Application of ahnst1 in negative regulation of inter-node length and plant height of peanut plant

Abstract

The application discloses application of AhNST1 in negative regulation of inter-node length and plant height of peanut plants. The application successfully clones a key candidate gene AhNST1 for regulating dwarf and close node of peanut on A02 chromosome of peanut, the gene codes a NAC family transcription factor, and the NAC family transcription factor is massively expressed in stems and stalks. Heterologous transformation of rice shows that AhNST1 negatively regulates the inter-node length and the plant height, and it is indicated that AhNST1 is a key gene for negatively regulating stem and inter-node development of crops such as peanut.

Description

Technical Field

[0001] This invention relates to crop breeding, specifically to the application of AhNST1 in negatively regulating internode length and plant height in peanut plants. Background Technology

[0002] Optimizing plant architecture is a core technical approach to breaking through the bottleneck of peanut yield per unit area (Peng Zhenying et al., 2019). Peanuts have a unique reproductive habit of "flowering above ground and setting pods underground," and pod yield is highly dependent on whether the pegs can successfully penetrate the soil. Plant height is the most important plant architecture parameter for peanuts. Excessive height can easily lead to lodging, difficulty in peg penetration, and a decrease in pod setting rate, while excessive height may restrict the peg attachment node and reduce the number of pods, both of which are detrimental to yield formation. Appropriately reducing peanut plant height can effectively optimize the plant's spatial structure, shorten the physical distance and time for the pegs to penetrate the soil, thereby improving the uniformity of peg penetration and pod setting efficiency. At the same time, the dense internodes at the base of the stem can promote a highly concentrated pod setting period, effectively improving the uniformity of pod size and maturity, reducing ineffective pod setting and nutrient waste, and effectively improving peanut yield per unit area and overall yield from the perspective of plant architecture (Zhao Feifei et al., 2025). Appropriately reducing plant height and shortening internode distance are key objectives for constructing an ideal peanut plant architecture (Li Guowei et al., 2020). Therefore, in-depth exploration of key genes regulating peanut plant height and analysis of their molecular mechanisms can not only reveal the genetic laws governing the synergistic development of peanut's unique underground pod-setting habit and aboveground plant architecture, but also provide theoretical support for breeding high-yield and stable-yield new peanut varieties.

[0003] With the continuous improvement of genomic data for wild and cultivated peanut species, preliminary progress has been made in the genetic mapping of peanut plant type. Early QTL mapping based on linkage analysis has successively reported multiple loci associated with plant height and branching. Shirasawa et al. (2012) used an F2 population to map three QTLs associated with main stem height and two QTLs associated with lateral branch length, explaining phenotypic variances of 4.80%–19.20% and 14.2%–21.1%, respectively. Fonceka et al. (2012) used 88 chromosome segment substitution lines for line × trait association analysis, identifying 15 QTLs associated with main stem height. Huang et al. (2016) mapped QTLs for 18 agronomic and quality-related traits, identifying three QTLs associated with main stem height and two QTLs associated with branching number. Furthermore, Li et al. (2019) used a recombinant inbred line population to map 11 QTLs associated with main stem height and 16 QTLs associated with primary lateral branch length, among which… qmsh-14-3 and qllb-11-1Stable expression explained 22.53% and 15.05% of phenotypic variation, respectively. Zhang et al. (2025) located three stable QTLs associated with main stem height, primary lateral branch length, and number of branches, located on chromosomes 9, 10, and 16, respectively. The candidate genes are involved in pathways such as hormone synthesis, signal transduction, and cell wall development.

[0004] Existing technologies have identified key genes in peanut plant architecture by resequencing the genomes of 390 natural peanut populations and using genome-wide association analysis. AhBSK1 Furthermore, it was confirmed that brassinolide regulates plant height and lateral branch number through the brassinolide pathway (Lu et al., 2024). The team of Academician Zhang Xinyou at the Henan Academy of Agricultural Sciences, using genome-wide association analysis and linkage analysis, identified a key gene controlling peanut plant architecture on chromosome 15. This gene encodes the MADS-box transcription factor family and exhibits three mutation types in natural populations (2 bp insertion, 1870 bp deletion, and MITE insertion), all linked to erect plant architecture (Zheng et al., 2024). These advances provide new clues for identifying key genes related to peanut plant architecture. However, most reported plant architecture-related QTLs suffer from large localization intervals and lack of functional verification of candidate genes, leaving the core regulatory network for peanut plant architecture unclear.

