Abnormal cotton chromosome segment capable of increasing seed kernel oil content, molecular marker and application thereof

Abstract

The application discloses an abnormal cotton chromosome fragment capable of improving cotton seed kernel oil content, a molecular marker and application thereof, the abnormal cotton chromosome fragment is located on the first chromosome of a cotton chromosome set, and comprises four pairs of SSR markers, namely, JAAS3611, JAAS1148, NAU3615 and NYS-14 markers; taking DNA of abnormal cotton as a template, the DNA of the cotton to be tested is amplified by using the four pairs of SSR markers simultaneously, and a chromosome fragment of a band containing the four pairs of SSR markers simultaneously is the abnormal cotton chromosome fragment A1-3; the abnormal cotton chromosome fragment and the molecular marker thereof can be applied to cotton high-oil breeding, can greatly improve the cotton seed kernel oil content, and improve the breeding efficiency of cotton.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular breeding, and particularly relates to an abnormal cotton chromosome fragment capable of improving the oil content of cotton seed kernel, and a molecular marker and application thereof. BACKGROUND

[0002] Cotton seeds are by-products in the production of cotton fibers and are also good oil resources. Cotton kernels contain more than 35% of cottonseed oil, and the content of linoleic acid in cottonseed oil is the highest in edible oil. The content of linoleic acid in cottonseed oil is as high as 55.6%, which is a high-grade health care oil with extremely high linoleic acid content. Therefore, tapping the genetic potential of cottonseed oil content and improving the total production of cottonseed oil in China have broad market prospects.

[0003] There is a large variation in cottonseed oil content among and within species, which provides rich germplasm resources for genetic improvement and gene mining of cottonseed oil content. With the increasing attention to the nutritional quality traits of cotton seeds, researchers have used linkage analysis and association analysis to mine some QTLs related to cottonseed oil content in different populations. So far, 335 QTLs of cottonseed oil content have been reported using sea-land introgression lines, recombinant inbred lines, chromosome segment substitution lines and natural populations as materials. Due to differences in population genetic background, marker type, positioning software and planting environment, there are large differences between the research results. Zhou Chaozhe et al. used the QTLs and molecular markers reported in the literature to construct a consistent physical map of the sites related to cottonseed oil content by comparing with the genome of the genetic standard line TM-1 of Gossypium hirsutum. A total of 109 QTLs of oil content and protein content were integrated. There are 23 QTLs of cottonseed oil content with a phenotypic variation rate of more than 10%, of which 3 are stable major QTLs. The oil use and feed value of cotton seeds have not been correctly recognized in the past, and the related genetic improvement research has lagged behind. The QTL research of cottonseed oil content is still in the initial positioning stage, the QTL positioning interval is large, and there is a lack of major loci and key genes. So far, there is no report on the improvement of cottonseed oil content using related markers in genetic populations. The reason is that based on traditional separation populations, the resolution is low, which cannot achieve fine mapping. The separation of markers and target genes will cause selection bias when marker-assisted selection, which affects the efficiency of marker-assisted selection.

[0004] Since cottonseed is merely a byproduct of cotton production, cotton breeding has primarily focused on cotton fiber yield and quality, with limited research reports on its nutritional quality. The oil content of cottonseed exhibits significant intraspecific and interspecific variation, and for a long time, improving its nutritional quality has not been a primary goal of cotton breeding. Decades of cotton breeding have essentially resulted in a process of natural selection for cottonseed nutritional traits. Genetic variations in oil content within the *Gossypium* genus have not been reflected in modern, commercially available varieties. Given the increasing scarcity of vegetable oil and protein resources in the international market, improving the nutritional quality of cottonseed should be a crucial objective of cotton breeding. Summary of the Invention

[0005] One objective of this invention is to provide an abnormal cotton (Gossypium anomalum) chromosome fragment A1-3, which can significantly increase the oil content of cottonseed kernels.

[0006] Another objective of this invention is to provide a molecular marker and primer sequence for an abnormal cotton chromosome fragment A1-3, which is closely linked to the high oil content site in cotton seed kernels of the abnormal cotton single fragment substitution line.

