CN117604139A

CN117604139A - Molecular marker related to soybean oil content on soybean chromosome 12 and application thereof

Info

Publication number: CN117604139A
Application number: CN202311332537.3A
Authority: CN
Inventors: 韩雪; 齐照明; 冯学珍; 辛大伟; 胡利民; 杨明亮; 刘春燕; 陈庆山; 武小霞
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2023-10-16
Filing date: 2023-10-16
Publication date: 2024-02-27

Abstract

The invention provides a soybean oil content-related molecular marker located on a soybean chromosome 12 and application thereof. Belonging to the field of plant identification. In order to rapidly and accurately screen high-oil-content high-quality soybean varieties. The invention provides a molecular marker related to soybean oil content, wherein the gene of the molecular marker is Glyma.12G018300, the nucleotide site at 847 is A or G, and application of the markers in preparation of a kit for detecting soybean high oil content and a screening method. The selection of characters is realized through the selection of marks, the breeding efficiency is greatly improved, and the effect of directionally improving soybean varieties is realized, so that the soybean varieties with high oil content can be selected.

Description

Molecular marker related to soybean oil content on soybean chromosome 12 and application thereof

Technical Field

The invention belongs to the field of plant identification, and particularly relates to a molecular marker related to soybean oil content on a soybean chromosome 12 and application thereof.

Background

The soybean has rich nutrition, and oil content is about 20%. People can eat soybeans to supplement needed nutrients, and can prevent cardiovascular diseases of human bodies, the soybeans are important oil crops at the same time, can be processed into edible oil, can meet the dietary requirements of people, and meanwhile, the soybeans mainly consist of five fatty acids, and the fatty acids can prevent heart diseases, cancers and the like. Along with the increasing living standard of people, more and more people pay more attention to the edible health and the nutritional value of food, so the demand for soybeans is great, but the soybeans in China are more dependent on import from other countries, so the soybean oil content is greatly improved in China, the high-oil soybean varieties are cultivated, and the daily needs of people are met.

The oil content of soybean grains is a quality-related property, a relatively complex quantitative property, is controlled by a plurality of genes, is limited by genetic characteristics and a breeding method, is too slow in a traditional method, and is proposed as technology is continuously advanced, molecular auxiliary selection is provided, molecular markers are closely linked with genes for determining target properties on the basis of the traditional hybridization breeding method, the selection of the properties is realized through the selection of the markers, the breeding efficiency is greatly improved, and the effect of directionally improving soybean varieties is realized, so that the soybean varieties with high oil content can be selected.

Disclosure of Invention

The invention aims to rapidly and accurately screen high-oil-content high-quality soybean varieties.

The invention provides a molecular marker related to soybean oil content, wherein the gene of the molecular marker is Glyma.12G018300, and the nucleotide site at 847 is A or G.

The invention provides a primer sequence for amplifying the molecular marker, wherein the forward primer sequence is shown as SEQ ID NO.2 or SEQ ID NO. 3; the reverse primer sequence is shown in SEQ ID NO. 1.

The invention provides a SNP locus related to soybean oil content, wherein the SNP locus is positioned at 1280727 position on chromosome 12 of soybean, and the base of the locus is A or G.

The invention provides a primer sequence for amplifying the SNP locus, and the forward primer sequence is shown as SEQ ID NO.2 or SEQ ID NO. 3; the reverse primer sequence is shown in SEQ ID NO. 1.

The invention provides an application of the molecular marker, the primer sequence, the SNP locus or the primer sequence in preparing a kit for identifying soybeans with high oil content or soybeans with low oil content.

The invention provides a kit for identifying soybeans with high oil content or soybeans with low protein content, which comprises the primer sequences.

Further defined, the kit further comprises a Master Mix and water.

The invention provides a method for identifying the content of soybean oil, which comprises the following specific steps:

step 1: extracting DNA of soybean to be detected;

step 2: performing PCR reaction by using the primer sequence of the molecular marker of claim 2 or the primer sequence of the SNP locus of claim 4, detecting that the soybean of the variety to be detected is AA genotype, and if the soybean of the variety to be detected is GG genotype, the soybean of the variety to be detected is high oil content soybean.

Further defined, the conditions of the PCR reaction in step 2 are: (1) Hot Start (Hot Start): maintaining at 95deg.C for 30s for 1 cycle; (2) gradual cooling (Touch down): at 95℃for 60s and then at 63℃for 20s, each cycle was cooled by 0.8℃and a total of 10 cycles were performed from 63℃to 55 ℃. (3) PCR amplification (PCR): the reaction was carried out at 95℃for 60s and at 55℃for 20s for 30 cycles. (4) Plate Read: the reaction was maintained at 37℃for 60s and 1 cycle was performed.

