Molecular marker related to sea-island cotton fiber quality and application thereof

Abstract

The invention discloses a molecular marker related to sea island cotton fiber quality and application thereof, and relates to the technical field of sea island cotton breeding, the key points of the technical scheme are as follows: the molecular marker is at least one of InDel-203, InDel-86 and InDel-423, the polymorphism of the molecular marker is a deletion / insertion sequence, and the molecular marker is at least one of InDel-203, InDel-86 and InDel-423. The molecular marker InDel-203 is obtained through amplification of a specific primer pair as shown in a nucleotide sequence SEQ NO.1-2, the molecular marker InDel-86 is obtained through amplification of a specific primer pair as shown in a nucleotide sequence SEQ NO.3-4, and the molecular marker InDel-423 is obtained through amplification of a specific primer pair as shown in a nucleotide sequence SEQ NO.5-6.

Description

Technical Field

[0001] The present invention relates to the technical field of cotton assisted breeding, and more specifically, to a molecular marker related to the fiber quality of sea-island cotton and its application. Background Art

[0002] Cotton fiber is the most important source of cellulose for the global textile industry. The continuous growth of the global population and the upgrading of the consumption structure have significantly expanded the demand scale for textiles. At the same time, the technological innovation of the textile industry and the improvement of the detection and evaluation system have put forward more stringent standards for the quality characteristics of cotton fiber materials. The cultivation of cotton varieties with excellent fiber quality and the improvement of germplasm are the common challenges faced by current cotton breeders.

[0003] Sea-island cotton (Gossypium barbadense) is known as "white gold in cotton". Due to its characteristics such as long fiber length, high strength, and fine fineness, it has become an important raw material for high-end textiles and occupies an irreplaceable position in the global high-end textile market. The yield of sea-island cotton is significantly lower than that of upland cotton (Gossypium hirsutum). Affected by climate change in traditional production areas, the yield per unit continues to decline, and the difficulty of mechanical harvesting leads to high labor costs, which poses a huge challenge for coordinated improvement.

[0004] A genetic marker refers to a biological characteristic that can be stably inherited and clearly reflects genetic polymorphism, which is used to distinguish research objects with different genetic backgrounds in genetic analysis. With the in-depth study of the genetic material DNA, especially the continuous iterative update of sequencing technology, along with the decline in sequencing costs, researchers can quickly obtain complete and accurate DNA information, thus understanding genetic polymorphism at the nucleotide sequence level, and molecular markers have also been developed accordingly. Molecular Markers are genetic markers based on the polymorphism of biological macromolecules, and the variation of nucleotide sequences in the genetic material between individuals can directly reveal genetic polymorphism at the DNA level.

[0005] Improving cotton fiber quality is one of the core objectives of cotton breeding. The application of molecular marker technology provides an important means for analyzing the genetic mechanism of fiber traits and accelerating the breeding process. Traditional breeding relies on self-crossing or backcrossing to fix recombinant haplotypes, which is time-consuming and costly, greatly limiting the breeding process of excellent cotton varieties. Moreover, traditional breeding relies on phenotypic selection, and it is necessary to wait until the cotton bolls open to detect fiber traits. The cycle is as long as 5-7 years, and it is easily affected by the environment, resulting in low efficiency. Molecular marker-assisted selection (MAS) significantly shortens the breeding cycle through early genotype screening. In recent years, breeders have constructed a multi-level QTL mapping technology system based on molecular marker data of biparental populations, multi-parental populations, and natural populations, making breakthrough progress in systematically analyzing the genetic mechanism of cotton fiber development, and laying an important theoretical foundation for the precise breeding of excellent cotton fiber quality traits. Molecular marker-assisted selection is a precise breeding technology based on genomic polymorphic loci. By establishing a marker-trait association model (MTAM), it can achieve the tracking of target genes, which is not affected by gene expression and environmental factors. It can break the linkage between target genes and unfavorable genes. During the backcross transfer process, it can significantly accelerate the stabilization of target traits and shorten the breeding period. Summary of the Invention

[0006] To solve the problems of insufficient molecular marker stability, difficulty in co-improving multiple traits, long selection cycle, and low efficiency in existing methods, the present invention provides a molecular marker of Gossypium barbadense and its application.