[0005] References:

[0006] 1. Li Guowei, Qin Shenghao, Liu Yiyang, Zhang Jialei, Han Yan, Wan Shubo (2020) Research progress on peanut plant type-related traits. Chinese Journal of Oil Crops , 42, 934-939. 2. Peng Zhenying, Shan Lei, Zhang Zhimeng, Li Xinguo, Wan Shubo (2019) Peanut plant type and high yield. Peanut Studies Report , 48, 69-72. 3. Zhao Feifei, Li Shaoxiong, Liu Hao, Li Haifen, Wang Runfeng, Huang Lu, Yu Qianxia, ​​Hong Yanbin, Chen Xiaoping, Lu Qing, Cao Yuman (2025) Association mapping and candidate gene analysis of internode length of peanut main stem and lateral branch. Crop Science Report , 51, 548-556. 4.Fonceka, D., Tossim, H.A., Rivallan, R., Vignes, H., Lacut, E., deBellis, F., Faye, I., Ndoye, O., Leal-Bertioli, S.C., Valls, J.F., Bertioli,D.J., Glaszmann, J.C., Courtois, B., Rami, J.F. (2012) Construction ofchromosome segment substitution lines in peanut ( Arachis hypogaea L.) using awild synthetic and QTL mapping for plant morphology. PLoS One , 7, e48642. 5.Huang, L., Ren, X., Wu, B., Li, X., Chen, W., Zhou, X., Chen, Y.,Pandey, M.K., Jiao, Y., Luo, H., Lei, Y., Varshney, R.K., Liao, B., Jiang, H.(2016) Development and deployment of a high-density linkage map identifiedquantitative trait loci for plant height in peanut ( Arachis hypogaea L.). Scitific Reports , 6, 39478. 6.Lu, Q., Huang, L., Liu, H., Garg, V., Gangurde, SS., Li, H.,Chitikineni, A., Guo, D., Pandey, MK, Li, S., Liu, H., Wang, R., Deng, Q.,Du, P., Varshney, RK, Liang, X., Hong, Y., Chen, X. (2003). (2024) A genomicvariation map provides insights into peanut diversity in China andassociations with 28 agronomic traits. Nature Genetics , 56 , 530–5 7.Shirasawa1, K., Koilkonda, P., Aoki, K., Hirakawa, H., Tabata, S.,Watanabe, M., Hasegawa, M., Kiyoshima, H., Suzuki, S., Kuwata, C., Naito, Y.,Kuboyama, T., Nakaya, A., Sasamoto, S., Watanabe, A., Kato, M., Kawashima,K., Kishida, Y., Kohara, M., Kurabayashi, A., Takahashi, C., Tsuruoka, H.,Wada, T., Isobe, S. (2012). BMC Plant Biology , 12 , 8. Zhang, S., Hu, X., Wang, F., Miao, H., Ye, C., Yang, W., Zhong, W., Chen, J. (2025) Identification of QTLs for plant height and branching-related traits in cultivated peanut. Journal of Integrative Agriculture , 24, 2511-2524. 9.Zheng, Z., Sun, Z., Qi, F., Fang, Y., Lin, K., Pavan, S., Huang, B., Dong, W., Du, P., Tian, ​​M., Shi, L., Xu, J., Han, S., Liu, H., Qin, L., Zhang, Z., Dai, X., Miao, L., Zhao, R., Wang, J., Liao, Y., Li, A., Ruan, J.,Delvento, C., Aiese Cigliano, R., Maliepaard, C., Bai, Y., Visser, RGF,Zhang, X. (2024) Chloroplast and whole-genome sequencing shed light on theevolutionary history and phenotypic diversification of peanuts. Nature Genetics , 56, 1975-1984. Summary of the Invention This invention focuses on internode length, constructing segregating populations using the dwarf peanut material ICG4601 and the tall peanut material Fesr-2. Through polarimetric pooled sequencing (BSA-Seq) combined with fine mapping of a large-scale F3 population, the key candidate gene AhNST1 (NAC Secondary WallThickening Promoting Factor 1), which regulates peanut dwarfism and dense internodes, was successfully cloned on chromosome A02. This gene encodes an NAC family transcription factor that is highly expressed in the stem. Heterologous transformation in rice showed that AhNST1 negatively regulates plant height and internode length, indicating that AhNST1 is a key gene negatively regulating peanut stem and internode development.

