A molecular marker related to peanut salt tolerance and application thereof

Abstract

This invention discloses a molecular marker related to peanut salt tolerance and its application, belonging to the fields of molecular biology and plant genetics and breeding technology. Based on the screening of molecular markers related to peanut salt tolerance, this invention establishes a molecular marker-assisted breeding method for salt-tolerant peanut varieties, cultivating new peanut varieties with potential for planting in saline-alkali land. The molecular marker is a SNP site molecular marker linked to peanut salt tolerance, located at position 77 of SEQ ID NO:1, with a base sequence of G / A. The molecular markers related to peanut salt tolerance obtained by this invention are used for screening salt-tolerant parents, thereby accelerating the genetic breeding and selection of salt-tolerant peanuts, improving the breeding efficiency of salt-tolerant lines, and increasing yields in saline-alkali land.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and plant genetics and breeding technology, specifically relating to a molecular marker related to peanut salt tolerance and its application. Background Technology

[0002] Peanuts (Arachis hypogaea L.) are an important global oilseed crop, protein source, and economic crop, playing an irreplaceable role in ensuring edible oil security and increasing farmers' income. However, soil salinization, an increasingly serious global problem, severely restricts peanut yields and the expansion of planting areas. Peanuts are moderately salt-tolerant crops; salt stress significantly inhibits seed germination, seedling growth, and pod development, ultimately leading to substantial yield reductions. Therefore, breeding and promoting new salt-tolerant peanut varieties is a fundamental way to address the challenges of salinization and ensure the sustainable development of the industry.

[0003] Traditional peanut salt tolerance breeding relies primarily on field phenotypic selection. This method requires observing and evaluating a large number of breeding materials across multiple growth cycles in natural saline-alkali land or artificially created salt stress environments, selecting salt-tolerant individual plants based on plant survival, growth vigor, and final yield. However, this method has significant limitations: First, the heterogeneity of salt distribution in the field and the variable climatic environment result in poor repeatability and low accuracy of phenotypic identification results; second, breeders must wait for plants to reach critical growth stages or even complete their entire growth cycle before effective evaluation can be conducted, leading to an extremely long breeding cycle, typically eight to ten years; finally, because salt tolerance is a complex quantitative trait controlled by multiple genes, traditional methods struggle to accurately identify individuals that combine excellent salt tolerance genes with high-yield and high-quality agronomic traits in the early stages, resulting in low selection efficiency.

[0004] The emergence of marker-assisted selection (MAG) technology has provided a powerful tool for overcoming traditional breeding bottlenecks. This technology, by detecting DNA molecular markers closely linked to target traits, enables precise and rapid genotyping at the seedling or even seed stage, unaffected by environmental interference and significantly shortening the breeding cycle. While this technology has been successfully applied in crops such as rice and maize, its development in peanut salt tolerance breeding has lagged behind. The main challenge lies in the scarcity of publicly disclosed molecular markers significantly associated with peanut salt tolerance and stably expressed under different genetic backgrounds. Many candidate regions or markers discovered in previous studies have not yet been translated into practical tools that are easy to detect in routine laboratories, inexpensive, and validated on a large scale using breeding materials. This lack of efficient molecular markers directly limits the large-scale application of this technology in the genetic improvement of peanut salt tolerance.

[0005] Therefore, the current peanut breeding field urgently needs a fully validated molecular marker that is closely related to salt tolerance and is easy and reliable to detect, in order to establish an efficient molecular marker-assisted selection system and accelerate the breeding process of salt-tolerant peanut varieties. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker related to peanut salt tolerance and its application. Specifically, based on the screening of molecular markers related to peanut salt tolerance, a molecular marker-assisted breeding method for peanut salt-tolerant varieties is established to cultivate new peanut varieties with potential for planting in saline-alkali land.

