KASP molecular marker for soybean plant height identification and application of KASP molecular marker

Abstract

The invention is suitable for the technical field of soybean breeding, and provides a KASP molecular marker for soybean plant height identification and application of the KASP molecular marker. The KASP molecular marker is based on an SNP variation site of a Glyma. 19G206100 gene between two parents of Chinese flat stem soybean and F02, the mutation position of the Glyma. 19G206100 gene is the 312th basic group on a CDS sequence, C of a reference genome is mutated into G, the 104th amino acid on a protein sequence is mutated into lysine from asparagine, and the 104th amino acid on the protein sequence is mutated into lysine from asparagine. The primer of the KASP molecular marker comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO.1-2 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.3. The invention further discloses a kit for detecting the KASP molecular marker. The KASP molecular marker is developed on the basis of the polymorphic site of Gm19G206100 in germplasm resources, the marker has the advantages of being high in resolution ratio and easy to detect, can be applied to plant height selection of early generations of soybean cross breeding, has the effects of reducing the breeding scale, reducing the workload and improving the breeding efficiency, and has important application value in production.

Description

Technical Field

[0001] This invention belongs to the field of soybean breeding technology, and in particular relates to a KASP molecular marker for identifying soybean plant height and its application. Background Technology

[0002] Soybean (Glycine max (L.) Merr.) is one of the world's most important food and oil crops and a significant source of plant protein for humans. Traditional soybean breeding techniques suffer from problems such as long cycles, low efficiency, and heavy workload. With the development of molecular biotechnology, combining molecular marker-assisted selection with traditional hybridization breeding techniques can improve breeding efficiency, shorten the breeding cycle, and reduce workload.

[0003] Plant height is a crucial factor influencing crop yield and quality, and it is significantly correlated with these traits. Furthermore, reducing plant height can effectively prevent lodging, thus ensuring stable soybean yields. Therefore, genetic and breeding research on plant dwarfing traits has always been a key focus for soybean researchers. Soybean plant height is a quantitative trait controlled by multiple genes. Currently, selection for soybean plant height in breeding mainly relies on phenotypic observation, which suffers from long cycles, low efficiency, and significant environmental influences. Molecular markers, on the other hand, offer advantages such as genetic stability, high throughput, high efficiency, and applicability to early-generation selection in breeding. Summary of the Invention

[0004] The purpose of this invention is to provide a KASP molecular marker for identifying soybean plant height, thereby addressing the problems mentioned in the background section.

[0005] This invention is implemented as follows: a KASP molecular marker for identifying soybean plant height. The KASP molecular marker is based on the SNP variation site of the Glyma.19G206100 gene between the two parents, Chinese flat-stemmed soybean and F02. The mutation location of the Glyma.19G206100 gene is the 312th base on the CDS sequence, where a C to G mutation occurs in the reference genome, resulting in the 104th amino acid on the protein sequence changing from asparagine to lysine. The primers for the KASP molecular marker include forward primers with nucleotide sequences as shown in SEQ ID NO. 1-2 and reverse primers as shown in SEQ ID NO. 3.

[0006] The F02 is a line with a unique plant type selected from the RIL population by constructing a recombinant inbred line population using Chinese flat-stem soybean and American flat-stem soybean as parents through the SSD method. Its plant height is less than 21cm and its plant type is compact.

[0007] Another objective of this invention is to provide an application of the KASP molecular marker for soybean plant height identification.

[0008] Based on the polymorphic site of Gm19G206100 in germplasm resources, this invention develops a KASP molecular marker. This marker has the characteristics of high resolution and easy detection. It can be used for plant height selection in the early generation of soybean hybrid breeding, which can reduce the scale of breeding, reduce workload, reduce costs, and improve breeding efficiency. It has important application value in production. Attached Figure Description

[0009] Figure 1 For the fine mapping of soybean plant height trait provided in Example 1 of this invention, a) the plant height gene was located on chromosome 19 using the BSA method; b) the candidate gene was further located between two markers, ka1942 and ka2144, using the BC1F2 population, excluding the possibility that the candidate gene is the DT1 gene; c) the candidate gene was further located between two markers, ka2057 and ka2061, using the BC1F2:3 population. The names of the parents and recombinants are listed on the left, and the phenotypic data are listed on the right. Yellow and green represent the genotypes of Chinese flat-stemmed plants and F02, respectively, and pink arrows represent the position and arrangement of candidate genes.

