181 related to the content of the soybean protein, otherwise, the variety to be detected does not have the QTL or the allele.

(V), the invention provides another QTL related to soybean protein content: located on linkage group H, the genomic position on the public map is 87.597cM, located by molecular marker Satt180, the 5 '-3' primer of molecular marker Satt180 is shown as SEQ ID NO.9, and the 3 '-5' primer is shown as SEQ ID NO. 10.

The QTL contained the allele Satt180-5 with an effect value of 8%, which allele was negatively associated with soybean protein content.

The invention also provides a method for obtaining the molecular marker of the QTL related to the soybean protein content, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.9, and the 3 '-5' primer is shown as SEQ ID NO. 10; taking a soybean variety, namely northern bean No.5 as a recurrent parent, hybridizing with biogeidolote or Aika 166 or Ruifeng No.2, and obtaining soybean individual genome DNA after twice hybridization and twice backcross as a template; performing PCR amplification on the template by using the 5 '-3' primer and the 3 '-5' primer, wherein the PCR amplification system and the procedure are the same as those of the first step; and (3) carrying out electrophoretic separation on the product obtained by the PCR amplification to obtain amplified fragments with the size of 350bp, 340bp or 300bp, namely the target molecular marker.

The invention also provides the molecular marker of the QTL related to the soybean protein content, which comprises the following components in percentage by weight: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.9, and the 3 '-5' primer is shown as SEQ ID NO. 10.

The invention also provides a method for soybean molecular breeding by using the molecular marker, which comprises the following steps: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.9, and the 3 '-5' primer is shown as SEQ ID NO. 10; the 5 '-3' primer and the 3 '-5' primer are used for carrying out PCR amplification on the genome DNA of the soybean variety to be detected, and the PCR amplification system and the procedure are the same; after the amplification products are subjected to electrophoretic separation, if amplification fragments with the size of 350bp, 340bp or 300bp exist in the amplification products, the soybean variety to be detected has QTLSatt180 related to the content of the soybean protein, if the amplification fragments with the size of 300bp exist in the amplification products, the soybean variety to be detected is a variety with allele Satt180-5 which reduces the content of the soybean protein in the QTLSatt180 related to the content of the soybean protein, otherwise, the variety to be detected does not have the QTL or the allele.

The electrophoretic separation is polyacrylamide gel electrophoresis or capillary electrophoresis. Knowing the effect value of the protein related gene, we can judge whether the soybean of a certain soybean variety is high-quality soybean with high protein content by checking whether the soybean variety has high-efficiency loci, thus realizing the practical application of the gene in molecular assisted breeding.

The invention determines important and effective QTLs in a large number of known soybean protein related QTLs, and determines the specific influence of each gene on the shape of the soybean protein: whether the QTL is positively or negatively correlated, and the QTL is utilized to realize breeding by means of molecular markers.

Drawings

FIG. 1 Soybean Satt683 band patterns in accession numbers 1-61 of the 250 pieces of resource soybeans shown in Table 3;

FIG. 2 Cascade analysis of resource alleles;

FIG. 3 hierarchical analysis of resource alleles for T-test;

FIG. 4 SSR analysis of alleles in a population;

FIG. 5 Cascade analysis of population alleles;

FIG. 6 hierarchical analysis of population alleles for T-test;

FIG. 7 rates of change of individual alleles in the source and the northern bean population;

FIG. 8 is a capillary electrophoresis diagram of the extended products of the genomic DNA of the population in which the female parent of the primer pair of Satt683 is northern bean No.5 male parent and is swan egg ZDD 02114;

FIG. 9 is a capillary electrophoresis diagram of the extended products of the genomic DNA of the population in which the female parent of the primer pair of Satt409 is northern bean No.5 male parent and is Hydrangeae aspera ZDD02114 or Zhonghuang;

FIG. 10 capillary electrophoresis chart of the genomic DNA extension product of the population with female parent of Satt584 as northern bean No.5 male parent and as swan egg ZDD 02114;

FIG. 11 is a capillary electrophoresis chart of the extended products of genomic DNA from a population of Satt181 with the female parent northern bean No.5 and the male parent soybean or Eika 166;

FIG. 12 capillary electrophoresis images of the genomic DNA extension products of the population with the female parent of Satt180 as North Bean No.5 and the male parent as biogeidolote or Eka 166 or Ruifeng No. 2.

