SSR sequence related to salt tolerance of abnormal cotton derived from cotton diploid wild species and application of SSR sequence

Abstract

The invention discloses a salt tolerance related SSR sequence derived from cotton diploid wild species abnormal cotton and application thereof, the SSR sequence belongs to a specific DNA fragment of an abnormal cotton introgression line CSSL44, and is located on a No.6 chromosome of an abnormal cotton chromosome group. The specific DNA fragment provided by the invention has an important application value in salt-tolerant cotton breeding, meanwhile, the 9 pairs of SSR molecular markers developed by the invention provide a molecular basis for cultivating salt-tolerant cotton varieties which can be applied to production, and by utilizing the molecular markers provided by the invention, the salt-tolerant character of a cotton plant can be quickly identified, and the application prospect is wide. And a technical foundation is laid for research on a cotton salt tolerance improving mechanism.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically relating to salt-tolerant SSR sequences derived from the diploid wild cotton species Abnormal Cotton and their applications. Background Technology

[0002] Cotton is an important economic crop. In recent years, with changes in the domestic and international economic situation and adjustments in national industrial policies, cotton production in my country has become more regionalized, with a significant decline in planting area in the Yangtze River and Yellow River basins. Xinjiang, with its higher level of mechanization, has become my country's main cotton-producing area. However, Xinjiang is also one of the regions with the widest distribution of saline-alkali land in my country, with saline-alkali soils accounting for about one-third of the country's total saline-alkali land area, mainly distributed in the Tarim Basin, Junggar Basin, and downstream river areas. Severe soil salinization severely restricts cotton yield and quality, and the existing cultivated upland cotton (Gossypium hirsutum) has a narrow genetic base and a scarcity of salt-tolerant germplasm resources. Although QTL mapping technology provides a method for discovering salt-tolerant traits, traditional QTL intervals are large, containing a large number of genes, and the cotton genome is complex, resulting in low efficiency in the precise mapping and functional verification of salt-tolerant genes. Gossypium anomalum, a type of cotton, is mainly distributed in arid and semi-arid regions of southwestern Africa, including Namibia, southern Angola, and northwestern South Africa. It exhibits strong resistance to various pests and diseases (such as bollworm, aphids, wilt, and verticillium wilt) as well as abiotic stresses such as drought and salinity. In recent years, introgression lines (ILs) have become ideal materials for QTL mapping and gene cloning. In our laboratory, using the hexaploid F1 generation obtained by doubling the cross between upland cotton and Gossypium anomalum with colchicine as the maternal parent and Su 8289 as the recurrent parent, we conducted four consecutive backcrosses, incorporating marker-assisted selection in each generation to obtain a batch of introgression lines derived from Gossypium anomalum (Xu et al., 2022). This provides crucial genetic resources for the continuous improvement of cultivated cotton varieties and their adaptation to future environmental changes.

[0003] Traditional salt-tolerance breeding relies on phenotypic screening, which is time-consuming and inefficient, and most existing salt-tolerance genes are derived from cultivated cotton or model plants. This invention identifies salt-tolerance QTLs and candidate genes from the diploid wild-type abnormal cotton introgression line CSSL44, combining high-density molecular markers and transcriptome analysis. This enables fine mapping of salt-tolerance QTLs and rapid cloning of functional genes, significantly shortening the breeding cycle and improving accuracy. Summary of the Invention

[0004] The purpose of this invention is to provide a salt tolerance-related SSR sequence derived from the diploid wild cotton species *G. anomalum* and its application.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] The salt tolerance-related SSR sequence is derived from the diploid wild cotton species *G. anomalum*. The SSR sequence consists of nine SSR markers: NAU1272, NAU2637, NAU905, DPL0365, NAU5373, NAU2397, JAAS6227, NBRI1275, and JAAS2480. The primer sequences and amplification fragments for these nine SSR markers are as follows:

[0007] NAU1272: The forward primer sequence is SEQ ID NO.10, the reverse primer sequence is SEQ ID NO.11, and the sequence for amplifying the target fragment in the abnormal cotton genome is as shown in SEQ ID NO.1;