[0007] Based on this, the present invention provides an application of AhNST1 to negatively regulate the internode length and plant height of crop plants, wherein the crop is peanut or rice.

[0008] This invention also provides the application of the AhNST1 gene and its primers in the identification and selection of peanut plant internode length and plant height traits. The AhNST1 gene sequence is shown in SIQID NO.1. The nucleotide sequence shown in SIQID NO.1 has an A / C base mutation at position 108944345 on peanut chromosome A02. This mutation site is significantly correlated with peanut plant internode length and plant height.

[0009] Optionally, the AhNST1 gene primers are: AhNST1-F: 5'-ATGCCAGAAAGCATGAGTAT-3'; AhNST1-R: 5'-CACTGACGTGTTTGACACGT-3'.

[0010] This invention also provides an application of the AhNST1 gene primer preparation kit for identifying and selecting peanut plant internode length and plant height traits. The AhNST1 gene sequence is shown in SIQID NO. 1. The nucleotide sequence shown in SIQID NO. 1 has an A / C base mutation at position 108944345 on peanut chromosome A02. This mutation site is significantly correlated with peanut plant internode length and plant height.

[0011] This invention further provides a peanut seed selection method, which includes the following steps: 1) Extract genomic DNA from the peanuts to be selected, detect and sequence to determine the genotype of the AhNST1 gene in the peanuts to be selected; the sequence of the AhNST1 gene is shown in SIQID NO.1; 2) Select plants with the AA genotype at position A / C of AhNST1 gene genotype 108944345 for seed production. Attached Figure Description

[0012] Figure 1 Differences between dwarf peanut ICG4601 and tall peanut Fesr-2 plants; (A) Photographs of dwarf peanut ICG4601 and tall peanut Fesr-2 plants; (B) Comparison of plant height between dwarf peanut ICG4601 and tall peanut Fesr-2.

[0013] Figure 2A The distribution of the ΔSNP index on chromosomes; Figure 2B Fine localization of candidate regions on chromosome A02; Figure 2C Transcriptome sequencing results of the internodes of stems from the parental ICG4601 and Fesr-2 lines; Figure 2DThe gene structure and variation of AhNST1; Figure 2E The frequency distribution of different AhNST1 genotypes in 390 peanut germplasms; Figure 2F Comparison of plant height and internode length between AhNST1AA and AhNST1CC genotypes.

[0014] Figure 3 (A) Functional validation of AhNST1 in plant height; (B) Subcellular localization of AhNST1; (C) Plant height phenotype of wild-type (WT) and AhNST1-overexpressing rice (OE#1, 2, 3); (D) Stem internode phenotype of wild-type and AhNST1-overexpressing rice; (E) Expression level of AhNST1 in wild-type and overexpressing rice; (FH) Comparison of plant height (F), first internode length (G), and second internode length (H) between wild-type and overexpressing rice. Detailed Implementation

[0015] Unless otherwise specified, the scientific and technical terms used herein are for the understanding of those skilled in the art. The technical solutions of the present invention will be further described in detail below with reference to specific embodiments.

[0016] Example 1: This embodiment uses a combination of QTL fine mapping, transcriptomics, and natural populations to pinpoint key genes involved in intersegmental development. AhNST1 To identify key genes regulating internode development in peanuts, this example uses the dwarf peanut material ICG4601 and the tall peanut material Fesr-2 (provided by the Crop Research Institute of Guangdong Academy of Agricultural Sciences, published in "Wang Runfeng et al., 2025, Evaluation of Pod Maturity and Screening of Early-Maturing Germplasm Resources of Peanut Core Germplasm Resources, Acta Agronomica Sinica, 51(2):395-404") as parents to construct a segregating population through hybridization. Figure 1 ).

[0017] First, BSA-seq analysis was performed on the mixed pool of extreme phenotypes from the parents and F2 generation. By calculating the ΔSNP-index, a QTL with a length of 3.30 Mb was located on chromosome A02. Figure 2A ).

