SNP (Single Nucleotide Polymorphism) marker related to soybean branching number gene QNE1 and application of SNP marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker related to a soybean branching number gene QNE1 and application thereof, and relates to the technical field of molecular genetics and crop genetic breeding, the SNP molecular marker is a nucleotide sequence as shown in SEQ ID NO.1 formed by single nucleotide mutation at the 686th site of the nucleotide sequence of the QNE1 gene in a soybean genome; the polymorphism of the single nucleotide mutation is C / G polymorphism. The method is used for assisting soybean breeding. The SNP marker provided by the invention can be used for molecular marker-assisted selection of soybean branch number related traits, and a key tool is provided for cultivating soybean varieties adapting to different planting requirements (such as a few-branch close planting type or a multi-branch high-yield type).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular genetics and crop genetic breeding, and particularly relates to a SNP marker related to a soybean branch number gene QNE1 and application thereof. BACKGROUND

[0002] Soybean is a crop for both food and oil, and its yield components are closely related to plant type. Branch number, as a core agronomic trait determining soybean plant type and yield, directly affects pod number per plant and population light energy utilization efficiency. Generally speaking, soybean with more branch numbers has higher yield potential per plant and is suitable for sparse planting; while soybean with fewer branch numbers and compact plant type is more suitable for high-density planting to realize high yield by increasing plant number per unit area.

[0003] In the era of molecular breeding, precise and efficient improvement of traits is the key. Although branch number can be observed in the middle and late growth stages, the use of functional molecular markers co-segregated with it can screen the genotypes of a large number of breeding materials at the seedling stage or even the previous generation, which can significantly shorten the breeding cycle, improve the selection efficiency, and overcome the interference caused by environmental variation. In addition, with the development of gene editing and other frontier technologies, breeding has entered the stage of precise operation of target genes, which urgently needs to accurately identify the genes and functional nucleotide sites that regulate key agronomic traits, thereby providing direct basis for designing editing targets.

[0004] Although some quantitative trait loci (QTL) related to soybean branch number have been located through genetic analysis, there are still a lack of key genes with clear functions and functional single nucleotide polymorphism (SNP) markers that can be directly used in molecular breeding practice. The applicant previously identified a gene QNE1 significantly associated with branch number on chromosome 6 through GWAS analysis. Further research found that different haplotypes of the gene are closely related to branch number variation, but it is still a technical problem to be solved to convert this scientific discovery into a key specific SNP marker that can be used for molecular marker-assisted selection (MAS) and provide clear targets for gene editing.

[0005] Therefore, there is an urgent need in the art to identify SNP molecular markers that can accurately distinguish different functional haplotypes of the QNE1 gene and can be directly used for assisted breeding and provide targets for gene editing, to provide efficient technical tools for the directional selection of soybean branch number traits. SUMMARY

[0006] The application aims to provide a specific SNP molecular marker related to the soybean branch number gene QNE1 and an application thereof.

[0007] The application provides a SNP molecular marker for identifying a soybean branch number trait, characterized in that the SNP molecular marker is a single nucleotide mutation at the 686th nucleotide of the nucleotide sequence of the QNE1 gene in the soybean genome, forming a nucleotide sequence as shown in SEQ ID NO. 1.

[0008] Further, the C / G polymorphism affects the soybean branch number, and the C / G polymorphism is a C>G mutation; wherein the number of soybean branches of the C genotype individual is less than that of the G genotype individual.

[0009] The application provides an application of the SNP molecular marker for identifying a soybean branch number trait.

[0010] Further, the soybean breeding assisted by the SNP molecular marker is achieved through early screening of the soybean branch number trait.

[0011] When the base of the SNP molecular marker is C, the soybean is a Hap1 haplotype soybean leading to a decrease in the number of soybean branches.

[0012] When the base of the SNP molecular marker is G, the soybean is a Hap2 / 3 haplotype soybean leading to no decrease in the number of soybean branches.

[0013] Further, if the breeding target is a soybean with a decreased branch number, a soybean with a base C of the SNP molecular marker or a Hap1 haplotype soybean is selected, and if the breeding target is a soybean with a normal branch number, a soybean with a base G of the SNP molecular marker or a Hap2 / 3 haplotype soybean is selected.

[0014] The kit for detecting the SNP molecular marker is characterized in that it comprises a primer pair or a probe or a sequencing primer or a combination thereof for specifically detecting the C / G polymorphism at the SNP site.

[0015] In addition, the present application provides a soybean marker-assisted breeding method. The method detects the genotype (i.e., the base at position 686 is C or G) of the above-mentioned SNP molecular marker or identifies the corresponding functional haplotype (Hap1 or Hap2 / 3) in a soybean sample, and selects the corresponding soybean plant according to the breeding goal: when the breeding goal is to breed a soybean variety with less branching and dense planting, select a plant with the SNP molecular marker genotype C (i.e., Hap1 haplotype); when the breeding goal is to breed a soybean variety with more branching and high yield, select a plant with the SNP molecular marker genotype G (i.e., Hap2 / 3 haplotype).