Detailed Description

In the following examples, the soy protein content was determined using a FOSS Infratec 1241 grain analyzer; capillary electrophoresis analysis all used a fully automated capillary electrophoresis apparatus, Advanced Analytical Technologies (AATI, USA), model Fragment Analyzer^TM。

Example 1 planting and protein content testing of experimental materials:

(1) planting and sampling of test materials

Resource materials: 250 parts of main-cultivated soybean varieties in China are selected. Table 3 shows the species name and the protein content of the granules of 250 parts of the resource.

Planting places: jilin province farm courtyard.

The planting method comprises the following steps: 1m rows long, 21 seeds per row.

The management method comprises the following steps: managing the same as the field.

The sampling method comprises the following steps: in the vegetative growth stage, the youngest leaf at the top of the plant is taken for DNA extraction and PCR reaction. At harvest, soybean kernels were harvested per individual plant for protein content determination.

And (3) verifying materials: the method is characterized in that a soybean variety, namely a northern bean 5, mainly planted in Heilongjiang province is taken as a recurrent parent, and is respectively hybridized with 19 varieties of soybeans, namely Dokatolisa, Hipposide yellow, Harosoy, green 75, Zhongte No.1, NOVA, Century, Dongnong 163, Dunageka, Amsoy, Zhongdou 27, 95-5383, Aika 166, Daiziandou, Dongshan 69, Ruifeng No.2, Zhonghuang, biogeidolote and Swan egg ZDD02114, and BC2F2 is harvested after two times of hybridization and two times of backcrossing. The population totaled 330 individuals.

Planting places: a research and breeding center for agricultural reclamation in Heilongjiang province.

The planting method comprises the following steps: 5m rows long, 100 seeds per row.

(2) Determination of protein content: the content of the grain protein of each soybean in the above-mentioned resource material and validation material was measured separately.

Procedure of use

1) Power to the Infratec 1241 grain analyzer was turned on.

2) And after the instrument is preheated by self-checking, finding the required model from the application model list column through up and down keys. There is an additional "STM" in the application model name.

3) The soybean kernels were poured into the sample tank, after which the holder was loaded into the docking unit, taking note that the side with the gear rack was on the right. The bracket is pressed down slightly to ensure that the rack and gear are properly engaged. The sample well holder is placed into the docking unit, at which time care is taken not to damage the internal pinion.

4) Pressing an analysis key in the keyboard area starts analysis.

The data measured by the FOSS Infratec 1241 grain analyzer is in the format of ". crd". It needs to be converted into EXCEL format before it can be opened. And a data exporting step:

the IFT folder contains two directories, Disk1 and Disk2, and a description file, and runs setup.exe under the Disk1 directory.

Begin → FOSS → IFT;

Result Data/CRD→TXT-format(columns)。

the path 20071030, crd is selected in the Source folder and check √ before the file. And then selecting an output path, such as Desktop, and clicking Convert selected.

And (5) naming the file and storing. The IFT is exited. A TXT file is obtained.

And newly building an Excel, opening a TXT file, selecting a separation symbol in a text import guide, clicking the next step, selecting a Tab key and a comma, clicking the next step, and completing clicking. The data for soy protein content as measured by the FOSS Infratec 1241 grain analyzer can be opened in Excel format.

TABLE 3250 parts Soybean resource names and their granulin protein content

Example 2 DNA extraction and PCR amplification:

(1) extraction of DNA from Soybean leaf (330 individual extracts of resource soybean and validation material 250 parts, each soybean extract separately)

1) The fresh leaves for DNA extraction and PCR reaction obtained in example 1 were placed in a 1.5ml centrifuge tube, and the centrifuge tube was placed on a centrifuge tube rack immersed in liquid nitrogen and cryogenically ground with a plastic drill until the sample became a white fine powder.