[0007] The present invention first provides a molecular marker for an SNP site linked to the salt tolerance trait of peanuts, wherein the SNP site molecular marker is located at position 77 of the sequence SEQ ID NO:1 and its base is G / A;

[0008] GCGGTTCTAGTTGGGTTGGAAAGCTAACATCCGGGGCTTCGAAATGATATAAGATTTTCTATGGTTGAACCACGCAGGGTGACGTACGCGCATAGTACGCGGACGCGCCGTTGCTGCCACCTGGTTCACTTAAAGCAAAACATGGCCAGCGAAT (SEQ ID NO: 1).

[0009] The sequence information of the primer pairs used to detect the above SNP sites is as follows:

[0010] Forward primer: 5'-GCGGTTCTAGTTGGGTTGGA-3' (SEQ ID NO:2);

[0011] Reverse primer: 5'-TAAGTGAACCAGGTGGCAGC-3' (SEQ ID NO:3);

[0012] In another aspect, the present invention provides an application of the molecular marker in screening peanut individuals with salt tolerance traits;

[0013] Another aspect of the present invention is to provide a method for screening salt-tolerant peanut seedlings, which involves screening individuals whose SNP loci are homozygous for GG.

[0014] The method involves amplifying the nucleic acid samples of selected individuals using PCR primers, and then typing or sequencing the amplification products.

[0015] The specific sequence information of the primer pairs used is as follows:

[0016] Forward primer: 5'-GCGGTTCTAGTTGGGTTGGA-3' (SEQ ID NO:2);

[0017] Reverse primer: 5'-TAAGTGAACCAGGTGGCAGC-3' (SEQ ID NO:3);

[0018] In another aspect, the present invention provides a method for cultivating salt-tolerant peanut seedlings, which uses the individuals selected above as parent seedlings as F0 generation for cultivation.

[0019] The present invention screens out molecular markers related to peanut salt tolerance traits and uses them to screen salt-tolerant parents, thereby accelerating the genetic breeding and selection of salt-tolerant peanuts, improving the breeding efficiency of salt-tolerant lines and the yield in saline-alkali land. Attached Figure Description

[0020] Figure 1 : Growth performance of salt-tolerant and sensitive peanut groups after planting in saline-alkali land;

[0021] Figure 2 Agarose gel electrophoresis image, where M is the marker, and 1, 2, 3, 4, and 5 are peanut DNA sample bands;

[0022] Figure 3 SNP locus typing map. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0024] Example 1: Screening of peanut salt-tolerant / sensitive groups based on field phenotypes in saline-alkali land

[0025] Under natural salt stress, through systematic field phenotypic identification, peanut plants with stable and extremely salt-tolerant or salt-sensitive phenotypes were screened out, providing reliable experimental materials for subsequent molecular marker discovery.

[0026] The experiment was conducted in the Yellow River Delta Agricultural High-tech Industrial Demonstration Zone, Dongying City, Shandong Province. This plot is a typical inland saline-alkali land. The basic physicochemical properties of the topsoil layer (0-20 cm) before sowing were: pH 8.3, total salt content 0.38% (mainly NaCl and Na2SO4), and organic matter content 12.1 g / kg.

[0027] One hundred and eighty peanut varieties and cultivars from diverse sources with significant differences in genetic background were selected as the baseline population. A randomized block design with three replicates was used. Except for the absence of special improvement measures such as salt leaching and salt suppression, other water and fertilizer management practices were consistent with those in local high-yield fields to ensure that the observed growth differences were mainly due to salt stress.

[0028] During the peanut seedling to pegging stage (35-50 days after sowing), when salt stress symptoms are most pronounced, three experienced technicians independently conducted two field surveys of the population.

[0029] The survey indicators include:

[0030] Plant growth vigor: divided into three levels: robust, moderate, and weak.

[0031] Salt damage symptoms: Record the severity of leaf yellowing, wilting, and edge scorching (0-5 levels, 0 for no symptoms).

[0032] Plant height and number of leaves: compared with control varieties in normal plots.