[0008] NAU2637: The forward primer sequence is SEQ ID NO.12, the reverse primer sequence is SEQ ID NO.13, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.2;

[0009] NAU905: The forward primer sequence is SEQ ID NO.14, the reverse primer sequence is SEQ ID NO.15, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.3;

[0010] DPL0365: The forward primer sequence is SEQ ID NO.16, the reverse primer sequence is SEQ ID NO.17, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.4;

[0011] NAU5373: The forward primer sequence is SEQ ID NO.18, the reverse primer sequence is SEQ ID NO.19, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.5;

[0012] NAU2397: The forward primer sequence is SEQ ID NO.20, the reverse primer sequence is SEQ ID NO.21, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.6;

[0013] JAAS6227: The forward primer sequence is SEQ ID NO.22, the reverse primer sequence is SEQ ID NO.23, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.7;

[0014] NBRI1275: The forward primer sequence is SEQ ID NO.24, the reverse primer sequence is SEQ ID NO.25, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.8;

[0015] JAAS2480: The forward primer sequence is SEQ ID NO.26, the reverse primer sequence is SEQ ID NO.27, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.9.

[0016] On the other hand, SSR marker primers for amplifying the above-mentioned molecular markers were also disclosed, and the sequences of the SSR marker primers are as follows:

[0017] NAU1272: The forward primer sequence is SEQ ID NO.10, and the reverse primer sequence is SEQ ID NO.11;

[0018] NAU2637: The forward primer sequence is SEQ ID NO.12, and the reverse primer sequence is SEQ ID NO.13;

[0019] NAU905: The forward primer sequence is SEQ ID NO.14, and the reverse primer sequence is SEQ ID NO.15;

[0020] DPL0365: The forward primer sequence is SEQ ID NO.16, and the reverse primer sequence is SEQ ID NO.17;

[0021] NAU5373: The forward primer sequence is SEQ ID NO.18, and the reverse primer sequence is SEQ ID NO.19;

[0022] NAU2397: The forward primer sequence is SEQ ID NO.20, and the reverse primer sequence is SEQ ID NO.21;

[0023] JAAS6227: The forward primer sequence is SEQ ID NO.22, and the reverse primer sequence is SEQ ID NO.23;

[0024] NBRI1275: The forward primer sequence is SEQ ID NO.24, and the reverse primer sequence is SEQ ID NO.25;

[0025] JAAS2480: The forward primer sequence is SEQ ID NO.26, and the reverse primer sequence is SEQ ID NO.27.

[0026] The present invention also discloses the application of the above-mentioned SSR marker primers in screening specific DNA fragments for breeding salt-tolerant cotton varieties or lines, wherein the specific DNA fragment is a fragment located on chromosome 6 of the abnormal cotton that contains at least the SSR molecular marker NAU1272 to the SSR molecular marker JAAS2480, and further, the specific DNA fragment is a fragment located on chromosome 6 of the abnormal cotton that contains at least the SSR molecular marker NAU1272 to the SSR molecular marker JAAS2480.

[0027] The specific DNA fragment includes the SSR molecular markers NAU1272, NAU2637, NAU905, DPL0365, NAU5373, NAU2397, JAAS6227, NBRI1275, and JAAS2480 located on the abnormal cotton chromosome 6.

[0028] The specific DNA fragment is identical to the sequence of the fragment located on chromosome 6 of the abnormal cottony chromosome, from the SSR molecular marker NAU1272 to the SSR molecular marker JAAS2480.

[0029] Plants containing the SSR molecular markers NAU1272, NAU2637, NAU905, DPL0365, NAU5373, NAU2397, JAAS6227, NBRI1275, and JAAS2480 on chromosome 6 of the abnormal cotton showed increased salt tolerance.

[0030] This invention also discloses the application of the aforementioned SSR molecular marker primers in identifying or assisting in the identification of salt-tolerant cotton varieties or lines. 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 salt-tolerant cotton variety or line. The detection can be performed using PCR, specifically using the aforementioned molecular marker primer pairs, or it can be performed using sequencing methods.