2) The water bath was opened and preheated to 65 ℃, while the CTAB extract (40 ml: mercaptoethanol was added to 10. mu.l) and heated to 65 ℃. After the temperature reached, 0.7ml of CTAB extract was added to each centrifuge tube, and after thorough mixing, it was water-bathed for 40 minutes and gently shaken intermittently.

3) An equal volume of chloroform was added to the centrifuge tube after the water bath, followed by centrifugation at 8000 (rpm) for 20 minutes. The supernatant was taken and an equal volume of chloroform was added again. Centrifuge 8000 (rpm) for 20 minutes.

4) The supernatant was added to 0.7ml of isopropanol which had been precooled in advance until DNA had precipitated out.

5) The DNA was washed with absolute ethanol, then air-dried, and dissolved in 80. mu.l of ultrapure water.

6) And detecting by 1% agarose gel electrophoresis. The voltage 100V and the current 100A run for 20 minutes.

Note that: 1) tip part of yellow pipette tip minus a small part to prevent shearing force from damaging DNA structure)

2) The liquid is transferred and the DNA is washed slowly and gently so as not to damage the DNA structure table 4 and the CTAB extracting solution formula for extracting the DNA of the soybean leaves

Name of medicine	Dosage (200ml)
		CTAB	4g
NaCl	13.364g
		EDTA	1.48g
Tris-HCl(pH8.0)	20ml
		Ultrapure water	The volume is up to 200ml

(2) PCR reaction

Selecting 29 pairs of primers distributed on 13 linkage groups of soybean DNA (see table 5), performing PCR amplification on the genomic DNA of 250 parts of resource soybean by using the selected 29 pairs of SSR primers, and analyzing by electrophoresis: in the system shown in Table 6, amplification was carried out according to the following procedure, and primers were synthesized from Shanghai.

PCR procedure: pre-denaturation at 94 ℃ for 5 min

Denaturation at 94 ℃ for 40 seconds

Annealing at 47 ℃ for 40 seconds

Extension at 72 ℃ for 40 seconds

Circulating for 35 times

Extension at 72 ℃ for 5 minutes

The reaction was terminated at 4 ℃.

TABLE 5 position and sequence of 29 primer pairs selected on 13 linkage groups of soybean DNA

TABLE 6 PCR reactions (10. mu.l system)

Medicine and food additive	Dosage (mu l)
		DNA template (stock diluted to 1.5 ng/. mu.l before use)	0.8
Primer (when in use, the stock solution is diluted to 0.5 mu mol/L)	1 (upstream and downstream primers 0.5)
		10Xbuffer Buffer	1
dNTP (diluted to 200mmol/L from stock solution before use)	0.6
		Taq enzyme (5U/. mu.l)	0.05
MgCl₂Solution (7.5. 10)^-3μmol/μl)	2
		Ultrapure water	4.55

Selecting the principle: 1) SSR primers are selected on each of 13 linkage groups as much as possible

2) The genetic distance of several primers selected from a linkage group is as far as possible

(3) Polyacrylamide gel electrophoresis: and (3) respectively carrying out polyacrylamide gel electrophoresis detection on the amplification products obtained in the step (2) to obtain PCR amplification product banding patterns of 29 pairs of primers of DNA of 250 resource varieties of soybeans and 330 verification resources shown in table 3, wherein partial banding patterns are shown in figure 1.

