A wheat root length taMYB85-b gene haplotype SNP molecular marker and application thereof

Abstract

The application discloses a SNP molecular marker for identifying a TaMYB85-B gene haplotype of wheat root length, namely SNP-M, a variation site of which is the 612th site of the TaMYB85-B gene, and the base of the site is C or A. Based on the haplotype SNP molecular marker, the application further develops a primer for detecting the haplotype SNP molecular marker, and prepares a kit containing the primer, and through PCR and sequencing analysis, the SNP and the genotype can be accurately identified, so that the excellent haplotype (long root length) and the non-excellent haplotype (short root length) of the wheat root length can be distinguished.

Description

Technical Field

[0001] This invention belongs to the field of wheat breeding technology and relates to a haplotype SNP molecular marker of the TaMYB85-B gene for identifying wheat root length and its application. Background Technology

[0002] As a major global food crop, wheat yield stability is crucial for national food security. However, water scarcity and frequent abiotic stresses severely restrict wheat production. Research indicates that root architecture (especially root length) is a key trait determining the efficiency of water and nutrient absorption in wheat. Improving root depth and distribution is considered an effective strategy to enhance wheat drought resistance and yield.

[0003] The wheat MYB transcription factor family plays a central role in growth and development, stress response, and metabolic regulation through a complex regulatory network. Previous studies have shown that the wheat MYB transcription factor family plays a key regulatory role in root development. In root architecture optimization, the TaMYB30-B gene can regulate the expression of related genes by modulating osmotic stress, maintaining root cell turgor pressure under drought stress, reducing root hair shedding, and promoting root hair elongation. Overexpression of TaMYB61 can enhance root tolerance to drought and salt stress, maintain the integrity of root xylem vessels, prevent stress-induced excessive root lignification, and ensure water transport efficiency (all of these functions are supported by corresponding gene knockout and overexpression experimental data, with related findings published in journals such as *CropScience*). Overall, published functional studies of wheat MYB genes confirm that this family of genes regulates root development in multiple dimensions, from root elongation and branching to stress adaptation, through hormonal regulation and stress response pathways, providing key gene resources for optimizing wheat root architecture, improving nutrient absorption, and enhancing stress resistance.

[0004] Studies have shown that some genes, such as TaSRL1 and the TaCEP15-TaCEPRL module, regulate root development through auxin signaling pathways or peptide hormones. Among them, the MYB transcription factor family (such as TapIMP1 and TaMYBSM151) has been confirmed to be involved in drought response and root morphogenesis, exhibiting extensive polymorphism and functional differentiation in the wheat genome. However, the specific mechanism and haplotype effect of the TaMYB85-B gene in root length regulation remain unclear. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a TaMYB85-B gene haplotype SNP molecular marker for identifying wheat root length and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. A haplotype SNP molecular marker for identifying wheat root length, namely SNP-M, with the variant site being position 612 of the TaMYB85-B gene, and the base at this site being C or A; wherein the nucleotide sequence of the TaMYB85-B gene is shown in SEQ ID NO.1.

[0008] As one of the preferred technical solutions, the genotype of the SNP-M locus is C / C, corresponding to the Hap1 haplotype, which has a shorter wheat root length; the genotype of the SNP-M locus is A / A, corresponding to the Hap2 haplotype, which has a longer wheat root length.

[0009] 2. Primers used to detect the aforementioned haplotype SNP molecular markers, comprising:

[0010] MYB85-stop-F: 5'-AACAGGGCTGAAAAGATGCG-3', as shown in SEQ ID NO.2; MYB85-stop-R: 5'-AAGGAGTTGTTGTGGTTGCC-3', as shown in SEQ ID NO.3.

[0011] As one of the preferred technical solutions, PCR amplification is performed using the primers. If the SNP-M site is C, it corresponds to the Hap1 haplotype; if the SNP-M site is A, it corresponds to the Hap2 haplotype.

[0012] 3. Application of the aforementioned primers in the preparation of the aforementioned detection kit for haplotype SNP molecular markers.

[0013] 4. A kit for detecting the aforementioned haplotype SNP molecular markers, containing the aforementioned primers.

[0014] 5. Application of the aforementioned haplotype SNP molecular markers, primers, or kits in identifying wheat root length.

[0015] 6. Application of the aforementioned haplotype SNP molecular markers, primers, or kits in wheat breeding.

[0016] 7. A method for identifying wheat root length, comprising detecting the aforementioned haplotype SNP molecular markers of the wheat to be tested, and identifying wheat root length based on genotype.

[0017] As one of the preferred technical solutions, the genotype of the SNP-M locus is C / C, corresponding to the Hap1 haplotype, which has a shorter wheat root length; the genotype of the SNP-M locus is A / A, corresponding to the Hap2 haplotype, which has a longer wheat root length.

