Abnormal cotton-derived salt-tolerant domesticated and improved lint-length-related molecular marker and application thereof

Abstract

The invention discloses a salt-tolerant and domesticated and improved lint-length-related molecular marker derived from abnormal cotton and application of the molecular marker. The invention provides any one of the following applications of a specific DNA fragment: cultivating or assisting in cultivating a cotton variety or strain with improved salt tolerance and / or improved lint percentage; the salt tolerance of the cotton is improved and / or the lint percentage of the cotton is improved; the specific DNA fragment is a fragment from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19 on the abnormal cotton chromosome 1; the specific DNA fragment is a fragment from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19; the nucleotide sequence of the SSR molecular marker NAU3615 is as shown in SEQ ID NO: 1; the nucleotide sequence of the InDel molecular marker NY1-19 is as shown in SEQ ID NO: 8. The chromosome segment provided by the invention has important application value in breeding of salt-tolerant cotton with domesticated and improved lint content.

Description

Technical Field

[0001] This invention relates to the field of cotton breeding, specifically to molecular markers related to salt tolerance and domestication-improved lint percentage derived from abnormal cotton and their applications. Background Technology

[0002] Cotton is not only an important economic crop but also the cornerstone of the healthy operation of the global textile industry, with its fiber production directly impacting the industry's sustainable development. my country's annual cotton consumption remains around 8 million tons, while its average annual production is only about 6 million tons, resulting in a significant shortage. Faced with the dual challenges of a rapidly growing global population and shrinking arable land, ensuring cotton production has become the primary task of cotton cultivation. Lint percentage, referring to the proportion of fiber (lint) weight in seed cotton to its total seed cotton weight, is a key parameter for assessing high-yield potential and a core indicator for measuring cotton yield composition. Exploring lint percentage-related loci has profound theoretical and practical value for cultivating high-yield cotton varieties and ensuring my country's cotton self-sufficiency.

[0004] Upland cotton ( Gossypium hirsutum *Cotton* L. is a widely cultivated cotton variety globally, but long-term artificial breeding has resulted in a narrow genetic base, posing a significant challenge to further improvements in yield and stress resistance. Wild species (close relatives) of the *Cotton* genus contain rich genetic variation, which can compensate for the superior alleles lost in modern breeding. Diploid wild species *Abnormal Cotton* (… G. anomalum Upland cotton (B1B1) originated in Africa and is mainly distributed along the Angolan coast, in southwestern Africa, and on the edge of the Sahara Desert. Compared with cultivated upland cotton, it has many superior traits, such as more prominent seed coat hairs and stronger stress resistance, making it a valuable resource for improving the yield and salt tolerance of upland cotton. Summary of the Invention

[0005] The purpose of this invention is to provide salt-tolerant and domesticated improved lint-related molecular markers derived from abnormal cotton and their applications.

[0006] In a first aspect, the present invention claims protection for the use of a specific DNA fragment in any of the following: (A1) To cultivate or assist in the cultivation of cotton varieties or lines with improved salt tolerance and / or improved lint percentage; (A2) Improve the salt tolerance of cotton and / or improve the lint percentage of cotton; The specific DNA fragment is the segment located on chromosome 1 of the abnormal cotton, from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19; The nucleotide sequence of the SSR molecular marker NAU3615 is shown in SEQ ID NO:1; The nucleotide sequence of the InDel molecular marker NY1-19 is shown in SEQ ID NO:8.

[0007] In the application, cotton containing the specific DNA fragment exhibits improved salt tolerance and / or improved lint content.

[0008] Secondly, the present invention claims protection for the use of a set of molecular markers in screening specific DNA fragments to cultivate or assist in the cultivation of cotton varieties or lines with improved salt tolerance and / or improved lint percentage. The set of molecular markers consists of the following (a1)-(a8); (a1) SSR molecular marker NAU3615: DNA molecule with nucleotide sequence as shown in SEQ ID NO:1; (a2) SSR molecular marker NAU2182: DNA molecule with nucleotide sequence as shown in SEQ ID NO:2; (a3) SSR molecular marker JAAS1148: DNA molecule with nucleotide sequence as shown in SEQ ID NO:3; (a4) SSR molecular marker NAU5100: DNA molecule with nucleotide sequence as shown in SEQ ID NO:4; (a5) SSR molecular marker NAU3714: DNA molecule with nucleotide sequence as shown in SEQ ID NO:5; (a6) SSR molecular marker NAU4045: DNA molecule with nucleotide sequence as shown in SEQ ID NO:6; (a7) SSR molecular marker NAU2083: DNA molecule with nucleotide sequence as shown in SEQ ID NO:7; (a8) InDel molecular marker NY1-19: DNA molecule with nucleotide sequence as shown in SEQ ID NO:8; The specific DNA fragment is the segment located on chromosome 1 of the abnormal cotton, from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19.

[0009] In the application, the salt tolerance and / or lint percentage of cotton with an abnormal cotton band type containing the SSR molecular markers NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, NAU2083 and NY1-19 are improved.