[0031] More specifically, the test detects whether the genomic DNA of the cotton to be tested contains a specific DNA fragment of the abnormal cotton introgression line CSSL44. Cotton containing the specific DNA fragment of the abnormal cotton introgression line CSSL44 is a salt-tolerant cotton variety or line.

[0032] The method for detecting whether the genomic DNA of the cotton to be tested contains a specific DNA fragment of the abnormal cotton introgression line CSSL44 is as follows: PCR amplification is performed on the genomic DNA of the cotton to be tested using SSR marker primers. If the fragments described in SEQ ID NO. 1 to 9 are present, then the genomic DNA of the cotton to be tested contains a specific DNA fragment of the abnormal cotton introgression line CSSL44; otherwise, the genomic DNA of the cotton to be tested does not contain a specific DNA fragment of the abnormal cotton introgression line CSSL44.

[0033] In the above applications, the specific DNA fragment can be used to replace the original chromosome fragment of the recipient parent to obtain cotton varieties or lines with improved salt tolerance.

[0034] This invention also discloses a breeding method for improving the salt tolerance of cotton. The method involves hybridizing upland cotton and abnormal cotton as parents to obtain a generation population. The generation population includes upland cotton progeny containing a specific DNA fragment of the abnormal cotton introgression line CSSL44. The salt tolerance 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 lines; any cotton variety or line that can be hybridized with abnormal cotton is acceptable.

[0035] In this invention, the salt tolerance is embodied in all or part of the following: germination rate, relative plant height, relative fresh weight of aboveground parts, and relative dry weight of aboveground parts.

[0036] In addition, the present invention also protects the use of a kit containing primers with the aforementioned nine SSR markers in identifying or assisting in the identification of salt tolerance in cotton.

[0037] The kit may also contain one or more of the following: PCR amplification buffer, double-distilled water, DNA polymerase, dNTPs, etc.

[0038] In a specific embodiment of the present invention, primer pair 1 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 9 and SEQ ID NO. 10; primer pair 2 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 11 and SEQ ID NO. 12; primer pair 3 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 13 and SEQ ID NO. 14; primer pair 4 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 15 and SEQ ID NO. 16; primer pair 5 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 17 and SEQ ID NO. 18; primer pair 6 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 19 and SEQ ID NO. 20; primer pair 7 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 21 and SEQ ID NO. 22; primer pair 8 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 23 and SEQ ID NO. 24; and primer pair 9 consists of two single-stranded DNA molecules as shown in SEQ ID NO. 25 and SEQ ID NO. 26.

[0039] This invention also discloses a method for constructing an upland cotton-abnormal cotton infiltration system, comprising the following steps:

[0040] (1) Triploid F1 was obtained by hybridization of upland cotton 86-1 as the female parent and abnormal cotton as the male parent, and hexaploid F1 hybrid was obtained by colchicine doubling (Zhang et al., 2014). Using hexaploid F1 as the female parent and upland cotton Su8289 as the recurrent parent, four consecutive backcrosses and two self-crosses were performed to the BC4F3 generation. In each generation, abnormal cotton-specific SSR markers covering the genome were used for assisted selection (Zhai et al., 2015) to obtain the single-segment introgression line CSSL44 of abnormal cotton chromosome 6 (Xu et al., 2022). The whole genome foreground and background of CSSL44 were identified using 230 SSR markers that uniformly cover the abnormal cotton chromosome set. Among them, the PCR amplification products of 9 SSR molecular markers differed between the single-segment introgression line CSSL44 and the recurrent parent Su8289.

