Molecular marker closely linked with relative root length of wheat and application of molecular marker
By detecting specific SNP sites in the wheat genome and using molecular markers to assist in selecting wheat varieties, the problem of difficult to identify and select relative root lengths of wheat varieties in the prior art is solved, and rapid and efficient screening of wheat varieties with strong salt tolerance is achieved, and its adaptability and yield to the salt environment is improved.
Patent Information
- Application Number
- CN202510291084.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to effectively identify and select the relative root length of wheat varieties, which affects their adaptability and yield to the salting environment.
By detecting the polymorphism or genotype of locus 617061314 of chromosome 3 in the Chinese spring wheat reference subgenome sequence, SNP molecular markers assist in the selection of wheat varieties with longer relative root lengths.
It has achieved rapid and efficient screening of wheat varieties with long relative root lengths, which has improved the adaptability and yield of wheat to the salt environment, and promoted the cultivation of new salt-tolerant wheat varieties.
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Figure CN120060544A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular biology, and in particular to a molecular marker closely linked to the relative root length of wheat and its application. Background Art
[0002] The wheat root system is a key organ for absorbing soil water and nutrients, and plays a decisive role in the growth and development of plants. Research shows that root length not only directly affects the absorption efficiency of soil water and nutrients by wheat, but is also an important indicator for identifying the salt tolerance of wheat. In a saline environment, wheat plants generally show phenotypic characteristics such as retarded growth and development, yellowing and withering of leaves, and poor root development, ultimately leading to a significant decrease in yield. Root depth directly affects the absorption capacity of wheat for deep soil water and nutrients, and thus determines the yield level and stress resistance performance. In addition, root length also determines the absorption range of soil nutrients by wheat, thereby affecting the overall growth status and final yield of the plant. Therefore, breeding wheat varieties with relatively longer root lengths is of crucial significance for increasing yield, cultivating salt-tolerant varieties, ensuring food security, and improving the utilization rate of saline-alkali land.
[0003] Molecular marker breeding is a new breeding method that utilizes the characteristic that molecular markers are closely linked to genes determining target traits. By detecting molecular markers, the presence of target genes can be detected, achieving the purpose of selecting target traits. It has the advantages of being fast, accurate, and not being interfered by environmental conditions.
[0004] Studying molecular markers closely linked to wheat root length helps improve the yield and stress resistance of wheat. By identifying and utilizing these markers, the breeding process can be accelerated, varieties with longer roots can be screened out, thereby improving the absorption capacity of water and nutrients, and enhancing the adaptability of wheat to saline-alkali soil environments. By establishing a rapid, accurate, and high-throughput identification system based on molecular markers, not only can the salt tolerance of wheat germplasm resources be efficiently evaluated, but also strong technical support is provided for wheat salt tolerance breeding. This strategy of combining molecular marker breeding with traditional breeding has opened up a new way for cultivating new high-yield and salt-tolerant wheat varieties. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a molecular marker closely linked to the relative root length of wheat and its application.
[0006] To solve the above technical problems, the following technical solutions are adopted in the present invention.
[0007] Application, wherein the application is the application of a substance for detecting the polymorphism or genotype at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat in the following A1)-A6):
[0008] A1) Identify or assist in identifying the relative root length of wheat;
[0009] A2) Prepare a product for identifying or assisting in identifying the relative root length of wheat;
[0010] A3) Screen or assist in screening wheat varieties with relatively longer root lengths;
[0011] A4) Prepare a product for screening or assisting in screening wheat varieties with relatively longer root lengths;
[0012] A5) Wheat breeding and / or assisting in breeding;
[0013] A6) Prepare a product for wheat breeding and / or assisting in breeding;
[0014] The 617061314th site on chromosome 3 of the reference A sub-genome sequence of Chinese Spring wheat is a SNP site in the wheat genome, which is the 905th nucleotide as shown in SEQ ID NO: 1, and the nucleotide type is G or C.
[0015] As a preferred technical solution of the present invention, the substance for detecting the polymorphism or genotype of the 617061314th site on chromosome 3 of the reference A sub-genome sequence of Chinese Spring wheat is a primer composition for amplifying a wheat genomic DNA fragment including the 617061314th site on chromosome 3 of the reference A sub-genome sequence of Chinese Spring wheat.
[0016] As a preferred technical solution of the present invention, the primer combination consists of the upstream primer F1 shown in SEQ ID NO: 2, the upstream primer F2 shown in SEQ ID NO: 3, and the downstream primer R shown in SEQ ID NO: 4.