[0010] Thirdly, the present invention claims protection for the use of primer sets or kits containing said primer sets in screening specific DNA fragments to cultivate or assist in the cultivation of cotton varieties or lines with improved salt tolerance and / or improved lint percentage. The primer set consists of the following (b1)-(b8); (b1) Primer pair 1: designed based on the nucleotide sequence of the SSR molecular marker NAU3615 shown in SEQ ID NO:1; (b2) Primer pair 2: designed based on the nucleotide sequence of the SSR molecular marker NAU2182 shown in SEQ ID NO:2; (b3) Primer pair 3: designed based on the nucleotide sequence of the SSR molecular marker JAAS1148 shown in SEQ ID NO:3; (b4) Primer pair 4: designed based on the nucleotide sequence of the SSR molecular marker NAU5100 shown in SEQ ID NO:4; (b5) Primer pair 5: designed based on the nucleotide sequence of the SSR molecular marker NAU3714 shown in SEQ ID NO:5; (b6) Primer pair 6: designed based on the nucleotide sequence of the SSR molecular marker NAU4045 shown in SEQ ID NO:6; (b7) Primer pair 7: designed based on the nucleotide sequence of the SSR molecular marker NAU2083 shown in SEQ ID NO:7; (b8) Primer pair 8: designed based on the nucleotide sequence of the InDel molecular marker NY1-19 shown in SEQ ID NO:8; The specific DNA fragment is the segment located on chromosome 1 of the abnormal cotton, from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19.

[0011] In some embodiments of the present invention, primer pair 1 consists of two single-stranded DNAs as shown in SEQ ID NO:9 and SEQ ID NO:10.

[0012] In some embodiments of the present invention, the primer pair 2 consists of two single-stranded DNAs as shown in SEQ ID NO:11 and SEQ ID NO:12.

[0013] In some embodiments of the present invention, primer pair 3 consists of two single-stranded DNAs as shown in SEQ ID NO:13 and SEQ ID NO:14.

[0014] In some embodiments of the present invention, primer pair 4 consists of two single-stranded DNAs as shown in SEQ ID NO:15 and SEQ ID NO:16.

[0015] In some embodiments of the present invention, primer pair 5 consists of two single-stranded DNAs as shown in SEQ ID NO:17 and SEQ ID NO:18.

[0016] In some embodiments of the present invention, primer pair 6 consists of two single-stranded DNAs as shown in SEQ ID NO:19 and SEQ ID NO:20.

[0017] In some embodiments of the present invention, the primer pair 7 consists of two single-stranded DNAs shown in SEQ ID NO:21 and SEQ ID NO:22.

[0018] In some embodiments of the present invention, primer pair 8 consists of two single-stranded DNAs as shown in SEQ ID NO:23 and SEQ ID NO:24.

[0019] In the application, the salt tolerance and / or lint percentage of cotton with an abnormal cotton band type containing the SSR molecular markers NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, NAU2083 and NY1-19 are improved.

[0020] Fourthly, the present invention claims protection for the use of a set of molecular markers or specific DNA fragments in the identification or auxiliary identification of cotton salt tolerance and / or lint content; The set of molecular markers consists of (a1) to (a8) described above; the specific DNA fragment is a segment located on chromosome 1 of the abnormal cotton, starting from the SSR molecular marker NAU3615 and ending at the InDel molecular marker NY1-19.

[0021] In the application, the salt tolerance and / or lint percentage of cotton with an abnormal cotton band type containing the SSR molecular markers NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, NAU2083 and NY1-19 are improved.

[0022] Fifthly, the present invention claims protection for the use of primer sets or kits containing said primer sets in identifying or assisting in the identification of cotton salt tolerance and / or lint content. The primer pair consists of the following as described above (b1)-(b8). In the application, the salt tolerance and / or lint percentage of cotton with an abnormal cotton band type containing the SSR molecular markers NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, NAU2083 and NY1-19 are improved.

[0023] In the aforementioned relevant aspects, the cotton is selected from upland cotton, atypical cotton, or generational populations with upland cotton and atypical cotton as parents, such as the BC4F4 generation or the BC4F5 generation, or the single-fragment introgression line CSSL5. Among these, the upland cotton includes, for example, Upland cotton 86-1 or Upland cotton Su 8289.

[0024] Sixthly, the present invention claims protection for any of the following methods: Method I: A method for improving the salt tolerance and / or improving the lint content of upland cotton, comprising the following steps: replacing a segment on chromosome 1 of upland cotton from SSR molecular marker NAU3615 to InDel molecular marker NY1-19 with the specific DNA segment described in the first aspect above. The nucleotide sequence of the SSR molecular marker NAU3615 is shown in SEQ ID NO:1; The nucleotide sequence of the InDel molecular marker NY1-19 is shown in SEQ ID NO:8.

[0025] Method II: A method for cultivating or assisting in the cultivation of cotton varieties or lines with improved salt tolerance and / or improved lint percentage, comprising the following steps: using upland cotton 86-1 as the female parent and abnormal cotton as the male parent, hybridizing to obtain a triploid; then doubling the triploid to a hexaploid (e.g., by doubling through colchicine); then using the hexaploid as the female parent and upland cotton Su 8289 as the recurrent parent, through multiple generations of backcrossing and self-pollination, obtaining a single-fragment introgression line containing the specific DNA fragment described in the first aspect above, thereby obtaining a cotton variety or line with improved salt tolerance and / or improved lint percentage.