[0007] This invention also protects the application of molecular markers and primers for the aforementioned abnormal cotton chromosome fragment A1-3.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] An abnormal cotton chromosome fragment A1-3, capable of increasing the oil content of cottonseed kernels, is derived from an abnormal cotton plant and located on chromosome 1 of the cotton chromosome set. It contains four pairs of SSR markers: JAAS3611, JAAS1148, NAU3615, and NYS-14. Using the abnormal cotton's DNA as a template, the DNA is simultaneously amplified using these four SSR markers. The chromosome fragment containing all four SSR marker target bands is identified as abnormal cotton chromosome fragment A1-3. The primer sequences for the four SSR markers and the lengths of the amplified fragments are as follows:

[0010] JAAS3611: The forward primer sequence is SEQ ID NO.1, the reverse primer sequence is SEQ ID NO.2, and the target fragment length is 241bp amplified in the abnormal cotton genome;

[0011] JAAS1148: The forward primer sequence is SEQ ID NO.3, the reverse primer sequence is SEQ ID NO.4, and the target fragment length is 168bp amplified in the abnormal cotton genome;

[0012] NAU3615: The forward primer sequence is SEQ ID NO.5, the reverse primer sequence is SEQ ID NO.6, and the target fragment length is 139bp amplified in the abnormal cotton genome;

[0013] NYS-14: The forward primer sequence is SEQ ID NO.7, the reverse primer sequence is SEQ ID NO.8, and the target fragment length is 163bp amplified in the abnormal cotton genome;

[0014] On the other hand, SSR marker primers for the abnormal cotton chromosome segment A1-3 were also disclosed, and the sequences of the marker primers are as follows:

[0015] The forward primer sequence for the SSR marker JAAS3611 is SEQ ID NO.1, and the reverse primer sequence is SEQ ID NO.2;

[0016] The forward primer sequence for the SSR marker JAAS1148 is SEQ ID NO.3, and the reverse primer sequence is SEQ ID NO.4.

[0017] The forward primer sequence for the SSR marker NAU3615 is SEQ ID NO.5, and the reverse primer sequence is SEQ ID NO.6;

[0018] The forward primer sequence for the SSR marker NYS-14 is SEQ ID NO.7, and the reverse primer sequence is SEQ ID NO.8.

[0019] This invention also discloses the application of the aforementioned SSR molecular marker primers in identifying or assisting in the identification of cotton varieties or lines with high oil content. In other words, the molecular markers of this invention can be used in future marker-assisted breeding. By extracting DNA from leaves during the seedling stage, the presence of the molecular markers of this invention can be detected, thereby identifying whether the cotton being tested is a cotton variety or line with high oil content. The detection can be performed using PCR, specifically using the aforementioned molecular marker primer pairs, or it can be performed using sequencing methods.

[0020] In addition, the abnormal cotton chromosome segment A1-3 can be detected in the genomic DNA of the cotton to be tested. Cotton containing the abnormal cotton chromosome segment A1-3 is a cotton variety or strain with high oil content.

[0021] Specifically, the method for detecting whether the cotton genomic DNA to be tested contains the abnormal cotton chromosome fragment A1-3 is as follows: PCR amplification is performed on the cotton genomic DNA to be tested using the SSR marker primers described in claim 2. If fragments with lengths of 241bp, 168bp, 139bp, and 163bp are present simultaneously, then the cotton genomic DNA to be tested contains the abnormal cotton chromosome fragment A1-3; otherwise, the cotton genomic DNA to be tested does not contain the abnormal cotton chromosome fragment A1-3.

[0022] This invention also discloses a cotton breeding method to increase the oil content of cottonseed kernels. The method involves hybridizing upland cotton and abnormal cotton as parents to obtain a generation population. The generation population includes upland cotton progeny containing chromosome segment A1-3 of the abnormal cotton, and the cottonseed kernel oil content of the upland cotton progeny is higher than that of the parent upland cotton. It should be noted that the upland cotton can be replaced by other commonly cultivated cotton varieties or strains; any cotton variety or strain that can hybridize with abnormal cotton is acceptable.