[0010] Figure 2 This is the genotyping detection result provided in Example 2 of the present invention;

[0011] Figure 3 The genotyping results provided in Example 3 of this invention. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0013] The Chinese flat-stemmed and American flat-stemmed soybeans used in this invention embodiment were donated by Tian Peizhan of the Jilin Provincial Breeding Center. They are biological materials that have been disclosed in the prior art. For details, please refer to: Research on improving soybean yield by changing the biological characteristics of common soybeans in Soybean Science, Vol. 17, No. 2; Jilin Agricultural Sciences, 2002, 27(5): 30-34; Design and breeding of super high-yielding soybean varieties; Jilin Agricultural Sciences, 2012, 37(2): 10-13; Research on improving yield by changing the biological characteristics of common soybeans; Northeast Agricultural Sciences, 2019, 44(3): 1-4; Effects of exogenous gibberellin on the growth and development of soybean dwarf line F03.

[0014] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0015] Example 1: Development of KASP molecular markers, specifically including the following process:

[0016] (1) Group building:

[0017] Using Chinese flat-stemmed soybean and American flat-stemmed soybean as parents, a recombinant inbred line (RIL) population was constructed using the SSD method. A line with a unique plant type was found in the RIL population, with a plant height of only 20.71 cm and a compact plant type, which will be named F02. Using F02 as the female parent and Chinese flat-stemmed soybean as the recurrent parent, backcrossing was performed to construct the BC1F2 population. BC1F2 individual plants were self-pollinated to derive the BC1F2:3 population. The frequency distribution of plant height trait in the tested BC1F2 population showed a bimodal distribution, and chi-square test confirmed that the plant height trait of this population conformed to a 3:1 segregation ratio.

[0018] (2) Gene localization (e.g.) Figure 1 (as shown)

[0019] From the BC1F2 population, 30 individual plants with a height greater than 110 cm were randomly selected, and their young leaves were harvested. DNA was extracted from these young leaves and mixed in equal amounts to construct a tall-stemmed DNA pool. Similarly, 30 individual plants with a height less than 40 cm were randomly selected, and their young leaves were harvested. DNA was extracted from these young leaves and mixed in equal amounts to construct a short-stemmed DNA pool. Young leaves from 5 individual plants were randomly selected from both the parental F02 and Chinese flat-stemmed plants to generate the parental pool. Genome resequencing and BSA analysis were performed on the samples from the four DNA pools. The soybean plant height gene was initially located within a 4.55 Mb region on chromosome 19. Further screening using 10 pairs of available polymorphic KASP markers narrowed the marker region to 1.79 Mb. Mb was used to exclude the soybean pod-setting habit gene Dt1; 10 pairs of available KASP markers were further developed, and the BC1F2:3 population was screened to narrow down the target region to ~53.7kb; through variation analysis of the localization interval bases, gene function annotation, gene expression analysis, and haplotype analysis, Glyma.19G206100 was further identified as a candidate gene, the sequence of which is shown in SEQ ID NO.4. Primers were developed based on the polymorphic sites of the two parents in the localization population on the Glyma.19G206100 gene.

[0020] (3) Molecular marker development:

[0021] Based on the SNP variation sites of the candidate gene Glyma.19G206100 between the two parents, KASP molecular markers were developed. The polymorphism of the markers between the parents was examined, and the linkage relationship between the markers and traits was analyzed. Three primers were used for the molecular markers, named qh2061.F1, qh2061.F2, and qh2061.R (see Table 1 for details). Each primer had a target fragment of 124 bp, used to distinguish between the C and G genotypes of the Glyma.19G206100 gene (CC for tall lines and flat-stemmed varieties; GG for dwarf lines and F02 varieties). The 3' ends of primers qh2061.F1 and qh2061.F2 contained the aforementioned non-synonymous mutation sites. Primer qh2061.F1 carried a FAM fluorescent tag, primer qh2061.F2 carried a HEX fluorescent tag, and qh2061.R was a universal downstream primer.

[0022] Table 1

[0023]

[0024] Example 2: The application of a KASP molecular marker for soybean plant height identification, specifically including the following steps:

[0025] Step 1: DNA extraction and purification of soybean material to be identified:

