Haploid molecular marker related to reproductive growth period of soybean and application of haplotype molecular marker

Abstract

The invention discloses a haplotype molecular marker related to the reproductive growth period of soybean and application of the haplotype molecular marker. By detecting the haplotype molecular marker (2020th nucleotide G / A of SEQ ID NO.1) remarkably related to the reproductive growth period of soybean, the characters of the reproductive growth period of soybean can be rapidly and accurately identified; the molecular marker provides a clear genetic target for soybean breeding, soybean varieties adapting to different photoperiods and regional requirements can be directionally bred by selecting a specific genotype (for example, a GG type can prolong the reproductive growth period, and an AA type can shorten the reproductive growth period), the breeding period is shortened, and the breeding accuracy is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a haplotype molecular marker related to the reproductive growth period of soybean and an application thereof. Background Art

[0002] Soybeans are an important source of vegetable oil and protein for humans. As a typical short-day crop, soybeans are highly sensitive to changes in the photoperiod, and therefore their flowering and maturity periods are affected by the photoperiod. This characteristic results in geographical restrictions on the cultivation of different varieties, limiting their planting range. Therefore, the response of the growing season to the photoperiod has become a key indicator for assessing the growth and development of soybeans. Soybean growth traits include flowering, reproductive growth, and maturity, and these traits are closely related to soybean quality, yield, adaptability, and planting range.

[0003] At present, the length of soybean reproductive growth period varies greatly among different soybean varieties and varieties, and the reproductive growth period of soybean is closely related to the quality, yield, adaptability and planting range of soybean. Therefore, it is of great significance to accurately and quickly detect and identify the reproductive growth period of soybean. Traditional methods mainly rely on field surveys of the growth period of soybean, but this survey method relies on the survey of the entire growth period of the material, which is time-consuming and labor-intensive, limiting the application of rapid identification of the reproductive growth period of soybean. Therefore, it is of great significance to develop a method for identifying the reproductive growth period of soybean based on haplotype molecular markers. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a haplotype molecular marker related to the reproductive growth period of soybean and an application thereof.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0006] Application, where the application is P1 or P2;

[0007] The P1 is an application of a substance for detecting the polymorphism or genotype of SNP in identifying or assisting in identifying the reproductive growth period of soybean or in preparing a product for identifying or assisting in identifying the reproductive growth period of soybean;

[0008] The P2 is an application of a substance for detecting the polymorphism or genotype of SNP in soybean breeding or preparing soybean breeding products;

[0009] The SNP is the 2020th nucleotide of SEQ ID NO.1 in the sequence list, which is G or A.

[0010] A product comprising the substance for detecting the polymorphism or genotype of soybean genome SNPs as claimed in claim 1, and being any one of the following G1) to G3):

[0011] G1) Products for detecting single nucleotide polymorphisms or genotypes related to soybean reproductive growth period;

[0012] G2) Identify or assist in identifying products in the reproductive growth stage of soybeans;

[0013] G3) Products used in soybean breeding.

[0014] A method for identifying or assisting in identifying the reproductive growth period of soybeans, by detecting the genotype of the SNP described in claim 1 in the soybeans to be tested, and identifying or assisting in identifying the reproductive growth period of soybeans according to the genotype of the soybeans to be tested: the reproductive growth period of soybeans with the SNP genotype of GG is higher or is a candidate to be higher than the reproductive growth period of soybeans with the genotype of AA, the genotype of the SNP GG is a homozygous type in which the nucleotide at position 2020 of SEQ ID NO.1 in the sequence listing is G; the genotype of the SNP AA is a homozygous type in which the nucleotide at position 2020 of SEQ ID NO.1 in the sequence listing is A.

[0015] Application of the above method in soybean breeding.

[0016] According to the above application, the above product, and the above method, it is characterized in that the purpose of the breeding includes cultivating or breeding soybeans with a longer reproductive growth period or a shorter reproductive growth period.

[0017] A soybean breeding method, comprising replacing the nucleotide at position 2020 of SEQ ID NO.1 in the genome of a soybean having a SNP genotype of AA with G, thereby obtaining target soybeans having a reproductive growth period longer than that of the soybean having the genotype of AA and a haplotype of GG; the SNP genotype of AA is a homozygous type in which the nucleotide at position 2020 of SEQ ID NO.1 in a sequence list is A; the SNP genotype of GG is a homozygous type in which the nucleotide at position 2020 of SEQ ID NO.1 in a sequence list is G.

