A kasp molecular marker associated with the flowering trait of upland cotton and application thereof

Abstract

The application belongs to the technical field of cotton molecular breeding, and particularly relates to a KASP molecular marker associated with the flowering trait of upland cotton and an application thereof. The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 27. The molecular marker contains a SNP site. The SNP site is located at the 14th bp from the 5' end of the sequence shown as SEQ ID NO. 27. The polymorphism is G / A. The A allele is a favorable allele for promoting early flowering. The application also develops a primer set for detecting the molecular marker. The early flowering germplasm resource of upland cotton can be screened, the flowering time of upland cotton can be predicted, or early flowering varieties of upland cotton can be assistedly selected by using the primer set. The application also develops a method for assisting in the selection of early flowering varieties of upland cotton. The method can shorten the breeding cycle of the flowering trait of cotton and improve the breeding efficiency of the early flowering trait.

Description

Technical Field

[0001] This invention belongs to the field of cotton molecular breeding technology, specifically relating to a KASP molecular marker associated with flowering traits of upland cotton and its application. Background Technology

[0002] Upland cotton ( Gossypium hirsutum Upland cotton is one of the world's most important natural fiber crops, and its precocity directly affects yield, fiber quality, and planting patterns. Flowering time is a key indicator of precocity in upland cotton; earlier flowering shortens the growth period, allows for adaptation to different ecological zones and planting systems, increases the multiple cropping index, and helps avoid adverse environmental conditions such as frost and drought later in the season. Therefore, identifying key genes regulating flowering time in upland cotton and developing molecular markers linked to flowering period are of great significance for molecular breeding of early-maturing cotton varieties.

[0003] Upland cotton is an allotetraploid crop with a large and complex genome, and its flowering regulation mechanism differs significantly from diploid model plants such as Arabidopsis thaliana. Previous studies have shown that flowering-related genes and their regulatory networks identified in model plants often exhibit different copy numbers, expression patterns, and functional differentiation in upland cotton, resulting in poor applicability of molecular markers developed from other species in upland cotton. Currently, some quantitative trait loci and molecular markers developed based on known flowering genes have been reported for flowering traits in upland cotton, but problems remain, including a limited number of markers, insufficient association strength between some markers and flowering traits, and unstable performance under different genetic backgrounds. These limitations make it difficult to fully meet the requirements of marker-assisted selection for accuracy, specificity, and universality.

[0004] KASP (competitive allele-specific PCR) markers have advantages such as high throughput, low cost, and accurate genotyping, making them a widely used type of functional marker in current crop molecular breeding. However, there is still a significant lack of KASP molecular markers specifically developed for upland cotton that are closely related to flowering traits and can be directly used for large-scale breeding screening, which limits the breeding efficiency of early-maturing cotton varieties.

[0005] Therefore, developing a stable and reliable KASP molecular marker based on the genome characteristics of upland cotton, which is highly correlated with flowering traits, and establishing its application method, has important theoretical and applied value for accelerating the breeding of new early-maturing upland cotton varieties and improving the level of cotton breeding technology. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a KASP molecular marker associated with flowering traits in upland cotton and its application, offering new genetic resources and efficient detection tools for the genetic improvement of early-maturing cotton varieties.

[0007] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of the present invention provides a KASP molecular marker associated with flowering traits of upland cotton, the nucleotide sequence of which is shown in SEQ ID NO.27, and the molecular marker contains a SNP site; the SNP site is located at the 14th bp from the 5' end of the sequence shown in SEQ ID NO.27, and the polymorphism is G / A.

[0008] Furthermore, the flowering trait refers to flowering time, and individuals with the AA genotype at the SNP locus flower earlier than individuals with the GG genotype.

[0009] A second aspect of the present invention provides a KASP primer set for detecting the KASP molecular marker, the primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30.

