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

Abstract

The invention discloses haplotypes related to cotton fiber strength and functional KASP molecular markers and detection application thereof, and relates to the technical field of plant molecular breeding and genetic markers. The haplotype is located in 5, 980, 849-5, 982 and 917 bp segments of a cotton reference genome D08 chromosome, is composed of seven SNP sites and can be divided into two types of Hap1 and Hap2, the KASP molecular marker is located at 5, 982 and 917 bp positions of the cotton reference genome D08 chromosome, and the basic group is A / T. The haplotype represented by the molecular marker is significantly related to the cotton fiber strength character, and a KASP detection primer developed based on the molecular marker has the advantages of accurate detection, simple operation, high flux, low cost and the like, can be used for screening early materials in cotton breeding, significantly improves the selection efficiency of the fiber strength character, and accelerates the breeding process of a new variety of high-quality cotton.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant molecular breeding and genetic marker technology, and more particularly to application of haplotype molecular marker related to cotton fiber strength in identifying cotton fiber strength. BACKGROUND

[0002] Cotton is an important economic crop, and the simultaneous improvement of yield and fiber quality has always been a difficult point in breeding work. Traditional breeding methods have made slow progress in improving fiber quality, and it is difficult to meet the demand of the textile industry for high-quality raw cotton. Therefore, improving cotton fiber quality has become one of the core goals of current cotton breeding.

[0003] Cotton fiber quality mainly includes fiber length, strength and fineness, etc. Among them, fiber strength is controlled by multiple genes and has a complex genetic mechanism, which is a key difficulty in quality improvement. Although multiple quantitative trait loci (QTLs) associated with fiber strength have been identified through linkage analysis and association mapping, effective molecular markers for practical breeding are still scarce.

[0004] Haplotype can more accurately define the genomic segment associated with traits by integrating specific combinations of linked SNPs. Its greatest advantage is that only a few "tag SNPs" are needed to identify the complete haplotype, which can achieve higher prediction accuracy with fewer markers, significantly reducing the cost of large-scale screening. The competitive allele-specific PCR (KASP) technology has the advantages of high throughput, low cost and high accuracy, and is an ideal platform for transforming haplotype markers. Therefore, developing KASP molecular markers based on key haplotypes can achieve efficient and accurate selection of cotton fiber strength traits at a lower genotyping cost, which is of great significance for promoting molecular marker-assisted breeding. SUMMARY

[0005] Therefore, the present application provides application of haplotype molecular marker related to cotton fiber strength in identifying cotton fiber strength.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The application of haplotype molecular marker related to cotton fiber strength in identifying cotton fiber strength, wherein the haplotype molecular marker is composed of 7 loci; including SNPD08_5980849, SNPD08_5981358, SNPD08_5981401, SNPD08_5981519, SNPD08_5981562, SNPD08_5982308 and SNPD08_5982917. The SNP D08_5980849 is located at 5980849 bp on chromosome D08 of the reference genome, and a C / T polymorphism exists; The SNP D08_5981358 is located at 5981358 bp on chromosome D08 of the reference genome, and a T / A polymorphism exists; The SNP D08_5981401 is located at 5981401 bp on chromosome D08 of the reference genome, and a A / T polymorphism exists; The SNP D08_5981519 is located at 5981519 bp on chromosome D08 of the reference genome, and a T / C polymorphism exists; The SNP D08_5981562 is located at 5981562 bp on chromosome D08 of the reference genome, and a G / A polymorphism exists; The SNP D08_5982308 is located at 5982308 bp on chromosome D08 of the reference genome, and a A / C polymorphism exists; The SNP D08_5982917 is located at 5982917 bp on chromosome D08 of the reference genome, and a T / A polymorphism exists.

[0007] As a preferred technical scheme, the identification of the cotton fiber strength comprises the following steps: S1: extracting cotton genomic DNA to be tested; S2: using a genome sequencing method, or amplifying the cotton to be tested based on a primer set for detecting the haplotype molecular marker, to obtain gene data of the cotton to be tested; S3: identifying and screening the gene data obtained in S2 through a variation site, to obtain a cotton fiber strength related haplotype; when the haplotype combination of the genotypes of the 7 sites is CCTTAATTGGAATT (SEQ ID NO. 4), the cotton fiber strength is greater than that of the cotton with the haplotype combination of TTAATTCCAACCAA (SEQ ID NO. 5).

