Soybean fatty acid character SNP (Single Nucleotide Polymorphism) marker positioning and application

Abstract

The invention relates to the technical field of soybean genetic breeding, in particular to positioning and application of a soybean fatty acid character SNP (Single Nucleotide Polymorphism) marker. Comprising the following steps: constructing a recombinant inbred line (RIL) group obtained by hybridizing a high-oleic-acid parent variety 'Glysoja-HO' and a low-oleic-acid parent variety 'Glysoja-LO'; performing whole genome re-sequencing on all individuals in the population to obtain SNP genotype data covering 20 chromosomes of the soybean; determining fatty acid composition phenotype data of individuals in the group in the three environments through a gas chromatographic method; performing whole genome association analysis on the SNP data and the phenotype data by adopting a linkage imbalance-linkage analysis integration model; screening an SNP (Single Nucleotide Polymorphism) marker% associated with the target fatty acid character; and determining a core function marker through haplotype block analysis and independent natural group verification. The invention provides a method for accurately positioning the soybean fatty acid character core SNP marker which is stable in environment, remarkable in effect and capable of being efficiently used for molecular breeding and variety identification.

Description

Technical Field

[0001] The present invention relates to the technical field of soybean genetic breeding, and in particular to the positioning and application of a soybean fatty acid trait SNP marker. Background Art

[0002] Soybean (Glycine max) is an important oil crop in the world. The fatty acid composition of its seed oil, especially the ratio of oleic acid, linoleic acid and linolenic acid, directly affects the nutritional value, oxidative stability and industrial use of the oil. Oils with high oleic acid, low linoleic acid and low linolenic acid are highly favored because of their healthier fatty acid profiles and longer shelf life. Traditional soybean fatty acid improvement mainly relies on phenotypic selection and conventional hybrid breeding, but fatty acid content is a quantitative trait and is easily affected by environmental factors (such as light, temperature, and moisture), and accurate phenotype measurement is time-consuming, labor-intensive and costly. This makes phenotypic-based selection inefficient, the breeding cycle long, and it is difficult to efficiently breed new soybean varieties with specific ideal fatty acid compositions.

[0003] Marker-assisted selection (MAS) technology can significantly improve breeding efficiency. However, existing technologies still face numerous challenges in the development and application of soybean fatty acid molecular markers. First, methods that can stably and efficiently simultaneously locate key loci regulating oleic, linoleic, and linolenic acid content and screen for core single nucleotide polymorphism (SNP) markers with significant effects and high explanatory power remain insufficient. Second, fatty acid content is highly sensitive to environmental changes, and existing marker mapping methods often fail to fully consider genotype-environment interactions across multiple environments, resulting in insufficient stability and predictive power of selected markers across diverse environments. Furthermore, there is a lack of core functional SNP markers that have undergone rigorous validation (such as haplotype analysis and natural population validation) and can be directly used in molecular breeding practices, particularly marker combinations that can simultaneously guide the increase of oleic acid content and the decrease of linoleic and linolenic acid content. Furthermore, existing technology systems still need to improve the stringency of marker screening (such as considering both significance thresholds and phenotypic variation explanatory power) and the potential of markers for derivative applications such as seed purity identification. Summary of the Invention

[0004] The present invention provides a method for accurately locating core SNP markers of soybean fatty acid traits that is environmentally stable, has significant effects, and can be efficiently applied to molecular breeding and variety identification.

