A snp molecular marker associated with gossypol suboleic acid of upland cotton and application thereof

Abstract

The present application relates to the technical field of molecular biology, in particular to a SNP molecular marker associated with linoleic acid of Gossypium hirsutum and application thereof, 98 SNP molecular markers associated with linoleic acid of Gossypium hirsutum are screened, these sites have significant influence on linoleic acid content, and the SNP molecular markers are used alone or in combination, so that germplasm materials with high and low linoleic acid content of Gossypium hirsutum can be identified through marker assistance, and the work of breeding improvement can be greatly saved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to an SNP molecular marker associated with linoleic acid in upland cotton and its applications. Background Technology

[0002] Vegetable oil is an important resource, a source of dietary nutrition, and a crucial raw material for biofuels, lubricants, and animal feed. Cotton (Gossypium spp.) is not only an important fiber crop but also a significant edible oilseed crop, ranking as the world's sixth largest source of vegetable oil and playing a vital role in ensuring my country's edible oil security. Linoleic acid is a polyunsaturated omega-6 fatty acid, an essential fatty acid for human and animal nutrition. In the human body, cholesterol must combine with linoleic acid to function properly and be metabolized. A deficiency in linoleic acid leads to cholesterol combining with saturated fatty acids, causing metabolic disorders, deposition on blood vessel walls, and the gradual formation of atherosclerosis, resulting in cardiovascular and cerebrovascular diseases. Therefore, linoleic acid has the effects of lowering blood lipids, softening blood vessels, lowering blood pressure, and promoting microcirculation, preventing or reducing the incidence of cardiovascular diseases. It is known as a "vascular cleanser" and has health benefits in preventing and treating atherosclerosis and cardiovascular diseases. Cottonseed oil contains approximately 55% linoleic acid. However, cotton research and production often focus solely on increasing yield and quality, neglecting research on linoleic acid in cottonseed. This results in low yield and quality of cottonseed oil, leading to poor market acceptance. Therefore, conducting research on linoleic acid in cotton is crucial for improving the nutritional composition and shelf life of cottonseed oil.

[0003] Genome-wide association study (GWAS) is an analysis that combines the genetic polymorphism of multiple individuals across the entire genome with the phenotypic diversity of a target trait. It can directly identify gene loci or marker loci closely related to phenotypic variations and possessing specific functions. Conducting a holistic study across the entire genome provides a comprehensive overview of desirable traits in a single instance, making it suitable for research aimed at identifying and cultivating desirable traits.

[0004] Molecular marker technology can quickly identify markers closely linked to QTLs related to linoleic acid (LA) content. Using molecular markers for LA selection can accelerate the breeding process for cottonseed oil quality. Researchers used molecular marker technologies such as SLAF and SSR to initially locate candidate LA genes in cotton. They found that the genetic distance between the candidate genes and the molecular markers was generally large, indicating that the molecular mechanism of LA formation is very complex and requires further in-depth research and exploration. Fully exploring and utilizing these genes controlling LA will enrich the genetic resources for fiber quality improvement and provide an important foundation for breeding new cotton varieties that meet various needs.

[0005] In recent years, with the rapid development of high-throughput DNA sequencing technology, the inventors have successfully completed the resequencing of 1286 cotton NAM population resources. Through bioinformatics data analysis and comparison, a large number of high-quality SNPs were obtained. These SNPs can be used to construct haplotype maps, genetic maps, association maps, and fingerprint maps, providing important support for molecular breeding, phylogenetics, and germplasm resource identification. This invention utilizes genome-wide association analysis to discover a batch of SNP molecular markers associated with linoleic acid in upland cotton, laying the foundation for marker-assisted selection and polymerase chain breeding to improve cottonseed oil quality. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention aims to provide a SNP molecular marker associated with linoleic acid in upland cotton and its application, thereby discovering a batch of SNP molecular markers associated with linoleic acid in upland cotton and laying the foundation for marker-assisted selection and polymerase chain breeding to improve the quality of cottonseed oil.

[0007] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides an SNP molecular marker associated with linoleic acid in upland cotton, wherein the SNP molecular marker includes ChrA05: 5575919, 5752302, 5805195, 5805241, 6019593, 6032696, and ChrA07: 9211, 11843, 115548, 117172, 149970, 201967, 299489, 426784, 445526, 493002, 706128, 833230, 855440, 857546, 879757, 881182, 881 183, 881485, 882869, 888057, 893621, 1819048, 1922003, 1951038, 2073729, 2073734, 2077136, 2080886, 2081161, ChrA10: 1038 38812, ChrD03: 5093442, ChrD09: 273576, ChrD10: 63489326, 63865511, 63874562, 63874962, 63880540, 64089088, 64093071, 640 95384, 64102888, ChrD13: 1640965, 1640985, 1641159, 1641274, 1641331, 1641553, 1659837, 1661189, 1661487, 1661488, 16619 12. 1663246, 1663790, 1664297, 1664971, 1665164, 1665276, 1665478, 1666611, 1666730, 1666785, 1666840, 1666922, 1667639, 1 668419, 1668627, 1669198, 1670934, 1671741, 1672003, 1694071, 1703958, 1723562, 1786049, 1788651, 1829859, 1829865, 1830029, 1830228, 1830925, 1831722, 1832090, 1832258, 1832602, 1832661, 1832677, 1834828, 1835885, 1837071, 1840257, 1840318.

[0008] Furthermore, the upstream and downstream 50bp sequences of the SNP molecular marker are as shown in SEQ ID No. 1-SEQ ID No. 98, respectively.

