Method for excavating genetic locus related to oil content and yield of soybean seeds by utilizing environmental difference, genetic locus, gene, protein and application of genetic locus, gene and protein

Abstract

The invention discloses a method for excavating genetic loci related to the oil content and yield of soybean seeds by utilizing environmental differences, the genetic loci, genes, proteins and application of the genetic loci, and belongs to the technical field of soybean breeding. A new site SO8 gene for regulating and controlling the oil content and the yield of the soybean seeds is excavated by utilizing environmental differences, a candidate gene GmAFP3 (Glyma. 08G246400) is further identified, the genetic site SO8 gene and the gene GmAFP3 can remarkably improve the oil content of the soybean seeds, the yield factor character levels such as the pod number of a single plant, the hundred-grain weight, the grain length, the grain width and the grain thickness are increased, and the yield of the soybean seeds is improved. The method can be used for creating soybean germplasm with different seed oil content and yield essential characters, has important theoretical value for soybean variety improvement, and also provides wide application prospects for improving the quality and yield of soybean oil.

Description

Technical Field

[0001] The present invention belongs to the technical field of soybean breeding, and particularly relates to a method for mining genetic loci related to soybean seed oil content and yield by using environmental differences, the genetic loci, genes, proteins and their applications. Background Art

[0002] Seed oil content and yield are classic agronomic traits that urgently need to be improved in soybeans. In recent years, with the increase in the number of genome-wide association studies (GWAS), many loci and genes affecting seed oil content and yield have been identified. Regarding the research on seed oil, such as the accumulation of oleic acid, linoleic acid and linolenic acid, can directly increase the seed oil content and simultaneously improve the soybean yield. At the same time, optimizing traits such as leaf epidermal hair morphology, branch number, and pod number per plant, and enhancing the plant's resistance to adversity can also increase the soybean yield. However, so far, a large number of loci and genes are still dormant and require specific environmental stimuli to activate their functions. Therefore, identifying the key genes regulating soybean seed oil and yield not only has important theoretical value for soybean variety improvement, but also provides broad application prospects for improving the quality and yield of soybean oil. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for mining genetic loci related to soybean seed oil content and yield by using environmental differences, and the mined genetic loci can significantly improve the levels of soybean seed oil content and yield-related traits.

[0004] Another purpose of the present invention is to provide a genetic locus SO8 gene related to soybean seed oil content and yield obtained by using the method.

[0005] Another purpose of the present invention is to provide a screening method for genes related to soybean seed oil content and yield.

[0006] Another purpose of the present invention is to provide a gene GmAFP3 related to soybean seed oil content and yield obtained by using the screening method.

[0007] Another purpose of the present invention is to provide a protein GmAFP3 related to soybean seed oil content and yield.

[0008] Another purpose of the present invention is to provide an application of the genetic locus SO8 gene, the gene GmAFP3 or the protein GmAFP3 in soybean germplasm improvement.

[0009] Another purpose of the present invention is to provide a method for increasing soybean seed oil content and / or yield.

[0010] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0011] The present invention provides a method for mining genetic loci related to soybean seed oil content and yield by using environmental differences. The method includes:

[0012] Introduce soybean varieties to be planted in areas with different environmental conditions from the original cultivation environment to obtain seed oil content data; perform genome-wide association analysis on the seed oil content and soybean 50K SNP gene chip data to obtain significant SNP loci; estimate the linkage disequilibrium intervals of the whole genome, determine the decay distance of the linkage disequilibrium intervals of the SNP loci, and this interval is the genetic locus related to soybean seed oil content and yield.

[0013] The present invention provides a genetic locus SO8 gene related to soybean seed oil content and yield obtained by the above method. The sequence of the genetic locus SO8 gene is from the 21,289,819th bp to the 21,486,468th bp of chromosome No. 8 of the soybean genome; the version of the soybean genome is Wm82.a2.v1.