[0017] The above primer composition.
[0018] A reagent containing the above primer composition.
[0019] A DNA molecule, and the nucleotide sequence of the DNA molecule is the DNA molecule shown in SEQ ID NO: 1.
[0020] Method for identifying or assisting in identifying relative root length of wheat, the method comprising using a substance for detecting the polymorphism or genotype at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat to detect the genotype at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat, and identifying or assisting in identifying the relative root length of wheat according to the genotype at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of the wheat to be tested; the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat is a SNP site in the wheat genome, which is the 905th nucleotide shown in SEQ ID NO: 1, and the nucleotide type thereof is G or C.
[0021] As a preferred technical solution of the present invention, the method for detecting the genotype at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat of the wheat to be tested comprises using the genomic DNA of the wheat to be identified as a template, performing PCR amplification with the primer composition described in claim 4 to obtain a PCR product; and determining the genotype at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat according to the sequencing result or fluorescence signal of the PCR product.
[0022] Method for wheat breeding, the method comprising selecting wheat with the genotype of CC at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat described in claim 1 as a parent for breeding, and the CC genotype represents a homozygous type in which the nucleotide type at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of the wheat genome is C.
[0023] A method for screening or assisting in screening wheat with different relative root lengths, comprising the following steps: detecting whether the genotype of the wheat to be tested is genotype TaWOX5-A-Hap I or genotype TaWOX5-A-Hap II, and the relative root length of wheat with genotype TaWOX5-A-Hap II > the relative root length of wheat with genotype TaWOX5-A-Hap I;
[0024] The wheat with genotype TaWOX5-A-Hap I is wheat with a homozygous GG genotype at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat;
[0025] The wheat with genotype TaWOX5-A-Hap II is wheat with a homozygous CC genotype at the 617061314th site of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat;
[0026] The 617061314th site on chromosome 3 in the reference A sub-genome sequence of Chinese spring wheat is the 905th nucleotide shown in SEQ ID NO: 1 in the wheat genome, and the nucleotide type is G or C.
[0027] The beneficial effects of adopting the above technical solution are as follows: The present invention provides KASP markers for identifying TaWOX5-A-Hap I and TaWOX5-A-Hap II allelic variations and their correlation with the relative root length, an important index of wheat salt tolerance. Applying the SNP molecular marker in the present invention to molecular marker-assisted selection of wheat relative root length characteristics can quickly and efficiently screen out wheat varieties (lines) with longer relative root length. Relative root length is an important index for identifying wheat salt tolerance ability, thus accelerating the breeding process of new salt-tolerant wheat varieties. The present invention has important theoretical significance and economic value for using molecular marker-assisted selection to obtain wheat germplasms or breeding offspring materials with longer relative root length and strong salt tolerance. Description of the Drawings
[0028] Figure 1 It is a schematic diagram of the detection results of genotyping of 100 wheat germplasms in a natural population; among them, the red dots indicate that the DNA sample to be tested is of the TaWOX5-A-Hap I type, that is, the GG homozygous type, and the blue dots indicate that the sample to be tested is of the TaWOX5-A-Hap II type, that is, the CC homozygous type;
[0029] Figure 2 It is a schematic diagram of the detection results of the relative root length of wheat with two different gene typings of TaWOX5-A-Hap I type and TaWOX5-A-Hap II type. Detailed Embodiments
[0030] The following examples illustrate the present invention in detail. All kinds of raw materials and equipment used in the present invention are conventional commercially available products and can be directly obtained through market purchase. In the description of the following examples, specific details such as specific system structures and technologies are put forward for the purpose of illustration rather than limitation, so as to thoroughly understand the embodiments of the present application. However, those skilled in the art should clearly understand that the present application can also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices and methods are omitted to avoid unnecessary details from interfering with the description of the present application.
[0031] It should be understood that when used in the specification and appended claims of this application, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their combinations. It should also be understood that the term "and / or" as used in the specification and appended claims of this application refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations. As used in the specification and appended claims of this application, the term "if" can be interpreted as "when", "once", "in response to determining" or "in response to detecting" according to the context. Similarly, the phrases "if determined" or "if [the described condition or event] is detected" can be interpreted as meaning "once determined", "in response to determining", "once [the described condition or event] is detected" or "in response to detecting [the described condition or event]" according to the context.