[0008] Another object of the present application is to provide the application of the above-mentioned cotton fiber strength related haplotype molecular marker in the genetic breeding of the cotton fiber strength trait, by selecting an individual with the haplotype combination of CCTTAATTGGAATT (SEQ ID NO. 4) as a parent, to enhance the fiber strength of the offspring.

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

[0010] Still another object of the present application is to provide an application of the reagent for detecting the haplotype molecular marker related to the cotton fiber strength in the genetic breeding of the cotton fiber strength trait.

[0011] Still another object of the present application is to provide a KASP primer set of a cotton molecular marker, wherein the molecular marker is located at 5,982,917 bp of chromosome D08 of a cotton reference genome, and the base is A / T. The nucleotide sequence of the KASP primer set is shown as SEQ ID NO. 1-SEQ ID NO. 3.

[0012] Still another object of the present application is to provide a detection kit, wherein the detection kit comprises the primer set.

[0013] Still another object of the present application is to provide an application of the KASP primer set or the detection kit, and the application is one of the following: A. identifying the cotton fiber strength trait; B. identifying the haplotype related to the cotton fiber strength; C. genetic breeding of the cotton with the fiber strength trait.

[0014] Still another object of the present application is to provide a method for identifying the cotton fiber strength, wherein the genomic DNA of the cotton to be tested is amplified and genotyped by using the KASP primer set or the detection kit, when the genotype is homozygous T / T, it represents excellent fiber strength, and when the genotype is homozygous A / A, it represents non-excellent fiber strength.

[0015] Still another object of the present application is to provide a cotton molecular assisted breeding method, wherein the genomic DNA of the cotton to be tested is amplified and genotyped by using the KASP primer set or the detection kit, and the material with the homozygous genotype T / T is reserved.

[0016] Beneficial effects: the present application provides the application of the haplotype molecular marker related to the cotton fiber strength in the identification of the cotton fiber strength, the identified haplotype is significantly related to the cotton fiber strength trait, 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 the screening of early materials in cotton breeding, significantly improves the selection efficiency of the fiber strength trait, and speeds up the breeding process of new high-quality cotton varieties. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only represent some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on the provided drawings.

[0018] Figure 1 The KASP marker typing result at D08_5,982,917 (A / T) site; wherein, blue represents genotype of homozygous T / T, red represents genotype of homozygous A / A, and green represents genotype of heterozygous T / A.

[0019] Figure 2 The identification result of KASP marker on fiber strength of genetic population in Example 1, ** and * represent that the numerical value reaches the extremely significant (P < 0.01) and significant (P < 0.05) difference level; Figure 3 The identification result of haplotype on fiber strength of natural population in Example 3, ** represents that the numerical value reaches the extremely significant (P < 0.01) difference level, and ns represents that the numerical value has no significant difference. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be described clearly and completely in the following with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments only represent some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present application pertains.

[0022] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available products unless otherwise specified.

[0023] Example 1 Haplotype identification and KASP marker development Fiber strength phenotype investigation: A multi-generation separation population constructed by crossing the parents of high-quality high-strength fiber upland cotton hybrid J1518 (Guosan cotton 20210023, fiber strength 33.45 cN / tex) was used as the material, and the families RIL131 (low strength) and RIL229 (high strength) with extremely significant difference in fiber strength were selected as the parents (Tang Liyuan et al., Whole genome identification of cotton FLA gene family and its association analysis with fiber strength, Cotton Science, 2018, 30(3): 175-185). GhFLA05F2 population and its derived F 2:3 The fiber strength phenotype (range 16.9-43.0 cN / tex) of the family was normally distributed and suitable for subsequent genetic mapping.

[0024] BSA-seq mapping of fiber strength QTLs: Based on the F2 and F 2:3 The fiber strength phenotype of the population was separated into two DNA pools, high (H) and low (L). Four DNA libraries were constructed from the two parents and the two pools. Sequencing was performed on the MGI-T7 sequencing platform of Huada, with a PE150 mode. The raw sequencing data was filtered using the software fastp, and the clean reads were aligned to the reference genome TM-1_V2.1 (http: / / cotton.zju.edu.cn / download.html) using the software BWA. A total of 445,666,210-571,842,720 clean reads were aligned, with an alignment rate of 98.30-99.65% (Table 1). GATK was used for variant detection, and a total of 2,200,956 SNPs and 1,208,780 InDels were detected.

