Haploid molecular marker related to soybean oil content character and application of haplotype molecular marker

Abstract

The invention provides a haplotype molecular marker related to soybean oil content traits and application thereof, and belongs to the technical field of molecular markers. The invention provides an application of a Glyma. 18G027100 gene and / or a Glyma. 03G021800 gene serving as a target spot in regulation and control or identification of the content of soybean oil components. The invention further provides an application of the Glyma. 18G027100 gene and / or the Glyma. 03G021800 gene. The haplotype of the gene Glyma. 18G027100 and the haplotype of the gene Glyma. 03G021800 show a remarkable difference in oil content (OC) in a natural soybean population. The average OC of the Hap1 (TAACACATT) of the Glyma. 18G027100 gene is obviously higher than that of the Hap2 (CGGTGGAC) of the Glyma. 18G027100 gene. The average OC of haplotypes Hap1 (AGGA) of the Glyma. 03G021800 gene is obviously higher than that of Hap2 (AGAC) and Hap3 (CAAC).

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to haplotype molecular markers related to soybean oil content traits and applications thereof. Background Art

[0002] Soybean (Glycine max (L.) Merr.) is an annual herbaceous plant of the genus Glycine in the Leguminosae family. The oil content of soybean directly affects the oil yield of soybean. Therefore, developing and screening molecular markers that are closely linked to the oil content of soybeans and improving the efficiency of soybean breeding are of great significance to soybean variety selection. Summary of the invention

[0003] The purpose of the present invention is to provide a haploid molecular marker related to the soybean oil content trait and its application. The haploid molecular marker of the present invention is closely linked to the soybean oil content and can improve the soybean breeding efficiency.

[0004] The present invention provides the use of Glyma.18G027100 gene and / or Glyma.03G021800 gene as a target in regulating or identifying soybean oil content.

[0005] The present invention also provides a haplotype molecular marker related to soybean oil content traits, including a first haplotype molecular marker and / or a second haplotype molecular marker;

[0006] The first haplotype molecular marker consists of Glyma.18G027100-SNP1, Glyma.18G027100-SNP2, Glyma.18G027100-SNP3, Glyma.18G027100-SNP4, Glyma.18G027100-SNP5, Glyma.18G027100-SNP6, Glyma.18G027100-SNP7 and Glyma.18G027100-SNP8;

[0007] Among them, Glyma.18G027100-SNP1 is located at bp 2149619 of the Glyma.18G027100 gene, and the base is T or C;

[0008] Glyma.18G027100-SNP2 is located at bp 2149789 of the Glyma.18G027100 gene, and the base is A or G;

[0009] Glyma.18G027100-SNP3 is located at bp 2150035 of the Glyma.18G027100 gene, and the base is A or G;

[0010] Glyma.18G027100-SNP4 is located at bp 2150825 of the Glyma.18G027100 gene, and the base is C or T;

[0011] Glyma.18G027100-SNP5 is located at bp 2150831 of the Glyma.18G027100 gene, and the base is A or G;

[0012] Glyma.18G027100-SNP6 is located at bp 2155101 of the Glyma.18G027100 gene, and the base is C or G;

[0013] Glyma.18G027100-SNP7 is located at bp 2155366 of the Glyma.18G027100 gene, and the base is A;

[0014] Glyma.18G027100-SNP8 is located at bp 2155379 of the Glyma.18G027100 gene, and the base is T or C;

[0015] When the haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 is TAACACAT, the soybean oil content is higher than that when the haplotype combination is CGGTGGAC;

[0016] The second haplotype molecular marker consists of Glyma.03G021800-SNP1, Glyma.03G021800-SNP2, Glyma.03G021800-SNP3 and Glyma.03G021800-SNP4;

[0017] Among them, Glyma.03G021800-SNP1 is located at bp 2354862 of the Glyma.03G021800 gene, and the base is A or C;

[0018] Glyma.03G021800-SNP2 is located at bp 2354994 of the Glyma.03G021800 gene, and the base is G or A;

[0019] Glyma.03G021800-SNP3 is located at bp 2357837 of the Glyma.03G021800 gene, and the base is G or A;

[0020] Glyma.03G021800-SNP4 is located at bp 2358021 of the Glyma.03G021800 gene, and the base is A or C;

[0021] When the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGGA, the soybean oil content is higher than that when the haplotype combination is AGAC and CAAC;

[0022] The reference genome of the soybean is the 'Zhonghuang 13' genome.