[0007] The present invention provides the following technical solutions: A molecular marker related to the fiber quality of Gossypium barbadense, wherein the molecular marker is at least one of InDel-203, InDel-86, and InDel-423. The polymorphism of the molecular marker is a deletion / insertion sequence. The molecular marker InDel-203 is amplified by a specific primer pair as shown in nucleotide sequences SEQ NO.1-2. The molecular marker InDel-86 is amplified by a specific primer pair as shown in nucleotide sequences SEQ NO.3-4. The molecular marker InDel-423 is amplified by a specific primer pair as shown in nucleotide sequences SEQ NO.5-6.

[0008] Application of any of the above molecular markers or their specific primer pairs in genotyping Gossypium barbadense materials.

[0009] Application of any of the above molecular markers or their specific primer pairs in the genetic breeding of Gossypium barbadense.

[0010] Application of any of the above molecular markers or their specific primer pairs in identifying or assisting in identifying the fiber quality of Gossypium barbadense.

[0011] A kit for detecting InDel molecular markers related to the fiber strength of sea-island cotton, wherein the kit contains any of the above specific primer pairs.

[0012] In summary, the present invention has the following beneficial effects: The present invention has developed three molecular markers, InDel-203, InDel-86, and InDel-423, which can be used alone or in combination to quickly and accurately detect the fiber strength genotypes of sea-island cotton population resources or recombinant inbred line materials. Using the above molecular markers, large-scale identification or auxiliary identification of breeding populations can be carried out through electrophoresis detection, accelerating the molecular breeding process, and being beneficial for application in improving the fiber quality breeding of sea-island cotton. Description of the Drawings

[0013] Figure 1 is the fine mapping map of the QTL for the fiber strength of sea-island cotton in the present invention;

[0014] Figure 2 is the result of genotyping and phenotypic analysis of the BC1F2 recombinant exchange population for realizing the present invention;

[0015] Figure 3 is the marker genotyping result of the RIL population in the embodiment of the present invention;

[0016] Figure 4 is the result of the analysis of the relationship between the genotype of the InDel-203 marker and fiber quality traits in the present invention;

[0017] Figure 5 is the result of the analysis of the relationship between the genotype of the InDel-86 marker and fiber quality traits in the present invention;

[0018] Figure 6 is the result of the analysis of the relationship between the genotype of the InDel-423 marker and fiber quality traits in the present invention;

[0019] Figure 7 is the result of the analysis of the effect of the InDel combined molecular marker gene pair on fiber strength in the present invention. Detailed Embodiments

[0020] The following further describes the present invention in detail with reference to embodiments.

[0021] The materials used in the following embodiments were from the BC1F2 secondary segregation population constructed by backcrossing the RIL material (RIL573) with the parent Pima S-7 by the research group in the early stage. 223 recombinant exchange materials were screened from this population using the markers at both ends of the QTL interval; 213 Fs derived from the sea-island cotton varieties PimaS-7 and 5917 5:6RIL population and 199 domestic and foreign sea island cotton resource varieties (lines) are experimental materials. Among them, 5917 is a material with high fiber strength, and Pima S-7 is a material with low fiber strength. The above sea island cotton germplasm resources are collected, preserved and provided by the Xinjiang Crop Biological Breeding Key Laboratory of the College of Agriculture, Xinjiang Agricultural University.

[0022] In the embodiment of the present invention, young and tender leaves of sea island cotton in seedling stage were selected, and DNA was extracted by CTAB method, and the specific steps were as follows:

[0023] (1) First, heat the DNA lysis buffer (containing 3% β-mercaptoethanol, Table 1) to 65°C in a water bath.

[0024] (2) Fresh cotton leaves were collected and placed in a 2 ml centrifuge tube filled with small steel balls. The collected samples were quickly frozen in liquid nitrogen and then crushed into powder using a tissue grinding instrument.

[0025] (3) Add 800 μl of lysis buffer to the centrifuge tube containing the sample, mix thoroughly and place in a 65°C water bath for 1 hour. Take out the centrifuge tube and mix thoroughly every 10 minutes to allow for complete lysis reaction.

[0026] (4) After the water bath, add 800 μl of a mixture of chloroform and isoamyl alcohol (V / V, 24:1) into the centrifuge tube for extraction. Invert the centrifuge tube for 10 min to mix well to form an emulsion. Place the tube in a refrigerated centrifuge set at a preset temperature of 4°C and centrifuge at 12,000 rpm / min for 10 min.

[0027] (5) Take 600 μL of supernatant and add 0.6 times volume of isopropanol (precooled at -20°C), let stand at 4°C for 30 min, mix thoroughly until white flocculent DNA precipitates from the tube wall, and centrifuge at 12,000 rpm / min for 10 min.