[0026] In some embodiments of the present invention, the hexaploid is used as the maternal parent and upland cotton Su8289 is used as the recurrent parent, and a total of 4 generations of backcrossing and 4 generations of selfcrossing are carried out.

[0027] In some embodiments of the present invention, the single-fragment introgression line can be screened and identified using the aforementioned set of SSR molecular markers or the aforementioned set of primer pairs. In this method, single-fragment introgression lines containing the SSR molecular markers NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, NAU2083, and InDel molecular marker NY1-19 are screened for anomalous cotton-band-like single-fragment introgression lines. Specifically, the single-fragment introgression line is the single-fragment introgression line CSSL5.

[0028] In the aforementioned aspects, salt tolerance may refer to salt tolerance during germination and / or salt tolerance during the seedling stage. In some embodiments of the present invention, salt stress is simulated using 200 mmol / L or 350 mmol / L NaCl solutions. In some embodiments of the present invention, the strength of salt tolerance is reflected by indicators such as relative germination rate, relative plant height, relative aboveground fresh weight, and / or relative aboveground dry weight.

[0029] Seventhly, the present invention claims protection for any of the following biological materials: (A1) The specific DNA fragment described in the first aspect above; (A2) An expression cassette, recombinant vector, or recombinant microorganism containing the specific DNA fragment described in (A1); (A3) The set of molecular markers described in the second aspect above; (A4) The primer pairs described in the third aspect above, or a kit containing the primer pairs or the primer pairs described above.

[0030] This invention provides loci and molecular markers related to salt tolerance and domestication improvement in diploid wild cotton species, which have important application value for molecular design breeding of salt-tolerant and high-yielding cotton. Attached Figure Description

[0031] Figure 1 The detection results of various cotton materials were obtained by utilizing the eight molecular markers of this invention.

[0032] Figure 2 Results of detection of SSR molecular markers NAU2182, NAU5100, NAU3714, NAU4045 and NAU2083 in the self-crossed progeny of the single-fragment introgression line CSSL5.

[0033] Figure 3This study analyzed the phenotypic and related traits of Su8289 and the single-segment introgression line CSSL5 under salt stress during germination and seedling stages. Specifically, a represents the phenotype of Su8289 and CSSL5 under salt stress during germination; b represents the relative germination rate of Su8289 and CSSL5 under salt stress during germination; c represents the phenotype of Su8289 and CSSL5 under salt stress during the seedling stage; d represents the relative plant height of Su8289 and CSSL5 under salt stress during the seedling stage; e represents the relative aboveground fresh weight of Su8289 and CSSL5 under salt stress during the seedling stage; and f represents the relative aboveground dry weight of Su8289 and CSSL5 under salt stress during the seedling stage. ** indicates a comparison with Su8289. P <0.01.

[0034] Figure 4 The proline content and peroxidase, superoxide dismutase, and catalase activities of SU8289 and the single-fragment introgression line CSSL5 after salt treatment for 0, 24, 72, and 144 h were compared. * indicates comparison with SU8289. P <0.05; ** indicates compared to Su-8289 P <0.01.

[0035] Figure 5 This is a comparison of the fabric percentage of the single-segment gradient dyeing lines CSSL5 and Su8289. ** indicates a comparison with Su8289. P <0.01. Detailed Implementation

[0036] The cotton of this invention, containing salt-tolerant and domesticated improved lint sites, is cultivated through the following steps: (1) Triploids were obtained by hybridization using upland cotton 86-1 as the female parent and abnormal cotton as the male parent. Hexaploid F1 fertile hybrids were obtained by treating the tender axillary buds of the triploid seedlings with 0.15% colchicine.

[0037] (2) Using a hexaploid as the maternal parent and upland cotton Su8289 as the recurrent parent, four backcrosses and three self-crosses were performed. In each generation, SSR markers covering the abnormal cotton genome developed by our research group were used for assisted selection to obtain the BC4F4 generation population. In the BC4F4 generation, whole-genome foreground and background identification of all recombinant individuals was performed using 230 SSR markers, and 74 introgression lines with clearly defined abnormal cotton chromosome segments were obtained. Among them, the single-segment introgression line CSSL5 showed strong salt tolerance (during germination and seedling stages) and low lint percentage.

[0038] (3) Single-fragment introgression line CSSL5, upland cotton 86-1, recurrent parent Su8289, anomalous cotton, and hexaploid F1 were planted. Young leaves were taken and DNA was extracted using the CTAB method. PCR amplification was performed using 8 molecular markers, including 7 SSR markers (NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, and NAU2083) and 1 InDel marker (NY1-19). Plants containing the anomalous cotton-specific bands of all 8 molecular markers were identified as single-fragment introgression line CSSL5.

