Quantitative trait loci related to soybean isoflavone content and their application

Abstract

The invention discloses a quantitative trait locus related to soybean isoflavone content and an application thereof, and provides a soybean molecular marker-assisted breeding method based on a high-density genetic linkage map. A cultivated soybean variety JD12ZDD23040 is selected to be hybridized with a wild soybean variety Y9ZYD02739, and a control group consisting of 185 recombinant inbred lines (RILs) from F8 sources is constructed by a single seed descent SSD method; genotyping is performed by sequencing and SNP calling, and map construction, QTL detection and QTL integration analysis are performed; based on statistical analysis, an integrated QTL significantly related to the soybean isoflavone content trait is obtained; based on the obtained integrated QTL, a molecular marker and a primer thereof are developed for soybean molecular marker-assisted breeding related to the soybean isoflavone content trait.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to a molecular breeding technology related to soybean isoflavone content. Background Art

[0002] Soybean (Glycine max L. Merr.) is a nutritious crop that produces seeds rich in protein and oil, which accounted for approximately 70% of the soybean meal and 28% of the vegetable oil consumed by humans and livestock in 2020. In recent years, the area cultivated and production of soybean have increased significantly as demand for food and feed has increased. In 2020, the area cultivated with soybean exceeded 127 million hectares, mainly in Brazil, the United States, Argentina and China (FAO data), while global soybean production reached 399 million tons, making soybean the fourth largest crop in terms of area and production in the world, after wheat, maize and rice. Therefore, releasing high-quality soybean varieties rich in protein and / or oil will remain a vital goal for soybean breeders around the world for the foreseeable future.

[0003] In addition to protein and oil, soybeans are able to produce large amounts of isoflavones, compounds that are useful in the adaptation of soybeans to different environments, as well as in maintaining human health and well-being. For example, recent studies have shown that isoflavones can reduce the risk of cancer and help prevent the onset of some chronic diseases. Therefore, there is a real and urgent need to breed soybean varieties that are rich in isoflavones and can produce high protein or high oil content to meet the needs of different groups. Although soybean seeds may contain high concentrations of isoflavones, there is a wide range of variation in this trait between soybean germplasm. For example, the variation in isoflavone concentrations among 1168 soybean varieties was found to range from about 700 μg g-1 to 5000 μg g-1, while other studies have also found relatively high heritability of isoflavone concentrations between soybean varieties. Overall, previous results suggest that efforts to breed or genetically engineer soybean varieties that consistently produce high concentrations of isoflavones have a reasonable chance of success.

[0004] Because isoflavones are synthesized via the amino acid (phenylpropanoid) pathway, potential relationships between isoflavone production and protein or oil have been investigated, although published reports have generated uncertainty due to significant disagreements in the results. One group of researchers found that isoflavone synthesis was negatively correlated with protein production and positively correlated with oil content. Others found that isoflavone content was negatively correlated with protein levels and had no significant correlation with oil production. In other work, decreased protein content was associated with increased isoflavone production in soybean LOX near-isogenic lines, suggesting that the isoflavone and protein synthesis pathways may share common genes or that these pathways include genes with pleiotropic effects. In short, all previous reports have consistently found correlations between protein and isoflavone production and unclear associations between isoflavone and oil synthesis, although the genetic components that may be involved in protein, oil, and isoflavone production remain to be determined.

[0005] Quantitative trait mapping is a method to study the genetic basis of quantitative traits using linkage maps constructed with molecular markers, and is widely used in the study of cereal crops such as rice, corn and wheat as well as beans. Isoflavone, protein and oil content are complex quantitative traits that are expected to be controlled by multiple loci that show highly flexible responses to environmental conditions. Previous studies have made great efforts in mapping markers for protein and oil production. However, research on isoflavones is still lacking. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a quantitative trait locus related to soybean isoflavone content and application thereof.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0008] A soybean molecular marker-assisted breeding method based on a high-density genetic linkage map, wherein the soybean molecular marker-assisted breeding is oriented to the isoflavone content trait of soybean, and the method comprises the following steps: selecting a cultivated soybean variety JD12 ZDD23040 and a wild soybean variety Y9 ZYD02739 for hybridization, and using a single seed descent SSD method to construct a control population consisting of 185 recombinant inbred lines (RILs) from F8 sources; the parental genotypes are planted in 6 replicates, and the RILs are planted in 3 replicates, and isoflavones are determined after maturity; then genotyping is performed by sequencing and SNP calling, DNA is isolated from soybean seedling leaf tissues using the cetyltrimethylammonium bromide (CTAB) method, the collected DNA samples are randomly cut, a library is constructed, and sequencing is performed on a platform for SNP detection, and the minimum SNP length is set to 4 bp; then map construction and QTL detection are performed, SNP data are analyzed in a chi-square test, the P value threshold is set to 0.05 to improve the map quality, the logarithm of the ratio LOD threshold is set to 2.5 during QTL detection, and in A permutation test was performed with a threshold P value set to 0.05 to estimate the LOD significance of 1000 iterations; then an integrated analysis of QTLs was performed to construct a composite genetic map based on the QTLs, all molecular markers present on the shared genetic map, and all coordinates of the consensus map markers, and significant QTLs associated with the target trait and neighboring markers located on the same position fragment were estimated based on the physical distance of the separation-related characteristics to construct a genetic map and a consensus map; finally, based on statistical analysis, integrated QTLs such as major effect QTLs / clustered QTLs / hotspot QTLs / preferred QTLs significantly associated with the soybean isoflavone content trait were obtained; based on the obtained integrated QTLs, molecular markers and their primers were developed for soybean molecular marker-assisted breeding related to the soybean isoflavone content trait.