[0023] This invention also discloses a method for constructing the abnormal cotton single-fragment replacement line CSSL3, the method comprising the following steps:

[0024] (1) Using upland cotton 86-1 as the female parent and abnormal cotton as the male parent, a hexaploid F1 hybrid was obtained by colchicine doubling. Morphological, cytological, and molecular marker techniques were used to identify and prove that we obtained a hexaploid hybrid that had successfully doubled (Zhang et al., 2014). Using the hexaploid F1 as the female parent and Su 8289 as the recurrent parent, we continued to backcross four times to the BC4 generation, and then self-crossed for four generations to produce BC4F4.

[0025] (2) Plant BC4F4 plants, the recurrent parent Su8289, and abnormal cotton were planted. Young leaves were taken and DNA was extracted using the CTAB method as a template. PCR amplification was performed using the four pairs of SSR markers (JAAS3611, JAAS1148, NAU3615, NYS-14) as primers. The molecular weights of the specific bands amplified by the four pairs of SSR markers in the abnormal cotton genome were 241bp, 168bp, 139bp, and 163bp, respectively. The chromosome segment containing the specific bands of the four molecular markers was the abnormal cotton chromosome segment A1-3, and the BC4F4 plant containing this segment was the abnormal cotton single-segment substitution line CSSL3.

[0026] (3) The oil content of cotton kernels of the abnormal cotton single fragment replacement line CSSL3 and the recurrent parent Su8289 was determined. The oil content of the abnormal cotton single fragment replacement line CSSL3 was significantly higher than that of Su8289, indicating that the abnormal cotton chromosome fragment A1-3 can significantly increase the oil content of cotton kernels.

[0027] The present invention has the following advantages:

[0028] This invention provides a single-segment substitution line that can increase the oil content of cottonseed kernels, creating important materials for promoting the fine localization of target genes and subsequent map-based cloning.

[0029] This invention provides an abnormal cotton chromosome fragment A1-3 that can significantly increase the oil content of cottonseed kernels. The single-fragment substitution line CSSL3 containing this fragment, after oil content determination, showed that the oil content of CSSL3 was significantly higher than that of Su8289. Therefore, the abnormal cotton chromosome fragment A1-3 contained in the abnormal cotton single-fragment substitution line CSSL3 can be applied to cotton molecular breeding, greatly increasing the oil content of cottonseed kernels and improving the efficiency of high-oil cotton breeding.

[0030] This invention provides four markers and their primer sequences for an abnormal cotton chromosome segment A1-3 that can significantly increase the oil content of cottonseed kernels. These markers can greatly improve the selection efficiency and breeding speed of high-oil-content cotton varieties. Molecular marker-assisted selection of target segments features early and rapid identification, as well as high accuracy and stability, thus accelerating the process of breeding new high-oil-content cotton varieties. Attached Figure Description

[0031] Figure 1 The present invention describes the amplification of molecular markers on four pairs of abnormal cotton chromosome fragments A1-3 in the genomes of abnormal cotton, CSSL3, and Su8289.

[0032] In the figure, lanes 1, 2, and 3 represent abnormal cotton, CSSL3, and Su8289, respectively, and the arrows indicate the target band specifically amplified by abnormal cotton.

[0033] Figure 2 This is a comparison of the oil content of cotton seed kernels from the abnormal single-sheet replacement line CSSL3 and the recurrent parent Su8289 in 2022 and 2023. Detailed Implementation

[0034] The present invention will now be described in detail through specific embodiments. These embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0035] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The following descriptions are preferred embodiments for carrying out the invention; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of the invention. The scope of protection of this invention is determined by the appended claims.

[0036] 1. Test materials

[0037] The upland cotton Su8289 (reincarnation parent) and the abnormal cotton (donor parent) used in this experiment were introduced by the Jiangsu Academy of Agricultural Sciences. Both are recorded in "Caijiao Zhai, Peng Xu, Xia Zhang et al. Development of Gossypium anomalum derived microsatellite markers and their use for genome-wide identification of recombination between the T. anomalum and G. hirsutum genomes. Theoretical and Applied Genetics, 2015, 128(8): 1531-1540". They can be obtained from the applicant and are only used to repeat the experiment of this invention. They may not be used for other purposes. They can also be obtained by purchasing.