[0026] Leaves of the material to be identified were removed and placed into numbered 2 mL centrifuge tubes. One steel ball was added to each tube, and the tubes were placed in liquid nitrogen and homogenized using a tissue homogenizer for 30 seconds. 800 μL of CTAB extraction buffer (preheated to 65 °C, 2% CTAB, with 2% β-mercaptoethanol added before use) was added, and the mixture was thoroughly mixed. The tubes were then incubated in a 65 °C water bath for 45 min, inverting the tubes every 10 min. After the water bath, the tubes were removed, and 800 μL of phenol:chloroform:isoamyl alcohol (volume ratio 25:24:1) was added. The mixture was thoroughly mixed, and the tubes were centrifuged at 12000 rpm for 10 min at 4 °C. After centrifugation, the supernatant was transferred to a new 2 mL centrifuge tube, and 600 μL of chloroform was added. The mixture was inverted and mixed, and the tubes were centrifuged at 12000 rpm for 10 min at 4 °C. After centrifugation, the supernatant was transferred to a new centrifuge tube, and 500 μL of chloroform was added. Pre-chilled isopropanol was mixed and placed in a -20°C freezer for 2 h to precipitate DNA. Then, the precipitate was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was discarded. The precipitate was washed 2-3 times with 75% ethanol solution. The DNA precipitate was air-dried at room temperature and then dissolved in an appropriate amount of ddH2O. Finally, the DNA quality was tested with 1% agarose and the concentration of extracted DNA was determined with NanodropOne. The precipitate was diluted to a concentration of 100 ng / μL and stored at -20°C for later use.

[0027] Step S2: Synthesize primers qh2061.F1, qh2061.F2, and qh2061.R;

[0028] Step S3: Sequence Amplification: Using the DNA of soybean parental materials Chinese Flat-stem and F02 as templates, PCR amplification was performed according to the following PCR reaction system: PCR reaction system 10 µL: 2 µL of 50 ng / μL DNA, 5 µL of 2×Master Mix for AS-PCRV1, 0.2 µL of primers qh2061.F1, 0.2 µL of primers qh2061.F2, 0.6 µL of primers qh2061.R, and 2 µL of ddH2O; PCR reaction program: pre-denaturation 94℃, 10 min; 10 cycles: denaturation 95℃, 20 s, annealing / extension 61℃-55℃ (-0.6℃ / cycle), 40 s; 30 cycles: denaturation 95℃, 20 s, annealing / extension 55℃, 40 s;

[0029] Step S4: KASP-labeled fluorescence scanning typing:

[0030] The KASP reaction products were identified using a fluorescence scanner, and the fluorescence signal measurement results were clustered using Matrix Master software to assist in identifying the genotypes of the plant height segregating population. Homozygous tall plants (Chinese flat-stemmed plants) produced FAM fluorescence signals, with a genotype of CC; homozygous dwarf plants (F02) produced HEX fluorescence signals, with a genotype of GG; and heterozygous plants produced both fluorescence signals, with a genotype of CG. Water was used as a negative control, and no fluorescence signal was detected. Genotypes were determined based on the different fluorescence signal types. The genotyping results are shown in the figure below. Figure 2 As shown.

[0031] Example 3: Genotyping of plant height gene in hybrid offspring population:

[0032] Two soybean lines, Chinese flat-stemmed soybean and American flat-stemmed soybean, were used as parents for hybridization. After multiple generations of self-pollination, a genetically stable line F02 was obtained. Using F02 as the female parent and Chinese flat-stemmed soybean as the donor parent, backcrossing was performed. BC1F1 generation single plants were self-pollinated to construct BC1F2, resulting in a BC1F2:3 segregating population. In the BC1F2:3 population, 337 single plants and parental plants were randomly selected. Using their genomic DNA as templates, the amplification system and procedure were as described in step 3 of Example 2. Homozygous tall plants produced FAM fluorescence signals, with a genotype of CC; homozygous dwarf plants produced HEX fluorescence signals, with a genotype of GG; and heterozygous (tall) plants produced both fluorescence signals, with a genotype of CG. Water was used as a negative control, and no fluorescence signals were detected.

[0033] Based on different fluorescence signal types, the plant height distribution of isolated individual plants was determined, and the genotyping detection results are as follows: Figure 3 As shown; genotyping results revealed that three genotypes were observed in the segregating population of the test population: genotype CC had (164) individual plants, genotype CG had (61) individual plants, and genotype GG had (112) individual plants. Plant height was measured at maturity, i.e., the height from the cotyledon node to the stem apex. Specific data and corresponding genotypes are shown in Tables 2-4. The plant height of the test materials was significantly correlated with genotype, as shown in Table 5.

[0034] Table 2. Genotypic and phenotypic statistics of homozygous tall plants in the segregating population.

[0035]

[0036]

[0037]

[0038] Table 3. Phenotypic statistics of heterozygous genotype lines in the segregating population

[0039]

[0040] Table 4. Genotypic and phenotypic statistics of homozygous dwarf lines in the segregating population

[0041]

[0042]

[0043]

[0044] Table 5. Association analysis between soybean Glyma.19G206100 C>G locus genotype and soybean plant height trait.