[0009] An integrated quantitative trait locus for soybean molecular marker-assisted selection breeding, wherein the soybean molecular marker-assisted selection breeding is oriented to the soybean isoflavone content trait, characterized in that the integrated quantitative trait locus is one or any combination of loci named qISO1, qISO6.1, qISO6.3, and qISO6.4;

[0010] Among them, qISO1 is located on soybean chromosome 1, with a genetic distance of 231.938-275.569 cM and a physical locus / interval of 01_51600690-01_54799844;

[0011] Among them, qISO6.1 is located on soybean chromosome 6, with a genetic distance of 179.84-191.072 cM and a physical locus / interval of 06_14871510-06_15642762;

[0012] Among them, qISO6.3 is located on soybean chromosome 6, with a genetic distance of 241.934-242.934 cM and a physical locus / interval of 06_35913434-36835583;

[0013] Among them, qISO6.4 is located on soybean chromosome 6, with a genetic distance of 271.344-273.163cM and a physical locus / interval of 06_42113786-06_43887351.

[0014] The quantitative trait locus is used to develop molecular markers and primers related to the isoflavone content trait of soybean.

[0015] The quantitative trait locus is used for molecular marker-assisted breeding related to soybean isoflavone content traits.

[0016] The beneficial effect of adopting the above technical scheme is that: the present invention uses a high-density genetic linkage map composed of 3943 SNP markers to analyze the genetic basis of soybean isoflavone production, the location of isoflavone markers is identified under this marker density, and the verification of the location markers can be applied to the breeding of soybean varieties rich in isoflavones or with reduced isoflavone content. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 . Comparison of quality and isoflavone traits between JD12 and Y9. (i) Photos of JD12 and Y9 seeds; (ii) Protein content; (iii) Oil content; (iv) Content of daidzein isoflavone derivatives (Ds); (v) Content of genistein isoflavone derivatives (Gs); (F) Content of glycidyl isoflavone derivatives (GLs); (G) Total isoflavone content (TIF). Bar graphs represent the mean standard deviation of 6 replicates. Asterisks indicate the significance of the difference between JD12 and J9 at the 0.05 (*), 0.01 (**), and 0.001 (***) significance levels by Student's t-test.

[0018] Figure 2 . Correlation analysis of isoflavone, protein, and oil contents. Histograms of trait distributions are located in diagonal cells. Correlation coefficients with significance levels are shown above the diagonal, while scatter plots with fitted curves are shown below the diagonal. Red asterisks indicate the significance of differences among RILs by Student's t-test at 0.05 (*), 0.01 (**), and 0.001 (***) significance levels.

[0019] Figure 3. Co-localization of isoflavone and protein loci identified in the current population with loci identified in previous studies. Colors and bold letters represent the identified loci. Red and black boxes represent loci for isoflavone and protein traits, respectively. The projected areas are highlighted in corresponding colors. Bold markers of different colors represent the boundaries of the corresponding loci, and green markers represent isoflavone and protein traits. The consensus map shows the isoflavone or protein production loci previously identified based on soybase data.

[0020] Figure 4 . Effect of combining favorable loci from qISO5 and qISO6.2 on total isoflavones (TIF), protein and oil content in RIL populations as described in Materials and methods. Letters indicate the significance of differences at the 0.01 significance level obtained by LSD-test. Different shapes represent different oil contents and different colors represent different protein contents. n represents lines in which no favorable loci were detected, while q5 and q6.2 represent lines with favorable loci qISO5 and qISO6.2, respectively. HP and HO indicate that the respective protein and oil contents in the RIL population are above the average. LP and LO indicate that the corresponding protein and oil contents in the RIL population are below the average.

[0021] Figure 5 . Correlation analysis of soybean isoflavones, protein, and oil traits in 2020. The histogram with the characteristic fitting curve is placed on the diagonal. Above the diagonal is the correlation coefficient with a significant level, and below the diagonal is the scatter plot with the fitting curve. The red asterisk indicates the significance of the RIL difference obtained by Student's t-test at the 5% (*), 1% (*), and 0.1% (**) levels.

[0022] Figure 6 . Correlation analysis of soybean isoflavones, protein, and oil traits in 2021. The histogram of the trait fitting curves is placed on the diagonal. Above the diagonal is the correlation coefficient with a significant level, and below the diagonal is the scatter plot with the fitting curve. The red asterisk indicates the significance of the difference between RILs at the 5% (*), 1% (*), and 0.1% (**) levels by Student's t-test.

[0023] Figure 7 . Correlation analysis of individual isoflavones with protein / oil traits. The histogram of the trait fitting curves is placed on the diagonal. Above the diagonal is the correlation coefficient with a significant level, and below the diagonal is the scatter plot with the fitting curve. The red asterisks indicate the significance of the differences between RILs at the 5% (*), 1% (*), and 0.1% (**) levels by Student's t-test.