[0023] The present invention also provides the use of the haplotype molecular marker described in the above scheme in identifying or assisting in identifying soybean oil content traits.

[0024] The present invention also provides a method for identifying or assisting in identifying soybean oil content traits using the haplotype molecular markers described in the above scheme, comprising the following steps:

[0025] Extracting genomic DNA of soybean materials to be tested;

[0026] The haplotype combinations of the Glyma.18G027100 gene and / or the Glyma.03G021800 gene in the genomic DNA were detected by gene sequencing. When the haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 was TAACACAT, the soybean oil content was higher than the soybean oil content when the haplotype combination was CGGTGGAC; and / or, when the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 was AGGA, the soybean oil content was higher than the soybean oil content when the haplotype combinations were AGAC and CAAC.

[0027] The present invention also provides an application of a reagent for detecting the haplotype molecular marker described in the above scheme in preparing a kit for identifying or assisting in identifying soybean oil content traits.

[0028] The present invention also provides the use of the haplotype molecular marker described in the above scheme as a target in regulating the oil content of soybean.

[0029] The present invention also provides the application of the haplotype molecular markers described in the above scheme in soybean molecular breeding.

[0030] Preferably, the soybean molecular breeding includes at least one of identifying the oil content trait of the soybean material, screening the soybean material with high oil content and screening the soybean material with low oil content.

[0031] The present invention also provides a method for soybean molecular breeding, comprising the following steps: when the target trait is high oil content, successive breeding of soybean materials in which the haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 is TAACACAT and / or the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGGA, and eliminating individuals of other genotypes;

[0032] When the target trait is low oil content, soybean materials whose haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 is CGGTGGAC and / or whose haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGAC or CAAC are selected for succession, and individuals with other genotypes are eliminated.

[0033] Preferably, the high oil content includes an oil content ≥ 21%; and the low oil content includes an oil content ≤ 17%.

[0034] The present invention provides the use of Glyma.18G027100 gene and / or Glyma.03G021800 gene as a target in regulating or identifying the oil content of soybean. The haplotypes of Glyma.18G027100 gene and Glyma.03G021800 gene show significant differences in oil content (OC) in natural soybean populations. For the Glyma.18G027100 gene, two haplotypes were identified based on SNPs at different positions in the exon region: Hap1 (TAACACAT) and Hap2 (CGGTGGAC), among which Hap1 had the highest frequency (82%). In the population used for haplotype analysis, the average OC of Hap1 was significantly higher than that of Hap2, and Hap1 of Glyma.18G027100 would increase OC. Three different haplotypes were identified from the analysis of Glyma.03G021800: Hap1 (AGGA), Hap2 (AGAC) and Hap3 (CAAC). Hap1 accounted for 51% of the population and its average OC was significantly higher than the other two haplotypes, making it an excellent haplotype for enhancing OC. In summary, Glyma.18G027100 and Glyma.03G021800 are candidate genes related to soybean OC. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0036] Figure 1 The distribution of important QTNs on 14 chromosomes; the important QTNs located in multiple environments are marked in red fonts ；

[0037] Figure 2 This is the expression heat map of 8 differentially expressed genes in 4 groups; blue represents down-regulated genes, red represents up-regulated genes, white represents no significant difference, gene names displayed in red fonts represent that the gene is up-regulated in all groups, and gene names displayed in blue fonts represent that the gene is down-regulated in all groups ；

[0038] Figure 3 The result of the correlation analysis between OC traits and WGCNA module is shown in Figure 2. Red indicates positive correlation, blue indicates negative correlation, * indicates significant correlation at the 0.05 level, ** indicates significant correlation at the 0.01 level, and *** indicates significant correlation at the 0.001 level.