[0016] The present application has the following advantages:

[0017] 1. Innovation: The present application first identifies a functional SNP marker at position 686 of the QNE1 gene, which has a direct causal relationship with the soybean branching number trait, overcoming the lack of functional SNP markers in the prior art.

[0018] 2. Practicality: The SNP marker can be used for early screening of the soybean branching number trait, and high-throughput genotype identification can be achieved at the seedling stage or seed stage, significantly shortening the breeding cycle, improving selection efficiency, and overcoming the influence of environmental variation.

[0019] 3. Precision: The marker provides a clear target for gene editing and other frontier technologies, which can be used for targeted improvement of the soybean branching number trait.

[0020] 4. Adaptability: By selecting different haplotypes, soybean varieties suitable for different planting needs (such as dense planting or high yield) can be flexibly cultivated to meet diverse production goals. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Manhattan plot and QQ plot of GWAS analysis based on 392 materials in 2014 data;

[0022] Figure 2 Manhattan plot and QQ plot of GWAS analysis based on 392 materials in 2015 data;

[0023] Figure 3 Local Manhattan plot and linkage disequilibrium heat map analysis plot around SNP peak of chromosome 6;

[0024] Figure 4 Association analysis plot of QNE1 haplotype and branch number;

[0025] Figure 5 RT-qPCR analysis plot of QNE1 different haplotype overexpression transgenic soybean;

[0026] Figure 6 Phenotype plot of QNE1 different haplotype overexpression transgenic soybean and non-transgenic soybean branch number. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the spirit of the present application will be described in detail below, and any person skilled in the art can make changes and modifications to the technology taught by the present application without departing from the spirit and scope of the present application after understanding the embodiments of the present application.

[0028] The schematic embodiments of the present application and the descriptions thereof are used to explain the present application, but not as a limitation of the present application.

[0029] In the following examples, the conventional methods are used unless otherwise specified.

[0030] The effect of the present application is verified by the following tests:

[0031] (1) GWAS analysis is used to locate soybean branch number gene QNE1 on chromosome 6

[0032] In this embodiment, GWAS analysis is performed on SNP data of 392 soybean germplasms and branch number phenotype data of 2014 and 2015, MAF<0.05 data filtering is performed on the SNP data to remove low-frequency variation sites, mixed linear model (MLM) of tassel software is used for association analysis, it is found that QNE1 gene on Chr6 exists allelic variation in CDS region, and SNP site and branch number are significantly associated (as shown in Figure 1 , 2 This embodiment performs LD block analysis near the significant siteFigure 3 Ultimately, the candidate region was narrowed down to 70kb. Gene annotation of the candidate region revealed two genes within it. One gene is the QNE1 gene, which encodes the TCP transcription factor. The TCP transcription factor plays an important role in branching regulation. This gene has allelic variations in the CDS region. The other gene does not have allelic variations in the CDS region.

[0033] (II) Association analysis between QNE1 gene haplotype and branch number

[0034] Analysis of the QNE1 gene sequence in 392 soybean germplasms revealed allelic variations at two loci in the CDS region of QNE1. Figure 4 A) The change in base sequence caused a change in the amino acid composition of the product, which is a missense mutation. Allelic variations at two sites constitute three genotype combinations: Hap1, Hap2, and Hap3. Association analysis between haplotype and branch number revealed that soybean varieties carrying the Hap1 haplotype had significantly fewer branches than those carrying the Hap2 and Hap3 haplotypes, while there was no difference in branch number between haplotypes Hap2 and Hap3. Figure 4 (B, C) Haplotypes Hap2 and Hap3 show sequence differences at the second locus in the CDS region, indicating that the difference at the second locus does not cause changes in gene function. Both haplotypes Hap2 and Hap3 show sequence differences from haplotype Hap1 at the first locus, and also differ in the number of branches, indicating that the difference at the first locus (686 bp) in the QNE1 gene's CDS region leads to changes in gene function.

[0035] (III) Construction of QNE1 gene overexpression vector

[0036] QNE1 -Hap1 The gene sequence QNE1 was cloned from the soybean variety Heinong 51. -Hap3The gene sequence was cloned from the soybean variety Williams 82. Using cDNA from leaves of Williams 82 and Heinong 51 soybean plants as templates, and QNE1-F1 (Seq ID No: 7) and QNE1-R1 (Seq ID No: 8) as primers, PCR amplification was performed using the high-fidelity enzyme FastPfu Fly DNA Polymeras purchased from Beijing TransGen Biotech Co., Ltd. PCR reaction conditions: 94℃ pre-denaturation for 5 min, (94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s) × 6 cycles, 94℃ denaturation for 30 s, 68℃ annealing for 30 s, 72℃ extension for 60 s, 26 cycles, followed by a final extension at 72℃ for 5 min. Gel recovery was performed using the Wizard® SV Gel and PCR Clean UpSystem purchased from Promega. The QNE1 gene was amplified by PCR, and an XbaI restriction enzyme site was introduced upstream of the QNE1 gene, followed by a SacI restriction enzyme site downstream. The resulting PCR product and vector pBA:35S:6×cmyc were simultaneously digested with XbaI and SacI, ligated overnight with T4 DNA ligase, and transformed into competent *E. coli* cells. The pBA:35S:6×cmyc:QNE1 vector contains the Bar selection marker gene (glufosinate resistance), and the QNE1 gene is driven by the 35S promoter.