[0010] A third aspect of the present invention provides a product for assisting in the breeding of early-flowering upland cotton varieties, the product comprising the aforementioned KASP primer set.

[0011] Furthermore, the products include diagnostic reagents and kits.

[0012] The fourth aspect of this invention provides an application of the KASP primer set or product, which is used for screening early-flowering germplasm resources of upland cotton, predicting the flowering time of upland cotton, or assisting in the breeding of early-flowering varieties of upland cotton.

[0013] The fifth aspect of this invention provides a method for assisting in the breeding of early-flowering upland cotton varieties, comprising the following steps: Genomic DNA was extracted from the upland cotton sample to be tested; Using the genomic DNA as a template, PCR amplification was performed using the KASP primer set to obtain PCR products; Genotyping of PCR products at SNP sites was detected, and early-flowering upland cotton varieties were assisted in the selection of genotyping results: when the genotype at the SNP site was AA, the upland cotton sample to be tested was determined to be an early-flowering upland cotton variety.

[0014] Further, each 10 μL of the PCR amplification reaction system contains 5 μL of DNA template and 5 μL of master reaction mixture; each 64 μL of the master reaction mixture contains 14 μL of primer mixture and 50 μL of Hi-Geno 2×Probe Mix genotyping premix; each 100 μL of the primer mixture contains 1150 ng to 1250 ng of primers with the sequence shown in SEQ ID NO. 28, 1150 ng to 1250 ng of primers with the sequence shown in SEQ ID NO. 29, and 2950 ng to 3050 ng of primers with the sequence shown in SEQ ID NO. 30, with ddH2O added to make up the difference.

[0015] Furthermore, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, 61℃~55℃ annealing extension for 40 s, 10 cycles with each cycle decreasing by 0.6℃; 95℃ denaturation for 20 s, 55℃ annealing for 40 s, 28~34 cycles.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses a KASP molecular marker associated with flowering traits in upland cotton and its application. The nucleotide sequence of the KASP molecular marker is shown in SEQ ID NO.27. The molecular marker contains a SNP site located at the 14th bp from the 5' end of the sequence shown in SEQ ID NO.27, with a polymorphism of G / A. The A allele is a favorable allele that promotes early flowering. Individuals with the AA genotype at the SNP site flower earlier than individuals with the GG genotype. This invention also develops a detection reagent-primer set for this molecular marker and a method for assisting in the breeding of early-flowering upland cotton varieties, which is beneficial for achieving rapid and accurate prediction of cotton flowering performance. Compared with traditional phenotypic identification, the method provided by this invention can shorten the breeding cycle of cotton flowering traits, improve the breeding efficiency of early-flowering traits, and provide a new technical tool and solution for molecular design breeding of early-flowering cotton varieties. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Phylogenetic analysis of TZP protein in 9 species: yellow area represents branch I, blue area represents branch II, and pink area represents branch III.

[0019] Figure 2 Analysis of the distribution and evolutionary relationship of TZP protein on chromosomes. Figure 2 In this context, A represents the chromosome distribution of GhTZP members; Figure 2 B in the figure represents the collinearity analysis of the GhTZP gene within the genome. Figure 2 C in the text represents the TZP gene in *Gossypium raymondii* (…). Gossypium raimondii ), upland cotton ( G. hirsutum ) and Asian cotton ( Gossypium arboreum Collinearity analysis in three different Gossypium genera.

