Application of haplotype molecular marker related to cotton fiber strength in identifying cotton fiber strength

Abstract

The application discloses a haplotype related to cotton fiber strength and a functional KASP molecular marker and detection application thereof, and relates to the technical field of plant molecular breeding and genetic markers. The haplotype is located in a 5,980,849-5,982,917 bp segment of a cotton reference genome D08 chromosome, is composed of 7 SNP sites, and can be divided into two types of Hap1 and Hap2. The KASP molecular marker is located at 5,982,917 bp of the cotton reference genome D08 chromosome, and the base is A / T. The haplotype represented by the molecular marker is significantly related to the cotton fiber strength, the KASP detection primer developed based on the haplotype has the advantages of accurate detection, simple operation, high throughput and low cost, and can be used for screening of early-stage materials in cotton breeding, significantly improves the selection efficiency of the fiber strength, and speeds up the breeding process of new cotton varieties with high quality.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding and genetic marker technology, and more specifically to the application of haplotype molecular markers related to cotton fiber strength in the identification of cotton fiber strength. Background Technology

[0002] As an important economic crop, simultaneously improving cotton yield and fiber quality has always been a challenge in cotton breeding. Traditional breeding methods have made slow progress in improving fiber quality, making it difficult to meet the textile industry's demand for high-quality raw cotton. Therefore, improving cotton fiber quality has become one of the core objectives of current cotton breeding.

[0003] Cotton fiber quality mainly includes indicators such as fiber length, strength, and fineness. Among these, fiber strength is controlled by multiple genes, and its genetic mechanism is complex, making it a key challenge for quality improvement. Although several quantitative trait loci (QTLs) associated with fiber strength have been identified through linkage analysis and association mapping, effective molecular markers that can be used for practical breeding remain scarce.

[0004] Haplotypes, through the integration of specific combinations of linked SNPs, can more precisely define genomic segments associated with traits. Their greatest advantage lies in the fact that only a few "tag SNPs" are needed to identify complete haplotypes, thus achieving higher predictive accuracy with fewer markers and significantly reducing the cost of large-scale screening. Competitive allele-specific PCR (KASP) technology, with its high throughput, low cost, and high accuracy, is an ideal platform for transforming haplotype markers. Therefore, developing KASP molecular markers based on key haplotypes can achieve efficient and precise selection of cotton fiber strength traits at a lower genotyping cost, which is of great significance for promoting marker-assisted breeding. Summary of the Invention

[0005] In view of this, the present invention provides the application of haplotype molecular markers related to cotton fiber strength in the identification of cotton fiber strength.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Application of haplotype molecular markers associated with cotton fiber strength in identifying cotton fiber strength, wherein the haplotype molecular markers consist of 7 sites; including SNPD08_5980849, SNPD08_5981358, SNPD08_5981401, SNPD08_5981519, SNPD08_5981562, SNPD08_5982308 and SNPD08_5982917; The SNPD08_5980849 is located at 5980849bp on chromosome D08 of the reference genome and exhibits C / T polymorphism. The SNPD08_5981358 is located at 5981358 bp on chromosome D08 of the reference genome and exhibits T / A polymorphism; The SNPD08_5981401 is located at 5981401 bp on chromosome D08 of the reference genome and exhibits A / T polymorphism; The SNPD08_5981519 is located at 5981519 bp on chromosome D08 of the reference genome and exhibits T / C polymorphism; The SNPD08_5981562 is located at 5981562bp on chromosome D08 of the reference genome and exhibits G / A polymorphism. The SNPD08_5982308 is located at 5982308 bp on chromosome D08 of the reference genome and exhibits A / C polymorphism; The SNPD08_5982917 is located at 5982917bp on chromosome D08 of the reference genome and exhibits T / A polymorphism.

[0007] As a preferred technical solution, the identification of cotton fiber strength includes the following steps: S1: Extract genomic DNA from the cotton sample; S2: Use genome sequencing or primer sets based on the above-mentioned haplotype molecular markers to amplify the cotton to be tested and obtain the genetic data of the cotton to be tested; S3: The gene data obtained in S2 were subjected to identification and screening of variant sites to obtain cotton fiber strength-related haplotypes; when the haplotype combination of the genotypes of the 7 loci is CCTTAATTGGAATT (SEQ ID NO.4), the cotton fiber strength is greater than that of cotton with the haplotype combination TTAATTCCAACCAA (SEQ ID NO.5).

[0008] Another object of the present invention is to provide the application of the above-mentioned haplotype molecular markers related to cotton fiber strength in the genetic breeding of cotton fiber strength traits, by selecting individuals with the haplotype combination CCTTAATTGGAATT (SEQ ID NO.4) as parents to enhance the fiber strength of offspring.