[0039] (4) Salt tolerance of the single-segment infiltration line CSSL5 and the recurrent parent Su8289 was assessed at the germination and seedling stages (two leaves and one heart). CSSL5 and Su8289 were planted in the field, and the lint percentage phenotype was investigated after normal harvest. Compared with the recurrent parent Su8289, CSSL5 showed stronger salt tolerance at the germination and seedling stages, and lower lint percentage under normal field conditions.

[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] The upland cotton 86-1 used in the following examples ( G. hirsutum var. 86-1), upland cotton Su8289 ( G . hirsutum var. Su8289), abnormal cotton ( Gossypium anomalum The hexaploid F1 and CSSL5 strains were introduced by the Jiangsu Academy of Agricultural Sciences: documented 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. hirsutumgenomes. Theoretical and Applied Genetics, 2015, 128(8): 1531-1540" and "Zhenzhen Xu, Jiedan Chen, Shan Meng, et al. Genome sequence of Gossypium anomalum The information provided in the paper, “facilitates interspecific introgression breeding [J]. Plant Communications, 2022, 3(5): 100350,” is available to the public from the applicant and may only be used for repeating experiments of this invention and may not be used for other purposes.

[0043] Example 1: Identification of salt tolerance and lint content-related fragments from abnormal cotton and development of related molecular markers. 1. DNA extraction Cotton DNA was extracted using a modified CTAB method combined with a novel plant genomic DNA extraction kit from Shanghai Pudi Biotechnology Co., Ltd. The specific steps are as follows: (1) Sampling: Select young cotton leaves (approximately 0.5 cm) 2 (Size) is placed in a 2 mL tube.

[0044] (2) Preparation of extraction solution: Preheat the DNA lysis buffer (Table 1) in a 65°C water bath, take 100 mL of buffer solution in an Erlenmeyer flask, add a spoonful of ascorbic acid and 1 mL of β-mercaptoethanol, and mix well.

[0045] (3) Grinding: Place steel balls in each EP tube, add 500 μL of extraction solution, and grind for 180 s using a fully automatic sample grinder at 60 Hz.

[0046] (4) DNA was extracted using the novel plant genomic DNA extraction kit from Shanghai Pudi Biotechnology Co., Ltd.

[0047] (5) Concentration detection. After DNA extraction, its concentration is detected using a multi-functional microplate reader. After passing the test, it is stored at -20℃.

[0048] Table 1. DNA lysis buffer formulation

[0049] 2. PCR method (1) Reagents and main instruments used The 2×3G Taq Master Mix for PAGE (Red Dye) used in the PCR reaction was a product of Nanjing Novizan Biotechnology Co., Ltd. The reagents used for the PAGE gel, including acrylamide, methylene acrylamide, Tris-base, boric acid, silver nitrate, sodium hydroxide, and TEMED, were products of Rongshengda Experimental Instrument Co., Ltd. The main instruments included an Applied Biosystems PCR instrument, an Eppendorf high-speed centrifuge, a water bath, a shaker, and an electrophoresis tank and electrophoresis apparatus manufactured by Beijing Liuyi Instrument Factory.

[0050] (2) PCR reaction system and amplification procedure The PCR reaction system consisted of 10 μL of the following components: 5.0 μL of 2×3G Taq Master Mix for PAGE (Red Dye), 2.0 μL of DNA template, 1.0 μL each of the front and rear primers, and 1.0 μL of ddH2O.

[0051] PCR reactions were performed on an Applied Biosystems PCR instrument. The reaction procedure was as follows: Step 1: 95℃ pre-denaturation for 5 min; Step 2: 94℃ denaturation for 30 s; Step 3: 55℃ annealing for 45 s; Step 4: 72℃ extension for 1 min (Steps 2 to 4 were repeated for 30 cycles); Step 5: 72℃ extension for 10 min; Step 6: storage at 16℃.

[0052] 3. Polyacrylamide gel electrophoresis Gel preparation and electrophoresis process: (1) Making the gel: Align the two glass plates of the set and place them flat, clamp them on both sides, and insert a 102-tooth comb halfway into the groove. Slowly pour the gel into the comb, insert the comb completely into the groove, and let it solidify.

[0053] (2) Placing the gel in the electrophoresis tank: Place 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 to cover the gel, and remove the comb.

[0054] (3) Spotting: The sample loading volume is 1 μL, and a 50 bp DNA marker is used for labeling.

[0055] (4) Electrophoresis: Electrophoresis at a constant voltage of 180 V for 1.5 hours, until the blue indicator mark is 2 cm below the gel.

[0056] (5) Staining: Carefully remove the gel from the glass plate and mark it. Place the gel in the staining solution and place it on a shaker at 55 rpm for 12 minutes.

[0057] (6) Development: Transfer the gel into the development solution and shake at 55 rpm until the stripe on the film is clearly visible. Discard the development solution, rinse with tap water 4 times, and place the film on a light box to take pictures.

