A molecular marker associated with soybean seed protein content and use thereof

Abstract

The application belongs to the technical field of molecular marker assisted breeding, and particularly relates to a molecular marker related to soybean seed protein content and application thereof, a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and is located in the interval of 32403844 bp-32403863 bp of the 16th chromosome of soybean. The QTL interval related to the soybean seed protein content identified by the application has a size of 0.076 Mb, the number of genes in the interval is small, and important foundation is provided for related gene mining. On the basis, one molecular marker related to the soybean seed protein content, namely SSR-16-1082, is developed in the interval, the molecular marker has high correlation degree with the soybean seed protein content, and important marker information is provided for molecular marker assisted selection breeding of the soybean seed protein content.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular marker assisted breeding, and particularly relates to a molecular marker related to soybean seed protein content and application thereof. BACKGROUND

[0002] Soybean quality traits are important indicators for measuring soybean, and increasing soybean seed protein content and breeding high-protein soybean varieties are important goals pursued by breeders. Soybean protein content is a quantitative genetic trait controlled by multiple genes, and is regulated by multiple genes and sensitive to environmental factors. Phenotypic variation is continuous and is greatly affected by the environment, which is a focus and difficulty in the study of soybean protein content. Therefore, soybean breeding has been in the traditional breeding mode for a long time. However, the traditional breeding process requires a long time and consumes a large amount of labor, does not have a clear breeding goal, and it is difficult to accurately breed new varieties from hybrid offspring. The diversification of contemporary breeding goals makes it more and more difficult for traditional breeding to make major breakthroughs, so traditional breeding cannot meet the development requirements of today. With the development and application of molecular markers, new markers are generated. After the completion of the first generation sequencing, the second generation sequencing technology is also developing and maturing, and the third generation sequencing technology is also in the process of development. The application of simplified sequencing and physical map is more widely used, which lays a foundation for molecular marker assisted selection breeding. This method can greatly improve the breeding efficiency and realize the directional improvement of soybean varieties by screening markers to regulate traits. SUMMARY

[0003] The application aims to provide a molecular marker related to soybean seed protein content, which solves the problems in the prior art.

[0004] The technical solution adopted by the application is as follows:

[0005] The application provides a molecular marker related to soybean seed protein content, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1 and located in the interval of 32403844bp-32403863bp of chromosome 16 of soybean.

[0006] The second aspect of the application provides an application of the molecular marker, and the molecular marker is used in any one of the following:

[0007] 1) detecting or assisting in detecting the protein content of soybean seeds;

[0008] 2) screening or assisting in screening soybean varieties;

[0009] 3) improving soybean varieties;

[0010] 4) Soybean breeding;

[0011] 5) Preparing a product for identifying or assisting in identifying the protein content of soybean seeds.

[0012] Preferably, the method for detecting the protein content of soybean seeds is as follows:

[0013] Extracting the genomic DNA of the soybean material;

[0014] Using the genomic DNA of the soybean material as a template, performing PCR amplification with the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3 to obtain a PCR product;

[0015] Performing polyacrylamide gel electrophoresis on the PCR product, and obtaining the protein content of the soybean seeds according to the band pattern after polyacrylamide gel electrophoresis.

[0016] Preferably, the genomic DNA of the soybean material is from the leaves of soybeans.

[0017] Preferably, the system for PCR amplification is:

[0018] 50 ng / µL of genomic DNA of the soybean material 1 µL, 2 mmol / µL of upstream primer 0.5 µL, 2 mmol / µL of downstream primer 0.5 µL, 10× Mg 2+ -containing Buffer 1 µL, 2.5 mmol / µL of dNTP 0.85 µL, 5 U / µL of Taq enzyme 0.15 µL, and 6 µL of sterile water.

[0019] Preferably, the program for PCR amplification is:

[0020] Pre-denaturation: 95℃ for 5 min;

[0021] Denaturation: 95℃ for 30 s; annealing: 53℃ for 30 s; extension: 72℃ for 30 s;

[0022] Final extension: 72℃ for 7 min.