[0033] Based on the survey results, two groups of materials with highly consistent and extreme phenotypes were rigorously selected. Figure 1 (1) Salt-tolerant group: 15 individual plants with the most robust growth, the least salt damage symptoms, and the least impact on plant height and number of leaves were selected. (2) Sensitive group: 15 individual plants with severely inhibited growth, severe salt damage symptoms, and obvious dwarfism were selected.

[0034] Following the second survey, fresh young leaves from the tips of 30 individual plants in these two groups were collected, immediately flash-frozen with liquid nitrogen, and then stored in an ultra-low temperature freezer at -80°C for DNA extraction.

[0035] Example 2: Discovery and validation of SNP molecular markers associated with salt tolerance

[0036] Thirty peanut leaf samples selected through field phenotypic identification in saline-alkali land in Example 1 were used, including 15 salt-tolerant individual plants and 15 salt-sensitive individual plants.

[0037] 2. Extraction of genomic DNA

[0038] Genomic DNA was extracted using a modified CTAB method, and the specific steps are as follows:

[0039] Grinding: Take about 100 mg of frozen leaves and grind them thoroughly into a fine powder in liquid nitrogen.

[0040] Lysis: Transfer the powder into a preheated centrifuge tube containing 600 μL of 2× CTAB extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 2% PVP-40) and 3 μL of β-mercaptoethanol. Vortex quickly to mix, and incubate in a 65°C water bath for 45 minutes, gently inverting the tube every 10 minutes to mix.

[0041] Remove protein: Remove and cool to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), gently invert and mix for 15 minutes to fully emulsify, then centrifuge at 4°C and 12,000 rpm for 15 minutes.

[0042] DNA precipitation: Carefully aspirate the supernatant into a new centrifuge tube, add 0.7 times the volume of pre-cooled isopropanol, and gently invert until a white, thread-like DNA precipitate appears.

[0043] Washing and dissolving: Use a glass rod to hook out the DNA precipitate, or wash the precipitate twice with 70% ethanol and dry it at room temperature to remove any residual ethanol. Dissolve the DNA precipitate in 100 μL of 1×TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0044] RNase treatment: Add 1 μL RNase A (10 mg / mL) and incubate at 37°C for 30 minutes to remove RNA.

[0045] Quality control: DNA concentration and purity were determined using Nanodrop 2000 (an A260 / A280 ratio between 1.8 and 2.0 is considered acceptable), and DNA integrity was assessed by 1% agarose gel electrophoresis. Figure 2 Qualified samples should be stored at -20℃ for future use.

[0046] 2.2 BSA-Seq analysis

[0047] DNA pool construction: The DNA of 15 individual plants from the salt-tolerant group was mixed in equal amounts to construct the "salt-tolerant pool" (T-pool); the DNA of 15 individual plants from the sensitive group was mixed in equal amounts to construct the "sensitive pool" (S-pool).

[0048] Library construction and sequencing: Two DNA pools were sent to a sequencing company to construct Illumina PE150 sequencing libraries and perform high-throughput sequencing on the NovaSeq 6000 platform. The effective sequencing depth of each pool reached more than 30X.

[0049] Bioinformatics analysis:

[0050] Data quality control: Fastp is used to filter the raw sequencing data to remove low-quality reads and adapter sequences.

[0051] Sequence alignment: High-quality clean reads were aligned to the peanut reference genome using BWA-MEM software.

[0052] SNP calling: SNP detection and genotyping are performed using the GATK standard procedure.

[0053] Association analysis: The SNP-index algorithm was used to calculate the difference in allele frequencies at each SNP locus between the two pools. An association threshold was set as Δ(SNP-index) > 0.6 and -log 10 (P-value) > 3 (i.e., P < 0.001), screening for genomic regions that are significantly associated with salt tolerance.