[0024] Figure 8. Correlation analysis between soybean isoflavones and protein / oil traits in 2020. The histogram of the trait fitting curves is placed on the diagonal. Above the diagonal is the correlation coefficient with a significant level, and below the diagonal is the scatter plot with the fitting curve. The red asterisk indicates the significance of the difference between RILs at the 5% (*), 1% (*), and 0.1% (**) levels by Student's t-test.

[0025] Fig. 9 . Correlation analysis between soybean isoflavones and quality traits in 2021. The histogram of the trait fitting curve is placed on the diagonal. Above the diagonal is the correlation coefficient with a significant level, and below the diagonal is the scatter plot with the fitting curve. The red asterisk indicates the significance of the difference between RILs at the 5% (*), 1% (*), and 0.1% (**) levels by Student's t-test.

[0026] Fig.10 . 20 linkages of the soybean high-density genetic map. The genetic distance scale is on the right.

[0027] Fig.11 . Correlation analysis between isoflavone, protein and oil traits, eliminating the effects of co-localized fragments on chromosomes 6, 8 and 9 in 2020. The histogram of the trait fitting curves is placed on the diagonal. Above the diagonal are the correlation coefficients with significant levels, and below the diagonal are the scatter plots with fitting curves. The red asterisks indicate the significance of the differences in RILs at the 5% (*), 1% (*) and 0.1% (**) levels by Student's t-test. DETAILED DESCRIPTION

[0028] The present invention is described in detail in the following examples. All raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained through market purchase.

[0029] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections. It should also be understood that the term "and / or" used in the present specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes these combinations. The reference "one embodiment" or "some embodiments" described in the present specification means that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Thus, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification are not necessarily all referring to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0030] Example 1. Plant materials and growth conditions

[0031] In this study, the cultivated soybean variety JD12 (ZDD23040), which produces relatively small amounts of isoflavones and proteins, was crossed with the wild soybean variety Y9 (ZYD02739), which produces relatively abundant isoflavones and proteins, and a control population consisting of 185 F8-derived recombinant inbred lines (RILs) was constructed using the single seed descent (SSD) method. The parents and populations were planted at the Shangdi Experimental Field (E114.48, N38.03) of the Institute of Cereals and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences in 2020 and 2021. The soil at the site is a river-type soil. The average temperature during the soybean growing season is traditionally about 22 degrees, and the average rainfall is about 500 milliliters. The physicochemical properties of the 25 cm soil surface layer were as follows: pH was 8.3; organic matter was 19.9 g kg-1; available N, P (Olsen-P) and K were 109.2 mg kg-1, 21.6 mg kg-1 and 193.4 mg kg-1, respectively. These field trials were designed using a randomized complete block design. The parental genotypes were planted in 6 replicates and the RILs were planted in 3 replicates. Each plot contained three 2 m long rows 0.5 m apart. Six plants were planted in each row. Seeds were harvested from each plot at maturity and dried at ambient temperature for subsequent isoflavone, protein and oil determinations.

[0032] Example 2: Extraction and Quantification of Isoflavones

[0033] Approximately 10 g of dry soybean seeds were selected and examined from each plot to ensure they were free from pests and diseases. Then, they were ground using a cyclone mill (CT 293 Cyclotec, FOSS, Denmark) and sieved through an 80-mesh filter. The method for extracting isoflavones described above was slightly modified. 20 mg (0.01 mg) of soybean powder was dissolved in 1 mL of 70% (v / v) ethanol and 0.1% (v / v) acetic acid in a 1.5 mL plastic tube. The mixture was shaken on an incubation shaker (INNOVA 42, Eppendorf, Germany) at 28 °C and 200 rpm for 12 hours. Then, the mixture in the test tube was centrifuged at 12,000 rpm for 10 minutes, and the supernatant (0.7 mL) was filtered through a 0.45-μm nylon syringe filter (Jinteng, Tianjin, China). Before analyzing the isoflavones, the filtered samples were refrigerated at 4 °C. According to the published method, high-performance liquid chromatography (HPLC) was used to identify and quantify the isoflavones. The chromatography was run on an Agilent 1260 HPLC system (Agilent 1260 HPLC system, Santa Clara, CA, USA), maintaining a 70-minute linear gradient of 13 - 35% acetonitrile (v / v) at 35 °C, with a solvent flow rate of 1.0 mL·min-1 and an injection volume of 20 μL. The chromatographic column used was a YMC-Pack ODS-AM-303 column (inner diameter 250 mm × 4.6 mm, S-5 μm, 120, YMC Kyoto, Japan), and isoflavones were detected by ultraviolet absorption at 260 nm with mobile phases A and B.

[0034] Twelve isoflavone standards were provided by the Chinese Academy of Agricultural Sciences. These isoflavones include daidzein (DE), daidzin (D), malonyldaidzein (MD), acetyl daidzein (AD), genistein (GE), genistin (G), malonyl genistein (MG), acetyl genistein (AG), glycitein (GLE), glycitin (GL), malonyl glycitin (MGL), and acetyl glycitin (AGL). Isoflavones were classified according to the aglycones they contain and were defined as follows: daidzein derivatives (Ds) were defined as the sum of De, D, MD, and AD; genistein derivatives (Gs) were defined as the sum of GE, G, MG, and AG; glycitein derivatives (GLs) were defined as the sum of GLE, GL, MGL, and AGL; and total isoflavones (TIF) were defined as the sum of the contents of all individual isoflavones. The concentration of isoflavones was determined based on the retention time and peak area. The peak area data were extracted in the R software package shiny_HPLC. V1.0 (https: / / git hub.com / Zhaoqing Songjia / shiny_HPLC).