[0058] 4. CSSL5 Genotyping Single-segment introgression line CSSL5, recurrent parent Su8289, upland cotton 86-1, aberrant cotton, and hexaploid F1 were planted. Young leaves were collected, and DNA was extracted using the CTAB method as a template. 230 pairs of SSR primers developed by the applicant, uniformly covering the chromosome set of aberrant cotton, were used to identify the whole-genome foreground and background of the single-segment introgression line CSSL5. The amplification products of the primers for the eight molecular markers shown in Table 2 differed between the single-segment introgression line CSSL5 and the recurrent parent Su8289. Plants containing the aberrant cotton-specific bands of all eight molecular markers were identified as the single-segment introgression line CSSL5 (the only difference between CSSL5 and the recurrent parent Su8289 is that the corresponding segment on chromosome 1 is introgressed by "the segment located on chromosome 1 of aberrant cotton from SSR molecular marker NAU3615 to InDel molecular marker NY1-19"; the rest of the genome is completely identical to the recurrent parent Su8289). The eight molecular markers are: NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, NAU2083, and NY1-19. The specific sequences of these eight molecular markers are as follows: SSR molecular marker NAU3615: nucleotide sequence as shown in SEQ ID NO:1.

[0059] SSR molecular marker NAU2182: nucleotide sequence as shown in SEQ ID NO:2.

[0060] SSR molecular marker JAAS1148: nucleotide sequence as shown in SEQ ID NO:3.

[0061] SSR molecular marker NAU5100: nucleotide sequence as shown in SEQ ID NO:4.

[0062] SSR molecular marker NAU3714: nucleotide sequence as shown in SEQ ID NO:5.

[0063] SSR molecular marker NAU4045: nucleotide sequence as shown in SEQ ID NO:6.

[0064] SSR molecular marker NAU2083: nucleotide sequence as shown in SEQ ID NO:7.

[0065] InDel molecular marker NY1-19: nucleotide sequence as shown in SEQ ID NO:8.

[0066] The primer sequences used to amplify the eight molecular markers are shown in Table 2.

[0067] Table 2. Primer sequences used to amplify the eight molecular markers

[0068] Figure 1 The results of testing various cotton materials using the aforementioned eight molecular markers (NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, NAU2083, and NY1-19) are presented. For each marker, lane 1 from left to right 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 CSSL5. Notably, the genotypes of CSSL5 in molecular markers NAU2182, NAU5100, NAU3714, NAU4045, and NAU2083 are identical to those of hexaploid F1. This is because abnormal cotton B1 is prone to recombination with the upland cotton At subgroup. Generally, the Dt subgroup of upland cotton also possesses a non-recombined SSR locus homologous to the At subgroup. During PCR amplification, primers bind to the SSR locus of the abnormal cotton B1 group, and also to the SSR locus of the Dt subgroup. To verify this conclusion, genotyping was performed on 20 self-crossed CSSL5 plants. The results showed that the genotypes of these 20 plants did not segregate, indicating that CSSL5 has been stably inherited and is a homozygous single-fragment introgression line. Figure 2 ).

[0069] Figure 2 The results of SSR molecular markers NAU2182, NAU5100, NAU3714, NAU4045, and NAU2083 in the BC4F5 generation population of CSSL5 are shown. From left to right, lane 1 represents the markers, lane 2 represents the genotype of Su8289, lane 3 represents the genotype of 86-1, lane 4 represents the genotype of abnormal cotton, lane 5 represents the genotype of hexaploid F1, and lanes 6-25 represent the genotypes of 20 self-pollinated single plants from CSSL5.

[0070] 5. Salt tolerance assessment of CSSL5 The cotton tested were the single-segment introgression line CSSL5 and the upland cotton Su8289, which served as the recurrent parent when CSSL5 was created.

[0071] (1) Salt tolerance assessment during germination A 200 mmol / L NaCl solution was used as the treatment group, and distilled water was used as the control group, with three replicates. The petri dishes were sterilized with 75% alcohol. After the seeds were thoroughly dried, uniformly sized and plump seeds were selected, soaked in 75% alcohol for 3 minutes, and then quickly rinsed three times with distilled water. Four layers of tissue paper were laid flat at the bottom of a glass petri dish, and 50 seeds were evenly arranged on top, covered with three layers of tissue paper, and approximately 35 mL of NaCl solution / distilled water was poured in. The dish was then placed in an incubator with a light / dark cycle of 16 h (28 ℃) / 8 h (23 ℃) and a relative humidity of 60%. Germination rate was recorded after 7 days.

[0072] Germination rate = (Number of germinated seeds / Total number of seeds) × 100%.

[0073] Relative germination rate = Germination rate under salt treatment / Germination rate under water treatment × 100%.

[0074] (2) Methods for identifying salt tolerance in seedlings Select plump seeds of uniform size, delint them with concentrated sulfuric acid, rinse several times with running water, and sun-dry them. Line the bottom of a glass petri dish with four layers of tissue paper, arrange the seeds evenly on top, cover with three more layers of tissue paper, pour in about 35 mL of tap water until the tissue paper is just soaked, cover the petri dish, label it, wrap the petri dish with newspaper, and place it in a 37 ℃ constant temperature incubator. Observe frequently; sow when the seeds show signs of germination. Mix the seedling substrate and vermiculite in a 4:1 ratio and set aside. Use a bamboo skewer to evenly poke five small holes in the bottom of a 250 mL paper cup, and fill the cup with the mixed substrate. Insert the germinated seeds vertically into the substrate to a depth of about 3-4 cm, cover with a thin layer of substrate, water thoroughly (ensuring no standing water in the tray), and cultivate in an environment of 60% humidity, 16 h (28 ℃) / 8 h (23 ℃). Once the plants reached the two-leaf-one-heart stage, select cotton seedlings with uniform growth for salt treatment. Each cup was treated with 70 mL of 350 mmol / L NaCl solution, while the control group was treated with 70 mL of water per cup. Three replicates were performed, with 10 plants in each replicate. After 7 days of treatment, photos were taken and salt tolerance-related indicators such as plant height, aboveground fresh weight, and aboveground dry weight were measured.