Further defined, the AA genotype in the step 2 is that the base of the SNP locus is A, and the GG gene is that the base of the SNP locus is G.

The beneficial effects are that: the invention utilizes 1029 resource materials from 5 resource hybridization populations as experimental populations. The mutation genes are initially screened by sequence comparison in the corresponding parents, effective molecular markers are selected by designing primers aiming at mutation sites, the KASP technology in the SNP molecular marker technology is adopted for verification in resource materials, and finally, the gene is re-verified and polymerized in a resequencing population by haplotype analysis. The aim is to determine effective functional markers and important candidate genes, and the main research results are as follows:

(1) 4 SNP markers related to oil content were obtained and developed: chr3:43490555, chr12:1280727, chr17:5830450, chr17:5831106.

(2) Important candidate genes near SNP markers related to soybean oil fraction are Glyma.03G219900, glyma.12G018300 and Glyma.17G074400.

Drawings

FIG. 1 is a diagram showing the sequence alignment of genes related to oil;

FIG. 2 is a graph showing the results of expression of candidate genes at SNP sites associated with oil components;

FIG. 3 is a graph showing the result of KASP genotyping of oil-related SNP markers in a parent material;

FIG. 4 is a histogram of protein and oil content distribution for different populations of materials;

FIG. 5 is a graph showing the result of KASP genotyping of oil-related SNP markers in resource materials;

FIG. 6 is a graph of the mean high oil haplotype and low oil haplotype phenotypes of oil-related SNP sites;

FIG. 7 is a graph of the average high oil and low oil phenotype results for an oil-related polymerization effect;

FIG. 8 is a distribution histogram of the resequencing population proteins and oil content.

Detailed Description

Example 1.

1. 1029 parts of material from 5 hybridized colony resource materials were utilized. First, seven varieties of Suilng 76, suilng 69, suilng 49, suilng 35, suilng 42, dongsheng No.1 and Suilk No.3 are selected as parents, and hybridized combination is carried out, wherein the characteristics of the varieties are shown in Table 1. The hybridization combination is Suizhong 69 XSuizhong 76, suiximang 35 XSuizhong 76, dongsheng 1 XSuiximang 76, suiximang 3 XSuiximang 42 and Suiximang 49 XSuiximang 76. 1209 soybean germplasm resources from the above 5 hybrid combination F6 generations are selected as an experimental group, planted in a saleization separation experimental field of the national academy of sciences of Heilongjiang province in 2022, and field management method is the same as field management. In the vegetative growth stage, three young leaves at the top of the plant are adopted for extracting DNA, KASP typing experiments are carried out, threshing is carried out after the seeds are mature, and the seeds are used for measuring the protein and oil content.

Next, the study performed haplotype analysis and gene polymerization using 643 parts of the finished genome resequencing material from soybean improvement genetics laboratory.

TABLE 1 quality characterization of hybrid parents

Variety of species	Quality traits
		Seiner 76	High protein variety, 46.78% protein and 16.86% fat
Suinong 69	Disease-resistant variety, protein content 40.57%, fat content 19.46%
		Suinong 49	Special variety (large grain variety), protein content 41.24%, fat content 21.57%
Suinong 35	High oil soybean with protein content of 42.17% and fat content of 22.00%
		Seism 42	High fatty acid soybean variety, protein content 40.68%, fat content 20.00%,
dongsheng No.1	Protein content 41.30%, fat content 19.97%
		No.3 of no fishy bean	No fishy bean variety, protein content 37.37%; fat content 21.81%

2. Important allele mining

Important allele mining is a method of screening for sites that are significantly associated with a trait of interest by analyzing the correlation between genomic data and the trait of interest, and then combining phenotypes to further determine the effect of the allele on the trait of interest. In this study, coincidence rate was used to represent the effect of a site.

In order to preliminarily screen candidate genes related to soybean oil protein, firstly, a soybean gene sequence in a SoyBase (https:// www.soybase.org /) data platform is downloaded as a template, the gene sequence of a hybrid combination parent is extracted from 634 parts of soybean core planting sequencing resources in northeast areas, DNAMAN is utilized for sequence comparison, SNP loci which are located in a CDS region and cause amino acid change are screened out, candidate genes are preliminarily screened out, and the functions of the candidate genes are preliminarily explored. Secondly, determining SNP loci corresponding to each pair of parents, designing specific primers aiming at the loci, verifying in parent materials by adopting KASP technology in SNP molecular marking technology, and screening effective primers capable of distinguishing alleles. Finally, preparing the effective primer before the resource material sample reacts; the KASP master mix comprises a kit of LGC company in UK, which contains two general quenching fluorescent probes FAM and HEX, and core components such as Taq enzyme; whereas the KASP analysis mixture contained two forward primers and one reverse primer, wherein the two forward primers were designed based on the sequence specificity before and after the SNP site. These primers bind to different alleles and bind to FAM and HEX fluorescent probes when subjected to the KASP procedure, producing different fluorescent results. If a given SNP genotype is homozygous, a green or blue fluorescent signal will be generated; if the genotype is heterozygous, the result shows a red fluorescent signal.