[0023] Preferably, the conditions for polyacrylamide gel electrophoresis are: 220 V voltage, 180 mA current, and electrophoresis for 150 min.

[0024] Preferably, after polyacrylamide gel electrophoresis is completed, staining is also required, and the staining time is 30 min.

[0025] Compared with the prior art, the beneficial effects of the present application are:

[0026] The application provides a molecular marker related to the protein content of soybean kernels, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the molecular marker is located in the interval of 32403844 bp-32403863 bp of the 16th chromosome of soybean. The QTL interval related to the protein content of soybean kernels identified by the application is 0.076 Mb in size, the number of genes in the interval is small, and important foundation is provided for related gene mining. On this basis, one molecular marker related to the protein content of soybean kernels, namely SSR-16-1082, is developed in the interval, and the molecular marker has high correlation degree with the protein content of soybean kernels, which provides important marker information for molecular marker assisted selection breeding of the protein content of soybean kernels. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Process diagram for constructing the CSSLs population.

[0028] Figure 2 Sequencing coverage depth distribution diagram of the recurrent parent SN14.

[0029] Figure 3 Distribution of the protein content of kernels in the CSSL-646 population.

[0030] Figure 4 Distribution diagram of the protein content phenotypes of the secondary separation populations R1 population and R2 population. A: R1 population; B: R2 population.

[0031] Figure 5 Fine mapping technology route.

[0032] Figure 6 Flow and results of fine mapping of the QTL related to the protein content of soybean kernels.

[0033] Figure 7 Results of polyacrylamide gel electrophoresis of different soybean materials. A: varieties 1-40 in table 3; B: varieties 41-80 in table 3.

[0034] Figure 8 Multiple comparison between different band types. DETAILED DESCRIPTION

[0035] The application will be further described below through specific examples, but the scope of the application is not limited. The details and forms of the technical solutions of the application can be modified or replaced without departing from the spirit and scope of the application, and the modifications or replacements all fall within the protection scope of the application.

[0036] The inventive concept of the application is as follows:

[0037] The application utilizes local cultivated variety Suinong 14 as recurrent parent, wild soybean variety ZYD00006 as donor parent, simultaneously utilizes whole genome sequencing technology for molecular assisted selection, and finally constructs a wild soybean genome backcross introduction line population. According to the resequencing results of the CSSLs, strains with homozygous background fragments, relatively less introduced fragments and relatively less heterozygous fragments are selected and planted; through the measurement of grain protein content, a set of primary mapping population is constructed to perform preliminary QTL positioning; according to the preliminary positioning interval, the remaining heterozygous line population is constructed by selecting the heterozygous single plants in the interval, and the target fragment is finally determined as Gm16:32403844bp~32480066bp, and the interval is the QTL interval related to the grain protein content detected by the application. Further, a molecular marker related to the soybean grain protein content is developed in the interval, which is used for molecular marker assisted selection breeding.

[0038] In order to make those skilled in the art better understand the technical solutions of the present application and implement them, the present application will be further described below in combination with specific examples. In the description of the present application, if not specially stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0039] The abbreviations used in the present application are as follows:

[0040] CSSLs: backcross introduction line population / Chromosome Segment Substitution Lines;

[0041] SN14: Suinong 14.

[0042] The materials and methods used in the present application are as follows:

[0043] 1. Construction of mapping population.

[0044] The present application uses Suinong 14 as recurrent parent and wild soybean ZYD00006 as donor parent, and constructs CSSLs through continuous backcrossing and selfing. The specific generation process is shown in Figure 1 .

[0045] 2. Genotype detection of population.

[0046] The population is detected for genotype by resequencing, and the steps include:

[0047] (1) The DNA of the parent and offspring strains was extracted by CTAB method. After the sample was qualified, the DNA was randomly broken by ultrasonic crushing method, and the DNA fragments were subjected to end repair, 3' end A addition, sequencing adapter addition, purification and PCR amplification to complete the construction of sequencing library. After the library was qualified, sequencing was performed by Illumina HiSeqTM sequencing platform.

