Biomarker for identifying upland cotton variety with high cottonseed oil content and application of biomarker

Abstract

The invention discloses a biomarker for identifying upland cotton varieties with high cottonseed oil content and application of the biomarker, and relates to the technical field of biology. The biomarker comprises a GhiD03G013530 gene, a GhiD03G013680 gene and a GhiD12G004870 gene, and is characterized in that the GhiD03G013530 gene, the GhiD03G013680 gene and the GhiD12G004870 The CDS sequences of the GhiD03G013530 gene, the CDS sequences of the GhiD03G013680 gene and the CDS sequences of the GhiD03G013680 gene and the CDS sequences of the GhiD12G004870 gene are shown as SEQ ID NO. 1 to SEQ ID NO. 3 respectively. The biomarker provided by the invention can be used for effectively identifying upland cotton varieties with high cottonseed oil content, provides a genetic mechanism basis for promoting genetic breeding of cottonseed oil and accelerating cultivation of high-oil cotton varieties, and has profound significance for further improving the utilization value of cotton seeds.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a biomarker for identifying upland cotton varieties with high cottonseed oil content and application thereof. Background Art

[0002] Cotton is one of the most important oil crops. The cottonseed oil it contains accounts for about 20% of the total seed weight and is the second most valuable component in cotton after fiber, indicating that cottonseed oil is an important agricultural product with development potential.

[0003] Cotton is widely grown in my country. Cottonseed oil is a by-product of cotton. Compared with other oil crops, cottonseed oil is rich in linoleic acid and plays an important role in the treatment of cardiovascular diseases. In addition, cottonseed oil is also an excellent frying oil and a raw material for biodiesel.

[0004] At present, cotton research focuses on fiber quality and yield, and cottonseed oil is not given enough attention, which may be the main reason for the fluctuation of cottonseed oil production. Therefore, accelerating the breeding of high-oil cotton varieties will help improve my country's oil self-sufficiency rate, ensure my country's oil safety, and break through the "bottleneck" problem in the seed industry. Summary of the invention

[0005] The purpose of the present invention is to provide a biomarker for identifying upland cotton varieties with high cottonseed oil content and its application, so as to solve the problems existing in the above-mentioned prior art. The biomarker provided by the present invention can effectively identify upland cotton varieties with high cottonseed oil content. The present invention provides a genetic mechanism basis for promoting the genetic breeding of cottonseed oil and accelerating the cultivation of high-oil cotton varieties, which has far-reaching significance for further improving the utilization value of cottonseed.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a biomarker for identifying upland cotton varieties with high cottonseed oil content, wherein the biomarker is a Ghir_D03G013530 gene, a Ghir_D03G013680 gene and a Ghir_D12G004870 gene;

[0008] The CDS sequence of the Ghir_D03G013530 gene is shown in SEQ ID NO.1;

[0009] The CDS sequence of the Ghir_D03G013680 gene is shown in SEQ ID NO.2;

[0010] The CDS sequence of the Ghir_D12G004870 gene is shown in SEQ ID NO.3.

[0011] The present invention also provides a primer pair for detecting the above-mentioned biomarker. When the biomarker is the Ghir_D03G013530 gene, the primer pair comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.5;

[0012] When the biomarker is the Ghir_D03G013680 gene, the primer pair includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.6 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.7;

[0013] When the biomarker is the Ghir_D12G004870 gene, the primer pair includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.8 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.9.

[0014] The present invention also provides the use of a reagent for detecting the expression amount of the above-mentioned biomarker in preparing a detection product for identifying upland cotton varieties with high cottonseed oil content.

[0015] Furthermore, the detection product is a test kit.

[0016] Furthermore, the reagent is the above-mentioned primer pair.

[0017] The present invention also provides a detection product for identifying upland cotton varieties with high cottonseed oil content, including a reagent for detecting the expression amount of the above-mentioned biomarker.

[0018] Furthermore, the detection product is a test kit.

[0019] Furthermore, the reagent is the above-mentioned primer pair.

[0020] The present invention also provides the use of the above-mentioned biomarkers, primer pairs or detection products in identifying upland cotton varieties with high cottonseed oil content.

[0021] The present invention also provides a method for identifying a high cottonseed oil content upland cotton variety, comprising the steps of detecting the expression level of the above-mentioned biomarker in the upland cotton sample to be tested, and identifying the high cottonseed oil content upland cotton variety according to the expression level detection result;

[0022] When the biomarker is the Ghir_D03G013530 gene, the lower the expression level of the Ghir_D03G013530 gene, the higher the cottonseed oil content of the upland cotton sample to be tested;

[0023] When the biomarker is the Ghir_D03G013680 gene, the lower the expression level of the Ghir_D03G013680 gene, the higher the cottonseed oil content of the upland cotton sample to be tested;

[0024] When the biomarker is the Ghir_D12G004870 gene, a higher expression level of the Ghir_D12G004870 gene indicates a higher cottonseed oil content in the upland cotton sample to be tested.

[0025] The present invention discloses the following technical effects:

[0026] The present invention is based on whole genome association analysis, haplotype analysis, phenotypic variation analysis, transcriptome expression level analysis and gene annotation. A total of three candidate genes involved in oil metabolism were identified, namely Ghir_D03G013530 (GhHAD), Ghir_D03G013680 (GhTPI) and Ghir_D12G004870 (GhRAN1), and the gene functions were preliminarily verified by qRT-PCR analysis. The results showed that Ghir_D03G013530 may reduce cottonseed oil content by inhibiting ABA response and hydrolyzing PA, and Ghir_D03G013680 may limit TAG assembly by catalyzing the mutual conversion between DHAP and GAP and reducing the substrate of G-3-P. qRT-PCR analysis showed that the above two genes were expressed at higher levels in low-oil varieties, which is consistent with the analysis results of the present invention. Ghir_D12G004870 may inhibit the expression of AAPT and increase the conversion of PC to DAG by activating ethylene response. qRT-PCR analysis showed that the expression level of this gene was higher in high-oil varieties, indicating that the expression of this gene can promote the synthesis of cottonseed oil.

