SNP molecular markers simultaneously associated with the traits of fiber length, elongation rate, micronaire value, strength, and boll weight of upland cotton and their applications

Abstract

The present invention discloses SNP molecular markers simultaneously associated with the traits of fiber length, elongation rate, micronaire value, strength and boll weight of upland cotton, and the SNP molecular markers are nucleotide sequences shown in any one of SEQ ID NO.1 - SEQ ID NO.91. The SNP molecular markers provided by the present invention, which are simultaneously associated with the fiber length, elongation rate, micronaire value, strength and boll weight of upland cotton, can be used for the early prediction and screening of the above-mentioned traits of cotton. It is directly presented in the form of DNA, can be detected in various tissues and at various developmental stages of cotton, is not restricted by seasons and environments, and there are no problems such as expression or non-expression; it is neutral and does not affect the expression of target traits; SNPs are suitable for rapid and large-scale screening. In genome screening, SNPs often only require + / − analysis, rather than analyzing the length of fragments, which is conducive to the development of automated technologies for screening or detecting SNPs.

Description

Technical Field

[0001] The present invention relates to SNP molecular markers related to fiber length, elongation rate, micronaire value, strength and boll weight on chromosome D11 of upland cotton and their applications, belonging to the fields of plant molecular breeding technology and bioinformatics. Background Art

[0002] Upland cotton is a fiber crop, and its output accounts for more than 90% of the total world cotton output, playing an important role in the world economy. Cotton yield and quality have always been one of the important target traits for improvement by cotton breeders. Traditional cotton breeding methods mainly rely on direct phenotypic selection, with low breeding efficiency and difficulty in meeting the needs of high-boll-weight cotton breeding. With the development of technology, SNP molecular markers, due to their large quantity and wide distribution in the genome, have become the most promising molecular markers at present and are suitable for large-scale automated detection. The SNP molecular marker technology can directly select the genotypes of quantitative traits, laying a foundation for marker-assisted breeding. Currently, it has been widely applied in the fields of medicine and biology, etc., but relatively less in cotton breeding research. Genome-wide association study (GWAS) is a method for overall association analysis of common genetic variations (single nucleotide polymorphisms and copy number) across the entire genome. This method takes natural populations as the research object and is based on the linkage disequilibrium (LD) between genes (loci) retained after long-term recombination. It combines the diversity of the target trait phenotype with the polymorphism of genes (or marker loci) for analysis, and can directly identify gene loci or marker loci that are closely related to phenotypic variation and have specific functions. Conducting an overall study across the entire genome can provide a contour overview of excellent traits at one time and is suitable for research such as exploring excellent traits.

[0003] With the completion of the whole-genome sequencing of upland cotton and the rapid development of high-throughput DNA sequencing technology, the inventor successfully completed the resequencing of 1,812 cotton core germplasm resources. Through comparison of sequencing data, a large number of high-quality SNPs were obtained. These SNPs can be used for the construction of genetic maps, association maps, and fingerprint maps, providing important guarantees for molecular breeding, systematic evolution, and germplasm resource identification. The present invention utilized genome-wide association analysis to discover a batch of SNP molecular markers associated with the quality indicators (fiber length, elongation rate, micronaire value, strength) and yield indicator (boll weight) of upland cotton, laying a foundation for subsequent marker-assisted selection and pyramiding breeding to improve cotton yield and quality. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the object of the present invention is to provide SNP molecular markers that are simultaneously associated with the traits of upland cotton fiber length, elongation, micronaire value, strength, and boll weight, and to apply these SNP molecular markers to the assistant selection of cotton fiber yield and quality, so as to improve the level of high-yield and high-quality cotton varieties in China as soon as possible.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] SNP molecular markers that are simultaneously associated with the traits of upland cotton fiber length, elongation, micronaire value, strength, and boll weight, and the SNP molecular markers are nucleotide sequences shown in any one of SEQ ID NO.1 - SEQ ID NO.91.

[0007] Furthermore, the SNP molecular markers are combinations of two or more SNP molecular markers.

[0008] The SNP molecular site mutates at the 51st bp of the sequence, and the mutation form of the SNP molecular marker is shown as follows:

[0009]

[0010]

[0011] A primer or reagent for detecting the SNP molecular marker.

[0012] A kit for detecting the SNP molecular marker.