[0054] 2.2 Primer Design and Synthesis

[0055] Based on the peanut reference genome sequence, 10 pairs of specific primers for amplifying candidate SNP sites were designed and synthesized within the region associated with chromosome 9 (genbank: NC_092044.1) indicated by preliminary BSA-Seq analysis. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Primer information is shown in Table 1.

[0056] Table 1: Information on some SNP primers for peanuts

[0057]

[0058] 2.3 PCR amplification and genotyping

[0059] The PCR reaction system consisted of 25 μL: 17.75 μL ddH2O, 2.5 μL 10×Buffer, 2.0 μL dNTPs (2.5 mMeach), 0.5 μL each of forward and reverse primers, 1.5 μL DNA template (50 ng / μL), and 1.25 U Taq DNA polymerase. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at 55-60℃ (adjusted according to the annealing temperatures of each primer in Table 1) for 30 s, extension at 72℃ for 45 s, for a total of 35 cycles; final extension at 72℃ for 10 min. The PCR products were sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing and genotyping.

[0060] 2.4 Data Analysis

[0061] The allele frequency, observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) of each SNP locus in the salt-tolerant and sensitive groups were calculated using POPGENE32 software. Association analysis between SNP loci and salt tolerance traits in the two populations was performed using SPSS 22.0 software. The chi-square test was used to compare differences in genotype distribution, with P < 0.01 considered highly significant.

[0062] The results showed that 31 alleles were detected at the 10 SNP loci in the salt-tolerant group, with an average of 3.1 alleles; and 21 alleles were detected in the sensitive group, with an average of 2.1 alleles. Polymorphism analysis is shown in Table 2.

[0063] Table 2: Information on polymorphisms of SNP sites related to salt tolerance in peanuts

[0064]

[0065] 2.5 Results of Association Analysis

[0066] General linear model (GLM) analysis revealed a highly significant association between the SNP locus AhSST10 and salt tolerance (P<0.01). Specifically, the GG genotype at the AhSST10 locus was significantly more prevalent in the salt-tolerant group than in the sensitive group (80% vs 13%), and individuals with this genotype had a significantly higher survival rate in saline-alkali soil than those with the GA and AA genotypes (P<0.01). The association analysis results between different genotypes and salt tolerance are shown in Table 3.

[0067] Table 3: Association analysis of different genotypes of SNP locus AhSST10 with salt tolerance trait

[0068]

[0069] Sequencing analysis results showed that the frequency of the GG genotype at the AhSST10 locus was significantly higher in the salt-tolerant group than in the sensitive group.

[0070] The SNP site is molecularly labeled as position 77 of SEQ ID NO: 1, with bases G / A ( Figure 3 );

[0071] GCGGTTCTAGTTGGGTTGGAAAGCTAACATCCGGGGCTTCGAAATGATATAAGATTTTCTATGGTTGAACCACGCAGGGTGACGTACGCGCATAGTACGCGGACGCGCCGTTGCTGCCACCTGGTTCACTTAAAGCAAAACATGGCCAGCGAAT (SEQ ID NO: 1).

[0072] Therefore, peanut individuals with high salt tolerance potential can be screened using upstream primer F: GCGGTTCTAGTTGGGTTGGA (SEQ ID NO: 2) and downstream primer R: TAAGTGAACCAGGTGGCAGC (SEQ ID NO: 3, i.e., the primers for AhSST10 in Table 1); among them, peanuts with the GG genotype have better salt tolerance potential.

[0073] Example 3: Application of SNP markers related to peanut salt tolerance traits

[0074] A widely cultivated peanut variety (genotype AA) with high salt sensitivity was selected as the female parent, and a peanut germplasm with high salt tolerance (genotype GG) was selected as the male parent. Hybridization was performed via artificial emasculation and pollination to obtain F1 hybrid seeds. After self-pollination, an F2 segregating population of 500 plants was obtained from the F1 generation, which was used for subsequent marker-assisted selection studies.

