A SNP molecular marker related to oil content of gossypium barbadense and application thereof

Abstract

The application discloses a SNP (Single Nucleotide Polymorphism) molecular marker related to oil content of island cotton and application thereof, relates to the technical field of molecular breeding, and has the technical scheme as follows: the SNP sites include Gbar_D07_49375283 and / or Gbar_D07_49956481, which are located at the 49375283th position and the 49956481th position of a D07 chromosome of island cotton reference genome 3-79_HAU respectively, and G / T and T / C single nucleotide polymorphisms exist respectively.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and more specifically, to an SNP molecular marker related to the oil content of sea island cotton and its application. Background Technology

[0002] Cotton (Gossypium L.) is an important economic crop in my country, serving as a core raw material for natural fiber and a significant source of oils and high-quality plant proteins. Its kernel oil content can reach 30-38%, and cottonseed oil, due to its rich nutritional value, is one of the commonly used vegetable oils, occupying an important position in my country's edible oil supply system. Since 2022, improving the utilization rate of cottonseed oil and conducting high-oil-content cotton breeding programs have become important ways to broaden vegetable oil sources, increase total oil production, and ensure the supply of edible oils.

[0003] Traditional cotton breeding for high oil content relies on phenotypic selection, requiring oil content testing only after harvest, resulting in a breeding cycle of 5-7 years. Furthermore, phenotypic data is easily affected by external environmental factors, leading to low selection efficiency. In contrast, marker-assisted selection (MAS) can significantly shorten the breeding cycle through precise early genotyping of plants. It can also effectively detect locus genes for the target trait and accurately analyze the genetic composition of individual plants within a population, facilitating the aggregation of superior agronomic traits. MAS has become a core technology for improving selection efficiency in cotton breeding. Currently, research on MAS in cotton focuses primarily on traits such as fiber quality and yield, while the development and application of molecular markers related to cottonseed oil content remain relatively lagging. There is an urgent need to identify molecular markers closely linked to cottonseed oil content to promote the efficient development of high-oil-content cotton breeding. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an SNP molecular marker for identifying the oil content of sea island cotton and its application, which has the advantages of accurate analysis and shortening the breeding cycle.

[0005] A molecular marker for SNPs associated with oil content in sea island cotton, wherein the SNP sites include Gbar_D07_49375283 and / or Gbar_D07_49956481, which are located at positions 49375283 and 49956481 on chromosome D07 of the sea island cotton reference genome 3-79_HAU, respectively, and exhibit G / T and T / C single nucleotide polymorphisms, respectively.

[0006] The primer pairs for detecting the above molecular markers include primer pair 1 for detecting Gbar_D07_49375283 and / or primer pair 2 for detecting Gbar_D07_49956481. Primer pair 1 includes the forward primers shown in SEQ ID NO.3-SEQ ID NO.4 and the universal primer shown in SEQ ID NO.5. Primer pair 2 includes the forward primers shown in SEQ ID NO.6-SEQ ID NO.7 and the universal primer shown in SEQ ID NO.8.

[0007] The above-mentioned SNP sites are used to locate genes closely linked to the oil content of sea island cotton.

[0008] The application of the above-mentioned SNP sites in the breeding of new high-oil-content varieties of island cotton through genetic engineering technology.

[0009] A biological detection product containing the above-mentioned SNP molecular marker or the above-mentioned primer pair.

[0010] Furthermore, the aforementioned biological detection products include detection kits.

[0011] Application of the above-mentioned SNP molecular markers, primer pairs, or biodetectors in early prediction and / or screening of oil content in sea island cotton.

[0012] A method for identifying the oil content of sea island cotton includes the following steps: Genomic DNA was extracted from the sample to be tested; using the above-mentioned sea island cotton genomic DNA as a template, PCR amplification was performed using the primer pair to obtain the amplification product; the amplification product was sequenced, and the oil content of the sea island cotton was determined based on the sequencing results.