TABLE 7 Polyacrylamide gel electrophoresis drugs

Medicine and food additive	Formulation of	Dosage of
			Loading Buffer coloring agent	Carboxamides	980ml
	EDTA(0.5M,Ph＝8.0)	20ml
				Bromophenol blue	2.5g
	Xylene green	2.5g
			TBE (10 times)	Tris	108g
	Boric acid	55g
				EDTA	7.435g
PA(6％)	Methylene bisacrylamide	3g
				Acrylamide	57g
	Urea	420.42g
				TBE (10 times)	50ml
APS	Ammonium persulfate	100g
			Silver nitrate	AgNO₃Crystal	1000g

Polyacrylamide gel electrophoresis procedure

The large plate was wiped clean with alcohol, and affinity silane (alcohol: affinity silane: glacial acetic acid 5 ml: 50. mu.l) was applied uniformly

The ear plate is put into an air draft cabinet, wiped clean by alcohol, and then uniformly smeared with stripping silane.

The clean edge strips are placed on the two sides of the large plate, and then the ear plates are covered. The side of the two plates coated with silane faces inward. And fixing by a clamp.

Add 200. mu.l APS and 50. mu.l TEMED to the prepared 6% PA gel. Uniformly poured between the large plate and the ear plate, taking care not to generate bubbles.

The smooth side of the comb is inserted into the space between the two plates by about 5 mm. And (5) leveling. Standing for about one hour for use

The plate was placed on, clamped, and the electrophoresis chamber was poured into buffer TBE (1-fold TBE in the upper chamber and 1/3-fold TBE in the lower chamber).

A small amount of coloring agent is dropped into the sample application groove, and the pre-electrophoresis is carried out for about 10 minutes at 1800V. During the period, a staining agent is added to the PCR product and denaturation treatment is carried out.

The comb was inserted into the spotting cell (teeth down) and spotted at 1800V for 90 minutes. A lower plate. And taking down the ear plate and the edge strip.

Silver staining: 1500. mu.l of silver nitrate solution was added to 1500ml of distilled water. And soaking the large plate in silver staining solution for 20-30 minutes.

Distilled water was washed for half a minute.

10) And (3) developing: 1500ml of distilled water, 1500. mu.l of formaldehyde, 3% NaOH and 6.25g of Na were added₂CO₃. And soaking the large plate in silver staining solution for 20-30 minutes.

11) Counting the tape type, recording the data, and scanning.

Table 8 fragment sizes amplified by partial primers in the hierarchy

Example 3 hierarchical evaluation of soybean protein content-associated alleles:

(1) hierarchical evaluation of soybean protein content-related alleles

The data of the protein content of each of the 250 resource varieties shown in table 3 obtained in example 1 corresponds to the band type data thereof obtained in example 2(3) one by one, and the first 40% and the last 40% of the protein content are selected to analyze the effect values of the alleles by using the carter test of MATLABR2009 a. The results were divided into 3 levels with alpha being 0.01 and 0.1 as thresholds for the levels. Where we consider the site to be the major site when a P value is tested to be less than 0.01 and the site to be the minor site when a P value is greater than 0.1, and referring to FIG. 2, there are Satt683-3, Satt578-4, Satt409-2 and Satt079-1 for alleles with P values less than 0.01 level (very significant). Satt181-2, Satt180-5, Satt126-1, Satt578-2, Satt584-5, Satt409-10, Sat _311-2, Sat _242-2, Satt578-6, Satt584-6, Satt713-1, Satt012-5, Sat _216-2, Sat _216-3, Satt584-2, Satt700-2 and Satt369-2 are among the significant levels between 0.1 and 0.01, with the remaining alleles all having significant levels greater than 0.1.

After the Minitab14.12T test and referring to FIG. 3, a total of ten alleles with significance levels of less than 0.01, in contrast to the 4 most significant alleles from the previous chi-square test, a total of three alleles were found to be very significant under both the chi-square test and the T-test, as measured by Satt683-3, Satt578-4 and Satt409-2, respectively. In addition to these three extremely significant levels of alleles, the remaining alleles either appeared to be generally significant or insignificant, or were inconsistent in significance levels in both assays. Major genes and minor genes can be preliminarily distinguished.