[0075] Plant height: The distance from the cotyledon node to the top of the main stem, in cm.

[0076] Fresh weight of aboveground parts: weight of fresh material in the part above the cotyledon node, in grams.

[0077] Aboveground dry weight: The part above the cotyledon node is dried in an oven at 100 ℃ to constant weight, in g.

[0078] Calculate the relative plant height, relative aboveground fresh weight, and relative aboveground dry weight respectively.

[0079] Relative plant height = Plant height under salt treatment / Plant height under water treatment × 100%.

[0080] Relative aboveground fresh weight = aboveground fresh weight under salt treatment / aboveground fresh weight under water treatment × 100%.

[0081] Relative aboveground dry weight = aboveground dry weight under salt treatment / aboveground dry weight under water treatment × 100%.

[0082] (3) Measurement of physiological and biochemical indicators On the day of salt treatment and at 24, 72, and 144 h after treatment, the second-to-last leaves of CSSL5 and Su8289 were selected for determination of relevant physiological and biochemical indicators. These included the content of proline (PRO), an osmotic regulator, and the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) in the antioxidant system. All measurements were performed using kits from Nanjing Jiancheng Bioengineering Institute, with three biological replicates.

[0083] (4) Results and Analysis The results of salt tolerance during germination are as follows: Both germinated normally under water treatment; however, under 200 mmol / L NaCl solution treatment, the relative germination rate of CSSL5 was 90.75%, while that of Su8289 was 64.10%. The relative germination rate of CSSL5 was significantly higher than that of Su8289. Figure 3 (a and b).

[0084] The results of salt tolerance during the seedling stage are as follows: All plants grew normally under water treatment, but after 7 days of treatment with 350 mmol / L salt, the growth of seedlings in both materials was inhibited to varying degrees, and the leaves were also damaged to varying degrees. Figure 3 (c). Among them, the cotyledons of Su8289 were wilted and yellowed, while the cotyledons of CSSL5 were in better condition and did not show wilting or yellowing. Figure 3 (c). After 7 days of NaCl treatment, compared with Su8289, CSSL5 showed a highly significant increase in relative plant height, relative aboveground fresh weight, and relative aboveground dry weight. Figure 3 (d, e, and f).

[0085] Based on the above results, the single-segment introgression line CSSL5 exhibits good salt tolerance during the germination and seedling stages.

[0086] The results of the physiological and biochemical index measurements are as follows: Compared with 0 h of salt treatment, the proline content and peroxidase activity of Su8289 and CSSL5 both increased to varying degrees after salt treatment. Figure 4 The proline content of CSSL5 was significantly higher than that of Su8289 at 72 h and 144 h, and the peroxidase activity was significantly higher than that of Su8289 at 24 h, 72 h, and 144 h. Figure 4 The activities of superoxide dismutase and catalase showed a trend of first increasing and then decreasing after salt treatment. Figure 4 The superoxide dismutase activity of CSSL5 was significantly increased at 24 h, 72 h, and 144 h, and the catalase activity was significantly increased at 24 h and 72 h. Figure 4 This indicates that the single-fragment introgression line CSSL5 exhibits good salt tolerance during the seedling stage, and can serve as an important material for cotton salt tolerance breeding and research on salt tolerance genetic mechanisms.

[0087] 6. Examination of the clothing phenotype of CSSL5 The cotton tested were the single-segment introgression line CSSL5 and the upland cotton Su8289, which served as the recurrent parent when CSSL5 was created.

[0088] During the boll opening stage, CSSL5 and Su8289 were harvested, and 30 mature bolls from the middle and upper parts were taken. The bolls were ginned using a ginning machine, and the lint percentage (lint percentage is gram weight of lint cotton / gram weight of seed cotton × 100%) was examined. Three replicates were set up.

[0089] The single-segment introgression line CSSL5 and the recurrent parent SU8289 were planted in four environments, and the pelvic traits were investigated. The results showed that the pelvic traits of the single-segment introgression line CSSL5 were 31.48%-38.72% in all four environments, all of which were highly significant. P <0.01) lower than Su 8289 ( Figure 5 Environment 1 (E1): Lishui Plant Science Base, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu Province, 2017; Environment 2 (E2): Lishui Plant Science Base, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu Province, 2018; Environment 3 (E3): Lishui Plant Science Base, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu Province, 2018; Environment 4 (E4): Korla, Xinjiang Province, 2018. Replication: Each experimental site was arranged in a randomized block design with three replicates. Planting density and production management were the same as in local field production.

