SNP sites for simultaneously regulating oil and protein contents in cotton seeds and application thereof

Abstract

The application discloses a SNP site for simultaneously regulating oil content and protein content in cotton seeds and application thereof. The application takes whole genome resequencing results of a natural population of Gossypium hirsutum as genotype data, takes oil content and protein content in two-year cotton seeds obtained by near infrared spectrum analysis technology as phenotype data, and identifies a SNP site capable of simultaneously regulating oil content and protein content in cotton seeds by using whole genome association analysis. The site is located at 3579341bp of Gossypium hirsutum D13 chromosome, is a single base mutation from T to C, and the oil content of cotton seeds of the TT type is significantly higher than that of cotton seeds of the CC type. Meanwhile, the protein content of cotton seeds of the CC type is significantly higher than that of cotton seeds of the TT type. The SNP site can be applied to identifying germplasm resource materials with high oil content or high protein content in Gossypium hirsutum, and molecular marker assisted breeding of oil content and protein content in cotton seeds, and has important application value and application prospect in related research on formation of cotton seed nutritional quality and cultivation of new varieties with improved cotton seed nutritional quality.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology application, and relates to a SNP site for simultaneously regulating oil content and protein content in cotton seeds and application thereof. BACKGROUND

[0002] Cotton is the most important fiber crop in the world, and its huge economic value makes it an important trade crop. For a long time, cotton breeding workers have focused on improving cotton yield and fiber quality. At present, a large number of genes or QTLs related to cotton yield and fiber quality traits have been identified, and cotton varieties suitable for cultivation conditions and natural environment in China have also been bred. In comparison, there are fewer studies on the nutritional components of cotton seeds.

[0003] Cotton seeds mainly include oil, protein, carbohydrates and gossypol, and the contents are 25%-40%, 30%-45%, 15% and 1% respectively, which are mainly affected by cotton varieties and determination methods. Cottonseed oil has a unique fatty acid structure, with a high content of unsaturated fatty acids, accounting for 69.23%-77.27% of the total fatty acid content, including oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and the like. The content of saturated fatty acids accounts for 21.41%-29.68% of the total fatty acid content, including palmitic acid (C16:0), stearic acid (C18:0) and the like. Cotton seeds also contain rich high-quality protein. The protein in cotton seeds contains a balanced proportion of amino acids, and the types of amino acids are comprehensive and rich. The content of amino acids in cotton seeds is about 40%, and especially includes eight essential amino acids for the human body. Therefore, cotton seeds are a good source of oil and protein.

[0004] The protein content in cotton seeds is significantly negatively correlated with the oil content, so it is difficult to simultaneously improve the protein content and the oil content in cotton seeds in the process of breeding new cotton varieties with high oil and high protein. The protein and oil contents in cotton seeds are complex quantitative traits, which are regulated by multiple genes and are easily affected by the environment. Therefore, analyzing the regulatory sites of the protein and oil contents in cotton seeds can lay a foundation for the formation mechanism research of the protein and oil contents in cotton seeds, and has important significance for further improving the economic value of cotton. SUMMARY

[0005] The purpose of the present application is to provide a SNP site for simultaneously regulating oil content and protein content in cotton seeds and application thereof to overcome the shortcomings of the prior art. The results of whole genome association analysis show that the SNP site is closely related to the oil content and the protein content in cotton seeds.

[0006] In order to achieve the above object, the technical scheme of the present application is to provide a SNP site for simultaneously regulating the oil content and protein content in cotton seeds and application thereof:

[0007] The SNP site for simultaneously regulating the oil content and protein content in cotton seeds according to the present application is located at the base of 3579341 bp on chromosome D13 of the reference genome (ZJU TM-1V2.1) of the genetic standard line TM-1 of Gossypium hirsutum, and the nucleotide base of the site is "TT" or "CC":

[0008] The DNA sequence of the amplification product of the SNP site with TT haplotype is shown in SEQ ID NO. 1.

[0009] The DNA sequence of the amplification product of the SNP site with CC haplotype is shown in SEQ ID NO. 2.

[0010] Further, the nucleotide sequences of the amplification primers of the SNP site with 200 bp upstream and downstream are shown in SEQ ID NO. 3 and SEQ ID NO. 4, and the DNA sequence of the amplification product is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0011] The SNP site for simultaneously regulating the oil content and protein content in cotton seeds is applied in the positioning of genes closely linked to the oil content or protein content in cotton seeds.

[0012] The SNP site for simultaneously regulating the oil content and protein content in cotton seeds is applied in the cultivation of new varieties of cotton seeds with high oil content or high protein content through genetic engineering technology.