[0009] Further, the genotypes of the SNP molecular markers are T / C, T / C, A / T, T / C, T / A, T / G, G / A, A / G, G / T, C / A, T / C, T / C, G / A, C / T, G / A, C / T, T / C, C / A, T / C, G / A, G / T. ,T / A,T / G,G / C,T / C,C / T,C / T,G / T,C / T,G / T,C / A,C / G,C / T,C / T,T / C,T / C,T / G,C / T,T / C,G / A,T / C,G / A,G / C,G / C,A / G,A / G,T / G, C / T, A / T, C / A, C / G, A / G, A / C, A / G, C / T, T / C, C / T, A / T, T / C, T / A, G / T, G / A, G / A, T / C, C / T, G / T, C / T, A / G, C / A, C / G, C / T, C / G, C / G, A / T, T / C, A / T, G / A, C / A, G / A, A / T, A / G, A / G, T / C, T / C, C / T, A / G, C / G, G / C, A / T, T / C, T / A, T / C, C / T, T / C, T / C, C / T, C / T, A / T.

[0010] Secondly, the present invention provides the application of the SNP molecular markers associated with linoleic acid in upland cotton in assessing the linoleic acid content of upland cotton.

[0011] Thirdly, the present invention provides the application of the SNP molecular markers associated with linoleic acid in upland cotton in the screening and breeding of upland cotton with high or low linoleic acid content.

[0012] Fourthly, the present invention provides the application of the reagent for detecting the SNP molecular marker associated with linoleic acid in upland cotton in the preparation of a reagent for detecting the linoleic acid content in upland cotton.

[0013] Fifthly, the present invention provides the application of the reagent for detecting the SNP molecular marker associated with linoleic acid in upland cotton in the preparation of screening and breeding reagents for upland cotton with high or low linoleic acid content.

[0014] The beneficial effects of this invention are as follows: This invention provides SNP molecular markers associated with the linoleic acid content of upland cotton seeds, which can be used alone or in combination. With the aid of markers, germplasm materials with high or low linoleic acid content in upland cotton seeds can be identified, which can greatly save the work of breeding improvement. Attached Figure Description

[0015] Figure 1 Manhattan plot of genome-wide association analysis of linoleic acid content in upland cotton seeds.

[0016] Figure 2 The linoleic acid content at 7 loci in a population of 1278 RILs was analyzed.

[0017] Figure 3 This is a KASP genotyping diagram for 4 SNPs.

[0018] Figure 4 The graph shows the linoleic acid content at four loci in a population of 1807 RILs. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments.

[0020] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0021] Example 1 (1) Determination of linoleic acid content in seeds Ten F1 hybrids were obtained through crossbreeding two upland cotton varieties (Zhongzhimian 3 and Jinke 178) and five high-quality Acala varieties (Acala 1517-77 BR, Acala 40(50-5), Acala SJ-3, Acala 1517-75, and Acala 1519-95). These were followed by multiple generations of self-pollination and single-seed propagation to obtain ten recombinant inbred line populations, comprising 1278 recombinant inbred lines. The populations were subjected to a 1-to-2 replication experiment in 2017 and 2018. The 1278 progeny were randomly arranged within and between subpopulations. Parents from each subpopulation were randomly added. Two controls were set up for the entire population: Zhongzhimian 3 and Jinke 178. These controls appeared sequentially every 15 materials in the population, eventually evenly covering the entire population. The experimental sites were: Anyang, Henan (AY) and Alar, Xinjiang (ALE). The experimental plots were planted in single-row sections with 2m rows, and each row contained 10-30 plants (depending on local cultivation practices). Sampling was conducted from September 20th to October 20th (depending on local frost dates and cultivation practices). Except for two plants at each end, bolls were collected from the middle of the remaining plants near the main stem in each plot, with 1-2 bolls collected from each plant, for a total of 20 bolls. The fatty acid content of cottonseed was determined using gas chromatography (GC-2030, SHIMADZU, JAPAN). The fatty acid content was determined by extracting seed oil with boiling petroleum ether, followed by methylation treatment of the seed oil with KOH-methanol solution. The chromatographic column used was SH-Rtx-65 (30 m × 0.25 mm × 0.50 μm), and the detector was FID. High-purity nitrogen (99.999%) was used as the carrier gas, with an injection volume of 1 μL (containing hexane solvent and seed fatty acid methyl ester sample) and a split ratio of 39:1. The temperature program was set as follows: (1) maintain column temperature at 100 °C and hold for 1 min, (2) increase from 100 °C to 210 °C at a rate of 4 °C / min, (3) maintain column temperature at 210 °C and hold for 4 min, (4) increase from 210 °C to 230 °C at a rate of 4 °C / min, and (5) maintain column temperature at 230 °C and hold for 5 min. The highest measurement temperature was set at 280 °C and the injection port temperature was set at 250 °C.

[0022] (2) Extract genomic DNA from the sample to be tested. Take about 3g of fresh leaves and place them in a 2ml centrifuge tube with steel balls. Quick-freeze in liquid nitrogen and grind into powder using a grinder. Add 800μl of preheated (65℃) CTAB lysis buffer (add vitamin C 100ml + 2g before use), mix well, and place in a 65℃ oven for 30-40 minutes, slowly inverting and mixing once every 10 minutes.

[0023] After warming in the bath, remove the centrifuge tube and add 800 μl of 24:1 (chloroform:isoamyl alcohol (24:1 by volume)). Slowly invert the tube until it is mixed and no longer separates into layers.

[0024] Centrifuge at 12000 rpm for 10 min.

[0025] The supernatant should be transferred to another 1.5 ml centrifuge tube using a pipette tip without a tip.

[0026] Add an equal volume of anhydrous ethanol, slowly invert the container several times until flocculent DNA forms, and let stand for 10 minutes. Use a pipette tip to pick up the DNA into a 1.5ml centrifuge tube (or centrifuge at 12000rpm for 1.5min and discard the supernatant), and wash once with 70% ethanol. Dry overnight, add ddH2O to dissolve the DNA until completely dissolved, and store at -20°C for later use.