[0045]

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A KASP molecular marker for soybean plant height identification, characterized in that, The KASP molecular marker is based on the SNP variation site of the Glyma.19G206100 gene between the Chinese flat stem soybean and F02 parents, the mutation position of the Glyma.19G206100 gene is the 312th base on the CDS sequence, which is mutated from C to G in the reference genome, resulting in the 104th amino acid on the protein sequence being mutated from asparagine to lysine, the primers of the KASP molecular marker include the forward primer with the nucleotide sequence as shown in SEQ ID NO. 1-2 and the reverse primer with the nucleotide sequence as shown in SEQ ID NO. 3; The F02 is a specific strain type screened from a RIL population by constructing a recombinant inbred line population by the SSD method with Chinese flat stem soybean and American flat stem soybean as parents, and the plant height of the specific strain type is lower than 21 cm, and the plant type is compact.

2. Application of the KASP molecular marker for identifying soybean plant height in soybean plant height identification according to claim 1.

3. Use according to claim 2, characterized in that, Comprising the following steps: Step 1, DNA extraction and purification of soybean materials to be identified: placing the leaves of the soybean materials to be identified in a centrifuge tube, and extracting genomic DNA by CTAB method; Step S2, synthesizing primers qh2061.F1, qh2061.F2 and qh2061.R, and the sequences thereof are shown in SEQ ID NO. 1-3; Step S3, sequence amplification: taking the DNA of soybean parent materials Chinese flat stem soybean and F02 as a template for PCR amplification; Step S4, KASP marker fluorescence scanning typing: using a fluorescence scanner to identify the fluorescence signal of the amplification product, and using Matrix Master software to perform cluster analysis on the fluorescence signal determination results, and identifying the genotype of the soybean segregating population materials, wherein homozygous tall plants produce FAM fluorescence signal, and the genotype thereof is CC, homozygous dwarf plants produce HEX fluorescence signal, and the genotype thereof is GG, and heterozygous plants produce two fluorescence signals, and the genotype thereof is CG, and water is used as a template for negative control, and the fluorescence signal cannot be detected.

4. Use according to claim 3, characterized in that, The specific process of step 1 is: The leaves are taken out and placed in a centrifuge tube, a steel ball is added, the centrifuge tube is placed in liquid nitrogen, and a tissue grinder is used to grind for 30 s; 800 μL of CTAB extraction solution is added, mixed thoroughly, and then placed in a 65°C water bath for 45 min, with inversion every 10 min; after the water bath, the centrifuge tube is taken out, 800 μL of phenol: chloroform: isoamyl alcohol (25:24:1) is added, mixed thoroughly, and centrifuged at 12,000 rpm for 10 min at 4°C; after centrifugation, the supernatant is transferred to a new centrifuge tube, 600 μL of chloroform is added, mixed thoroughly, and centrifuged at 12,000 rpm for 10 min at 4°C; after centrifugation, the supernatant is transferred to a new centrifuge tube, 500 μL of pre-cooled isopropanol is added, mixed thoroughly, and then placed in a -20°C refrigerator for 2 h to precipitate the DNA; then, the mixture is centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant is discarded; the precipitate is rinsed with 75% ethanol solution multiple times; the DNA precipitate is air-dried at room temperature, and then an appropriate amount of ddH2O is added to dissolve the precipitate; finally, the quality of the DNA is detected using 1% agarose, the concentration of the extracted DNA is determined using a Nanodrop One, the DNA is diluted to a concentration of 100 ng / μL, and then stored at -20°C for later use.

5. Use according to claim 3, characterized in that, In step 3, the F02 is a specific strain type with a plant height of less than 21 cm and a compact plant type, which is screened from a RIL population constructed by using Chinese flatstem soybeans and American flatstem soybeans as parents through an SSD method.

6. Use according to claim 3, characterized in that, In step 3, the reaction system for PCR amplification is 10 μL, including 50 ng / μL of DNA 2 μL, 2×Master Mix for AS-PCR V1 5 μL, primer qh2061.F1 0.2 μL, primer qh2061.F2 0.2 μL, primer qh2061.R 0.6 μL, and ddH2O 2 μL. The reaction program for PCR amplification is: pre-denaturation at 94°C for 10 min; denaturation at 95°C for 20 s, annealing / extension at 61-55°C for 40 s, 10 cycles; denaturation at 95°C for 20 s, annealing / extension at 55°C for 40 s, 30 cycles.

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