A SNP molecular marker related to nitrogen high-efficiency utilization of cotton and application thereof

Abstract

The application discloses a SNP molecular marker related to nitrogen high-efficiency utilization of cotton and application thereof, and belongs to the field of agricultural biotechnology.The SNP molecular marker is located at the 50,814,137th base of chromosome 4 of a D subgenome of cotton, that is, the 725th base upstream of a transcription start point of a GhGLN1.3 gene, and T / C polymorphism exists.The expression amount of the GhGLN1.3 gene is significantly increased under low nitrogen stress, and the GhGLN1.3 gene is involved in regulation of nitrogen high-efficiency utilization of cotton.The SNP molecular marker has genotypes TT and CC, and the lint yield of cotton germplasm material with the genotype TT is significantly higher than that of material with the genotype CC.The SNP molecular marker provided by the application can be used for early screening of the nitrogen high-efficiency utilization trait of cotton at the seedling stage, has the advantages of simple operation, accurate detection and the like, and is suitable for molecular marker assisted selection breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to an SNP molecular marker related to efficient nitrogen utilization in cotton and its application. Background Technology

[0002] Cotton (Gossypium hirsutum L.) is one of the world's most important natural fiber crops. Nitrogen is the mineral nutrient element most needed during cotton growth and development, playing a crucial role in cotton yield and quality. However, excessive application of nitrogen fertilizer is widespread in cotton production, with a nitrogen fertilizer utilization rate of only 30%–40%, significantly lower than the theoretical potential. Excessive nitrogen fertilizer application not only increases production costs but also leads to serious environmental problems such as soil degradation and eutrophication of water bodies. Therefore, breeding nitrogen-efficient cotton varieties to improve nitrogen fertilizer utilization efficiency and effectively increase cotton's economic yield is of great significance for achieving the green and sustainable development of the cotton industry.

[0003] Glutamine synthetase (GLN) is a key enzyme in the assimilation and re-assimilation of nitrogen in higher plants, responsible for the catalytic conversion of ammonium ions into glutamine, playing a central role in plant nitrogen metabolism. Studies have shown that the GLN gene family plays an important role in regulating crop nitrogen use efficiency. GLN genes all play crucial roles in the absorption, assimilation, transport, and remobilization of nitrogen in plants. Systematic identification and functional analysis of the cotton GLN gene family revealed that the GhGLN1 gene responds to nitrogen-induced treatment, and its silencing affects nitrogen accumulation and nitrogen use efficiency in cotton.

[0004] The GhGLN1.3 gene, as a member of the GLN family, may also play an important role in nitrogen metabolism in cotton, but the association between natural variation in its promoter region and nitrogen-efficient use traits has not been reported. Nitrogen-efficient use is a typical quantitative trait, regulated by multiple genes and easily influenced by environmental factors. Traditional breeding relies on phenotypic selection, which has limitations such as long cycles, low efficiency, and significant environmental interference. Single nucleotide polymorphism (SNP) markers, as third-generation molecular markers, have advantages such as large numbers, wide distribution, rich polymorphism, and convenient detection, and have been widely used in marker-assisted breeding of crops. Developing SNP molecular markers related to nitrogen-efficient use in cotton is of great significance for accelerating the breeding process of nitrogen-efficient cotton varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a SNP molecular marker related to efficient nitrogen use in cotton and its application, in order to solve the problem of the lack of effective molecular markers related to efficient nitrogen use traits in cotton in the prior art. The SNP molecular marker provided by this invention can be used for early screening of efficient nitrogen use traits in cotton during the seedling stage. It has the advantages of simple operation and accurate detection, and is suitable for molecular marker-assisted selection breeding.

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

[0007] First, this invention provides a SNP molecular marker related to efficient nitrogen utilization in cotton. The SNP molecular marker is located at bases 50, 814, 137 on chromosome 4 of the cotton D subgenome (based on the upland cotton reference genome version Gossypium hirsutum(AD1)'TM-1'genome CRI_v1), specifically 725 bases upstream of the transcription start point of the GhGLN1.3 gene, and exhibits T / C polymorphism. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1. This sequence consists of 200 bp flanking fragments upstream and downstream of the target SNP site, with base T or C at position 201.

[0008] Furthermore, the SNP molecular markers include genotypes TT and CC; among them, the lint percentage of cotton material with genotype TT is significantly higher than that of cotton material with genotype CC.