[0090] Based on the experimental results of this invention, it is evident that the segment from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19 derived from chromosome 1 of the abnormal cotton in the single-segment introgression line CSSL5 is associated with salt tolerance and lint percentage. This invention yields the salt-tolerant and low-lint-percentage abnormal cotton single-segment introgression line CSSL5, providing a material basis for map-based cloning of genes related to salt tolerance and lint percentage. Using the eight molecular markers provided by this invention (NAU3615, NAU2182, JAAS1148, NAU5100, NAU3714, NAU4045, NAU2083, and NY1-19) and their developed primers (see Table 2 above), target chromosome segments can be rapidly screened to identify cotton salt tolerance and lint percentage traits, laying an important foundation for molecular design breeding of salt-tolerant and high-yielding cotton.

[0091] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of a specific DNA fragment in any one of the following: (A1) breeding or assisting in breeding of a cotton variety or line with improved salt tolerance and / or lint improvement; (A2) improving salt tolerance and / or improving lint of cotton; said specific DNA fragment is a fragment located on chromosome 1 of Gossypium arboreum from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19; the nucleotide sequence of the SSR molecular marker NAU3615 is shown as SEQ ID NO: 1; the nucleotide sequence of the InDel molecular marker NY1-19 is shown as SEQ ID NO:

8.

2. Use of a set of molecular markers in screening a specific DNA fragment for breeding or assisting in breeding of a cotton variety or line with improved salt tolerance and / or lint improvement; said set of molecular markers consists of (a1)-(a8) as follows: (a1) SSR molecular marker NAU3615: a DNA molecule with the nucleotide sequence shown as SEQ ID NO: 1; (a2) SSR molecular marker NAU2182: a DNA molecule with the nucleotide sequence shown as SEQ ID NO: 2; (a3) SSR molecular marker JAAS1148: a DNA molecule with the nucleotide sequence shown as SEQ ID NO: 3; (a4) SSR molecular marker NAU5100: a DNA molecule with the nucleotide sequence shown as SEQ ID NO: 4; (a5) SSR molecular marker NAU3714: a DNA molecule with the nucleotide sequence shown as SEQ ID NO: 5; (a6) SSR molecular marker NAU4045: a DNA molecule with the nucleotide sequence shown as SEQ ID NO: 6; (a7) SSR molecular marker NAU2083: a DNA molecule with the nucleotide sequence shown as SEQ ID NO: 7; (a8) InDel molecular marker NY1-19: a DNA molecule with the nucleotide sequence shown as SEQ ID NO: 8; said specific DNA fragment is a fragment located on chromosome 1 of Gossypium arboreum from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19.

3. Use of a set of primer pairs or a kit containing the set of primer pairs in screening a specific DNA fragment for breeding or assisting in breeding of a cotton variety or line with improved salt tolerance and / or lint improvement; said set of primer pairs consists of (b1)-(b8) as follows: (b1) primer pair 1: designed according to the nucleotide sequence of the SSR molecular marker NAU3615 shown as SEQ ID NO: 1; (b2) primer pair 2: designed according to the nucleotide sequence of the SSR molecular marker NAU2182 shown as SEQ ID NO: 2; (b3) primer pair 3: designed according to the nucleotide sequence of the SSR molecular marker JAAS1148 shown as SEQ ID NO: 3; (b4) primer pair 4: designed according to the nucleotide sequence of the SSR molecular marker NAU5100 shown as SEQ ID NO: 4; (b5) primer pair 5: designed according to the nucleotide sequence of SSR molecular marker NAU3714 shown in SEQ ID NO: 5; (b6) primer pair 6: designed according to the nucleotide sequence of SSR molecular marker NAU4045 shown in SEQ ID NO: 6; (b7) primer pair 7: designed according to the nucleotide sequence of SSR molecular marker NAU2083 shown in SEQ ID NO: 7; (b8) primer pair 8: designed according to the nucleotide sequence of InDel molecular marker NY1-19 shown in SEQ ID NO: 8; The specific DNA fragment is a fragment located on chromosome 1 of Abnormal cotton, from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19.

4. The use according to claim 3, characterized in that: the primer pair 1 consists of two single-stranded DNAs shown in SEQ ID NO: 9 and SEQ ID NO: 10; the primer pair 2 consists of two single-stranded DNAs shown in SEQ ID NO: 11 and SEQ ID NO: 12; the primer pair 3 consists of two single-stranded DNAs shown in SEQ ID NO: 13 and SEQ ID NO: 14; the primer pair 4 consists of two single-stranded DNAs shown in SEQ ID NO: 15 and SEQ ID NO: 16; the primer pair 5 consists of two single-stranded DNAs shown in SEQ ID NO: 17 and SEQ ID NO: 18; the primer pair 6 consists of two single-stranded DNAs shown in SEQ ID NO: 19 and SEQ ID NO: 20; the primer pair 7 consists of two single-stranded DNAs shown in SEQ ID NO: 21 and SEQ ID NO: 22; the primer pair 8 consists of two single-stranded DNAs shown in SEQ ID NO: 23 and SEQ ID NO:

24.