[0038] 2. Test Methods

[0039] 2.1 Methods for extracting genomic DNA

[0040] CSSL3, Su8289, and abnormal cotton seeds were planted and genomic DNA was extracted. The method for extracting genomic DNA was as follows:

[0041] (1) Place 5g of frozen leaves (or fresh leaves) in a pre-cooled mortar and grind with liquid nitrogen. Add 10ml of freshly prepared extraction buffer (Table 1) in two portions, transfer to 50ml centrifuge tubes, vortex to mix, and incubate on ice for 10min. Centrifuge at 4000rpm for 20min (4℃) and discard the supernatant;

[0042] (2) Add 15 ml of lysis buffer (preheated at 65℃) to the precipitate, loosen it with copper wire, vortex mix it, and incubate it in a water bath at 65℃ for 30 min.

[0043] (3) Add 15 ml of chloroform:isoamyl alcohol (24:1, volume ratio) mixture, invert more than 50 times, centrifuge at 4000 rpm for 20 min (15℃), transfer the supernatant to a 50 ml centrifuge tube, add 0.6 volume of pre-cooled isopropanol, slowly invert 30 times to mix, let stand for 10 min, centrifuge at 4000 rpm for 10 min (room temperature), discard the supernatant, add 2 ml of 70% ethanol to the precipitate to wash, and transfer to a 10 ml centrifuge tube, centrifuge at 10000 rpm for 5 min. Discard the supernatant and air dry for 20 min;

[0044] (4) Add 3 ml of TE buffer (pH=8.0, 2.5 ml of 1.0 M Tris-HCl solution and 0.5 ml of 0.5 M EDTA solution mixed, and distilled water to a final volume of 250 ml for sterilization) to dissolve, incubate at 65℃ for 10-30 min, add an equal volume of chloroform:isoamyl alcohol (24:1, volume ratio), slowly invert 50 times to mix, let stand at room temperature for 5 min, and centrifuge at 10000 rpm for 10 min;

[0045] (5) Transfer the supernatant to a 10ml centrifuge tube, add 0.1 volume of 3M sodium acetate (pH 5.2), add an equal volume of isopropanol, invert 30 times, let stand for 30 min, and centrifuge at 10000 rpm for 5 min. Discard the supernatant, add 2ml of 70% ethanol to wash the DNA clumps, and centrifuge at 10000 rpm for 5 min. Discard the supernatant and air dry for 20 min.

[0046] (6) Dissolve in 3 ml of TE buffer and incubate at 65°C for 10–30 min;

[0047] (7) Add 5 μl of RNAase A (10 mg / ml) and incubate at 37°C for 30–60 min. Add an equal volume of chloroform:isoamyl alcohol (24:1, volume ratio), slowly invert 50 times to mix, let stand at room temperature for 5 min, and centrifuge at 10,000 rpm for 10 min.

[0048] (8) Transfer the supernatant to a 10ml centrifuge tube, add 0.1 volume of 3M sodium acetate (pH 5.2), add an equal volume of isopropanol, and invert 30 times.

[0049] (9) Transfer the flocculent precipitate into a 1.5 ml centrifuge tube containing 800 μl of 70% ethanol, and centrifuge at 10,000 rpm for 5 min. Discard the supernatant and allow the DNA clumps to air dry naturally.

[0050] (10) Dissolve DNA in 200 μl TE buffer [10 mM Tris / HCl (pH 8.0), 1 mM EDTA (pH 8.0)] (4℃, 1-2 days), and store at -20℃.

[0051] (11) Using 5 μL of Dalian Baosheng Biotechnology 50bp DNA Ladder as a control, the DNA was diluted 5 times, 10 times and 20 times in sequence, and the concentration, purity and integrity of the DNA were determined by 1% agarose gel electrophoresis.

[0052] (12) Based on the concentration of extracted DNA, dilute the DNA to 20 ng / μl working solution with TE and mix well for later use.