[0018] The beneficial effect of adopting the above technical solution is that: the present invention can quickly and accurately identify the reproductive growth period traits of soybean by detecting the haplotype molecular marker (nucleotide G / A at position 2020 of SEQ ID NO.1) that is significantly associated with the reproductive growth period of soybean. This molecular marker provides a clear genetic target for soybean breeding. By selecting specific genotypes (such as GG type can extend the reproductive growth period, and AA type can shorten the reproductive growth period), soybean varieties that adapt to different photoperiods and regional requirements can be cultivated in a targeted manner, shortening the breeding cycle and improving breeding accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the frequency distribution diagram of the reproductive growth period of the RIL population;

[0020] Figure 2 It is a frequency distribution diagram of the reproductive growth period of natural populations;

[0021] Figure 3 It is the QTL locus associated with the 3-year reproductive growth period traits of the RIL population;

[0022] Figure 4 It is the Manhattan plot and QQ plot of the average data of the reproductive growth period over 3 years;

[0023] Figure 5 This is the LD Block diagram of chromosome 4 during the reproductive growth period;

[0024] Figure 6 It is a haplotype analysis of reproductive growth period phenotype;

[0025] Figure 7 These are photos of the planting site of RIL group and natural group materials. DETAILED DESCRIPTION

[0026] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained through commercial purchase. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0027] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, integers, steps, operations, elements and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or collections thereof.

[0028] It will also be understood that the term "and / or" used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0029] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in one or more embodiments of the present application. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in other embodiments" appearing in various places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more but not all embodiments," unless otherwise specifically emphasized. The terms "including," "comprising," "having," and variations thereof all mean "including but not limited to," unless otherwise specifically emphasized.

[0030] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0031] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1. Materials and methods

[0033] 1.1 Test materials

[0034] This study used F10 high-generation recombinant inbred line populations (RILs) constructed by hybridization of Jidou 12 and wild soybean Y9 and natural populations constructed from 290 resource materials with a broad genetic basis as experimental materials for the study of soybean flowering and maturity. The RIL population contains 180 families, of which the parent Jidou 12 comes from a nationally approved variety bred by the Hebei Academy of Agricultural Sciences, and Y9 comes from a wild species. The natural population materials come from a wide range of sources, including 16 provinces in China (200, 68.97%), 6 states in the United States (83, 38.62%), and many other countries (7, 2.38%), and have rich genetic diversity. Figure 7 As shown, RIL and natural populations were planted from 2021 to 2023 at the Dishang Experimental Station in Shijiazhuang, Hebei Province (38°04′N, 114°28′E). The experiment used a randomized block design, with two replicates planted in 3-meter-long rows, three rows, and 0.5-meter row spacing. The plant spacing for the RIL population was 0.2 meters, while the plant spacing for the natural population was 0.1 meters. Conventional local management practices were followed.

[0035] 1.2 Field survey

[0036] First, the flowering and maturity dates of RIL and natural populations were investigated. Flowering Days (FD) were recorded at the R1 stage (the number of days from sowing to 50% flowering in 50% of soybeans). Maturity Days (MD) were recorded at the R8 stage (when 95% of the pods in 50% of the plants had turned mature and the seeds within the pods were swaying). Reproductive Days (RD) were recorded as R8 minus R1 (RD = R8 – R1).

[0037] 1.3 DNA extraction

[0038] The soybean leaf genome was extracted using the Kangwei DNA kit. The specific method is as follows:

[0039] (1) About 100-500 mg of fresh and expanded leaves from soybean plants were picked and placed in a 2 ml centrifuge tube. The tube was cooled with liquid nitrogen and then quickly ground with a plastic grinding rod to ensure that the sample was fully crushed.

[0040] (2) Add 400 μl of Buffer LP1 and 6 μl of RNase A (10 mg / ml) to the sample centrifuge tube. Vortex the sample for 1 minute to mix thoroughly. Let it stand at room temperature for 20-30 minutes to allow the soybean leaf tissue to fully lyse.

[0041] (3) Add 130 μl of Buffer LP2 to the lysed sample and mix thoroughly. Centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to a newly labeled 2 ml centrifuge tube.

[0042] (4) After centrifugation, transfer the supernatant to a new centrifuge tube. Then add 1.5 times the original volume of Buffer LP3 (containing anhydrous ethanol) and mix thoroughly.

[0043] (5) Pour the mixed solution into the adsorption column, centrifuge it at 12000 rpm for 1 minute, and discard the filtrate.

[0044] (6) Add 500 μl of Buffer GW (containing anhydrous ethanol) to the adsorption column and centrifuge at 12,000 rpm for 1 min. Discard the filtrate again and return the adsorption column to the centrifuge tube. Repeat this process once.