[0041] (2) To verify whether the genotype of the abnormal cotton introgression line CSSL44 identified by SSR was homozygous, young leaves were taken from self-pollinated plants of the BC4F4 generation of the single-segment introgression line CSSL44, the recurrent parent Su8289, upland cotton 86-1, abnormal cotton, and hexaploid plants, and DNA was extracted using the CTAB method. Using the DNA from the recurrent parent Su8289, upland cotton 86-1, abnormal cotton, hexaploid plants, and the self-pollinated plants of the BC4F4 generation of the single-segment introgression line CSSL44 as templates, PCR amplification was performed using nine SSR markers (NAU1272, NAU2637, NAU905, DPL0365, NAU5373, NAU2397, JAAS6227, NBRI1275, and JAAS2480). The results showed that the genotypes did not segregate, indicating that the abnormal cotton introgression line CSSL44 had been stably inherited and was a homozygous introgression line.

[0042] (3) The abnormal cotton chromosome 6 single-segment introgression line CSSL44 exhibits stronger salt tolerance during the seedling and germination stages compared to the recurrent parent Su8289. This indicates that the abnormal cotton introgression line CSSL44 can significantly improve the salt tolerance of cotton.

[0043] The present invention has the following advantages:

[0044] (1) This invention combines distant hybridization and marker-assisted selection to replace a fragment on chromosome 6 of the abnormal cotton (located between SSR markers NAU1272 and JAAS2480) with the genetic background of upland cotton Su8289, thus obtaining a salt-tolerant cotton line with the abnormal cotton chromosome fragment. Therefore, the specific DNA fragment developed in this invention (the fragment on chromosome 6 of the abnormal cotton located between SSR markers NAU1272 and JAAS2480) has important application value in future salt-tolerant cotton breeding.

[0045] (2) Molecular marker-assisted target fragment selection has the characteristics of early identification, rapid identification and high accuracy and stability. Therefore, the nine SSR molecular markers and their primer pairs provided by this invention are expected to greatly improve the selection efficiency and breeding speed of salt tolerance traits in cotton breeding, which is of great significance for accelerating the breeding process of salt-tolerant new cotton varieties.

[0046] (3) The salt-tolerant cotton line provided by the present invention can be used for fine mapping of salt-tolerant genes and cloning and functional analysis of related genes through SSR molecular markers and their primer pairs. This can not only elucidate the molecular genetic mechanism of salt tolerance in cotton, but also provide materials and molecular basis for breeding salt-tolerant varieties that can be applied to production. Attached Figure Description

[0047] Figure 1 This involves a banding analysis of nine polymorphic markers within the CSSL44 interval;

[0048] Figure 2 This is a diagram of the CSSL44 genotype based on SSR markers and resequencing;

[0049] Figure 3 Salt tolerance identification during the germination period of Su8289 and CSSL44;

[0050] Figure 4 This study identified the salt tolerance of seedlings of Su8289 and CSSL44. Detailed Implementation

[0051] 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.

[0052] 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.

[0053] 1. Test materials

[0054] The gossypium 86-1 (G. hirsutum var. 86-1), gossypium 8289 (G. hirsutum var. Su8289), and gossypium anomalum used in this experiment are all described 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 G. anomalum and G. hirsutum genomes. Theoretical and Applied Genetics, 2015, 128(8): 1531-1540”. They are available to the public from the applicant and may only be used to repeat the experiments of this invention and may not be used for other purposes.

[0055] 2. Test Methods

[0056] 2.1 DNA Extraction

[0057] Cotton DNA was extracted using a modified CTAB (Paterson et al., 1993) combined with a novel plant genomic DNA extraction kit from Shanghai Pudi Biotechnology Co., Ltd. The specific steps are as follows:

[0058] (1) Sampling. Select a young shoot (approximately 0.5 cm) from each plant. 2 Place the sample (size) in a 2mL EP tube and briefly place it in an ice box to await extraction;

[0059] (2) Preparation of extraction solution. Preheat DNA buffer in a 65°C water bath. Take 100 mL of buffer into an Erlenmeyer flask, add half a spoonful of ascorbic acid and 1 mL of β-mercaptoethanol, and mix well.

[0060] (3) Sample grinding. Place steel balls in each EP tube, add 500 μL of extraction solution, and grind for 180 s using a fully automated rapid sample grinder at a frequency of 55 Hz.

