A molecular marker related to soybean leaf sucrose content and application thereof

Abstract

The application discloses a soybean leaf sucrose content related molecular marker and application thereof, and belongs to the technical field of plant breeding. In order to quickly and accurately screen a soybean variety with high leaf sucrose content, the application provides a soybean leaf sucrose content related molecular marker, wherein the gene of the molecular marker is Glyma.14G029100, and the nucleotide site at 2131531 is T or C. The marker has high practical value in actual production process, can be used for early screening of materials at the seedling stage, and is helpful to shorten the breeding time, but the effect of the single marker is limited, and more related molecular markers can be developed for assisting breeding of excellent soybean germplasm in the later period.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically relating to a molecular marker related to sucrose content in soybean leaves and its application. Background Technology

[0002] Soybean (Glycine Max [L] Merr.) originated in China and has a cultivation history of over 5,000 years. It is an annual herbaceous plant belonging to the genus Glycine in the legume family. It is also one of the world's earliest cultivated economic crops, rich in plant protein and oil, making it an important oilseed and feed crop with promising development prospects. With the improvement of people's living standards, the demand for soybeans is increasing. However, my country's current soybean production is limited and cannot meet domestic demand, leading to a rising dependence on imports. This is mainly due to the low yield per unit area at present. Improving yield per unit area is currently the focus of breeding work. Therefore, to meet the needs of my country's soybean industry development, it is urgent to breed high-yielding varieties. Biomass yield is the foundation of economic yield. A relatively large biomass yield is formed during the seedling stage, laying a good foundation for the final biomass yield of soybeans. Soybean seedlings mainly undergo vegetative growth. As the vegetative body increases in size, more light energy is converted into chemical energy, promoting the accumulation of dry matter and laying a solid foundation for reproductive growth.

[0003] Sucrose is an important intermediate product in the conversion of light energy into chemical energy, and most of the photosynthetic products required for plant growth and development are supplied and transported in the form of sucrose. Studying the changes in sucrose content in soybean seedling leaves is of significant reference value for the breeding of superior soybean varieties. Traditional detection methods are too slow, and a rapid, accurate, and convenient method is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to quickly and accurately screen soybean varieties with high sucrose content in their leaves.

[0005] This invention provides a molecular marker related to sucrose content in soybean leaves. The gene of the molecular marker is Glyma.14G029100, and the nucleotide position at position 2131531 is T or C.

[0006] The present invention provides a primer sequence for amplifying the above-mentioned molecular marker, wherein the forward primer sequence is shown in SEQ ID NO.4 or SEQ ID NO.5; and the reverse primer sequence is shown in SEQ ID NO.6.

[0007] This invention provides a SNP locus related to sucrose content in soybean leaves, the SNP locus being located at position 2131531 on chromosome 14, and the base of the locus being T or C.

[0008] This invention provides primer sequences for amplifying the above-mentioned SNP sites, with the forward primer sequence shown in SEQ ID NO.4 or SEQ ID NO.5 and the reverse primer sequence shown in SEQ ID NO.6.

[0009] This invention provides the application of the above-mentioned molecular marker, primer sequence, SNP site, or primer sequence in the preparation of a kit for identifying soybean leaves with high or low sucrose content.

[0010] This invention provides a kit for identifying soybean leaves with high sucrose content. The kit includes the primer sequences described above. If the soybean variety to be tested is found to have the TC genotype, then the soybean leaves of the variety to be tested are high in sucrose content.

[0011] This invention provides a kit for identifying soybeans with high seedling biomass, the kit including the primer sequences described above; if the soybean variety to be tested is found to be of the TC genotype, then the variety to be tested is a soybean with high seedling biomass.

[0012] This invention provides a method for identifying soybean leaves with high sucrose content, the specific steps of which are as follows:

[0013] Step 1: Extract DNA from the soybeans to be tested;

[0014] Step 2: Perform a PCR reaction using the primer sequences of the molecular markers or the primer sequences of the SNP sites mentioned above. If the soybean variety to be tested is of the TC genotype, the soybean leaves of the variety to be tested are of high sucrose content; if it is of the CC genotype, the soybean leaves of the variety to be tested are of low sucrose content.