[0045] (7) Pour out the waste liquid in the tube, put the adsorption column back into the collection tube and leave it empty for 2 minutes to remove the residual ethanol components.

[0046] (8) Transfer the adsorption column to a new 1.5 ml centrifuge tube and let it stand at room temperature for 10-20 minutes to completely dry the adsorption membrane.

[0047] (9) Add 50-100 μl of Buffer GE reagent to the center of the dried adsorption column adsorption membrane and let it sit for several minutes.

[0048] (10) Place the adsorption column in a centrifuge tube and centrifuge at 12,000 rpm for 1 min. Retain the DNA in the tube and test the quality of the DNA extraction by 0.8% agarose gel electrophoresis. Quantitative analysis of the DNA is performed using an ultraviolet spectrophotometer. Qualified DNA is stored in a -20°C refrigerator until use.

[0049] 1.4 Genotype identification

[0050] The RIL population established by Jidou 12 and Ye 9 was sequenced using GBS. The specific steps are as follows: (1) Place different samples and adapters containing different barcodes in pairs on a plate; (2) Use ApeKI restriction enzyme for enzymatic digestion; (3) Use T4 ligase to connect the adapters to the sticky ends generated by enzyme digestion at both ends of the fragment; (4) Mix the samples containing different barcodes and then pass them through a fragment length screening column to filter out the adapters that have not reacted; (5) Add PCR primers and perform PCR amplification.

[0051] 290 natural populations were resequenced using the following steps:

[0052] (1) Construction of resequencing library using The DNA Library Prep Kit for ILM is used to construct a resequencing library from DNA that has passed quality inspection. The steps are as follows:

[0053] ① Take 200ng of DNA that has passed the quantitative and quality inspection and place it in a 0.2mL PCR tube, and add 4μL End Repair Buffer and 2.6 μL End Repair Enzyme was added with water to 20 μL, and the mixture was placed in an ABI 9700 PCR instrument and incubated at 37°C for 20 min, followed by denaturation at 72°C for 20 min to complete DNA fragmentation, end repair, and A-tailing.

[0054] ②Add 2 μL Ultra DNA Ligase, 8 μL Ultra DNA Ligase Buffer and 4 μL Add 40 μL of Adapter for ILM and place in an ABI 9700 PCR instrument at 22°C for 60 minutes to complete the ligation of sequencing adapters. Then, add 48 μL of GenoPrep DNA Clean Beads to the ligation product for purification. After purification, perform fragment selection using 0.68x + 0.2x magnetic beads, retaining ligation products with inserts between 300 and 350 bp.

[0055] ③ Add 10 μL of sequencing adapter with barcode sequence and 10 μL of PCR MasterMix was added and water was added to 20 μL; amplification was performed using an ABI 9700 PCR instrument with the following program: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 40 s, 5 cycles, and extension at 72°C for 4 min.

[0056] ④ Add 20 μL of GenoPrep DNA Clean Beads to the second-round PCR product, place on a magnetic stand until the solution is clear, discard the supernatant and add 100 μL of 80% ethanol to wash the magnetic beads, and add 35 μL of 10 mM Tris-HCl to obtain the purified DNA library.

[0057] (2) After library construction, preliminary quantification was performed using Qubit2.0, and the effective concentration of the library was accurately quantified using qPCR to ensure library quality. After the library quality test was qualified, sequencing was performed using the BGI-2000 / MGI-T7 sequencing platform in the PE150 mode.

[0058] (3) The SNP sites were quality controlled using vcftools software, and the obtained VCF was used for genome-wide association study (GWAS).

[0059] 1.5 QTL mapping and genome-wide association analysis

[0060] To map QTLs in the RIL population, a genetic map was constructed using QTLICIMaping V4.2 software based on GBS sequencing results from the RIL population. The reference genome was Williams 82.a2.v1. The map was 6,626.06 cM in length and included 6,288 markers with specific length amplified fragments (FLAGS) covering 20 linkage groups (LGs). The average inter-marker distance was 1.81 cM. QTL analysis for soybean reproductive traits was performed using the ICIM-ADD method within QTLICIMaping V4.2 software. QTLs were identified based on a significance threshold of LOD > 2.5. QTLs were named using the formula: q + FD + chromosome number + QTL number.