[0039] Figure 4 The correlation between the 8 significantly different genes and the modules; red indicates positive correlation, blue indicates negative correlation, * indicates significant correlation at the 0.05 level, ** indicates significant correlation at the 0.01 level, and *** indicates significant correlation at the 0.001 level;

[0040] Figure 5 The haplotype information of Glyma.18G027100 and Glyma.03G021800 and the phenotypic analysis results in natural populations are shown in FIG. ， A is the haplotype information of Glyma.18G027100 and the corresponding number of individuals; B is the box plot of the lipid content of Hap1 and Hap2 haplotypes of Glyma.18G027100 in 121 materials; C is the haplotype information of Glyma.03G021800 and the corresponding number of individuals; D is the haplotype information of Glyma.03G021800 in 141 materials.

[0041] Box plot of lipid content of Hap1, Hap2 and Hap3 haplotypes of Glyma.03G021800 in the materials. DETAILED DESCRIPTION

[0042] The present invention provides the use of Glyma.18G027100 gene and / or Glyma.03G021800 gene as a target in regulating or identifying soybean oil content.

[0043] In the present invention, the Glyma.18G027100 gene (LOC100784534: XM_003552305.5) and the Glyma.03G021800 gene (LOC100527561: NM_001250951.2) are located at Chr18: 2033838..2038697 bp reverse and Chr03: 2265528..2269179 bp forward in the Glycine max Wm82.a2.v1 reference sequence, respectively, when Williams 82 is used as the reference genome. When Zhonghuang 13 is used as the reference genome, the Glyma.18G027100 gene and the Glyma.03G021800 gene are located at Chr18: 2149555..2155433bp (-strand) and Chr03: 2354584..2358026bp (+strand) in the ZH13 reference sequence, respectively.

[0044] The present invention also provides a haplotype molecular marker related to soybean oil content traits, including a first haplotype molecular marker and / or a second haplotype molecular marker; the first haplotype molecular marker is composed of Glyma.18G027100-SNP1, Glyma.18G027100-SNP2, Glyma.18G027100-SNP3, Glyma.18G027100-SNP4, Glyma.18G027100-SNP5, Glyma.18G027100-SNP6, Glyma.18G027100-SNP7 and Glyma.18G027100-SNP8; wherein Glyma.18G027100-SNP1, Glyma.18G027100-SNP2, Glyma.18G027100-SNP3, Glyma.18G027100-SNP4, Glyma.18G027100-SNP5, Glyma.18G027100-SNP6, Glyma.18G027100-SNP7 and Glyma.18G027100-SNP8; 27100-SNP1 is located at the 2149619 bp of the Glyma.18G027100 gene, and the base is T or C; Glyma.18G027100-SNP2 is located at the 2149789 bp of the Glyma.18G027100 gene, and the base is A or G; Glyma.18G027100-SNP3 is located at the 2150035 bp of the Glyma.18G027100 gene, and the base is A or G; Glyma.18G027100-SNP4 is located at the 2150825 bp of the Glyma.18G027100 gene, and the base is C or T; Glyma.18G027100-SNP5 Located at the 2150831bp of the Glyma.18G027100 gene, the base is A or G; Glyma.18G027100-SNP6 is located at the 2155101bp of the Glyma.18G027100 gene, the base is C or G; Glyma.18G027100-SNP7 is located at the 2155366bp of the Glyma.18G027100 gene, the base is A; Glyma.18G027100-SNP8 is located at the 2155379bp of the Glyma.18G027100 gene, the base is T or C; Glyma.18G027100-SNP1~Glyma.18G02 When the haplotype combination corresponding to 7100-SNP8 is TAACACAT, the soybean oil content is higher than that when the haplotype combination is CGGTGGAC; the second haplotype molecular marker is composed of Glyma.03G021800-SNP1, Glyma.03G021800-SNP2, Glyma.03G021800-SNP3 and Glyma.03G021800-SNP4; among them, Glyma.03G021800-SNP1 is located at the 2354862bp of the Glyma.03G021800 gene, and the base is A or C; Glyma.03G021800-SNP2 is located at the 2354862bp of the Glyma.03G021800 gene, and the base is A or C;The base of Glyma.03G021800-SNP3 is located at the 2354994bp of the Glyma.03G021800 gene, which is G or A; Glyma.03G021800-SNP3 is located at the 2357837bp of the Glyma.03G021800 gene, which is G or A; Glyma.03G021800-SNP4 is located at the 2358021bp of the Glyma.03G021800 gene, which is A or C; when the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGGA, the soybean oil content is higher than the soybean oil content when the haplotype combination is AGAC and CAAC; the reference genome of the soybean is the 'Zhonghuang 13' genome. .