[0013] In summary, the present invention has the following beneficial effects: This invention provides a molecular marker related to the oil content of sea island cotton. The SNP sites include Gbar_D07_49375283 and / or Gbar_D07_49956481, which are located at positions 49375283 and 49956481 on chromosome D07 of the sea island cotton reference genome 3-79_HAU, respectively, and exhibit G / T and T / C single nucleotide polymorphisms, respectively. When primer pair 1 amplifies Gbar_D07_49375283 and the 101st position of the product is T, it is identified as a high-oil-content Sea Island cotton variety; when the 101st position of the amplified product is G, it is identified as a low-oil-content Sea Island cotton variety. Similarly, when primer pair 2 amplifies Gbar_D07_49956481 and the 101st position of the product is C, it is identified as a high-oil-content Sea Island cotton variety; when the 101st position of the amplified product is T, it is identified as a low-oil-content Sea Island cotton variety. These methods can also be used in combination: when the two loci are GG and TT respectively, it is identified as a low-oil-content Sea Island cotton variety; when the two loci are TT and CC respectively, it is identified as a high-oil-content Sea Island cotton variety.

[0014] The SNP molecular markers of this invention are applied to marker-assisted breeding, which can help shorten the breeding cycle. Furthermore, the aforementioned molecular markers can be detected throughout the entire life cycle of the plant and are not affected by the external environment. They can not only effectively detect the locus genes of the target trait and accurately analyze the genetic composition of individual plants in the population, but also help shorten the time for constructing linkage genetic maps of new populations. This is beneficial for the purposeful selection and utilization of superior materials, thereby achieving the aggregation of excellent agronomic traits in the same material. Attached Figure Description

[0015] Figure 1 This invention relates to the haplotype analysis of cottonseed oil content-related SNPs on chromosome D07 of the RIL population, wherein: A: Manhattan plots of oil content traits in five environments in Awat in 2022 and 2023; B: SNPs associated with chromosome D07; C: LD analysis of chromosome D07.

[0016] Figure 2 This invention relates to the analysis of the difference between haplotypes at the Gbar_D07_49956481 locus and cottonseed oil content;

[0017] Figure 3 This invention relates to the analysis of the difference between haplotype at the Gbar_D07_49375283 locus and cottonseed oil content;

[0018] Figure 4This invention relates to the analysis of the difference in cottonseed oil content between the Gbar_D07_49956481 locus and the RIL population and the natural population of sea island cotton; wherein, A: the genotyping of the marker in the RIL population; B: the genotyping of the marker in the natural population; C: the difference in oil content between the marker and the RIL population; D: the difference in oil content between the marker and the natural population.

[0019] Figure 5 This invention relates to the analysis of the difference in cottonseed oil content between the Gbar_D07_49375283 locus and the RIL population and the natural population of sea island cotton; wherein, A: the genotyping of the marker in the RIL population; B: the genotyping of the marker in the natural population; C: the difference in oil content between the marker and the RIL population; D: the difference in oil content between the marker and the natural population.

[0020] Figure 6 This invention relates to the analysis of the differences in cottonseed oil content among four haplotypes of combined molecular markers and the RIL population of sea island cotton;

[0021] Figure 7 This invention relates to the analysis of the differences in cottonseed oil content among four haplotypes of combined molecular markers and natural populations of sea island cotton. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the embodiments.

[0023] All raw materials used in the following examples are commercially available. Unless otherwise specified, the test methods used in the following examples are conventional methods.

[0024] In this specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0025] The term “and / or” as used in this specification and the appended claims refers to any combination of one or more of the associated listed items, as well as all possible combinations, and includes such combinations.