[0053] Table 1 DNA Extraction Buffer Formulation

[0054]

[0055] Table 2 Lysis buffer formulation

[0056]

[0057] 2.2 PCR Method

[0058] (1) Reagents and main instruments used

[0059] The Taq polymerase and dNTPs used in the PCR reaction were purchased from Dalian Takara Bio Engineering Co., Ltd. Reagents used in the PAGE gel, including acrylamide, methylene acrylamide, Tris-base, boric acid, silver nitrate, sodium hydroxide, and TEMED, were purchased from Rongshengda Experimental Instrument Co., Ltd. The main instruments included an Applied Biosystems PCR instrument, an Eppendoff high-speed refrigerated centrifuge, a water bath, a shaker, and an electrophoresis tank and electrophoresis apparatus manufactured by Beijing Liuyi Instrument Factory.

[0060] (2) PCR reaction system and amplification procedure

[0061] The PCR reaction system is shown in Table 3.

[0062] Table 3 PCR reaction system

[0063]

[0064] The PCR reaction was performed on an Applied Biosystems PCR instrument, and the reaction program was as follows:

[0065]

[0066] Preparation of electrophoresis solution:

[0067] The amplified products were subjected to non-denaturing polyacrylamide gel electrophoresis: gel concentration 9%, electrophoresis buffer 0.5×TBE, electrophoresis at 180y constant voltage for 1.5 to 2 hours.

[0068] 9% PAGE Gel: 43.5g acrylamide, 1.5g methylene acrylamide, 100ml 5×TBE, add distilled water to a final volume of 500ml, store at 4℃.

[0069] 10% Ammonium Persulfate: Dissolve 10g of ammonium persulfate in 100ml of distilled water and store at 4℃.

[0070] Sample loading buffer: 0.25g bromophenol blue + 0.25g xylene cyanide + 40g sucrose, distilled water to a final volume of 100ml.

[0071] 5×TBE: 54g Tris-base, 27.5g boric acid, 20ml 0.5M EDTA (pH=8.0), distilled water to a final volume of 1L, store at room temperature.

[0072] Staining solution: 1g silver nitrate + 500ml distilled water.

[0073] Colorimetric solution: 7.5g sodium hydroxide + 750μL formaldehyde + 500ml distilled water.

[0074] Gel preparation and electrophoresis process:

[0075] (1) Wash the glass plate, adhesive strip and comb with clean water and let them dry.

[0076] (2) Install the glass plate, adhesive strip and comb as required, and seal the bottom with 1% agarose gel. After the gel solidifies, fix it on the electrophoresis tank.

[0077] (3) Pour the prepared 9% PAGE gel into the conical flask, add 10% AP and TEMED, pour the gel quickly, insert the comb after filling, and remove the comb after 15-20 minutes to prepare for electrophoresis.

[0078] (4) Before electrophoresis, add 2 μL of loading buffer to the PCR amplification product, remove the comb, and add electrophoresis buffer (1×TBE) to the positive and negative electrode electrophoresis tanks. The buffer level should be above the short glass plate. Load 2 μL of sample into each well and electrophoresis at a constant voltage of 180V for 1.5–2 hours. Stop when the blue indicator is 2 cm below the gel. Carefully remove the glass plate, remove the gel, and label the gel.

[0079] Staining and developing process:

[0080] (1) Fixation: Place the removed gel in the fixative (10% ethanol + 0.5% glacial acetic acid) for 12 min.

[0081] (2) Staining: After fixation, pour out the fixative and pour the staining solution into (0.2% silver nitrate aqueous solution). After 12 minutes, rinse 3 times with distilled water.

[0082] (3) Color development: Add 1.5% sodium hydroxide + 0.4% formaldehyde, shake, and stop when the stripe on the film is clearly visible. Pour off the color development solution, rinse with tap water 4 times, and place the film on a light box to take pictures.

[0083] 2.3 Methods for determining oil content

[0084] The oil content of cottonseed kernels was determined by Soxhlet extraction, and the specific method was in accordance with GB / T 5512285.