[0045] In the present invention, the first haplotype molecular marker is located in the soybean Glyma.18G027100 gene, specifically located at 2149619 bp to 2155379 bp of the soybean Glyma.18G027100 gene, with a total length of about 5760 bp.

[0046] In the present invention, the second haplotype molecular marker is located in the soybean Glyma.03G021800 gene, specifically located at 2354862 bp to 2358021 bp of the soybean Glyma.03G021800 gene, with a total length of about 3159 bp.

[0047] The present invention also provides the use of the haplotype molecular marker described in the above scheme in identifying or assisting in identifying soybean oil content traits.

[0048] The present invention also provides a method for identifying or assisting in identifying soybean oil content traits using the haplotype molecular markers described in the above scheme, comprising the following steps: extracting genomic DNA of a soybean material to be tested; detecting the haplotype combination on the Glyma.18G027100 gene and / or the Glyma.03G021800 gene in the genomic DNA by gene sequencing, when the haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 is TAACACAT, the soybean oil content is higher than the soybean oil content when the haplotype combination is CGGTGGAC; and / or, when the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGGA, the soybean oil content is higher than the soybean oil content when the haplotype combinations are AGAC and CAAC.

[0049] The present invention also provides an application of a reagent for detecting the haplotype molecular marker described in the above scheme in preparing a kit for identifying or assisting in identifying soybean oil content traits.

[0050] The present invention also provides the use of the haplotype molecular marker described in the above scheme as a target in regulating the oil content of soybean.

[0051] The present invention also provides the application of the haplotype molecular markers described in the above scheme in soybean molecular breeding.

[0052] In the specific implementation process of the present invention, the soybean molecular breeding includes at least one of identifying the oil content trait of soybean materials, screening soybean materials with high oil content and screening soybean materials with low oil content.

[0053] The present invention also provides a method for soybean molecular breeding, comprising the following steps: when the target trait is high oil content, successive breeding of soybean materials in which the haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 is TAACACAT and / or the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGGA, and eliminating individuals of other genotypes;

[0054] When the target trait is low oil content, soybean materials whose haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 is CGGTGGAC and / or whose haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGAC or CAAC are selected for succession, and individuals with other genotypes are eliminated.

[0055] In the specific implementation of the present invention, the high oil content includes an oil content ≥ 21%; the low oil content includes an oil content ≤ 17%.

[0056] To further illustrate the present invention, the haplotype molecular markers related to soybean oil content traits and their applications provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0057] Materials: Kenfeng 14, Kenfeng 15 and Kenfeng 19 were bred by Heilongjiang Agricultural Reclamation Research Institute; Heinong 48 was bred by Heilongjiang Academy of Agricultural Sciences; Kenfeng 14, Kenfeng 15, Kenfeng 19 and Heinong 48 were all provided by the Soybean Research Institute of Northeast Agricultural University;

[0058] The FW-RIL population was obtained by (Kenfeng 14 x Kenfeng 15) x (Heinong 48 x Kenfeng 19). This population was provided by the Soybean Research Institute of Northeast Agricultural University.

[0059] Example 1

[0060] Example 1

[0061] 1. Materials

[0062] In this example, a population of 144 FW-RILs obtained by a four-way hybridization of the parental lines Kenfeng 14 (OC 20.88%), Kenfeng 15 (OC 21.66%), Kenfeng 19 (OC 18.33%) and Heinong 48 (OC 17.76%) was used.

[0063] 144 FW-RILs and 4 parental lines were grown in 10 environments (hereafter denoted as E1-E10) at different locations and years (Table 1).