[0027] The invention provides a genetic mechanism basis for promoting genetic breeding of cottonseed oil and accelerating the cultivation of high-oil cotton varieties, and has far-reaching significance for further improving the utilization value of cottonseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1Distribution diagram of cottonseed oil content in different ecological zones and breeding periods; a is the cottonseed oil content in different cotton zones; b is the cottonseed oil content in different breeding periods; the letters above the violin plot indicate the degree of difference between different cotton zones, the same letters indicate no difference, and different letters indicate significant difference;

[0030] Figure 2 The population structure diagram of 146 natural populations of upland cotton; a is the principal component analysis diagram, circles of different colors represent germplasms from different cotton regions, yellow circles represent G1 subpopulations, and green circles represent G2 subpopulations; b is the phylogenetic analysis diagram; c is the population structure analysis when K=2, 3, yellow represents the first subpopulation, green represents the second subpopulation, and purple represents the third subpopulation;

[0031] Figure 3 Figure 2 is a haplotype analysis and expression verification diagram of the candidate gene Ghir_D03G013530; a is the local Manhattan plot of chromosome D03 and the strongly linked LD block region under the BLUP environment, as well as the gene structure of Ghir_D03G013530 and the number of different haploid SNP sites and germplasm; b is a box plot of cottonseed oil content and fatty acid content under different haplotypes; c is a genetic diversity analysis diagram of Ghir_D03G013530 in four breeding stages; d is a haplotype distribution diagram of Ghir_D03G013530 in four ecological cotton areas; e is a tissue-specific expression profile of Ghir_D03G013530; f is an expression analysis diagram of Ghir_D03G013530 in ovules of upland cotton varieties with different oil contents;

[0032] Figure 4 Figure 2 is a haplotype analysis and expression verification diagram of the candidate gene Ghir_D03G013680; a is a local Manhattan plot of chromosome D03 under the BLUP environment; b is a strongly linked LD block region of qOC-D03-3; c is a boxplot of cottonseed oil content, palmitic acid content, oleic acid content and linoleic acid content under different haplotypes; d is a haplotype distribution map of Ghir_D03G013680 in four ecological cotton areas; e is a genetic diversity analysis map of Ghir_D03G013680 at four breeding stages; f is a tissue-specific expression profile of Ghir_D03G013680; g is an expression analysis map of Ghir_D03G013680 in ovules of upland cotton varieties with different oil contents;

[0033] Figure 5Figure 2 is a haplotype analysis and expression verification diagram of the candidate gene Ghir_D12G004870; a is a local Manhattan plot of D12 staining and a strongly linked LD block region under the E4 environment, as well as the gene structure of Ghir_D12G004870 and the number of different haploid SNP sites and germplasm; b is a box plot of cottonseed oil content under different haplotypes; c is a genetic diversity analysis diagram of Ghir_D12G004870 at four breeding stages; d is a haplotype distribution diagram of Ghir_D12G004870 in four ecological cotton areas; e is a tissue-specific expression profile of Ghir_D12G004870; f is an expression analysis diagram of Ghir_D12G004870 in ovules of upland cotton varieties with different oil contents;

[0034] Figure 6 Schematic diagram of the functional hypothesis of candidate genes. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The CDS sequence of the Ghir_D03G013530 gene referred to in the present invention is shown as SEQ ID NO.1; the CDS sequence of the Ghir_D03G013680 gene is shown as SEQ ID NO.2; and the CDS sequence of the Ghir_D12G004870 gene is shown as SEQ ID NO.3.

[0041] Example 1

[0042] 1. Materials and Methods

[0043] 1.1 Phenotypic analysis of oil content in upland cotton seeds

[0044] 1.1.1 Test materials

[0045] 146 cotton germplasm resources in the cotton breeding laboratory of Zhejiang A&F University were selected as experimental materials (Table 1). In 2021, they were planted in Liaocheng City, Shandong Province (36°48′N, 115°41′E) and Huanggang City, Hubei Province (30°57′N, 114°92′E), and named E1 and E2. The seeds from 2020 to 2022 were harvested in Sanya City, Hainan Province (18°36′N, 109°17′E), and named E3 and E4. Cotton harvested under different environments was delinted and washed with concentrated sulfuric acid after ginning. After drying, the damaged and shriveled cotton seeds were removed, and the smooth seeds with no fiber attachment were obtained and stored in the seed bank for the subsequent determination of cottonseed oil content and fatty acid content.

[0046] Three high-oil varieties carrying different haplotypes, namely "CRI16", "CRI20", and "Xinluzhong4", and one low-oil variety, "CRI27", were planted in the farm garden of Zhejiang A&F University (30°20′N, 119°34′E). According to previous reports, the period of rapid accumulation of cottonseed oil is 20DPA-30DPA. Therefore, the present invention sets three sampling periods, with the flowering day as 0DPA, and samples are taken at 0DPA, 10DPA, and 20DPA, respectively. Three biological replicates are set, and eight cotton bolls are taken from each replicate, which are quickly frozen in liquid nitrogen. The ovules are then taken out and stored in a -80°C refrigerator.

[0047] Table 1146 Natural varieties of upland cotton

[0048]

[0049]

[0050]

[0051] 1.1.2 Field trial design

[0052] The planting method adopted randomized block design. The E1 material was planted in the experimental station of the Cotton Research Center in Liaocheng City, Shandong Province, with a row length of 5m, a row spacing of 0.6m, and a plant spacing of 0.3m. The E2 material was planted in the Modern Agricultural Science and Technology Demonstration Park in Huanggang City, Hubei Province, with a row length of 6m, a row spacing of 0.8m, and a plant spacing of 0.4m. The E3 and E4 materials were planted in the Yacheng Base of the China Cotton Research Institute in Sanya City, Hainan Province, with film-covered planting, a row length of 3m, a row spacing of 0.5m, and a plant spacing of 0.2m (Table 2).

[0053] Table 2146 Natural Population Field Experiment Design

[0054]

[0055] 1.1.3 Determination of cottonseed oil content and fatty acid content

[0056] The cottonseed oil content was determined according to the national standard GB / T 14488.1-200; the fatty acid content was determined according to the method in the reference literature “Yang Yan, Wang Xianlei, Li Guan. Analysis of fatty acid composition of cottonseeds of six Xinjiang upland cotton varieties [J]. Biotechnology, 2009, 19(04): 54-56”.