[0018] Subsequently, using a population of 4000 F3 strains and 20 SNP markers developed within the region (see Table 1, sequences as shown in SEQ ID NO: 8–67), fine mapping was performed, successfully narrowing the target region to 795.799 Kb, containing 34 candidate genes. Figure 2B ).

[0019] Table 1. Marker Primer Sequences

[0020] To further narrow down the scope, a combined analysis was performed using RNA-seq data from the internodes of the parental stems. A total of 15 genes were detected within the aforementioned fine-mapping region of 795.799 kb, among which… Ahy_A02g009643 , Ahy_ A02g009657 and Ahy_A02g009671 The expression levels of three genes were significantly upregulated in YY215. Figure 2C Functional annotations indicate Ahy_A02g009671 Encoding an NAC family transcription factor (named AhNST1 Its Arabidopsis homolog has been reported to be a key factor regulating secondary cell wall thickening and lignin deposition. Sequence alignment shows that... AhNST1 A specific A>C nonsynonymous mutation exists in the third exon of the gene, causing the encoded amino acid to change from lysine (Lys) to glutamine (Gln). Figure 2D ). Ahy_A02g009643 , Ahy_A02g009657 No variations were found in the coding region of the gene.

[0021] By analyzing 390 peanut resequencing results previously published by the team, it was discovered that... AhNST1 AA There are 106 genotypes (sequences shown in SEQ ID NO:1). AhNST1 CC There are 272 accessions of the genotype (sequence shown in SEQ ID NO:2). Figure 2E Further correlation with plant height and internode length revealed that it carried... AhNST1 AA The plant height and internode length of the genotype were significantly lower than those of the carrier. AhNST1 CC Genotype germplasm ( Figure 2F ).

[0022] The above multi-dimensional results indicate that AhNST1 It is a key candidate gene for regulating peanut plant height.

[0023] Example 2: AhNST1 (shown as SEQ ID NO:1) negatively regulates plant height and internode length To preliminarily elucidate the biological function of AhNST1, this example conducted studies on expression patterns, subcellular localization, and gene function.

[0024] The specific research method is as follows: Construction of the 35S:AhNST1-GFP vector: targeting AhNST1Specific primers were designed based on the CDS sequence, and the primer sequences are shown in Table 2. Total RNA was extracted from dwarf peanut ICG4601 using a plant RNA extraction kit (purchased from Hunan Aike Rui Biotechnology Co., Ltd., catalog number No. AG21019). Using the total RNA as a template, cDNA was synthesized using a reverse transcription kit (purchased from Beijing TransGen Biotech Co., Ltd., catalog number No. AE311). Using the above cDNA as a template, the target gene was amplified by PCR using AhNST1-specific primers AhNST1-F and AhNST1-R and high-fidelity DNA polymerase. After the PCR reaction, agarose gel electrophoresis was performed. The size of the target gene was determined based on the DNA marker, and the correctly sized bands were quickly excised from the gel. The target gene fragment was purified and recovered using a universal DNA purification and recovery kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number No. DP214). A one-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., trade name ClonExpress Ⅱ One Step Cloning Kit) was used to ligate the purified target gene fragment with the enzyme-digested pCambia1300-35S-EGFP-Golgi vector at a molar ratio of 2:1. The recombination reaction was carried out at 37℃ for 30 minutes to complete the ligation of the target gene fragment into the linear vector. The nucleotide sequence of the pCambia1300-35S-EGFP-Golgi vector is SEQ ID NO:3.

[0025] The ligation product was added to *E. coli* competent cells DH5α, gently aspirated and mixed, and placed on ice for 20 minutes. The cells were then incubated in a 42°C water bath for 90 seconds, immediately placed on ice for 2 minutes, and 700 µL of LB liquid medium was added. The cells were then incubated at 37°C with shaking for 30 minutes. The bacterial culture was then spread onto a plate containing 50 μg / mL of LB liquid medium. -1 Incubate kanamycin on LB agar plates inverted at 37°C overnight. Select single colonies from the overnight plates for colony PCR verification. After PCR, perform agarose gel electrophoresis on the products, and select single colonies with the correct bands to add 50 μg / mL of [agarose gel]. -1 The culture was carried out in LB broth containing kanamycin at 28°C and 220 rpm for 20 hours. Sequencing of the cultured bacterial culture was performed, and the sequencing results were identical to the target gene sequence, indicating that the AhNST1 gene had been successfully cloned into the overexpression vector pCambia1300-35S-EGFP-Golgi.