[0026] To address the issues of long marker selection cycles and low efficiency in marker-assisted breeding of oil content in sea island cotton, this invention develops SNP molecular markers closely linked to the oil content of sea island cotton. Using the sea island cotton RIL population as research material, the invention achieves this through core steps including genome-wide association analysis (GWAS) to locate target SNP sites, extraction of high-quality genomic DNA using the CTAB method, specific primer design, PCR amplification and electrophoresis detection, and verification of marker effectiveness in multiple environments. Ultimately, the invention obtains the SNP molecular markers Gbar_D07_49375283 and Gbar_D07_49956481, which can be stably used for high oil content breeding screening of sea island cotton. The technical process is logically closed-loop, the operation is repeatable, and the markers have high specificity and high detection efficiency.

[0027] Example 1: Genome-wide association analysis of oil content trait in RIL population

[0028] This study uses a population of 120 recombinant inbred lines (RILs) constructed from the superior Sea Island cotton variety Xin Hai 21 (female parent) and the high-yielding, high-lint percentage Pima cotton variety 06E2062 (male parent) as the core research material. The F1 generation was obtained through hybridization in 2012, and after continuous self-pollination in southern China until 2024, a genetically stable F12 generation was obtained, ensuring the accuracy of the correlation analysis between experimental phenotypic and genotypic data. All Sea Island cotton germplasm resources used in this invention were collected, preserved, and provided by the Key Laboratory of Crop Genetic Improvement and Germplasm Innovation, College of Agriculture, Xinjiang Agricultural University.

[0029] Phenotypic data collection: Phenotypic data on oil content of the RIL population were collected under five environments in 2022, 2023, 2023, and 2024 (Awat, Yuepuhu, and Yuepuhu). Descriptive statistical analysis was performed on the correlation traits of cottonseed oil content and cottonseed size of the parental lines "Xinhai 21" (XH21), "06E2062" and the RIL population. The results showed that the coefficient of variation of oil content ranged from 3.44% to 4.23%, indicating that the oil content trait was relatively stable.

[0030] Table 1. Descriptive statistical results of oil content properties in sea island cotton.

[0031] Genome-wide association analysis of the RIL population revealed 60 quantitative genetic loci associated with cottonseed oil content located in the 41.0-51.4 Mb region of chromosome D07 in cotton sea island cotton. Figure 1 (B) This interval is the core correlation region for cottonseed oil content.

[0032] Chaining imbalance detection was performed on the marked interval of 41.0-51.4Mb, and R was found in the sub-interval of 49.5-50.5Mb. ²The average value is greater than 0.8 (P<0.001), indicating extremely strong linkage disequilibrium. Figure 1 C), indicating that the genetic loci in this sub-region are highly linked to the oil content trait.

[0033] Within the 49.5-50.5 Mb high LD range, SNP sites Gbar_D07_49375283 and Gbar_D07_49956481, which are significantly associated with the oil content of Sea Island cotton, were screened out. These two sites are located at positions 49375283 and 49956481 on chromosome D07 of the Sea Island cotton reference genome (reference genome is Sea Island cotton 3-79_HAU, https: / / yanglab.hzau.edu.cn / CottonMD / blast.1), respectively, and they exhibit G / T and T / C single nucleotide polymorphisms, respectively.

[0034] Example 2: Development of SNP Molecular Markers

[0035] Tender leaves from 90 RIL populations of sea island cotton and 100 seedlings of natural sea island cotton were selected, and total genomic DNA was extracted from cotton using the CTAB method. The procedure is as follows:

[0036] (1) First, heat the DNA lysis buffer (containing 3% β-mercaptoethanol, Table 1) to 65°C in a water bath.

[0037] (2) Fresh cotton leaves were placed in a 2 ml centrifuge tube containing small steel balls. The collected samples were quickly frozen in liquid nitrogen and then crushed into powder using a tissue grinder.

[0038] (3) Add 800 μl of lysis buffer to the centrifuge tube containing the sample, mix thoroughly, and then heat in a water bath at 65°C for 1 hour. Take out the centrifuge tube every 10 minutes and mix thoroughly to allow for complete lysis reaction.

[0039] (4) After water bath, add 800 μl of chloroform:isoamyl alcohol (V / V, 24:1) mixture to the centrifuge tube for extraction. Invert the centrifuge tube and mix for 10 min to form an emulsion. Place it in a refrigerated centrifuge at a preset temperature of 4℃ and centrifuge at 12000 rpm / min for 10 min.