[0005] The technical solution adopted by the present invention is: a method for locating SNP markers of soybean fatty acid traits, comprising the following steps:

[0006] Step 1: construct a recombinant inbred line (RIL) population obtained by crossing the high oleic acid parent variety "Glysoja-HO" with the low oleic acid parent variety "Glysoja-LO";

[0007] Step 2: Perform whole-genome resequencing on all individuals in the population to obtain SNP genotype data covering 20 soybean chromosomes;

[0008] Step 3: Determine the fatty acid composition phenotype data of the individuals in the group under three environments by gas chromatography, including the content of oleic acid, linoleic acid, and linolenic acid;

[0009] Step 4: Use the linkage disequilibrium-linkage analysis integrated model, with environmental effects as fixed factors and parental genetic background as random factors, to conduct genome-wide association analysis on SNP data and phenotypic data;

[0010] Step 5: Screening SNP markers associated with target fatty acid traits, with a significance threshold of P < 1 × 10 -7 The phenotypic variation explanation rate R 2 >15%;

[0011] Step 6: Through haplotype block analysis and independent natural population verification, SNP-OL-10A located at GmChr10:38,156,789-38,157,255 and SNP-LL-15B located at GmChr15:72,834,112-72,834,598 were identified as core functional markers.

[0012] As a further improvement of the present invention, the recombinant inbred line (RIL) population in step 1 is an F7 generation or above population, and the population size is ≥200 families; the oleic acid content of the parent "Glysoja-HO" is ≥85%, and the oleic acid content of "Glysoja-LO" is ≤25%.

[0013] As a further improvement of the present invention, in step 2, the average depth of whole genome resequencing is ≥10×, SNP calling adopts the GATK process, and the filtering criteria are: deletion rate <20%, minor allele frequency >5%.

[0014] As a further improvement of the present invention, the fixed factors in step four include: environmental temperature gradient (15°C / 25°C / 35°C) and soil moisture gradient (40% / 60% / 80% field capacity); the random factors adopt the kinship matrix derived from the parental pedigree.

[0015] As a further improvement of the present invention, when screening the associated SNPs in step 5, a multi-environment joint analysis (MET) is performed simultaneously, requiring the target SNP to reach a significance threshold (P < 1×10 -7 ).

[0016] As a further improvement of the present invention, the haplotype block analysis in step six is ​​performed using Haploview software, and the block is defined as: D' value between SNPs > 0.8 and LOD > 2; the natural population verification covers 100 soybean germplasm resources, requiring that the direction of the allele effect of the target marker in the population is consistent with that of the RIL population.

[0017] An application of core functional markers obtained based on the above-mentioned positioning method, wherein the core functional markers SNP-OL-10A and SNP-LL-15B are used for:

[0018] Application 1: Soybean molecular marker-assisted selection breeding to increase or decrease the content of oleic acid, linoleic acid, and linolenic acid in soybean seeds;

[0019] Application 2: Screening and creating soybean germplasm resources and varieties with target fatty acid composition;

[0020] Application three: Identifying genotypes in soybean seeds and plants that are related to the fatty acid traits described in claim 1.

[0021] As a further improvement of the present invention, the application of molecular marker-assisted selection breeding is specifically: using the SNP-OL-10A marker to screen soybean plants or lines carrying superior alleles that increase oleic acid content; using the SNP-LL-15B marker to screen soybean plants and lines carrying superior alleles that reduce linoleic acid and linolenic acid content; and simultaneously using the SNP-OL-10A and SNP-LL-15B markers to carry out gene aggregation breeding to create new soybean germplasms and varieties with significantly increased oleic acid content and significantly reduced linoleic acid and linolenic acid contents.

[0022] As a further improvement of the present invention, the third application of identifying soybean seed and plant genotypes is specifically: using specific primers or probes designed based on SNP-OL-10A and SNP-LL-15B sites, through PCR, fluorescent quantitative PCR, high-throughput sequencing, and gene chip technology, the allele type of the soybean sample to be tested at the GmChr10:38,156,789-38,157,255 and GmChr15:72,834,112-72,834,598 sites is detected to predict the potential of its oleic acid, linoleic acid, and linolenic acid content.

[0023] As a further improvement of the present invention, the core functional markers SNP-OL-10A and SNP-LL-15B are also used for soybean seed purity and authenticity identification, which is achieved by detecting whether the genotype of the target seed at the marker site conforms to the characteristic allele type of the expected parent and variety.