[0061] (4) Collect the supernatant. Incubate in a 65℃ water bath for 30 min, shaking and turning the tube several times every 10 min. After the water bath is complete, centrifuge at 12000 rpm for 15 min, and transfer 250 μL of the supernatant to a new batch of 1.5 mL centrifuge tubes.

[0062] (5) Extract DNA using the kit. Add 500 μL of reagent FC to a centrifuge tube, vortex to mix, and pour the mixture, along with the precipitate, into the adsorption column in the kit. Centrifuge at 12000 rpm for 1 min. Discard the filtrate. Add 800 μL of reagent GW to the adsorption column and centrifuge at 12000 rpm for 1 min. Discard the filtrate and centrifuge at 12000 rpm for 1 min. Place the adsorption column into a new 1.5 mL centrifuge tube, open the cap, and let it stand until the alcohol in reagent GW evaporates completely. Add 100 μL of sterile water at 65°C to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the adsorption column, and label the centrifuge tube. DNA extraction is complete.

[0063] (6) Concentration detection. After DNA extraction, its concentration is detected using a multi-functional microplate reader. Samples that pass the test are stored at -20℃. Samples with insufficient concentration are marked and need to be extracted again.

[0064] 2.2 PCR reaction and polymorphic primer screening

[0065] Using 208 newly developed SSR primer pairs, PCR reactions were performed on upland cotton (Su-mian 8289), abnormal cotton, and F1 as templates to screen for polymorphic primers. The 2×3G Taq Master Mix for PAGE (Red Dye) used in this experiment, containing 3GT Taq DNA Polymerase, dNTP Mix, a visualization red dye, and an optimized buffer system, was purchased from Nanjing Novizan Biotechnology Co., Ltd. The PCR reaction system is shown in Table 1.

[0066] Table 1 PCR reaction system

[0067]

[0068] The PCR reaction was performed on an Applied Biosystems PCR instrument, and the reaction program was as follows:

[0069]

[0070] 2.3 Polyacrylamide gel electrophoresis

[0071] (1) Applying the adhesive: Align the two glass plates of the set, lay them flat, clamp them on both sides, and place them on the flat plate. Insert a 104-tooth comb halfway into the groove. Slowly pour 8% polyacrylamide gel (29:1) into the comb, fully insert the comb into the groove, and let it stand for 2 hours;

[0072] (2) Placing the glass plate with gel into the electrophoresis tank, pour in 1×TBE electrophoresis buffer, and tighten it with clamps on both sides. Continue to add electrophoresis buffer until the gel is submerged, and then remove the comb.

[0073] (3) Sample loading: The sample loading volume is 1 μL, 96 samples are loaded per plate, and 50 bp DNA marker is used for labeling;

[0074] (4) Electrophoresis: Electrophoresis was performed at a constant voltage of 180V for 1.5 hours;

[0075] (5) Staining and development process: a. Fixation: Place the removed gel in the fixative (10% ethanol + 0.5% glacial acetic acid) for 12 min; b. Staining: After fixation, discard the fixative and pour in the staining solution (0.2% silver nitrate aqueous solution). After 12 min, rinse 3 times with distilled water. c. Development: Add 1.5% sodium hydroxide + 0.4% formaldehyde, shake, and stop when the bands on the film are clearly visible. Discard the development solution, rinse 4 times with tap water, and place the film on a light box for photography.

[0076] 2.4 Reading tape

[0077] Place the gel on an X-ray viewing lamp and record the band pattern. The same band pattern as Su Cotton 8289 is read as "1", the same band pattern as Abnormal Cotton is read as "2", the heterozygous band pattern is read as "3", and the missing band is read as "-".

[0078] 2.5 Selection and Identification of Markers

[0079] 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 markers were used to identify and prove that we obtained a hexaploid hybrid that had successfully doubled (Xia Zhang, Caijiao Zhai, Linchi He, Qi Guo, Xianggui Zhang, Peng Xu, Hongmei Su, Yuanyong Gong, Wanchao Ni, Xinlian Shen. Morphological, cytological and molecular analyses of a synthesized hexaploid derived from an interspecific hybrid between Gossypium hirsutum and G. anomalum. The Crop Journal, 2014, 2(5): 272-277.).