[0014] The present invention also provides a method for screening genes related to soybean seed oil content and yield. Gene annotation is performed on the genetic loci obtained by the above method, and genes with significantly high expression and haplotype differences are screened, which are the genes related to soybean seed oil content and yield.

[0015] The present invention also provides a gene GmAFP3 related to soybean seed oil content and yield obtained by the above screening method. The sequence of the gene GmAFP3 is as shown in SEQ ID NO.1.

[0016] The present invention also provides a protein GmAFP3 related to soybean seed oil content and yield. The protein GmAFP3 is encoded by the gene GmAFP3.

[0017] The present invention also provides an application of the genetic locus SO8 gene or the gene GmAFP3 or the protein GmAFP3 in soybean germplasm improvement.

[0018] Preferably, the soybean germplasm improvement includes creating soybean germplasms with different seed oil content and / or yield element trait levels.

[0019] Preferably, the yield element traits include one or more of the number of pods per plant, 100-seed weight, seed length, seed width, and seed thickness.

[0020] Preferably, by regulating the expression level of the genetic locus SO8 gene or the gene GmAFP3, or regulating the content of the protein GmAFP3, soybean germplasms with different seed oil content and / or yield element trait levels are created.

[0021] Preferably, reducing the expression level of the genetic locus SO8 gene or the gene GmAFP3, or reducing the content of the protein GmAFP3, can reduce the soybean seed oil content and / or yield element trait levels; increasing the expression level of the genetic locus SO8 gene or the gene GmAFP3, or increasing the content of the protein GmAFP3, can increase the soybean seed oil content and / or yield element trait levels.

[0022] The present invention also provides a method for increasing the soybean seed oil content and / or yield, the method comprising: expressing the genetic locus SO8 gene or the gene GmAFP3 in soybean, or increasing the expression amount of the genetic locus SO8 gene or the gene GmAFP3 in soybean, or increasing the content of the protein GmAFP3 in soybean, or increasing the activity of the protein GmAFP3 in soybean, so as to increase the soybean seed oil content and / or yield.

[0023] Preferably, by introducing the genetic locus SO8 gene or the gene GmAFP3 or the protein GmAFP3 into soybean, the soybean seed oil content and / or yield is increased.

[0024] Advantages of the present invention:

[0025] The present invention provides a method for mining genetic loci related to soybean seed oil content and yield using environmental differences. Different environmental condition factors shape the adaptability of soybean through different selection pressures. Introducing soybean varieties into regions with different cultivation environments can activate previously dormant loci and genes, and can be identified by genome-wide association study (GWAS).

[0026] The present invention mines a new locus SO8 that regulates soybean seed oil and yield using environmental differences, and further identifies a candidate gene GmAFP3 (Glyma.08G246400). The genetic locus SO8 gene, the gene GmAFP3 or the protein GmAFP3 can significantly increase the soybean seed oil content, and increase the yield element trait levels such as the number of pods per plant, 100-seed weight, seed length, seed width and seed thickness. It can be used to create soybean germplasms with different seed oil contents and yield element traits, has important theoretical value for soybean variety improvement, and also provides a broad application prospect for improving the quality and yield of soybean oil. Description of the Drawings

[0027] Figure 1Identification of the soybean genetic locus SO8 locus and GMAFP3 haplotype analysis in Example 1; where a: Manhattan plot of GWAS results of seed oil content of 1466 soybean varieties; b: QQ plot of seed oil content; c: Linkage disequilibrium (LD) analysis; d: Candidate genes within the linkage interval, where GmAFP3 (Glyma.08G246400) is marked in red; e: Expression heatmap of candidate genes in different tissue parts; f: Schematic diagram of GmAFP3 gene mutation sites for haplotype analysis, with the A in the start codon ATG as the first base; g: GmAFP3 gene mutation position information for haplotype analysis, with the A in the start codon ATG as the first base; h: Statistical analysis of oil content of four haplotypes, different letters indicate statistically significant differences obtained by one-way analysis of variance (ANOVA) at P<0.05.