[0015] Further specifying, the PCR reaction in step 2 is as follows: 95 °C, 10 min; 95 °C, 20 sec, 61 °C, 45 sec, 10 cycles at -0.6 °C; 95 °C, 20 sec, 30 cycles; 55 °C, 45 sec; 37 °C, 1 min, 1 cycle.

[0016] Further specifying, in step 2, the base of the SNP site for the TC genotype is T; and the base of the SNP site for the CC genotype is C.

[0017] Beneficial Effects: Significant differences were observed in biomass yield and leaf sucrose content among different varieties in the soybean KASP marker development population, with large variation ranges. A significant positive correlation was found between soybean seedling biomass yield and leaf sucrose content. A KASP marker, Chr14_2131531, was developed to determine soybean seedling leaf sucrose content. The KASP marker validation population confirmed that Chr14_2131531 has excellent genotyping effects, dividing 337 germplasm resources into two categories: varieties carrying the TC genotype and varieties carrying the CC genotype. Furthermore, the Chr14_2131531 marker showed a 70.14% selection effect on varieties with high leaf sucrose content. Attached Figure Description

[0018] Figure 1 Histogram of biomass frequency distribution for soybean KASP marker development population; Note: X-axis is the biomass of soybean KASP marker development population in 2022 and 2023, in g; Y-axis is the number of varieties corresponding to this biomass; Figure A is the production in 2023;

[0019] Figure 2 Correlation analysis results of soybean KASP marker development population biomass; Note: X-axis is the biomass of the 2023 KASP marker development population in g, Y-axis is the biomass of the 2022 soybean KASP marker development population in g;

[0020] Figure 3 Histogram of frequency distribution of sucrose content in leaves of soybean KASP marker development population; Note: X-axis is the sucrose content in leaves of soybean KASP marker development population in 2022 and 2023, in mg / g, Y-axis is the number of varieties corresponding to this sucrose content; Figure A shows the sucrose content in 2023;

[0021] Figure 4 Correlation analysis results of leaf sucrose content in soybean KASP marker development population; Note: X-axis represents leaf sucrose content in soybean KASP marker development population in 2023, in mg / g; Y-axis represents leaf sucrose content in soybean KASP marker development population in 2022, in mg / g.

[0022] Figure 5 A statistical diagram showing the variation sites on the key gene Glyma.14G029100;

[0023] Figure 6 Image showing the genotyping results of the key gene Glyma.14G029100;

[0024] Figure 7Figure A shows the differential results of different superior haplotypes of the Glyma.14G029100 gene; Figure B is a heatmap of the differential analysis of sucrose content in leaves of two haplotypes; Figure B is a violin plot of sucrose content in leaves carrying different haplotypes.

[0025] Figure 8 The image shows the screening results for variant sites in the Glyma.14G029100 gene.

[0026] Figure 9 The distribution of sucrose content in the leaves of soybean KASP marker validation population is shown in the figure. Note: The X-axis represents the sucrose content of 337 soybean KASP marker validation populations in 2023, in mg / g, and the Y-axis represents the number of varieties corresponding to that sucrose content.

[0027] Figure 10 This is a graph showing the signal distribution of Chr14_2131531; Note: Blue and green represent two different alleles.

[0028] Figure 11 Figure showing the differences in leaf sucrose content among different genotypes of Chr14_2131531. Detailed Implementation

[0029] Example 1.

[0030] 1. Field sampling

[0031] This study investigated soybeans. Samples were collected during the seedling stage, in June 2022 and June 2023. Sampling was conducted between 4 PM and 5 PM when the fourth trifoliate leaf was fully expanded (V4 stage). Seven plants of consistent growth were collected from each variety. The middle leaf of the third trifoliate leaf was placed in an EP tube for leaf sucrose content determination, while the aboveground part of the plant at the cotyledon node was used for biomass yield determination. After sampling, the EP tubes containing the samples were quickly placed in liquid nitrogen for short-term preservation. After sampling, the tubes were stored at -80 °C for subsequent research.

[0032] 2. Determination of biomass yield

[0033] First, place the aboveground soybean samples in an oven at 105 ℃ for 30 min to kill the green, then dry them at 85 ℃ to constant weight. Use a balance to determine the biomass of the aboveground parts of the plant and record it in time.