[0024] The beneficial effects of the present invention are as follows: (1) The present invention effectively overcomes the problem that fatty acid content is easily affected by environmental interference by constructing a RIL population (≥F7) of extreme phenotypic parents, combining multi-environmental gradient (temperature / moisture) phenotypic determination and a linkage disequilibrium-linkage analysis integrated model (fixed environmental effect + random genetic background), and significantly improves the accuracy of association analysis.

[0025] (2) The present invention confirmed the core functions of SNP-OL-10A (regulating oleic acid) and SNP-LL-15B (regulating linoleic acid / linolenic acid) through haplotype block analysis (D'>0.8, LOD>2) and verification of independent natural populations (100 germplasms).

[0026] (3) The genotype identification technology developed by the present invention based on core markers (such as specific primers / probes combined with PCR, chips, etc.) can not only accurately predict the fatty acid trait potential of soybean materials and accelerate resource screening and breeding processes, but can also be used for soybean seed purity and authenticity identification. By detecting whether the genotype of the target site conforms to the variety characteristics, it provides reliable technical support for variety intellectual property protection and market regulations. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, this application is further described in detail below in conjunction with the embodiments. It should be understood that the embodiments described herein are only used to explain this application and are not intended to limit this application.

[0028] The present invention provides a method for locating SNP markers of soybean fatty acid traits, comprising the following steps:

[0029] Step 1: construct a recombinant inbred line (RIL) population obtained by crossing the high oleic acid parent variety "Glysoja-HO" with the low oleic acid parent variety "Glysoja-LO";

[0030] Step 2: Perform whole-genome resequencing on all individuals in the population to obtain SNP genotype data covering 20 soybean chromosomes;

[0031] Step 3: Determine the fatty acid composition phenotype data of the individuals in the group under three environments by gas chromatography, including the content of oleic acid, linoleic acid, and linolenic acid;

[0032] Step 4: Use the linkage disequilibrium-linkage analysis integrated model, with environmental effects as fixed factors and parental genetic background as random factors, to conduct genome-wide association analysis on SNP data and phenotypic data;

[0033] Step 5: Screening SNP markers associated with target fatty acid traits, with a significance threshold of P < 1 × 10 -7The phenotypic variation explanation rate R 2 >15%;

[0034] Step 6: Through haplotype block analysis and independent natural population verification, SNP-OL-10A located at GmChr10:38,156,789-38,157,255 and SNP-LL-15B located at GmChr15:72,834,112-72,834,598 were identified as core functional markers.

[0035] In step 1 of the present invention, the recombinant inbred line (RIL) population is an F7 generation or above population, with a population size of ≥200 families; the oleic acid content of the parent "Glysoja-HO" is ≥85%, and the oleic acid content of "Glysoja-LO" is ≤25%.

[0036] In the step 2 of the present invention, the average depth of whole genome resequencing is ≥10×, and SNP calling adopts the GATK process with the filtering criteria of: missing rate <20%, minor allele frequency >5%.

[0037] The fixed factors in step 4 of the present invention include: environmental temperature gradient (15°C / 25°C / 35°C) and soil moisture gradient (40% / 60% / 80% field capacity); the random factors use the kinship matrix derived from the parental pedigree.

[0038] In the step 5 of the present invention, when screening associated SNPs, a multi-environment joint analysis (MET) is performed simultaneously, requiring the target SNP to reach a significance threshold (P < 1×10 -7 ).

[0039] The haplotype block analysis in step 6 of the present invention uses Haploview software, and the block is defined as: D' value between SNPs>0.8 and LOD>2; the natural population verification covers 100 soybean germplasm resources, requiring that the direction of the allele effect of the target marker in the population is consistent with that of the RIL population.

[0040] An application of core functional markers obtained based on the above-mentioned positioning method, wherein the core functional markers SNP-OL-10A and SNP-LL-15B are used for:

[0041] Application 1: Soybean molecular marker-assisted selection breeding to increase or decrease the content of oleic acid, linoleic acid, and linolenic acid in soybean seeds;

[0042] Application 2: Screening and creating soybean germplasm resources and varieties with target fatty acid composition;

[0043] Application three: Identifying genotypes in soybean seeds and plants that are related to the fatty acid traits described in claim 1.