[0032] In addition, in the description of the specification and appended claims of this application, the terms "first", "second", "third", etc. are only used for distinguishing descriptions and cannot be understood as indicating or implying relative importance. The reference to "one embodiment" or "some embodiments" etc. described in the specification of this application means that a specific feature, structure or characteristic described in connection with the embodiment is included in one or more embodiments of this application. Thus, the statements "in one embodiment", "in some embodiments", "in other some embodiments", "in still other embodiments" etc. that appear in different places in this specification do not necessarily all refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "comprising", "including", "having" and their variants all mean "including but not limited to", unless otherwise specifically emphasized in other ways.
[0033] Example 1. TaWOX5-A gene
[0034] A molecular marker closely linked to the relative root length of wheat, the molecular marker is located at the 617061314th position of chromosome 3 in the reference A sub-genome sequence of Chinese Spring wheat. When the base at this site is G, the genotyping result of the wheat is TaWOX5-A-Hap I, and its relative root length is shorter, indicating its salt intolerance; when the base at this site is C, the genotyping result of the wheat is TaWOX5-A-Hap II, and its relative root length is longer, indicating its salt tolerance. The TaWOX5-A gene sequence is as shown in SEQ ID NO: 1.
[0035] Example 2. Selection of experimental materials and determination of related indicators
[0036] Using 100 wheat germplasm resources from different sources as experimental materials. Select plump and uniform seeds, place 50 seeds in a petri dish lined with moistened germination paper, add an equal amount of distilled water to each dish for cultivation to ensure germination rate and uniformity. Place the petri dishes in a 4°C refrigerator for 3 days to ensure germination rate and uniformity, then place them at room temperature for dark treatment until the seeds germinate. After germination, place the petri dishes in an artificial climate chamber for 2 days (the artificial climate chamber is set at 23°C / 16 hours of light, 18°C / 8 hours of darkness). Select seedlings with consistent growth after 2 days of germination and transfer them to a hydroponic box, and add 1 / 2 Hoagland nutrient solution at the same time. Set up a control group and a salt treatment group, and change the nutrient solution every 2 days. On the 7th day after germination, start the 250 mM NaCl treatment for the salt treatment group, and the control group continues normal cultivation. On the 7th day of salt treatment, select seedlings with consistent growth, and investigate the root length traits of the control group and the salt treatment group respectively, and compare and analyze their relative root lengths (relative root length = root length of salt treatment group / root length of control group) to evaluate the effect of salt stress on wheat growth.
[0037] Example 3, Extraction of DNA
[0038] ① Place a 2 cm wheat leaf sample and 6 mm steel beads in a 1.5 mL centrifuge tube, quickly freeze and grind them in a liquid nitrogen environment.
[0039] ② Add 600 μL of CTAB to dissolve the DNA significantly, and water bath in a 65°C water bath for 30 minutes, and gently shake and mix it every 10 minutes during this period.
[0040] ③ Add 600 μL (equal volume to CTAB) of chloroform-isoamyl alcohol solution prepared in a ratio of 24:1 in a fume hood, and gently shake for 1 minute.
[0041] ④ Centrifuge at 10000 rpm for 10 minutes at room temperature in a centrifuge.
[0042] ⑤ Pipette 450 μL of the supernatant, add 450 μL (equal volume to the supernatant) of isopropanol frozen at -20°C and gently mix. At this time, white flocculent precipitates appear.
[0043] ⑥ Let it stand for 30 minutes at 4°C, or let it stand for 20 minutes at -20°C.
[0044] ⑦ Centrifuge at 10000 rpm for 10 minutes at 4°C in a centrifuge.
[0045] ⑧ Take 1 mL of 75% ethanol to wash the precipitate twice, gently shake the precipitate up by hand each time, and centrifuge at 10000 rpm for 2 minutes at room temperature in a centrifuge.
[0046] ⑨ Discard the supernatant, use medium and small pipette tips to suck out the residual liquid, and place it in a fume hood to blow until it becomes a gummy state.
[0047] ⑩ Dissolve the DNA in 100 μL of ddH2O and store it in a -20℃ refrigerator for later use.
[0048] Example 4. KASP marker primer design and marker detection
[0049] The primer set for detecting molecular markers includes two left primers of SEQ ID NO: 1 and SEQ ID NO: 2 and a right primer of SEQ ID NO: 3; different fluorescent markers are respectively set at the 5' ends of the two front primers, and the specific sequences are as follows:
[0050] Allele-specific primer 1-FAM: 5'-FAM-GGCAATGGAGGCGCTGAGCGGGCGC-3' (SEQ ID NO: 2);
[0051] Allele-specific primer 2-HEX: 5'-HEX-GGCAATGGAGGCGCTGAGCGGGCGG-3' (SEQID NO: 3);
[0052] Common, reverse primer: 5'-CGTCGGGTTCACCGCCCGCACTTCACTC-3' (SEQ ID NO: 4).