According to the principle, a corresponding upstream and downstream 50bp base sequence can be selected on the SNP locus obtained by screening, and a KASP primer is designed by utilizing Premier5.0 software. The primers comprise two specific forward primers (F1/F2) and a common reverse primer (R). The forward primer not only has the characteristic of identifying different alleles, but also has fluorescent labels FAM (GAAGGTGACCAAGTTCATGCT) and HEX (GAAGGTCGGAGTCAACGGATT) with different colors connected to one end so as to realize the distinction of PCR amplification products. The primer sequences are shown in Table 2. The PCR reaction uses 384-well plate as carrier, adds the chemical substance needed by the reaction, and uses the Roche Light Cycler 480 II real-time fluorescence quantitative PCR instrument to make the reaction. The reaction procedure was divided into the following parts: (1) Hot Start (Hot Start): maintaining at 95deg.C for 30s for 1 cycle; (2) gradual cooling (Touch down): at 95℃for 60s and then at 63℃for 20s, each cycle was cooled by 0.8℃and a total of 10 cycles were performed from 63℃to 55 ℃. (3) PCR amplification (PCR): the reaction was carried out at 95℃for 60s and at 55℃for 20s for 30 cycles. (4) Plate Read: the reaction was maintained at 37℃for 60s and 1 cycle was performed. After the PCR reaction is completed, we need to read the end fluorescent signal.

Table KASP reaction System

TABLE 2 primer sequences

Results: in order to preliminarily determine candidate genes related to soybean oil fraction content, the study screens important genes which are collected and arranged by a subject group, are related to soybean oil fraction channels and are obtained by combining with MateQTL analysis, and the important genes are referred to in mining breeding evaluation and screening of excellent soybean protein and oil fraction alleles of a published article Meta-analysis and transcriptome profiling reveal hub genes for soybean seed storage composition during seed development of the subject group. Firstly, extracting the gene sequence of an important gene related to soybean oil content by utilizing a SoyBase (https:// www.soybase.org /) data platform, extracting the sequences of hybrid combined parents of Suilnong 76, suilnong 69, suilnong 49, suilnong 35 and Suilnong 43 from the soybean core germplasm sequencing resource in 634 northeast regions, and carrying out sequence comparison by utilizing DNAMAN according to the gene sequences related to Dongsheng No.1 and Suilnon-fishy bean No.3 in published articles, screening SNP loci which are positioned in a CDS region and cause amino acid change, and initially screening candidate genes.

GO notes and biological processes involving 109 genes of soybean oil fraction. Relates to the functions of fatty acid synthesis, lipid metabolism, various fatty acid desaturases and the like. For the 109 important genes, soybean reference genome from American variety Williams 82 downloaded on SoyBase platform is compared with extracted parent sequence in DNAMAN, wherein one SNP locus comparison result is shown as figure 1, and the female parent seiner 35 in the 35X seiner 76 hybridization combination is mutated from genotype T to A in the gene, and causes mutation of amino acid.

Finally, screening 62 SNP loci, namely 28 candidate genes related to oil, wherein 5 SNP loci are arranged on the gene Glyma.17G074400, and the number of the SNP loci is large; the other sites are distributed on the gene more uniformly. Preliminary studies of functional annotation of 27 candidate genes, such as Gprobable glycerol-3-phosphate acyltransferase, are membrane-associated enzymes that play a key role in the production of triacylglycerols and phospholipids; 1-stearoyl-acyl carrier protein desaturase plays an important role in unsaturated fatty acid biosynthesis; omega-6 fatty acid desaturases are capable of saturating omega-6 fatty acids in tandem, while 3delta8-sphingolipid desaturase is involved in the process of sphingoid metabolism, etc. The candidate genes including a plurality of genes including Glyma.01G120400, glyma.02G106300 and Glyma.07G151300 are related to encoding lipid related enzymes and proteins and play a key role in the synthesis, decomposition and transportation of intracellular lipids (as shown in Table 3).