[0028] Figure 2 Results of seed oil content and yield determination of W82 and its mutants in Example 2; where a: Nucleotide changes in GmAFP3 mutant materials, with the A in the start codon ATG as the first base; b: Seed oil content of W82 and its mutants planted in Sanya, Hainan in 2023 (n≥5 biologically independent samples); c: Seed oil content of W82 and its mutants planted in Jize, Hebei in 2024 (n≥5 biologically independent samples); d: Number of pods per plant of W82 and its mutants planted in Jize, Hebei in 2024 (n = 5 biologically independent samples); e: 100-seed weight of W82 and its mutants planted in Jize, Hebei in 2024 (n = 5 biologically independent samples); f: Seed length of W82 and its mutants planted in Jize, Hebei in 2024 (n = 25 biologically independent samples); g: Seed width of W82 and its mutants planted in Jize, Hebei in 2024 (n = 25 biologically independent samples); h: Seed thickness of W82 and its mutants planted in Jize, Hebei in 2024 (n = 25 biologically independent samples). Detailed implementation mode

[0029] The present invention provides a method for mining genetic loci related to soybean seed oil content and yield by using environmental differences, and the method includes:

[0030] Introduce soybean varieties to be planted in an area with different environmental conditions from the original cultivation environment to obtain seed oil content data; perform genome-wide association analysis on the seed oil content and soybean 50K SNP gene chip data to obtain significant SNP loci; estimate the linkage disequilibrium intervals of the whole genome, determine the decay distance of the linkage disequilibrium intervals of the SNP loci, and this interval is the genetic locus related to soybean seed oil content and yield.

[0031] In the present invention, the area with different cultivation environmental conditions from the original cultivation environment is preferably an area with better cultivation environmental conditions than the original cultivation environment. The soybean 50K SNP gene chip data is preferably the Illumina Infinium SoySNP50K Bead Chip data downloaded from the Soybase database.

[0032] The present invention also provides a genetic locus SO8 gene related to soybean seed oil content and yield obtained by using the above method. The sequence of the genetic locus SO8 gene is from the 21,289,819th bp to the 21,486,468th bp of chromosome No. 8 of the soybean genome. The version of the soybean genome is Wm82.a2.v1.

[0033] The growth and development of crops are deeply affected by their cultivation environment. As two major agricultural producing countries, China and the United States exhibit very different environmental conditions. These different environmental condition factors have shaped the adaptability of crops through different selection pressures. Introducing Chinese soybean varieties into the cultivation environment in the United States may activate previously dormant loci and genes, which can be identified through genome-wide association analysis (GWAS). The present invention utilizes environmental differences to mine new loci regulating soybean seed oil and yield. Among them, Seed oil 8 (SO8) is a newly unnamed locus, which can significantly increase the soybean seed oil content and increase the yield-related trait elements such as the number of pods per plant, 100-seed weight, seed length, seed width, and seed thickness.

[0034] The present invention also provides a screening method for genes related to soybean seed oil content and yield. Gene annotation is performed on the genetic locus obtained by the above method, and genes with significantly high expression and haplotype differences are screened, which are genes related to soybean seed oil content and yield.

[0035]

[0036] The present invention further screens among the obtained genetic locus SO8 gene, and annotates 15 protein-coding genes within this interval range, including the GmAFP3 gene. The GmAFP3 gene shows significantly higher expression during seed development and can control the oil content and yield of soybean seeds.

[0037] The present invention also provides a protein GmAFP3 related to the oil content and yield of soybean seeds, and the protein GmAFP3 is encoded by the gene GmAFP3.

[0038] The present invention also provides an application of the genetic locus SO8 gene, the gene GmAFP3 or the protein GmAFP3 in the improvement of soybean germplasm.