[0053] The genomic DNA of the wheat sample to be tested extracted as above was used as a template and the designed primer set was used to perform PCR amplification.
[0054] The PCR reaction system is: 5 μL genomic DNA (50 ng / μL) of the wheat sample to be tested, 5 μL HiGeno2x ProbeMix (Beijing Jiacheng Biotechnology Co., Ltd.) and 0.14 μL SNP-Specific Primers, and add ddH2O to make up to 10 μL. The preparation method of SNP-Specific Primers is as follows: 12 μL of 100 μM Allele-specific primer 1-FAM, 12 μL of 100 μM Allele-specific primer 2-HEX left primer and 30 μL of 100 μM right primer (Common, reverse primer) are mixed, and ddH2O is added to make up to 100 μL;
[0055] The PCR amplification procedure is as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing and extension at 61 - 55°C for 40 s, for 10 cycles, with a decrease of 0.6°C per cycle; denaturation at 95°C for 20 s, annealing and extension at 55°C for 40 s, for 28 cycles. Store at 4°C. The PCR reaction was carried out on an S1000 TM Thermal Cycler PCR instrument (Bio-Rad Laboratories Inc.). After the reaction was completed, the obtained PCR amplification products were genotyped according to the fluorescence signal on a real-time quantitative instrument (QuantStudio 3 Real-Time PCR System), and the PCR amplification products were scanned. The excitation wavelength of FAM was 485 nm and the emission wavelength was 520 nm; the excitation wavelength of HEX was 535 nm and the emission wavelength was 556 nm, and the excitation wavelength of the system reference fluorescence ROX was 575 nm and the emission wavelength was 610 nm. The genotyping data was read using fluorescence genotyping software (QuantStudio Design & Analysis Software).
[0056] The PCR amplification products of the left primer Allele-specific primer 1-FAM carrying the fluorescence sequence FAM and the right primer Common,reverseprimer were analyzed by software and represented by blue dots, indicating that the DNA sample to be tested was of the TaWOX5-A-Hap II type; the relative root length of the wheat in this sample was longer.
[0057] The PCR amplification products of the left primer Allele-specific primer 2-HEX carrying the fluorescence sequence HEX and the right primer Common,reverseprimer were analyzed by software and represented by red dots, indicating that the DNA sample to be tested was of the TaWOX5-A-Hap I type; the relative root length of the wheat in this sample was shorter.
[0058] Example 5. Results
[0059] Select seedlings with consistent growth of the above-ground parts and lines, and investigate the root length traits of the control group and the salt treatment group respectively. Compare and analyze their relative root lengths (relative root length = root length of the salt treatment group / root length of the control group), and genotype these 100 wheat samples. After KASP marker detection, among the 100 wheat natural population materials, 30 germplasms were of the TaWOX5-A-Hap I allele type and 70 germplasms were of the TaWOX5-A-Hap II allele type. The obtained relative root length data and genotyping results are shown in Table 1.
[0060] Table 1 Relative root length data and genotyping results of 100 wheat natural population materials
[0061]
[0062]
[0063] Note: HapⅠ represents TaWOX5-A-HapI type, and HapⅡ represents TaWOX5-A-HapII type.
[0064] The fluorescence genotyping software (QuantStudio Design&Analysis Software) reads the genotyping results of some wheat samples in Table 1 as Figure 1 shown. Among them, the red dots indicate that the DNA sample to be tested is of TaWOX5-A-Hap I type, and the blue dots indicate that the sample to be tested is of TaWOX5-A-Hap II type.
[0065] Table 2 Results of the correlation analysis between the genotyping of the natural population and the relative root length
[0066]
[0067] The detection results of the relative root lengths of wheat with two different genotypes, TaWOX5-A-Hap I type and TaWOX5-A-Hap II type, are as Figure 2 shown. The statistical results show that the mean relative root length of the wheat sample materials carrying the allele TaWOX5-A-Hap II is higher than that of the wheat sample materials carrying the allele TaWOX5-A-Hap I, and the difference between the two is extremely significant (P<0.01). The TaWOX5-A-Hap II haplotype has a larger relative root length than the TaWOX5-A-Hap I haplotype. A longer relative root length indicates stronger salt tolerance.
