KASP marker related to detection of soybean grain fat content and application of KASP marker

Abstract

The invention belongs to the technical field of soybean genetic breeding, and particularly relates to a KASP marker related to detection of soybean grain fat content and application of the KASP marker. The invention provides a KASP marker S1045008520 related to detection of the soybean grain fat content, the KASP marker S1045008520 is located at the 45008520th basic group position of a No.10 chromosome of a soybean reference genome Glycine max Wm82.a2.v1 and has nucleotide polymorphism A / T, the grain fat content of a soybean sample with the KASP marker genotype being TT is remarkably higher than that of a soybean sample with the KASP marker genotype being AA, and the TT has the fat content increased by 0.67%-1.17% relative to the AA fat content. The KASP marker technology is used for performing genotype analysis on soybean samples, the detection is accurate and efficient, the sample genotyping is convenient and stable, the fat content of soybean seeds is rapidly pre-judged in the early growth stage of soybeans, molecular marker-assisted selection with high fat content as a breeding target is realized, the breeding selection precision is improved, and the breeding efficiency is improved. The method has important application value in accelerating the soybean fat content molecular breeding process.

Description

Technical Field

[0001] This invention belongs to the field of soybean genetic breeding technology, specifically relating to a KASP marker for detecting soybean seed fat content and its application. Background Technology

[0002] Soybean (Glycine max (L.) Merr.) is a globally important oilseed crop, providing abundant vegetable oil resources for humankind. Soybean oil accounts for 29% of the world's edible oil consumption. Soybean fat is rich in unsaturated fatty acids such as oleic acid, linoleic acid, and linolenic acid, which can, to some extent, prevent coronary heart disease and cardiovascular diseases, and regulate inflammatory responses. In recent years, the contradiction between soybean supply and demand in my country has become increasingly prominent. Imported soybeans now account for more than 85% of my country's soybean consumption, making my country the world's largest consumer and importer of soybeans, posing a severe challenge to the long-term stable supply of soybeans. Therefore, increasing the fat content of soybean kernels has become one of the important goals of soybean quality breeding, which is not only crucial for human health but also of great strategic significance for ensuring my country's food security.

[0003] Traditional breeding methods for improving soybean fat content mainly rely on conventional hybridization and phenotypic selection. However, soybean fat content is a complex quantitative trait controlled by multiple genes and is easily affected by environmental conditions (such as temperature, light, fertilization, and irrigation). Furthermore, accurate phenotypic determination requires a lot of time and labor, which makes phenotypic selection for soybean fat content very difficult, inefficient, time-consuming, and costly, and cannot efficiently improve soybean fat content.

[0004] By identifying molecular markers closely linked to soybean fat content, the genotypes of related loci / genes can be rapidly and accurately identified, allowing for the screening of superior plants containing the target gene. Molecular marker-assisted selection (MAS) technology avoids the blindness and uncertainty of traditional selection, ensuring the accuracy of offspring selection and significantly improving breeding efficiency and predictability. KASP (Kompetitive Allele-Specific Polymerase Chain Reaction) is a genotyping technique based on fluorescence detection after PCR amplification, accurately identifying biallelic genotypes at SNP (Single Nucleotide Polymorphism) sites. KASP is a next-generation SNP genotyping marker with high sensitivity and specificity, enabling large-scale detection of SNP marker genotypes and improving breeding efficiency.

[0005] Currently, there are many reports on loci / genes related to soybean fat content. However, soybean fat content is highly sensitive to environmental changes, and the identified key loci with reproducibility and stability are few and have large intervals. The molecular markers screened by existing technologies are far from the genes controlling fat content, and their stability and predictability under different environments are insufficient. This makes it difficult to promote molecular marker-assisted breeding in most laboratories and breeding units. Further exploration of relevant loci / genes and development of molecular markers closely linked to soybean fat content for molecular marker-assisted selection breeding are still needed to reduce the workload of soybean breeding and accelerate the soybean breeding process. Summary of the Invention

[0006] The purpose of this invention is to provide a KASP marker for detecting soybean seed fat content and its application, which can accurately, efficiently and cost-effectively detect the level of soybean fat content, screen soybean breeding materials with high fat content, and can be used for soybean molecular marker-assisted selection breeding.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] This invention is achieved through the following technical solution:

[0009] The first objective of this invention is to provide a KASP marker for detecting the fat content of soybean seeds. The KASP marker is S10_45008520, located at base position 45008520 on chromosome 10 of the soybean reference genome Glycine max Wm82.a2.v1. The base difference of the KASP marker is A / T. Soybean samples with base A have lower seed fat content, while soybean samples with base T have higher seed fat content.