[0020] Figure 3 Analysis of GhTZPs expression patterns. Figure 3 In the figure, A represents the relative transcriptional level of 16 GhTZP genes analyzed by RNA-seq. The blue dashed line indicates FC≥2, and the yellow dashed line indicates FC≤0.67. Figure 3 In the image, B is a heatmap showing four candidate genes in TM-1. GhTZP3 , GhTZP11 , GhTZP10 , GhTZP13 The tissue expression pattern is represented by 22 tissues / organs arranged vertically, from top to bottom: root, stem, leaf, bract, sepal, petal, filament, anther, pistil, receptacle, and ovules (0 dpa, 10 dpa, 15 dpa, 20 dpa, 25 dpa) and fibers (10 dpa, 15 dpa, 20 dpa, 25 dpa) at different developmental stages. Figure 3 C in the text represents four candidate genes. GhTZP3 , GhTZP11 , GhTZP10 , GhTZP13 The relative expression levels (RT-qPCR) were detected in six cotton varieties, of which ZM113, JK161, and JK1125 are early-maturing cotton varieties, and EKM8, KSN4, and LIU8 are late-maturing cotton varieties. Different lowercase letters a, b, c, d, and e indicate significant differences between groups.

[0021] Figure 4 for GhTZP3 Silence leads to delayed flowering. Figure 4 In the image, A represents the flower bud formation phenotype, and the top row of images are magnified views of the parts within the white frame of the bottom row of images. Figure 4 B in the text is GhTZP3 Flower bud formation time under gene silencing, * indicates P <0.05. Figure 4 C in the image represents the flowering phenotype, and the top row of images are enlarged views of the parts within the white frame in the bottom row. Figure 4 D in GhTZP3 Flowering time under gene silencing, ** indicates P <0.01. Figure 4E in the figure represents a gene related to promoting flowering. GhAP1 , GhCOL2 , GhCAL , GhLFY and GhFT exist GhTZP3 Expression levels in silent plants, * indicates P <0.05, ** indicates P <0.01.

[0022] Figure 5 upland cotton GhTZP3 Allelic analysis of genes. Figure 5 In this invention, A represents the clone obtained through cloning. GhTZP3 Gene structure; Figure 5 B in the figure is a scatter plot of KASP genotyping at the SNP locus. The horizontal axis represents Allele A (A allele), and the vertical axis represents Allele G (G allele). Red dots represent homozygous A / A genotype (Homozygous Allele A / A), blue dots represent homozygous G / G genotype (Homozygous Allele G / G), and crosses represent negative controls, using water samples as templates. Figure 5 The image shows the Sanger sequencing results for the SNP locus (C). The top row represents late-flowering varieties, from left to right: Yanzao275 (80.6 d), Xinluzhong3 (80.8 d), Jinmian36 (81.3 d), Chuiliugmian (81.5 d), and Xiangmian10 (82.1 d). The bottom row represents early-flowering varieties, from left to right: Jiucai1hao (62.3 d), jiumian9 (65.3 d), CCR136 (66.8 d), Xinluzao16 (68.8 d), and Long9529 (70.8 d). Sequence alignment shows that late-flowering varieties have the G allele at this locus, while early-flowering varieties have the A allele. Figure 5 In the bar chart, D represents the frequency distribution of alleles in the Min-50 and Max-50 populations. The horizontal axis represents the Min-50 and Max-50 populations from left to right, and the vertical axis represents the frequency. The blue bars represent the G allele, and the pink bars represent the A allele. Figure 5 E in the figure represents the box plot of flowering time for alleles G and A. The horizontal axis represents the G and A genotypes from left to right, and the vertical axis represents the flowering time (d). The flowering time of genotype G is significantly longer than that of genotype A. Figure 5In the diagram, F represents the box plot of gene expression levels for genotypes G and A. The horizontal axis represents genotypes G and A from left to right, and the vertical axis represents gene expression level (FPKM). The expression level of genotype A is significantly higher than that of genotype G. * indicates... P <0.05, ** indicates P <0.01. Detailed Implementation