TABLE 3 candidate genes near oil-related SNP markers

The results of the expression pattern analysis using the RNA-seq dataset in SoyBase (https:// www.soybase.org /) were mapped using TBtool software (FIG. 2), with orange to green color indicated the expression level from high to low. 14 candidate genes in 28 genes are expressed in each stage of the development of the seed grains, wherein the expression level of 9 genes such as Glyma.02G106300, glyma.09G195400, glyma.12G027300 and the like is low; the gene Glyma.12G018300 has higher expression quantity, especially the highest expression quantity in flowers; the difference in the expression levels of 4 genes, such as Glyma.07G030000, glyma.08G212900, glyma.14G095400, and Glyma.18G120400, is most remarkable. Gene Glyma.07G030000 shows the lowest expression level of Seed 14DAF in the Seed development period, and the peak value of Seed 28DAF is 2.5 times that of Seed 14 DAF; the gene Glyma.08G212900 has the lowest expression level of Seed 25DAF in the Seed grain development period, and the peak value of the Seed 21DAF is 2.4 times of the Seed 21 DAF; the gene Glyma.14G09540 has the lowest expression quantity of Seed 10DAF in the Seed grain development period, and the peak value of Seed 42DAF is 8.8 times of that of Seed 21 DAF; gene Glyma.18G120400 showed the lowest amount of expressed Seed 25DAF in its Seed stage, and the peak of Seed 10DAF was 4.5 times that of Seed 25 DAF.

To preliminarily determine SNP loci associated with soybean oil fraction, KASP primers were designed for the base sequences of 50bp upstream and downstream of the extraction site for the 62 SNP loci of the 28 soybean oil fraction-related candidate genes of Table 1 using Primer5.0 software (http:// www.premierbiosoft.com/index. Html). And verifying the primer in the parent corresponding to the hybridization group, and repeating the test at least three times for each pair of parent of the primer to improve the reliability of the result. FIG. 4 is one of many results, in which green and blue represent two different homozygous genotypes and red is heterozygous genotype. When the primer can be stably displayed as different homozygous genotypes in parents, the primer is judged to have a better typing effect. According to the KASP typing result, 17 excellent primers with better typing effect were finally screened out as shown in Table 2.

TABLE 4 SNP molecular marker loci associated with oil content

SNP numbering	Gene number	Base group	Chromosome of the human body	Position of
					23	Glyma.02G106300	T/A	Chr2	10109326
54	Glyma.02G106300	G/A	Chr2	10109331
					30	Glyma.02G274900	T/A	Chr2	47624936
31	Glyma.02G274900	T/C	Chr2	47625857
					63	Glyma.03G219900	T/C	Chr3	43490555
39	Glyma.07G030000	T/G	Chr7	2392752
					40	Glyma.07G151300	G/T	Chr7	18219120
42	Glyma.08G212900	G/C	Chr8	17252809
					33	Glyma.09G250400	C/T	Chr9	47499468
29	Glyma.11G106300	G/A	Chr11	8085118
					16	Glyma.12G018300	C/A	Chr12	1280727
79	Glyma.14G119000	A/G	Chr14	33480275
					18	Glyma.14G119000	A/C	Chr14	33480631
19	Glyma.17G074400	T/C	Chr17	5830450
					50	Glyma.17G074400	T/C	Chr17	5831106
51	Glyma.17G074400	C/A	Chr17	5830396
					52	Glyma.17G074400	G/T	Chr17	5831106

The specific distribution number of these 17 excellent sites on 20 chromosomes of soybean is Chr02 (4), chr03 (1), chr07 (2), chr08 (1), chr09 (1), chr11 (1), chr12 (1), chr14 (2), chr17 (4), and the greatest number of SNPs distributed on Chr07 chromosomes can be seen. Specific distributions in different hybridization combinations are 69×seism 76 (6), 1×seism 76 (10), 35×seism 76 (11), 3×seism 42 (3), 49×seism 76 (9), and it can be seen that there are many polymerized SNPs in 35×seism 76 hybridization combinations.

Kasp typing experiment

Adding the prepared KASP reaction system into a 384-well plate, obtaining experimental results through a Roche Light Cycler 480 II instrument, and then importing the results into an Excel table, and processing and analyzing by combining phenotype data, wherein the basic method comprises the following steps:

(1) Classifying materials according to soybean seed protein or oil phenotype, calculating the average value and standard deviation of each group of data, and determining a critical value according to the result of adding and subtracting the standard deviation from the average value;

(2) Materials above this value are referred to as high protein or high oil materials, and below this value are referred to as low protein or low oil materials, and the criterion is used to calculate the gene locus compliance, i.e., the proportion of materials that meet a phenotypic characteristic in the population.