[0040] (5) Take 600 μL of supernatant and add 0.6 times the volume of isopropanol (pre-cooled at -20℃), let stand at 4℃ for 30 min, mix thoroughly until white flocculent DNA precipitates from the tube wall, and centrifuge at 12000 rpm / min for 10 min.

[0041] (6) Discard the supernatant and keep the white DNA flakes at the bottom of the centrifuge tube. Add 300 μL of 75% alcohol (pre-cooled at -20°C). Use a pipette to repeatedly blow and wash the flakes. After removing the 75% alcohol, add the same volume of anhydrous ethanol to wash once more. Open the centrifuge tube and air dry the residual alcohol in a clean bench.

[0042] (7) Add 200 μL of sterile ddH2O and shake to dissolve the DNA. Use a UV-Micro Spectrophotometer (Nano Drop2000) to detect the concentration and quality of the extracted genomic DNA. The OD260 / 280 ratio is between 1.8 and 2.2, and there is no protein or visible impurity contamination.

[0043] (8) Dilute the DNA stock solution to 200 ng / ul as a working solution for subsequent experiments.

[0044] Table 2 Composition of DNA lysis buffer

[0045] For the molecular markers Gbar_D07_49375283 and Gbar_D07_49956481 obtained through screening, the flanking sequences of the above SNP molecular markers were determined using the reference genome (3-79_HAU) data of cotton sea island, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0046] Among them, SEQ ID NO.1: AATATTAACAACACTTTCACTTAAATCTTCATCCAATCCACTTGAACCTGGCGTTGCATTGGTCCCAAGAGAAACTACTAAAAGTGAAACCAGTAATAGA[G>T]TACCTAAAACTACAGAGAGGAAACGTTTAAAAGCTGCATCTTTGAAAC GGGGTTTCTTGTAATTAGCCATAGTACAGAACACAGACGACGTCAGCCAAGTTGCAAGTCAAAGCTTGCTCTCTCCAAAAACAAGCATAAGAAAAGCTAAAAACAATTCACATTCATAGACAATCAAACGGCAAAGAGTAACAGCAACCCAT;

[0047] SEQ ID NO.2: TTTAGTGCATTTAGTTCGTTGTAAAGGTTTTTAGTTCATTAAAGAAAAATGTTGGGTCGTCCTGTGTCCTATAGAACTCGAAGCTCTAGGCCAGAGAACC[T>C]GGGAAAAAATTCTTTACATATGATTGCCAATATTTGTTTAACTATAT TCGTAATAGGAGCTCTAGTGGTCACTATTATAGCCGCTACTTATGAACCAGATGACCCATTATTCCAGTCTTCTACCAAGATCACAACTTTCCTCACTTCCATGTCAAATTCTACGTTTCAATCGAATAATACTGTCAGTAGAACCGGGGAG.

[0048] For the specific sites of the molecular markers Gbar_D07_49375283 and Gbar_D07_49956481, specific amplification primer pair 1 and primer pair 2 were designed respectively. Each primer pair includes two forward primers and one universal primer. When the above sites are GG and TT respectively, the F1+RPCR product has a band. When the above sites are TT and CC respectively, the F2+RPCR product has a band.

[0049] Primer pair 1 is: Gbar_D07_49375283-F1: CTACTAAAAGTGAAACCAGTAATAGAG (SEQ ID NO.3); Gbar_D07_49375283-F2: CTACTAAAAGTGAAACCAGTAATAGAT (SEQ ID NO.4); Gbar_D07_49375283-R: ATGGGTTGCTGTTACTCTTTG (SEQ ID NO. 5).

[0050] Primer pair 2 is: Gbar_D07_49956481-F1: AAGCTCTAGGCCAGAGAACCT (SEQ ID NO.6); Gbar_D07_49956481-F2: AAGCTCTAGGCCAGAGAACCC (SEQ ID NO.7); Gbar_D07_49956481-R: CCTCCCCGGTTCTACTGACA (SEQ ID NO. 8).