[0061] GWAS was conducted using TASSEL 2.1 software based on mixed linear models (MLM) combined with natural population phenotype and genotype data. The LOD value significant threshold of all SNP sites in the GWAS was set to -log10(P) ≥ 6 for comparative analysis. Quantile-Quantile scatter plots were drawn using the ggplot2 drawing tool. Manhattan plots were drawn using qqman in the R software package (e.g. Figure 4 shown).

[0062] 1.6 Candidate gene prediction and haplotype analysis

[0063] The Soybase database (https: / / www.soybase.org / sbt / ) was used to extract all genes within 50 kb before and after the co-localized site. Genetic enrichment (GO) analysis was performed using the GO enrichment tool within Soybase. Candidate genes regulating growth-stage traits were predicted based on soybean genome annotation data. Haplotype block analysis was performed within the 50 kb candidate region using Haploview 4.2 software.

[0064] Example 2, Results and Analysis

[0065] 2.1 Phenotypic analysis during reproductive growth

[0066] The results of phenotypic identification of the RIL population from 2021 to 2023 showed that the reproductive growth period was between 40 and 65 days. The coefficient of variation of the reproductive growth period was 6.15%. The skewness was 0.02 and the kurtosis was 0.21. The reproductive growth period of the natural population was between 40 and 90 days, with a coefficient of variation of 9.37%. The skewness was -0.01 and the kurtosis was 0.36. Figure 1 、 Figure 2 As shown, the reproductive growth period of both populations was normally distributed.

[0067] 2.2 QTL mapping and analysis of reproductive growth period in RIL population

[0068] Based on the flowering and maturity phenotypes of the RIL population, the length of the reproductive growth period was calculated. A total of 14 QTLs related to the reproductive growth period were detected using the ICIM method. These QTLs were distributed on 10 different chromosomes ( Figure 3 Table 1). qRD4-2 on chromosome 4, qRD7-2 on chromosome 7, and qRD16 on chromosome 16 were reproducibly detected in both environments, indicating that they are three stably expressed QTLs. qRD16 is the reported FT5a gene. qRD8 on chromosome 8, qRD13 on chromosome 13, and qRD17 on chromosome 17 all overlap or are adjacent to known growth-stage-associated loci. The LOD values ​​for qRD3, qRD10, qRD13, and qRD17 ranged from 2.52 to 4.08, with phenotypic contributions of 2.58% to 4.07%. These loci are minor QTLs detected in the one-year environment with LOD values ​​and PVE (%) values ​​≤ 5.

[0069] Table 4. QTL mapping results for reproductive growth traits in RIL population

[0070]

[0071] 2.3 GWAS of reproductive growth in natural populations

[0072] By conducting GWAS on natural populations, a total of 88 SNP sites with LOD values ​​≥ 6 associated with reproductive growth period were found. Among them, 30 SNP sites had LOD values ​​≥ 6.50, which were distributed on 15 chromosomes ( Figure 4 (Table 2). Chr10_45248537 is adjacent to the reported E2 gene, located 121 kb away. Four QTNs (Chr06_6230952, Chr09_43772632, Chr15_38546523, and Chr18_24364093) are all located 96 to 242 kb from known SNPs controlling soybean growth period. In addition, 15 novel SNPs were identified that were significantly different from major QTLs or reported QTNs. The molecular mechanisms underlying these novel loci require further investigation.

[0073] Table 2 GWAS results of reproductive growth traits in natural populations

[0074]

[0075]

[0076] 2.4 Analysis of common location sites for reproductive growth traits

[0077] Linkage analysis and GWAS association analysis each have their limitations. The former has low resolution, while the latter may have the problem of false positives. Therefore, when these two methods are combined, the advantages of each method can offset the limitations of the other method, resulting in more accurate and precise QTL positioning. In this study, the QTL effect intervals detected by linkage analysis were compared with the significant loci associated with GWAS. The results showed that there were two SNP loci overlapping with the stable QTL (qRD4-1, qMD19-2) intervals. They are Chr04_16576138 and Chr19_45573720, which are distributed on chromosomes 4 and 19. Among them, Chr19_45573720 is located near the known SNP that controls the growth period of soybean, and Chr04_16576138 is a new locus that has not been reported in the co-location.