[0009] Another object of the present invention is to provide the application of reagents for detecting the above-mentioned haplotype molecular markers related to cotton fiber strength in the identification of cotton fiber strength.

[0010] Another object of the present invention is to provide the application of reagents for detecting the above-mentioned haplotype molecular markers related to cotton fiber strength in the genetic breeding of cotton fiber strength traits.

[0011] Another object of the present invention is to provide a KASP primer set for cotton molecular markers, wherein the molecular markers are located at 5,982,917 bp on chromosome D08 of the cotton reference genome and have the bases A / T; The nucleotide sequences of the KASP primer set are shown in SEQ ID NO.1-SEQ ID NO.3.

[0012] Another object of the present invention is to provide a detection kit, characterized in that the detection kit includes the primer set described above.

[0013] Another object of the present invention is to provide an application of the above-described KASP primer set or the above-described detection kit, characterized in that the application is one of the following: A. Identifying the strength properties of cotton fibers; B. Identification of haplotypes related to cotton fiber strength; C. Cotton genetic breeding based on fiber strength trait.

[0014] Another objective of this invention is to provide a method for identifying cotton fiber strength, which involves amplifying and genotyping the genomic DNA of the cotton to be tested using the aforementioned KASP primer set or the aforementioned detection kit. When the genotype is homozygous T / T, it indicates excellent fiber strength, and when the genotype is homozygous A / A, it indicates non-excellent fiber strength.

[0015] Another objective of this invention is to provide a cotton molecular-assisted breeding method, which uses the above-mentioned KASP primer set or the above-mentioned detection kit to amplify and genotype the genomic DNA of the cotton to be tested, and retains materials with homozygous T / T genotype.

[0016] Beneficial effects: This invention provides the application of haplotype molecular markers related to cotton fiber strength in the identification of cotton fiber strength. The identified haplotypes are significantly correlated with cotton fiber strength traits. The KASP detection primers developed based on them have the advantages of accurate detection, simple operation, high throughput, and low cost. They can be used for screening early-stage cotton breeding materials, significantly improving the selection efficiency of fiber strength traits and accelerating the breeding process of high-quality new 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 Genotyping results for KASP markers at the D08_5,982,917 (A / T) locus; where blue represents homozygous T / T genotype, red represents homozygous A / A genotype, and green represents heterozygous T / A genotype.

[0019] Figure 2 The results of KASP marker identification of fiber strength in the genetic population in Example 1 are shown. ** and * represent extremely significant (P<0.01) and significant (P<0.05) differences, respectively. Figure 3 The results of haplotype identification of natural population fiber strength in Example 3 are shown. ** indicates that the value reached a highly significant (P<0.01) level of difference, and ns indicates that the value had no significant difference. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0022] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0023] Example 1: Haplotype Identification and KASP Marker Development Fiber strength phenotypic survey: Using a multi-generational segregating population constructed from two parents of the high-quality, high-strength upland cotton hybrid Ji 1518 (national approved cotton 20210023, fiber strength 33.45 cN / tex), the families RIL131 (low strength) and RIL229 (high strength), which showed extremely significant differences in fiber strength, were selected as parents (Tang Liyuan et al., whole-genome identification of the cotton FLA gene family and...). GhFLA05Functional analysis of cotton fiber development [J], Chinese Agricultural Science, 2023, 56(23):4602-4620), constructing a secondary segregating population. An F2 population containing 384 individuals and its derived F... 2:3 The family fiber strength phenotype (range 16.9–43.0 cN / tex) exhibited a continuous normal distribution, which is suitable for subsequent genetic mapping.

[0024] BSA-seq positioning fiber strength QTL: based on F2 and F 2:3 The fibrillation strength phenotype of the population segregation was used to construct two DNA pools, one for high strength (H) and one for low strength (L). Four DNA libraries were constructed from the two parents and the pools, and sequenced using the BGI-T7 sequencing platform in PE150 mode. The raw sequencing data were filtered using the FASTP software, and the clean reads after quality control filtering were aligned with the reference genome TM-1_V2.1 (http: / / cotton.zju.edu.cn / download.html) using the BWA software. 445,666,210–571,842,720 clean reads were found, with an alignment rate of 98.30–99.65% (Table 1). GATK was used for variant detection, identifying a total of 2,200,956 SNPs and 1,208,780 InDels.