[0064] Table 1 Field planting information

[0065] environment Place Longitude and Latitude years E1 Acheng 126.50°E, 45.27°N 2016 E2 Twin Cities 126.18°E, 45.22°N 2016 E3 Harbin 126.63°E, 45.75°N 2016 E4 Keshan 125.52°E, 48.02°N 2016 E5 Harbin 126.63°E, 45.75°N 2017 E6 Harbin 126.63°E, 45.75°N 2018 E7 Keshan 125.52°E, 48.02°N 2018 E8 Keshan 125.52°E, 48.02°N 2019 E9 Harbin 126.63°E, 45.75°N 2020 E10 Harbin 126.63°E, 45.75°N 2021

[0066] All materials under each environment were planted in a completely randomized block design with 3 replicates. The ridge length was 5m and the ridge width was 0.7m. The management of the experimental plots was consistent with local soybean production. When mature, 10 plants in each line were randomly harvested and threshed. The OC of dry seeds (about 10% moisture) of each line was measured by a near-infrared analyzer (Infratec 1241, Foss, Denmark) at the Key Laboratory of Soybean Biology, Ministry of Education, Northeast Agricultural University, China. The calibration regression technique used for the near-infrared analyzer is partial least squares (PLS), which involves combining spectral data with laboratory data (Kjeldahl method) to calculate seed OC as a percentage of seed weight. The calculation results are shown in Table 2. The phenotypic values ​​of each parent and FW-RIL individual used in this example are the average of three replicates.

[0067] Table 2 Descriptive statistics of OC data in 10 environments of the parental and FW-RIL populations

[0068]

[0069] 2. Phenotypic data analysis

[0070] Phenotypic data analysis included determination of mean, standard deviation, range, minimum, maximum, skewness, kurtosis, and coefficient of variation (CV) values, analysis of variance (ANOVA), and broad-sense heritability (h 2 ) analysis. All statistical analyses were performed in SAS 9.2 (SAS Institute, Cary, USA). Multi-environment ANOVA was performed jointly using the generalized linear model (GLM) method, and the variance components were estimated using the mixed linear model (MLM). Then, h was calculated by the following formula 2 :

[0071]

[0072] in and denote the variance of genotype, genotype-environment interaction, and error variance, respectively; r denotes the number of replicates in each environment; and n denotes the number of environments.

[0073] 3. Genotype, population structure and linkage disequilibrium

[0074] Genomic DNA was extracted from young leaves of parental and FW-RIL plants by the CTAB method. DNA concentration was determined using a UV752N spectrophotometer (Shanghai Jingke Scientific Instrument Co., Ltd.), and DNA was diluted to 100 ± 1 ng in deionized water. SNP genotyping was based on the SoySNP660KBeadChip method at Beijing Boao Biotechnology Co., Ltd. 109,676 SNPs were selected for GWAS analysis through quality screening with a standard maximum deletion site ratio of <10% and a minimum allele frequency (MAF) >5%.

[0075] By analyzing the population structure, the FW-RIL population was divided into two subgroups, and the Q matrix was obtained and used for multi-site GWAS analysis. The LD region decayed fastest before 200 kb and then tended to be flat, so the 200 kb region above and below the significant SNP was determined to search for potential candidate genes.

[0076] 4. Genome-wide association study (GWAS)

[0077] GWAS analysis of soybean seed OC traits was performed using five multi-locus GWAS methods in the mrMLM GUI (version 3.0) software: mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB, and ISISEM-BLASSO. The critical P value parameters of these methods were set to 0.01 in the first stage, except for FASTmrEMMA, whose critical P value was set to 0.005; for the significant quantitative trait nucleotides (QTNs) in the final stage, the critical LOD score was set to 3 and the P value was set to 0.0002. The parameters were set as likelihood = "REML", SearchRadius = 100, CriLOD = 3, SelectVariable = 143, and Bootstrap = FALSE. The kinship matrix was also obtained using the mrMLM GUI 3.0 software.

[0078] Significant SNPs that meet one of the following conditions are considered important QTNs: (1) the same SNP marker is detected; (2) the attenuation intervals of different SNP markers intersect (the same QTN detected repeatedly by multiple methods is only calculated once).

[0079] QTNs are named as follows: q+trait name-chromosome name-QTN number, where q represents QTN, the trait name is OC, and the QTN number represents the ranking of the QTN on the corresponding chromosome.

[0080] QTNs detected by multiple GWAS methods or in multiple settings were considered important QTNs.