[0026] Table 2 Primers used in the examples

[0027] Subcellular localization of AhNST1: The constructed 35S:AhNST1-GFP vector was transformed into Agrobacterium strain GV3101-P19. A resuspension containing 10 mM MES (2-morpholinoethanesulfonic acid, pH 5.7), 10 mM MgCl2, and 100 μM acetylsuccinone was prepared. The 35S:AhNST1-GFP Agrobacterium cells were resuspended in the resuspension to OD200. 600 The concentration was 0.9–1.0, and the cells were kept in the dark for 2–3 hours. Healthy *Nicotiana benthamiana* plants approximately 4 weeks old were selected, and the resuspended mycelium was injected into the tobacco leaves from the lower epidermis using a syringe. The plants were then cultured normally in a light incubator for 3 days. Afterward, the fluorescence signal was observed and photographed under a laser confocal microscope (Leica TCS SP8SR, Germany). GFP was excited using a 488 nm solid-state laser, and the GFP signal was detected in the range of 498–540 nm. mCherry was excited using a 552 nm solid-state laser, and the mCherry signal was detected in the range of 590–640 nm. Results are shown below. Figure 3 B in the middle.

[0028] 35S:AhNST1-GFP overexpression rice genetic transformation, screening, and identification: Rice grains without mold spots and with normal bud openings (Nipponbare) were selected, disinfected with 75% alcohol for 1 min, and rinsed with sterile water for 1 min each time; disinfected with 15% sodium hypochlorite for 20 min, and rinsed with sterile water 3 times for 1 min each time; the disinfected rice grains were inoculated into N6D induction medium (N6 basic medium + 30 g / L sucrose + 2.5~3.0 mg / L 2,4-D + 500 mg / L proline + 300 mg / L casein hydrolysate + coagulant such as Phytagel, pH 5.8), and cultured at 26℃ for 20 days; rice grains pre-treated with 50 μg·mL⁻¹ of sodium hypochlorite were selected. -1 Kanamycin with 20 μg·mL -1 Monoclonal Agrobacterium, activated by streaking on rifampicin-containing LB agar plates and identified by PCR as containing the 35S:AhNST1-GFP target plasmid, was inoculated in AAM infection medium (nitrogen-free macro-elements [KCl 2950 mg / L, MgSO4·7H2O 250 mg / L, CaCl2·2H2O 150 mg / L, NaH2PO4·2H2O 150 mg / L] + MS trace elements + MS iron salts + vitamins [inositol 100 mg / L, etc.] + amino acids [L-glutamine 876 mg / L, L-aspartic acid 266 mg / L, L-arginine 228 mg / L, glycine 7.5 mg / L] + 68.5 g / L sucrose + 36.0 g / L glucose + 100~200 μM AS, pH adjusted to 5.2) to prepare OD.600 = 0.2% Agrobacterium resuspension; pick callus grains into Erlenmeyer flasks, add 20 ml of Agrobacterium resuspension, infect for 10-15 min and discard the bacterial suspension, inoculate the callus into N6 co-culture medium (N6 basic medium + 10 g / L glucose + 2.0-3.0 mg / L 2,4-D + 100~200 μM AS (acetylsyleugenol) + coagulant, pH 5.2), co-culture at 20℃ for 48-72 h; after co-culture, wash and blot dry the surface moisture with sterile filter paper, inoculate the callus into N6S selection medium (N6 basic medium + 30 g / L sucrose + 2.5 mg / L 2,4-D + 50 mg / L Hygromycin + 400 mg / L Cefotaxime sodium + ... (Coagulant), cultured in the dark at 26℃ for 20-30 days; positive calluses that survive normally and continuously proliferate to produce fresh, dense, pale yellow granular new calluses are inoculated onto N6S selection medium. During callus selection, single-clone calluses must be selected. Cultured in the dark at 26℃ for 7-10 days; then, positive calluses are inoculated onto MS differentiation medium (MS basal medium + 30 g / L sucrose + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 50 mg / L hygromycin + 200 mg / L cephalosporin + coagulant, pH 5.8), and cultured under light at 25-27℃ for 15-20 days. After buds differentiate to 2-5 cm, they are inoculated onto 1 / 2 MS rooting medium (1 / 2 MS basal medium + 30 g / L sucrose + 50 mg / L hygromycin + coagulant, pH 5.8). (using a coagulant), cultured at 30℃ under light for 7-10 days, to obtain T0 generation 35S:AhNST1-GFP overexpressing transgenic rice complete regenerated plants.