[0025] Table 1. Statistics of sequencing data and alignment results with reference genome

[0026] Using the SNP-index algorithm, fiber strength QTLs were located on chromosomes A05, A06, and D08, with two candidate intervals located on chromosome D08 (Table 2).

[0027] Table 2. Statistical analysis of candidate intervals for fiber strength localization on chromosome D08 based on the SNP-index algorithm.

[0028] Haplotype analysis and molecular marker development: To resolve the genetic variation patterns of this candidate region, we performed haplotype analysis on the genomic data covering the target region. Specifically, seven high-quality linked SNPs were identified within the 5,980,849–5,982,917 bp region of chromosome D08. These loci collectively constitute two distinct haplotypes (Hap1 and Hap2, Table 3).

[0029] Table 3. Haplotype structure of chromosome D08 in the 5,980,849–5,982,917 bp region.

[0030] Based on the sequence specificity within the region, we further selected the A / T variant at 5,982,917 bp as a key marker site to distinguish and represent the two haplotypes, Hap1 and Hap2, and designed KASP genotyping primers accordingly: Primer_AlleleX: 5'-gaaggtgaccaagttcatgctAGCAAGCATTTACCAAACCTCTA-3' (SEQ ID NO. 1); Primer_AlleleY: 5'-gaaggtcggagtcaacggattAGCAAGCATTTACCAAACCTCTC-3' (SEQ ID NO. 2); Primer_Common: 5'-cgcggcATATTTCAAATTCTACCTG-3' (SEQ ID NO. 3).

[0031] Using the KASP genotyping primers, when the genotype is homozygous T / T, it indicates that the person carries haplotype Hap1 and has high fiber strength. When the genotype is homozygous A / A, it indicates that the person carries haplotype Hap2 and has low fiber strength.

[0032] Example 2: Validation of KASP molecular markers in a genetic population Using the KASP primers developed in Example 1, the above-mentioned F1 gene containing 384 families was analyzed. 2:3 KASP typing was performed on the segregating progeny population, and the results showed that 377 markers could be detected using this set of markers, with a detection rate of 98.2% (see appendix). Figure 1 and attached Figure 2 The results indicate that this marker can effectively classify cotton. Phenotypic and locus association results show that individuals with the T allele at locus D08_5,982,917 have significantly higher fiber strength than those with the A allele. This further indicates that haplotype Hap1 has significantly better fiber strength than haplotype Hap2, and this haplotype can be divided into two categories: haplotype Hap1 (superior fiber strength) and haplotype Hap2 (non-superior fiber strength).

[0033] Example 3: Haplotype Verification in Cotton Germplasm Resources Forty-two cotton germplasm accessions from different sources were collected (Table 4) and planted in Shijiazhuang, Weixian, and Haixing in 2023 and 2024, respectively. A randomized block design was used, with conventional field management. Twenty bolls from the middle of each plot were taken to determine fiber strength, and the best linear unbiased estimator (BLUE) was used as the phenotypic value.

[0034] Table 4. Names of 462 cotton germplasm accessions

[0035] SNP detection and haplotype classification: Genomic DNA was extracted from young leaves of each material listed in Table 4, and whole-genome resequencing with a depth ≥10× was performed. After aligning the sequencing results to the reference genome TM-1_V2.1 (http: / / cotton.zju.edu.cn / download.html), the genotypes of the seven target SNP loci on chromosome D08 (Table 3) were extracted, and haplotypes were classified. The results showed that all samples could be classified using the haplotype classification method of this invention, with 188 samples being homozygous for the Hap1 type, 255 samples being homozygous for the Hap2 type, and 19 samples being heterozygous for the Het type.

[0036] Association analysis between haplotype and fiber strength: The fiber strength of materials corresponding to different haplotypes was statistically analyzed. The average fiber strength of samples with genotype Hap1 was 34.3 cN / tex; the average fiber strength of samples with genotype Hap2 was 32.8 cN / tex, which was significantly lower than that of samples with genotype Hap1; the average fiber strength of samples with genotype heterozygous Het was 33.4 cN / tex, which was not significantly different from samples with genotypes Hap1 and Hap2. The results show that the fiber strength of Hap1 material is significantly higher than that of Hap2 material, verifying the predictive effect of this haplotype on fiber strength in natural populations, and it can be used for molecular marker-assisted selection breeding (see appendix). Figure 3 ).

[0037] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0038] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention.