[0075] During the F2 seedling stage (three-leaf stage), a suitable amount of young leaf samples were taken from each plant, and high-quality genomic DNA was extracted using a modified CTAB method. PCR amplification was performed using AhSST10-labeled specific primers (SEQ ID NO:2 and SEQ ID NO:3). The total reaction volume was 25 μL, containing 2.5 μL of 10×Buffer, 2.0 μL of dNTP mixture, 0.5 μL each of forward and reverse primers, 1.5 μL of template DNA, and 1.25 U of Taq DNA polymerase. The PCR reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; final extension at 72℃ for 10 min. Sequencing analysis of the PCR products determined the AhSST10 genotype of each individual plant.

[0076] From a segregating population of 500 F2 individuals, 128 individuals with the GG genotype were obtained through screening using the AhSST10 molecular marker, achieving a selection efficiency of 25.6%. The genotype distribution conformed to Mendelian segregation, with 128 individuals (25.6%) of the GG type, 254 individuals (50.8%) of the GA type, and 118 individuals (23.6%) of the AA type. This indicates that the AhSST01 marker possesses stable discriminative ability under different genetic backgrounds.

[0077] GG genotype plants obtained through molecular marker screening were co-planted with randomly selected F2 plants in a natural saline-alkali experimental field (soil pH 8.2, total salt content 0.35%) in the Yellow River Delta region of Shandong Province. A randomized block design was used, with three replicates for each genotype and 30 plants per replicate. Salt damage symptoms were investigated during key peanut growth stages (seedling stage, pegging stage, and pod-setting stage), and salt damage indices were recorded according to a 0-5 scale. Survival rate was calculated at maturity, and yield traits such as pod weight per plant were measured after harvest.

[0078] The results of the saline-alkali land experiment (Table 4) showed that there were highly significant differences in salt tolerance and yield traits among different AhSST10 genotype populations (P<0.01). The GG genotype population exhibited the strongest salt tolerance, with an average salt damage index of only 1.2, significantly lower than the GA type (2.8) and AA type (4.5); its survival rate reached 90.2%, and the weight of a single pod was 18.5 grams, which were 3.15 times and 2.72 times that of the AA genotype, respectively.

[0079] Table 4: Agronomic performance of different genotypes in saline-alkali land

[0080]

[0081] All 10 F2:3 families exhibited stable and consistent salt tolerance in saline-alkali soil environments. The coefficient of variation within each family was less than 10%, indicating high uniformity in agronomic traits. Eight families showed significantly higher pod weight per plant than their salt-tolerant parents (P<0.05), demonstrating good yield potential. The salt damage index of each family remained between 1.0 and 1.5, with a survival rate exceeding 85%, indicating that salt tolerance is stably inherited by offspring without significant segregation.

[0082] The molecular marker AhSST10 can be effectively used for early selection in peanut salt-tolerant breeding. The GG genotype plants selected using this marker exhibit strong salt tolerance and good yield traits in saline-alkali soil environments, and this salt tolerance trait can be stably inherited. The molecular marker-assisted selection system established in this study can significantly improve the efficiency of peanut salt-tolerant breeding and shorten the breeding cycle, and has important value for widespread application.

Claims

1. A method for screening salt-tolerant peanut seedlings, characterized in that, The method involves screening individuals with a GG homozygous genotype among SNP molecular markers linked to the peanut salt tolerance trait; the sequence of the SNP molecular marker is shown in SEQ ID NO:1, and a G / A mutation exists at its 77th base.

2. The method as described in claim 1, characterized in that, The method involves amplifying the nucleic acid samples of individuals to be screened using PCR primers, and then performing genotyping or sequencing on the amplified products for screening. The upstream primer sequence is SEQ ID NO:2, and the downstream primer sequence is SEQ ID NO:

3.

3. A method for cultivating salt-tolerant peanut seedlings, characterized in that, The method involves using the seedlings screened by the method of claim 1 as the F0 generation for cultivation.

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