[0081] Results: In this example, 23 important QTNs were found (important QTNs refer to QTNs detected in multiple GWAS methods or in multiple environments) (Table 3 and Figure 1 ). Twenty-one common QTNs were detected by at least two methods in one environment (listed in black font in Table 3); the LOD values ​​ranged from 3.24 to 7.78, and the proportion of PVE in each QTN ranged from 4.22% to 17.68%. Two common QTNs were identified across multiple environments by single or multiple methods, and the detailed information of these two QTNs (qOC-7-1, qOC-18-1) is shown in bold black font in Table 3; the LOD values ​​ranged from 4.83 to 5.20, and the proportion of PVE in QTN ranged from 5.52% to 9.20%. For all 23 common QTNs, the direction of the effect (positive or negative) of each QTN was consistent across different methods or environments. No important QTNs were located on the remaining six chromosomes (chromosomes 06, 12, 13, 15, 19, and 20).

[0082] Table 3 23 important QTNs related to soybean oil content identified by different methods or in different environments

[0083]

[0084]

[0085] b ,mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB and ISIS EM-BLASSO represent 1 to 5 respectively.

[0086] 5. Transcriptome sequencing analysis

[0087] The soybean materials used for transcriptome sequencing were Keshan 01 (KS01), Kedou 31 (KD31), Kedou 48 (KD48) and Kedou 57 (KD57). KS01 and KD31 are high-oil varieties with OC contents of 21.82% and 21.26%, respectively; KD48 and KD57 are low-oil varieties with OC contents of 15.78% and 16.44%, respectively. These varieties were provided by the Keshan Branch of Heilongjiang Academy of Sciences.

[0088] Uniform seeds of four varieties were selected and potted (5 seeds / pot, 25×18 cm pots), and after seed germination, the number of seedlings per pot was reduced to 1. At 25 days after flowering (rapid oil accumulation stage), seeds were collected and frozen in liquid nitrogen (three replicates) and stored in an ultra-low temperature freezer at -80°C until transcriptome sequencing analysis.

[0089] Transcriptome sequencing analysis was performed on an Illuminahis Q2500 / 4000 system by Guangzhou Kidio Biotechnology Co., Ltd. (Guangzhou, China).

[0090] 6. Real-time fluorescence quantitative PCR verification

[0091] Total RNA extraction, cDNA synthesis, and qRT-PCR analysis of each sample were performed as described by Zhang et al. (see [Zhang, W. et al. A cation diffusion facilitator, GmCDF1, negatively regulates salt tolerance in soybean. PLoS Genet. 15 (2019).]), with Acint7 as the internal reference gene, and data analysis using 2 -ΔΔCT The primers used for qRT-PCR are shown in Table 4.

[0092] Table 4 qRT-PCR primers

[0093]

[0094]

[0095] 7. Finding potential candidate genes

[0096] According to the LD decay, the 100 kb intervals on both sides of each important QTN were set as the range for searching for potential candidate genes. All genes within the interval were found through the phytozome website (https: / / phytozome.jgi.doe.gov). Based on the soybean seed development stage transcriptome dataset (GES42871) downloaded from GEO (Gene Expression Omnibus), genes highly expressed in seeds during oil formation were subsequently selected. All highly expressed genes were then subjected to pathway analysis through the KEGG website (http: / / www.kegg.jp), after which genes in lipid anabolism-related pathways were identified. Finally, based on the transcriptome sequencing results, differentially expressed genes (DEGs) between high-oil and low-oil varieties were identified in lipid anabolism-related pathways. Potential candidate genes were identified by comparing these DEGs with the weighted gene correlation network analysis (WGCNA) results and gene annotation information.

[0097] The results are as follows:

[0098] There were 441 genes in the 23 important QTN intervals, of which 196 genes were highly expressed in the process of seed lipid formation. Metabolic pathway analysis of the 196 genes showed that 132 genes were enriched in 163 pathways, of which only 15 were related to lipid anabolism, including glycolysis / gluconeogenesis, citric acid cycle (TCA cycle), fructose and mannose metabolism, galactose metabolism, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, glyoxylate and dicarboxylate metabolism, propionate metabolism and inositol phosphate metabolism, belonging to the carbohydrate metabolism pathway; oxidative phosphorylation, photosynthetic antenna protein, nitrogen metabolism and sulfur metabolism, belonging to energy metabolism; glycerolipid metabolism, belonging to the lipid metabolism pathway (Table 5).