[0034] This study investigated 112 KASP marker-developed populations from 2022 to 2023. Samples were taken when the fourth trifoliate leaf was fully expanded (V4 stage). The biomass distribution is shown below. Figure 1 As shown, A exhibits a distribution pattern of high in the middle and low at both ends, demonstrating a good normal distribution characteristic.

[0035] result: Figure 1 Figure A shows the biomass distribution of the KASP marker development population in 2023. There were 9 varieties with a biomass range of 2–3 g, 20 varieties with a range of 3–4 g, 33 varieties with a range of 4–5 g, 26 varieties with a range of 5–6 g, 11 varieties with a range of 6–7 g, 10 varieties with a range of 7–8 g, 2 varieties with a range of 8–9 g, and 1 variety with a range of 9–10 g. The variety with a biomass range of 4–5 g was the most numerous, accounting for 29.46% of the total, while the variety with a range of 9–10 g was the least numerous. The biomass distribution of the KASP marker development population in 2022 was as follows: 8 varieties had a biomass range of 2–3 g; 25 varieties had a biomass range of 3–4 g; 36 varieties had a biomass range of 4–5 g; 23 varieties had a biomass range of 5–6 g; 12 varieties had a biomass range of 6–7 g; 6 varieties had a biomass range of 7–8 g; 1 variety had a biomass range of 8–9 g; and 1 variety had a biomass range of 9–10 g. The largest number of varieties (32.14%) had a biomass range of 4–5 g, while the fewest varieties had biomass ranges of 8–9 g and 9–10 g.

[0036] Table 1 presents a statistical analysis of seedling biomass in 112 KASP marker development populations from 2022 to 2023. In the 2023 KASP marker development populations, the variety with the highest biomass was Kenfeng 22 (9.12 g), and the variety with the lowest was Ha 12-3510 (2.07 g). The maximum biomass was 4.41 times the minimum, the mean was 4.89 g, the standard deviation was 1.55, the coefficient of variation was 31.70%, the skewness was 0.64, and the kurtosis was -0.05. In the 2022 KASP marker development populations, the variety with the highest biomass was Henong 85 (9.07 g), and the variety with the lowest was Ha 12-3510 (2.08 g). The maximum biomass was 4.36 times the minimum, the mean was 4.79 g, the standard deviation was 1.31, the coefficient of variation was 28.60%, the skewness was 0.58, and the kurtosis was 0.09.

[0037] right Figure 1 A comprehensive analysis of Table 1 revealed that the coefficient of variation of soybean seedling biomass was relatively large between the two years of 2022 and 2023, with a clear multiple relationship between the maximum and minimum values. This indicates that there are significant differences and a wide range of variations in the seedling biomass of different soybean varieties, and that there is rich genetic variation in biomass within this population.

[0038] Table 1. Statistical analysis of biomass yield in soybean KASP marker-developed populations

[0039]

[0040] Figure 2 A correlation analysis of seedling biomass data from 112 soybean KASP marker development populations from 2022 to 2023 revealed a high positive correlation between the biomass of the 2022 and 2023 soybean KASP marker development populations, with a coefficient of determination R0. 2 =0.833, indicating that the biomass yield was highly consistent over the two years and the population biomass yield was stable. The linear correlation proved the usability of the experimental data.

[0041] 3. Determination of sucrose content in leaves

[0042] The sucrose content of soybean leaves was determined using the resorcinol method. The specific steps are as follows:

[0043] (1) Preparation of extract (mother liquor). The leaves were dried in an 80 ℃ oven until constant weight and then ground. 0.02 g of sample was accurately weighed and poured into a 2 mL graduated centrifuge tube. 600 µL of 80% ethanol was added and the tube was placed in an 80 ℃ water bath with continuous stirring for 30 min. After centrifugation at 3000 r for 10 min, the supernatant was collected. The residue was extracted twice with 600 µL of 80% ethanol. The supernatants were combined and 150 mg of activated carbon was added. The tube was decolorized in an 80 ℃ water bath for 30 min and centrifuged at 5000 r for 10 min. The filtrate was diluted to 2 mL with 80% ethanol for subsequent determination of sucrose content.