[0044] The application of molecular marker-assisted selection breeding described in the present invention specifically includes: using the SNP-OL-10A marker to screen soybean plants or lines carrying superior alleles that increase oleic acid content; using the SNP-LL-15B marker to screen soybean plants and lines carrying superior alleles that reduce linoleic acid and linolenic acid contents; and simultaneously using the SNP-OL-10A and SNP-LL-15B markers to carry out gene cluster breeding to create new soybean germplasms and varieties with significantly increased oleic acid content and significantly reduced linoleic acid and linolenic acid contents.

[0045] The third application of the present invention for identifying soybean seed and plant genotypes is specifically as follows: using specific primers or probes designed based on the SNP-OL-10A and SNP-LL-15B sites, PCR, fluorescent quantitative PCR, high-throughput sequencing, and gene chip technology are used to detect the allele type of the soybean sample to be tested at the GmChr10:38,156,789-38,157,255 and GmChr15:72,834,112-72,834,598 sites to predict the potential of its oleic acid, linoleic acid, and linolenic acid content.

[0046] The core functional markers SNP-OL-10A and SNP-LL-15B described in the present invention are also used for soybean seed purity and authenticity identification by detecting whether the genotype of the target seed at the marker site conforms to the characteristic allele type of the expected parent and variety.

[0047] Example:

[0048] (1) Material preparation and group construction

[0049] Parent selection: The high oleic acid parent "Glysoja-HO" (oleic acid content 86.2%) was hybridized with the low oleic acid parent "Glysoja-LO" (oleic acid content 23.5%).

[0050] Construction of RIL population: Through continuous self-pollination to the F8 generation through single-seed propagation, a recombinant inbred line population containing 218 families was obtained, and 10 plants were planted in each family to ensure biological replication.

[0051] (2) Genotype data analysis

[0052] Whole-genome resequencing: Whole-genome resequencing (Illumina NovaSeq platform) was performed on 218 RIL individuals and their parents, with an average sequencing depth of 12×.

[0053] SNP calling and filtering: (1) The GATK4.1 standard process was used for variant detection (including HaplotypeCaller, VQSR, and other steps); (2) Filtering criteria: missing rate <15%, minor allele frequency (MAF) >5%, depth ≥5×, and finally 1,203,467 high-quality SNP sites were obtained.

[0054] (III) Multi-environment phenotyping

[0055] Environmental design: Three environmental combinations (temperature × moisture) were set: (1) E1: 15°C / 40% field water capacity; (2) E2: 25°C / 60% field water capacity; (3) E3: 35°C / 80% field water capacity.

[0056] Phenotype collection: At the maturity stage of R8 generation seeds, 5 seeds were collected from each family in each environment, and the contents of oleic acid (OA), linoleic acid (LA), and linolenic acid (LNA) (unit: %) were determined by gas chromatography (GC-2030, Shimadzu).

[0057] Data statistics: Phenotypic data showed continuous distribution (oleic acid: 25.8%-82.3%), broad heritability H 2 =0.73 (calculated based on three environmental variance components).

[0058] (IV) Association analysis and marker screening

[0059] Model construction: The LD-LA integrated model (R package GAPIT) was used, with the environmental gradient (E1-E3) as the fixed factor and the kinship matrix constructed based on SNP data as the random factor.

[0060] Multi-environment analysis (MET): A genome-wide scan identified 32 SNPs significantly associated with fatty acid traits (P < 1 × 10 -7 ).

[0061] Core site screening: further requirements for R 2 >15% and significant in at least two environments, ultimately locked:

[0062] GmChr10:38,156,932 (designated as SNP-OL-10A): Regulates oleic acid (OA), P = 3.2 × 10 -10 (E1), 7.8×10 -9 (E2), R 2 =18.7%. Allele "A" increases oleic acid by 21.3% (mean).

