SNP molecular markers associated with upland cotton fiber length and their applications

Abstract

The present invention discloses SNP molecular markers associated with upland cotton fiber length and their applications, wherein the SNP molecular marker is at least one of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.16. The present invention planted 1,812 cotton materials for 2 years, in 5 locations, and in 10 natural environments, and detected and analyzed the fiber length of these varieties. The genome of these 1,812 cotton varieties was resequenced 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 parents, and an average sequencing depth of more than 4X for offspring. A total of 21 calculated values were accumulated through GWAS analysis, and 16 SNP molecular markers associated with upland cotton fiber length that appeared stably in at least three or more environments were obtained. The SNP molecular markers associated with upland cotton fiber length provided by the present invention can be used for early prediction and screening of cotton fiber length traits, and can also be used for breeding long-fiber cotton varieties.

Description

Technical Field

[0001] The present invention relates to a SNP molecular marker associated with upland cotton fiber length and an application thereof, and belongs to the fields of molecular biology and bioinformatics. Background Art

[0002] Cotton fiber is a vital commodity in my country and plays a crucial economic role in the textile industry. Fiber length, strength, micronaire value, and elongation are key indicators of cotton fiber quality. Currently, the overall quality of Chinese cotton is inferior to that of cotton fibers from foreign countries, such as Australian cotton. This is primarily due to a mismatch between these quality indicators, with fiber lengths mostly concentrating between 27-29 mm and poor spinnability. Therefore, producing long fibers that meet the demands of the textile market is a primary research goal for improving cotton fiber quality.

[0003] Previous studies have shown that cotton fiber length is controlled by multiple genes, and there is a negative correlation between fiber length and yield. The use of conventional breeding methods to improve cotton fiber quality has been slow. Molecular marker technology can quickly find markers that are closely linked to cotton fiber length QTLs. Using molecular markers to assist in the selection of cotton fiber length can accelerate the breeding process of long-fiber cotton quality. Researchers used molecular marker technologies such as SLAF and SSR to preliminarily locate candidate genes for cotton fiber length and found that the genetic distance between candidate genes and molecular markers is generally far, indicating that the molecular mechanism of cotton fiber length formation is very complex and needs to be further studied and explored. Fully tapping and utilizing these genes that control fiber length will enrich the genetic resources for fiber quality improvement and provide an important foundation for breeding new cotton varieties that meet various needs.

[0004] In recent years, with the rapid development of high-throughput DNA sequencing technology, the inventors have successfully resequenced 1,812 core cotton germplasm resources. Through bioinformatics data analysis and comparison, a large number of high-quality single-nucleotide polymorphisms (SNPs) were obtained. These SNPs can be used to construct haplotype maps, genetic maps, association maps, and fingerprint maps, providing important support for molecular breeding, phylogenetic evolution, and germplasm resource identification. Using genome-wide association analysis, the present invention has discovered a group of SNP molecular markers associated with upland cotton fiber length, laying the foundation for molecular marker-assisted selection and polygenic breeding to improve cotton fiber quality. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a group of SNP molecular markers associated with upland cotton fiber length and their applications.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A SNP molecular marker associated with upland cotton fiber length, wherein the SNP molecular marker is at least one of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.16.

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

[0009]

[0010]

[0011] An application of the SNP molecular marker in early prediction and screening of upland cotton fiber length specifically comprises the following steps:

[0012] (1) Extracting genomic DNA from the sample to be tested;

[0013] (2) Using the extracted DNA as a template, primers were designed based on the SNP molecular markers and PCR amplification was performed respectively;

[0014] (3) Analyze the fiber length of upland cotton based on the PCR amplification products.

[0015] A primer or reagent for detecting the SNP molecular marker.

[0016] A kit for detecting the SNP molecular marker.

[0017] A gene chip containing the SNP molecular marker.

[0018] Beneficial effects of the present invention:

[0019] The present invention planted 1812 cotton materials in 5 locations and 10 natural environments for 2 years, and detected and analyzed the fiber length of these varieties. The genome of these 1812 cotton varieties was resequenced by the Illumina HiSeq sequencing platform to obtain high-quality clean data with a data volume of 20.47Tb, an average sequencing depth of 35X for parents, and an average sequencing depth of more than 4X for offspring. A total of 21 calculated values were accumulated through GWAS analysis (a total of 10 environments in 5 test points over 2 years, and the BLUP value of all 10 environments was recorded as 1; the breeding values of the 5 test points each year totaled 10, and a total of 20 in 2 years; the above total was 21 calculated values), and 16 SNP molecular markers associated with upland cotton fiber length that appeared stably in at least three or more environments were obtained.

[0020] The SNP molecular markers associated with upland cotton fiber length provided by this invention can be used for early prediction and screening of cotton fiber length traits and for the breeding of long-fiber cotton varieties. They are directly expressed in DNA and can be detected in all tissues and developmental stages of cotton, unaffected by seasonal or environmental constraints or expression issues. They are neutral and do not affect the expression of target traits. These SNPs are suitable for rapid, large-scale screening. Genomic screening of SNPs often requires only a plus / minus analysis, rather than fragment length analysis, facilitating the development of automated technologies for SNP screening or detection. DETAILED DESCRIPTION

[0021] The following examples further illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: A Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0022] Example 1: Acquisition of SNP molecular markers

[0023] (1) Fiber length determination:

[0024] The population was tested with 2 replicates at 5 sites (1 replicate at some sites) in 2017 and 2018. A total of 1,799 progeny and 13 parents, totaling 1,812 materials, were randomly arranged within and between subpopulations. The parents of the subpopulation were randomly added to the subpopulation. Three controls were set up for the entire population, namely the parents of the population, ZZM3, Lumianyan 28, and Jinke 178. The three controls appeared in the population every 15 materials and eventually evenly covered the entire population. The five test sites are: Anyang, Henan (AY), Anqing, Anhui (AQ), Xingtai, Hebei (XT), Shihezi, Xinjiang (SHZ), and Alaer, Xinjiang (ALE). Each experimental site was planted in single rows (except for Alar, Xinjiang, which had double rows) with a row length of 2 m. The number of plants per row ranged from 10 to 30 (depending on local cultivation practices). Sampling occurred between September 20 and October 20 (depending on local frost season and cultivation practices). Except for two plants at the ends, bolls were sampled from the middle of the remaining plants, close to the main stem, for a total of 20 bolls. To reduce errors, a unique barcode numbering system was used, and the number plates were placed in the boll weight bags. To reduce errors, all boll weight materials were ginned at the Cotton Research Institute, Anyang Farm, Chinese Academy of Agricultural Sciences, using a Xinxiang MPSY-20A gin. Lint samples were obtained from 20 bolls harvested from each plot. The 2.5% span length (fiber length) was measured using an HFT9000 (HVICC calibration level) instrument from the Cotton Fiber Quality Supervision and Inspection Center (Anyang) of the Ministry of Agriculture and Rural Affairs according to the manufacturer's instructions. The best linear unbiased predictor of fiber length (mm) was estimated (two replicates over two years) using the R package lme4 (https: / / github.com / lme4 / lme4).

[0025] (2) SNP detection:

[0026] A total of 1,812 upland cotton samples were collected for genome resequencing, including 13 parental lines and 1,799 recombinant inbred lines (RILs). For sampling, seeds of each line were sown in an incubator, and young leaves of the cotton plants were collected. 5 μg of high-quality cotton genomic DNA was extracted from each sample using the CTAB method. This extracted genomic DNA was sent to Shenzhen BGI Genomics Technology Co., Ltd. for genome resequencing. Sequencing yielded 20.47 Tb of high-quality clean data, with an average sequencing depth of 35x for the parents and over 4x for the progeny. Sequence mapping was performed using the genome of the high-quality tetraploid cotton (G. hirsutum 'Texas Marker 1') as the reference genome. Prior to mapping, all unassembled contigs were ligated to a pseudochromosome (designated "ChrUN"). Short sequences from each of the 1,812 samples were mapped to the reference genome using BWA (v.0.7.12) software, removing all unaligned reads and low-quality reads (mapping quality less than 20). GATK UnifiedGenotyper (v.3.8.0) was then used to identify variants in each sample, and the variant files for all samples (n=1812) were merged into a single VCF file. Finally, 11,856,129 high-quality SNPs and 4,543,742 high-quality indels were identified. VCFtools was used to further filter variant sites based on minor allele frequencies greater than 0.05 and deletion rates less than 0.2, resulting in 1,855,955 high-quality SNPs and 1,309,084 high-quality indels for subsequent genome-wide association analysis. The effects of all variants were annotated using ANNOVAR.

[0027] (3) Genome-wide association analysis of fiber length traits in upland cotton:

[0028] The fiber length trait of upland cotton was subjected to genome-wide association scanning (GWAS) positioning. The fiber length trait results obtained in step (1) and the genotype data obtained in step (2) were statistically analyzed using a mixed linear model using the Efficient Mixed-Model Association Expedited (EMMAX) statistical analysis software. For details, please refer to: http: / / csg.sph.umich.edu / kang / emmax / download / index.html. The statistical model is:

[0029] y=Xα+Zβ+Wμ+e

[0030] y is the phenotypic trait, X is the indicator matrix of fixed effects, α is the estimated parameter of fixed effects; Z is the indicator matrix of SNPs, β is the effect of SNPs; W is the indicator matrix of random effects, μ is the predicted random individual, e is the random residual, and it obeys e~(0,δ e 2 ). In this model, the population analysis was corrected by adding a kinship matrix to μ. The analysis found that a total of 16 SNPs were significantly associated with the fiber length trait of upland cotton. The allele loci of the SNP markers are shown in Table 1. The reference sequence is the upland cotton cultivar TM-1, reference genome version number G.hirsutum_TM-1_ICR: (http: / / grand.cricaas.com.cn / page / download / download). The nucleotide sequences 50 bp upstream and downstream of these SNP sites are shown in SEQ ID NO.1-SEQ ID NO.16.

[0031] Table 1 SNP molecular markers associated with upland cotton fiber length

[0032]

[0033] (4) Verification: The effects of the above SNPs were verified using the fiber length BLUP values (best linear unbiased prediction value) of 1812 cotton multi-parent populations under 10 environments with 5 points over 2 years. The results showed that 100% of the SNPs showed a significant effect on the variation of fiber length traits in upland cotton.

[0034] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Application of a SNP molecular marker combination in early prediction and screening of upland cotton fiber length, characterized in that: The SNP molecular marker combination includes the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.16; The SNP molecular site mutated at the 51 bp position of the sequence, and the mutation form of the SNP molecular marker is as follows:

2. The use according to claim 1, characterized in that The following steps are involved: (1) Extracting genomic DNA from the sample to be tested; (2) Using the extracted DNA as a template, primers were designed according to the SNP molecular marker combination and PCR amplification was performed separately; (3) Analyze the fiber length of upland cotton based on the PCR amplification products.

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