[0068] In the above embodiments, the descriptions of the respective embodiments have their own focuses. For parts not detailed or recorded in a certain embodiment, reference may be made to the relevant descriptions of other embodiments.
[0069] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the same; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.
Claims
1. Application, characterized in that, The application is the application of the material for detecting the polymorphism or genotype of the 617061314th site of chromosome 3 in the reference A subgenome sequence of Chinese spring wheat in the following A1)-A6): A1) Identify or assist in identifying the relative root length of wheat; A2) preparing products for identifying or assisting in identifying the relative root length of wheat; A3) Screening or assisting in the selection of wheat varieties with relatively long root length; A4) preparing products for screening or assisting in screening wheat varieties with relatively long root length; A5) Wheat breeding and / or assisted breeding; A6) preparing wheat breeding and / or breeding-assisted products; The 617061314th position of chromosome 3 in the Chinese spring wheat reference A subgenome sequence is a SNP site in the wheat genome, such as the 905th nucleotide shown in SEQ ID NO: 1, and its nucleotide type is G or C.
2. The use according to claim 1, characterized in that: The material for detecting the polymorphism or genotype of the 617061314th site of chromosome 3 in the Chinese spring wheat reference A subgenome sequence is a primer composition for amplifying a wheat genomic DNA fragment including the 617061314th site of chromosome 3 in the Chinese spring wheat reference A subgenome sequence.
3. The use according to claim 2, characterized in that: The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 2, an upstream primer F2 shown in SEQ ID NO: 3, and a downstream primer R shown in SEQ ID NO:
4.
4. The primer composition according to any one of claims 2 to 3.
5. A reagent comprising the primer composition according to claim 4.
6. A DNA molecule, wherein the nucleotide sequence of the DNA molecule is a DNA molecule shown in SEQ ID NO:
1.
7. A method for identifying or assisting in identifying the relative root length of wheat, characterized in that: The method comprises the following steps: using a material for detecting the polymorphism or genotype of the 617061314th site of chromosome 3 in the Chinese spring wheat reference A subgenome sequence to detect the genotype of the 617061314th site of chromosome 3 in the Chinese spring wheat reference A subgenome sequence, and identifying or assisting in identifying the relative root length of wheat according to the genotype of the 617061314th site of chromosome 3 in the Chinese spring wheat reference A subgenome sequence of the wheat to be tested; the 617061314th site of chromosome 3 in the Chinese spring wheat reference A subgenome sequence is a SNP site in the wheat genome, is the 905th nucleotide shown in SEQ ID NO: 1, and the nucleotide type thereof is G or C.
8. The method according to claim 7, characterized in that The method for detecting the genotype of the 617061314th site of chromosome 3 in the Chinese spring wheat reference A subgenomic sequence of claim 1 of the wheat to be tested comprises using the genomic DNA of the wheat to be identified as a template and performing PCR amplification using the primer combination described in claim 4 to obtain a PCR product; The genotype of the 617061314th site of chromosome 3 in the Chinese spring wheat reference A subgenome sequence is determined according to the sequencing result of the PCR product or the fluorescent signal.
9. A method for wheat breeding, characterized in that: The method comprises selecting wheat having a genotype of CC at position 617061314 of chromosome 3 in the Chinese spring wheat reference A subgenome sequence in claim 1 as a parent for breeding, wherein the CC genotype indicates that the nucleotide type at position 617061314 of chromosome 3 in the Chinese spring wheat reference A subgenome sequence in the wheat genome is a homozygous type of C.
10. A method for screening or assisting in screening wheat with different relative root lengths, characterized in that: The method comprises the following steps: detecting whether the genotype of the wheat to be tested is the genotype TaWOX5-A-Hap I or the genotype TaWOX5-A-Hap II, and the relative root length of the wheat of the genotype TaWOX5-A-Hap II is greater than the relative root length of the wheat of the genotype TaWOX5-A-Hap I; The wheat of the genotype TaWOX5-A-Hap I is a wheat of homozygous GG genotype at the 617061314th site of chromosome 3 based on the reference A subgenome sequence of Chinese spring wheat; The wheat of the genotype TaWOX5-A-Hap II is a wheat of CC homozygous genotype at position 617061314 of chromosome 3 based on the reference A subgenome sequence of Chinese spring wheat; The 617061314th position of chromosome 3 in the Chinese spring wheat reference A subgenome sequence is the 905th nucleotide shown in SEQ ID NO: 1 in the wheat genome, and the nucleotide type is G or C.