[0048] (2) The sequencing reads obtained by resequencing were repositioned on the reference genome for subsequent variation analysis. The short sequences obtained by high-throughput sequencing were aligned with the reference genome by using BWA software. The positions of Clean Reads on the reference genome were located by alignment, the sequencing depth and genome coverage of each sample were counted, and the variation detection was performed. The genome coverage depth of the recurrent parent SN14 is shown in Figure 2 .

[0049] (3) For the results obtained by BWA alignment, the Mark Duplicate tool of Picard was used to remove duplicates and shield the influence of PCR duplication. GATK was used for InDel Realignment, i.e. local realignment of sites near the alignment results with insertions or deletions to correct alignment errors caused by insertions or deletions. GATK was used for base quality recalibration to correct the quality of bases. GATK was used for variation detection, mainly including SNP and InDel. SNP was strictly filtered, including SNP cluster filtering, SNP filtering near InDel and adjacent InDel filtering, and finally 580524 SNP tags were screened.

[0050] (4) Using the obtained 580524 SNPs, a window of 17 SNPs and a step of 1 SNP were used to slide on the chromosome. When the number of SNPs with aa genotype in the sliding window was greater than 12, the genotype was aa; when the number of SNPs with bb genotype in the sliding window was greater than 14, the genotype was bb; otherwise, the genotype was ab. In this way, the genotype was filled and corrected.

[0051] (5) After filling and correction, Bin division was performed according to the recombination of offspring. The samples were arranged in order according to the physical position of the chromosome. When any sample showed a change in genotype, it was considered that a recombination breakpoint occurred, and then the SNPs between the recombination breakpoints were divided into Bin. After Bin screening, finally 3196 bins were used as markers for mapping.

[0052] 3. Phenotype data acquisition.

[0053] This invention primarily utilizes the FOSS-1241TM near-infrared grain quality analyzer to measure the protein content of soybean seeds. The FOSS-1241TM near-infrared grain quality analyzer operates on the principle of near-infrared transmission technology, collecting infrared spectral information and comparing it with a calibration database to obtain high-precision protein content data. The FOSS-1241TM near-infrared grain quality analyzer is a highly efficient experimental instrument for measuring cereal crops such as wheat, soybeans, and rice. During the measurement process, the moisture content of the soybean seeds is kept within a safe range. Each sample is measured three times, and the average of the three measurements is taken as the final data for the soybean seed protein content phenotype.

[0054] 4. Fine localization of QTLs related to soybean seed protein content.

[0055] A single plant, CSSL-646, was selected from the CSSLs population to construct the CSSL-646 line as the initial QTL mapping population. The maximum grain protein content in the CSSL-646 population was 44.56%, and the minimum was 39.53%. The phenotypic segregation of grain protein content was obvious and followed a normal distribution. Figure 3 .

[0056] Forty extreme phenotypic materials, including high-protein and low-protein materials from the CSSL-646 population, were selected and genotyped using polyacrylamide gel electrophoresis. Candidate intervals were preliminarily determined by comparing genotypic changes among extreme phenotypes. Results showed significant differences in the genotype of Gm016 among the extreme phenotypes. The QTL affecting soybean seed protein content was preliminarily identified as being located at Gm16: 26705080bp~33180908bp, and named Gm016. qSPJ_1 .

[0057] Choose from F2 qSPJ_1 Heterozygous plants within the specified intervals, namely 646-2-14, 646-2-15, 646-2-21, and 646-2-25, were planted individually in rows to construct the R1 population. Genotypic analysis was performed on 20 accessions each of high and low protein content from the R1 population. The results are shown below. Figure 4 .exist qSPJ_1 The interval is encrypted by marking, and single-marker analysis is used to narrow the interval to 32403844bp~33428851bp.

[0058] According to the genotype in R1 population, the target fragment heterozygous single strain was selected, and the offspring of 646-2-15-14, 646-2-21-20, 646-2-21-23 and 646-2-21-24 four single strains were selected, and a total of 182 were constructed into R2 population. Genotype analysis was performed on 40 extreme phenotype materials in R2 population. It was shown that the genotype of the target segment was separated, and the target fragment was separated into two parts. Continue to select single strains that are heterozygous in two intervals and homozygous in other positions, and continue to construct RHL population. Single marker analysis was performed on the primers in the two fragments, and the target fragment was obtained on chromosome 16, which was 32403844bp~32480066bp interval, and the fine mapping process and results were shown in Figure 5 and Figure 6 .