[0013] An application of the SNP molecular marker, primer or reagent, and kit in identifying the traits of cotton fiber length, elongation, micronaire value, strength, and boll weight. Specifically, it includes the following steps:

[0014] (1) Extract the genomic DNA of the sample to be tested;

[0015] (2) Using the extracted DNA as a template, design primers according to the SNP molecular marker and perform PCR amplification respectively;

[0016] (3) Analyze the cotton fiber length, elongation, micronaire value, strength, and boll weight according to the PCR amplification products.

[0017] An application of the SNP molecular marker, primer or reagent, and kit in cotton assistant breeding.

[0018] An application of the SNP molecular marker, primer or reagent, and kit in the improvement of cotton germplasm resources.

[0019] The beneficial effects of the present invention:

[0020] The present invention planted 1,812 cotton materials in 10 natural environments at 5 locations over 2 years, and measured the performance values of boll weight, fiber length, elongation rate, micronaire value, and strength for each cotton material in each natural environment as required. The genomes of these 1,812 cotton varieties were re-sequenced using the Illumina Hiseq sequencing platform to obtain high-quality clean data with a data volume of 20.47 Tb, an average sequencing depth of 35X for the parents, and an average sequencing depth of more than 4X for the offspring. Through GWAS analysis, a total of 21 calculated values were accumulated (10 environments in total for 2 years at 5 test sites, with the breeding values of all 10 environments recorded as 1; 10 breeding values for each of the 5 test sites each year, a total of 20 for 2 years; the above totals to 21 calculated values), and 91 SNP molecular markers associated with fiber length, elongation rate, micronaire value, strength, and boll weight in upland cotton that stably appeared in at least one or more environments were obtained.

[0021] The SNP molecular markers provided by the present invention that are simultaneously associated with fiber length, elongation rate, micronaire value, strength, and boll weight in upland cotton can be used for the early prediction and screening of the above traits in cotton. It is directly expressed in the form of DNA, can be detected in various tissues and at various developmental stages of cotton, is not restricted by seasons and environments, and there are no problems such as expression or non-expression; it is neutral and does not affect the expression of target traits; SNPs are suitable for rapid and large-scale screening. In genomic screening, SNPs often only require + / - analysis without analyzing the fragment length, which is conducive to the development of automated technologies for screening or detecting SNPs. Specific Embodiments

[0022] The following further elaborates on the specific embodiments of the present invention in conjunction with the examples.

[0023] Example 1. Obtaining SNP Molecular Markers

[0024] (1) Determination of fiber length, elongation rate, micronaire value, and strength:

[0025] The population was subjected to a 5-point 2-replicate (1 replicate at some locations) experiment in 2017 and 2018. A total of 1812 materials, including 1799 progenies and 13 parents, were randomly arranged within and between subpopulations. The two parents of each subpopulation were randomly added within the subpopulation. Three controls were set for the whole population, namely the parents ZZM3, Lumianyan 28, and Jinke 178 of this population. The three controls appeared successively every 15 materials in the population and finally evenly covered the whole population. The five experimental sites were Anyang, Henan (AY); Anqing, Anhui (AQ); Xingtai, Hebei (XT); Shihezi, Xinjiang (SHZ); and Alar, Xinjiang (ALE). Each experimental site was planted in single-row plots (double-row in Alar, Xinjiang), with a row length of 2 m. The number of plants per row was between 10 and 30 (specifically according to the local cultivation pattern). The sampling time varied from September 20th to October 20th (specifically according to the local frost period and cultivation pattern). Except for the two plants at both ends, the middle bolls near the main stem of the remaining plants in each plot were taken, and 1 - 2 bolls were taken from each plant, with a total of 20 bolls taken. To reduce errors, the unique barcode numbering system of the present invention was adopted, and the number plates were placed in the boll weight bags. To reduce errors, all boll weight materials from various locations were ginned with a Xinxiang MPSY-20A gin at the Anyang Farm of the Cotton Research Institute, Chinese Academy of Agricultural Sciences to obtain lint samples from the 20-boll samples harvested, and the cotton fiber length, elongation, micronaire value, and strength were measured using the HV1900 (HVICC calibration level) of the Cotton Fiber Quality Supervision and Inspection Center (Anyang), Ministry of Agriculture and Rural Affairs. The best linear unbiased prediction values (repeated twice in 2 years) were estimated using the R software package lme4 (https: / / github.com / lme4 / lme4).

[0026] Determination of boll weight: 1812 cotton materials were planted for 2 years at 5 locations, a total of 10 natural environments. For each cotton material in each natural environment, 30 evenly developed open bolls were randomly picked, air-dried, weighed, and the average boll weight (g) was calculated.