[0009] Meanwhile, the present invention provides a specific primer set for detecting the SNP molecular marker, the primer set including an upstream primer as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.3.

[0010] The present invention also provides a method for identifying the nitrogen-efficient use trait of cotton using the SNP molecular marker, comprising the following steps: extracting genomic DNA from the cotton to be tested, and detecting the genotype of the SNP molecular marker; the lint percentage of cotton plants with genotype TT is significantly higher than that of cotton plants with genotype CC.

[0011] Furthermore, the method includes:

[0012] (1) Extract genomic DNA from the cotton sample;

[0013] (2) Using the genomic DNA as a template, perform PCR amplification using the primer set to obtain PCR amplification products;

[0014] (3) Perform genotyping detection on the PCR amplification products to determine the genotype of the SNP molecular marker;

[0015] (4) Determine the nitrogen-efficient use trait of the cotton plant to be tested based on the genotype; cotton plants with genotype TT have a higher or higher lint percentage than cotton plants with genotype CC.

[0016] This invention also provides the application of the SNP molecular marker in the breeding of high nitrogen-efficiency cotton varieties, by extracting genomic DNA from the cotton to be tested, detecting the genotype of the SNP molecular marker, and selecting cotton plants with the TT genotype as breeding parents or for screening.

[0017] The present invention also provides the application of the SNP molecular marker in identifying or assisting in the identification of traits related to high nitrogen utilization efficiency in cotton.

[0018] This invention discloses a SNP molecular marker associated with efficient nitrogen use in cotton and experimentally confirms its application effect. A genotyping and lint percentage survey of 379 natural cotton germplasm resources showed significant differences in lint percentage among different genotypes (TT, CC) of this marker. The average lint percentage of the TT genotype population was significantly higher than that of the CC genotype population, indicating a close association between this SNP molecular marker and lint percentage.

[0019] Experiments using different nitrogen levels—low nitrogen (LN, 0.25 mM), control (CK, 2.5 mM), high nitrogen 1 (HN1, 5 mM), and high nitrogen 2 (HN2, 10 mM)—showed that the expression level of the GhGLN1.3 gene was higher under LN treatment than under other treatments. This indicates that the gene is induced by low nitrogen stress and participates in the adaptive response of cotton to low nitrogen environments, further validating the functional relevance of this SNP molecular marker in the regulation of nitrogen-efficient use.

[0020] Using the SNP molecular markers described in this invention for marker-assisted selection can identify and screen for nitrogen-efficient use traits in the early stages of cotton growth, which can significantly shorten the breeding cycle, reduce the workload of field screening, improve breeding efficiency, and reduce breeding costs. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the GhGLN1.3 gene structure and SNP sites.

[0022] Figure 2 A comparative graph showing the differences in lint percentage among the CC and TT genotypes of cotton in different pilot areas over three consecutive years. CC and TT represent two genotypes; n represents the number of experimental materials for each genotype group. This indicates a statistically significant difference in calyx percentage between the two groups (P<0.05); A, B, and C correspond to the pilot projects in Alar, Anyang, and Kuitun, respectively, all of which were conducted in field trials over three consecutive years; A1, A2, and A3 are the calyx percentage data from the Alar pilot project over three years; B1, B2, and B3 are the calyx percentage data from the Anyang pilot project over three years; and C1, C2, and C3 are the calyx percentage data from the Kuitun pilot project over three years.

[0023] Figure 3 The graph shows the expression levels of the GhGLN1.3 gene under different nitrogen levels (CK, LN, HN1, HN2). CK (control, 2.5 mM), LN (low nitrogen, 0.25 mM), HN1 (high nitrogen 1, 5 mM), and HN2 (high nitrogen 2, 10 mM). Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1: Discovery and Identification of SNP Sites

[0026] 1. Test materials.

[0027] The test material consisted of a natural population of 379 cotton germplasm resources, including the main varieties promoted in my country's major cotton-producing areas and regional trial materials. This population had a wide range of sources, rich genetic variation, and covered the superior germplasm widely used in cotton breeding in my country.

[0028] 2. DNA extraction

[0029] Genomic DNA was extracted from the test materials. DNA was extracted from young cotton leaves using the CTAB method. The specific steps are as follows:

[0030] (1) Take about 0.5g of tender cotton leaves and grind them into powder in liquid nitrogen;

[0031] (2) Transfer the powder to preheated CTAB extraction buffer (2% CTAB, 1.4M NaCl, 20mM EDTA, 100mM Tris-HCl, pH 8.0, 1% β-mercaptoethanol), and incubate in a water bath at 65°C for 30-60 minutes, inverting occasionally to mix.