[0035] Example 3. Protein and Oil Determination

[0036] Using the constructed model, the protein and oil content was measured with a near infrared spectrometer (NIS). About 10 g of disease-free dry mature soybean seeds were placed in a measuring cup and then scanned with a NIS analyzer (MATRIX-I, BRUKER, Germany). The reflectance spectrum information was converted into log values ​​by calculation and stored. The data was then analyzed in the WINISI software (WINISI1.02) developed by INFROSOFT, and the protein and oil content of the soybean seeds was finally output.

[0037] Example 4: Genotyping by sequencing and SNP calling

[0038] DNA was isolated from soybean seedling leaf tissue using the cetyltrimethylammonium bromide (CTAB) method. The collected DNA samples were randomly cut to a length of approximately 350 bp using a Covaris crusher. Libraries were constructed using the TruSeq library construction kit (Novogene, China) and sequenced on the Illumina HiSeq platform. Sequencing data were compared with the reference genome (G.max Wm82.a2) using the BWA software using the following parameters: mem-t 4-k 32-M. Format conversion and SNP detection were performed using the SAMtools software with the minimum SNP length set to 4 bp and the minimum quality value (MQ) set to 20.

[0039] Example 5: Map construction and QTL detection

[0040] To improve the quality of the constructed map, SNP data were analyzed in the chi-square test with a P value threshold set to 0.05 to allow for inclusion of SNPs in subsequent steps. Redundant markers were then removed using the bin function in the software QTL IciMapping 4.1 run with default parameters. A genetic map was constructed in MSTmap (Linux version). The parameters used were as follows: cutoff p value was set to 10–20, on-map distance was set to 15, no map size was set to 2, pre-clustering estimation was set to yes, and other values ​​were default. To detect QTLs associated with protein, oil, and isoflavone content, mapqtl 6.0 (https: / / www.kyazma.nl / index.php / MapQTL / ) was run using the multi-model (MQM) approach. The logarithm of the odds ratio (LOD) threshold was set to 2.5 to verify the presence of QTLs. A permutation test was performed to estimate the LOD significance for 1000 iterations with a threshold P value set to 0.05.

[0041] Example 6. QTL Integration

[0042] The co-localized fragments of isoflavones and protein or oil yield identified in this study were compared with previously published QTLs associated with the same traits. A composite genetic map was constructed using all downloaded QTLs, all molecular markers present on the consensus genetic map, and all coordinates of consensus map markers using the G. max Wm82.a2 genome version present on the soybase website ( https: / / soybase.org / ).Significant QTLs associated with the target trait and neighboring markers located on the same position fragment were evaluated for the physical distance separating the associated traits.Genetic maps and consensus maps were constructed in MapChart2.32 software.

[0043] Example 7: Statistical analysis

[0044] A total of 14 traits were analyzed, including 12 isoflavone traits and protein and oil traits. Since only trace amounts of DE, GE, GLE, and AG were detected in the observation population, these compounds were excluded from further analysis. Parental importance was analyzed using Student's t-test in the basic R package. Population genetic variation was analyzed using the R package psychometrics. Broad-sense heritability (h2b) was calculated using the R package LME4 (https: / / github.com / lme4) according to the following formula: h2b = VG / (VG+VE), where VG is the variance between RILs and VE is the variance within RILs. Correlation analysis was performed using the R package perfanalysis.

[0045] Example 8. Comparison of isoflavones, protein and oil between parents

[0046] The parents used in this study include the representative soybean variety JD12 (ZDD23040) and the wild soybean variety Y9 (ZYD02739). The genotypes of the two parents differed significantly in seed size, seed color and other seed traits ( Figure 1 A), and more importantly, there were significant differences in isoflavone, protein and lipid content ( Figure 1 The total isoflavone and protein contents of wild soybean parent Y9 were much higher than those of cultivated parent JD12, but the oil content was lower than that of cultivated parent JD12 ( Figure 1 ). This suggests that the genetics favors the production of isoflavones and proteins in Y9, while the genetics of JD12 favors oil synthesis. By dividing the aglycones in isoflavones into three groups, Ds, Gs, and GL can be analyzed separately in the two parents. Here, we found that the content of Ds, Gs, and GLs in Y9 was significantly higher than that in JD12, with the concentrations in Y9 being 169%, 161%, and 119% higher than those in JD12, respectively ( Figure 1 DF).