[0085] 1. Slice

[0086] Cut the filter paper into 8cm x 8cm pieces, fold them into paper packets with one side unsealed, and number them sequentially with a hard pencil. Arrange them in petri dishes in order. Transfer the petri dishes containing the filter paper packets to an oven at 105±2℃ and dry for 2 hours. Remove them and place them in a desiccator to cool to room temperature. Place each filter paper packet in the same weighing bottle in order and weigh them (recorded as a). The relative humidity in the room must be below 70% during weighing.

[0087] 2. Packaging and drying

[0088] Place approximately 3g of finely ground sample into the weighed filter paper packet, seal the packet, and dry it in an oven at 105±2℃ for 3 hours. Then, transfer it to a desiccator to cool to room temperature. Weigh the sample into the weighing bottle in sequence (referred to as b).

[0089] 3. Extraction

[0090] Place the filter paper packet containing the sample into the extraction tube using long forceps, and inject anhydrous diethyl ether to completely submerge the sample packet. Connect all parts of the extractor, turn on the cooling water flow, and perform extraction in a constant temperature water bath. Adjust the water temperature between 70 and 80°C so that the condensed ether drips in a continuous stream (120-150 drops / min or reflux more than 7 times / h). Extract until no oil residue is found when a drop of ether is tested with filter paper in the extraction tube (approximately 6-12 hours). After extraction, remove the filter paper packet using expanded forceps and allow the ether to evaporate in a ventilated area (the extraction room temperature should ideally be 12-25°C). The ether in the extraction flask should be recovered separately.

[0091] 4. Weighing

[0092] After the ether evaporates, place the filter paper package in an oven at 105±2℃ and dry for 2 hours. Then, place it in a desiccator to cool until constant weight is achieved (denoted as c).

[0093] 5. Results and Calculations

[0094] Crude fat content (%) = (bc) / (ba) × 100%, where a: weight of the weighing bottle with filter paper (g); b: weight of the weighing bottle with filter paper and dried sample (g); c: weight of the weighing bottle with filter paper and dried residue after extraction (g). 3. Screening of labels and determination of oil content.

[0095] (1) Using upland cotton 86-1 as the female parent and abnormal cotton as the male parent, a hexaploid F1 hybrid was obtained by colchicine doubling. Morphological, cytological, and molecular marker techniques were used to identify and prove that we obtained a hexaploid hybrid that had successfully doubled (Zhang et al., 2014). Using the hexaploid F1 as the female parent and Su 8289 as the recurrent parent, we continued to backcross four times to the BC4 generation, and then self-crossed for four generations to produce BC4F4.

[0096] (2) BC4F4 plants, the recurrent parent Su8289, and abnormal cotton were planted. Young leaves were collected, and DNA was extracted using the CTAB method as a template. Genotyping of BC4F4 plants was performed using 230 pairs of SSR primers developed by our research group that uniformly cover the abnormal cotton genome. Only the molecular weights of the amplification products of the four pairs of SSR molecular marker primers shown in Table 4 differed between the BC4F4 plant (F5016) and the recurrent parent Su8289. The molecular weights of the amplification products of the remaining SSR molecular marker primers did not differ between the BC4F4 plant (F5016) and the recurrent parent Su8289. Figure 1 The sequences of the bands are shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively, and are located on chromosome 1 of the cotton chromosome set. Through marker identification, we determined that the target fragments of four molecular markers could be amplified in the BC4F4 plant numbered F5016 (named CSSL3). The chromosome fragment containing the specific bands of the four molecular markers is the abnormal cotton chromosome fragment A1-3, that is, the abnormal cotton single fragment replacement line CSSL3 contains the abnormal cotton chromosome fragment A1-3.