[0027] (3) Detection of SNPs A total of 1286 upland cotton samples were collected, and genomic DNA was extracted for genome resequencing. This included 8 parental samples and 1278 RILs. During sample collection, seeds of each variety were sown in an incubator, and young leaves of cotton plants were collected. 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. The sequencing yielded 20.47 Gb of high-quality clean data, with an average sequencing depth of 35X for parents and over 4X for progeny. Sequence mapping was performed using the upland cotton cultivar TM-1, with the reference genome version number G.hirsutum_TM-1_ICR (http: / / grand.cricaas.com.cn / page / download / download). The short sequences from each of the 1286 samples were mapped to the reference genome using BWA (v.0.7.12) software, and all misaligned reads and low-quality reads (mapped quality less than 20) were removed. Then, variant identification was performed on each sample using GATK UnifiedGenotyper (v.3.8.0), and the variant files of all samples (n=1286) were merged into a single VCF file. Finally, 11,856,129 and 4,543,742 high-quality SNPs and indels were identified, respectively. Based on a minor allele frequency greater than 0.05 and a deletion rate less than 0.2, VCFtools was used to further filter variant sites, identifying 1,855,955 and 1,309,084 high-quality SNPs and indels, respectively, for subsequent genome-wide association analysis. The impact of all variants was annotated using ANNOVAR.

[0028] (4) Genome-wide association analysis of linoleic acid content in upland cotton seeds The obtained upland cotton phenotypic results and genotypic data were statistically analyzed using a mixed linear model in the Efficient Mixed-Model Association Expedited (EMMAX) statistical analysis software. For details, please refer to (http: / / csg.sph.umich.edu / kang / emmax / download / index.html). The statistical model is as follows: y = Xα + Zβ + Wμ + e y represents the phenotypic trait, X is the indicator matrix for fixed effects, α is the estimated parameter for fixed effects; Z is the indicator matrix for the SNP, β is the effect of the SNP; W is the indicator matrix for random effects, μ is the predicted random individual, and e is the random residual, following the order e ~ N(0, δ). e2In this model, the population analysis was corrected by adding a kinship matrix to μ. The analysis revealed a total of 98 SNPs that were simultaneously and significantly correlated with the linoleic acid content trait in upland cotton seeds (Table 1). Figure 1 ).

[0029] Table 1. SNP molecular markers associated with linoleic acid content in upland cotton seeds.

[0030] (5) 50bp sequences upstream and downstream of SNP sites associated with linoleic acid content in upland cotton seeds >A05:5575869-5575969 (SEQ ID No.1): ATGTATAATTTAATTCACTCCAAAAAAATTTCTAACTTCACTCTTGTCCTTAAATCATGTTTCTCAATCCGTATTCTAAACCTAATCCTTATTATAAATAA.

[0031] >A05:5752252-5752352 (SEQ ID No.2): CCCACATGTCATTTACTTAATATGTACATACAACTCTACACGGCCCAAGTCGGGCTCTAATAAACTTATAGGCCCGTTTAATAAGTTTGGATTCGGTCCAA.

[0032] >A05:5805145-5805245 (SEQ ID No.3): AAAAGTATTTTTTGACAGCAATGCTAAACACTCAAATTTTAAACTAAACATTTCAAAAGCACTTCACTTTTCCACAGCATTTTTTAAAAGTATTGTCAAAC.

[0033] >A05:5805191-5805291 (SEQ ID No.4): AACATTTCAAAAGCACTTCACTTTTCCACAGCATTTTTTAAAAGTATTGTCAAACTAGCCCTAAGTACATGTCAAACTAAGTCCAAATCTATATCCTAGAG.

[0034] >A05:6019543-6019643(SEQ ID No.5): TCCAAATTAAAAGGGTTTTATAGAATCTATTTGAATAAAGATTTTTTAAATTTTGGTTCAATTTAAAATACTATCAATTATAATTGCATTGGACATGT。

[0035] >A05:6032646-6032746(SEQ ID No.6): ACTCTCATCTTTGCATGTGTCAAGGCAGAAAATTTAGAAGCTGCAATGCTTTTTTTTAAGGAAATGAATGTCAGACACTTATTTGATGATTGAGATTTGGT。

[0036] >A07:9161-9261(SEQ ID No.7): CCCCACCTAATCCCTTTAAATAATTTAATAATCTTATAAATGTGCTAATGGATTTTAACCAATGGCCATGTCTGAAAATTGCTTATTGAAAAAAGAACAAAG。

[0037] >A07:11793-11893(SEQ ID No.8): TACGGATATTGCCAAACTTCACAACCATAGCATACCCCAAGAAGTATATATTACTTGCTGCCCATCCCCCATAATACCTAAAGTTTACACATATCAATAGC。

[0038] >A07:115498-115598(SEQ ID No.9): ATATGATTAGCAGCTGAACCTGTGCTTTCTGCAGGAATATTATGTAAAGGTTTTCCAGAAAGAACCATCTGTCAAGACAATGATAGGTGAAATTTAAAAAA。

[0039] >A07:117122-117222(SEQ ID No.10): AATTCTAACCACTTTCAGGTTAGATATGTTTCATTTCCAATAAAACTTACAATAAGTGGTGATTTTGGTGTAATGTGGATAACCCATGCCTTCGAGTTTTA。

[0040] >A07:149920-150020(SEQ ID No.11): ACAAAATGCCCCTAAATCTTAGTATTTCTGCAATTTAGTCAGGTAGGTTTGTATAACTTGAATGAGCATATAAATATGTCAATTTAAATATTATATTGTAT。

[0041] >A07:201917-202017(SEQ ID No.12): CTGTTAAGATCTGTTTTCTGATTTTCGGTAACGCCTTGTCCTATTCCGGTGACGGTTACGGGATAGGGGTGTTACAGTGTTTGGTTGGATCTCTGCAATGT。