[0039] In the present invention, the improvement of soybean germplasm preferably includes creating soybean germplasms with different levels of oil content and / or yield element traits. The yield element traits preferably include one or several of the number of pods per plant, 100-seed weight, seed length, seed width, and seed thickness.

[0040] In the present invention, it is preferably to create soybean germplasms with different seed oil content and / or yield element trait levels by regulating the expression level of the genetic locus SO8 gene or the gene GmAFP3, or regulating the content of the protein GmAFP3. As an alternative embodiment, reducing the expression level of the genetic locus SO8 gene or the gene GmAFP3, or reducing the content of the protein GmAFP3, can reduce the soybean seed oil content and / or yield element trait levels; the method for reducing the expression level of the genetic locus SO8 gene or the gene GmAFP3 can be conventionally selected according to actual needs, such as specifically silencing or mutating the genetic locus SO8 gene or the gene GmAFP3 to reduce the expression level of the genetic locus SO8 gene or the gene GmAFP3. In some embodiments, mutating the gene GmAFP3 reduces the soybean seed oil content and / or yield element trait levels; the mutated site can be conventionally selected according to actual needs. For example, taking the A in the start codon ATG in SEQ ID NO.1 as the first base, mutating one or several of the -17th, 326th, 563rd, 3092nd, 3152nd, 3308th, 3373rd, 3391st and 3537 - 3539th bases to obtain soybean germplasms with reduced soybean seed oil content and / or yield element trait levels; preferably, the -17th base is mutated from C to T; the 326th base is mutated from T to A; the 563rd base is mutated from A to T; the 3092nd base is mutated from G to A; the 3152nd base is mutated from C to T; the 3308th base is mutated from G to A; the 3373rd base is mutated from G to A; the 3391st base is mutated from G to A; the 3537 - 3539th bases are mutated from TCT to -. As another alternative embodiment, increasing the expression level of the genetic locus SO8 gene or the gene GmAFP3, or increasing the content of the protein GmAFP3, can increase the soybean seed oil content and / or yield element trait levels.

[0041] The present invention also provides a method for increasing the soybean seed oil content and / or yield, the method comprising: expressing the genetic locus SO8 gene or the gene GmAFP3 in soybean, or increasing the expression level of the genetic locus SO8 gene or the gene GmAFP3 in soybean, or increasing the content of the protein GmAFP3 in soybean, or increasing the activity of the protein GmAFP3 in soybean to increase the soybean seed oil content and / or yield.

[0042] In the present invention, the method for increasing the expression level of the genetic locus SO8 gene or the gene GmAFP3 in soybeans, or increasing the content of the protein GmAFP3 in soybeans can be conventionally selected according to actual needs. For example, techniques such as gene amplification and gene editing can be used to specifically promote the expression of the genetic locus SO8 gene or the gene GmAFP3, or an activator of the genetic locus SO8 gene or the gene GmAFP3 or the protein GmAFP3 can be used; the activator includes substances that can promote the expression of the genetic locus SO8 gene or the gene GmAFP3 or the protein GmAFP3, including but not limited to nucleic acid molecules, nucleic acid constructs, inorganic compounds, or organic compounds, etc.; the nucleic acid molecules include the genetic locus SO8 gene or the gene GmAFP3, specific microRNAs of the genetic locus SO8 gene or the gene GmAFP3, nucleic acid molecules that activate the promoter of the genetic locus SO8 gene or the gene GmAFP3, etc.; the nucleic acid construct is a gene fragment containing the gene encoding the above nucleic acid molecule and can express the nucleic acid molecule. As an alternative embodiment, by introducing the genetic locus SO8 gene or the gene GmAFP3 or the protein GmAFP3 into soybeans, the oil content and / or yield of soybean seeds can be increased.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] In the following embodiments, unless otherwise specified, all are conventional methods.