[0080] Using hexaploid F1 as the maternal parent and Su8289 as the recurrent parent, four consecutive backcrosses and two self-crosses were performed until the BC4F3 generation. In each generation, assisted selection was performed using a genome-specific SSR marker covering Gossypium anomalum (Zhai et al. 2015), resulting in the single-segment introgression line CSSL44 of Gossypium anomalum chromosome 6 (Zhenzhen Xu, Jiedan Chen, Shan Meng, Peng Xu, Caijiao Zhai, Fang Huang, Qi Guo, Liang Zhao, Yonggang Quan, Yixin Shangguan, Zhuang Meng, Tian Wen, Ya Zhang, Xianggui Zhang, Jun Zhao, Jianwen Xu, Jianguang Liu, Jin Gao, Wanchao Ni, Xianglong Chen, Wei Ji, Nanyi Wang, Xiaoxi Lu, Shihong Wang, Kai Wang, Tianzhen Zhang, Xinlian Shen. (2022) Genome sequence of Gossypium anomalum facilitates interspecific introgression breeding.PlantCommunications, https: / / doi.org / 10.1016 / j.xplc.2022.100350).

[0081] Young leaves from BC4F3 plants of the single-fragment introgression line CSSL44, upland cotton 86-1, the recurrent parent Su8289, and the hexaploid F1 of the abnormal cotton were collected. DNA was extracted using the CTAB method and used as a template. Foreground and background identification of CSSL44 was performed using 230 SSR markers uniformly covering the abnormal cotton chromosome set, developed previously by our research group. The results showed that the amplification products of 9 SSR molecular markers differed between the single-fragment introgression line CSSI44 and the recurrent parent Su8289. Figure 1 ). Figure 1The results of detecting the above nine SSR molecular markers in the BC4F3 generation of the single-fragment introgression line CSSL44 are shown in the table. For each marker, lane 1 represents the marker, lane 2 represents the genotype of upland cotton Su8289, lane 3 represents the genotype of upland cotton 86-1, lane 4 represents the genotype of abnormal cotton, lane 5 represents the genotype of hexaploid F1, and lane 6 represents the genotype of CSSL44. The genotype of CSSL44 is the same as that of hexaploid F1 because abnormal cotton B1 and upland cotton At subgroups are prone to recombination. Generally, upland cotton Dt subgroup also contains an SSR locus homologous to the At subgroup that has not undergone recombination. During PCR amplification, the primers bind to the SSR locus of abnormal cotton B1 and also to the SSR locus of Dt subgroup; therefore, the genotype of CSSL44 is the same as that of hexaploid F1.

[0082] Therefore, the abnormal cotton chromosome segment containing all nine molecular marker-specific bands is the abnormal cotton single-segment introgression line CSSL44. The specific sequences of these nine molecular markers (NAU1272, NAU2637, NAU905, DPL0365, NAU5373, NAU2397, JAAS6227, NBRI1275, JAAS2480) are as follows:

[0083] SSR molecular marker NAU1272: DNA molecule with nucleotide sequence as shown in SEQ ID NO.1;

[0084] SSR molecular marker NAU2637: DNA molecule with nucleotide sequence as shown in SEQ ID NO.2;

[0085] SSR molecular marker NAU905: DNA molecule with nucleotide sequence as shown in SEQ ID NO.3;

[0086] SSR molecular marker DPL0365: DNA molecule with nucleotide sequence as shown in SEQ ID NO.4;

[0087] SSR molecular marker NAU5373: DNA molecule with nucleotide sequence as shown in SEQ ID NO.5;

[0088] SSR molecular marker NAU2397: DNA molecule with nucleotide sequence as shown in SEQ ID NO. 6;

[0089] SSR molecular marker JAAS6227: DNA molecule with nucleotide sequence as shown in SEQ ID NO.7;

[0090] SSR molecular marker NBRI1275: DNA molecule with nucleotide sequence as shown in SEQ ID NO.8;

[0091] SSR molecular marker JAAS2480: DNA molecule with nucleotide sequence as shown in SEQ ID NO.9.