(3) The sample data obtained from the KASP result are counted according to the high protein/oil component material and the low protein/oil component material, and the two data are added to obtain the total number, and the coincidence rate is obtained by dividing the coincidence number by the total number. Then, the high protein/oil material and the low protein/oil material were used as rows, the x-containing allele and the y-containing allele were used as columns, and finally a four-grid table (see table 5) of coincidence rates was constructed. According to the data in the four-grid table, the accuracy and the reliability of the detection method, and the possible misjudgment condition are analyzed, and necessary improvements are carried out.

(4) Judging whether the SNP locus is related to the soybean protein oil content by using hypothesis test: the original assumption is that H0 indicates that the content size is independent of the x/y allele, while HA indicates that there is a correlation between these two variables. By calculation we can get the coincidence rates P1 and P2 and determine whether the H0 hypothesis needs to be rejected and the HA hypothesis accepted based on P1, P2 and the set significance level α (60%).

Table 5 four grid table of compliance rates

	High protein/oil material	Low protein/oil material
			Containing x alleles	a	c
Containing y alleles	b	d
			Total number of	M	N

Note that: x and y are genotypes of KASP (kaSP) typing of SNP locus design primers, a is the number of x alleles in a non-sequencing high-protein or high-oil material typing result, b is the number of x alleles in a non-sequencing low-protein or low-oil material typing result, c is the number of y alleles in a non-sequencing high-protein or high-oil material typing result, d is the number of y alleles in a non-sequencing low-protein or low-oil material typing result, M is the total number of non-sequencing high-protein or high-oil materials, and N is the total number of non-sequencing low-protein or low-oil materials.

Results:

the study was directed to F from 5 hybridization populations ₅ The material is planted in the area of the seismosis in 2022. Wherein, the group 28 is the hybridization of the seiner 69 and the seiner 76, the male parent is a high-protein variety, the protein content is 46.78%, the female parent is a disease-resistant variety, 390 plants are planted together, and the phenotype data 245 plants are harvested and measured. Population 122 is the hybridization of Suinon 35 with Suinon 76, the male parent is highThe protein variety, the protein content of 46.78, the female parent of high oil variety, the oil content of 22.00%, the 390 plants are planted, and the phenotype data 274 plants are harvested and measured. Group 163 is the hybridization of the non-fishy bean 3 of the seiid with the seiid 42, the male parent is the non-fishy bean variety, the female parent is the high oleic acid variety, 390 plants are planted for harvesting and 205 plants are measured, and the like. Other cross-combining information is shown in Table 6, with final offspring co-harvested and measured for 1029 strains.

TABLE 6 Soybean resource Material information

Group numbering

Female parent

Protein content

Oil content

Father parent

Protein content

Oil content

Quantity of materials

28

Suinong 69

40.57％

19.64％

Seiner 76

46.78％

16.86％

245

119

Dongsheng 1

41.30％

19.97％

Seiner 76

46.78％

16.86％

212

122

Suinong 35

42.17％

22.00％

Seiner 76

46.78％

16.86％

274

163

Fishy smell of seiid 3

37.37％

21.81％

Seism 42

40.68％

20.00％

205

167

Suinong 49

42.17％

19.97％

Seiner 76

46.78％

16.86％

272

Soy protein, oil phenotype data were measured by a Foss grain analyzer for 2022 and were descriptive statistically analyzed using SPSS software. The maximum protein content of the 2022 material is 46.6 percent, 31.53 percent and the average value is between 36.5 percent and 40.5 percent; the maximum oil content is 25.64%, 16.16% and the average value is 18.46% -21.49%. The phenotype data are widely distributed and obviously different, the quantitative trait genetic characteristics are met, and the protein oil content of the material is moderately distributed in a bias manner through analysis of kurtosis and bias discovery, so that the material is suitable for subsequent research.

Analysis of the different hybridization populations revealed from Table 7 that among the five populations, the 49X 76 hybrid protein content was highest, the maximum was 46.63% and the average was 40.49%; the content of the combined oil of the hybridization of the 3 XSuinon 42 with no fishy smell is the highest, the maximum value is 25.64%, and the average value is 21.48%. The standard deviation of the protein content is between 1.24 and 2.40, and the variation coefficient is between

3.39 to 5.92 percent; the standard deviation of the oil content is between 0.72 and 1.37, the variation coefficient is between 3.27 and 6.84 percent, the total standard deviation is smaller, and no larger amplitude is generated.