[0025] Table 1 Statistics of sequencing data and alignment to the reference genome

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

[0027] Table 2 Statistics of fiber strength candidate intervals located on chromosome D08 based on the SNP-index algorithm

[0028] Haplotype analysis and molecular marker development: To understand the genetic variation pattern of the candidate interval, we performed haplotype analysis on the genomic data covering the target region. Specifically, we identified 7 high-quality linked SNP sites within the 5,980,849-5,982,917 bp region on chromosome D08, which together constitute two distinct haplotypes (Hap1 and Hap2, Table 3).

[0029] Table 3 Haplotype structure of the 5,980,849-5,982,917 bp interval on chromosome D08

[0030] According to the sequence specificity in the region, we further selected the A / T variation at 5,982,917 bp as a key marker site to distinguish and represent Haplotype 1 and Haplotype 2, and designed KASP typing primers as follows: 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 typing primer for detection, when the genotype is homozygous T / T, it represents carrying Haplotype 1, and the fiber strength is higher, when the genotype is homozygous A / A, it represents carrying Haplotype 2, and the fiber strength is lower.

[0032] Example 2 KASP molecular marker verification in genetic population The KASP primers developed in Example 1 were used to KASP genotype the F 2:3 offspring segregation population containing 384 families, and the results showed that 377 could be detected by using this set of markers, and the detection rate was 98.2% (see Appendix Figure 1 and Appendix Figure 2 ), indicating that the marker can effectively genotype and detect cotton. The results of phenotype and site association showed that the fiber strength of individuals with T allele at D08_5,982,917 site was significantly higher than that of A allele, further indicating that the fiber strength of Haplotype 1 material was significantly better than that of Haplotype 2, and the haplotype could be divided into two types of excellent fiber strength haplotype Hap1 and non-excellent fiber strength haplotype Hap2.

[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 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.

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: Use genome sequencing or a primer set based on the haplotype molecular marker described in claim 1 to amplify the cotton to be tested, and obtain the genetic data of the cotton to be tested; S3: The gene data obtained from S2 were analyzed and screened for variant sites to obtain cotton fiber strength-related haplotypes; the cotton fiber strength was greater when the haplotype combination of the genotypes of the 7 loci was CCTTAATTGGAATT than when the haplotype combination was TTAATTCCAACCAA.

3. The application of the haplotype molecular marker related to cotton fiber strength as described in claim 1 in the genetic breeding of cotton fiber strength traits, characterized in that, The fiber strength of offspring can be enhanced by selecting individuals with the haplotype combination CCTTAATTGGAATT as parents.

4. The application of the reagent for detecting the haplotype molecular markers related to cotton fiber strength as described in claim 1 in the identification of cotton fiber strength.

5. The application of the reagent for detecting the haplotype molecular markers related to cotton fiber strength as described in claim 1 in the genetic breeding of cotton fiber strength traits.

6. A cotton molecularly labeled KASP primer set, characterized in that, The molecular marker is located at 5,982,917 bp on chromosome D08 of the cotton reference genome, with the base sequence A / T. The nucleotide sequences of the KASP primer set are shown in SEQ ID NO.1-SEQ ID NO.

3.

7. A test kit, characterized in that, The detection kit includes the primer set as described in claim 6.

8. The application of the KASP primer set of claim 6 or the detection kit of claim 7, 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 as a trait.

9. A method for determining the strength of cotton fibers, characterized in that, Using the KASP primer set described in claim 6 or the detection kit described in claim 7, the genomic DNA of the cotton to be tested is amplified and genotyped. When the genotype is homozygous T / T, it represents excellent fiber strength, and when the genotype is homozygous A / A, it represents non-excellent fiber strength.

10. A method for molecular-assisted breeding of cotton, characterized in that, The genomic DNA of the cotton to be tested is amplified and genotyped using the KASP primer set of claim 6 or the detection kit of claim 7, and materials with homozygous T / T genotype are retained.

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