[0092] The specific sequence is as follows:

[0093] SEQ ID NO.1:

[0094] SEQ ID NO.2:

[0095]

[0096] SEQ ID NO.3:

[0097] SEQ ID NO.4:

[0098] SEQ ID NO.5:

[0099] SEQ ID NO.6:

[0100] SEQ ID NO.7:

[0101] SEQ ID NO.8:

[0102]

[0103]

[0104] SEQ ID NO.9:

[0105]

[0106] The primer pairs used to amplify the above nine SSR molecular markers are shown in Table 2.

[0107] Table 2 shows the SSR polymorphism marker information used to detect CSSL44 recombination types.

[0108]

[0109] Using hexaploid F1 as the maternal parent and Su 8289 as the recurrent parent, four consecutive backcrosses were performed, with marker-assisted selection in each generation, resulting in a batch of introgression lines derived from abnormal cotton (Xu et al., 2022). Among them, the single-segment introgression line CSSL44 on chromosome A6 exhibited stronger salt tolerance during the seedling and germination stages compared to the recurrent parent. Previous genome-wide SSR analysis identified CSSL44 as a single-segment introgression line on chromosome 6, with the marker interval NAU1272-JAAS2480. Resequencing analysis of CSSI44 using the Illumina sequencing platform revealed that, except for an 88.66 Mb abnormal cotton fragment in the region 11,304,496–99,962,242 bp on chromosome 6, the rest of the genetic background of the recurrent parent Su 8289 was present, consistent with the SSR molecular identification results. Figure 2 (Table 3).

[0110] Table 3. CSSL44 Infiltration System SSR Marking Information

[0111]

[0112] Example 2: Salt tolerance assessment of CSSL44, a gradient line derived from abnormal cotton.

[0113] 2.1 Identification of Salt Tolerance During Seed Germination of the Abnormal Cotton Single-Fragment Introgression Line CSSL44

[0114] Salt tolerance of the abnormal single-fragment introgression line CSSL44 and the recurrent parent Su8289 was assessed at the germination and seedling stages in a culture laboratory. It was found that CSSL44 showed salt tolerance at 200 mmol·L⁻¹. 1 Under NaCl solution treatment, it exhibited a high relative germination rate of 72.5%, which was significantly higher than that of the recurrent parent strain Su8289. Figure 3 ).

[0115] 2.2 Identification of Salt Tolerance in Seedlings of the Abnormal Cotton Single-Fragment Introgression Line CSSL44

[0116] When the plant grows to the stage of two leaves and one bud, use 350 mmol·L⁻¹ 1 After treatment with NaCl solution for 7 days, the relative plant height of CSSL44 was 63%, the relative fresh weight of aboveground parts was 76%, and the relative dry weight of aboveground parts was 72%, all of which were significantly higher than those of the recurrent parent, Su8289. Figure 4 The above results indicate that CSSL44 exhibits strong salt tolerance during both the germination and seedling stages.

[0117] 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. The application of salt tolerance-related SSR sequences derived from the diploid wild cotton species *Abnormal cotton* in improving cotton salt tolerance or breeding salt-tolerant cotton varieties or lines, characterized in that... The SSR sequence consists of nine SSR markers: NAU1272, NAU2637, NAU905, DPL0365, NAU5373, NAU2397, JAAS6227, NBRI1275, and JAAS2480. The primer sequences and amplification fragments of the nine SSR markers are as follows: NAU1272: The forward primer sequence is SEQ ID NO.10, the reverse primer sequence is SEQ ID NO.11, and the sequence for amplifying the target fragment in the abnormal cotton genome is as shown in SEQ ID NO.1; NAU2637: The forward primer sequence is SEQ ID NO.12, the reverse primer sequence is SEQ ID NO.13, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.2; NAU905: The forward primer sequence is SEQ ID NO.14, the reverse primer sequence is SEQ ID NO.15, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.3; DPL0365: The forward primer sequence is SEQ ID NO.16, the reverse primer sequence is SEQ ID NO.17, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.4; NAU5373: The forward primer sequence is SEQ ID NO.18, the reverse primer sequence is SEQ ID NO.19, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.5; NAU2397: The forward primer sequence is SEQ ID NO.20, the reverse primer sequence is SEQ ID NO.21, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.6; JAAS6227: The forward primer sequence is SEQ ID NO.22, the reverse primer sequence is SEQ ID NO.23, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.7; NBRI1275: The forward primer sequence is SEQ ID NO.24, the reverse primer sequence is SEQ ID NO.25, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.8; JAAS2480: The forward primer sequence is SEQ ID NO.26, the reverse primer sequence is SEQ ID NO.27, and the sequence for amplifying the target fragment in the abnormal cotton genome is SEQ ID NO.