[0037] The constructed vector pBA:35S:6×cmyc:QNE1 was transferred into the genome of soybean variety Williams 82 via Agrobacterium tumefaciens-mediated transfer of cotyledonary nodes, resulting in transgenic soybean lines overexpressing the QNE1 gene.

[0038] (II) Molecular identification of transgenic soybeans overexpressing the QNE1 gene

[0039] 1. Through genetic transformation of soybean, using a glufosinate application experiment with 160 mg / L, two different haplotype transformation events (35S: QNE1) were observed in the T1 generation to obtain glufosinate resistance. -Hap1 1. 35S: QNE1 -Hap1 2. 35S: QNE1 -Hap3 1. 35S: QNE1 -Hap3 2) RNA was extracted from mature soybean leaves of these four transformation events. The RNA extraction method can be found in the Invitrogen TRIzol kit manual.

[0040] 2. The total RNA extracted in step 1 was treated with DNase. The RNA content and quality of the treated total RNA were determined using NanoDrop, which was purchased from Toyobo.

[0041] 3. Following step 2, 2µg of total RNA was collected after reaching the standard using NanoDrop assay. This was then analyzed using TransScript RNA sorbed from Beijing TransGen Biotech Co., Ltd. One-Step gDNA Removal and cDNA Synthesis SuperMix Kit, and the dT included with the kit 18 Primers were used to synthesize cDNA according to the kit's instruction manual;

[0042] 4. Take 1 µl of the 30-fold diluted cDNA from step 3 as a qPCR template. Use qQNE1-F1 (Seq ID No: 9) and qQNE1-R1 (Seq ID No: 10) as primers, and the primer sequences for the soybean internal reference gene TUA5 are qTUA5 (Seq ID No: 11 and Seq ID No: 12). Follow the TransStart Top Green qPCR Super Mix Kit instructions, using SYBR Green I as dye, and employ a three-step PCR reaction. qPCR reaction conditions: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, 40 cycles, using 2... –ΔΔCT The algorithm calculates the relative expression level of the GmQNE1 gene, and the results are as follows: Figure 5 As shown, the expression level of the QNE1 gene was significantly higher in soybeans than in non-GMO soybeans in both independent transformation events. This indicates that the QNE1 gene was successfully overexpressed in soybeans.

[0043] (iv) Phenotypic analysis of branch number in transgenic soybeans overexpressing the QNE1 gene

[0044] T2 generation 35S: QNE1 -Hap1 35S: QNE1 -Hap3 Transgenic soybeans with overexpressing the gene and the non-transgenic control variety Williams 82 were sown in pots, with 10 plants per line, and placed under natural light (long day). The phenotype of the plants was investigated in detail. The results showed that QNE1... -Hap1 The number of branches in transgenic soybeans with overexpression of the QNE1 gene was significantly reduced. -Hap3 There was no significant difference in the number of branches compared to the non-GMO control variety Williams 82. Figure 6 ).

Claims

1. A SNP molecular marker for identifying the soybean branch number trait, characterized in that, The SNP molecular marker is a single nucleotide mutation at the 686th nucleotide of the nucleotide sequence of the QNE1 gene in the soybean genome, forming a nucleotide sequence as shown in SEQ ID NO. 1; the single nucleotide mutation polymorphism is C / G polymorphism. 2.The SNP molecular marker for identifying the soybean branch number trait according to claim 1, wherein, The C / G polymorphism affects the branch number of soybean, and the C / G polymorphism is a mutation of C>G; the branch number of soybean of an individual with C genotype is less than that of an individual with G genotype.

3. The use of a SNP molecular marker for identifying the soybean branch number trait according to claim 1 or 2, characterized in that, The SNP molecular marker is used for assisting soybean breeding.

4. Use according to claim 3, characterized in that, The assisting soybean breeding is realized through early screening of the branch number of soybean; the screening is completed by the following method: When the base of the SNP molecular marker is C, the soybean is Hap1 haplotype soybean which causes the branch number of soybean to decrease; When the base of the SNP molecular marker is G, the soybean is Hap2 / 3 haplotype soybean which does not cause the branch number of soybean to decrease.

5. Use according to claim 4, characterized in that, If the breeding target is soybean with decreased branch number, the soybean with base C of the SNP molecular marker or Hap1 haplotype soybean is selected, and if the breeding target is soybean with relatively more branch number, the soybean with base G of the SNP molecular marker or Hap2 / 3 haplotype soybean is selected.

6. A kit for detecting the SNP molecular marker of claim 1 or 2, characterized in that, A primer pair or a probe or a sequencing primer or a combination thereof for specifically detecting the C / G polymorphism of the SNP site is comprised.

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