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0024] This invention first performed a genome-wide identification of the upland cotton TZP gene family, discovering 16 GhTZPs members, among which... GhTZP3 The expression difference was most significant between early-maturing and late-maturing varieties. Virus-induced gene silencing (VIGS) experiments confirmed the suppression of gene expression. GhTZP3 Expression significantly delays the budding and flowering time of upland cotton, indicating GhTZP3 It is a positive regulator of flowering in upland cotton. Further analysis revealed that... GhTZP3 A molecular marker significantly associated with flowering time exists in the exon region. The nucleotide sequence of this marker is shown in SEQ ID NO.27. A G / A base variation exists at position 14bp, where the "A" allele is linked to the early flowering trait and exhibits a high frequency in cotton varieties from the Northwest inland cotton-growing region. Its frequency gradually increases from early-maturing varieties to modern varieties, suggesting that this locus may have undergone artificial or natural selection. Based on this molecular marker, this invention develops a primer set for detecting it and a method for assisting in the breeding of early-flowering upland cotton varieties, which can be used for the auxiliary selection of flowering time traits in upland cotton.

[0025] Example 1: Development of molecular markers associated with flowering traits in upland cotton 1. Screening and identification of candidate genes To elucidate the evolutionary relationships within the TZP family, this invention identified 94 TZP proteins from nine species—Gossypium arboreum (8 members), Gossypium barbadense (16 members), Gossypium raimondii (8 members), Gossypium hirsutum (16 members), soybean (Glycine max (15 members), Arabidopsis thaliana (6 members), Theobromacacao (7 members), rice (Oryza sativa (6 members), and maize (Zea mays (12 members)—based on Pfam ID PF03126 and the PLUS3 domain—).

[0026] Figure 1 Phylogenetic analysis revealed that these proteins can be divided into three distinct branches: branch III is the largest, containing 65 members; branch I is the second largest, with 17 members; and branch II is the smallest, containing only 12 members. Furthermore, each of these three branches contains members from all nine species, indicating that TZP genes existed before the corresponding species diverged and exhibit a relatively conserved evolutionary path in both dicotyledonous and monocotyledonous plants.

[0027] To determine the physical distribution of the GhTZP gene family (TZP family) in the upland cotton genome, its 16 members were mapped to chromosomes. The results showed that these genes were unevenly distributed across 14 chromosomes. Figure 2 In the A group, the At and Dt subgenomes each have 8 members.

[0028] Collinearity analysis showed that all GhTZP genes were located in blocks collinear with the ancestral diploid genome, suggesting that they may have originated from an allopolyploidization event. Figure 2 (B in the text) By comparing the collinearity of diploid Asian cotton (At), Gossypium raymondii (Dt), and their allopolyploid progeny, upland cotton (AD), a total of 36 orthologous gene pairs were detected. Among them, 16 pairs were identified between upland cotton and Asian cotton, and between upland cotton and Gossypium raymondii, accounting for 44.4% of the total; 4 pairs were identified between the two diploid ancestral species, accounting for 11.1%. Figure 2 (C in the middle).

[0029] 2. GhTZP3 Candidate gene identification To investigate the relationships among GhTZP family members during flowering, RNA sequencing (RNA-seq) transcriptome data were used to compare the expression levels of 16 GhTZP genes in the late-flowering cultivar GXM11 and the early-flowering cultivar ZMS50. The results showed that... GhTZP3 , GhTZP10 , GhTZP11 and GhTZP13 Genes undergo significant changes during the flowering period ( Figure 3 (A in the middle).

[0030] By organizing expression patterns, it was discovered that GhTZP3 and GhTZP11 It is mainly expressed in leaves and stems, while GhTZP10 and GhTZP13 Enriched in ovules ( Figure 3 (B) Comparing gene expression levels between early-flowering and late-flowering varieties, it was found that... GhTZP3 The expression level was consistently high in the four early-flowering varieties: Zhongmian113 (ZM113), Jinken1161 (JK1161), Jinmian11 (JIN11), and Zhongmian125 (ZM125), especially in ZM113. In contrast, GhTZP10 , GhTZP11 and GhTZP13 High expression was observed in four late-flowering varieties: Ekangmian8 (EKM8), Keyuan4 (KEY4), Lumian1 (LU), and Jimian8 (JIU8). Figure 3 (C in the middle).