9.

2. SSR marker primers for amplifying the salt tolerance-related SSR sequence derived from the diploid wild-type cotton, *Abnormal Cotton*, as described in claim 1, characterized in that... The sequence of the SSR marker primer is as follows: NAU1272: The forward primer sequence is SEQ ID NO.10, and the reverse primer sequence is SEQ ID NO.11; NAU2637: The forward primer sequence is SEQ ID NO.12, and the reverse primer sequence is SEQ ID NO.13; NAU905: The forward primer sequence is SEQ ID NO.14, and the reverse primer sequence is SEQ ID NO.15; DPL0365: The forward primer sequence is SEQ ID NO.16, and the reverse primer sequence is SEQ ID NO.17; NAU5373: The forward primer sequence is SEQ ID NO.18, and the reverse primer sequence is SEQ ID NO.19; NAU2397: The forward primer sequence is SEQ ID NO.20, and the reverse primer sequence is SEQ ID NO.21; JAAS6227: The forward primer sequence is SEQ ID NO.22, and the reverse primer sequence is SEQ ID NO.23; NBRI1275: The forward primer sequence is SEQ ID NO.24, and the reverse primer sequence is SEQ ID NO.25; JAAS2480: The forward primer sequence is SEQ ID NO.26, and the reverse primer sequence is SEQ ID NO.

27.

3. The application of the SSR marker primers according to claim 2 in screening specific DNA fragments for breeding salt-tolerant cotton varieties or lines, characterized in that, The specific DNA fragment is a chromosomal segment located on chromosome 6 of the abnormal cotton chromosome, from the SSR molecular marker NAU1272 described in claim 1 to the SSR molecular marker JAAS2480.

4. The application according to claim 3, characterized in that, The test detects whether the genomic DNA of the cotton sample contains a specific DNA fragment of the abnormal cotton introgression line CSSL44. Cotton containing the specific DNA fragment of the abnormal cotton introgression line CSSL44 is a salt-tolerant cotton variety or line.

5. The application according to claim 4, characterized in that, The method for detecting whether the cotton genomic DNA to be tested contains the specific DNA fragment is as follows: PCR amplification of the cotton genomic DNA to be tested is performed using the SSR marker primers described in claim 2. If the fragments described in SEQ ID NO. 1 to 9 are present, then the cotton genomic DNA to be tested contains the specific DNA fragment; otherwise, the cotton genomic DNA to be tested does not contain the specific DNA fragment.

6. The application according to claim 5, characterized in that, The cotton was selected from a generational population with upland cotton and variegated cotton as parents.

7. A breeding method for improving the salt tolerance of cotton, characterized in that, The method is as follows: hybridization is carried out between upland cotton and atypical cotton as parents to obtain a generation population, wherein the generation population simultaneously contains upland cotton offspring with the specific DNA fragment described in claim 3, and the salt tolerance of the upland cotton offspring is higher than that of the parent upland cotton.

8. The method according to claim 7, characterized in that, The salt tolerance is reflected in all or part of the following: germination rate, relative plant height, relative fresh weight of aboveground parts, and relative dry weight of aboveground parts.

9. The use of a kit containing primers with the nine SSR markers described in claim 1 in identifying or assisting in the identification of salt tolerance in cotton.

Images