5. Use of a set of molecular markers or a specific DNA fragment in identifying or assisting in identifying salt tolerance and / or lint percentage of cotton; The set of molecular markers consists of (a1)-(a8) as follows: (a1) SSR molecular marker NAU3615: a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 1; (a2) SSR molecular marker NAU2182: a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 2; (a3) SSR molecular marker JAAS1148: a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 3; (a4) SSR molecular marker NAU5100: a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 4; (a5) SSR molecular marker NAU3714: a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 5; (a6) SSR molecular marker NAU4045: a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 6; (a7) SSR molecular marker NAU2083: a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 7; (a8) InDel molecular marker NY1-19: a DNA molecule with the nucleotide sequence shown in SEQ ID NO:

8. (a7) the SSR molecular marker NAU2083: a DNA molecule with a nucleotide sequence as set forth in SEQ ID NO: 7; (a8) the InDel molecular marker NY1-19: a DNA molecule with a nucleotide sequence as set forth in SEQ ID NO: 8; The specific DNA fragment is a fragment located on chromosome 1 of Gossypium anomalum from the SSR molecular marker NAU3615 to the InDel molecular marker NY1-19.

6. Use of a primer pair or a kit containing the primer pair in identifying or assisting in identifying salt tolerance and / or lint percentage of cotton; The primer pair consists of (b1)-(b8) as follows: (b1) primer pair 1: designed according to the nucleotide sequence of the SSR molecular marker NAU3615 as set forth in SEQ ID NO: 1; (b2) primer pair 2: designed according to the nucleotide sequence of the SSR molecular marker NAU2182 as set forth in SEQ ID NO: 2; (b3) primer pair 3: designed according to the nucleotide sequence of the SSR molecular marker JAAS1148 as set forth in SEQ ID NO: 3; (b4) primer pair 4: designed according to the nucleotide sequence of the SSR molecular marker NAU5100 as set forth in SEQ ID NO: 4; (b5) primer pair 5: designed according to the nucleotide sequence of the SSR molecular marker NAU3714 as set forth in SEQ ID NO: 5; (b6) primer pair 6: designed according to the nucleotide sequence of the SSR molecular marker NAU4045 as set forth in SEQ ID NO: 6; (b7) primer pair 7: designed according to the nucleotide sequence of the SSR molecular marker NAU2083 as set forth in SEQ ID NO: 7; (b8) primer pair 8: designed according to the nucleotide sequence of the InDel molecular marker NY1-19 as set forth in SEQ ID NO:

8.

7. Use according to claim 6, characterized in that: The primer pair 1 consists of two single-stranded DNAs as set forth in SEQ ID NO: 9 and SEQ ID NO: 10; The primer pair 2 consists of two single-stranded DNAs as set forth in SEQ ID NO: 11 and SEQ ID NO: 12; The primer pair 3 consists of two single-stranded DNAs as set forth in SEQ ID NO: 13 and SEQ ID NO: 14; The primer pair 4 consists of two single-stranded DNAs as set forth in SEQ ID NO: 15 and SEQ ID NO: 16; The primer pair 5 consists of two single-stranded DNAs as set forth in SEQ ID NO: 17 and SEQ ID NO: 18; The primer pair 6 consists of two single-stranded DNAs as set forth in SEQ ID NO: 19 and SEQ ID NO: 20; The primer pair 7 consists of two single-stranded DNAs as set forth in SEQ ID NO: 21 and SEQ ID NO: 22; The primer pair 8 consists of two single-stranded DNAs as set forth in SEQ ID NO: 23 and SEQ ID NO:

24.

8. Use according to any one of claims 1 to 7, characterized in that: The cotton is selected from a generation population with upland cotton and Gossypium anomalum as parents.

9. Any one of the following methods: Method I: A method for improving salt tolerance and / or lint percentage of upland cotton, comprising the following steps: replacing the fragment on chromosome 1 of upland cotton from SSR molecular marker NAU3615 to InDel molecular marker NY1-19 with the specific DNA fragment in claim 1; The nucleotide sequence of the SSR molecular marker NAU3615 is shown as SEQ ID NO: 1; The nucleotide sequence of the InDel molecular marker NY1-19 is shown as SEQ ID NO: 8; Method II: A method for breeding or assisting breeding of cotton varieties or lines with improved salt tolerance and / or lint percentage, comprising the following steps: obtaining a triploid by crossing upland cotton 86-1 as female parent and Gossypium anomalum as male parent; then doubling the triploid to a hexaploid; taking the hexaploid as female parent and upland cotton Su8289 as recurrent parent, and obtaining a single fragment introgression line containing the specific DNA fragment in claim 1 by multiple backcrossing and selfing, and further obtaining a cotton variety or line with improved salt tolerance and / or lint percentage.

10. Any one of the following biological materials: (A1) the specific DNA fragment in claim 1; (A2) an expression cassette or a recombinant vector or a recombinant microorganism containing the specific DNA fragment in (A1); (A3) the complete set of molecular markers in claim 2; (A4) the complete set of primer pairs in claim 3 or a kit containing the primer pairs or the complete set of primer pairs.

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