[0059] 5、Molecular marker identification.

[0060] Using the existing soybean variety, a molecular marker related to soybean seed protein content was identified in the 32403844bp~32480066bp interval, and a molecular marker related to soybean seed protein content was obtained, which was located in the 32403844bp~32403863bp interval of soybean chromosome 16, and was named SSR-16-1082, the nucleotide sequence was shown in SEQ ID NO. 1.

[0061] SEQ ID NO. 1, SSR-16-1082: TATATATATATATATATATA.

[0062] Example 1

[0063] A molecular marker related to soybean seed protein content and its application, specifically as follows:

[0064] 1. Obtaining of QTL and molecular marker related to soybean seed protein content.

[0065] 1.1. Preliminary positioning of QTL related to soybean seed protein content.

[0066] CSSL-646 strain was selected in the CSSLs population to construct CSSL-646 strain, which was used as QTL preliminary positioning population. In the CSSL-646 population, the maximum value of seed protein content was 44.56%, and the minimum value was 39.53%, the phenotypic separation of seed protein content was obvious and showed normal distribution, as shown in Figure 3 .

[0067] Forty extreme phenotypic materials were selected from the CSSL-646 population, including 20 high-protein materials and 20 low-protein materials. Genotyping was performed using polyacrylamide gel electrophoresis. Candidate intervals were preliminarily determined by comparing genotypic changes among extreme phenotypes. The results showed that the genotype of Gm016 differed significantly among extreme phenotypes. The QTL affecting soybean seed protein content was preliminarily identified as being located at Gm16: 26705080bp~33180908bp, and named Gm016. qSPJ_1 .

[0068] 1.2 Fine localization of QTLs related to soybean seed protein content.

[0069] Choose from F2 qSPJ_1 Heterozygous plants within the interval: 646-2-14, 646-2-15, 646-2-21, and 646-2-25, were planted individually in rows to construct an R1 population. See [link to R1 population]. Figure 4 Genotyping was performed on 20 accessions each from the R1 population, 20 with high protein content and 20 with low protein content. qSPJ_1 The interval is encrypted by marking, and single-marker analysis is used to narrow the interval to 32403844bp~33428851bp.

[0070] Based on the genotypes in the R1 population, heterozygous individuals for the target fragment were selected. The offspring of four individuals (646-2-15-14, 646-2-21-20, 646-2-21-23, and 646-2-21-24) totaling 182 were selected to construct the R2 population. Genotyping analysis of 40 extreme phenotypic materials in the R2 population revealed segregation of the target fragment genotype, resulting in two parts. Individuals heterozygous at these two positions and homozygous at other positions were selected to construct the RHL population. Single-marker analysis of primers within the two separated parts of the target fragment yielded the final target fragment on chromosome 16, ranging from 32403844bp to 32480066bp. The fine mapping procedure is described in [link to detailed procedure]. Figure 5 and Figure 6 .

[0071] 1.3 Development of molecular markers.

[0072] Using existing soybean germplasm resources, molecular markers related to soybean seed protein content were identified in the 32403844bp~32480066bp region of chromosome 16. One SSR molecular marker related to seed protein content was identified. This SSR molecular marker is located in the 32403844bp~32403863bp region of soybean chromosome 16, named SSR-16-1082, with the nucleotide sequence TATATATATATATATTATA, and is denoted as SEQ ID NO.1.

[0073] 2. Use of the molecular marker in breeding of soybean seed protein content traits.

[0074] (1) Extracting genomic DNA of soybean material.