[0032] (3) Add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1), gently invert to mix, and let stand at room temperature for 10 minutes;

[0033] (4) Centrifuge at 12,000 rpm for 10 minutes, and transfer the supernatant to a new centrifuge tube;

[0034] (5) Add 2 volumes of pre-cooled anhydrous ethanol and let stand at -20°C for 30 minutes to precipitate DNA;

[0035] (6) Centrifuge at 12000 rpm for 10 minutes, discard the supernatant, and wash the precipitate twice with 70% ethanol;

[0036] (7) After drying at room temperature, add an appropriate amount of TE buffer to dissolve the DNA and store it at -20℃ for later use.

[0037] DNA integrity was detected by agarose gel electrophoresis, and DNA concentration and purity (OD260 / OD280 between 1.8 and 2.0, OD260 / OD230 greater than 2.0) were detected by NanoDrop spectrophotometer.

[0038] 3. Genotyping

[0039] Genotyping was performed on 379 tested materials using sequencing. A SNP site was found at positions 50,814,137 on chromosome 4 of the cotton D subgenome (based on the upland cotton reference genome version Gossypium hirsutum(AD1)'TM-1'genome CRI_v1), specifically 725 bases upstream of the transcription start point of the GhGLN1.3 gene. Figure 1 ), which corresponds to the 201st bit from the 5' end of SEQ ID NO.1.

[0040] SEQ ID NO.1: GAGAAAAATATAAATACTCTAAGATACCACCTACTAAGGGTTTTCTTTTCATTATCAAAATCTTAATACGTTAAACAGTACTTCGAGTTCTTTTATTTATCTAGTTTTTGATTCTAGAACGATTGAATTGACCTCTTTTCATATCATCCCTTTCCTTTTCACTTGTGTTAATATTGTGTAACAATAATGGTACGGTGAA [T / C]ATGGACCAAACACGCGACAAAAGAATACAAGAAAGACGTATAACATTCAAAAAAACACTAATTATATTTTATCTCGAAATTCACTTGATTACTTGGTTTTAGATATTTACAAATTTTACAACTTTCCACAACCAACTAATTTTTTCAATTTTTTTTATTGCTTGGATGATTTTTATTAGCTATTTTGTTTATTTGTTGA.

[0041] The genotype of the SNP locus with T / T is denoted as TT genotype, and the genotype with C / C is denoted as CC genotype.

[0042] 4. Survey of Clothing Characteristics

[0043] The lint percentage (the percentage of lint weight to seed cotton weight) of each tested material was investigated at maturity. Ten representative plants were randomly selected from each material, and the lint percentage was calculated after harvesting and ginning. The average value was taken as the phenotypic data of the material. Lint percentage (%) = lint weight / seed cotton weight × 100%.

[0044] 5. Statistical Analysis

[0045] Analysis was performed using a combination of genotype and pelvic distribution phenotypic data. In 379 samples, the pelvic distribution of the TT genotype was significantly higher than that of the CC genotype (P<0.05). Figure 2 The SNP molecular markers (A1, A2, A3, B1, B2, B3, C1, C2, C3) indicate that this SNP molecular marker is closely associated with pelvic trait.

[0046] Example 2: Application of SNP molecular markers in breeding

[0047] 1. Primer design

[0048] Based on the SNP site information of the promoter region of the GhGLN1.3 gene on chromosome 4 of the cotton D subgenome, specific primers were designed. The primer sequences are as follows:

[0049] Forward primer F (SEQ ID NO.2): 5'-TTCCTTTTTCACTTGTGTTAATATTGTG-3';

[0050] Reverse primer R (SEQ ID NO.3): 5'-CGTCTTTCTTGTATTCTTTTGTCGCGTG-3'.

[0051] 2. DNA extraction

[0052] Genomic DNA was extracted from the cotton material to be tested using the CTAB method (same as in Example 1). The concentration and purity of the DNA were detected using a NanoDrop spectrophotometer. The DNA concentration was diluted to 50~100 ng / μL and stored at -20℃ for later use.

[0053] 3. PCR amplification

[0054] Using the cotton genomic DNA to be tested as a template, PCR amplification was performed using the primers shown in SEQ ID NO.2 and SEQ ID NO.3. The PCR amplification system was 25 μL, including: 1 μL of cotton genomic DNA to be tested (50~100 ng / μL), 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 12.5 μL of 2×TaqPlus Master Mix, and ddH2O to a final volume of 25 μL.