[0031] Based on the above expression profile analysis results GhTZP3 The gene was identified as a core candidate gene regulating the flowering time of upland cotton.

[0032] Used for detection GhTZP3 The primer set sequences for real-time quantitative PCR of gene expression are shown in SEQ ID NO.1 and SEQ ID NO.2, and are used for detection. GhTZP10 The primer sets for real-time quantitative PCR of gene expression are shown in SEQ ID NO.3 and SEQ ID NO.4; used for detection. GhTZP11 The primer sets for real-time quantitative PCR of gene expression are shown in SEQ ID NO. 5 and SEQ ID NO. 6; used for detection. GhTZP13 The primer sequence for real-time PCR of gene expression is shown in SEQ ID NO.7 and SEQ ID NO.8.

[0033] qGhTZP3 --F:5'-GCACAACAAAGTTCCAACAGGT-3', SEQ ID NO.1; qGhTZP3 -R: 5'-TCCACCAGTCCTTGTGGTTG-3', SEQ ID NO.2; qGhTZP10 -F: 5'-GATAGGCCTAGCCGGTCACA-3', SEQ ID NO.3; qGhTZP10 -R: 5'-CCCCGGTTGCCAGAACTA-3', SEQ ID NO.4; qGhTZP11 -F: 5'-ATCACTCACACAAGGGCCAC-3', SEQ ID NO.5; qGhTZP11 -R: 5'-GTTAGTCAGCTCAAGTCCGGC-3', SEQ ID NO.6; qGhTZP13 -F: 5'-CCAATAGCCGCGAGATCAGT-3', SEQ ID NO.7; qGhTZP13 -R: 5'-CGGCTTCTCCAGGTCTTGTT-3', SEQ ID NO. 8.

[0034] 3. GhTZP3 Functional verification of genes In order to analyze GhTZP3 To investigate the function of gene silencing in regulating flowering of upland cotton, the early-flowering variety ZM113 was used as background material, and the gene was silenced using virus-induced gene silencing (VIGS) technology.

[0035] The primer sequences used to construct the VIGS silencing vector are as follows: GhTZP3-VIGS-F: 5'-AAAAGGTGGGGCTTTGAGCA-3', SEQ ID NO.9; GhTZP3-VIGS-R: 5'-CGATGCAATCTTTGGCCGAG-3', SEQ ID NO. 10.

[0036] Phenotypic identification results showed that, compared with the TRV:00 empty vector control plants, the budding and flowering times of the TRV:GhTZP3 silent plants were significantly delayed by 3.1 days and 5.2 days, respectively. Figure 4 (A~D in the text) indicates GhTZP3 Positively regulates plant flowering.

[0037] Molecular-level analysis revealed that in GhTZP3-silenced plants, five flowering-promoting genes were... GhAP1 , GhCOL2 , GhCAL , GhLFY and GhFT The expression of flowering inhibitors was significantly downregulated, while the expression of flowering inhibitors was significantly downregulated. GhSVP Then significantly upregulated ( Figure 4 (E in the text).

[0038] The above results indicate that GhTZP3 It may promote the flowering process by regulating the expression of core regulatory factors in the flowering regulatory network.