[0027] (2) Detection of SNPs:

[0028] A total of 1812 upland cotton samples were collected to extract genomic DNA for genome resequencing, including 13 parental lines and 1799 recombinant inbred lines (RILs). When collecting samples, the seeds of each line were sown in an incubator, and young leaves of cotton plants were collected. High-quality cotton genomic DNA (5 μg each) of all samples was extracted using the CTAB method. The genomic DNA extracted above was sent to BGI-Shenzhen for genome resequencing. High-quality clean data were obtained by sequencing, with a data volume of 20.47 Gb, an average sequencing depth of 35X for the parental lines, and an average sequencing depth of more than 4X for the offspring. The sequence was mapped using the genome of the high-quality tetraploid cotton (G. hirsutum 'Texas Marker 1') as the reference genome. Before mapping, all unassembled contigs were joined into a pseudo-chromosome (named "ChrUN"). The short sequences of 1812 samples were mapped to the reference genome using BWA (v.0.7.12) software respectively, and all unmapped reads and low-quality reads (mapping quality less than 20) were removed. Then, GATK UnifiedGenotyper (v.3.8.0) was used to identify variations for each sample respectively, and the variation files of all samples (n = 1812) were merged into a total VCF file. Finally, 11,856,129 high-quality SNPs and 4,543,742 Indels were identified respectively. Based on a minor allele frequency greater than 0.05 and a missing rate less than 0.2, VCFtools was used to further filter the variant sites to screen out 1,855,955 high-quality SNPs and 1,309,084 Indels for subsequent genome-wide association studies. The effects of all variations were annotated by ANNOVAR.

[0029] (3) Genome-wide association study of upland cotton fiber length, elongation rate, micronaire value, strength and boll weight traits

[0030] The mixed linear model of the (efficient mixed-model association expedited) (EMMAX) statistical analysis software was used to perform statistical analysis on the upland cotton trait results obtained in step (1) and the genotype data obtained in step (2) respectively. For details, please refer to: http: / / csg.sph.umich.edu / kang / emmax / download / index.html. The statistical model is:

[0031] y = Xα + Zβ + Wμ + e

[0032] y is a phenotypic trait, X is an indicator matrix of fixed effects, α is an estimated parameter of fixed effects, Z is an indicator matrix of SNPs, β is the effect of SNPs, W is an indicator matrix of random effects, μ is a predicted random individual, and e is a random residual, where e ~ (0, δ e 2 ). In this model, the population analysis is corrected by adding a kinship matrix to μ. Analysis found that a total of 91 SNPs were significantly associated with the traits of fiber length, elongation, micronaire value, strength, and boll weight in upland cotton. The allele locus information of SNP markers is shown in Table 1. The reference sequence is the upland cotton cultivar TM-1, and the reference genome version number is G.hirsutum_TM-1_ICR (http: / / grand.cricaas.com.cn / page / download / download). The nucleotide sequences of 50 bp upstream and downstream of these SNP loci are shown in SEQ ID NO.1 - SEQ ID NO.91.

[0033] Table 1 SNP molecular markers simultaneously associated with fiber length, elongation, micronaire value, strength, and boll weight in upland cotton

[0034]

[0035]

[0036]

[0037] (4) Verification: The effects of the above SNPs were verified using the BLUP values (Best Linear Unbiased Predictors) of fiber length, elongation, micronaire value, fiber strength, and boll weight of 1812 cotton multi-parent populations in 10 environments with 5 locations in 2 years. The results showed that 95.6% of the SNPs had a significant impact on the variation of the above traits in upland cotton.

[0038] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. SNP molecular markers simultaneously associated with the traits of fiber length, elongation, micronaire value, strength, and boll weight in upland cotton, characterized in that, the SNP molecular markers are a combination of the nucleotide sequences shown in SEQ ID NO.1 - SEQ ID NO.91; a mutation occurs at the 51st bp from the 5' end of the nucleotide sequences shown in SEQ ID NO.1 - 91, and the mutation form is as follows:

2. Use of the SNP molecular marker according to claim 1 in identifying the traits of cotton fiber length, elongation, micronaire value, strength, and boll weight.

3. The use according to claim 2, characterized in that, comprises the following steps: (1) Extract the genomic DNA of the sample to be tested; (2) Using the extracted DNA as a template, design primers according to the SNP molecular markers and perform PCR amplification respectively; (3) Analyze the cotton fiber length, elongation, micronaire value, strength, and boll weight according to the PCR amplification products.

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