[0045] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0046] Example 1

[0047] 1. Obtain the data on the oil content of seeds of 1466 soybean varieties (collected in China and planted in the United States) from the website of the United States Department of Agriculture (USDA) (https: / / npgsweb.ars-grin.gov / gringlobal / search) (Table 1);

[0048] Table 1 Oil content of seeds of 1466 soybean varieties

[0049]

[0050]

[0051]

[0052]

[0053] 2. Download the Illumina Infinium SoySNP50K Bead Chip data from the Soybase database (https: / / www.soybase.org / snps / ) and use it to genotype the population;

[0054] 3. In TASSEL v5.0, import the seed oil content and SoySNP50K Bead Chip data for genome-wide association analysis (GWAS), and calculate the P-value of each SNP marker through the F-test. P ≤ 1×10 -6 (i.e., -log 10 P ≥ 6) is a significant SNP. At the same time, estimate the genome-wide linkage disequilibrium (LD) interval through PopLDdecay v3.42, calculate the paired squared allele frequency correlation (r 2 ) between the known genomic positions of SNPs. When r 2 = 0.2, the LD decay distance was determined to be 196.65 kb, that is, a total of 196.65 bk intervals on both sides of each SNP locus are the corresponding genetic loci. The results showed that a total of 6 significant SNP loci were detected on chromosomes 5, 8, and 15, and 5 of these SNP loci coincided with the previously reported seed oil genetic loci or genes (ss715591641 / GmMFT, ss715601154 / Seed oil 24-1, ss715621777 / GmSWEET39, ss715620444 / Seed oil 27-2, ss715637321 / Seed oil 2-1). Among them, ss715600679 is a newly reported locus, and we named the corresponding interval Seed oil 8 (SO8) ( Figure 1 as shown in a-c); SO8 is located at positions 21,289,819 bp to 21,486,468 bp on chromosome 8 of version Wm82.a2.v1;

[0055] 4. Fifteen protein-coding genes were annotated within the 196.65 kb (Wm82.a2.v1; Chr08: 21289819...21486468) interval of SO8 ( Figure 1 as shown in d), and only Glyma.08G246400 showed significantly higher expression during seed development ( Figure 1 as shown in e). Using the analysis website https: / / yanglab.hzau.edu.cn / SoyMD / # / variation, import the gene sequence of Glyma.08G246400 for haplotype analysis, and use the two-tailed t-test to determine statistical significance. The results showed haplotype differences ( Figure 1as shown in f-h of the figure; Glyma.08G246400 is a homologous gene of Arabidopsis thaliana ABI5-binding protein 3 (AFP3), so Glyma.08G246400 was named GmAFP3, and the sequence is as shown in SEQ ID NO.1.

[0056] Example 2

[0057] 1. Using iSoybean (http: / / isoybean.org / index.php / search / ), multiple mutant materials (W82M-309, W82M-160, W82M-412, W82M-036, W82M-170) that only mutated in the GmAFP3 (Glyma.08G246400) gene of Williams 82 (W82) were identified and obtained. The nucleotide mutation sites of the mutant materials are as Figure 2 shown in a of the figure (the 264bp-266bp in SEQ ID NO.1 is the start codon ATG. Taking the A in the start codon ATG as the first base, the 563rd base of W82M-309 mutated from A to T, the 3092nd base of W82M-160 mutated from G to A, the 3152nd base of W82M-412 mutated from C to T, the 3308th base of W82M-036 mutated from G to A, and the 3373rd base of W82M-170 mutated from G to A). The mutant materials were provided by Nanjing Agricultural University;

[0058] 2. In 2023, W82 and its mutants were planted in Sanya, Hainan. The planting method is as follows:

[0059] (1) Land preparation: Use a rotary tiller to fully till the land, and the soil thickness is 20-30 cm;

[0060] (2) Sowing: The plant spacing is 10-15 cm;

[0061] (3) Management: No fertilizer is applied during the whole growth process of soybeans, and only pesticides for killing spider mites, aphids, and Riptortus pedestris are sprayed;

[0062] (4) Harvesting: Harvest when it is fully mature.