[0029] Wild-type cultivation: Select plump wild-type Nipponbare seeds, soak them in 30℃ warm water for 48 hours, and then germinate them in the dark at 30-32℃ for 24-48 hours until the seeds "show white". Then sow them in seedling trays and move them into an artificial climate chamber (28-30℃ during the day / 24-25℃ at night, 14 hours of light / 10 hours of darkness, 75% humidity) for cultivation. When the seedlings have grown 3-4 true leaves, transplant them into larger clay pots for conventional water and fertilizer management.

[0030] RT-qPCR primers for AhNST1, AhNST1-RT-qPCR-F and AhNST1-RT-qPCR-R (primer sequences are shown in Table 2), were designed to identify positive transgenic rice at the RNA level. The results are as follows: Figure 3 As shown in E, the AhNST1 gene is overexpressed in all positive plants.

[0031] Tissue-specific transcriptome data analysis showed that AhNST1 High expression is mainly found in stems and pods. Figure 3 The A protein in the sample is highly consistent with its expected function of regulating stem development; subcellular localization results confirmed that the protein is specifically located in the cell nucleus. Figure 3 The B in the formula conforms to the characteristics of a transcription factor. Based on this, a [structure / system] was constructed. AhNST1 The expression vector was overexpressed and introduced into rice for heterologous functional verification. Figure 3 (E in the text). Phenotypic identification results showed that, compared with wild-type WT, AhNST1 The plant height of the overexpressing transgenic lines (OE#1, 2, 3) was significantly reduced. Figure 3 In C and F), the internode length of the stem is drastically shortened ( Figure 3 (D, G, H in the text). This result demonstrates that AhNST1 has a biological function of negatively regulating crop internode length and plant height.

[0032] Instruction manual nucleotide sequence SEQ ID NO:1 ( Ahy_A02g009657 AhNST1 sequence; AhNST1 AA Gene) SEQ ID NO:2 ( Ahy_A02g009657 AhNST1 Mutant sequence; AhNST1 CC Gene) SEQ ID NO:3 (pCambia1300-35S-EGFP-Golgi vector sequence)

Claims

1. Application of AhNST1 in negative regulation of internode length and plant height in peanut plants.

2. Application of the AhNST1 gene and its primers in the identification and selection of peanut plant internode length and plant height traits. The AhNST1 gene sequence is shown in SIQID NO.

1. The nucleotide sequence shown in SIQID NO.1 has an A / C base mutation at position 108944345 on peanut chromosome A02. This mutation site is significantly correlated with peanut plant internode length and plant height.

3. The application according to claim 2, characterized in that, The primers are: AhNST1-F: 5'-ATGCCAGAAAGCATGAGTAT-3'; AhNST1-R: 5'-CACTGACGTGTTTGACACGT-3'.

4. Application of the kit for identifying and selecting peanut plant internode length and plant height traits using AhNST1 gene primers. The AhNST1 gene sequence is shown in SIQID NO.

1. The nucleotide sequence shown in SIQID NO. 1 has an A / C base mutation at position 108944345 on peanut chromosome A02. This mutation site is significantly correlated with peanut plant internode length and plant height.

5. A method for selecting peanut seeds, characterized in that, The method includes the following steps: 1) Extract genomic DNA from the peanuts to be selected, detect and sequence to determine the genotype of the AhNST1 gene in the peanuts to be selected; the sequence of the AhNST1 gene is shown in SIQID NO.1; 2) Select plants with the AA genotype at position A / C of AhNST1 gene genotype 108944345 for seed production.

6. Application of AhNST1 in negative regulation of internode length and plant height in rice plants.

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