[0010] A second objective of this invention is to provide a primer set for detecting the KASP marker S10_45008520 as described in claim 1, comprising two specific primers with 3' end bases matching different alleles and one universal primer: a forward primer F1 with the nucleotide sequence shown in SEQ ID NO.1 (5′-GCTAACAACCATAGTTTATTACTTCAA-3′), a forward primer F2 with the nucleotide sequence shown in SEQ ID NO.2 (5′-GCTAACAACCATAGTTTATTACTTCAT-3′), and a reverse primer R with the nucleotide sequence shown in SEQ ID NO.3 (5′-TAATTGACTAATGGTAGTGGTTATGC-3′);

[0011] The forward primer F1 is attached to the 5′ end with a universal tag of the FAM fluorescent adapter sequence (5′-GAAGGTGACCAAGTTCATGCT-3′), and the forward primer F2 is attached to the 5′ end with a universal tag of the VIC fluorescent adapter sequence (5′-GAAGGTCGGAGTCAACGGATT-3′).

[0012] A third objective of this invention is to provide a kit for detecting KASP markers associated with the fat content of soybean seeds as described in claim 1, comprising the primer set described in claim 2.

[0013] The fourth objective of this invention is to provide a method for detecting the fat content of soybean seeds using the KASP marker described in claim 1, comprising the following steps:

[0014] (1) Using the soybean DNA to be detected as a template, quantitative real-time PCR amplification was performed using the primer set targeting the KASP marker;

[0015] (2) Determine the genotype and fat content of soybean samples based on the fluorescence detection results after amplification;

[0016] Specifically, the soybean sample with base A at the KASP marker site has a lower fat content, while the soybean sample with base T has a higher fat content.

[0017] Furthermore, the primer set is the primer set described in claim 2.

[0018] Further, the reaction system for the real-time PCR amplification is as follows: 2.5 μL of 2 × KASP Master mix, 1.25 μL of KASP Assay mix, 1.25 μL of DNA template at a concentration of 20 ng / μL, and a total reaction volume of 5 μL; wherein the KASP Assay mix is ​​a mixture of forward primer F1 shown in SEQ ID NO.1, forward primer F2 shown in SEQ ID NO.2, and reverse primer R shown in SEQ ID NO.3 in a volume ratio of 1:1:3.

[0019] Furthermore, the procedure for the quantitative PCR amplification is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, annealing and extension at 61℃ for 60 s, repeated 10 times, with the annealing / extension temperature decreasing by 0.6℃ each time; denaturation at 95℃ for 20 s, annealing / extension at 55℃ for 60 s, repeated 26 times; fluorescence signal acquisition at 25℃ for 30 s.

[0020] Furthermore, determining the genotype and fat content of soybean samples based on the amplified fluorescence detection results includes:

[0021] If the universal tag FAM fluorescent adapter sequence is detected, the genotype is AA, and the corresponding soybean sample has a low seed fat content.

[0022] If the universal tag VIC fluorescent adapter sequence is detected, and the genotype is TT, the corresponding soybean sample has a high fat content in the kernels.

[0023] A fifth object of the present invention is to provide the application of the KASP marker or the primer set in any of the following aspects:

[0024] (a) Application in identifying or screening soybean varieties or lines with high seed fat content;

[0025] (b) Application in genetic analysis or fine mapping of soybean seed fat content genes;

[0026] (c) Application in detecting and predicting the fat content of soybean seeds;

[0027] (d) Application in marker-assisted selection breeding of soybean;

[0028] (e) Application in improving soybean germplasm resources.

[0029] The present invention has the following advantages over the prior art:

[0030] 1. This invention provides a KASP marker S10_45008520 associated with the detection of soybean seed fat content. S10 is located at base position 45008520 on chromosome 10 of the soybean reference genome Glycine max Wm82.a2.v1, exhibiting a nucleotide polymorphism A / T. Soybean samples with the KASP marker genotype TT have significantly higher seed fat content than soybean samples with the KASP marker genotype AA, with the increase in fat content between TT and AA ranging from 0.67% to 1.17%.