[0057] 1.1.4 Statistical analysis of cottonseed oil content phenotypic variation data

[0058] The best linear unbiased prediction (BLUP) of the four environments was calculated by the “lme4” R package, descriptive statistics and normal distribution test were statistically analyzed by the “pastecs” R package, variance analysis was performed using the LSD multiple test using the “agricolae” R package, environmental correlation analysis and phenotypic variation analysis were visualized using the “ggplot2” R package, and the screening of excellent varieties was obtained by cluster analysis using the “stats” R package.

[0059] 1.2 Genome-wide association analysis of cottonseed oil content in upland cotton

[0060] 1.2.1 Development of SNP markers

[0061] The present invention utilized the genotyping work completed by Novogene and the resequencing data already available in the laboratory. The Illumina Hiseq4000 platform was used for double-end sequencing during data collection to ensure that the average sequencing depth was not less than 10×. The experimental steps include the following key steps: First, use FastQC (version 0.11.9) to check the quality of the sequencing data, and then use Fastp (version: 0.23.2) to evaluate the quality of the paired-end reads of 146 germplasms; then, use Trimmomatic (version: 0.38) for quality control to obtain high-quality reads; then, all high-quality reads are aligned to the TM-1 reference genome HAU_v1.1 using BWA (version 0.7.17-r1188); then, use Picard to sort the alignment results and convert them to BAM format, and remove duplicate data; then, use GATK (version: 4.2.6.1)

[121] bcftools (version: 1.9) was used to identify variant sites and filter SNPs, and the filtering conditions included: "QD<2.0, QUAL<30.0, FS>60.0, MQ<40.0, MQ Rank Sum<-12.5, Read Pos Rank Sum<-8.0"; finally, VCFtools (version: 0.1.16) was used to screen out high-quality SNPs with a minimum allele frequency (MAF)>5% and a missing rate (missing rate)<20%. These steps together constitute the data processing and analysis process of the present invention.

[0063] 1.2.2 Annotation of SNP markers

[0064] According to the TM-1 reference genome and the reference genome annotation file in GFF3 format, the present invention uses ANNOVAR

[124] The SNPs obtained after screening were annotated and classified. The annotation of the variant sites covers the intergenic region, upstream / downstream region, intronic region and exonic region. In these regions, the upstream and downstream regions refer to the regions within 2Kb from the transcription start site and the transcription end site. SNPs in the exonic region are classified as nonsynonymous mutations (nonsynonymous SNV) and synonymous mutations (synonymous SNV).

[0065] 1.2.3 Analysis of population structure of 146 natural populations

[0066] Using MAF>5% and missing rate<20% as filtering conditions, 2222327 high-quality SNPs were screened out, and 6823 quadruple degenerate sites (4D SNP, base positions in DNA or RNA sequences, the third base at these positions can change but does not affect the corresponding amino acid sequence) were selected from them through python scripts as data sets for principal component analysis, phylogenetic analysis and population structure analysis.

[0067] 1.2.3.1 Principal Component Analysis

[0068] The principal component analysis of 6823 4D SNPs was performed using GCTA (v1.94.1) and Plink (v1.90b6.21) software. The first 20 principal components were selected to generate Eigenvec (eigenvector used to refer to square matrices in linear algebra) files. The principal component analysis was visualized using the “ggplot2” R package.

[114] .

[0069] 1.2.3.2 Phylogenetic analysis

[0070] The VCF file containing 6823 4D SNPs was used to calculate the distance matrix between each sample using VCF2Dis (v1.47) software (https: / / github.com / BGI-shenzhen / VCF2Dis), and then submitted to the online tool ATGC FastME2.0 (http: / / www.atgc-montpellier.fr / fastme / ) to calculate the evolutionary tree of biological sequences. The phylogenetic tree was constructed using the neighbor joining method, and the nwk file was generated, which was finally visualized using the "ggtree" R package.

[0071] 1.2.3.3 Group structure analysis

[0072] Admixture (version 1.3.0) software was used to analyze the population structure of 146 natural populations of upland cotton using 6823 4D SNPs, with k values ​​ranging from 1 to 10. The “ggplot2” R package was used to visualize the population structure analysis.

[0073] 1.2.4 Genome-wide association analysis

[0074] The 2222327 high-quality SNPs screened by filtration were subjected to genome-wide association analysis with the oil content (OC) of cotton seeds planted in four environments. The mixed linear model (MLM) of GEMMA (version: 0.98.5) was used to perform association analysis on the 2222327 high-quality SNP data. The threshold was set according to the Bonferroni correction, that is, P = 1 / n (n is the number of SNPs), and the threshold of the present invention should be P = 1 / 2222327 = 4.49979E -07 However, at this threshold, the screening condition is too strict and may filter out important genes related to oil content, so the threshold is set to 1E -05 (-log 10 (P) = 5), the circular Manhattan plot was visualized using the “CMplot” R package, and the linkage disequilibrium heat map (LD block) was drawn using the “LDheatmap” R package.

[0075] 1.2.5 Candidate gene analysis

[0076] The obtained high-quality SNPs were divided into QTL intervals according to the LD decay distance. In order to determine the candidate genes in the QTL interval, the genes 200 Kb upstream and downstream of the significant SNP sites in the strongly linked QTL interval were regarded as the interval of the candidate genes. Based on the HAU_v1.1 version, the amino acid sequences of the candidate genes were downloaded from the Cottongen database (https: / / www.cottongen.org / ), and the Arabidopsis amino acid sequences were downloaded from the TAIR database (https: / / www.arabidopsis.org / download / ). Subsequently, the amino acid sequences of the candidate genes were compared with the Arabidopsis amino acid sequence database using the local BLASTP program, and the significance threshold was set to E value <1×10 -20 After obtaining homologous genes, agriGO v2.0 was used for gene annotation. Genes with annotation information related to lipid synthesis were screened as candidate genes. The gene structure was visualized using the GSDS online tool (version: 2.0) (http: / / gsds.gao-lab.org / ).

[0077] In order to mine excellent SNP sites and perform haplotype analysis, the present invention uses Haploview software to determine the haplotype of the gene, analyzes the distribution of different haplotypes in domestic cotton areas, and uses VCFtools (version: 0.1.16) to calculate the nucleotide diversity values ​​of different breeding periods. The differences between haplotypes with different oil contents are visualized using the "ggplot2" R package, and the phenotypic differences between haplotypes are calculated using the "agricolae" R package.