[0018] 8. A wheat breeding method, wherein the genotype of the aforementioned haplotype SNP molecular markers in a natural wheat population is detected, and superior haplotype wheat varieties with longer root lengths are selected for breeding.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention provides a haplotype SNP molecular marker for identifying wheat root length in the TaMYB85-B gene, namely SNP-M, with the variant site at position 612 of the TaMYB85-B gene, where the base is C or A. Based on this haplotype SNP molecular marker, this invention also develops primers for detecting this haplotype SNP molecular marker and prepares a kit containing these primers. Through PCR and sequencing analysis, the SNP and genotype can be accurately identified, thereby distinguishing between superior haplotypes (long root length) and non-superior haplotypes (short root length) in wheat.

[0021] The results of testing on existing wheat varieties show that the superior haplotypes detected using this molecular marker have significantly longer root lengths than the non-superior haplotypes. Therefore, this molecular marker can be used to rapidly screen wheat lines with longer root lengths from different populations in the early stages of wheat breeding, improving breeding efficiency and accelerating the process of high-yield wheat breeding. Attached Figure Description

[0022] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0023] Figure 1 A schematic diagram of the SNP molecular markers for the TaMYB85-B gene haplotype.

[0024] Figure 2 Comparison of sequence differences among different haplotypes of the TaMYB85-B gene.

[0025] Figure 3 Comparison of root lengths among different haplotypes of the TaMYB85-B gene in a natural population. Detailed Implementation

[0026] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0027] Example 1: Detection of Superior Haplotypes of the TaMYB85-B Gene

[0028] The differences between the superior haplotype (Hap2) and the non-superior haplotype (Hap1) of the TaMYB85-B gene in this invention are as follows: Figure 1 As shown, this differential haplotype includes one SNP site, named SNP-M, located at position 466076174 on chromosome 1B of the wheat Chinese Spring reference genome (IWGSC v1.1). This nucleotide is either C or A, corresponding to position 612 of SEQ ID NO.1. In SEQ ID NO.1, m represents C or A.

[0029] Two primers were designed based on SEQ ID NO.1 to detect different genotypes at the SNP-M site: MYB85-stop-F: 5'-AACAGGGCTGAAAAGATGCG-3', as shown in SEQ ID NO.2; MYB85-stop-R: 5'-AAGGAGTTGTTGTGGTTGCC-3', as shown in SEQ ID NO.3. The specific procedure for detecting the superior haplotype of the TaMYB85-B gene using this primer set is as follows:

[0030] 1.1 Extraction of genomic DNA

[0031] Genomic DNA was extracted from wheat leaves using the CTAB method;

[0032] 1.2 PCR amplification and detection

[0033] Using genomic DNA as a template, PCR amplification was performed using MYB85-stop-F / MYB85-stop-R primers.

[0034] High-fidelity PCR amplification system: The high-fidelity Taq enzyme used was Tks Gflex™ DNA Polymerase (catalog number: R060A) from Takara. The amplification system is shown in Table 1.

[0035] Table 1

[0036] Components Dosage 2X GC Buffer 5 μL Tks Gflex DNA Polymerase 0.2 μL Primer (2 μM) 2 μL DNA 100 ng <![CDATA[ddH2O]]> Add to 10 μL

[0037] The specific reaction procedure was as follows: 94°C pre-denaturation for 5 min; 98°C for 10 s, 58°C for 15 s, 68°C for 1 min, 35 cycles; extension at 68°C for 7 min to obtain the PCR product. Agarose gel electrophoresis confirmed that approximately 500 bp of PCR amplification product was obtained. This PCR product was sent to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The sequencing results were observed using SnapGene software (e.g., ...). Figure 2 As shown in the figure, by comparing the sequence differences of different haplotypes of the TaMYB85-B gene, it was found that the nucleotide at the SNP-M site of Hap1 is C, while the nucleotide at the SNP-M site of Hap2 is A.

[0038] Example 2: Screening of natural wheat populations using the superior TaMYB85-B allele.

[0039] The primers (MYB85-stop-F / ) obtained in Example 1 for detecting superior alleles (SNP-M locus genotype) were used.

[0040] The MYB85-stop-R assay was used to detect a natural population containing 410 bred varieties (preserved in this laboratory and available to the public upon application; used solely for replication purposes; reference: Wang X, Guan P, Xin M, Wang Y, Chen X, Zhao A, Liu M, Li H, Zhang M, Lu L, Zhang J, Ni Z, Yao Y, Hu Z, Peng H, Sun Q. Genome-wide association study identifies QTL for thousand grain weight in winter wheat under normal- and late-sown stressed environments. Theor ApplGenet. 2021 Jan;134(1):143-157. doi: 10.1007 / s00122-020-03687-w. Epub 2020Oct 8. PMID: 33030571.). The results of the detection of some wheat lines and their haplotypes are shown in Table 2.