[0039] The primer sequences for quantitative real-time PCR used to detect the expression of flowering-related genes are as follows: qGhAP1-F: 5'-ATGTGGAGCAGGCAATTGG-3', SEQ ID NO.11; qGhAP1-R: 5'-TCATCCATGGCAGCAAGACG-3', SEQ ID NO. 12; qGhCAL-F: 5'-GCGATGCTGATGTTGCTTTGA-3', SEQ ID NO. 13; qGhCAL-R: 5'-ATTCAGAACCAGTTGGGGCAT--3', SEQ ID NO.14; qGhCOL2-F: 5'-GCCTGGATTACGATGCCTCA-3', SEQ ID NO. 15; qGhCOL2-R: 5'-TGTACCTTTAGGCGGCCTTG-3', SEQ ID NO. 16; qGhFT-F: 5'-TGCTCCAAGTCCAAGTGACC-3', SEQ ID NO. 17; qGhFT-R: 5'-ACGATGGATACCGACCGTTG-3', SEQ ID NO. 18; qGhLFY-F: 5'-TGGCAAAAAGAACGGCCTTG-3', SEQ ID NO. 19; qGhLFY-R: 5'-TAGTGGGGGCATTTTTCGCCA-3', SEQ ID NO. 20; qGhSVP-F: 5'-ATTGGGTTGAGCCGTGTGAT-3', SEQ ID NO. 21; qGhSVP-R: 5'-GGTAACTTGTCTTGGCCGT-3', SEQ ID NO. 22; The primer sequences for the internal reference gene are shown in SEQ ID NO.23 and SEQ ID NO.24.

[0040] qGhActin-F: 5'-ATCCTCCGTCTTGACCTTG-3', SEQ ID NO. 23; qGhActin-R: 5'-TGTCCGTCAGGCAACTCAT-3', SEQ ID NO. 24; 4. Identification of SNP loci associated with flowering traits This invention first obtains by cloning GhTZP3 The gene (Gene ID: GH_D09G2378, gene version number GH_D09G2378), its complete negative strand nucleotide sequence is shown in SEQ ID NO.25, with a full length of 2967 bp, containing four exons and three introns. Figure 5 (A in the sequence). For ease of sequence description, the present invention also provides a positive strand reference sequence of the genomic region where the gene is located, as shown in SEQ ID NO.26.

[0041] SEQ ID NO.25:

[0042] SEQ ID NO.26:

[0043] By conducting correlation analysis on cotton germplasm resources with different flowering time patterns, this invention discovered... GhTZP3 The genomic sequence variation of the gene is significantly associated with the flowering time trait, and there is a single nucleotide polymorphism (SNP) site in the exon region of the gene that is significantly associated with flowering time. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.27. The SNP site is located at the 14th bp from the 5' end of the sequence shown in SEQ ID NO.27, and there is a G / A base variation.

[0044] SEQ ID NO.27 (5'→3'): CTAAAGCCCTAACrTTCTTCTCTCTTTAACTTTCGTTGTCAACTCTTCCTCTGATGGAATTTTTCCACCAGTCCTTGTGGTTGCGA, r represents G / A.

[0045] Example 2: Design and Development of KASP Molecular Marker Primers 1. Primer design Based on the molecular marker with nucleotide sequence as shown in SEQ ID NO.27 mined in Example 1, a primer set for detecting this molecular marker was designed and synthesized using the principle of competitive allele-specific PCR (KASP) technology. The primer set contains two allele-specific primers and one universal primer.

[0046] Specific primer 1 (Fam marker): D09_50028094-Fam: 5'-GAAGGTGACCAAGTTCATGCTAAGTGACTAAAGCCCTAACG-3', SEQ ID NO.28; Specific primer 2 (Hex label): D09_50028094-Hex: 5'-GAAGGTCGGAGTCAACGGATTAAAAGTGACTAAAGCCCTAACA-3', SEQ ID NO.29; Common primer: D09_50028094-Com: 5'-CTCGCAACCACAAGGACT-3', SEQ ID NO.30.

[0047] 2. Validation of KASP marker genotyping Genotyping of 90 upland cotton materials was performed using the aforementioned KASP primers. The bioinformatics of the 90 upland cotton materials can be found in the paper Guo X, Yang J, Li D, Zhang X, Yuan W, Li Y, Wang F, Ma Q, Wang C, Su J (2025) Genome-wide association study reveals novel SNP loci and candidate genes linked to flowering time in upland cotton. Theor Appl Genet 138:214.