[0078] 2.5 Candidate gene prediction and haplotype analysis of co-localized loci

[0079] This study conducted candidate gene prediction and haplotype analysis on the common loci of growth-related traits in two populations ( Figure 5 ). qRD4-1 (located between 15855447bp and 16974748bp) located in the RIL population and Chr04_16576138 identified in the natural population are loci significantly associated with the reproductive growth period. qMD19-2 (located between 45523455 and 45322411) and Chr19_45573720 located in the natural population are loci significantly associated with the maturity period. Since the co-localized loci regulating soybean maturity on chromosome 19 are located near the known SNPs that control soybean growth period discovered by previous researchers, this study only performed candidate gene prediction and haplotype analysis on the co-localized loci on chromosome 4 related to the reproductive growth period. Haplotype analysis within a 50 kb region surrounding Chr04_16576138 revealed three candidate genes: Glyma.04g125400, Glyma.04g125500, and Glyma.04g125600. These candidate genes encode products including GRF zinc-finger binding protein, histone lysine N-methyltransferase, histone methyltransferase, and zinc-binding protein. However, these predicted candidate genes require further screening and functional validation to determine their actual roles in regulating soybean growth stages.

[0080] The predicted full length of the Glyma.04G125500 gene is 4444 bp. Further analysis revealed a key mutation in the coding region (CDS) of the Glyma.04G125500 gene: a base change from G to A at 2020 bp, a synonymous mutation. These mutations together produced two different haplotypes, Hap1 and Hap2. Figure 6 ).

[0081] A total of 323 materials were analyzed this time, of which some had poor sequencing quality (for example, the gene was not homozygous, the heterozygosity rate of the sequenced data was too high or the deletion rate was too high. According to quality control, the poor quality materials were deleted), and some materials did not amplify effective bands. After removing the above problematic materials, a total of 161 Hap1 materials (GG) and 35 Hap2 materials (AA) were detected.

[0082] Table 7 Phenotypic data of Hap1 and Hap2 haplotypes during reproductive growth period

[0083]

[0084]

[0085]

[0086] Through the analysis of 161 Hap1 materials and 35 Hap2 materials, e.g. Figure 6 As shown in Tables 7 and 8, combined with the phenotypic data, the average reproductive growth period of the materials with haplotype Hap1 was 67.74 days; the average reproductive growth period of the materials with haplotype Hap2 was 63.89 days; the reproductive growth period of the materials with haplotype Hap1 was significantly longer than that of the materials with haplotype Hap2, and the difference in reproductive growth period between the two haplotypes reached a significant level (P<0.05).

[0087] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0088] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0089] The embodiments described above 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. 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 various embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. Application, characterized in that, The application is P1 or P2; The P1 is an application of a substance for detecting the polymorphism or genotype of SNP in identifying or assisting in identifying the reproductive growth period of soybean or in preparing a product for identifying or assisting in identifying the reproductive growth period of soybean; The P2 is an application of a substance for detecting the polymorphism or genotype of SNP in soybean breeding or preparing soybean breeding products; The SNP is the 2020th nucleotide in SEQ ID NO. 1 in the sequence list, which is G or A.

2. The product is characterized in that The product contains the substance for detecting the polymorphism or genotype of soybean genome SNP as claimed in claim 1, and is any one of the following G1)-G3): G1) Products for detecting single nucleotide polymorphisms or genotypes related to soybean reproductive growth period; G2) Identify or assist in identifying products in the reproductive growth stage of soybeans; G3) Products used in soybean breeding.

3. A method for identifying or assisting in identifying the reproductive growth phase of soybean, characterized in that: By detecting the genotype of the SNP described in claim 1 in the soybean to be tested, the reproductive growth period of the soybean is identified or assisted in identification according to the genotype of the soybean to be tested: the reproductive growth period of soybeans with the SNP genotype of GG is higher or candidate to be higher than the reproductive growth period of soybeans with the genotype of AA, the genotype of the SNP GG is a homozygous type in which the nucleotide at position 2020 of SEQ ID NO.1 in the sequence listing is G; the genotype of the SNP AA is a homozygous type in which the nucleotide at position 2020 of SEQ ID NO.1 in the sequence listing is A.

4. Application of the method according to claim 3 in soybean breeding.

5. The use according to claim 1 or 4, the product according to claim 2, and the method according to claim 3, characterized in that: The purpose of the breeding includes cultivating or breeding soybeans with a longer reproductive growth period or a shorter reproductive growth period.

6. A soybean breeding method, characterized in that: The method comprises replacing the 2020th nucleotide of SEQ ID NO. 1 in the soybean genome with the SNP genotype of AA with G, thereby obtaining target soybeans having a reproductive growth period longer than that of the soybeans with the genotype of AA and a haplotype of GG; the genotype of the SNP AA is a homozygous type in which the 2020th nucleotide of SEQ ID NO. 1 in the sequence list is A; the genotype of the SNP GG is a homozygous type in which the 2020th nucleotide of SEQ ID NO. 1 in the sequence list is G.

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