[0041] Table 2

[0042] Serial Number Material Name haplotype 1 German Selection No. 1 Hap1 2 Huanqun No. 4 Hap1 3 Jimai 19 Hap1 4 Jimai 20 Hap1 5 Jimai 21 Hap1 6 Taimai No. 1 Hap1 7 Thai Wheat 18 Hap1 8 Jinan 16 Hap1 9 Jining No. 3 Hap1 10 Jining No. 16 Hap1 11 Lumai No. 7 Hap1 12 Lumai 13 Hap1 13 Shan Nong 23 Hap1 14 Shannong Radiation 63 Hap1 15 Early wheat Hap1 16 Huapei No. 3 Hap1 17 Huayu 116 Hap1 18 Huayu 198 Hap1 19 Jimai 06039 Hap1 20 Ji 729 Hap1 21 Northwest A&F University 979 Hap2 22 68g of fresh wheat Hap2 23 Henan Education No. 6 Hap2 24 Henan Agricultural University 201 Hap2 25 Zhengmai 583 Hap2 26 Shaanxi Agricultural 66 Hap2 27 Copper Wheat 3 Hap2 28 Northwest A&F University 928 Hap2 29 Zhengpinmai No. 8 Hap2 30 Zhongyu 7410 Hap2 31 Zhoumai 27 Hap2 32 Luo PH02 Hap2 33 Henan Agricultural University 416 Hap2 34 Luohan 13 Hap2 35 Red Old Wheat Hap2 36 Xia Zhuang 30 Hap2

[0043] Twenty uniform and plump seeds were selected from each wheat variety in this population. Each seed was disinfected by soaking in 5 ml of 1% hydrogen peroxide for 10 minutes, then rinsed 3-4 times with distilled water. The seeds were then neatly arranged in petri dishes (lined with two layers of filter paper and a small amount of distilled water) with the ventral groove facing down. After being placed at 4℃ in the dark for 2 days, they were cultured at room temperature for 2 days. Seedlings of uniform growth were transplanted into hydroponic culture boxes, with 16-20 seedlings transplanted into each box. The seedlings were cultured for 7 days under normal conditions of 22℃, 16 h light / 8 h dark, and 60%-70% humidity, and the taproot length was recorded. The root length data, combined with haplotype analysis, yielded the following results: Figure 3 As shown, the root length of wheat lines with the Hap1 haplotype (genotype C / C at the SNP-M locus, i.e., homozygous for SNP-M at the C locus in the wheat genome) in the natural population was significantly shorter than that of wheat lines with the superior Hap2 haplotype (genotype A / A at the SNP-M locus, i.e., homozygous for SNP-M at the A locus in the wheat genome) (p<0.001), and the proportion of superior haplotypes in this population was relatively small (22.2%).

[0044] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A haplotype SNP molecular marker for identifying wheat root length, namely SNP-M, with the variant site at position 612 of the TaMYB85-B gene, where the base is either C or A; wherein, The nucleotide sequence of the TaMYB85-B gene is shown in SEQ ID NO.

1.

2. The haplotype SNP molecular marker according to claim 1, characterized in that, The genotype at the SNP-M locus is C / C, corresponding to the Hap1 haplotype, which has shorter wheat roots; the genotype at the SNP-M locus is A / A, corresponding to the Hap2 haplotype, which has longer wheat roots.

3. Primers for detecting the haplotype SNP molecular marker of claim 1, characterized in that, Include: MYB85-stop-F: 5'-AACAGGGCTGAAAAGATGCG-3', as shown in SEQ ID NO.2; MYB85-stop-R: 5'-AAGGAGTTGTTGTGGTTGCC-3', as shown in SEQ ID NO.

3.

4. The use of the primers described in claim 3 in the preparation of the aforementioned detection kit for haplotype SNP molecular markers.

5. A kit for detecting the haplotype SNP molecular marker of claim 1, characterized in that, It contains the primers described in claim 3.

6. The application of the haplotype SNP molecular marker of claim 1, the primer of claim 3, or the kit of claim 5 in the identification of wheat root length.

7. The application of the haplotype SNP molecular marker of claim 1, the primer of claim 3, or the kit of claim 5 in wheat breeding.

8. A method for determining the root length of wheat, characterized in that, The haplotype SNP molecular markers of the wheat to be tested as described in claim 1 were detected, and the root length of the wheat was identified based on the genotype.

9. The method according to claim 8, characterized in that, The genotype at the SNP-M locus is C / C, corresponding to the Hap1 haplotype, which has shorter wheat roots; the genotype at the SNP-M locus is A / A, corresponding to the Hap2 haplotype, which has longer wheat roots.

10. A method for breeding wheat, characterized in that, Genotyping was performed on the aforementioned haplotype SNP molecular markers in natural wheat populations, and superior haplotype wheat varieties with longer root lengths were selected for breeding.

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