[0044] (2) Determination of sucrose in the sample. Take 50 µL of the extract, add 50 µL of 2 mol / L NaOH, boil in a water bath for 5 min, cool, add 700 µL of 30% HCl and 200 µL of 0.1% resorcinol, shake well, keep warm in a water bath at 80℃ for 10 min, cool, and then measure the OD value at 480 nm.

[0045] (3) Construction of the standard curve. Take 100 µL of standard solutions with concentrations of 0, 20, 40, 60, 80, and 100 µg / mL, respectively, and proceed as above. Finally, construct the sucrose concentration-OD value curve. Plot the standard curve with concentration (x) on the x-axis and absorbance value (y) on the y-axis to obtain the sucrose standard curve. Calculate the sucrose content of the sample based on the standard curve.

[0046] result: Figure 3 The study subjects were 112 soybean KASP marker development populations from 2022 to 2023. The sucrose content of the third trifoliate leaf was measured at the V4 stage. Figure 3In the figure, A represents the distribution of sucrose content in the leaves of 112 KASP marker development populations in 2023. All of them show a distribution characteristic of high in the middle and low at both ends, which shows a good normal distribution.

[0047] Figure 3 Figure A shows the leaf sucrose content distribution of the KASP marker development population in 2023. Five varieties had sucrose contents in the range of 4–6 mg / g, 11 varieties in the range of 6–8 mg / g, 17 varieties in the range of 8–10 mg / g, 28 varieties in the range of 10–12 mg / g, 23 varieties in the range of 12–14 mg / g, 14 varieties in the range of 14–16 mg / g, 9 varieties in the range of 16–18 mg / g, 3 varieties in the range of 18–20 mg / g, and 2 varieties in the range of 20–22 mg / g. The largest number of varieties (25%) had sucrose contents in the range of 10–12 mg / g, while the smallest number had sucrose contents in the range of 20–22 mg / g. The distribution of sucrose content in the leaves of the germplasm population in 2022 was as follows: 4 varieties had a sucrose content range of 4–6 mg / g, 12 varieties had a range of 6–8 mg / g, 26 varieties had a range of 8–10 mg / g, 31 varieties had a range of 10–12 mg / g, 23 varieties had a range of 12–14 mg / g, 11 varieties had a range of 14–16 mg / g, 3 varieties had a range of 16–18 mg / g, and 2 varieties had a range of 18–20 mg / g. The largest number of varieties (27.68%) had a sucrose content range of 10–12 mg / g, while the smallest number had a range of 18–20 mg / g.

[0048] Statistical analysis was performed on the sucrose content in leaves of 112 soybean KASP marker development populations from 2022 to 2023 (Table 2). In 2023, the variety with the highest sucrose content in leaves among the soybean KASP marker development populations was Heinong 48, with a sucrose content of 20.67 mg / g, while the variety with the lowest was Jiyu 76, with a sucrose content of 4.29 mg / g. The maximum value was 4.81 times the minimum value. The mean was 11.85 mg / g, the standard deviation was 3.46, the coefficient of variation was 29.20%, the skewness was 0.33, and the kurtosis was -0.08. In 2022, the variety with the highest sucrose content in leaves among the soybean KASP marker development populations was Heinong 48, with a sucrose content of 18.22 mg / g, while the variety with the lowest was Jiyu 76, with a sucrose content of 4.60 mg / g. The maximum value was 3.96 times the minimum value, and the mean was 10.96 mg / g. mg / g, standard deviation 2.85, coefficient of variation 26.00%, skewness 0.18, kurtosis -0.08.

[0049] right Figure 3A comprehensive analysis of Table 2 revealed that the coefficient of variation of sucrose content in soybean seedling leaves was relatively large among varieties during the two years from 2022 to 2023, with a clear multiple relationship between the maximum and minimum values, indicating that there are significant differences and a wide range of variation in sucrose content in the leaves of different soybean varieties.

[0050] Table 2 Statistical analysis of sucrose content in leaves of soybean KASP marker development population

[0051]

[0052] Figure 4 Correlation analysis of leaf sucrose content data from 112 spring soybean accessions widely planted in Northeast China during 2022 and 2023, using KASP marker-developed populations, revealed a high positive correlation between leaf sucrose content and the 2022 and 2023 soybean KASP marker-developed populations, with a coefficient of determination R0. 2 =0.827, indicating that the sucrose content in the leaves was very consistent over the two years, and the sucrose content data in the leaves of the seedling group was stable.