[0063] GmChr15:72,834,305 (named SNP-LL-15B): Regulates linoleic acid (LA) and linolenic acid (LNA), P(LA) = 1.5×10 -8 (E2), 4.1×10 -8 (E3); P(LNA)=9.2×10 -9 (E1), 2.7×10 -8 (E3), R 2 (LA)=16.2%,R 2 (LNA) = 17.5%. The "T" allele reduced linoleic acid by 18.5% and linolenic acid by 22.1% (mean).

[0064] (5) Marking Verification and Functional Confirmation

[0065] Haplotype block analysis (Haploview 4.2): With SNP-OL-10A as the core, a haplotype block was defined in the GmChr10:38.15-38.16Mb region (D'=0.92, LOD=35), including five strongly linked SNPs (r 2 The block where SNP-LL-15B is located (GmChr15:72.83-72.84Mb, D'=0.88, LOD=28) contains three linked SNPs.

[0066] Natural population verification: 100 soybean germplasm resources (including cultivated varieties and landraces) were selected to detect the SNP-OL-10A and SNP-LL-15B genotypes.

[0067] Results: (1) The frequency of the "A" allele at SNP-OL-10A was significantly positively correlated with oleic acid content (r=0.71, P<0.001). (2) The frequency of the "T" allele at SNP-LL-15B was negatively correlated with linoleic acid (r=-0.68) and linolenic acid (r=-0.73) (P<0.001), and the direction of the effect was consistent with that in the RIL population.

[0068] (VI) Demonstration of molecular breeding applications

[0069] Genetic aggregation breeding: A RIL individual (family RIL-147) carrying the SNP-OL-10A-"A" (high oleic acid) and SNP-LL-15B-"T" (low linoleic / linolenic acid) mutations was selected as a donor and crossed with the high-yielding variety "Jidou28." Prospect selection using the KASP marker was performed in the BC2F3 generation to generate an aggregated strain.

[0070] Phenotypic effect: The oleic acid content of the polymer strain reached 78.5% (142% higher than that of the recipient parent), linoleic acid dropped to 9.8% (61% lower), and linolenic acid dropped to 3.2% (58% lower).

[0071] (VII) Application of seed purity identification

[0072] Twenty market samples of the commercial variety "Gaolidou No. 1" (known genotype: SNP-OL-10A-"A" / SNP-LL-15B-"T") were tested: (1) the target site genotypes of 18 samples were completely matched (purity ≥ 99%); (2) 2 samples showed the "C" allele at the SNP-LL-15B site (purity only 85%) and were judged to be adulterated.

[0073] As can be seen from the above-described embodiments, a kind of soybean fatty acid trait SNP marker positioning and application of the present invention has successfully solved the problem that fatty acid content is susceptible to environmental interference, and significantly improved the accuracy of association analysis. By building RIL colony, carrying out whole genome resequencing, multi-environment phenotyping, association analysis and marker screening, marker verification and function confirmation and other steps, core function markers SNP-OL-10A and SNP-LL-15B have been accurately located. These marks can not only be used for soybean molecular marker assisted selection breeding, screening and creation of soybean germplasm resources and variety, identification of soybean seeds and plant genotype with target fatty acid composition, but also can effectively carry out soybean seed purity and authenticity identification, for soybean breeding and seed market specification provide powerful technical support. In the future, it is expected that based on this technology, soybean fatty acid trait will be further optimized, and more high-quality soybean varieties that meet market demand will be cultivated.