TABLE 7 descriptive analysis of different populations of protein, oil quality traits

Drawing frequency distribution histograms of protein oil phenotype data of five groups by utilizing GraphPad Prism 8 software, wherein the protein content distribution is from 30% to 48%, and the group spacing is 1; the oil content distribution is 16% -25% and the group distance is 0.5. As can be seen from FIG. 4, the soybean seed protein oil content values of the respective populations were measured to show continuous distribution, and the normal distribution trend was evident. Secondly, the figure shows that the total protein content of the 49X-seiner 76 hybridization group and the 69X-seiner 76 hybridization group of the seiner is higher and concentrated to more than 40%, and the oil content of the seiner is relatively lower; the oil content of the 3 XSuinon 42 hybridization group without fishy smell is higher and concentrated to more than 21%, and the protein content is lower as a whole; the 35X seiner 76 hybridized colony protein content is concentrated at 38% -41%, and the oil content is concentrated at 19% -20.5%.

4. Verification of SNP loci associated with oil content

To verify the excellent allele associated with oil, the 17 primers screened were typed by the KASP platform, the fluorescent signal was shown green or blue if the genotype of a given SNP was homozygous, the fluorescent signal result was shown red if the genotype was heterozygous, and the KASP results for the oil-associated SNP locus shown in fig. 5.

Analysis of KASP results in combination with oil phenotype: at Chr3:43490555 (fig. 5 (5)): the high-oil material has 21 parts of TT genotype, the coincidence rate is 53.85%, the low-oil material has 26 parts of AA genotype, and the coincidence rate is 78.79%; at Chr12:1280727 (fig. 5 (12)): the high oil content material has 23 parts of GG genotype, the coincidence rate is 88.64%, the low oil content material has 16 parts of AA genotype, and the coincidence rate is 55.14%; at Chr17:5830450 (fig. 5 (16)): 45 parts of TT genotypes are adopted for the high-oil-content material, the coincidence rate is 55.56%, 56 parts of CC genotypes are adopted for the low-oil-content material, and the coincidence rate is 67.47%; at Chr17:5831106 (17 in fig. 5): the high oil content material had 27 parts of AA genotype, the compliance rate was 54.00%, and the low oil content material had 30 parts of GG genotype, the compliance rate was 88.83%. The four SNP markers can successfully carry out typing and show different genotypes in high and low proteins, and can better distinguish high and low protein materials, as shown in Table 8.

TABLE 8 screening results of SNP markers related to soybean seed oil

Note that: "shows excellent SNP locus with better screening effect

Finally, 4 SNP loci which are determined in resource materials and are related to soybean oil content are positioned in 3 important candidate genes: glyma.03G219900 (Chr 3: 43490555), glyma.12G018300 (Chr 12: 1280727), glyma.17G074400 (Chr 17: 5830450), glyma.17G074400 (Chr 17: 5831106) are as shown in Table 9, wherein the hybrid combination seism 69×seism 76 contains 2 mutation sites at SNP numbers 51, 52; the hybrid combination seism 35 x seism 76 contains 2 mutation sites at SNP numbers 51 and 52; the hybrid combination Dongsheng 1 XSuinon 76 contains 2 mutation sites at SNP numbers 63 and 79; the hybrid combination amblyseius free 3 x amblyseius 42 contains 4 mutation sites at SNP numbers 63, 79, 51 and 52; the hybrid amblyseius 49×amblyseius 76 contains 2 mutation sites at SNPs 63, 51. The hybridization combination seiid no fishy 3 x seiner 42 is polymerized with the most oil related SNP loci, the oil content is the highest in 5 groups, the oil content is concentrated to more than 21%, the highest value is 25.64%, and the phenotype is consistent with the genotype.

TABLE 9 SNP genotypes associated with Soybean oil

SNP numbering	Gene number	Position of	Maternal genotype and phenotype	Male parent genotype and phenotype
					63	Glyma.03G219900	Chr3:43490555	T (high oil content)	A (Low oil content)
79	Glyma.12G018300	Chr12:1280727	A (Low oil content)	G (high oil content)
					51	Glyma.17G074400	Chr17:5830450	T (high oil content)	C (Low oil content)
52	Glyma.17G074400	Chr17:5831106	A (high oil content)	G (Low oil content)

5. Haplotype analysis

Verifying the mined candidate genes in a resequencing population, and carrying out haplotype analysis on the candidate genes in 643 resequencing materials by using software, wherein the specific method is as follows:

(1) The SoyBase (https:// www.soybase.org /) data platform is utilized to extract the genome sequence of the soybean protein oil candidate genes, and the genome information of the resequencing population is combined to search all candidate genes, and the important candidate genes of the SNP loci are screened out.