[0053] 4. Trait Data Analysis

[0054] A large amount of soybean population data was initially organized using Excel 2010, then statistically analyzed using SPSS 26.0, and finally the relevant images were completed using Origin 2023.

[0055] Results: Correlation analysis was performed on the seedling biomass and leaf sucrose content data of soybean KASP marker-developed populations in 2022 and 2023 (see Table 3). The correlation coefficient of biomass between 2022 and 2023 was 0.913, indicating a significant positive correlation between the two years. The correlation coefficient of leaf sucrose content between 2022 and 2023 was 0.909, also indicating a significant positive correlation between the two years.

[0056] The correlation coefficient between soybean seedling biomass and leaf sucrose content in 2023 was 0.791, indicating a significant positive correlation. In 2022, the correlation coefficient was 0.683, also showing a significant positive correlation. This demonstrates a close relationship between seedling biomass and leaf sucrose content. Therefore, we will utilize biomass as a supporting indicator for leaf sucrose content to develop relevant molecular markers. The results are shown in Table 3.

[0057] Table 3. Correlation analysis between soybean seedling biomass and leaf sucrose content

[0058]

[0059] Note: * and ** indicate significance at the 0.05 and 0.01 levels, respectively (Pearson correlation analysis).

[0060] 5. KASP Tag Development Method

[0061] (1) DNA extraction and detection

[0062] The main test reagents include CTAB solution, chloroform, isoamyl alcohol, anhydrous ethanol, DD water, etc., and the main test instruments include tissue grinder, water bath, centrifuge, vortex mixer, NanoDrop 2000, etc.

[0063] The CTAB method was used to extract DNA from plant leaves. First, 0.5 g of plant tissue was placed in a 2 ml centrifuge tube with two small steel balls added, and then ground at low temperature using a tissue grinder. 800 µL of 2% CTAB extraction buffer, preheated to 65 °C, was added and thoroughly mixed. The mixture was then incubated in a 65 °C water bath for 30-60 min, gently inverting and shaking periodically. 800 µL of chloroform:isoamyl alcohol (24:1) solution was added to the centrifuge tube, and the mixture was vigorously shaken for 3 min to mix. The tube was then centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a 1.5 mL centrifuge tube; an equal volume of chloroform:isoamyl alcohol (24:1) solution was added, and the tube was centrifuged at 12000 rpm for 10 min. The supernatant was then transferred to a new 1.5 mL centrifuge tube; 0.6 times the volume of isopropanol was added, and the tube was precipitated at -20 °C for 30 min. Centrifuge at 12000 rpm for 10 min, discard the supernatant, wash twice with 500 µL of 75% ethanol; air dry at room temperature, dissolve in 50 µL of DD water, detect DNA concentration and quality using NanoDrop 2000 (Thermo Fisher Scientific), and dilute to approximately 100 ng / µL for later use, and store at -20 ℃.

[0064] (2) KASP reaction

[0065] Primer and probe design: First, two forward PCR primers were designed for the specific SNP, with the 3' end of each primer adjusted to correspond to an allele of the SNP. Second, a tag sequence was added to the 5' end of each forward primer. Furthermore, fluorescent probes corresponding to the tag sequences were designed.

[0066] Primer amplification: The DNA template is first bound to universal primers, and then ligated with fluorescent probe sequences to obtain complete specific primers and amplify on a large scale. The specific reaction system is shown in Table 4, and the reaction procedure is shown in Table 5.

[0067] Amplification product signal reading: Genotyping and allele type identification were performed using an Abi Prism 7500 real-time quantitative PCR instrument (Thermo Fisher Scientific, USA).