[0042] >A07:299439-299539(SEQ ID No.13): ATGGTACTGAGTACAAACGATCCATTTCTACAAGTATGCCAAGATGAGGGTATTGTGCGACACTTCACTGTTCGGGATACACCACAGCAATTGGGGTG。

[0043] >A07:426734-426834(SEQ ID No.14): TTCTTTGGTATTGAAGCGTCACCTTGAAAGTGCTGAGGCCTAATCGAAACAGAAGATCTTCGTGGATGTGGTTCAATCTGTACTTGCGCATGCGGAGGCGT。

[0044] >A07:445476-445576(SEQ ID No.15): ATACAATTCTATTAATGACATTTAGATTTTAATCATTTTTACATAAATTGCCAACACATGAGACACCATTTTTAGTTTTTAGTTTACTTAATGTGTACACA。

[0045] >A07:492952-493052(SEQ ID No.16): CAAATTAAATGAAAAAAAAGTAGATTAATTAGATGGGTATTTAAGATTACGTTGTTGAAGTAACTCTGATTAATCTATTTTTAATTATATTATTCAATGCA。

[0046] >A07:706078-706178(SEQ ID No.17): AATTCAAGATTTTAATATTTCAGCACATAATAATAATAATAAACAATTGTTGTGGCTAAATTGCATGGAAAGCTTTAAAAGACTTCAAAAAAAGAATGAGA。

[0047] >A07:833180-833280(SEQ ID No.18): GTGAAAAATATACTTTTTATACAAAATGACTTTCAAAGGAAAGTGGACACCAAACTGGGAAGGAACTTATGTAGTGAAGAATGCCCGTTGTAACACCCCCT。

[0048] >A07:855390-855490(SEQ ID No.19): ACACAAAAGTGTAGGAGTTAAAATGAAAAAAAAAAAGAGATATTATGTCTTTTTTTTAATCATTCAACTATAAAAAAATTGTAAAATAGACACTTAATTAT。

[0049] >A07:857496-857596(SEQ ID No.20): AATGATAAGCGTCAATCCATTAACTGCATTTTTAGATAGTAATATGTAGGAATAATAAGTTGATAATACATTACACATATGATAATAATATTTACCATGTC。

[0050] >A07:879707-879807(SEQ ID No.21): AAAAGAAAAAAGAAAGAAGGTGATTTTTGTGAAAATGATTGGTGAATTCGCCTTCATGAAAAGGGCTCATTGCTTTCCATTTCAAGCTTTTGAAATTTAAC。

[0051] >A07:881132-881232(SEQ ID No.22): GAGTAGCTTAAGCATAGTATTTAAAGCTACATTAATATCATTATAATTATTGGTGAAGTTTGAATTTCTTTTGGTGGATCAATATTGACTCTCAAAATTTTA。

[0052] >A07:881133-881233(SEQ ID No.23): AGTAGCTTAAGCATAGTATTTAAAGCTACATTAATATCATTATAATTATTGGTGAAGTTTGAATTTCTTTTGGTGGATCAATATTGACTCTCAAAATTTTAG。

[0053] >A07:881435-881535(SEQ ID No.24): GAACATCATCATATTTGAAAAAAATATTAAATATAATTTTGTATACAGAGTAATAAATATTAATACGAGGATATTTTTATCCTTTTATGTTTTTTAAAATAA。

[0054] >A07:882819-882919(SEQ ID No.25): CCAACAATTAATTAAGAAAGACCATTATGAAGTAAATTAATAAAAATATTTAGTCTTCAATACTTGCACATTCCATCAAATTAACGAAAACAATGACATAAT。

[0055] >A07:888007-888107(SEQ ID No.26): GGCTACCCAATATTTCATGTAGTAGGTACACTTATGTTTACTAATTGGTCAACATATTTTATATACTTATACTTTGAAGTTCTGTGATTGATTTAAAATCT。

[0056] >A07:893571-893671(SEQ ID No.27): TGATTTTTTTTATGTAAGATTTTGAGTTTAAATTTTGCGTATAAAAAAACTTAAAAATTATATTATTTTGTAAATTTATTTAATTTAATTTTAGATTTAGG。

[0057] >A07:1818998-1819098(SEQ ID No.28): AAACTTATAATGAAATTATTCAAATAAAAAAAATATTTCGATAAACTCAGATAAAAAGTGATTTTATAGGGGGACAAAGAATCAAATTAGCCCAAGAAT。

[0058] >A07:1921953-1922053(SEQ ID No.29): ACCTCAAGGCGCCCTGAAGAAGAAAGCTCTGGAAACGGGTATCTGGGCTCATCATCAGAAACAGGTTGCTTTTGCTTATTATCAGAAACTTTACCGCCACA。

[0059] >A07:1950988-1951088(SEQ ID No.30): TATCAATATGTACTTTTACAATTCAGGTACCATATTGGACTTTTTCAAAGTAGAAGCACCACTATGTTTTTTTAAAAAGTACGAGTACCATATTTGACAAT。

[0060] >A07:2073679-2073779(SEQ ID No.31): AGAAGTTGAAACCCCCTAAAACCATAATTCTTAGTAGATATATAAATATCGTAACTCTAAAATTATTCATAACTTCTCATTAGCCTTTAAATAAAAAATAA。

[0061] >A07:2073684-2073784(SEQ ID No.32): TTGAAACCCCCTAAAACCATAATTCTTAGTAGATATATAAATATCGTAACTCTAAAATTATTCATAACTTCTCATTAGCCTTTAAATAAAAAATAAATGTA。

[0062] >A07:2077086-2077186(SEQ ID No.33): ATTGATAAAGTTTAACAACATCTTAATGAATGGAATGGAATGATGTTTCTAAGAATAACCAGATAACACTAGCTAAACTGACACTATTATCTTATTCCTA。