[0028] (6) Discard the supernatant and keep the white DNA pellet at the bottom of the centrifuge tube. Add 300ul of 75% alcohol (precooled at -20℃) and use a pipette to repeatedly blow and wash the pellet. After aspirating the 75% alcohol, add the same volume of anhydrous ethanol to wash it again. Open the centrifuge tube and air-dry the residual alcohol in an ultra-clean workbench.

[0029] (7) Add 200 μl of sterile ddH2O and shake to dissolve the DNA. Use a UV-micro spectrophotometer (Nano Drop 2000) to detect the concentration and quality of the extracted genomic DNA. The OD260 / 280 ratio is between 1.8 and 2.2, and there is no protein or visible impurity contamination.

[0030] (8) Dilute the DNA stock solution to 200 ng / ul as working solution for subsequent experiments.

[0031] Development of Molecular Markers in Gossypium barbadense L. cv. Sea Island Cotton - Example 1

[0032] According to the fiber strength QTL interval located on chromosome D08 of Gossypium barbadense L. cv. Sea Island Cotton in previous work, a secondary F2 segregation population was constructed for fine mapping of the fiber strength QTL in Gossypium barbadense L. cv. Sea Island Cotton. As Figure 1 shown, the QTL interval was narrowed down to 47997612 - 49510025. The above QTL locus explained an average phenotypic variation rate of 10.42%. Three molecular markers, InDel - 423, InDel - 86, and InDel - 203, that were tightly linked to fiber strength were developed within this interval. The information is shown in Table 1.

[0033] Table 1 Information of InDel Molecular Markers

[0034]

[0035] The method for designing InDel primers was as follows: Based on the whole - genome sequencing data of parents Pima S - 7 and 5917, the DNA sequences of 300 bp upstream and downstream of each Indel variation site were extracted using TBtools software. Primer3 was used to design InDel primers within the target QTL interval. The design parameters were: selecting a base difference greater than 10 bp, a primer annealing temperature of 55℃ - 60℃, and an amplification range of 100 - 500 bp.

[0036] Development of InDel Primer Polymorphism Verification and Population Verification - Example 2

[0037] In the present invention, 8% non - denaturing polyacrylamide gel electrophoresis was used to detect PCR products for marker genotyping. The PCR amplification system was as follows:

[0038]

[0039]

[0040] The PCR amplification program was as follows:

[0041]

[0042] First, the designed markers were verified for polymorphism in parents Pima S - 7 and 5917. Second, verification was carried out in the BC1F2 population, RIL population, and Gossypium barbadense L. cv. Sea Island Cotton resource materials.

[0043] The A / B / H method was used to record the detected band patterns, that is, the band pattern consistent with the genotype of Pima S - 7 (P1) was denoted as "A", the band pattern consistent with the genotype of 5917 (P2) was denoted as "B", the heterozygous genotype was denoted as "H", and the missing band pattern was denoted as "-1".

[0044] The analysis method combining molecular markers with phenotypic data is as follows: The genotyping results of InDel markers in three populations are statistically analyzed respectively. In each population, the phenotypic data of the materials carrying the B genotype are compared with those of the materials carrying the A genotype, and the results are analyzed by plotting.

[0045] By associating 3 polymorphic primers with the fiber quality phenotypic data of the recombinant exchange population in the BC1F2 population, it was found that the primers InDel-203, InDel-86, and InDel-423 could divide the recombinant exchange population into three categories: A, H, and B. The fiber strength of the materials carrying the B genotype distinguished by the primers InDel-203, InDel-86, and InDel-423 was significantly different from that of the materials carrying the A genotype. According to the genotyping results of the recombinant exchange population, it can be preliminarily judged that these primers are linked to fiber quality traits.

[0046] After genotyping the above 3 pairs of polymorphic primers InDel-203, InDel-86, and InDel-423 in 213 RIL populations and combining with the phenotypic data of fiber quality for 5 years, as Figure 3 shown, the fiber strength of the materials carrying the B genotype distinguished by the primers InDel-203, InDel-86, and InDel-423 was different from that of the materials carrying the A genotype in multiple years and environments, and there were significant differences in some years.