[0013] The SNP site for simultaneously regulating the oil content and protein content in cotton seeds is applied in the identification of Gossypium hirsutum varieties with high oil content or high protein content: detecting the SNP site, selecting cotton with T as the base at the position of 3579341 bp on chromosome D13 as the cotton variety with high oil content, and selecting cotton with C as the base at the position of 3579341 bp on chromosome D13 as the cotton variety with high protein content.

[0014] Further, the primers for detecting the SNP site are as follows: an upstream primer is shown in SEQ ID NO. 3, and a downstream primer is shown in SEQ ID NO. 4.

[0015] The beneficial effects of the present application are as follows:

[0016] The present application identifies a SNP site closely related to the oil content and protein content in cotton seeds through resequencing data of a natural population of cotton varieties (lines) and whole genome association analysis, and the site can be used for molecular marker assisted breeding of the oil content and protein content in cotton seeds. The SNP site provided by the present application has good application value in breeding of new varieties with improved oil content and protein content in cotton seeds.

[0017] The SNP site provided by the present application can be applied to screening of cotton germplasm with high oil content or high protein content, and lays a foundation for breeding of new varieties with improved oil content and protein content in cotton seeds.

[0018] The SNP site provided by the present application can be applied to fine mapping of genes related to the oil content and protein content in cotton seeds, cloning of regulatory genes and whole genome selection of breeding materials, and improves the efficiency of cotton molecular breeding. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a frequency distribution histogram of the oil content and protein content in a natural population of upland cotton, wherein (a) is a frequency distribution histogram of the oil content, and (b) is a frequency distribution histogram of the protein content.

[0020] Figure 2 is a GWAS result graph of the oil content and protein content traits of cotton seeds, wherein (a) is a GWAS result graph of the oil content of cotton seeds, and (b) is a GWAS result graph of the protein content of cotton seeds.

[0021] Figure 3 is a box plot of the oil content and protein content of TT haplotypes and CC haplotypes, wherein (a) is a box plot of the oil content of TT haplotypes and CC haplotypes, and (b) is a box plot of the protein content of TT haplotypes and CC haplotypes.

[0022] Figure 4 is a GWAS scatter plot and a linkage disequilibrium heat map near the associated site. DETAILED DESCRIPTION

[0023] The present application will be described in detail below through specific embodiments. These embodiments are provided so that the present application can be more thoroughly understood and the scope of the present application can be completely conveyed to those skilled in the art.

[0024] As used throughout this specification and claims, "comprising" or "comprise" is intended to mean that the specification and claims can encompass one or more elements, features or steps in addition to those expressly recited. The description herein is intended to be illustrative of the best mode of the application and is not intended to limit the scope of the application. The scope of the present application is as set forth in the appended claims.

[0025] The Gossypium hirsutum genome version used in the present application is Gossypium hirsutum genetic standard line TM-1 reference genome ZJU TM-1 V2.1 (Hu et al. Nature Genetic, 2019).

[0026] In 2016 and 2017, a set of Gossypium hirsutum natural population was planted in Korla experimental field in Xinjiang, which contained 486 Gossypium hirsutum varieties or lines, and each material was planted in three replicates. After the cotton bolls were matured, the lint was ginned and the seeds were de-cottoned, the oil content and protein content in each cotton seed were determined by Near Infrared Reflectance Spectroscopy (NIRS) analysis technology: 10 grams of cotton seeds were taken from each sample and placed in a circular analysis element with a diameter of 35 mm quartz window, FOSS NIRSystems 5000 near infrared grain analyzer was used to collect the reflection intensity every 2 nm in the wavelength range of 1100-2498 nm, each whole grain cotton seed sample was repeated three times for scanning and the average value was taken, and then the oil and protein content data in the cotton seed were obtained by substituting into the near infrared calibration model. The results showed that the average oil content in the cotton seeds of the natural population was 31.70%, and the average protein content was 46.11%, which conformed to the normal distribution model. Figure 1

[0027] ​Meanwhile, the natural population of upland cotton was whole-genome resequenced, and the average depth of sequencing was ~10.51X. The clean data was aligned to the cotton upland cotton TM-1 reference genome ZJUTM-1V2.1 using BWA (Version 0.1.17) software (Yan Hu, Jiedan Chen, Lei Fang, Zhiyuan Zhang, Wei Ma, Yongchao Niu, et al. Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton [J]. Nature Genetics, 2019, 51: 739-748.), and the SNP variation information of the whole genome was identified using bioinformatics software such as SAMtools (Version 1.6) and BCFtools (Version 1.6). The filtering of SNP variation was carried out with the screening criteria of deletion rate ≤0.2, heterozygosity ≤0.2, minimum allele frequency >0.05, and SNP markers matching only in the cotton genome, and finally the high-quality SNP variation information of the population was obtained.