[0047] Example 9: Genetic and phenotypic variation results of RIL population

[0048] To further investigate the genetic basis of isoflavone production in soybean seeds, recombinant inbred lines (RILs) of F8:10 progeny of JD12, Y9, and 185 were evaluated for 12 isoflavone traits as well as protein and oil contents in a two-year field trial. Consistent with previous results, aglycones of DE, GE, GLE, and acetylated glycosides of AG were not detected in this study. In addition, large genetic variations in other isoflavone contents as well as protein and oil contents were detected in parents and RILs (Table 1). The average CVs for isoflavone, protein, and oil contents during the two-year observation period were 34.44%, 13.12%, and 6.90%, respectively, indicating that the genetic variation in isoflavone production was greater than that in protein or oil production. Widely exceeded heritability was observed for each observed trait except oil in 185 RILs. The population means for most traits were between the parental values, while the maximum and minimum values ​​exceeded the parental limits, indicating that both parents could contribute to phenotypic variation (Table 1). Based on the kurtosis and skewness values ​​present in the trait histograms, the detected contents of isoflavones, protein, and oil followed a normal and continuous distribution throughout the population, indicating that these traits were inherited as quantitative traits ( Tables 1 and Figure 1 ). In addition, the broad-sense heritability of the traits observed in the two-year experiment ranged from 0.72 to 0.87 (Table 1), indicating that the phenotypic variation mainly came from genetic variation rather than environmental variation. Therefore, it is feasible to locate the trait locus. The heritability of protein and oil content was 0.81 and 0.87, respectively, both higher than the heritability of isoflavones, indicating that the production of isoflavones is more sensitive to environmental fluctuations than the synthesis of protein or oil.

[0049] Example 10. Correlation analysis results of isoflavones, protein and oil contents in soybean seeds

[0050] In order to investigate the potential relationships among the observed traits, correlation analyses were performed, and the correlation coefficients, histograms, and scatter plots for isoflavone, protein, and oil contents are summarized in Figure 2 After two years of experimentation, the protein and oil contents of harvested soybean seeds showed a consistent negative correlation (r = -0.79***), and TIF was positively correlated with Ds, Gs, and GLs ( Figure 2 Total isoflavone content was highly correlated with Ds (r = 0.96***) and Gs (r = 0.98***), which is consistent with the additional observed correlations between TIF and D, MD, G, and MG ( Figure 7 ). Meanwhile, the correlation between TIF and GLs was relatively low (r=0.79***), indicating that Ds and Gs may be the main contributors to soybean TIF ( Figure 2). Isoflavones were significantly correlated with both protein and oil content, with TIF being negatively correlated with protein content (r=-0.36***) and TIF being weakly positively correlated with oil (r=0.067*)( Figure 2 ). When the results for 2020 and 2021 were analyzed separately, similar results were observed, with annual TIF contents showing correlations with protein content of -0.42*** and -0.34***, and with oil content of 0.09* and 0.005, respectively ( Figure 5-Figure 6 ). These results suggest that isoflavone and protein traits may be coordinately inherited, whereas oil production appears to be more independent.

[0051] Example 11: Further map construction and verification

[0052] The chromosomal locations of loci affecting isoflavone, protein, and oil contents were evaluated using 226,186 SNPs identified between parents by GBS analysis. Assuming that markers segregate in a 1:1 ratio, 6072 (2.68%) SNP markers remained after the chi-square test in the QTL software IciMapping 4.1 run with default parameters, and 4075 (1.8%) SNP markers remained after removing redundant markers that fell into the common bin (Meng et al., 2015). These screened SNP markers generated a high-density linkage map containing 3943 SNP markers spanning approximately 938Mb of the 1.1Gb soybean genome, covering 6307cM, and the average distance between adjacent SNP markers was 1.6cM. The average number of SNPs in each linkage group was 197, with the most in linkage group 13 (314 SNPs) and the least in linkage group 2 (138 SNPs) ( Fig.10 Table S 1). To verify the accuracy of the map, the seed coat color trait was located on the constructed map, and the dominant QTL was located between 8.2 and 8.4 Mb on chromosome 8, with a high LOD value of 39.87, which was consistent with the previously published GWAS data. Therefore, the constructed high-density and high-quality linear image map was considered suitable for further research.

[0053] Example 12. QTL identification results for isoflavones, protein and oil content in soybean seeds

[0054] With the appropriate high-density linkage map, potential QTLs for isoflavone content were identified each year. A total of 176 QTLs exceeded the LOD and permutation detection thresholds, which could then be integrated into 25 loci based on genetic and physical distances (Table 2, Table S2). The LOD values ​​of the isoflavone loci ranged from 2.52 to 11.34, the PVE values ​​ranged from 6.1% to 24.8%, and the ADD values ​​were mostly negative, indicating that the alleles from males promoted the increase in isoflavone content, which is consistent with the high isoflavone content in Y9. On the other hand, several loci favoring isoflavone production were found on chromosomes 8, 9, and 20 of JD12. In the two years of the experiment, 14 stable loci were consistently detected, accounting for 56% of the total isoflavone variation (qISO1, qISO5, qISO6.1, qISO6.2, qISO6.3, qISO6.4, qISO8.1, qISO10.3, qISO11, qISO12, qISO14, qISO17, qISO19.2, qISO19.3). These loci are located on chromosomes 1, 5, 6, 8, 9, 10, 11, 12, 14, 17 and 19 (Table 2).