(2) And then, dividing the similar SNP loci into a group for haplotype analysis, and analyzing the relationship between haplotype and phenotype in the important candidate genome sequence information.

(3) The boxplot was drawn using GraphPad Prism 8 software and the significance differences between the different haplotypes and their phenotypes in each important candidate gene were analyzed. Significance analysis the variance alignment was detected and multiple comparisons made using the Least Significant Difference (LSD) method in the one-way ANOVA model.

For haplotype analysis, subsequent studies were performed using the phenotype data of the 643 re-sequencing resource population provided by the present laboratory for two years 2018, 2019, and the population re-sequencing genotype data. The BIUP value of the protein oil content for two years is shown in figure 8, and the 2 quality character variation coefficients are between 4.7% and 4.9%, so that the BIUP value is stable and has no larger amplitude; the protein property of the protein is in medium bias distribution, and the oil property of the protein is in high bias distribution, so that the protein is suitable for subsequent experiments.

To further determine the relationship between important candidate genes and soybean oil content, haplotype analysis was performed on the SNP loci of the 3 important candidate genes, and joint analysis was performed by grouping the proximity loci into groups, each group producing a different haplotype. The proportion of haplotypes in 643 sequenced materials was analyzed, and the phenotypic mean of the different haplotypes was calculated for analysis of variance. The final analysis resulted in 2 groups of high and low oil haplotypes with large differences in average oil phenotype (see figure 6).

Analysis at gene Glyma.17G074400 gave high oil content excellent haplotype hap_4 (TCAGTCCCG) and low oil content excellent haplotype hap_5 (TTAGTCCCG), the high oil content excellent haplotype accounting for 55.1% of the oil content average value was 21.1%, the low oil content split haplotype accounting for 10.1% of the oil content average value was 20.4%; analysis at gene Glyma.12G018300 gave high oil content excellent haplotype Hap_1 (TCGAGGGAGGTCAA) and low oil content excellent haplotype Hap_7 (CCAAGGGAGGTCAC), the high oil content excellent haplotype was 61.7%, the average oil content was 21.1%, the low oil content haplotype was 14.2%, and the average oil content was 20.4%. Both genes achieved significant differences in oil content. Analysis on the gene Glyma.03G219900 gave a high oil content excellent haplotype Hap_2 (CCGAGTTAGC) and a low oil content haplotype Hap_1 (CCGAGTAAGC), the high oil content excellent haplotype was 6.7%, the average oil content was 21.1%, the low oil content haplotype was 84.3%, and the average oil content was 20.8%.

To further determine if there was synergy of the superior haplotypes in the high oil material, the material was subjected to a polymerization analysis in 643 heavy sequencing populations (as in Table 10). 130 parts of high-oil-content material with the oil content higher than 21.5% is selected, the haplotype ratio is counted and the polymerization effect is analyzed: analysis at gene Glyma.12G018300 gave 110 parts of material containing high oil genotype Hap_1 (TCGAGGGAGGTCAA), 84.6% of high oil material; analysis at gene Glyma.17G074400 shows that 106 parts of the material contains high oil genotype hap_4 (TCAGTCCCG) accounting for 80.8% of the high oil material; as a result of analysis at the gene Glyma.03G219900, 11 parts of the material contained high oil genotype Hap_2 (CCGAGTTAGC) accounting for 7.1% of the high oil material. Of 130 parts of material, 89 parts of material polymerized both Glyma.12G018300Hap_1 (TCGAGGGAGGTCAA) and Glyma.17G074400Hap_4 (TCAGTCCCG) high oil genotypes, accounting for 68.5% of the high oil material, were judged to have higher polymerization effects.

Selecting 180 parts of low-oil-content material with oil content lower than 20.5%, counting haplotype ratio and analyzing polymerization effect: analysis at gene Glyma.12G018300 gave 38 parts of material containing low oil genotype Hap_7 (CCAAGGGAGGTCAC), 21.1% of low oil material; analysis at gene Glyma.17G074400 shows that 25 parts of the material contains low oil genotype Hap_5 (TTAGTCCCG) accounting for 13.9% of the low oil material; analysis at gene Glyma.03G219900 gave 149 parts of material containing low oil genotype Hap_1 (CCGAGTAAGC) at 82.8% of the low oil material. Of 180 parts of the material, 37 parts of the material were polymerized with both Glyma.12G018300Hap_7 (CCAAGGGAGGTCAC) and Glyma.03G219900Hap_1 (CCGAGTAAGC) low oil genotypes, accounting for 20.6% of the low oil material, which was judged to have higher polymerization effects.