[0063] >A07:2080836-2080936(SEQ ID No.34): CATATTTTTGTCTCGGTTTTATTAAATAGTATGTACTATGATTTGTTAACACCTCGTACCCTGTTCCGGCGATGGATACGGGGAAGGGGTGTTACATTAGT。

[0064] >A07:2081111-2081211(SEQ ID No.35): ATAAATTTAATTAATTAAAAATTTTTTAAACCCCAAATCTTAGTTAAAAAATCCTTCATCTCCCCCTTTTTTTTAGTTTTCTTTTTCAATTGACTAAAAAAAC。

[0065] >A10:103838762-103838862(SEQ ID No.36): ATCACACTAAGTTCAATTCCCAGGAAAGAGAGGTAGATCACAAAGATCATTTAAATACCAAGTCTTTCCTAACCAGAATATTCCTCTACAGTAATTTAATA。

[0066] >D03:5093392-5093492(SEQ ID No.37): ATTTTAAAAAATTGGCCTAATTAAATAATATTATAATGACTATATTGTATAATTAATTTTACCTTGTTACGAGTGGGAAAGTACAAGGGTAGTTGGCTTGG。

[0067] >D09:273526-273626(SEQ ID No.38): CTAAAGAAACATCCATGTACAGTTTGCTAACTCTAACAGATTTCCCCAACGGGCTCAGCACAGTCACCTCACTAGAAGTACTCTCAACCAAAATCCCTAGG。

[0068] >D10:63489276-63489376(SEQ ID No.39): ATTCCAAAGGTCACTGAACTACTAGTAAGTTTACGTTTTGGTCAATTAATTTCAAAAAATTACAAAATGACCACTAAACTATTGAATATTTTTATTTAAGT。

[0069] >D10:63865461-63865561(SEQ ID No.40): AGGGTTTTGGGCCATTTTTTGGGCCTTGGGCAAATTTGGGCTTATACATGGTTTACTTAGGGTCTGTTTGATTGACGGAATTGATTTTTCGGAAAATCGTT。

[0070] >D10:63874512-63874612(SEQ ID No.41): AATTTGTCATTTATTTTTTTTTAAATATTTTATTGTTTATTTATAGGATACTTGTCAATTTATTTACGAAGTCTAAAAACTCTACTAACAGTATACCAA。

[0071] >D10:63874912-63875012(SEQ ID No.42): TGTTGATTTATGAGCTTGTTCGTATTTAATTTGTATTTATTTATTTAAAGTTAAGATAAATTATATTTAAGGTCATTAAATTATTAGTATATTTATATTTT。

[0072] >D10:63880490-63880590(SEQ ID No.43): GGCTTAGGATAGATATTTCTTGCCATAAGCTTTCAATTTCATAACAAAGGGATTCTATTTGATTAATTAATTTTTTTAATAATTTTATTGCAAGTTTTGG。

[0073] >D10:64089038-64089138(SEQ ID No.44): AGGTCAACGAAGTAGCCTATGTGGTGTAACACCCCAAACCCGGCCTAAAGGTTATGGCCAAATCTGACAATATCACATGATAGTGCTTTGCTTTTCGATAA。

[0074] >D10:64093021-64093121(SEQ ID No.45): TCATGTCGATGAAGGCATCAACATAGTAAATGGGGGAGTGTCATGGTCTACAATTTACTATGTTAATTATCATGGTTGACCTTTCCCCAAATGGTCTTGAT。

[0075] >D10:64095334-64095434(SEQ ID No.46): ATGGTCATAATATGAGCTCTCGAACTCAAAATCGAGTGATTCAAAATGTATTTCGAAGCTAAGAAATAGATCTTCAAACACCATGAGACATCTCTACCCAG。

[0076] >D10:64102838-64102938(SEQ ID No.47): CATATCAAGAGAAAAAAATGCTCCTTCTCCACCTACATATTCAGTATTTTTTTTAATGCCTATTCCTCTCCTATTGGTTGACGTATTATATGTTCATCATGT。

[0077] >D13:1640915-1641015(SEQ ID No.48): GAACATGTGTTAAATCATAATAATAATTAATTATAATTTAAAAGAATGAACACTTTTATAATTTCATAACAGAGTTGGTAACCCGAATTAAGGATGATGCTAA。

[0078] >D13:1640935-1641035(SEQ ID No.49): TAATATTAATTATAATTTAAAAGAATGAACACTTTTATAATTTCATAACAGAGTTGGTAACCCGATTAAGGATGATGCTAACTCAGTAAAATACTAAGTAT。

[0079] >D13:1641109-1641209(SEQ ID No.50): TAATTATATGGGTTGTGCAAAAAGTATTGTGGGTTATTTGTTTTAAGTCCTTTGTGTTGGAGATCGACCCGATTAAGTAACAAACAAGTAAAAAATAACGG。

[0080] >D13:1641224-1641324(SEQ ID No.51): TTAAACACACAAATTTAACGTGGAAAAACATCTCTAAAGAGGATAAAACTCATGGATAAAGATAATTTTACTATAATGACAAAAGAACGAAAAGTACAAA。

[0081] >D13:1641281-1641381(SEQ ID No.52): TAAAGATAATTTTACTATAATGACAAAAGAACGAAAAGTACAAAAGATGAAGATAAAAACTAAACTCTGAAACCCAAAAATAAAACCCTCAAAACGTAA。

[0082] >D13:1641503-1641603(SEQ ID No.53): ATCTTTTAGGAAATAAAATATATTATGTTTAACTTGACTTGCAAGCAATATCTTATTTTATTGAACAGGATTTTGAGGCACACAAGACTAACAAATTGTCT。