[0075] The genomic DNA of soybean material was obtained by using CTAB method to extract the genomic DNA of soybean leaves, and the specific steps were as follows:

[0076] Take 4 g of soybean leaves and put them into a 1.5 mL centrifuge tube, add 4 steel balls with a diameter of 2 mm, freeze in liquid nitrogen, and then grind at 30 times / s for 1 min. After grinding, add 700 μL of preheated CTAB extraction solution at 65°C and mix well. Put the centrifuge tube with added CTAB extraction solution into a 65°C water bath, and water bath for 60 min, and mix well every 15 min. Add 700 μL of chloroform solution to the centrifuge tube after water bath, mix well, and centrifuge at 12000 rpm / min for 15 min. Take the supernatant and put it into another prepared 1.5 mL centrifuge tube, add 700 μL of chloroform again, mix gently up and down, and then centrifuge at 12000 rpm / min for 15 min. Take the supernatant and put it into another prepared 1.5 mL centrifuge tube, add 700 μL of chloroform again, mix gently up and down, and then centrifuge at 12000 rpm / min for 15 min. Take another new 1.5 mL centrifuge tube, add 70 μL of pre-cooled isopropanol at -20°C, take the supernatant of the centrifuged solution, and slowly drop it into the centrifuge tube containing isopropanol solution, centrifuge at 8000 rpm / min for 2 min. Pour out the supernatant and reserve the precipitate. Add 700 μL of anhydrous ethanol and blow several times, then aspirate the anhydrous ethanol, add 700 μL of 75% v / v ethanol and blow several times again, aspirate the 75% ethanol, and dry the DNA in a ventilated place. Add 100 μL of sterile water to the dried DNA, put it into a 4°C refrigerator until the DNA is completely dissolved, and then transfer it into a -20°C refrigerator for storage.

[0077] (2) PCR amplification of the genomic DNA of soybean material using amplification primers to obtain PCR products.

[0078] According to the gene sequence of SSR-16-1082, the amplification primers were designed, the upstream primer was SSR-16-1082F, and the downstream primer was SSR-16-1082R, and the sequences of SSR-16-1082F and SSR-16-1082R were shown in SEQ ID NO. 2 and SEQ ID NO. 3.

[0079] SSR-16-1082F: 5'-CAATGGTTGGATGGTTGGAT-3', recorded as SEQ ID NO. 2;

[0080] SSR-16-1082R: 5′-TGGACCACACCTTTTGAACA-3′, denoted as SEQ ID NO.3.

[0081] The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0082] Table 1 PCR reaction system

[0083]

[0084] Table 2 PCR reaction procedure

[0085]

[0086] Note: In Tables 1 and 2, "-" indicates that this item is not present.

[0087] (3) Perform electrophoretic analysis on the PCR products to identify the electrophoretic banding of the PCR products.

[0088] The banding pattern was identified using polyacrylamide gel electrophoresis, and the specific steps are as follows:

[0089] 1) Clean the plate and ear plate with alcohol. Place the gel strip on the ear plate, then place the plate on top, clamping the four corners to prevent gel leakage. Pour 30 mL of acrylamide gel into a beaker, add 300 µL of APS catalyst and 30 µL of TEMED accelerator, and stir quickly with a glass rod until homogeneous. Pour the mixture into a glass plate and allow it to solidify. First, fill the electrophoresis tank two-thirds full with electrophoresis buffer, then place the glass plate into the tank and fill it completely with 0.5 × TBE electrophoresis buffer.

[0090] 2) Add 2.5 μL of bromophenol blue solution to the PCR product above, and then pipette 2.5 μL into the sample well. After sample addition, control the voltage at 220V and the current at 180mA, and stop electrophoresis after about 150 minutes.

[0091] 3) Staining: Add 20 μL of 10000×Gel-red dye to 400 mL of water to prepare a dye dilution solution; carefully remove the gel from the glass plate and place it in the dye dilution solution, and stain for 30 min.

[0092] Table 3 shows the statistical analysis of existing phenotypes and banding patterns used for SSR-16-1082 molecular marker identification. The results of polyacrylamide gel electrophoresis are as follows: Figure 7The amplified primer based on the gene sequence of SSR-16-1082 amplifies the DNA of the soybean material, and there are six band types in total, wherein the average protein content of the material of the first band type is 40.83%; the average protein content of the material of the second band type is 42.38%; the average protein content of the material of the third band type is 41.62%; the average protein content of the material of the fourth band type is 46.31%; the average protein content of the material of the fifth band type is 46.60%; and the average protein content of the material of the sixth band type is 46.02%. Figure 7 The lanes 1-40 shown in the A figure of FIG. 1 correspond to the soybean varieties represented by the numbers 1-40 in Table 3 one by one; Figure 7 The lanes 41-80 shown in the B figure of FIG. 1 correspond to the soybean varieties represented by the numbers 41-80 in Table 3 one by one.