[0068] Table 4 KASP Reaction System

[0069]

[0070] Table 5 KASP Reaction Procedure

[0071]

[0072] Result: Development of the KASP tag

[0073] Key genes associated with biomass and leaf sucrose content were identified in the CSSLs (Chromosome segment substitution lines) constructed from SN14 and ZYD00006 (described in the literature "Construction of Chromosome Segment Substitution Lines and Inheritance of Seed-Pod Characteristics in Wild Soybean"). Glyma.14G029100The function was initially validated, and the gene was used to develop a KASP marker. First, based on population sequencing data, 7 TB of data was obtained through resequencing on the Illumina sequencing platform. The raw data volume obtained from the sequencing was ≥29,008,308, and after filtering, the minimum effective data volume was 27,178,342, with the effective data accounting for up to 98.88% of the raw data. Based on the principle that reads with a quality value Q≤19 exceeding half are considered low-quality reads, 0.33~2.34% of each sample were removed. According to the principle that reads containing more than 5% N bases in a sample need to be removed, the proportion to be removed varied in each sample, with the highest removal rate around 0.04%. The Q30 of the raw data for each sample was above 88.88%, while the Q30 of the filtered samples was above 89.7%, indicating that the base content and data quality met the requirements and could be further analyzed. Based on the reference genome alignment results, approximately 1.2 million SNP loci were integrated. Following the principles of a SNP deletion rate ≤10% within the population sample and a MAF (Minor allele frequency) ≥5%, approximately 1.2 million high-quality SNP loci were selected. Further screening based on linkage disequilibrium yielded approximately 200,000 SNP loci for haplotype analysis and marker development. Key genes located on chromosome 14 were extracted. Glyma.14G029100 Analysis of all SNP sites within the range revealed that, in key genes Glyma.14G029100 There are a total of 16 SNP sites within the range, such as Figure 5 As shown, it can be divided into two superior haplotypes, namely H001 and H002. Figure 6 As shown.

[0074] Will Glyma.14G029100 Two superior haplotypes identified in the KASP marker development population were combined with sucrose content in seedling leaves for analysis, as shown below. Figure 7 As shown, Figure 7 Figure A shows a significant difference in sucrose content in the leaves of the two haplotypes H001 and H002. Figure B clearly shows that the sucrose content in the leaves of the H002 haplotype population during the seedling stage is significantly higher than that of the H001 population.

[0075] Figure 8 The association analysis between phenotypic values ​​and variant sites identified four variant sites with sufficiently high effect values. Three of these variant sites, located in the CDS region and exhibiting the highest association with phenotypic values, were selected for KASP marker development. Specific primer information is shown in Table 6.

[0076] Table 6 Glyma.14G029100 Primer sequences for KASP gene markers

[0077]

[0078] Example 2. Validation of KASP markers in soybean populations

[0079] one, Figure 9 The study focused on 337 soybean germplasm resources from the KASP marker validation population, which had a relatively large number of varieties in 2023. The leaf sucrose content in this population exhibited a distribution pattern of high levels in the middle and low levels at both ends, conforming to a normal distribution. This indicates that the accumulation of sucrose content in soybean leaves during the seedling stage follows the genetic laws of quantitative traits. (See the sucrose content distribution map of the KASP marker validation population, e.g., [image of KASP marker validation population]). Figure 9 As shown in the figure, there were 5 varieties with sucrose content ranging from 2 to 4 mg / g, 13 varieties with 4 to 6 mg / g, 39 varieties with 6 to 8 mg / g, 70 varieties with 8 to 10 mg / g, 80 varieties with 10 to 12 mg / g, 55 varieties with 12 to 14 mg / g, 33 varieties with 14 to 16 mg / g, 14 varieties with 16 to 18 mg / g, 5 varieties with 18 to 20 mg / g, 2 varieties with 20 to 22 mg / g, and 1 variety with 22 to 24 mg / g. The variety with sucrose content in the range of 10 to 12 mg / g was the most numerous, accounting for 23.74% of the total, while the variety with 22 to 24 mg / g was the least numerous.

[0080] Table 7 presents a statistical analysis of the sucrose content in the leaves of 337 soybean KASP marker validation populations in 2023. The maximum sucrose content in soybean leaves in 2023 was 23.63 mg / g, and the minimum was 3.12 mg / g, with the maximum being 7.57 times the minimum. The average was 10.99 mg / g, the standard deviation was 3.34, the coefficient of variation was 30.39%, the skewness was 0.47, and the kurtosis was 0.63. Compared with the previous 112 KASP marker development populations, the range of variation in leaf sucrose content is wider, indicating richer genetic variation.