[0074] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for locating SNP markers for soybean fatty acid traits, characterized in that: The following steps are involved: Step 1: Construct a population of recombinant inbred lines (RILs) obtained by crossing the high oleic acid parent variety "Glysoja-HO" with the low oleic acid parent variety "Glysoja-LO"; Step 2: Perform whole-genome resequencing on all individuals in the population to obtain SNP genotype data covering 20 soybean chromosomes; Step 3: Determine the fatty acid composition phenotype data of the individuals in the group under three environments by gas chromatography, including the content of oleic acid, linoleic acid, and linolenic acid; Step 4: Use the linkage disequilibrium-linkage analysis integrated model, with environmental effects as fixed factors and parental genetic background as random factors, to conduct genome-wide association analysis on SNP data and phenotypic data; Step 5: Screening SNP markers associated with target fatty acid traits, with a significance threshold of P < 1 × 10 -7 The phenotypic variation explanation rate R 2 >15%; Step 6: Through haplotype block analysis and independent natural population verification, SNP-OL-10A located at GmChr10:38,156,789-38,157,255 and SNP-LL-15B located at GmChr15:72,834,112-72,834,598 were identified as core functional markers.

2. A soybean fatty acid trait SNP marker positioning method according to claim 1, characterized in that, In the step 1, the recombinant inbred line (RIL) population is an F7 generation or above population, and the population size is ≥200 families; the oleic acid content of the parent "Glysoja-HO" is ≥85%, and the oleic acid content of "Glysoja-LO" is ≤25%.

3. A soybean fatty acid trait SNP marker positioning method according to claim 1, characterized in that, In the second step, the average depth of whole genome resequencing was ≥10×, and SNP calling was performed using the GATK process with the filtering criteria of: missing rate <20%, minor allele frequency >5%.

4. A soybean fatty acid trait SNP marker positioning method according to claim 1, characterized in that, The fixed factors in step 4 include: environmental temperature gradient (15°C / 25°C / 35°C) and soil moisture gradient (40% / 60% / 80% field capacity); the random factors use the kinship matrix derived from the parental pedigree.

5. A soybean fatty acid trait SNP marker positioning method according to claim 1, characterized in that, When screening the associated SNPs in step 5, a multi-environment joint analysis (MET) was performed simultaneously, requiring the target SNP to reach a significance threshold (P < 1×10 -7 ).

6. The method for locating SNP markers for soybean fatty acid traits according to claim 1, wherein: The haplotype block analysis in step 6 was performed using Haploview software, and the block was defined as: D' value between SNPs > 0.8 and LOD > 2; the natural population validation covered 100 soybean germplasm resources, requiring that the direction of the allele effect of the target marker in the population be consistent with that of the RIL population.

7. An application of a core function marker obtained based on any positioning method of claims 1-7, characterized in that: The core functional markers SNP-OL-10A and SNP-LL-15B are used to: Application 1: Soybean molecular marker-assisted selection breeding to increase or decrease the content of oleic acid, linoleic acid, and linolenic acid in soybean seeds; Application 2: Screening and creating soybean germplasm resources and varieties with target fatty acid composition; Application three: Identifying genotypes in soybean seeds and plants that are related to the fatty acid traits described in claim 1.

8. The use according to claim 7, characterized in that The application of molecular marker-assisted selection breeding specifically includes: using the SNP-OL-10A marker to screen soybean plants or lines carrying superior alleles that increase oleic acid content; using the SNP-LL-15B marker to screen soybean plants and lines carrying superior alleles that reduce linoleic acid and linolenic acid content; and simultaneously using the SNP-OL-10A and SNP-LL-15B markers to carry out gene aggregation breeding to create new soybean germplasms and varieties with significantly increased oleic acid content and significantly reduced linoleic acid and linolenic acid contents.

9. The use according to claim 7, characterized in that The third application for identifying soybean seed and plant genotypes is specifically as follows: using specific primers or probes designed based on the SNP-OL-10A and SNP-LL-15B sites, PCR, fluorescent quantitative PCR, high-throughput sequencing, and gene chip technology are used to detect the allele type of the soybean sample to be tested at the GmChr10:38,156,789-38,157,255 and GmChr15:72,834,112-72,834,598 sites to predict the potential of its oleic acid, linoleic acid, and linolenic acid content.

10. The use according to claim 7, characterized in that The core functional markers SNP-OL-10A and SNP-LL-15B are also used for soybean seed purity and authenticity identification by detecting whether the genotype of the target seed at the marker site conforms to the characteristic allele type of the expected parent and variety.