[0083] >D13:1659787-1659887(SEQ ID No.54): TTTCAACAATGTAGCATTCACATAATACACTTGCACTCATTTCCCAAGCATGATCTTATATGTCTACAAAAAATCCTCTCTAGAGTCTCTTTCAAATTCAA。

[0084] >D13:1661139-1661239(SEQ ID No.55): ACTCTTGTCCATGCATGCCACTTTGGTTGGTTGGAAAAATAGACATGCACCCCCACGATAATCTCTTTTTCTTCTTGTCATCTTGTTTGTCCATTCTTGGT。

[0085] >D13:1661437-1661537(SEQ ID No.56): TTTTTAGTCCCTCTCAGATATTAATAAGTTAATTTAAGCTTTTTATGCTTCGGTTGGTTACTAGTATAATAATATGTGGAAAAGACTTATCTGATACACAT。

[0086] >D13:1661438-1661538(SEQ ID No.57): TTTTAGTCCCTCTCAGATATTAATAAGTTAATTTAAGCTTTTTATGCTTCGGTTGGTTACTAGTATAATAATATGTGGAAAAGACTTATCTGATACACATT。

[0087] >D13:1661862-1661962(SEQ ID No.58): CTTTTTACATGTAGTAATGGTAGGGGAAGAATTATGGTTAAAAACCAAGATGTTTAAAGCCAACATTGATCCACTCTCCATACCTTCATAACTTTGAAGAC。

[0088] >D13:1663196-1663296(SEQ ID No.59): TTGAAATGATGATTTAACGTGCCACATCAGTTTATTGTTACACCGTTAATAGAAACTAACGGCTCAGTGACTAAAATGTTACAACGTGATAACATAAGTGA。

[0089] >D13:1663740-1663840(SEQ ID No.60): CGAATATTTATAATTTAATTCCAATCCCGGAAATCTTTTAGCGTCCCCTTTATATTTTAATCATATCATGTATTGATATTAGGATGGTATTTGATGGGTTG。

[0090] >D13:1664247-1664347(SEQ ID No.61): TAAATGGGACATAAATGGTAACCTAAGTTGGAACCTTTAATTAGGAAGGGAAATTGATGAAAGGAAGGTTAGATTAGGAAATAGACCCAAAGCTTGATTGA。

[0091] >D13:1664921-1665021(SEQ ID No.62): CAACCTTGAACAAGTCTACTCATTACCAATATAACATACAATGTAAACTGAAGTTTCTACACATTTTTATAAAATTTTAAATCCGACCACCATATTTTACG。

[0092] >D13:1665114-1665214(SEQ ID No.63): GTCCAAACTCTTATTATCCCAACAGCTTGTCCAAATAGATGTTCGAACAGGGAATGGAACAGAGTGTTTCAACAGACAATCCAAAGAAATATTCGACCAGA。

[0093] >D13:1665226-1665326(SEQ ID No.64): TAGTCATTTTAGACAACTAAGGTCTCAATAATTGTGACTCGGATGTATATATAATATTTTTTAATTTTAATTATATACATTTAAAAAAATTTAAATAGGTAAA。

[0094] >D13:1665428-1665528(SEQ ID No.65): ATCCAAAATCAAATATGAAATCGGGACGAAAAAAATTCAATTACTAAATCGAACATTGAAGACAAACTCAAATACTGAATAATATATTAACCCAATATAAT。

[0095] >D13:1666561-1666661(SEQ ID No.66): TCCTCGTTTCCTTCCAAATGCACTTGTTGTTGCAATAGCGCAATGATGGGGTGCAATATCAGTCAACCATAGACTCCAGTCACTGGATCTAATCCTCCATG。

[0096] >D13:1666680-1666780(SEQ ID No.67): ATATTTTGACCAATTCAAGGTAAAAAATGAGGCTGCTCCCTCGACAAAACTGGGATAATATTAAGCCAAAAGATCACGATTCAAGATAAATTCATGGGGAA。

[0097] >D13:1666735-1666835(SEQ ID No.68): TAATATTAAGCCAAAAGATCACGATTCAAGATAAATTCATGGGGAAGTGAGATCTCTTTAGAGTCTTCTCCAACATTTAGAAATTAGTTTATCGATAAAGA。

[0098] >D13:1666790-1666890(SEQ ID No.69): CTTTAGAGTCTTCTCCAACATTTAGAAATTAGTTTATCGATAAAGATATCTGTACTTGATGCCCCGCCCAAGAAAACCAGCTGCTTCACTTTTAGCGGGGC。

[0099] >D13:1666872-1666972(SEQ ID No.70): GCTTCACTTTTAGCGGGGCCTCTTCAATCCAAGACCTAGCTGATTTAAACAGCTTCTCTTCCTGAGCTATACCATAGGTGGCTGTATGCGTCTTCCTAAAT。

[0100] >D13:1667589-1667689(SEQ ID No.71): CCTTACATTTTTAAAAATTTGAAATTTTAATATTTACACAAATGATAGTCGCTAATCCATTAACAGAATTTTTAGCGAGTAATATATAGAAATAACAAGTG。

[0101] >D13:1668369-1668469(SEQ ID No.72): TGATGGAGAAGAAGAACAAGCAAACCAGACAGACAACGAGGAGCAACAACAAGGGACCAACAGCAGCAGGTGTTCGTCGTCTTGTTCGAATGAAGCAAAAT。

[0102] >D13:1668577-1668677(SEQ ID No.73): AAGCTGCATTTGGACAATGTGGAGAGGGATTGTCGGATTTGTCATCTCACCTTGGATCCAAGTAACCAAGAATGTGATTTGCCTATTGAGTTGGGGTGTTC。