[0081] Table 7 Statistical analysis of sucrose content in leaves of soybean KASP marker validation population

[0082]

[0083] Table 8 shows the cluster analysis results of sucrose content in the leaves of soybean KASP marker validation populations at the seedling stage in 2023. The quantitative trait of sucrose was qualitatively classified. The 337 soybean KASP marker validation populations in 2023 were divided into two categories (as shown in Table 8): Category 1, characterized by low sucrose content, had a minimum of 3.12 mg / g, a maximum of 11.37 mg / g, an average of 8.74 mg / g, and a coefficient of variation of 21.40%, including 193 varieties and accounting for 57.27% of the entire population; Category 2, characterized by high sucrose content, had a minimum of 11.41 mg / g, a maximum of 23.63 mg / g, an average of 14.03 mg / g, and a coefficient of variation of 16.46%, including 144 varieties and accounting for 42.73% of the entire population.

[0084] Table 8. Clustering of sucrose content in leaves of soybean KASP marker validation populations.

[0085]

[0086] II. KASP marker typing in soybean populations

[0087] Candidate genes associated with seedling biomass and leaf sucrose content phenotypes in the early stage. Glyma.14G029100 Three SNP sites were obtained and validated in a soybean KASP marker validation population of 337 samples. The detection rate of the three SNP sites reached 100% (as shown in Table 9). The next step is to genotype the soybean KASP marker validation population based on these three SNP sites.

[0088] Table 9 shows the development of SNP sites in the soybean KASP marker validation population.

[0089]

[0090] A total of 337 KASP marker validation populations selected in 2023 were used as genotyping validation materials. The three selected loci were validated in the population. Table 10 shows the genotyping results of the developed SNP loci in the soybean population. We successfully developed three markers: Chr14_2130137, Chr14_2131531, and Chr14_2131853. The results showed that only the marker Chr14_2131531 had a good genotyping effect in the KASP marker validation population. Using this locus, the 337 KASP marker validation populations were divided into two parts: a population carrying the TC genotype and a population carrying the CC genotype. Among the soybean materials, 162 varieties had the TC genotype, accounting for 48.10%, and 175 varieties had the CC genotype, accounting for 51.90%. The genotype segregation ratio was approximately 1:1, indicating that the developed marker Chr14_2131531 had a very good genotyping effect.

[0091] Table 10. Genotyping of the developed SNP loci in the population

[0092]

[0093] To verify the accuracy of KASP markers, genotyping of the target locus was performed using KASP markers. Only Chr14_2131531 yielded clear genotyping results, and its signal point distribution is shown below. Figure 10 As shown, the genotypes of the samples were clearly divided into two categories using this marker. The blue signal dots clustered near the y-axis showed competitive amplification of the primers linking the FAM fluorescent tag sequence, indicating the CC genotype with low sucrose content. The green signal dots clustered in the middle showed both FAM and HEX fluorescent signals, indicating the heterozygous TC genotype. The squares near the origin represent the negative control NTC, which remained clustered together near the base and did not produce any fluorescent signal.

[0094] Comparative analysis of the clustering results, comparing the total number of varieties in type 1 and the number of CC varieties in type 1, and the total number of varieties in type 2 and the number of TC varieties in type 2, revealed that: Type 1 has 193 varieties, of which 129 have the CC genotype; therefore, the CC genotype explains 66.84% of the low sucrose content. Type 2 has 144 varieties, of which 101 have the TC genotype; therefore, the TC genotype explains 70.14% of the high sucrose content. This indicates that the marker performs well in the population and can be widely applied. The KASP marker genotyping results and phenotypic genotyping results show high consistency, successfully screening germplasm resources with high sucrose content.

[0095] 3. Differences in sucrose content in leaves of different genotypes using KASP markers

[0096] Based on the phenotypic data of leaf sucrose content, the seedling leaf sucrose phenotypic data of the two genotypes TC and CC, distinguished by Chr14_2131531, were analyzed and organized, such as... Figure 11 As shown, the average sucrose content in the leaves of the TC genotype population was 14.54 mg / g, while the average sucrose content in the leaves of the CC genotype population was 8.38 mg / g. The sucrose content in the leaves of the TC genotype population was significantly higher than that of the CC genotype population, and the difference in sucrose content in the leaves during the seedling stage was significant.