[0055] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 35 cycles; 72℃ final extension for 5 min; and storage at 4℃.

[0056] The PCR amplification products were subjected to 1.5% agarose gel electrophoresis to confirm that the amplification product bands were clear, single, and the fragment size was correct.

[0057] 4. Genotyping

[0058] The PCR amplification products were sent for sequencing, and the genotype of the SNP locus was determined based on the sequencing peak pattern. If the locus was T / T homozygous, the genotype of the material was TT; if it was C / C homozygous, the genotype of the material was CC.

[0059] 5. Breeding Applications

[0060] Using the SNP molecular markers provided by this invention for marker-assisted selection allows for the identification and screening of nitrogen-efficient use traits in the early stages of cotton growth. This significantly shortens the breeding cycle, reduces field screening workload, improves breeding efficiency, and lowers breeding costs. Cotton plants with the TT genotype exhibit significant nitrogen-efficient use, with a significantly higher lint percentage than cotton plants with the CC genotype, indicating that this marker can be used for early screening of nitrogen-efficient use-related traits.

[0061] Example 3: Expression analysis of the GhGLN1.3 gene

[0062] 1. Material handling

[0063] Representative cotton materials were selected, and four nitrogen level treatments were set up: low nitrogen (LN, 0.25 mM), control (CK, 2.5 mM), high nitrogen 1 (HN1, 5 mM), and high nitrogen 2 (HN2, 10 mM). Cotton seedlings were cultivated in a greenhouse until they reached the three-leaf stage, and then different nitrogen treatments were started. Seven days after treatment, leaf samples were collected, quickly frozen in liquid nitrogen, and then transferred to a -80℃ freezer for storage.

[0064] 2. RNA extraction

[0065] Total RNA was extracted from the leaves of each treated sample using the TRIzol method. The specific steps are as follows:

[0066] (1) Take about 100 mg of leaf tissue and grind it into powder in liquid nitrogen;

[0067] (2) Add 1 mL of TRIzol reagent, shake well to mix thoroughly, and let stand at room temperature for 5 minutes;

[0068] (3) Add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes;

[0069] (4) Centrifuge at 12000 rpm and 4℃ for 15 minutes, and transfer the upper aqueous phase to a new centrifuge tube;

[0070] (5) Add an equal volume of isopropanol, mix gently, and let stand at -20°C for 30 minutes;

[0071] (6) Centrifuge at 12000 rpm and 4℃ for 10 minutes, then discard the supernatant;

[0072] (7) Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 7500 rpm and 4℃ for 5 minutes, and discard the supernatant;

[0073] (8) After drying at room temperature, add 30 μL of DEPC-treated water to dissolve the RNA and store at -80℃ for later use.

[0074] RNA concentration and purity (OD260 / OD280 between 1.9 and 2.1) were detected using a NanoDrop spectrophotometer, and RNA integrity (28S and 18S rRNA bands were clear and bright) was detected using 1.5% agarose gel electrophoresis.

[0075] 3. Reverse transcription to synthesize cDNA

[0076] Using the extracted total RNA as a template, cDNA was synthesized using a reverse transcription kit. The reverse transcription system consisted of 20 μL: 1 μg total RNA, 4 μL 5× reverse transcription buffer, 2 μL dNTP mixture (10 mM), 1 μL Oligo(dT) primer (10 μM), 1 μL reverse transcriptase, 0.5 μL RNase inhibitor, and DEPC-treated water to a final volume of 20 μL. The reverse transcription program was: incubation at 37°C for 15 minutes, denaturation at 85°C for 5 seconds, and storage at 4°C.

[0077] 4. Real-time quantitative PCR (qRT-PCR)

[0078] Using cDNA obtained through reverse transcription as a template and cotton GhActin or GhUBQ7 as an internal reference gene, the expression level of the GhGLN1.3 gene under different nitrogen treatments was detected by real-time quantitative PCR. The qPCR primer sequences used to detect the expression level of the GhGLN1.3 gene are as follows:

[0079] Forward primer F (SEQ ID NO.4): 5'-AACTAAGTGGCCTCTTGGATGGC-3';

[0080] Reverse primer R (SEQ ID NO.5): 5'-GCATTTTCTGATCGATGTCATG-3'.