[0103] >D13:1669148-1669248(SEQ ID No.74): TAATTTACTCTTTGATTTAACGTACATAGATCAATTTATCCTATTTTTTAGTAGAAGGGGCAAAATGCAATCTTACAATTAATATAGGTGCCTTGATTGGTA。

[0104] >D13:1670884-1670984(SEQ ID No.75): GAACTTGAAGGATCTCTAATCTCAAATTCATCCTCTTCAGCATCATAACTGCATATCACGACATAATGACCTACAACAAGACCAAATTTCTCAACGATGTG。

[0105] >D13:1671691-1671791(SEQ ID No.76): CAGAGTAGTTCGGGTTAGCTCCAAATGTTACAGTGTAATAGGAAAACCTAACGGAAAACTTTCGCAGTAAATAAGCGAGATCAACGGTCCAAATGCTGCAA。

[0106] >D13:1671953-1672053(SEQ ID No.77): ATCAAAACACAAGCAAGTCCACAGTCCCAATAGAACAGCTGGTTAATATGTGGAACCTGTAAACACCAAACAAACCTCATCATCGAAACATAAACAAAATA。

[0107] >D13:1694021-1694121(SEQ ID No.78): TGATCCCACATTGCATTAAAATCACTCAAGTAAATTAAACATATGTAAACTGTCTTCTCATCTCGAGGTAAACAAAAATGTTTCGTCCCATAAGTTAGGAC。

[0108] >D13:1703908-1704008(SEQ ID No.79): TCCATGCTGCCTTTTGAGAAAGGAAAAGACATAGAATTGTATGAATTCTGGATGTCACCGTTTTGGTTAAGGGTTTACAATATCCCTATTGAACTTATGGA。

[0109] >D13:1723512-1723612(SEQ ID No.80): TCATGGCTGCTCATGCTTACTGGTCTCAGCATCAAACTAAACAACAAGGAAAGCATCATCCCGGAACTTTGAGAATTACTGACTCCCAAGCTGACAGAATC。

[0110] >D13:1785999-1786099(SEQ ID No.81): TGACACAATACCCAGAAATACCAATAATTCCTCTCCAATCCATAAATAAAAGGATAATACATTTTTATGCACTTGAATTCACATCTTTTTATATTGGCAAC。

[0111] >D13:1788601-1788701(SEQ ID No.82): TCGTCTTTTAAAAAAATACATAAATTTATATAAGTAAGGGTGAGCAATTAGTTAATGTGGCCAGTTCGGTCGATTTTTTCTCCAACTTGACCGACTTATAT。

[0112] >D13:1829809-1829909(SEQ ID No.83): GTCAGAGTGGTGTTTCCCCACATCGGTTAAATCCGAGTTTGCTTGCAGTTTTATATTGAAGTTTGATCTCTTGTTGCCAATCGGTTTTAGGTTGAATGCTT。

[0113] >D13:1829815-1829915(SEQ ID No.84): GTGGTGTTTCCCCACATCGGTTAAATCCGAGTTTGCTTGCAGTTTTATATTGAAGTTTGATCTCTTGTTGCCAATCGGTTTTAGGTTGAATGCTTTAACAT。

[0114] >D13:1829979-1830079(SEQ ID No.85): AGAAACATGCAAAGTTCATTTAGGGGAGAGAATATGAACTTGTTTCCATCATTAAATGATTCAGAGGTTATTTTAATTGATTCATGTAATTACTCTTAAAG。

[0115] >D13:1830178-1830278(SEQ ID No.86): TGAAATAATCCCTATCCATTCCAATCAAATGTCCCACATTATTTCTTAGAGTGAAATTTTGTCTAAAAAGTTTGATTTTTCTTATTCTCTGTAAAACACTA。

[0116] >D13:1830875-1830975(SEQ ID No.87): TAGATACAAGCACTTTGTTAATTCTAGTGAAAATTACTAGATACAACGCCAGTTACAACTTAAGGAGATTATTGTAACTTGCCTTATATGGTTGGCATGTT。

[0117] >D13:1831672-1831772(SEQ ID No.88): TTATAACTATTTTTACAAATAAAGAACTTTCGCATTTACAAATATCAAGTTTTCACATCAAAATATTATACATAATTTTAAATTTTGAAATTATTTCA。

[0118] >D13:1832040-1832140(SEQ ID No.89): AATTCTCAGTCTACCATCAAGTTGAAGAATGAACATTTTAGGTAGAAAATATGTAAATCAGCACAAGACAATTTGTTTATAGTTTTTCTTAAGAAAGGA。

[0119] >D13:1832208-1832308(SEQ ID No.90): TTTAAGCTTTCTAGAAATATAAATCTTCACCAAATCTGGGTTTTGCATAAGAAGGAATCATTTCTTGGCCAGTTATGGCAATGTAAGTAATTCAGACAA。

[0120] >D13:1832552-1832652(SEQ ID No.91): ATGTAAGAGGATTATGGGTTTGAGTGCGTGAAAGTGTATTATCCTTTTTTTTGGGTTGGGGCTATAGATAATTACAAAATGATCACTCAACTATTCA。

[0121] >D13:1832611-1832711(SEQ ID No.92): TTGGGGCTATAGATAATTACAAAATGATCACTCAACTATTCAATTTTATTTTTTTTGGTCACCAACGGATAACATAAAATGAAGATACCCAAAAATCGA。

[0122] >D13:1832627-1832727 (SEQ ID No.93): TTACAAAATGATCACTCAACTATTCATTTATTTTTTTTGGTCACCAACGGGATAAAAAAAATGAGATACCCAAATCGAGATAGTTAGATGATC。

[0123] >D13:1834778-1834878 (SEQ ID No.94): TAAAAATTAGCATGACTGACGGAATAAACATAATTTCACATGATATCACGTGTATCTCATGTTGATGTCAAAAACAGCTTTTAACATAAAAAATG。