[0097] Table 11 shows the independent samples t-test of phenotypic data of two genotypes using KASP markers. The t-value was 22.07, and P was ***, indicating that the difference between the two genotypes was extremely significant. The mean ± standard deviation of genotype TC was 14.54 ± 2.70, and the mean ± standard deviation of genotype CC was 8.38 ± 2.43, indicating that the developed KASP marker is reliable.

[0098] Table 11 Independent Samples t-Test of Sucrose Content in Two Genotype Materials

[0099]

[0100] Note: *** indicates P<0.001.

[0101] Using the Chr14_2131531 marker, 337 germplasm resources were divided into two categories: varieties carrying the TC genotype and varieties carrying the CC genotype. Phenotypic data on leaf sucrose content revealed that the average leaf sucrose content of varieties carrying the TC genotype was 14.54 mg / g, while that of varieties carrying the CC genotype was 8.38 mg / g. This indicates that at the Chr14_2131531 locus, the seedling leaf sucrose content of varieties carrying the TC genotype was higher than that of varieties carrying the CC genotype. The explanatory power of varieties carrying the TC genotype in the high sucrose content category was 70.14%, while that of varieties carrying the CC genotype in the low sucrose content category was 66.84%. These results can provide parental materials for breeding high-quality, high-yield soybeans. High soybean leaf sucrose content indicates high seedling biomass, thus leading to the acquisition of high-yielding soybean varieties.

[0102] Example 2.

[0103] I. A reagent kit for screening soybeans with high sucrose content:

[0104] The forward primer sequence for amplifying the molecular marker is shown in SEQ ID NO.4 or SEQ ID NO.5; the reverse primer sequence is shown in SEQ ID NO.6.

[0105] Screening method: Select soybean seedling samples with unknown sucrose content, and use the PCR amplification procedure described in step one for screening high-protein soybeans, as shown in Table 12:

[0106] Table 12

[0107]

[0108] The steps are as follows:

[0109] II. A method for identifying soybeans with high sucrose content, wherein the specific steps of the method are as follows:

[0110] (1) Extract DNA from the leaves of soybean seedlings to be tested;

[0111] (2) PCR reaction is performed using primers with molecular markers. If the soybean variety to be tested is of the TC genotype, then the soybean variety to be tested is a soybean with high sucrose content in the leaves, and this soybean has high biomass yield in the seedling stage. If it is of the CC genotype, then the soybean variety to be tested is a soybean with low sucrose content in the leaves, and this soybean has low biomass yield in the seedling stage.

[0112] Results: Samples with unknown soybean sucrose content were identified as having the TC genotype using genotyping markers. Soybean samples with high sucrose content were consistent with the genotypes obtained from marker detection. Soybean samples with low sucrose content were also consistent with the genotypes obtained from marker detection.

Claims

1. The application of a KASP primer combination for detecting molecular markers related to sucrose content in soybean seedling leaves in the preparation of a kit for identifying soybean seedling leaves with high or low sucrose content, characterized in that... The KASP primer combination consists of a forward primer and a reverse primer, wherein the forward primers are shown in SEQ ID NO.4 and SEQ ID NO.5, and the reverse primer is shown in SEQ ID NO.

6.

2. The application according to claim 1, characterized in that, If the soybean genotype is TC, the soybean has a high sucrose content; if the soybean genotype is CC, the soybean has a low sucrose content.

3. A method for identifying soybean seedling leaves with high sucrose content, characterized in that, The specific steps of the method are as follows: Step 1: Extract DNA from the soybeans to be tested; Step 2: Using the KASP primer combination of molecular markers described in claim 1, perform a PCR reaction to detect the genotype. If the soybean genotype is TC, the soybean has a high sucrose content; if the soybean genotype is CC, the soybean has a low sucrose content.

4. The method according to claim 3, characterized in that, In step 2, the PCR reaction was performed at 95 °C for 10 min; 95 °C for 20 sec, 61 °C for 45 sec, with each cycle at -0.6 °C for 10 cycles; 95 °C for 20 sec, 55 °C for 45 sec, for 30 cycles; and 37 °C for 1 min, for 1 cycle.

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