[0097] Table 4. SSR molecular markers and their sequences on chromosome segments A1-3

[0098]

[0099] Specifically, the band sequences corresponding to each molecular marker JAAS3611, JAAS1148, NAU3615, and NYS-14 are as follows:

[0100] SEQ ID NO.9:

[0101]

[0102] SEQ ID NO.10:

[0103]

[0104] SEQ ID NO.11:

[0105]

[0106] SEQ ID NO.12:

[0107]

[0108]

[0109] (3) In 2022 and 2023, the abnormal cotton single-petal replacement line CSSL3 and the recurrent parent Su8289 were planted at the Lishui Plant Science Base of Jiangsu Academy of Agricultural Sciences. Each material was planted in 3 rows with 12 seedlings per row, and 3 replicates were performed. After maturity, cotton bolls that opened normally in the middle of the plant were harvested. After sulfuric acid delinting, the oil content of the cotton kernels was measured using Soxhlet extraction. In 2022, the average oil content of the abnormal cotton single-petal replacement line CSSL3 was 38.2%, and the average oil content of the recurrent parent Su8289 was 31.9%. In 2023, the average oil content of the abnormal cotton single-petal replacement line CSSL3 was 37.4%, and the average oil content of the recurrent parent Su8289 was 31.3%. The results of the two years showed that the oil content of the abnormal cotton single-petal replacement line CSSL3 was significantly higher than that of the recurrent parent Su8289. Figure 2 ).

[0110] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A molecular marker for the abnormal cotton chromosome segment A1-3 that can increase the oil content of cottonseed kernels, characterized in that, The molecular markers consist of four SSR markers: JAAS3611, JAAS1148, NAU3615, and NYS-14. The primer sequences and amplified fragment lengths of the four SSR markers are as follows: JAAS3611: The forward primer sequence is SEQ ID NO.1, the reverse primer sequence is SEQ ID NO.2, and the target fragment length is 241bp amplified in the abnormal cotton genome; JAAS1148: The forward primer sequence is SEQ ID NO.3, the reverse primer sequence is SEQ ID NO.4, and the target fragment length is 168bp amplified in the abnormal cotton genome; NAU3615: The forward primer sequence is SEQ ID NO.5, the reverse primer sequence is SEQ ID NO.6, and the target fragment length is 139bp amplified in the abnormal cotton genome; NYS-14: The forward primer sequence is SEQ ID NO.7, and the reverse primer sequence is SEQ ID NO.

8. The target fragment length is 163bp amplified in the abnormal cotton genome.

2. An SSR marker primer for amplifying the molecular marker of the abnormal cotton chromosome segment A1-3, which can increase the oil content of cottonseed kernels as described in claim 1, characterized in that, The sequence of the SSR marker primer is as follows: JAAS3611: The forward primer sequence is SEQ ID NO.1, and the reverse primer sequence is SEQ ID NO.2; JAAS1148: The forward primer sequence is SEQ ID NO.3, and the reverse primer sequence is SEQ ID NO.4; NAU3615: The forward primer sequence is SEQ ID NO.5, and the reverse primer sequence is SEQ ID NO.6; NYS-14: The forward primer sequence is SEQ ID NO.7, and the reverse primer sequence is SEQ ID NO.

8.

3. The application of the SSR marker primers as described in claim 2 in the identification or auxiliary identification of cotton varieties or lines with high oil content.

4. The application according to claim 3, characterized in that, The test detects whether the genomic DNA of the cotton sample contains the abnormal cotton chromosome fragment A1-3. Cotton containing the abnormal cotton chromosome fragment A1-3 is a cotton variety or strain with high oil content.

5. The application according to claim 4, characterized in that, The method for detecting whether the cotton genomic DNA to be tested contains the abnormal cotton chromosome fragment A1-3 is as follows: PCR amplification is performed on the cotton genomic DNA to be tested using the SSR marker primers described in claim 2. If fragments with lengths of 241bp, 168bp, 139bp, and 163bp are present simultaneously, then the cotton genomic DNA to be tested contains the abnormal cotton chromosome fragment A1-3; otherwise, the cotton genomic DNA to be tested does not contain the abnormal cotton chromosome fragment A1-3.

6. A cotton breeding method for increasing the oil content of cottonseed kernels, characterized in that, The method is as follows: hybridization is carried out between upland cotton and abnormal cotton as parents to obtain a generation population, wherein the generation population also includes upland cotton offspring with chromosome segment A1-3 of abnormal cotton as described in claim 1, and the oil content of the cottonseed kernels of the upland cotton offspring is higher than that of the parent upland cotton.

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