Claims

1. The application of haplotype molecular markers related to cotton fiber strength in identifying cotton fiber strength, characterized in that, The haplotype molecular marker consists of the following 7 sites: SNPD08_5980849, SNPD08_5981358, SNPD08_5981401, SNPD08_5981519, SNPD08_5981562, SNPD08_5982308 and SNPD08_5982917; The SNPD08_5980849 is located at 5980849bp on chromosome D08 of the reference genome and exhibits C / T polymorphism. The SNPD08_5981358 is located at 5981358 bp on chromosome D08 of the reference genome and exhibits T / A polymorphism; The SNPD08_5981401 is located at 5981401 bp on chromosome D08 of the reference genome and exhibits A / T polymorphism; The SNPD08_5981519 is located at 5981519 bp on chromosome D08 of the reference genome and exhibits T / C polymorphism; The SNPD08_5981562 is located at 5981562bp on chromosome D08 of the reference genome and exhibits G / A polymorphism. The SNPD08_5982308 is located at 5982308 bp on chromosome D08 of the reference genome and exhibits A / C polymorphism; The SNPD08_5982917 is located at 5982917bp on chromosome D08 of the reference genome and exhibits T / A polymorphism; When the haplotype combination of the genotypes at the 7 loci is CCTTAATTGGAATT, the cotton fiber strength is greater than that of cotton with the haplotype combination TTAATTCCAACCAA. The reference genome is TM-1_V2.

1.

2. The application according to claim 1, characterized in that, The determination of cotton fiber strength includes the following steps: S1: Extract genomic DNA from the cotton sample; S2: Obtain the genetic data of the cotton to be tested using genome sequencing methods; S3: The gene data obtained from S2 is analyzed, screened, and judged based on the identification of variant sites.

3. The application of haplotype molecular markers related to cotton fiber strength in the genetic breeding of cotton fiber strength traits, characterized in that, The fiber strength of offspring is enhanced by selecting individuals with the haplotype combination CCTTAATTGGAATT as parents. The haplotype molecular marker consists of the following 7 sites: SNPD08_5980849, SNPD08_5981358, SNPD08_5981401, SNPD08_5981519, SNPD08_5981562, SNPD08_5982308 and SNPD08_5982917; The SNPD08_5980849 is located at 5980849bp on chromosome D08 of the reference genome and exhibits C / T polymorphism. The SNPD08_5981358 is located at 5981358 bp on chromosome D08 of the reference genome and exhibits T / A polymorphism; The SNPD08_5981401 is located at 5981401 bp on chromosome D08 of the reference genome and exhibits A / T polymorphism; The SNPD08_5981519 is located at 5981519 bp on chromosome D08 of the reference genome and exhibits T / C polymorphism; The SNPD08_5981562 is located at 5981562bp on chromosome D08 of the reference genome and exhibits G / A polymorphism. The SNPD08_5982308 is located at 5982308 bp on chromosome D08 of the reference genome and exhibits A / C polymorphism; The SNPD08_5982917 is located at 5982917bp on chromosome D08 of the reference genome and exhibits T / A polymorphism; When the haplotype combination of the genotypes at the 7 loci is CCTTAATTGGAATT, the cotton fiber strength is greater than that of cotton with the haplotype combination TTAATTCCAACCAA. The reference genome is TM-1_V2.

1.

4. A method for determining the strength of cotton fibers, characterized in that, Genomic DNA was extracted from young cotton leaves and whole-genome resequencing was performed at a depth of ≥10×. The sequencing results were aligned to the reference genome TM-1_V2.

1. The genotypes of 7 target SNP loci on chromosome D08 were extracted and classified into haplotypes. When the haplotype combination of the genotypes of the 7 target SNP loci was CCTTAATTGGAATT, the cotton fiber strength was greater than that of cotton with the haplotype combination TTAATTCCAACCAA. The seven target SNP sites are SNPD08_5980849, SNPD08_5981358, SNPD08_5981401, SNPD08_5981519, SNPD08_5981562, SNPD08_5982308, and SNPD08_5982917. The SNPD08_5980849 is located at 5980849 bp on chromosome D08 of the reference genome and exhibits C / T polymorphism; The SNPD08_5981358 is located at 5981358 bp on chromosome D08 of the reference genome and exhibits T / A polymorphism; The SNPD08_5981401 is located at 5981401 bp on chromosome D08 of the reference genome and exhibits A / T polymorphism; The SNPD08_5981519 is located at 5981519 bp on chromosome D08 of the reference genome and exhibits T / C polymorphism; The SNPD08_5981562 is located at 5981562bp on chromosome D08 of the reference genome and exhibits G / A polymorphism. The SNPD08_5982308 is located at 5982308 bp on chromosome D08 of the reference genome and exhibits A / C polymorphism; The SNPD08_5982917 is located at 5982917bp on chromosome D08 of the reference genome and exhibits T / A polymorphism.

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