[0078] The public transcriptome data of TM-1 were obtained from the SRA module of NCBI (https: / / www.ncbi.nlm.nih.gov / sra / ) (database number: PRJNA490626), and expression analysis was performed in different tissues (roots, stems, leaves, rings, sepals, bracts, filaments, pistils, anthers) and developmental stages (ovules and fibers 10 to 25 days after flowering). The data were processed using salmon software (version: 0.13.1) to quantify gene expression levels, which were then normalized to TPM values. The normalization formula took into account the read counts and exon length of each gene to ensure accurate reflection of gene expression levels in different samples. The normalized data were logarithmically transformed (log2(x+0.01)) and visualized using GraphPad Prism software (version: 10.0.3).

[0079] 1.2.6 qRT-PCR analysis

[0080] In order to verify the expression level of candidate genes, three high-oil varieties carrying different haplotypes, namely "CRI16", "CRI20", "Xinluzhong4" and one low-oil variety, "CRI27", were planted in the agricultural garden of Zhejiang Agricultural and Forestry University (30°20′N, 119°34′E). The day of flowering was 0 DPA, and samples were taken at 0 DPA, 10 DPA and 20 DPA, respectively. Three biological replicates were set, and eight cotton bolls were taken from each replicate, which were quickly frozen in liquid nitrogen. The ovules were then taken out and stored in a -80°C refrigerator. Total RNA was extracted using an RNA kit (Novozyme RC411), and after reverse transcription to obtain cDNA, real-time fluorescence quantitative PCR was performed. The reaction system is shown in Table 3, the reaction procedure is shown in Table 4, and the primer sequences are shown in Table 5.

[0081] qRT-PCR calculation method: GhActin4 (LOC107890338) was used as the internal reference gene, and the gene transcription level was quantified using 2 -ΔΔCT The expression data were visualized by GraphPad Prism software (version: 10.0.3).

[0082] Table 3 Real-time fluorescence quantitative PCR reaction system

[0083]

[0084] Table 3 Real-time fluorescence quantitative PCR reaction conditions

[0085]

[0086] Table 4 Real-time fluorescence quantitative PCR primers

[0087]

[0088] 2. Results and Analysis

[0089] 2.1 Analysis of phenotypic variation of cottonseed oil content in upland cotton

[0090] The cottonseed oil content of 146 natural populations of upland cotton collected from four environments was statistically analyzed. As shown in Table 5, the range of cottonseed oil content was between 12.13% and 30.66%, among which the average cottonseed oil content in the E4 (Sanya, Hainan 2021-2022) environment was the highest, at 20.27±2.69%. According to the Shapiro-Wilk test, the cottonseed oil content was nearly normally distributed. The coefficient of variation of cottonseed oil content was between 13.25% and 18.15%, indicating that the degree of phenotypic variation in the population was large and the genetic diversity within the population was high. The cottonseed oil content in the four environments was subjected to correlation analysis, and the correlation range was between 0.75 and 0.82. The higher correlation between environments indicated that the cottonseed oil content in different environments was stable. Combined with the variance analysis (Table 6), it was shown that the genotype had a significant effect on the cottonseed oil content (P<0.001), and the broad heritability (H 2 ) was 96.00%, indicating that genotype is the main factor determining the oil content of cottonseed, and it is suitable for the next step of genome-wide association analysis of this trait.

[0091] Table 5 Phenotypic variation of cottonseed oil content in 146 natural populations

[0092]

[0093] Table 6 Variance analysis of cottonseed oil content in 146 natural populations

[0094]

[0095] The experimental materials of the present invention are widely distributed. In order to explore the changes in cottonseed oil content from different cotton regions and different breeding periods, the cotton regions are first divided into five, namely Northwest Inland Region: NIR, 40 materials, Northern Specific Early-maturity Region: NSER, 7 materials, Yellow River Region: YRR, 63 materials, Yangtze River Region: YZRR, 23 materials and Foreign cotton regions (Foreign, 7 materials). Variance analysis was performed on cottonseed oil content in different cotton regions, such as Figure 1As shown in a, there is no significant difference in cottonseed oil content among different cotton ranges.

[0096] The present invention divides the breeding periods into four, namely the initial stage of cotton improvement (S1: 1920-1949, 4 materials), the cotton fiber yield increase breeding stage (S2: 1950-1979, 8 materials), the cotton fiber yield, fiber quality and disease resistance comprehensive improvement breeding stage (S3: 1980-1999, 21 materials), cotton pursuit of high-quality fiber, use of transgenic technology, and adaptation to mechanized harvesting breeding stage (S4: 2000-2020, 80 materials). Since the germplasm from S1 and S2 periods in the present invention is relatively small (a total of 12 materials), S1 and S2 are combined into one period, which is divided into three breeding periods in total (S1+S2, S3, S4). According to the cottonseed oil content performance of materials in different breeding stages, such as Figure 1 As shown in middle b, there was no significant difference in cottonseed oil content during the S1-S3 periods, but compared with the previous three periods, the cottonseed oil content decreased significantly in the S4 period. It is speculated that breeders had different breeding goals in different breeding periods, and cottonseed oil as a by-product of cotton may also be indirectly affected, so the cottonseed oil content showed a downward trend.

[0097] 2.2 Genome-wide association analysis and candidate gene screening for cottonseed oil content

[0098] 2.2.1 Distribution of GWAS population markers

[0099] The natural population library was resequenced using the Illumina HiSeq4000 platform with 150 bp paired-end reads. A total of 2222327 high-quality SNPs were obtained under the conditions of MAF>5% and missing rate<20%, which were used for GWAS of cottonseed oil content in 146 cotton germplasms, of which the At subgenome and the Dt subgenome contained 1382437 and 839890 SNPs, respectively, with average SNP densities of 12418.59 SNP / Mb and 13306.24 SNP / Mb, respectively, as shown in Table 7. The proportion of SNPs in each chromosome ranged from 3.59% in A04 to 18.78% in A08. Overall, A06 and A08 had the highest SNP density. Most of the SNPs in the At and Dt subgenomes are located in the intergenic regions, accounting for 89.2% and 79.2% respectively. The Dt subgenome has more SNPs located in the upstream and downstream regions of the gene (15.3%) than the At subgenome (7.8%), while the exon and intron genomic regions account for the least, accounting for only 0.7% and 2.2% in the At subgenome and 1.2% and 4.3% in the Dt subgenome, respectively. Among the SNPs in the coding region (coding sequence: CDS) of the At subgenome, synonymous mutations account for 66.2% and non-synonymous mutations account for 33.8%, while the SNPs in the coding region of the Dt subgenome account for 65.3% and non-synonymous mutations account for 34.7%.