[0031] 2. This invention provides KASP markers for detecting soybean seed fat content and their applications. Utilizing KASP marker technology for genotyping soybean samples results in accurate and efficient detection, convenient and stable sample genotyping, and rapid prediction of soybean seed fat content in the early stages of soybean growth. This enables marker-assisted selection with high fat content as a breeding target, improving the accuracy of breeding selection and having significant application value for accelerating the molecular breeding process for soybean fat content. Attached Figure Description

[0032] Figure 1This is a QTL mapping of soybean fat content in the FG-RIL population in this embodiment of the invention, including genome-wide LOD value distribution curves for five environments (HA2018, HA2019, JN2019, HA2020 and HA2021) and the mean (Mean).

[0033] Figure 2 This is a mapping map of the major QTL (qOil-10-3) for soybean fat content located on chromosome 10 in the FG-RIL population of this invention, including QTL mapping results for five environments (HA2018, HA2019, JN2019, HA2020, and HA2021) and the mean.

[0034] Figure 3 This is a genotyping diagram of the KASP marker S10_45008520 in the soybean FG-RIL population in this embodiment of the invention; the horizontal axis represents the homozygous genotype AA with FAM fluorescence, the vertical axis represents the homozygous genotype TT with VIC fluorescence, the middle area has an equal proportion of the two fluorescence types, which is the heterozygous genotype AT; the black square in the lower left corner is the blank control (NTC) without added DNA.

[0035] Figure 4 This is a box plot showing the difference in fat content between AA and TT genotype families distinguished by the KASP marker S10_45008520 in this embodiment of the invention. It includes five environments (HA2018, HA2019, JN2019, HA2020, and HA2021) and the mean. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments.

[0037] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0038] Example 1: QTL Locating Analysis of Fat Content in Soybean Seeds

[0039] 1.1 Material Preparation and Group Building

[0040] Parental selection: The low-fat parent "Feidong Yellow Soybean" (fat content 16.69%) and the high-fat parent "Guanyun Da Sili" (fat content 19.19%) were selected for hybridization.

[0041] Population construction: A soybean recombinant inbred line (FG-RIL) population containing 201 families was obtained by continuous self-pollination to the F6 generation using the single-seed method.

[0042] 1.2 Genotype analysis and genetic map construction

[0043] High-throughput sequencing was performed using ddRAD-Seq (Double Digest Restriction-Site Associated DNA Sequencing) technology. Genomic DNA from the FG-RIL population and parents was digested with restriction endonucleases EcoRI and NlaIII, respectively, and EcoRI and NlaIII adapters were ligated. After agarose gel electrophoresis, 400–600 bp ligation products were recovered. DNA quantification of the recovered products was performed using Qubit 3.0 (Life Technology). Twenty-four samples were mixed in equal volumes, and DNA libraries were constructed using the Illumina TruSeq kit. Sequencing was performed on the Illumina HiSeq 2500 sequencing platform. ddRAD tags were developed in both parents and offspring using read clustering, and polymorphic ddRAD tags in the parents were identified. Using Glycine max Wm82.a2.v1 as the soybean reference genome, BWA software was used to align the quality-controlled sequencing data to the reference genome, and GATK software was used to detect SNPs, ultimately obtaining 42,974 high-quality SNPs. 11,220 SNPs were selected, and the MAP function of QTL IciMapping v4.2 software was used to construct a high-density SNP marker genetic map, containing 20 linkage groups, with a total map distance of 2507.62 cM and an average genetic distance of 0.22 cM between markers.

[0044] 1.3 Multi-environment phenotyping

[0045] The FG-RIL population and its parents were planted in five environments: the Huai'an Academy of Agricultural Sciences Research and Innovation Base (HA2018, HA2019, HA2020, HA2021) from 2018 to 2021, and the Jiaxiang Experimental Base in Jining City, Shandong Province (JN2019) in 2019. A randomized block design was used in the field trials, with row lengths of 1 m and row spacing of 0.5 m. The grain fat content of the FG-RIL population and its parents was measured using a Botong DA7250 near-infrared cereal analyzer. Phenotypic data showed a continuous distribution, with a variation range of 15.26%–20.15%, and a broadly heritable rate of 92.34%.

[0046] 1.4 QTL Location Analysis and Tag Filtering

[0047] Using QTL IciMapping v4.2 software, the Inclusive Composite Interval Mapping (ICIM) method was employed to perform 1000 permutation tests to determine the LOD threshold (significance level 0.05). Based on the high-density SNP marker genetic map, QTL mapping and additive effect analysis were performed on the soybean seed fat content phenotype in 5 environments and the mean phenotype.