Table 10 Excellent haplotypes involved in polymerization in relation to oil

Gene	High oil genotype	Material	Low oil genotype	Material
					Glyma.12G018300	Hap_1(TCGAGGGAGGTCAA)	110	Hap_7(CCAAGGGAGGTCAC)	38
Glyma.17G074400	Hap_4(TCAGTCCCG)	106	Hap_5(TTAGTCCCG)	25
					Glyma.03G219900	Hap_2(CCGAGTTAGC)	11	Hap_1(CCGAGTAAGC)	149

The oil content phenotype after final polymerization is shown in fig. 7, with the highest oil content of 23.11%, the lowest 20.511% and the average value of 21.88% in 89 parts of material polymerized in the high oil genotype; the oil content of the 37 parts of the material polymerized in the low oil-based form was 20.46% at the highest, 17.87% at the lowest, and the average value was 19.73%.

Example 2.

1. A kit for screening high-oil soybean:

the forward primer sequence of the amplified molecular marker is shown as SEQ ID NO.2 or SEQ ID NO. 3; the reverse primer sequence is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer of the amplification SNP1 is shown as SEQ ID NO. 1;

2. the screening method comprises the following steps:

selecting a sample with unknown soybean oil content, and performing a PCR amplification procedure by using the kit for screening high-oil soybean in the first step: (1) Hot Start (Hot Start): maintaining at 95deg.C for 30s for 1 cycle; (2) gradual cooling (Touch down): at 95℃for 60s and then at 63℃for 20s, each cycle was cooled by 0.8℃and a total of 10 cycles were performed from 63℃to 55 ℃. (3) PCR amplification (PCR): the reaction was carried out at 95℃for 60s and at 55℃for 20s for 30 cycles. (4) Plate Read: the reaction was maintained at 37℃for 60s and 1 cycle was performed. The steps after KASP analysis are as follows:

the invention also provides a method for identifying the soybean with high oil content, which comprises the following specific steps:

(1) Extracting DNA of soybean to be detected;

(2) And (3) carrying out PCR reaction by using SEQ ID NO.2 or SEQ ID NO.3 and SEQ ID NO.1, wherein the soybean of the to-be-detected variety is detected to be of AA genotype, the soybean of the to-be-detected variety is detected to be of low oil content, and the soybean of the to-be-detected variety is detected to be of high oil content if the soybean of the to-be-detected variety is of GG genotype.

Results: the soybean oil content in the sample with unknown soybean protein content is detected, the primer label is used for marking GG genotype, and the soybean high oil content is consistent with the genotype obtained by marking detection. The low oil content of soybean is consistent with the genotype detected by the marker.

Claims

1. A soybean oil content-related molecular marker, wherein the gene of the molecular marker is Glyma.12g018300, and the nucleotide site at position 847 is a or G.

2. Amplifying the primer sequence of the molecular marker of claim 1, wherein the forward primer sequence is shown in SEQ ID NO.2 or SEQ ID NO. 3; the reverse primer sequence is shown in SEQ ID NO. 1.

3. A soybean oil content-related SNP locus, wherein the SNP locus is located at 1280727 on chromosome 12 of soybean, and the base of the locus is A or G.

4. Amplifying the primer sequence of the SNP locus according to claim 3, wherein the forward primer sequence is shown in SEQ ID NO.2 or SEQ ID NO. 3; the reverse primer sequence is shown in SEQ ID NO. 1.

5. Use of the molecular marker of claim 1, the primer sequence of claim 2, the SNP site of claim 3 or the primer sequence of claim 4 for the preparation of a kit for identifying high-oil content soybeans or low-oil content soybeans.

6. A kit for identifying high oil content soybeans or low protein content soybeans, comprising the primer sequence of claim 2 or claim 4.

7. The kit of claim 6, further comprising a Master Mix and water.

8. A method for identifying the component content of soybean oil, which is characterized by comprising the following specific steps:

step 1: extracting DNA of soybean to be detected;

9. The method according to claim 8, wherein the conditions for the PCR reaction in step 2 are: (1) Hot Start (Hot Start): maintaining at 95deg.C for 30s for 1 cycle; (2) gradual cooling (Touch down): at 95℃for 60s and then at 63℃for 20s, each cycle was cooled by 0.8℃and a total of 10 cycles were performed from 63℃to 55 ℃. (3) PCR amplification (PCR): the reaction was carried out at 95℃for 60s and at 55℃for 20s for 30 cycles. (4) Plate Read: the reaction was maintained at 37℃for 60s and 1 cycle was performed.

10. The method according to claim 8, wherein the AA genotype of the step 2 is the base of the SNP site is A, and the GG gene is the base of the SNP site is G.