[0081] The qRT-PCR reaction system consisted of 20 μL: 2 μL cDNA template (diluted 5-fold), 10 μL 2×SYBR Green Master Mix, 0.5 μL each of upstream primer (10 μM) and downstream primer (10 μM), and ddH2O to a final volume of 20 μL. Three replicates were set up for each sample.

[0082] The qRT-PCR reaction program was as follows: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds; annealing and extension at 60℃ for 30 seconds, for a total of 40 cycles; after the reaction, melting curve analysis was performed (fluorescence signal was collected every 0.5℃ from 65℃ to 95℃) to verify the specificity of the amplified products.

[0083] qRT-PCR analysis showed that the expression level of the GhGLN1.3 gene under LN treatment was higher than that under CK, HN1, and HN2 treatments. Figure 3 This indicates that the gene is induced by low nitrogen stress and is involved in the adaptive response of cotton to low nitrogen environment.

[0084] In summary, this invention discloses a SNP molecular marker related to nitrogen-efficient use in cotton and its applications. Through genotyping and lint percentage analysis of cotton germplasm resources, this invention discovered that the SNP site located in the promoter region of the GhGLN1.3 gene is associated with nitrogen-efficient use, with the lint percentage of TT genotype materials significantly higher than that of CC genotype materials. Simultaneously, the expression level of the GhGLN1.3 gene significantly increased under low nitrogen stress. This invention also provides a primer set for detecting this SNP. This SNP molecular marker can be used for marker-assisted selection breeding of nitrogen-efficient use-related traits in cotton, and has broad application prospects.

[0085] 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 SNP molecular marker related to efficient nitrogen utilization in cotton, characterized in that, The SNP site is located at bases 50, 814, 137 on chromosome 4 of the cotton D subgenome, which is 725 bases upstream of the transcription start point of the GhGLN1.3 gene, and exhibits T / C polymorphism. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.

1. This sequence is a 200bp flanking fragment upstream and downstream of the target SNP site, with base T or C at position 201.

2. The SNP molecular marker according to claim 1, characterized in that, The SNP molecular markers include genotypes TT and CC; among them, cotton materials with genotype TT have a significantly higher lint percentage than cotton materials with genotype CC.

3. A specific primer set for detecting the SNP molecular marker of claim 1 or 2, characterized in that, The primer set includes an upstream primer as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.

3.

4. A molecular marker method for identifying nitrogen-efficient utilization traits in cotton, characterized in that, Identification using the SNP sites described in claim 1 or 2 includes the following steps: Genomic DNA was extracted from the cotton plants to be tested, and the genotypes of the SNP sites were detected. Cotton plants with the TT genotype showed significantly high nitrogen utilization efficiency, and their lint percentage was significantly higher than that of cotton plants with the CC genotype.

5. The method according to claim 4, characterized in that, Includes the following steps: (1) Extract genomic DNA from the cotton sample; (2) Using the genomic DNA as a template, perform PCR amplification using the primer set described in claim 3 to obtain PCR amplification products; (3) Perform genotyping detection on the PCR amplification products to determine the genotype of the SNP molecular marker; (4) Determine the nitrogen-efficient use trait of the cotton plant to be tested based on the genotype; cotton plants with genotype TT show obvious nitrogen-efficient use, and their lint percentage is significantly higher than that of cotton plants with genotype CC.

6. The method according to claim 5, characterized in that, The PCR amplification reaction system described in step (2) is 25 μL, including: 1 μL of cotton genomic DNA to be tested at a concentration of 100 ng / μL, 1 μL each of upstream and downstream primers at a concentration of 10 μM, 12.5 μL of 2×Taq Plus Master Mix, and the remainder is ddH2O; The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 35 cycles; 72℃ final extension for 5 min.

7. The application of the SNP molecular marker as described in claim 1 or 2 in the breeding of nitrogen-efficient cotton varieties, characterized in that, The genotype of the SNP molecular marker described in claim 2 is detected, and cotton plants with the genotype TT are selected as breeding parents or screened.

8. The application of the SNP molecular marker as described in claim 1 or 2 in identifying or assisting in the identification of traits related to efficient nitrogen use in cotton.

9. A method for breeding a high-nitrogen-efficiency cotton variety, characterized in that, Extract genomic DNA from the cotton plant to be tested, detect the genotype of the SNP molecular marker described in claim 2, and select cotton plants with the TT genotype as breeding parents or for screening.

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