[0099] Table 5 Annotation information of 13 genes and their corresponding metabolic pathway information in KEGG

[0100]

[0101] A total of 13 genes were associated with these 15 pathways, and compared with the DEGs in the KD48-vs-KS01, KD57-vs-KS01, KD48-vs-KD31, and KD57-vs-KD31 groups, 8 genes were differentially expressed between high-OC and low-OC soybean varieties ( Tables 5 and Figure 2 ). The eight DEGs were Glyma.09G173200, Glyma.03G021800, Glyma.14G218800, Glyma.16G213200, Glyma.18G025500, Glyma.18G027100, Glyma.11G093200, and Glyma.18G027200.

[0102] For WGCNA analysis based on transcriptome sequencing data, genes with similar expression patterns were clustered into 20 modules ( Figure 3 ). Analysis of the relationship between each module and OC phenotype showed that MM.darkseagreen4 (p = 4e-07), MM.violet (p = 2e-04), MM.darkred (p = 0.01) and MM.floralwhite (p = 0.04) were significantly positively correlated with OC traits. The module with a significant negative correlation with OC traits was MM.brown 4 (p = 0.003) ( Figure 3). The 8 DEGs were further correlated with the above 5 modules, and 7 DEGs (except Glyma.16G13200) were significantly correlated with one or more of the 5 modules. Glyma.11G093200 and Glyma.18G027100 were significantly or extremely significantly positively correlated with the 4 modules ( Figure 4 The red font in the figure showed that the DEGs were positively correlated with OC traits, Glyma.18G027200 was significantly positively correlated with MM.darkred, and extremely significantly positively correlated with MM.floralwhite. Glyma.03G021800, Glyma.09G173200, Glyma.18G025500, and Glyma.14G218800 were significantly or extremely significantly positively correlated with the MM.brown4 module (a module negatively correlated with oil content traits). Therefore, the seven DEGs (except Glyma.16G213200) were considered as potential candidate genes for further analysis of haplotype analysis.

[0103] 8. Haplotype analysis

[0104] Haplotype analysis of genes in 141 soybean materials was performed using existing genotype data (https: / / ngdc.cncb.AC.cn / soy omics). Haplotypes were constructed using Haploview software based on SNPs in untranslated regions (UTRs) and exon regions.

[0105] The results are as follows:

[0106] Haplotypes were classified through natural populations, including 141 soybean materials with genotype data (https: / / ngdc.cncb.AC.cn / soy omics). Through haplotype analysis of the above 7 DEGs, it was found that the haplotypes of Glyma.18G027100 and Glyma.03G021800 both showed significant differences in oil content (OC) in natural soybean populations. For Glyma.18G027100, two haplotypes were identified based on SNPs at different positions in the exon region: Hap1 (TAACACAT) and Hap2 (CGGTGGAC) ( Figure 5 A in Figure 1). Hap1 has the highest frequency (82%). In the population used for haplotype analysis, the average OC of Hap1 is significantly higher than that of Hap2 ( Figure 5 B). These results suggest that Hap1 (TAACACAT) of Glyma.18G027100 increases OC. Three different haplotypes were identified from the analysis of Glyma.03G021800: Hap1 (AGGA), Hap2 (AGAC), and Hap3 (CAAC) ( Figure 5 C in the figure). Hap1 accounts for 51% of the population, and its average OC is significantly higher than that of the other two haplotypes, making it an excellent haplotype for enhancing OC ( Figure 5 In summary, it can be predicted that Glyma.18G027100 and Glyma.03G021800 are candidate genes related to soybean OC.

[0107] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Glyma.18G027100 gene and / or Glyma.03G021800 gene as targets in regulating or identifying soybean oil content.