[0124] >D13:1835835-1835935 (SEQ ID No.95): GTGGTCACATGAATTCATAGAAGAGATAGAGCTAAGCTCAAACAATCTCTTAGTCCTAAAATGATTGTTTCTAATCAATCGACCCCAATCCT。

[0125] >D13:1837021-1837121 (SEQ ID No.96): TAAATTCCATTTTTGATTGGTGGAATAGCTCCTATCCGTAGTTCTTGCTAAATTTAAACTTCTCAAAGCTTTCAAAATTACTCTTTCTCTAACAAGGAC。

[0126] >D13:1840207-1840307 (SEQ ID No.97): TATTTTCCCCGACTTTGTAAATTATTCATTCAAAAATT。

[0127] >D13:1840268-1840368 (SEQ ID No.98): TATTTTAACTTTAAAAACTTATTTGAAATATTCAAAAATTCTTATTTACATTATTGAATTATTAAAATTATTATTGTATGATTTTTTCTGTTCGCGCTGCG.

[0128] Based on the locations of 98 SNPs and the LD decay distance of the population, the SNPs were divided into 7 QTLs located on 7 chromosomes. Haplotypes were performed on the population at each QTL locus based on the SNP genotyping, and the linoleic acid content of each haplotype was determined. The results are as follows: Figure 2 As shown, significant differences in linoleic acid content were found between the two subtypes at each locus. The differences in linoleic acid content at the seven loci (A05, A07, A10, D03, D09, D10, and D13) were 2.07, 2.33, 0.16, 0.30, 1.16, 1.89, and 1.73 percentage points, respectively. This indicates that these loci significantly influenced the linoleic acid content, which is of great significance for improving the fatty acid content of cotton.

[0129] To verify the fitness of these loci, we randomly selected 4 loci from 7 loci and developed KASP markers using 4 SNPs with Primer 3.0 (Table 2). Then, we performed genotyping using these 4 KASP markers in a multi-parent population containing 1807 offspring. Figure 3 ), and analyze the degree of influence of these four sites on linoleic acid content, such as Figure 4 As shown, the differences in linoleic acid content at the four sites A05, A07, D10, and D13 were 2.99, 3.16, 2.05, and 1.91 percentage points, respectively, indicating that these sites do have a significant impact on linoleic acid content and verifying the association between these SNP sites and linoleic acid content.

[0130] Table 2. KASP primers for the four SNPs

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A SNP molecular marker associated with linoleic acid in upland cotton, characterized in that, The SNP molecular markers include ChrA05: 5575919, 5752302, 5805195, 5805241, 6019593, 6032696; ChrA07: 9211, 11843, 115548, 117172, 149970, 201967, 299489, 426784, 445526, 493002, 706128, 833230, 855440, 857546, 879757, 881182, 881183, 881485, 882869, 8880. 57, 893621, 1819048, 1922003, 1951038, 2073729, 2073734, 2077136, 2080886, 2081161, ChrA10: 103838812, ChrD03: 509344 2. ChrD09: 273576, ChrD10: 63489326, 63865511, 63874562, 63874962, 63880540, 64089088, 64093071, 64095384, 64102888, ChrD13: 1640965, 1640985, 1641159, 1641274, 1641331, 1641553, 1659837, 1661189, 1661487, 1661488, 1661912, 1663246, 1663790, 1664297, 1664971, 1665164, 1665276, 1665478, 1666611, 1666730, 1666785, 1666840, 1666922, 1667639, 1668419, 1668627, 1669198, 1670934, 1671741, 1672003, 1694071, 1703958, 1723562, 1786049, 1788651, 1829859, 1829865, 1830029, 1830228, 1830925, 1831722, 1832090, 1832258, 1832602, 1832661, 1832677, 1834828, 1835885, 1837071, 1840257, 1840318.

2. The SNP molecular marker associated with linoleic acid in upland cotton according to claim 1, characterized in that, The upstream and downstream 50bp sequences of the SNP molecular marker are as follows: SEQ ID No. 1-SEQ ID No.

98.

3. The SNP molecular marker associated with linoleic acid in upland cotton according to claim 1, characterized in that, The genotypes of the SNP molecular markers are T / C, T / C, A / T, T / C, T / A, T / G, G / A, A / G, G / T, C / A, T / C, T / C, G / A, C / T, G / A, C / T, T / C, C / A, T / C, G / A, G / T, T / A, T / G, G / C, T / C, C / T, C / T, G / T, C / T, G / T, C / A, C / G, C / T, C / T, T / C, T / C, T / G, C / T, T / C, G / A, T / C, G / A, G / C, G / C, A / G, A / G, T / G, C / T, A / T, C / A, C / G, A / G, A / C, A / G, C / T, T / C, C / T, A / T, T / C, T / A, G / T, G / A, G / A, T / C, C / T, G / T, C / T, A / G, C / A, C / G, C / T, C / G, C / G, A / T, T / C, A / T, G / A, C / A, G / A, A / T, A / G, A / G, T / C, T / C, C / T, A / G, C / G, G / C, A / T, T / C, T / A, T / C, C / T, T / C, T / C, C / T, C / T, A / T.

4. The application of the SNP molecular marker associated with linoleic acid in upland cotton as described in claim 1 in assessing the linoleic acid content of upland cotton.

5. The application of the SNP molecular marker associated with linoleic acid in upland cotton as described in claim 1 in the screening and breeding of upland cotton with high or low linoleic acid content.

6. The application of the reagent for detecting the SNP molecular marker associated with linoleic acid in upland cotton as described in claim 1 in the preparation of a reagent for detecting the linoleic acid content in upland cotton.

7. The application of the reagent for detecting the SNP molecular marker associated with linoleic acid in upland cotton as described in claim 1 in the preparation of screening and breeding reagents for upland cotton with high or low linoleic acid content.

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