[0100] Table 7 Distribution and frequency of single nucleotide polymorphisms (SNPs) in Upland cotton

[0101]

[0102]

[0103] 2.2.2 Analysis of Population Structure of 146 Natural Populations of Upland Cotton

[0104] The intersection of 2222327 high-quality SNPs and the quadruple degenerate sites in the whole genome was taken to obtain a total of 6823 4DSNPs. The present invention divides 146 natural populations of upland cotton into five major ecological zones: foreign, northwest inland, northern early maturing, Yangtze River Basin and Yellow River Basin. Figure 2 In a), the five ecological zones can be divided into two subgroups, among which the germplasms from the Northwest Inland Cotton Area and the Northern Extra Early Cotton Area are clustered together and named G1, while the germplasms from the Yangtze River Basin and the Yellow River Basin can be clearly clustered together and named G2. Foreign germplasms are distributed in two clusters and tend to be clustered with the germplasms of G1. Through phylogenetic analysis ( Figure 2b), it was found that the experimental materials of the present invention were mainly divided into two groups. The green circles in the evolutionary tree were mixed with some orange circles, and the orange circles were mixed with some green circles, which means that there may be a certain amount of mutual introduction and hybrid breeding between different regions between G1 and G2, resulting in the mutual penetration of genes and genetic loci of different types of upland cotton, but G1 and G2 were obviously clustered together, indicating that the genetic diversity of germplasm between regions is relatively stable, which is consistent with the principal component analysis. Finally, the population structure analysis of 146 upland cotton germplasm resources was carried out, and it was found that compared with K=3, the results were more ideal when K=2, and it can be clearly divided into two subgroups ( Figure 2 In c), there were 44 G1 accessions and 22 G2 accessions in the first subgroup, and 13 G1 accessions and 62 G2 accessions in the second subgroup. The results were consistent with principal component analysis and phylogenetic analysis.

[0105] 2.2.3 Genome-wide association analysis of cottonseed oil content traits in upland cotton

[0106] The present invention uses a total of 2222237 high-quality SNP loci to perform genome-wide association analysis with cottonseed oil content collected from four environments and its best linear unbiased estimate. The threshold of significant SNP used in the present invention is -log 10 (P) = 5, a total of 639 significant SNP sites were detected, 82 of which were distributed on the At subgenome. Except for chromosome A11, they were distributed on other chromosomes, with the largest distribution on chromosomes A04 and A09, 24 and 22 respectively. 557 were distributed on the Dt subgenome, and no SNPs were distributed on chromosome D11. The largest number of SNPs were distributed on chromosomes D03 and D12, 119 and 332 respectively. It is worth noting that there are 8 significant SNP sites on the Dt subgenome that are associated with all four environments (E1, E3, E4, BLUP), five on chromosome D03, two on chromosome D04, and one on chromosome D06. Secondly, 37 significant SNP sites were associated with three environments, 54 significant SNP sites were associated with two environments, and 540 SNP sites were only detected in a single environment. The SNP on chromosome D03 was the most detected in multiple environments, with a total of 45, -log 10 The range of (P) was between 5.01 (rsGhir_D03_44403700) and 7.30 (rsGhir_D03_44422716), and the PVE was 12.79%-18.49%. The second was chromosome D04, where 20 stable SNPs were detected, -log 10The range of (P) was between 5.22 (rsGhir_D04_4510014) and 7.11 (rsGhir_D04_3847520), and the PVE was 13.14%-18.02%. The most single-environment SNPs were detected on chromosome D12, with a total of 310, -log 10 The range of (P) was between 5.01 (rsGhir_D12_4177835) and 7.37 (rsGhir_D12_7289061), and the PVE was 12.59%-18.67%. The number of SNPs detected in different environments was different (Table 8). The most sites were detected in the E4 environment, and the PVE value in this environment was the highest. The cottonseed oil content measured in this environment was also higher than that in the other three environments.

[0107] Table 8 SNP distribution statistics under different environments

[0108]

[0109] Note: BLUP stands for Best Linear Unbiased Prediction.

[0110] 2.2.4 QTL positioning

[0111] According to LD block analysis, 2222327 significant SNPs were delineated into QTL intervals with a range of 300Kb (Table 9), and a total of 77 QTL intervals were divided, of which 48 were in the At subgenome, 10 were detected in multiple environments, and 38 were detected in a single environment; 29 were in the Dt subgenome, 8 were detected in multiple environments, and 21 were detected in a single environment. The present invention refers to the QTL interval located in two or more environments as a stable QTL. The present invention obtained a total of 18 stable QTLs (Table 10), which were located on 14 chromosomes: A02 (qOC-A02-1), A03 (qOC-A03-1), A04 (qOC-A04-1 and qOC-A04-10), A06 (qOC-A06-1), A07 (qOC-A07-3), A09 (qOC-A09-4), A100 (qOC-A100-5), A110 (qOC-A110-6), A120 (qOC-A120-7), A130 (qOC-A130-8), A140 (qOC-A140-9), A150 (qOC-A150-11), A160 (qOC-A160-12), A170 (qOC-A170-13), A180 (qOC-A180-14), A190 (qOC-A180-15), A201 (qOC-A180-16), A211 (qOC-A180-17), A221 (qOC-A180-18), A230 (qOC-A180-19), A240 (qOC-A240-2 A09-2 and qOC-A09-3), A10 (qOC-A10-3), A13 (qOC-A13-1), D03 (qOC-D03-3), D04 (qOC-D04-1), D 05 (qOC-D05-1), D06 (qOC-D06-1), D09 (qOC-D09-1 and qOC-D09-3) and D12 (qOC-D12-3 and qOC-D12-4). -log 10The range of (P) is between 5.06 (qOC-A07-3) and 7.96 (qOC-D09-1). The present invention found that a large number of significant SNPs were detected in chromosomes D03 and D12 under multiple environments, which were 119 and 322, respectively. Therefore, the present invention will further explore candidate genes in the QTL intervals of these two chromosomes.