[0055] Two major stable loci with average PEV values ​​exceeding 15% were mapped to narrow genomic fragments (Table 4). The qISO5 locus was located on the 1.05MB physical section (41042159–42098680) and was identified as a strong major isoflavone locus for TIF, DS, GS, GLS, D, MD, G, MG, GL, and MGL. In all experiments, the LOD and PVE values ​​of qISO5 ranged from 3.1 to 11.11% and 7.5 to 24.4%, respectively. In addition, within the qISO5 locus, the individual QTLs of qDs5, qTIF5, and qGs5 all produced relatively high LOD values ​​(11.11, 9.94, and 7.12, respectively) and PEV values ​​(24.4%, 22.1%, and 16.4%, respectively), indicating that these loci may be the main controllers of Ds, TIF, and Gs synthesis (Table 4). Another locus, qISO6.2, which is closely related to the production of TIF, GL and MGL, is located at a narrow physical distance of 0.95 Mb (18449150-19395795), with LOD and PVE values ​​ranging from 2.94 to 10.15% and 7.1% to 24.8% during the experiment, respectively. The average LOD and PVE values ​​of the combined sections of qGLs6.2 and qMGL6.2 within qISO6.2 were 10.59% and 23.4%, respectively, indicating that this locus is the main control site for GLs synthesis (Table 4).

[0056] A total of 42 protein and oil QTLs were detected, including 20 protein QTLs and 22 oil QTLs. The LOD values ​​ranged from 2.51 to 10.30, and the PVE values ​​ranged from 6.10% to 22.80% (Table 3). Seven stable protein QTLs were significant in both years, including qPC6.1, qPC8, qPC9, qPC15, qPC20.1, qPC20.2, and qPC20.3. Except for qPC6.1, the QTL LOD values ​​of each locus were greater than 5. The maximum protein LOD value returned by locus qPC20.2 (22632082bp) was 9.41, and the maximum PVE value was 21.10%. Eight QTLs for oil were detected in each year, including qOC6.1, qOC8, qOC13.1, qOC15.1, qOC20.1, qOC20.3, qOC20.4, and qOC20.5. Except for qOC6.1, qOC13, and qOC20.5, each locus returned an LOD value greater than 5. The maximum average LOD value for the qOC20.1 locus (5834525bp-6101553bp) was 10.13, and the maximum average PEV was 22.55%. For protein production, all QTLs returned negative ADD values ​​except the QTL on chromosome 6, indicating that Y9 contributed to favorable protein inheritance in addition to the locus on chromosome 6. In contrast, JD12 provided favorable alleles for oil production in addition to the alleles on chromosome 6. Based on genetic distance and physical distance, the combined protein and oil loci can be integrated into 15 quality loci. These 10 loci contained both protein QTL and oil QTL (Table 3). This is consistent with previous results, in which protein yield in soybean was strongly negatively correlated with oil yield ( Figure 2 ). Overall, several stable major QTLs associated with isoflavone, protein, and oil contents were detected in the observed RIL populations.

[0057] Example 13. Co-localization results of soybean isoflavone, protein and oil content sites

[0058] This study found three overlapping regions of co-localized protein and isoflavone QTLs on chromosomes 6, 8, and 9. Among them, qISO6.2 and qPC6.1 were located at the same position, qISO8.1 and qPC8 were located at the same position, and qISO9.1 and qISO9.2 were both located at the same position as qPC9 ( Figure 3 JD12 provided alleles that increased protein and decreased isoflavones on colocalized segments on chromosome 6, as well as alleles that decreased protein and increased isoflavones on colocalized segments on chromosomes 8 and 9 (Table 2, Table 3). This is consistent with the overall negative correlation between isoflavone and protein content observed in this study ( Figure 2 ).

[0059] Not surprisingly, markers for previously reported protein and isoflavone QTLs were found in the colocalized segments of this study. On chromosome 6, the region of colocalized isoflavone and protein loci identified in this study was also close to previously identified isoflavone loci SeedIsoflavone1-2, SeedIsoflavone8-1, and protein loci SeedProtein28-1, SeedProtein29-1, and Protein35-2. On chromosome 8, the colocalized region identified in this study was very close to previously reported isoflavone loci SeedIsoflavone7-1, SeedIsoflavone7-7, SeedIsoflavone7-10, and SeedIsoflavone6-7, as well as protein loci SeedProtein30-4, cqSeedProtein013, cqSeedProtein016, SeedProtein34-4, SeedProtein34-5, and Protein26-1. On chromosome 9, the colocalization region defined in this study is close to the previously identified isoflavone locus Seed isoflavone 12-6, and the previously identified protein loci Seed protein 36-27, Seed protein 24-3, Seed protein 41-7, and protein 40-3 ( Figure 3 ).

[0060] More interestingly, previously identified isoflavone and protein loci were located in the same region as the major isoflavone and protein loci identified in this study. Close to qISO5 lay the previously identified isoflavone loci Seed isoflavone 1-1, Seed isoflavone 6-1, and Seed isoflavone 7-5, as well as the previously identified protein loci Seed protein 34-1, Seed protein 9-1, protein 2-1, cqSeed protein 011, and protein 12-1. Meanwhile, the region close to qPC15.1 contained the previously identified isoflavone loci Seed isoflavone 6-3, Seed isoflavone 9-2, and Seed isoflavone 11-10, as well as the protein loci Seed protein 30-3, Seed protein 4-5, Seed protein 39-2, Seed protein 3-6, cqSeed protein 001, and protein 5-1 ( Figure 3 ). All these results clearly indicate that the production of soy isoflavones and protein is coordinated genetically, which may contribute to effectively increasing the content of soy isoflavones as well as the content of protein or oil.