2. A haplotype molecular marker associated with soybean oil content, characterized in that: including a first haplotype molecular marker and / or a second haplotype molecular marker; The first haplotype molecular marker consists of Glyma.18G027100-SNP1, Glyma.18G027100-SNP2, Glyma.18G027100-SNP3, Glyma.18G027100-SNP4, Glyma.18G027100-SNP5, Glyma.18G027100-SNP6, Glyma.18G027100-SNP7 and Glyma.18G027100-SNP8; Among them, Glyma.18G027100-SNP1 is located at bp 2149619 of the Glyma.18G027100 gene, and the base is T or C; Glyma.18G027100-SNP2 is located at bp 2149789 of the Glyma.18G027100 gene, and the base is A or G; Glyma.18G027100-SNP3 is located at bp 2150035 of the Glyma.18G027100 gene, and the base is A or G; Glyma.18G027100-SNP4 is located at bp 2150825 of the Glyma.18G027100 gene, and the base is C or T; Glyma.18G027100-SNP5 is located at bp 2150831 of the Glyma.18G027100 gene, and the base is A or G; Glyma.18G027100-SNP6 is located at bp 2155101 of the Glyma.18G027100 gene, and the base is C or G; Glyma.18G027100-SNP7 is located at bp 2155366 of the Glyma.18G027100 gene, and the base is A; Glyma.18G027100-SNP8 is located at bp 2155379 of the Glyma.18G027100 gene, and the base is T or C; When the haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 is TAACACAT, the soybean oil content is higher than that when the haplotype combination is CGGTGGAC; The second haplotype molecular marker consists of Glyma.03G021800-SNP1, Glyma.03G021800-SNP2, Glyma.03G021800-SNP3 and Glyma.03G021800-SNP4; Among them, Glyma.03G021800-SNP1 is located at bp 2354862 of the Glyma.03G021800 gene, and the base is A or C; Glyma.03G021800-SNP2 is located at bp 2354994 of the Glyma.03G021800 gene, and the base is G or A; Glyma.03G021800-SNP3 is located at bp 2357837 of the Glyma.03G021800 gene, and the base is G or A; Glyma.03G021800-SNP4 is located at bp 2358021 of the Glyma.03G021800 gene, and the base is A or C; When the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGGA, the soybean oil content is higher than that when the haplotype combinations are AGAC and CAAC; The reference genome of soybean is the 'Zhonghuang 13' genome.

3. Use of the haplotype molecular marker according to claim 2 in identifying or assisting in identifying soybean oil content traits.

4. The method for identifying or assisting in identifying soybean oil content traits using the haplotype molecular marker according to claim 2, characterized in that: The following steps are involved: Extracting genomic DNA of soybean materials to be tested; By detecting the haplotype combinations of the Glyma.18G027100 gene and / or the Glyma.03G021800 gene in the genomic DNA through gene sequencing, when the haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 is TAACACAT, the soybean oil content is higher than the soybean oil content when the haplotype combination is CGGTGGAC; and / or, when the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 is AGGA, the soybean oil content is higher than the soybean oil content when the haplotype combinations are AGAC and CAAC.

5. Use of a reagent for detecting the haplotype molecular marker according to claim 2 in preparing a kit for identifying or assisting in identifying soybean oil content traits.

6. Use of the haplotype molecular marker according to claim 2 as a target in regulating soybean oil content.

7. Use of the haplotype molecular marker according to claim 2 in soybean molecular breeding.

8. The use according to claim 7, characterized in that The soybean molecular breeding includes at least one of identifying the oil content trait of soybean materials, screening soybean materials with high oil content, and screening soybean materials with low oil content.

9. A method for soybean molecular breeding, characterized in that: The following steps are involved: When the target trait is high oil content, soybean materials with the haplotype combination corresponding to Glyma.18G027100-SNP1 to Glyma.18G027100-SNP8 being TAACACAT and / or the haplotype combination corresponding to Glyma.03G021800-SNP1 to Glyma.03G021800-SNP4 being AGGA are selected for successive generations, and individuals with other genotypes are eliminated; When the target trait is low oil content, soybean materials with the haplotype combination corresponding to Glyma.18G027100-SNP1~Glyma.18G027100-SNP8 being CGGTGGAC and / or the haplotype combination corresponding to Glyma.03G021800-SNP1~Glyma.03G021800-SNP4 being AGAC or CAAC are selected for succession, and individuals with other genotypes are eliminated.

10. The method according to claim 9, characterized in that The high oil content includes an oil content ≥ 21%; the low oil content includes an oil content ≤ 17%.

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