[0112] Table 9 Distribution of QTL on chromosomes

[0113]

[0114]

[0115] The previous studies on cottonseed oil content QTL were summarized, and a total of 351 QTLs were found, with 183 QTLs in the At subgenome and 168 QTLs in the Dt subgenome. The QTLs intervals distributed in the At subgenome are more than those in the Dt subgenome, which is consistent with the present invention. The QTLs obtained by the present invention were compared with those studied by the previous researchers, and it was found that a total of 6 QTLs intervals had been reported by the previous researchers (Table 10). The newly identified sites of the present invention overlap with the QTL intervals associated with the previous researchers, indicating that the results of the whole genome association analysis of the present invention are credible, and the newly discovered QTL intervals also narrow the scope for the following exploration of candidate genes and excellent sites related to cottonseed oil content. The present invention will select QTL intervals that can be detected in multiple environments, with strong stability and significance, such as qOC-D03-3, qOC-D09-1 and qOC-D12-3, to mine candidate genes.

[0116] Table 10 Stable QTLs identified by genome-wide association analysis in multiple environments

[0117]

[0118]

[0119] Note: All means all environments are detected.

[0120] 2.2.5 Mining and identification of candidate genes

[0121] The present invention regards the QTL intervals identified in multiple environments as stable QTLs, and identifies 692, 511, 425, 771 and 559 candidate genes in E1, E2, E3, E4 and BLUP, respectively. Genes identified in multiple environments are considered stable candidate genes, and the present invention identifies a total of 479 stable candidate genes, which are then further annotated.

[0122] The amino acid sequences of candidate genes in all stable QTL intervals were compared with the Arabidopsis amino acid sequence database by local Blast, and the significance threshold was set at an E value < 1 × 10 -20 , obtained Arabidopsis homologous genes and annotated biological processes mainly include cellular processes, metabolic processes and single organism processes. Cellular components mainly include cell parts, cells and intracellular. Molecular functions mainly include catalytic activity, binding and transferase activity.

[0123] Oil metabolism is a complex biological process. Plant hormones and stress responses during plant growth will affect cottonseed oil synthesis and decomposition. The process of oil metabolism occurs in the plastid, endoplasmic reticulum and its membrane, and the binding and decomposition of ATP will occur during this process. Plant hormones such as abscisic acid and gibberellins may indirectly affect oil metabolism. At the same time, oil metabolism is also a secondary metabolic process. The homologous genes obtained by GO analysis were compared with the gene library related to oil in Arabidopsis (http: / / aralip.plantbiology.msu.edu / downloads). It was found that 11 Arabidopsis genes were related to oil metabolism. Corresponding to the candidate genes, a total of 15 upland cotton genes were involved in the synthesis and decomposition of oil.

[0124] Comprehensive analysis of all QTLs showed that significant SNPs were clustered on chromosomes D03 and D12. LD linkage analysis revealed that qOC-D03-3 and qOC-D12-3 were strongly linked and could be identified in multiple environments, so they were the main focus of research.

[0125] 2.2.5.1 Analysis of candidate genes on chromosome D03

[0126] The present invention found that a 1.092Mb QTL interval named qOC-D03-3 on chromosome D03 contains 117 significant SNPs, of which 67 (57%) significant SNPs overlapped in physical position with previous research reports on cottonseed oil candidate gene mining, proving that qOC-D03-3 is a reliable QTL interval related to cottonseed oil content and has a candidate gene for regulating cotton oil content. It is located between 43920725-45013278bp on chromosome D03, and the most significant SNP is rsGhir_D03_44422716, -log 10(P) was 7.3, located downstream of the gene, and was detected under E1, E3, E4 and BLUP. A total of 77 genes were distributed in qOC-D03-3, of which 21 candidate genes were annotated to have direct or indirect relationships with oil metabolism. Tissue-specific expression analysis and LD block linkage analysis were performed using public transcriptome data. Two genes were expressed in multiple tissues, and the expression level in ovules was also higher than that in fibers, and they were located in the strongly linked LD block region. Therefore, the present invention conducted further research on these two genes.

