SNP molecular markers associated with cottonseed kernel oil content and protein content in upland cotton and uses thereof

Abstract

This invention discloses SNP molecular markers simultaneously associated with oil and protein content in upland cotton seed kernels and their applications. The SNP molecular markers are at least one of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.57. The SNP molecular markers related to oil and protein content in upland cotton seed kernels provided by this invention can be used for early prediction and screening of these traits. These molecular markers exist in DNA form, can be detected in various tissues and developmental stages of cotton, are unaffected by season or environment, and do not involve expression issues. These markers have no effect on the expression of the target trait, exhibiting neutrality. In genome screening, typically only + / - analysis is required, without analyzing fragment length, which facilitates the development of automated technologies for SNP screening or detection. Validation experimental results show that all SNP sites screened by this invention have a significant impact on the variation of oil and protein content traits in upland cotton seed kernels.

Description

Technical Field

[0001] This invention relates to SNP molecular markers that are simultaneously associated with the oil and protein content of upland cottonseed kernels and their applications, belonging to the fields of molecular biology and bioinformatics. Background Technology

[0002] Cotton is not only a fiber crop vital to national welfare and people's livelihood, but also an important oilseed and protein crop. Cottonseed is a significant byproduct of cotton processing, accounting for approximately 60% of the weight of seed cotton. Cottonseed is rich in cottonseed protein and cottonseed oil; the former is a high-quality animal protein feed, and the latter is an important edible vegetable oil. Fully utilizing cottonseed resources is crucial for establishing a modern cotton industry system and increasing cotton farmers' income. Studies have shown a strong negative correlation between protein and oil content in cottonseed. During cottonseed maturation, the carbon source required for the biosynthesis of both proteins and oils comes from carbohydrates synthesized through cotton photosynthesis, leading to competition between them for carbon sources. In some cotton germplasm resources collected by breeders, the protein and oil content of cottonseed has exceeded 65%, indicating significant room for improvement in both protein and oil content. Identifying and recognizing molecular markers related to cottonseed protein and oil traits to guide the breeding of high-oil, high-protein cottonseed has significant application value and contributes to the sustainable and healthy development of the cotton industry.

[0003] Genome-wide association analysis (GWAS) is a method for analyzing the genetic basis of traits at the population level, aiming to discover the association between genetic variation and specific traits. By analyzing genomic and phenotypic data from a large number of cotton germplasm resources, genetic variation sites closely related to these traits can be identified, thereby screening candidate genes or regulatory elements. These molecular markers can be used in molecular-assisted selection (MAS) breeding to accelerate the selection of traits such as cotton seed yield and quality, and to breed new varieties that meet market demands.

[0004] With the rapid development of high-throughput DNA sequencing technology, we performed high-throughput DNA sequencing on 852 cotton population resources, obtaining a large amount of single nucleotide polymorphism (SNP) data. Using this SNP data, GWAS analysis of cottonseed kernel oil and protein content revealed SNP molecular markers simultaneously associated with both oil and protein content. Utilizing these SNPs for assisted breeding selection can rapidly and accurately screen cotton varieties with high oil and protein content, meeting market demand for high-quality cotton products. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a batch of SNP molecular markers that are simultaneously associated with the oil and protein content of upland cotton seed kernels and their applications.

[0006] To achieve the above objectives, one aspect of the technical solution of the present invention is to provide an SNP molecular marker that is simultaneously associated with the oil and protein content of cottonseed kernels of upland cotton, wherein the SNP molecular marker is at least one of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.57.

[0007] Furthermore, the SNP molecular site undergoes a mutation at the 51st bp of the sequence, and the mutation form of the SNP molecular marker is as follows:

[0008]

[0009]

[0010] On the other hand, the technical solution of the present invention is to provide an application of the SNP molecular marker in the early prediction and screening of oil and protein content in upland cotton seed kernels.

[0011] On the other hand, the technical solution of the present invention is to provide an application of the SNP molecular marker in molecular marker-assisted breeding of upland cotton with high cotton seed oil content and high protein content.

[0012] On the other hand, the technical solution of the present invention is to provide an application of the SNP molecular marker in the improvement of upland cotton germplasm resources.

[0013] On the other hand, the technical solution of the present invention is to provide an application of the SNP molecular marker in identifying upland cotton varieties with high cotton seed oil content and high protein content.

[0014] On the other hand, the technical solution of the present invention is to provide a product for detecting the SNP molecular marker, wherein the product is a primer, reagent or kit.