(2) Validation of key alleles in northern bean No.5 population

All primers with extremely significant levels and other primers with higher significant levels are selected according to the resource stratification data. Chi-square and T-tests were performed. Meanwhile, in order to verify the establishment of the layering effect, a primer with an extremely low significance level is selected. Two analyses were performed as well.

The alleles Satt683-3, Satt578-4 and Satt409-2 were selected at very significant levels, the alleles Satt180-5, Satt181-2 and Satt584-5 were selected at a generally significant level for the chi-square detection and a very significant level for the T-detection, and primers for a total of 7 alleles for the allele Sat-174-2 (Table 5) at which neither chi-square nor T-detection was significant were selected, and genomic DNAs of 330 individuals of the population having northern bean number 5 as the recurrent parent were used as templates, and PCR amplification was performed in the system shown in Table 6 according to the following procedure, respectively, and analyzed by electrophoresis. The primer is synthesized by Shanghai. A portion of the electrophoretogram is shown in FIG. 4. And (5) counting the band type.

PCR procedure: pre-denaturation at 94 ℃ for 5 min;

the denaturation was carried out at 94 ℃ for 40 seconds,

annealing is carried out at the temperature of 47 ℃ for 40 seconds,

the elongation was carried out at 72 ℃ for 40 seconds,

circulating for 35 times;

the extension was carried out at 72 ℃ for 5 minutes,

the reaction was terminated at 4 ℃.

The same method as that of the present example (1) for processing banding pattern data of resources, the chi-square test and the T test were performed on the banding pattern data of 7 pairs of primers in the northern bean No.5 population:

the protein content data of the imported northern bean No.5 population individuals (330 verification resources) and the banding pattern data are in one-to-one correspondence

The protein content is used as a key word for full sequencing. Selecting the first 40% and the last 40%

Chi-square test using MATLAB R2009a

Minitab14.12 was opened for the T-test. And verifying the card party checking result.

The results are shown in FIGS. 5 and 6.

From the above two figures, Satt683-3 was still very significant in both assays, indicating that this allele indeed belongs to the major gene of soybean kernel protein content. Satt409-2 was still very significant in the T test, and remained at a higher level despite the reduction in chi-square test results. Satt180-5 and Satt584-5 were shown to be extremely significant in both tests, and together with Satt181-2, which performed consistently in the previous round of stratification evaluation, it was determined that these four alleles should belong to the high-efficiency gene, but the effect was not as significant as that of Satt 683-3. Sat _174-2 is completely consistent with the results of the previous round and is not significant, which indicates that the gene has low effect value in increasing protein content. It was unexpected that each allele of primer Satt578, which was significantly high in resources, was significantly low in northern bean populations, even though allele Satt578-4 was not found. The extreme phenomenon may be caused by a systematic error caused by, for example, the lack of the gene in the genome of 20 parents of the northern bean No.5 introgression line population, or the lack of accuracy of a measuring instrument.

Thus, although the stratification assessments in the two populations are slightly different, the differences are not large, and the overall stratification is still more evident, i.e. the significance level in the resource is higher, still higher in the northern bean No.5 population, and the roles of the alleles of the different primers in the two populations are consistent. Thus, it is considered that the results of allele-stratification evaluation of the 29 primers are accurate.

Example 4 analysis of allele additive effects using phenotypic rate of change validation:

the invention expresses whether the change of the allele is contained or not by the change rate, the change rate is based on the phenotype value difference of the maturity period of individuals in the resource or the group, if a certain allele has additive effect on the protein content of the soybean kernel in a positive direction, the phenotype data of the resource or the group of individuals containing the allele is higher than that of the resource or the individuals not containing the allele, but the influence of the interaction between the markers and the interaction between the alleles is not excluded. The calculation method of the change rate comprises the following steps:

where Rate of change is the Rate of change, Value A represents the average of the protein content phenotype values for individuals or resources containing the A allele, and Value B represents the average of the protein content phenotype values for individuals or resources containing the B allele or not containing the A allele.