[0127] Ghir_D03G013530 (GhHAD), located in the range of 44467318-44472381 bp on chromosome D03 ( Figure 3 ), and its Arabidopsis homologous gene was determined to be AT5G59480 through sequence similarity analysis. This gene is a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily and is involved in abscisic acid response. There are five significant SNPs on this gene, namely rsGhir_D03_44466802 located in the upstream region of the gene and rsGhir_D03_44469197, rsGhir_D03_44469223, rsGhir_D03_44469360, and rsGhir_D03_44474072 located in the intron region of the gene. The -log 10 The range of (P) was between 5.01 and 5.81, and the PVE was 12.60%-14.29%, indicating that this gene can affect the metabolism of cottonseed oil. By drawing the LD heatmap ( Figure 3 In a), it is noted that the five SNPs are located in a 7.6Kb strongly linked LDblock region. In order to mine excellent SNP sites, haplotype analysis was performed, and the haplotype of Ghir_D03G013530 was determined using Haploview software. There are three haplotypes in total, namely Hap1: GAGGC (87), Hap2: AGCAT (49), and Hap3: GAGGT (6). There are large base differences between Hap2 and Hap1 and Hap3, but there is only a difference in rsGhir_D03_44474072 (Hap1: C, Hap3: T) between Hap1 and Hap3. The change of bases may affect the function of the gene. Therefore, the present invention analyzes the phenotypes under different haplotypes ( Figure 3b), the germplasm carrying Hap2 has higher cottonseed oil content and oleic acid content than the germplasm carrying Hap1 and Hap3, while there is no significant difference in the content of palmitic acid and linoleic acid between the three haplotypes. It can be concluded that the difference in oil content between cottonseeds carrying different haplotypes may be caused by oleic acid content. In addition, since the base difference between Hap1 and Hap3 is not large, there may be no significant difference in the phenotype between the two haplotypes. Hap2 has less germplasm than Hap1, but has higher oil content and two fatty acid contents. Therefore, the present invention believes that Hap2 is an excellent haplotype. The present invention speculates that rsGhir_D03_44466802, rsGhir_D03_44469197, rsGhir_D03_44469223, and rsGhir_D03_44469360 are the main effect SNPs of Ghir_D03G013530. By analyzing the genetic diversity of Ghir_D03G013530 at four breeding stages ( Figure 3 c), and found that as the breeding years went by, its genetic diversity decreased. The genetic diversity of germplasm cultivated before 1950 was much higher than that of germplasm cultivated after 2000, indicating that the gene was gradually selected. In order to further understand the relationship between the genetic characteristics and geographical distribution of the three haplotypes contained in Ghir_D03G013530 ( Figure 3 d), according to the geographical distribution of the present invention, the superior haplotype Hap2 is most widely distributed in the northern early-maturing cotton area, which is consistent with the result that high-oil varieties are more distributed in the northern early-maturing cotton area, and the northern early-maturing cotton area does not contain Hap3; the proportion of the three haplotypes in the other three cotton areas is: Hap1>Hap2>Hap3, indicating that the current cotton varieties are still mainly carrying Hap1. The tissue-specific expression analysis of Ghir_D03G013530 was performed, and it can be found from the results that ( Figure 3 e), Ghir_D03G013530 is expressed in all tissues. By comparing the expression levels between ovules and fibers, it can be clearly observed that at 10DPA, 15DPA, and 25DPA, the expression level of this gene in ovules is higher than that in fibers. Finally, the present invention uses "CRI16" (high oil material) and "CRI27" (low oil material) to perform qRT-PCR analysis on Ghir_D03G013530 ( Figure 3 (f) The results showed that the low-oil material "CRI27" carrying Hap1 had a higher expression level at each stage than the high-oil material "CRI16" carrying Hap2 (P<0.05), indicating that this gene may play a negative regulatory role in oil metabolism.

[0128] rsGhir_D03_44748621 is located in the strongly linked LD block region of qOC-D03-3 ( Figure 4 a, b), and are correlated in both E1 and BLUP, -log 10 (P) ranged from 5.00 to 5.26, and PVE was 12.58% to 13.26%. After genotyping of the associated population, it was found that rsGhir_D03_44748621 had two haplotypes, of which 44 germplasms contained AA haplotype and 100 germplasms contained GG haplotype. By comparing the oil content and fatty acid composition of germplasms carrying different haplotypes ( Figure 4 In (c), it can be found that germplasm carrying AA haplotype has higher oil content and oleic acid content, while germplasm carrying GG haplotype phenotypes have higher palmitic acid levels. The distribution results of AA and GG haplotypes in geographical environments show ( Figure 4 In (d), AA haplotypes are more distributed in NSER, indicating that high-oil varieties mainly come from NSER, which is consistent with the previous study. Therefore, rsGhir_D03_44748621 is a SNP site that affects cottonseed oil content and is worth studying. There are 32 genes within 200Kb upstream and downstream of this SNP site. According to gene annotation, Ghir_D03G013680 (GhTPI), which is 14343bp away from rsGhir_D03_4474862, may be a candidate gene involved in oil metabolism. According to homologous sequence alignment, the Arabidopsis homologous gene AT3G55440 was obtained. This gene is a triosephosphate isomerase. According to GO annotation, it is involved in biological processes such as phosphorus-containing compound metabolism (GO: 0006796), phosphorus metabolism (GO: 0006793), phosphorylation (GO: 0016310), and is related to the biosynthesis of glycerol triphosphate. By analyzing the genetic diversity of Ghir_D03G013680 at four breeding stages ( Figure 4 e), and found that its genetic diversity decreased with the passage of breeding years. Cotton varieties released before the 21st century showed greater diversity than varieties bred after the 21st century, indicating that this gene was gradually selected. Combined with public transcriptome data for analysis ( Figure 4 f), it was found that Ghir_D03G013680 was only expressed in leaves and ovules, and at 20DPA, the expression level in ovules was higher than that in fibers, but qRT-PCR analysis ( Figure 4 g), the results showed that the expression level of Ghir_D03G013680 in the "CRI20" high-oil variety was significantly lower than that in the "CRI27" low-oil variety at 10DPA and 20DPA (P<0.05). Therefore, the present invention speculates that Ghir_D03G013680 plays a negative regulatory role in the accumulation of cottonseed oil content.

[0129] 2.2.5.2 Analysis of candidate genes on chromosome D12

[0130] The present invention found a stable LD linkage region of 138.9 Kb on chromosome D12 ( Figure 5 a), detected simultaneously under E1, E4 and BLUP, named qOC-D12-3, containing a total of 302 significant SNPs associated with cottonseed oil content, -log 10 The range of (P) was between 5.03 (rsGhir_D12_7258064) and 7.37 (rsGhir_D12_7289061), and the PVE was 12.66%-18.68%. A total of 41 genes were included in this QTL interval. According to the homologous annotation of Arabidopsis thaliana, the present invention found that Ghir_D12G004870 (GhRAN1) may be a candidate gene for regulating the oil content of cottonseed.