[0051] The PCR amplification system is shown in Table 3 below: Table 3 PCR amplification system

[0052] The PCR amplification procedure is shown in Table 4 below: Table 4 PCR Amplification Program

[0053] Based on GWAS analysis, the correlation between polymorphisms at Gbar_D07_49375283 and Gbar_D07_49956481 and oil content was further validated. The results showed that the G / T single nucleotide polymorphism at Gbar_D07_49375283, corresponding to two haplotypes (GG and TT), and the T / C single nucleotide polymorphism at D07_49956481, corresponding to two haplotypes (TT and CC), exhibited highly significant differences in oil content across RIL populations from five environments: 2022 Awati, 2023 Awati, 2023 Yuepuhu, 2024 Awati, and 2024 Yuepuhu (P<0.01). Figure 2 , Figure 3 This indicates that the above-mentioned sites are not affected by environmental factors and are stably linked to the oil content trait of sea island cotton.

[0054] Based on the LD analysis results, the R value of the above-mentioned sites is located in the 49.5-50.5 Mb interval. ² With a value >0.8, strong genetic stability, and no obvious recombination, this site has specificity and stability as a molecular marker. Therefore, Gbar_D07_49956481 and Gbar_D07_49375283 were identified as the core SNP molecular markers for oil content in sea island cotton.

[0055] Example 3: Validation of SNP molecular markers

[0056] To verify the practical application value of the two SNP molecular markers in island cotton breeding, genotyping and oil content correlation were verified in RIL populations and natural island cotton populations under multiple environments and large sample sizes.

[0057] For Gbar_D07_49956481:

[0058] RIL population validation: 90 RIL population materials from five environments (Awat in 2022, Awat in 2023, Yuepuhu in 2023, Awat in 2024, and Yuepuhu in 2024) were genotyped. Results showed 50 TT-type materials, 11 CC-type materials, and 29 heterozygous materials. There was a significant difference in oil content between TT and CC-type materials, with the CC-type being the highest oil content material. Figure 4C), the high oil content phenotype is stably associated with the CC genotype.

[0059] Natural population validation: One hundred accessions of Sea Island cotton from four environments (Korla in 2022, Awat in 2022, Korla in 2023, and Awat in 2023) were selected for validation. Genotyping results showed 51 accessions of the TT type, 32 accessions of the CC type, and 17 accessions of the heterozygous type. The oil content of the CC type was significantly higher than that of the TT type. Figure 4 D), consistent with the RIL population validation results;

[0060] For Gbar_D07_49375283:

[0061] RIL population validation: 90 RIL population materials from five environments (Awat in 2022, Awat in 2023, Yuepuhu in 2023, Awat in 2024, and Yuepuhu in 2024) were genotyped. Results showed 19 GG-type materials, 20 TT-type materials, and 57 heterozygous materials. There was a significant difference in oil content between GG and TT-type materials, with the TT-type being the highest oil content material. Figure 5 C), the high oil content phenotype is stably associated with the TT genotype.

[0062] Natural population validation: One hundred natural population materials of Sea Island cotton from four environments (Korla in 2022, Awat in 2022, Korla in 2023, and Awat in 2023) were selected for validation. Genotyping results showed 50 GG type, 39 TT type, and 11 heterozygous types. The oil content of TT type materials was significantly higher than that of GG type materials. Figure 5 D), consistent with the RIL population validation results.