[0061] Example 14: Effect of isoflavone site combination on isoflavone and protein / oil content

[0062] Two major favorable loci, including qISO5, which mainly targets Ds and Gs, and qISO6.2, which mainly targets GL, were selected to further analyze the effects of combined isoflavone loci on isoflavone, protein, and oil contents. The isoflavone contents of progenies without favorable loci (N group) were significantly lower than those with favorable loci, and the additive effect of qISO5 (q5 group) was greater than that of qISO6.2 (q6.2 group). The progenies carrying both qISO5 and qISO6.2 (q5q6.2 group) had the highest isoflavone concentration, indicating that the two loci had a cumulative effect on isoflavone content. There were more dispersed progenies with high protein contents (pink dots) in the members of the N and q5 groups, while there were more dispersed lines with high oil contents (round dots) in the q6.2 and q5q6.2 groups. This leads to the speculation that breeding soybeans for high isoflavone and high oil content may be simpler than breeding soybeans for high isoflavone and high protein content, which is consistent with the rest of the results of this study, in which isoflavone content was negatively correlated with protein content and weakly positively correlated with oil content ( Figure 2 We also found that some protein-rich lines in the q5q6.2 group were dispersed among other members, suggesting that it may be possible to breed high-protein, high-isoflavone lines in soybeans through targeted breeding efforts ( Figure 4 ).

[0063] Embodiment 15, application prospects

[0064] Improved living standards have driven an increase in demand for healthy nutrition, which has led to an increase in demand for crop agricultural products such as soybeans to provide an optimal combination of protein and oil content. In addition, soybean seeds may also contain high isoflavone content, which has associated health functions such as reducing the risk of cancer and several chronic diseases. Therefore, in modern soybean breeding programs, there is currently an urgent need to develop superior soybean varieties that can produce both high isoflavone content and high protein or high oil content.

[0065] The importance of optimizing protein, oil, and isoflavone contents in soybean using a quantitative trait locus approach has long been recognized and studied. However, the understanding of the relationship between isoflavone, protein, and oil production in soybean remains limited. Here, the relationship between seed isoflavone, protein, and oil content was investigated using QTLs associated with isoflavone, protein, and oil yield in two years of field trials. The h2b for isoflavone content in these trials was 0.81 (Table 1), corresponding to the high heritability reported previously. Although genetic variation for isoflavone production exists in soybean, environmental influences are also known to affect seed isoflavone concentrations. As a result, 11 of the 25 loci, including five previously reported loci, were detected in only one year, while six new loci were identified, designated qISO2.2, qISO3, qISO10.1, qISO10.2, qISO19.1, and qISO20 (Table 2). On the other hand, 14 of the 25 loci, including 10 previously reported loci, were detected in both trials (Table 2), making four significant loci named qISO1, qISO6.1, qISO6.3, and qISO6.4 the loci that have not been registered in the corresponding composite map at www.soybase.org. Interestingly, we found that the gene Glyma.01g172900 on chromosome 1 (51020047-51025420) close to qISO1 (51600690-54799844) (Table 2) was predicted to be an isoflavone reductase-like protein, which may be the gene controlling the isoflavone content at the qISO1 locus. The reconfirmation of QTLs in multiple trials suggests that not only the regions associated with isoflavone production detected in this study are reliable, but also new stable QTLs may be detected in different populations.

[0066] Previously, a major QTL at the lower end of chromosome 5 was detected in five different populations, which was narrowed down to a 611.4 kb fragment on chromosome 5 between bp positions 38434171 and 39045620 in the G.maxWm82.a1 reference genome. In the present study, we also detected a major QTL marker qTIF5 in a 373.6 kb fragment between bp positions 41042159 and 41415752 on chromosome 5, which explained 16.1%-22.1% of the variation in isoflavone content observed in the two-year included field trial. Near this fragment, QTLs qD5, qMD5, qDs5, qG5, qMG5, qGs5, qGL5, qMGL5, qgl5 were also found, indicating that this region may control the production of multiple individual isoflavones (Table 4), consistent with previous reports.

[0067] The low correlation coefficients of glycyrrhizin derivative isoflavones with other isoflavones in this study are consistent with previous reports and suggest that Ds and Gs may be produced in similar metabolic pathways that share metabolic enzymes, while GL may be produced in another more isolated metabolic pathway that shares few metabolic enzymes with other isoflavone pathways. This means that the production of GLs may involve new genes or loci.

[0068] In this study, we fine-mapped a locus labeled qISO6.2, which is located in a 946.3 kb segment on chromosome 6 between positions 18449510 and 19395795 BP. In this region, higher PVE values ​​(mean: 23.4% vs. 9.5%) and LOD (mean: 10.59% vs. 3.4%) were observed for the qGLs6.2 and qML6.2 segments than for qTIF6.2, indicating that qISO6.2 mainly controls the content of GL. Using 480 SNP markers, a locus associated with GL production was found near BARC-031337-07051 (physical position: 16679945) on chromosome 6, with an associated LOD value of 4.2. The LOD values ​​of the loci detected on chromosome 6 in this study were higher than those in previous studies, which is more conducive to gene cloning and marker-assisted selection (MAS).