[0131] The Arabidopsis homologous gene of Ghir_D12G004870 is AT5G44790, which is a member of the copper-transporting ATP hydrolase (RAN1) and is involved in the ethylene-activated signal transduction pathway, stomatal movement regulation, and the response to ethylene and copper ion homeostasis. Ethylene plays an important role in plant growth and development and physiological processes, and may indirectly affect the accumulation of cottonseed oil content. The upstream location of Ghir_D12G004870 ( Figure 5 In a), three significant SNPs (rsGhir_D12_7322034, rsGhir_D12_7322539, rsGhir_D12_7322849) were located, two significant SNPs (rsGhir_D12_7321013, rsGhir_D12_7321002) were located on UTR5, two significant SNPs (rsGhir_D12_7318248, rsGhir_D12_7318170) were located on the intron, and four significant SNPs (rsGhir_D12_7314392, rsGhir_D12_7314404, rsGhir_D12_7314742, rsGhir_D12_7315340) were located downstream, -log 10 The range of (P) was between 5.88-6.69, and the PVE was 14.9%-16.9%. The 11 significant SNPs were divided into two haplotypes: Hap1 (AGTAACTGGAT, 136 materials) and Hap2 (GAAGGACATGC, 8 materials), indicating that the germplasm carrying Hap1 was mainly used in the present invention. However, through phenotypic analysis, it can be found that ( Figure 5b), although the number of germplasms carrying the Hap2 haplotype is small, the oil content is significantly higher than that of the germplasm carrying the Hap1 haplotype (P<0.01), and there is no significant difference in the palmitic acid content, oleic acid content and linoleic acid content between the two haplotypes. Genetic diversity analysis of Ghir_D12G004870 at four breeding stages ( Figure 5 The results show that the genetic diversity of this gene in varieties cultivated from 2000 to the present is significantly lower than that in the other three breeding stages. The geographical distribution of the SNP of Ghir_D12G004870 was explored, and the results showed that ( Figure 5 d) Hap1 accounts for up to 100% on NSER and YZRR, and up to 92.5% and 91.9% on NIR and YRR. Tissue-specific analysis showed that Ghir_D12G004870 was expressed in all tissues ( Figure 5 In addition, qRT-PCR analysis showed that during the rapid oil accumulation period (20DPA-30DPA), the expression level of Ghir_D12G004870 in the high-oil material "Xinluzhong4" carrying Hap2 was significantly higher than that in the low-oil material "CRI27" carrying Hap1 at 10DPA and 20DPA ( Figure 5 f). Therefore, it can be considered that Hap2 carried by Ghir_D12G004870 has a positive regulatory effect on cottonseed oil content and is an excellent haplotype worthy of further in-depth study.

[0132] 2.2.6 Functional hypothesis of candidate genes in lipid metabolism pathway

[0133] Based on the known lipid metabolism pathway, the present invention proposes a working model to illustrate the functional role of three candidate genes in regulating lipid synthesis and accumulation in different subcellular compartments ( Figure 6 ), Kennedy pathway is known to be a key link in TAG assembly. GhTPI is located in the plastid, where FA is synthesized de novo. The expression of GhTPI converts DHAP to GAP, reducing important substrates in the Kennedy pathway, resulting in a decrease in cottonseed oil content. ER is an organelle that produces membrane lipids, epidermal lipids, and triglycerides (TAG). GhHAD and GhRAN1 are located on the cell membrane of ER. The expression of GhHAD inhibits TAG assembly by inhibiting ABA response and PA synthesis, while the expression of GhRAN1 activates the ETR1 ethylene response element, further increasing the expression of CTR1, and ultimately inhibiting AAPT synthesis, thereby increasing the conversion efficiency of PC Pool to DAG, and ultimately promoting the accumulation of cottonseed oil.

[0134] 2.2.7 Screening of materials carrying excellent haplotypes

[0135] Among the 146 upland cotton materials in the present invention, 21 high-oil materials were screened, and the haplotypes carried by them were analyzed (Table 11). The results showed that a total of 15 high-oil materials carried at least one excellent haplotype, among which "Xinlu Zao No. 6", "Sizi Cotton 2B", "Heishan Cotton No. 1", "Zhongmian Institute No. 91", "Jinmian No. 6", "Zhongmian Institute No. 30", "X25TF", "Zhongmian Institute No. 7" all carried excellent haplotypes of GhHAD and GhTPI. It is worth noting that the high-oil materials "Zhongmian Institute No. 16", "Zhongmian Institute No. 19" and "Zhongmian Institute No. 20" all carried 3 excellent haplotypes.

[0136] Table 11 High oil varieties carrying excellent haplotypes

[0137]

[0138] Note: All represents GhRAN1, GhTPI and GhHAD.

[0139] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biomarker for identifying upland cotton varieties with high cottonseed oil content, It is characterized in that The biomarkers are Ghir_D03G013530 gene, Ghir_D03G013680 gene and Ghir_D12G004870 gene; The CDS sequence of the Ghir_D03G013530 gene is shown in SEQ ID NO.1; The CDS sequence of the Ghir_D03G013680 gene is shown in SEQ ID NO.2; The CDS sequence of the Ghir_D12G004870 gene is shown in SEQ ID NO.

3.

2. A primer pair for detecting the biomarker according to claim 1, It is characterized in that When the biomarker is the Ghir_D03G013530 gene, the primer pair includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.5; When the biomarker is the Ghir_D03G013680 gene, the primer pair includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.6 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.7; When the biomarker is the Ghir_D12G004870 gene, the primer pair includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.8 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.

9.

3. Use of a reagent for detecting the expression amount of the biomarker according to claim 1 in preparing a detection product for identifying upland cotton varieties with high cottonseed oil content.

4. The use according to claim 3, It is characterized in that The detection product is a test kit.

5. The use according to claim 3, It is characterized in that The reagent is the primer pair according to claim 2.

6. A detection product for identifying upland cotton varieties with high cottonseed oil content, It is characterized in that Comprising a reagent for detecting the expression amount of the biomarker according to claim 1.

7. The detection product according to claim 6, It is characterized in that The detection product is a test kit.

8. The detection product according to claim 6, It is characterized in that The reagent is the primer pair according to claim 2.

9. Use of the biomarker according to claim 1, the primer pair according to claim 2, or the detection product according to any one of claims 6 to 8 in identifying upland cotton varieties with high cottonseed oil content.

10. A method for identifying upland cotton varieties with high cottonseed oil content, It is characterized in that The method comprises the steps of detecting the expression level of the biomarker according to claim 1 in the upland cotton sample to be tested, and identifying the upland cotton variety with high cottonseed oil content according to the expression level detection result; When the biomarker is the Ghir_D03G013530 gene, the lower the expression level of the Ghir_D03G013530 gene, the higher the cottonseed oil content of the upland cotton sample to be tested; When the biomarker is the Ghir_D03G013680 gene, the lower the expression level of the Ghir_D03G013680 gene, the higher the cottonseed oil content of the upland cotton sample to be tested; When the biomarker is the Ghir_D12G004870 gene, a higher expression level of the Ghir_D12G004870 gene indicates a higher cottonseed oil content in the upland cotton sample to be tested.

Images