[0028] The data of oil content and protein content in cotton seeds and SNP variation information data were analyzed by whole-genome association study (GWAS) based on mixed linear model using EMMAX analysis software. The significant association sites were screened with the threshold of -log10(P) = 6. The association site of oil content in cotton seeds was located on the D13 chromosome of cotton, and the significantly associated SNP information was D13:3579341. Similarly, the significantly associated site of protein content in cotton seeds was also D13:3579341. That is, the association site can regulate the oil content and protein content in cotton seeds at the same time, and its SNP information is D13:3579341bp. Figure 2

[0029] ​At the base of 3579341bp on D13 chromosome, the site is single base mutation from T to C, and the genotypes include two homozygous types of "TT" or "CC". From the natural population, 247 cotton materials of haplotype TT and 168 cotton materials of haplotype CC are identified. The two SNP haplotypes are subjected to single base association analysis with oil content and protein content, and it is found that the average oil content of TT type cotton is 32.56%, the average oil content of CC type is 30.30%, and the oil content of TT type cotton is significantly higher than that of CC type (t-test, P<0.01); at the same time, the average protein content of TT type cotton is 45.25%, the average protein content of CC type is 47.53%, and the protein content of CC type cotton is significantly higher than that of TT type cotton (t-test, P<0.01) Figure 3 ). That is, the TT type cotton material is suitable for extracting cottonseed oil, and the CC type cotton material is suitable for extracting cottonseed protein. As shown in Table 1, the names of part of the materials of high oil and high protein content haplotype are shown:

[0030] Table 1: Names of part of the materials of high oil and high protein content haplotype

[0031]

[0032]

[0033]

[0034]

[0035] The linkage disequilibrium in the 500Kb interval upstream and downstream of the SNP site D13:3579341bp (i.e. 3079341bp to 4079341bp on D13 chromosome) is analyzed, and it is found that there is strong linkage disequilibrium on D13 chromosome from 3347580bp to 3699582bp. Therefore, the QTL candidate segment associated with oil content and protein content is reduced to the interval from 3347580bp to 3699582bp on D13 chromosome Figure 4 ), and the SNP variation D13:3579341bp is located in the region where the strong linkage disequilibrium decays.

[0036] Therefore, the site is further proved to be significantly associated with the oil content and the protein content in cotton seeds, and the TT haplotype and the CC haplotype of the site can be applied to the screening of cotton materials with the oil content and the protein content in cotton seeds: the cotton with the base T at the position of 3579341bp on the D13 chromosome is a high-oil cotton variety; the cotton with the base C at the position of 3579341bp on the D13 chromosome is a cotton variety with high protein content. The genome amplification primers are designed at both ends thereof, the upstream primer is shown as SEQ ID NO. 3, and the downstream primer is shown as SEQ ID NO. 4.

[0037] Obviously, the above embodiments are only examples for clearly illustrating the present application, and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. All the embodiments are not required to be enumerated, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. The application of a primer for detecting SNP sites that simultaneously regulate oil and protein content in cottonseed in the breeding of new upland cotton varieties with high oil or high protein content through genetic engineering, characterized in that... The SNP site is located at 3579341 bp on chromosome D13 of the reference genome of the upland cotton genetic standard line TM-1, and the nucleotide base of the site is "T" or "C". The DNA sequence of the amplification product of the SNP site as the TT haplotype is shown in SEQ ID NO.1; the DNA sequence of the amplification product of the SNP site as the CC haplotype is shown in SEQ ID NO.2; the oil content of the TT type cotton is significantly higher than that of the CC type cotton, and the protein content of the CC type cotton is significantly higher than that of the TT type cotton.

2. The application of a primer for detecting SNP sites that simultaneously regulate oil and protein content in cottonseed in the identification of upland cotton varieties with high oil or high protein content, characterized in that... The SNP site is located at 3579341 bp on chromosome D13 of the reference genome of the upland cotton genetic standard line TM-1, and the nucleotide base of the site is "T" or "C". The DNA sequence of the amplification product of the SNP site as the TT haplotype is shown in SEQ ID NO.1; the DNA sequence of the amplification product of the SNP site as the CC haplotype is shown in SEQ ID NO.2; the oil content of the TT type cotton is significantly higher than that of the CC type cotton, and the protein content of the CC type cotton is significantly higher than that of the TT type cotton.

3. The application according to claims 1 and 2, characterized in that, The primers for detecting the SNP site are as follows: the upstream primer is shown in SEQ ID NO.3, and the downstream primer is shown in SEQ ID NO.4.

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