[0048] In the FG-RIL population, the major QTL controlling soybean grain fat content was located on soybean chromosome 10 and named qOil-10-3. Figure 1 , Figure 2 This QTL was stable in different environments, with LOD values ​​ranging from 5.07 to 27.15 and phenotypic variation contributions ranging from 7.30% to 19.91%. The location information of this QTL is shown in Table 1. Based on the average phenotypic data of fat content, qOil-10-3 was located within the 0.89 cM interval of the SNP markers Chr10_44729744 - Chr10_45008520, where the marker Chr10_45008520 was closely linked to soybean grain fat content in multiple environments.

[0049] Table 1. Genetic effects and flanking markers of the main QTL qOil-10-3 on soybean seed fat content.

[0050]

[0051] Example 2: Development and application of KASP markers related to soybean seed fat content

[0052] The SNP marker Chr10_45008520, which is tightly linked to the major QTL qOil-10-3 associated with soybean seed fat content, was developed into a high-throughput KASP marker S10_45008520. The specific process is as follows:

[0053] 2.1 KASP Tag Development

[0054] The SNP marker site is located at nucleotide position 45008520 on chromosome 10 of the soybean reference genome Glycine max Wm82.a2.v1, with an A / T polymorphism. Primers were designed based on the flanking sequences of this SNP site to develop the KASP marker S10_45008520. This KASP marker consists of three primers: two specific forward primers, including forward primer F1 (nucleotide sequence shown in SEQ ID NO. 1) and forward primer F2 (nucleotide sequence shown in SEQ ID NO. 2); and one universal reverse primer, reverse primer R (nucleotide sequence shown in SEQ ID NO. 3). The primer sequences are as follows:

[0055] S10_45008520_ SEQ ID NO. 1 (F1):

[0056] 5′-GCTAACAACCATAGTTTATTACTTCAA-3′

[0057] S10_45008520_ SEQ ID NO. 2 (F2):

[0058] 5′-GCTAACAACCATAGTTTATTACTTCAT-3′

[0059] S10_45008520_ SEQ ID NO. 3 (R):

[0060] 5′-TAATTGACTAATGGTAGTGGTTATGC-3′

[0061] The forward primer F1 is attached to the 5′ end with a universal tag of the FAM fluorescent adapter sequence (5′-GAAGGTGACCAAGTTCATGCT-3′), and the forward primer F2 is attached to the 5′ end with a universal tag of the VIC fluorescent adapter sequence (5′-GAAGGTCGGAGTCAACGGATT-3′).

[0062] 2.2 Genotypic Analysis of KASP Markers

[0063] Genomic DNA was extracted from the FG-RIL population and its parents using the CTAB method, and the DNA concentration of the samples to be tested was diluted to 20 ng / μL.

[0064] Amplification and fluorescence reading were performed on a QuantStudio™ 7 Flex real-time quantitative PCR instrument. The reaction system for the quantitative PCR amplification was as follows: 2.5 μL of 2 × KASP Master mix, 1.25 μL of KASP Assay mix, and 1.25 μL of DNA template at a concentration of 20 ng / μL, for a total reaction volume of 5 μL. The KASP Assay mix consisted of forward primer F1 (SEQ ID NO. 1), forward primer F2 (SEQ ID NO. 2), and reverse primer R (SEQ ID NO. 3) mixed in a volume ratio of 1:1:3.

[0065] The procedure for quantitative real-time PCR amplification is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, annealing and extension at 61℃ for 60 s, for 10 cycles, with the annealing / extension temperature decreasing by 0.6℃ each time; denaturation at 95℃ for 20 s, annealing / extension at 55℃ for 60 s, for 26 cycles; fluorescence signal acquisition at 25℃ for 30 s.

[0066] Genotyping analysis of the KASP marker S10_45008520 in the FG-RIL population and its parents revealed that the genotyping results of the KASP marker were consistent with the ddRAD-Seq sequencing results.

[0067] According to the results of the real-time PCR instrument, samples clustered near the horizontal axis are homozygous genotype AA, linked with the FAM fluorescent tag sequence, and shown as red dots; samples clustered near the vertical axis are homozygous genotype TT, linked with the VIC fluorescent tag sequence, and shown as blue dots; the middle area has an equal proportion of the two fluorescent groups, representing the heterozygous genotype AT; the black square in the lower left corner represents the blank control (NTC) without added DNA. Figure 3 ).