[0015] On the other hand, the technical solution of the present invention is to provide a gene chip containing the SNP molecular marker.

[0016] On the other hand, the technical solution of the present invention is to provide the application of the product or the gene chip in the early prediction and screening of the oil and protein content of upland cotton kernels, molecular marker-assisted breeding of upland cotton with high oil and protein content, improvement of upland cotton germplasm resources, or identification of upland cotton varieties with high oil and protein content.

[0017] On the other hand, the technical solution of the present invention is to provide a method for analyzing the oil content and protein content of upland cottonseed kernels using the SNP molecular markers, comprising the following steps:

[0018] (1) Extract genomic DNA from the sample to be tested;

[0019] (2) Using the extracted DNA as a template, primers were designed based on SNP molecular markers, and PCR amplification was performed respectively;

[0020] (3) Based on the PCR amplification products, predict the oil content and protein content of upland cottonseed kernels.

[0021] The beneficial effects of this invention are:

[0022] This invention provides SNP molecular markers associated with oil and protein content in upland cotton seed kernels, which can be used for early prediction and screening of these traits. These molecular markers exist in DNA form, are detectable in various tissues and developmental stages of cotton, are unaffected by season or environment, and do not involve expression issues. These markers have no effect on the expression of the target trait, exhibiting neutrality. SNP markers are suitable for rapid, large-scale screening. In genomic screening, typically only + / - analysis is required, without analyzing fragment length, which facilitates the development of automated technologies for SNP screening or detection. Validation experimental results show that all SNP sites screened by this invention have a significant impact on the variation of oil and protein content traits in upland cotton seed kernels. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0024] Example: Obtaining SNP molecular markers

[0025] (1) Determination of oil and protein content in cottonseed kernels of cotton population materials:

[0026] In 2022, 852 cotton population materials were tested at two locations with three replicates at the experimental base. All population materials were randomly planted before the experiment. Experimental plots were planted in single-row sections with 2m rows, and each row contained 10-30 plants. Field management followed local field production practices. All cottonseed samples were harvested between September 20th and October 20th (specific dates depended on local frost dates and cultivation methods). Excluding the two cotton plants at each end of each row, 1-2 bolls were taken from the middle of each plant near the main stem, for a total of 20 normally developed and generally uniform bolls. These bolls were then ginned, dried, and stored in a 41℃ seed storage cabinet. The oil and protein content of the harvested cottonseed samples from each plot was determined using a near-infrared rapid component analyzer (FOSSDanmark) from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. Specifically, after delinting, cottonseed samples were placed in an environment with a temperature of 25℃ and a relative humidity of 60% for moisture equilibration. Then, the protein and oil content of the cottonseed was determined using a near-infrared rapid component analyzer. Each sample was scanned three times, and the average value was taken as the protein and oil content value of that cottonseed sample. The results were analyzed using a t-test. A p-value less than 0.05 indicated a significant difference.

[0027] (2) Detection of SNP molecular markers:

[0028] 852 upland cotton samples were collected for genome resequencing. First, seeds of various varieties were sown in an incubator, and young leaves from cotton plants were collected as samples. Then, 5 μg of high-quality cotton genomic DNA was extracted from each sample using the CTAB method. The extracted genomic DNA was sent to BGI Genomics Co., Ltd. in Shenzhen for genome resequencing. A high-quality upland cotton TM-1 (G. hirsutum 'Texas Marker 1') was selected as the reference genome, and all unassembled contigs were ligated to a pseudo-chromosome (named "ChrUN") before mapping. Using BWA (v.0.7.12) alignment software, the short sequences of the 852 samples were mapped to the reference genome, and all misaligned reads and low-quality reads with a mapping quality less than 20 were removed. Then, GATK UnifiedGenotyper (v.3.8.0) was used to identify variants in each sample, and the variant files of all 852 samples were merged into a single VCF file. Based on the criteria of minor allele frequency greater than 0.05 and deletion rate less than 0.2, we further filtered variant sites using VCFtools, identifying 1,247,929 high-quality SNPs for subsequent genome-wide association analysis. Finally, we annotated all variants using ANNOVAR.