[0063] The near-infrared spectroscopy method (GBT24870-2010) was used to measure and statistically analyze the seed oil content. The results are as Figure 2 shown in b of the figure. It can be seen that the seed oil content of W82 is higher than that of the mutants, and there are significant differences from the mutants W82M-160, W82M-036, and W82M-170.

[0064] 3. In 2024, W82 and its mutants were planted in Jize, Hebei. The planting method is the same as the above method.

[0065] The near-infrared spectroscopy method (GBT24870-2010) was used to measure and statistically analyze the seed oil content and yield-related traits such as the number of pods per plant, 100-seed weight, seed length, seed width, and seed thickness. The results are shown as Figure 2 shown in c-h below. It can be seen that the overall trends of the seed oil content, the number of pods per plant, 100-seed weight, seed length, seed width, and seed thickness of W82 are significantly higher than those of the mutants. This indicates that the gene GmAFP3 helps to significantly increase the soybean seed oil content and the levels of yield-related traits.

[0066] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for mining genetic loci related to soybean seed oil content and yield by using environmental differences, characterized in that, The method includes: Introducing a soybean variety to be planted in an area with different original cultivation environmental conditions to obtain seed oil content data; performing a genome-wide association analysis on the seed oil content data and soybean 50K SNP gene chip data to obtain significant SNP loci; estimating the linkage disequilibrium intervals of the whole genome and determining the decay distance of the linkage disequilibrium intervals of the SNP loci, and the interval determined by this distance is the genetic locus related to soybean seed oil content and yield.

2. The genetic locus SO8 gene related to the oil content and yield of soybean seeds obtained by the method according to claim 1, characterized in that, The sequence of the genetic locus SO8 gene is from the 21,289,819th bp to the 21,486,468th bp on chromosome 8 of the soybean genome; the version of the soybean genome is Wm82.a2.v1.

3. A screening method for genes related to soybean seed oil content and yield, characterized in that, Performing gene annotation on the genetic locus obtained by the method of claim 1, and screening genes with significantly high expression and haplotype differences, which are the genes related to soybean seed oil content and yield.

4. The gene GmAFP3 related to the oil content and yield of soybean seeds obtained by the screening method according to claim 3, characterized in that The sequence of the gene GmAFP3 is as shown in SEQ ID NO.

1.

5. A protein GmAFP3 related to the oil content and yield of soybean seeds, characterized in that, The protein GmAFP3 is encoded by the gene GmAFP3 of claim 4.

6. Use of the genetic locus SO8 gene of claim 2, or the gene GmAFP3 of claim 4, or the protein GmAFP3 of claim 5 in soybean germplasm improvement.

7. The application according to claim 6, wherein The soybean germplasm improvement includes creating soybean germplasms with different levels of seed oil content and / or yield element traits.

8. The application according to claim 7, characterized in that, The yield element traits include one or several of the number of pods per plant, 100-seed weight, seed length, seed width, and seed thickness.

9. The application according to claim 7, characterized in that By regulating the expression level of the genetic locus SO8 gene or the gene GmAFP3, or regulating the content of the protein GmAFP3, creating soybean germplasms with different levels of seed oil content and / or yield element traits.

10. A method for increasing the oil content and / or yield of soybean seeds, characterized in that, The method includes: expressing the genetic locus SO8 gene of claim 2 or the gene GmAFP3 of claim 4 in soybean, or increasing the expression level of the genetic locus SO8 gene of claim 2 or the gene GmAFP3 of claim 4 in soybean, or increasing the content of the protein GmAFP3 of claim 5 in soybean, or increasing the activity of the protein GmAFP3 of claim 5 in soybean, so as to increase the soybean seed oil content and / or yield.

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