[0048] Genomic DNA was extracted from 90 samples of upland cotton using the CTAB method. The DNA concentration and purity were detected using an ultra-micro spectrophotometer, and the samples were uniformly diluted to 10 ng / µL for later use.

[0049] The three primers SEQ ID NO.28 to SEQ ID NO.30 were diluted to 100 ng / µL, and primer mixtures were prepared according to the proportions shown in Table 1.

[0050] Table 1 KASP primer configuration Since the amount of primers used for each SNP site detection in a single reaction is small and is usually not included in the total volume of the final PCR system, to ensure sample loading accuracy and reduce experimental errors, this invention performs a second primer preparation to obtain the working solution: 14 μL of the initially prepared primer solution is thoroughly mixed with 50 μL of Hi-Geno2×Probe Mix genotyping premix to prepare the master reaction mixture for subsequent experimental detection. 5 μL of DNA template and 5 μL of master mixture are added to each reaction system for amplification.

[0051] Amplification and fluorescence detection were performed on an Applied Biosystems Quant Studio 5 real-time quantitative PCR instrument (Thermo Fisher Scientific, USA).

[0052] The reaction program was set as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, followed by annealing and extension at 61℃~55℃ for 40 s, repeated 10 times with a 0.6℃ decrease in temperature per cycle; then denaturation at 95℃ for 20 s, followed by annealing at 55℃ for 40 s, repeated 32 times; finally, fluorescence signal was acquired at 35℃ for 30 s. After amplification, the fluorescence signal was visualized and genotyped using Quant Studio 5 software.

[0053] Fluorescence signals showed that the SNP genotypes of the 90 upland cotton materials could be divided into two categories, corresponding to A and G homozygous genotypes, respectively. Figure 5 The B marker in the sample confirms that the marker can effectively distinguish between the two genotypes.

[0054] 3. Sanger sequencing verification To verify the accuracy of KASP genotyping, five representative samples from each of the earliest flowering group (Min-50) and the latest flowering group (Max-50) were selected for Sanger sequencing validation. Specific varietal information is as follows:

[0055] Late flowering group: Yanzao275 (flowering time 80.6 days), Xinluzhong3 (flowering time 80.8 days), Jinmian36 (flowering time 81.3 days), Chuiliugmian (flowering time 81.5 days), Xiangmian10 (flowering time 82.1 days); Early flowering group: Jiucai1hao (flowering time 62.3 days), jiumian9 (flowering time 65.3 days), CCR136 (flowering time 66.8 days), Xinluzao16 (flowering time 68.8 days), Long9529 (flowering time 70.8 days).

[0056] Sequencing results show that ( Figure 5 In the C-type SNP, late-flowering materials all carried the G allele at this SNP locus, while early-flowering materials all carried the A allele at this SNP locus, which is completely consistent with the KASP typing results, proving that this KASP molecular marker has high accuracy and reliability.

[0057] Example 3: Application of KASP molecular markers in the selection-assisted selection of flowering traits in upland cotton 1. Association analysis between SNP locus genotype and flowering time To determine whether SNP loci variation is related to flowering time, a statistical analysis was performed on the flowering time of 619 upland cotton germplasm resources. Information on the 619 upland cotton germplasm resources and flowering phenotypes can be found in Guo X, Yang J, LiD, Zhang X, Yuan W, Li Y, Wang F, Ma Q, Wang C, Su J (2025) Genome-wide association study reveals novel SNP loci and candidate genes linked to flowering time in upland cotton. Theor Appl Genet 138:214.

[0058] The results showed that varieties carrying the A allele flowered significantly earlier than varieties carrying the G allele. P <0.01)( Figure 5 (E in the text). Further analysis of the relationship between allele type and gene expression level revealed that in varieties carrying the A allele... GhTZP3 The abundance of transcripts was significantly higher in varieties carrying the G allele ( P <0.05)( Figure 5 (F in the text).