[0029] (3) Genome-wide association analysis of oil and protein content traits in upland cottonseed kernels:

[0030] Genome-wide association analysis was performed on the traits of oil content and protein content in upland cottonseed kernels. Using the phenotypic data of oil content and protein content in upland cottonseed kernels obtained in step (1) and the genotypic data obtained in step (2), statistical analysis was conducted using a mixed linear model in the Efficient Mixed-Model Association Expedited (EMMAX) software (http: / / csg.sph.umich.edu / kang / emmax / download / index.html). The statistical model is as follows:

[0031] y = Xα + Zβ + Wμ + e

[0032] y represents the phenotypic trait, X is the indicator matrix for fixed effects, and α is the estimated parameter for fixed effects; Z is a design matrix indicating SNP effects, and β is the parameter for SNP effects; W is the indicator matrix for random effects, μ is the predicted random individual, and e is the random residual, following the order e ~ (0, δ). e 2 ).

[0033] In this model, population analysis was corrected by incorporating a kinship matrix into μ. Analysis revealed a total of 57 SNPs that were simultaneously and significantly associated with oil and protein content in upland cotton seed kernels. The allele loci information for these SNPs is shown in Table 1. The reference sequence is for the upland cotton cultivar TM-1, with the reference genome version number G.hirsutum_TM-1_ICR, available at http: / / grand.cricaas.com.cn / page / download / download. The nucleotide sequences 50 bp upstream and downstream of these SNP sites are shown in SEQ ID NO.1-SEQ ID NO.57.

[0034] Table 1. SNP molecular markers that are simultaneously associated with oil and protein content in upland cotton seed kernels.

[0035]

[0036]

[0037] We used Student's t-test to assess the differences in protein content before and after mutation at each of the 57 SNP sites listed in the table above across 852 upland cotton populations. The results showed that all p-values ​​were less than 0.05, indicating a significant difference in protein content before and after mutation. Similarly, we used the same test method for cottonseed oil content, and found that the statistical p-values ​​for all SNP markers were also less than 0.05, demonstrating a significant difference in cottonseed oil content before and after mutation.

[0038] Continued from Table 1

[0039]

[0040]

[0041] (4) Verification: The effect of the above SNP loci was further verified by using the BLUP values ​​(best linear unbiased prediction values) of cottonseed kernel oil content and protein content in 10 environments at 5 locations over 2 years from another 1279 upland cotton core germplasm resources. The results showed that 100% of the SNP loci had a significant impact on the phenotypic variation of cottonseed kernel oil content and protein content in upland cotton.

Claims

1. The application of a combination of SNP molecular markers in the early prediction and screening of oil and protein content in upland cotton seed kernels, characterized in that, The SNP molecular marker combination is the nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.57; the mutation sites of the SNP molecular markers are located at the 51st bp of the sequences SEQ ID NO.1-SEQ ID NO.57, and the mutation forms of the SNP molecular markers are as follows: 。 2. The application of an SNP molecular marker combination in marker-assisted breeding of upland cotton with high cottonseed oil content or high protein content, characterized in that, The SNP molecular marker combination is the nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.57; the mutation sites of the SNP molecular markers are located at the 51st bp of the sequences SEQ ID NO.1-SEQ ID NO.57, and the mutation forms of the SNP molecular markers are as follows: 。 3. The application of a combination of SNP molecular markers in identifying upland cotton varieties with high cottonseed oil content or high protein content, characterized in that... The SNP molecular marker combination is the nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.57; the mutation sites of the SNP molecular markers are located at the 51st bp of the sequences SEQ ID NO.1-SEQ ID NO.57, and the mutation forms of the SNP molecular markers are as follows: 。 4. The application of a product or gene chip containing a combination of SNP molecular markers in the early prediction and screening of upland cottonseed oil and protein content, marker-assisted breeding of upland cotton with high cottonseed oil or high protein content, or in the identification of upland cotton varieties with high cottonseed oil or high protein content, characterized in that... The product is a primer, reagent, or kit; the SNP molecular marker combination is the nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.57; the mutation sites of the SNP molecular markers are located at the 51st bp of the sequences SEQ ID NO.1-SEQ ID NO.57, and the mutation forms of the SNP molecular markers are as follows: 。 5. A method for analyzing the oil and protein content of upland cottonseed kernels using SNP molecular marker combinations, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the extracted DNA as a template, primers were designed based on SNP molecular markers, and PCR amplification was performed respectively; (3) Based on the PCR amplification products, predict the oil content and protein content of upland cottonseed kernels; The SNP molecular marker combination is the nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.57; the mutation sites of the SNP molecular markers are located at the 51st bp of the sequences SEQ ID NO.1-SEQ ID NO.57, and the mutation forms of the SNP molecular markers are as follows: 。

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