[0069] A total of 42 protein and oil QTLs were detected, and 15 QTLs detected in the two-year experiment were basically consistent with the QTLs identified in previous studies (Table 3). The major locus qPC20.3 is located between 32.38Mb and 33.29Mb on chromosome 20, close to the previously cloned gene Glyma.20g085100 located at the physical position of 31.77Mb on chromosome 20. In addition, the qOC15.1 major locus was found to be located between 2.32Mb and 4.37Mb on chromosome 15, close to GMsweet 39 (Gly ma.15g 049200) at 3.87Mb on chromosome 15 (mio et al., 2020). These results show that the high-density map generated in this paper accurately locates the relevant genes in the important QTL region (Table 3, Table S3).

[0070] Three of the 14 loci associated with protein content colocalized with the isoflavone loci on three linkage groups (chromosomes 6, 8, and 9) ( Figure 3). In these co-localized fragments, the additive effects on isoflavone and protein content seem to be opposite. For example, the additive effect of qISO6.2 on protein content is negative, with a minimum additive value of -515.976, while the contribution of the qPC6.1 site located in the same fragment is positive, with a maximum additive value of 1.13 (Table S2, Table S3). This may be the reason why soybean isoflavone content is significantly negatively correlated with protein content (-0.36***), which is consistent with previous reports.

[0071] Phenotypic correlations between traits could be due to genetic proximity or pleiotropy in the linkage group. To verify that the observed phenotypic correlations were due to colocalization, the 2020 data were analyzed to understand the effect of eliminating the three colocalized segments. In this permutation, the correlation between protein and isoflavone content was reduced (-0.12) and became non-significant ( Fig.11 ), further demonstrating that the observed correlation is due to colocalization. In addition, previously mapped isoflavone and protein sites were found in all three deleted fragments ( Figure 3 Although only one major isoflavone locus (qISO5) was located on chromosome 5 in this study, isoflavone and protein loci have been found on the same segment in previous studies ( Figure 3 ). The additive effect of this fragment on isoflavone content was found to be opposite to that on protein content. Similarly, although only one major protein locus was detected on chromosome 15 in this study, both protein and isoflavone loci have been previously localized to the same region ( Figure 3 ). These results strongly suggest that seed isoflavone and protein content may be coordinately inherited through colocalization of influential loci in the soybean genome.

[0072] Mainly due to the colocalization of protein and isoflavone loci, isoflavone production may be negatively correlated with protein content. However, some loci, such as qPC15.1 and qISO5, are thought to control only protein or isoflavone content, so breeding soybean varieties rich in isoflavones and protein or oil may still be a possibility. In this study, no favorable isoflavone loci were found to cluster with high protein loci ( Figure 4 ), although there was enough separation to suggest that it might be possible to breed soybeans with high levels of isoflavones and protein. For example, when qISO5 and qISO6.2 were combined, some progeny lines contained high levels of isoflavones and protein. Conversely, in some cases, such as when providing infant nutrition, low-isoflavone varieties may be desired. In this study, we elucidated the genetic basis for protein, oil, and isoflavone production, which can be further explored to produce combinations with optimal isoflavone, protein, and oil content for various uses.

[0073] In conclusion, the studies conducted and presented in this paper identified and located stable and major QTLs for soybean isoflavone, protein, and oil yields in a high-density genetic map. Some loci were consistent with previous studies, while other novel isoflavone-related loci were described here for the first time. The consistency with previous results, the significant correlation, and the elimination of the effects of significant loci from the analysis all demonstrated the accuracy and novelty of the results. In addition, the loci for isoflavone and protein production were mapped to co-localized regions, which further demonstrated that the correlation between isoflavone and protein content observed in soybean seeds may be generated by the coordinated inheritance of linked loci rather than genetic pleiotropy. Overall, these results provide a theoretical basis for the coordinated improvement of isoflavone and protein quality in future soybean breeding efforts.

[0074] Example 16, table summary

[0075] Table 1

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[0077] Table 2

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[0081] Table 3

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[0084] Table 4

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[0087] Table s1

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[0090] Table s2

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[0096] Table s3

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[0099] In the above embodiments, the description of each embodiment has its own emphasis. For the part that is not described or recorded in detail in a certain embodiment, please refer to the relevant description of other embodiments. The above embodiments are only used to illustrate the technical solution of the present invention, not to limit it; although the present invention is described in detail with reference to the above embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above embodiments, or replace some of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention.

Claims

1. An integrated quantitative trait locus for soybean molecular marker-assisted selection breeding, wherein the soybean molecular marker-assisted selection breeding is oriented to the soybean isoflavone content trait, characterized in that: The integrated quantitative trait locus is one or any combination of the loci named qISO1, qISO6.1, qISO6.3, and qISO6.4; Among them, qISO1 is located on chromosome 1 of the soybean reference genome G. max Wm82.a2, with a genetic distance of 231.938-275.569 cM and a physical locus / interval of 01_51600690-01_54799844; Among them, qISO6.1 is located on chromosome 6 of the soybean reference genome G. max Wm82.a2, with a genetic distance of 179.84-191.072 cM and a physical locus / interval of 06_14871510-06_15642762; Among them, qISO6.3 is located on chromosome 6 of the soybean reference genome G. max Wm82.a2, with a genetic distance of 241.934-242.934 cM and a physical locus / interval of 06_35913434-36835583; Among them, qISO6.4 is located on chromosome 6 of the soybean reference genome G. max Wm82.a2, with a genetic distance of 271.344-273.163 cM and a physical locus / interval of 06_42113786-06_43887351.

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