[0093] The single factor variance analysis is used to analyze the significant difference of the protein content among the band types, and the multiple comparison results are shown in Table 4 and Figure 8 The protein content of the first band type and the second band type is significantly lower than that of the fourth band type, the fifth band type and the sixth band type, the protein content of the first band type and the second band type is significantly different, the protein content of the fourth band type, the fifth band type and the sixth band type is not significantly different, and the protein content of the third band type is significantly lower than that of the fourth band type and the fifth band type.

[0094] In summary, the present application provides a new method for identifying the protein content of soybean seeds, that is:

[0095] The genomic DNA of the soybean material is extracted, the primer shown in SEQ ID NO. 2 and SEQ ID NO. 3 is used for amplification, and then the band type is identified by electrophoresis. If the electrophoresis band type of the material to be identified is consistent with the fourth band type, the fifth band type or the sixth band type of the present application, it is a soybean material with high protein content; if it is consistent with the first band type, it is a material with low protein content; and if it is consistent with the second band type or the third band type, it is a material with medium protein content.

[0096] Table 3: Phenotype and band type statistics of existing varieties used for identifying soybean protein content molecular markers

[0097]

[0098] Table 4: Statistics of different band types of soybean protein content related molecular markers

[0099]

[0100] The technical features of the above-described embodiments can be combined in any manner. In order to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not exist, they should be considered as the scope of the present application.

[0101] The above-described embodiments only express several implementation manners of the present application, which are described in a more specific and detailed manner, but should not be understood as a limitation on the scope of the patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. An application of a primer pair for detecting SSR molecular markers related to soybean seed protein content, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3; the nucleotide sequence of the SSR molecular marker related to soybean seed protein content is shown in SEQ ID NO.1, which is located in the 32403844bp~32403863bp interval of soybean chromosome 16. The application refers to at least one of the following: 1) To detect or assist in the detection of protein content in soybean seeds; 2) Screening or assisting in the screening of soybean varieties with high protein content in the grains; 3) Prepare products for identification or auxiliary identification of soybean seed protein content; The method for detecting the protein content of soybean seeds is as follows: Genomic DNA was extracted from soybean materials; Using soybean genomic DNA as a template, PCR amplification was performed using the primers shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain PCR products; The PCR products were subjected to polyacrylamide gel electrophoresis, and the protein content of soybean seeds was obtained based on the banding pattern after polyacrylamide gel electrophoresis.

2. The application as described in claim 1, characterized in that, The genomic DNA of the soybean material was derived from soybean leaves.

3. The application as described in claim 1, characterized in that, The PCR amplification system is as follows: Genomic DNA from 50 ng / µL soybean material: 1 µL, 2 mmol / µL upstream primer: 0.5 µL, 2 mmol / µL downstream primer: 0.5 µL, 10× Mg2+ primer: 2+ The solution contained 1 µL of buffer, 0.85 µL of 2.5 mmol / µL dNTP, 0.15 µL of 5 U / µL Taq enzyme, and 6 µL of sterile water.

4. The application as described in claim 1, characterized in that, The PCR amplification procedure is as follows: Pre-denaturation: 95℃ for 5 min; Denaturation: 95℃ for 30s; Annealing: 53℃ for 30s; Extension: 72℃ for 30s; Final extension: 72℃ for 7 minutes.

5. The application as described in claim 1, characterized in that, The conditions for the polyacrylamide gel electrophoresis were: 220V voltage, 180mA current, and electrophoresis for 150 min.

6. The application as described in claim 1, characterized in that, After polyacrylamide gel electrophoresis, staining is required, and the staining time is 30 minutes.

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