Further validation of key allele additive effects using phenotypic rate of change

Theoretically, if the protein content of the soybean variety with a certain allele is higher than that of the soybean variety without the allele, the quantitative trait of the allele relative to the protein content is shown as a positively regulated positive correlation gene. Conversely, the allele is a negatively related gene. The invention selects 5 extremely significant alleles obtained by two times of hierarchical evaluation and screening, and selects a small northern bean No.5 population containing the 5 alleles (Table 9). Verification was performed by the rate of change of important primer alleles in the resource and northern bean No.5 population to protein content. The results are shown in FIG. 7. After screening, several primers and allele corresponding validation populations were obtained. The female parent of the population is Beidou No.5, and the male parent is shown in Table 9.

Table 9 screening of populations for Key alleles

It is clear from the table that the effect of these 5 alleles on the protein content of soybean kernels in resources and populations is consistent. The change rates of the three alleles of Satt683-3, Satt409-2 and Satt584-5 in the two populations are positive, which indicates that the alleles are positively correlated with the characteristic expression of the soybean protein content, i.e., the alleles play a role in promoting the increase of the soybean protein content in soybean kernels. While the rates of change of Satt181-2 and Satt180-5 were negative in both populations, demonstrating that the two alleles appear to be negatively correlated with respect to soybean protein content, i.e., the role of the alleles in soybean kernels is to inhibit the increase in large protein content.

Example 5 application of allelic loci in molecular marker breeding

Primers of QTL site Satt683 (SEQ ID NO.1 and SEQ ID NO.2), primers of QTL site Satt409 (SEQ ID NO.3 and SEQ ID NO.4), primers of QTL site Satt584 (SEQ ID NO.5 and SEQ ID NO.6), primers of QTL site Satt181 (SEQ ID NO.7 and SEQ ID NO.8) and primers of QTL site Satt180 (SEQ ID NO.9 and SEQ ID NO.10), and genomic DNAs of 330 individuals of a population taking northern bean No.5 as a recurrent parent are used as templates, and PCR amplification is performed according to the following procedures respectively in a system shown in Table 6, and analysis is performed by capillary electrophoresis (steps and parameter reference instrument description). The primer is synthesized by Shanghai.

PCR procedure: pre-denaturation at 94 ℃ for 5 min;

the denaturation was carried out at 94 ℃ for 40 seconds,

annealing is carried out at the temperature of 47 ℃ for 40 seconds,

the elongation was carried out at 72 ℃ for 40 seconds,

circulating for 35 times;

extension at 72 ℃ for 5 minutes;

the reaction was terminated at 4 ℃.

The result of capillary electrophoresis analysis shows that the genome DNA of the population of which the female parent of the primer pair of the Satt683 is the northern bean No.5 male parent and the swan egg ZDD02114 expands a molecular marker fragment with the size of 410bp (figure 8), which indicates that the genome DNA of the population contains the Satt683-3 allele for improving the content of the soybean protein;

the 330bp molecular marker fragment is expanded from the genome DNA of the population of which the female parent of the primer pair Satt409 is a northern bean No.5 male parent and is a swan egg ZDD02114 or medium yellow (figure 9), which indicates that the genome DNA of the population contains Satt409-2 allele for improving the content of soybean protein;

the genome DNA of the population of which the female parent of the primer pair of Satt584 is the northern bean No.5 male parent and the swan egg ZDD02114 expands a molecular marker fragment with the size of 300bp (figure 10), which indicates that the genome DNA of the population contains Satt584-5 allele for improving the content of the soybean protein;

the genome DNA of the population of which the female parent of the Satt181 is northern bean No.5 male parent and soybean seed or Eika 166 expands a molecular marker fragment with the size of 280bp (FIG. 11), which indicates that the genome DNA of the population contains Satt181-2 allele with reduced soybean protein content;

the genome DNA of the population of which the female parent of the primer pair of the Satt180 is the northern bean No.5 male parent and is the biogeidote or the Erca 166 or Ruifeng 2 expands a molecular marker fragment with the size of 300bp (figure 12), which indicates that the genome DNA of the population contains the Satt180-5 allele for reducing the content of the soybean protein.