[0063] Combinatorial verification:

[0064] By combining the oil content data analysis results of multi-year, multi-location RIL populations and natural populations of Sea Island cotton, four haplotypes were identified. As shown in Table 5, in the Sea Island cotton RIL population, there were 18 haplotypes of Hap1 (GG / TT), 10 of Hap2 (TT / CC), 1 of Hap3 (GG / CC), and 9 of Hap4 (TT / TT). In the natural population, there were 27 haplotypes of Hap1 (GG / TT), 26 of Hap2 (TT / CC), 6 of Hap3 (GG / CC), and 23 of Hap4 (TT / TT). A significance analysis of the four haplotypes with the oil content traits of the Sea Island cotton RIL population revealed that HAP1 represented the lowest oil content, while HAP2 represented the highest, and the difference was significant. Figure 6Significance analysis of the oil content traits of the four haplotypes and the sea island cotton resource population revealed that HAP1 represented the lowest oil content, while HAP2 represented the highest oil content, and the difference was significant. Figure 7 ).

[0065] Table 5 Combination Types of Molecular Markers

[0066] As shown above, the SNP molecular marker Gbar_D07_49375283 can stably distinguish between high-oil-content (CC type) and low-oil-content (TT type) Sea Island cotton materials in both RIL and natural populations, under various conditions and for many years. The SNP molecular marker Gbar_D07_49956481 can also stably distinguish between high-oil-content (TT type) and low-oil-content (GG type) Sea Island cotton materials in both RIL and natural populations, under various conditions and for many years. Combining these two markers yields four haplotypes, with HAP2 representing the Sea Island cotton material with the highest oil content and HAP1 representing the lowest.

[0067] The aforementioned molecular markers exhibit high specificity, stability, and environmental tolerance, and can be directly used for marker-assisted breeding of high-oil-content varieties of island cotton.

[0068] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A SNP molecular marker related to the oil content of sea island cotton, characterized in that, The SNP sites include Gbar_D07_49375283 and / or Gbar_D07_49956481, which are located at positions 49375283 and 49956481 on chromosome D07 of the reference genome 3-79_HAU of cotton sea island, respectively, and exhibit G / T and T / C single nucleotide polymorphisms, respectively.

2. A primer pair for detecting SNP molecular markers related to the oil content of sea island cotton, characterized in that, This includes primer pair 1 for detecting Gbar_D07_49375283 and / or primer pair 2 for detecting Gbar_D07_49956481; primer pair 1 includes the forward primers shown in SEQ ID NO.3-SEQ ID NO.4 and the universal primer shown in SEQ ID NO.5; primer pair 2 includes the forward primers shown in SEQ ID NO.6-SEQ ID NO.7 and the universal primer shown in SEQ ID NO.

8.

3. The application of SNP loci in locating genes closely linked to the oil content of sea island cotton, characterized in that... The SNP sites include Gbar_D07_49375283 and / or Gbar_D07_49956481, which are located at positions 49375283 and 49956481 on chromosome D07 of the reference genome 3-79_HAU of cotton sea island, respectively, and exhibit G / T and T / C single nucleotide polymorphisms, respectively.

4. The application of SNP loci in the breeding of new high-oil-content varieties of island cotton through genetic engineering technology, characterized in that... The SNP sites include Gbar_D07_49375283 and / or Gbar_D07_49956481, which are located at positions 49375283 and 49956481 on chromosome D07 of the reference genome 3-79_HAU of cotton sea island, respectively, and exhibit G / T and T / C single nucleotide polymorphisms, respectively.

5. A bioassay product comprising the SNP molecular marker as described in claim 1 or the primer pair as described in claim 2, characterized in that, The biological testing product is used to detect the oil content of sea island cotton.

6. The biological detection product according to claim 5, characterized in that, The biological detection products include detection kits.

7. The application of the SNP molecular marker as described in claim 1, the primer pair as described in claim 2, or the biodetection product as described in claim 5 in the early prediction and / or screening of oil content in sea island cotton.

8. A method for identifying the oil content of sea island cotton, characterized in that, Includes the following steps: Genomic DNA was extracted from the sample to be tested; using the genomic DNA of sea island cotton as a template, PCR amplification was performed using the primer pair as described in claim 2 to obtain the amplification product; The amplified products were sequenced, and the oil content of the sea island cotton was determined based on the sequencing results.

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