[0059] 2. Analysis of allele distribution frequency in extreme populations To assess the contribution of favorable alleles to flowering time, allele frequencies were compared between the Min-50 (earliest flowering) and Max-50 (latest flowering) groups. The results showed that allele A was significantly enriched in the Min-50 group. Figure 5 The D allele was dominant in the Max-50 group, while the G allele was dominant in the Max-50 group.

[0060] 3. Identification and application of favorable alleles Based on the above analysis, the A allele was defined as a favorable allele variation that promotes early flowering. This KASP molecular marker can be used in the following areas:

[0061] (1) Screening of early-flowering upland cotton germplasm resources: The KASP primers provided in this invention are used to perform genotyping on the upland cotton materials to be tested, and materials carrying A / A homozygous genotypes are screened. This can quickly identify early-flowering germplasm resources and shorten the breeding cycle.

[0062] (2) Molecular marker-assisted selection breeding: In the process of hybrid breeding of upland cotton, by detecting the genotype of offspring materials and selecting individuals carrying the A allele, the selection efficiency of early flowering traits can be improved, and the flowering time can be improved in a targeted manner.

[0063] (3) Early prediction of flowering time: Genotype can be detected by KASP markers during the seedling stage to predict the flowering performance of materials, without waiting for the plants to flower before phenotypic identification, which greatly improves breeding efficiency.

[0064] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0065] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A KASP molecular marker associated with flowering traits in upland cotton, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.27, and the molecular marker contains a SNP site; the SNP site is located at the 14th bp from the 5' end of the sequence shown in SEQ ID NO.27, and the polymorphism is G / A.

2. The KASP molecular marker according to claim 1, characterized in that, The flowering trait refers to the flowering time; individuals with the AA genotype at the SNP locus flower earlier than individuals with the GG genotype.

3. A KASP primer set for detecting the KASP molecular marker of claim 1, characterized in that, The primer set consists of primers with nucleotide sequences as shown in SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.

30.

4. A product for assisting in the breeding of early-flowering upland cotton varieties, characterized in that, The product includes the KASP primer set as described in claim 3.

5. The product according to claim 4, characterized in that, The products include testing reagents and kits.

6. An application of the KASP primer set of claim 3 or the product of claim 4, characterized in that, The applications are for screening early-flowering germplasm resources of upland cotton, predicting the flowering time of upland cotton, or assisting in the breeding of early-flowering varieties of upland cotton.

7. A method for assisting in the breeding of early-flowering upland cotton varieties, characterized in that, Includes the following steps: Genomic DNA was extracted from the upland cotton sample to be tested; Using the genomic DNA as a template, PCR amplification was performed using the KASP primer set described in claim 3 to obtain PCR products; Genotyping of PCR products at SNP sites was detected, and early-flowering upland cotton varieties were assisted in the selection of genotyping results: when the genotype at the SNP site was AA, the upland cotton sample to be tested was determined to be an early-flowering upland cotton variety.

8. The method according to claim 7, characterized in that, Each 10 μL of the PCR amplification reaction system contains 5 μL of DNA template and 5 μL of master reaction mixture; each 64 μL of the master reaction mixture contains 14 μL of primer mixture and 50 μL of Hi-Geno 2×Probe Mix genotyping premix; each 100 μL of the primer mixture contains 1150 ng to 1250 ng of primers with the sequence shown in SEQ ID NO. 28, 1150 ng to 1250 ng of primers with the sequence shown in SEQ ID NO. 29, and 2950 ng to 3050 ng of primers with the sequence shown in SEQ ID NO. 30, with ddH2O added to make up the volume.

9. The method according to claim 7, characterized in that, The PCR amplification reaction program is as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, 61℃~55℃ annealing extension for 40 s, 10 cycles with a decrease of 0.6℃ per cycle; 95℃ denaturation for 20 s, 55℃ annealing for 40 s, 28~34 cycles.

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