Claims

1. A method for obtaining a molecular marker of QTL related to soybean protein content is characterized in that: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.3, and the 3 '-5' primer is shown as SEQ ID NO. 4; taking the soybean variety northern bean No.5 as a recurrent parent, hybridizing with a swan egg ZDD02114 or yellow, and obtaining soybean individual genome DNA after twice hybridization and twice backcross as a template; carrying out PCR amplification on the template by using the 5 '-3' primer and the 3 '-5' primer, and carrying out electrophoretic separation on a product obtained by the PCR amplification to obtain amplified fragments with the size of 360bp, 330bp or 300bp, namely the target molecular marker; the QTL related to the soybean protein content is positioned on the linkage group A2, the genomic position of a public map is 145.565cM, the QTL is positioned by a molecular marker Satt409, a 5 '-3' primer of the molecular marker Satt409 is shown as SEQ ID NO.3, and a 3 '-5' primer is shown as SEQ ID NO. 4; the QTL comprised an allele Satt409-2 with an effect value of 10%, which allele was positively correlated with soybean protein content.

2. A molecular marker of QTL related to soybean protein content is used for soybean molecular breeding, wherein a 5 '-3' primer of the molecular marker is shown as SEQ ID NO.3, and a 3 '-5' primer is shown as SEQ ID NO. 4; carrying out PCR amplification on the genome DNA of the soybean variety to be detected by using a 5 '-3' primer and a 3 '-5' primer, carrying out electrophoretic separation on an amplification product, wherein if the amplification product has an amplification fragment with the size of 360bp, 330bp or 300bp, the soybean variety to be detected has QTLSatt409 related to the content of soybean protein, if the amplification product has an amplification fragment with the size of 330bp, the soybean variety to be detected is a variety with allele QTLSatt409-2 which is related to the content of soybean protein and increases the content of soybean protein in the QTLSatt409, and otherwise, the variety to be detected does not have the QTL or the allele.

3. A method for obtaining a molecular marker of QTL related to soybean protein content is characterized in that: the 5 '-3' primer of the molecular marker is shown as SEQ ID NO.5, and the 3 '-5' primer is shown as SEQ ID NO. 6; taking the soybean variety northern bean No.5 as a recurrent parent, hybridizing with a swan egg ZDD02114, and obtaining soybean individual genome DNA after twice hybridization and twice backcross as a template; carrying out PCR amplification on the template by using the 5 '-3' primer and the 3 '-5' primer, and carrying out electrophoretic separation on a product obtained by the PCR amplification to obtain amplified fragments with the size of 400bp, 350bp or 300bp, namely the target molecular marker; the QTL related to the soybean protein content is positioned on the linkage group N, the genomic position of a public map is 29.37cM, the QTL is positioned by a molecular marker Satt584, a 5 '-3' primer of the molecular marker Satt584 is shown as SEQ ID NO.5, and a 3 '-5' primer is shown as SEQ ID NO. 6; the QTL contained the allele Satt584-5 with an effect value of 10%, which allele was positively correlated with the soy protein content.

4. A molecular marker of QTL related to soybean protein content is used for soybean molecular breeding, wherein a 5 '-3' primer of the molecular marker is shown as SEQ ID NO.5, and a 3 '-5' primer is shown as SEQ ID NO. 6; carrying out PCR amplification on the genome DNA of the soybean variety to be detected by using a 5 '-3' primer and a 3 '-5' primer, carrying out electrophoretic separation on an amplification product, wherein if the amplification product has an amplification fragment with the size of 400bp, 350bp or 300bp, the soybean variety to be detected has QTLSatt584 related to the content of the soybean protein, if the amplification product has an amplification fragment with the size of 300bp, the soybean variety to be detected is a variety with allele Satt584-5 which is related to the content of the soybean protein and increases the content of the soybean protein in the QTLSatt584, and otherwise, the variety to be detected does not have the QTL or the allele.