[0047] After detecting the above 3 pairs of polymorphic primers InDel-203, InDel-86, and InDel-423 in 199 Gossypium barbadense resource materials, it was found that the primer InDel-203 showed differences in fiber strength in all 9 years; for fiber length, spinning index, and fiber uniformity, there were significant differences in other years except in the environment of 2023; the primer InDel-86 showed differences in fiber strength and fiber length in multiple years and environments, and there were differences in fiber uniformity in multiple years and environments; for the primer InDel-423 in Gossypium barbadense resource materials, the genotyping results of fiber strength were better, with significant differences in all years, and for the genotyping results of fiber length, there were also significant differences in other years except in 2022 and 2023, which was consistent with fiber uniformity and spinning index. Therefore, these 3 pairs of primers can be used to screen the fiber quality traits of Gossypium barbadense.

[0048] Example 3: Combining molecular marker genotyping and associating with fiber quality phenotypes

[0049] Through the analysis of fiber strength data of the Gossypium barbadense resource population in multiple years and locations, the genotyping results of the above 3 markers were combined, and a total of 8 genotyping types were obtained, as shown in Table 2 below.

[0050] Table 2: Combined types of combined molecular markers

[0051]

[0052] After associating these 8 combination types with the phenotypic data of fiber quality traits, it was found that in multiple years of environments, the materials carrying the genotypes of Hap4, Hap6, Hap7, and Hap8 had good genotyping in fiber strength, and they had significant differences compared with other types of materials. In terms of fiber length, the materials carrying the genotypes of Hap6, Hap7, and Hap8 had significantly longer fiber lengths than other types of materials. In the spinning index, the materials carrying the genotypes of Hap7 and Hap8 had significantly higher spinning indices than the materials of Hap1 - Hap4 types. In fiber evenness, the materials carrying the types of Hap7 and Hap8 had significantly higher fiber evenness than the materials of Hap1 type. Generally speaking, the combined molecular markers are superior to single markers in screening materials.

[0053] Example 4 Cluster Analysis of Fiber Quality Traits and Joint Analysis of Combined Markers for Sea Island Cotton Resource Materials

[0054] Based on the comprehensive BLUP values of the fiber quality phenotypic data of sea island cotton for 9 years, cluster analysis was carried out on the fiber quality phenotypic data. The results showed that these sea island cotton resource materials were divided into 3 groups: The first group included a total of 107 materials, accounting for 45.53%, mainly materials with medium fiber length and strength; the second group had 52 materials, accounting for 26.13%, mainly materials with low fiber elongation rate and high spinning consistency index; the third group included a total of 40 materials, accounting for 20.10%, mainly materials with excellent comprehensive traits.

[0055] Combined with the genotyping results of 3 InDel markers, through the genotyping results of combined molecular markers and phenotypic clustering, it was found that there were 17 materials with intersections. As shown in Table 3 below.

[0056] Table 3 Phenotypes of Excellent Fiber Quality Materials of Sea Island Cotton

[0057]

[0058]

[0059] As shown in Table 3, the materials carrying the genotype of Hap7 (B / B / A) had good performances in fiber length and fiber strength, and the materials carrying the genotype of Hap8 (B / B / B) had excellent performances in fiber length, fiber strength, and spinning consistency index. A total of 7 materials were screened, which can provide references for the breeding improvement of excellent fiber quality of sea island cotton.

[0060] This specific embodiment is only an interpretation of the present invention and is not a limitation thereof. After reading this specification, those skilled in the art may make modifications to this embodiment that do not contribute creatively as needed, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A molecular marker related to the fiber quality of sea-island cotton, characterized in that, The molecular marker is at least one of InDel-203, InDel-86, and InDel-423. The polymorphism of the molecular marker is a deletion / insertion sequence. The molecular marker InDel-203 is amplified by a specific primer pair as shown in nucleotide sequences SEQ NO.1-2. The molecular marker InDel-86 is amplified by a specific primer pair as shown in nucleotide sequences SEQ NO.3-4. The molecular marker InDel-423 is amplified by a specific primer pair as shown in nucleotide sequences SEQ NO.5-6.

2. Use of the molecular marker or its specific primer pair as described in claim 1 in genotyping of sea-island cotton materials.

3. Use of the molecular marker or its specific primer pair as described in claim 1 in genetic breeding of sea-island cotton.

4. Use of the molecular marker or its specific primer pair as described in claim 1 in identifying or assisting in identifying the fiber quality of sea-island cotton.

5. A kit for detecting InDel molecular markers related to sea-island cotton fiber strength, the kit comprising any one of the specific primer pairs as described in claim 1.

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