[0068] 2.3 Analysis of Differences in Fat Content Among Different Genotypes of KASP Markers

[0069] The marker genotyping results of KASP marker S10_45008520 in the FG-RIL population showed that the grain fat content of 94 families with genotype AA in the five environments (HA2018, HA2019, JN2019, HA2020, and HA2021) and the mean was 16.67%, 17.93%, 18.00%, 17.72%, 16.72%, and 17.42%, respectively; while the grain fat content of 97 families with genotype TT in the five environments and the mean was 17.34%, 19.10%, 18.97%, 18.68%, 17.68%, and 18.36%, respectively. The difference in grain fat content between the AA and TT genotypes was extremely significant (P < 0.0001), with the TT genotype having a significantly higher fat content than the AA genotype. Figure 4 ).

[0070] As can be seen from the above embodiments, the KASP marker of the present invention can effectively identify the fat content of soybean grains, and can be used for molecular marker-assisted selection breeding of soybean grain fat content, predicting, screening and creating soybean germplasm resources, and breeding high-quality new soybean varieties that meet market demands.

[0071] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A KASP marker for detecting the fat content of soybean seeds, characterized in that, The KASP marker is S10_45008520, located at base position 45008520 on chromosome 10 of the soybean reference genome Glycine max Wm82.a2.v1. The base difference of the KASP marker is A / T. Soybean samples with base A have lower seed fat content, while soybean samples with base T have higher seed fat content.

2. A primer set for detecting the KASP marker S10_45008520 as described in claim 1, characterized in that, It includes two specific primers with 3' end bases matching different alleles and one universal primer: forward primer F1 with nucleotide sequence as shown in SEQ ID NO. 1, forward primer F2 with nucleotide sequence as shown in SEQ ID NO. 2, and reverse primer R with nucleotide sequence as shown in SEQ ID NO. 3; The forward primer F1 is attached to the FAM fluorescent adapter sequence universal tag at its 5′ end, and the forward primer F2 is attached to the VIC fluorescent adapter sequence universal tag at its 5′ end.

3. A kit for detecting KASP markers related to the fat content of soybean seeds as described in claim 1, characterized in that, Includes the primer set as described in claim 2.

4. A method for detecting the fat content of soybean seeds using the KASP marker as described in claim 1, characterized in that, Includes the following steps: (1) Using the soybean DNA to be detected as a template, quantitative real-time PCR amplification was performed using the primer set targeting the KASP marker; (2) Determine the genotype and fat content of soybean samples based on the fluorescence detection results after amplification; Specifically, the soybean sample with base A at the KASP marker site has a lower fat content, while the soybean sample with base T has a higher fat content.

5. The method according to claim 4, characterized in that, The primer set is the primer set described in claim 2.

6. The method according to claim 4 or 5, characterized in that, The reaction system for the quantitative real-time PCR amplification was as follows: 2.5 μL of 2× KASP Master mix, 1.25 μL of KASP Assay mix, and 1.25 μL of DNA template at a concentration of 20 ng / μL, for a total reaction volume of 5 μL. The KASP Assay mix consisted of forward primer F1 (SEQ ID NO.1), forward primer F2 (SEQ ID NO.2), and reverse primer R (SEQ ID NO.3) mixed in a volume ratio of 1:1:

3.

7. The method according to claim 6, characterized in that, The procedure for quantitative real-time PCR amplification is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, annealing and extension at 61℃ for 60 s, for 10 cycles, with the annealing / extension temperature decreasing by 0.6℃ each time; denaturation at 95℃ for 20 s, annealing / extension at 55℃ for 60 s, for 26 cycles; fluorescence signal acquisition at 25℃ for 30 s.

8. The method according to claim 7, characterized in that, Determining the genotype and fat content of soybean samples based on the amplified fluorescence detection results includes: If the universal tag FAM fluorescent adapter sequence is detected, the genotype is AA, and the corresponding soybean sample has a low seed fat content. If the universal tag VIC fluorescent adapter sequence is detected, and the genotype is TT, the corresponding soybean sample has a high fat content in the kernels.

9. The use of the KASP marker of claim 1 or the primer set of claim 2 in any of the following aspects: (a) Application in identifying or screening soybean varieties or lines with high seed fat content; (b) Application in genetic analysis or fine mapping of soybean seed fat content genes; (c) Application in detecting and predicting the fat content of soybean seeds; (d) Application in marker-assisted selection breeding of soybean; (e) Application in improving soybean germplasm resources.

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