CN116334268A - SNP molecular marker related to upland cotton fiber length and application thereof - Google Patents
SNP molecular marker related to upland cotton fiber length and application thereof Download PDFInfo
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- 229920000742 Cotton Polymers 0.000 title claims abstract description 51
- 244000299507 Gossypium hirsutum Species 0.000 title claims abstract description 41
- 235000009429 Gossypium barbadense Nutrition 0.000 title claims abstract description 23
- 235000018322 upland cotton Nutrition 0.000 title claims abstract description 23
- 239000003147 molecular marker Substances 0.000 title claims abstract description 22
- 238000012216 screening Methods 0.000 claims abstract description 7
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- 108090000623 proteins and genes Proteins 0.000 claims description 8
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- 238000012408 PCR amplification Methods 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
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- 102000054766 genetic haplotypes Human genes 0.000 description 1
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Abstract
The invention discloses an SNP molecular marker related to the length of upland cotton fibers and application thereof, wherein the SNP molecular marker is at least one of nucleotide sequences shown as SEQ ID NO.1-SEQ ID NO. 16. The invention plants 1812 parts of cotton materials in 2 years, 5 places and 10 natural environments, and detects and analyzes the fiber length of the varieties. Genome re-sequencing is carried out on 1812 cotton varieties through an IlluminaHiseq sequencing platform, so that high-quality clean data is obtained, the data size is 20.47Tb, the parent average sequencing depth is 35X, and the offspring average sequencing depth is more than 4X. And accumulating 21 calculated values through GWAS analysis to obtain 16 SNP molecular markers which are stably appeared in at least three and more environments and are related to the length of the upland cotton fiber. The SNP molecular marker related to the length of the upland cotton fiber can be used for early prediction and screening of the length character of the cotton fiber and can also be used for breeding of long fiber cotton varieties.
Description
Technical Field
The invention relates to an SNP molecular marker related to the length of upland cotton fibers and application thereof, belonging to the fields of molecular biology and bioinformatics.
Background
Cotton fiber is an important material in China and has important economic status in textile industry. Fiber length, strength, micronaire, elongation, etc. are important indicators for determining cotton fiber quality. At present, the cotton in China has lower overall fiber quality compared with cotton fibers in foreign countries such as Australian cotton and the like, is mainly characterized by mismatch among quality indexes, has the fiber length of 27-29mm in many cases and has poor spinning adaptability. Thus, the production of long fibers meeting the demands of the textile market is one of the main research objectives for improving the quality of cotton fibers.
Previous studies have shown that cotton fiber length is controlled by multiple genes and that there is a negative correlation between fiber length and yield, and improvement of cotton fiber quality by conventional breeding methods has progressed slowly. The molecular marking technology can quickly find out the markers closely linked with the cotton fiber length QTL, and the molecular markers can assist in selecting the cotton fiber length, so that the breeding process of the long fiber cotton quality can be accelerated. Researchers adopt SLAF, SSR and other molecular marking technologies to initially position candidate genes of cotton fiber length, find that the genetic distance between the candidate genes and molecular markers is generally far, indicate that the molecular mechanism of forming the cotton fiber length is very complex, and fully excavate and utilize the genes for controlling the fiber length to enrich the gene resources for improving the fiber quality, thereby providing an important basis for cultivating new cotton varieties meeting various requirements.
In recent years, with the rapid development of high throughput DNA sequencing technology, the inventors successfully completed re-sequencing 1812 parts of cotton core germplasm resources. The SNPs with high quality can be used for constructing haplotype patterns, genetic patterns, association patterns and fingerprint patterns, and provide important guarantee for molecular breeding, system evolution and germplasm resource identification. The invention utilizes whole genome association analysis to discover a batch of SNP molecular markers associated with the length of upland cotton fibers, and lays a foundation for molecular marker assisted selection and polymerization breeding improvement of cotton fiber quality.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a batch of SNP molecular markers related to the length of upland cotton fibers and application thereof.
In order to achieve the above object, the technical scheme of the present invention is as follows:
and the SNP molecular marker is at least one of nucleotide sequences shown as SEQ ID NO.1-SEQ ID NO. 16.
The SNP molecular locus is mutated at the 51bp position of the sequence, and the mutation form of the SNP molecular marker is shown as follows:
an application of the SNP molecular marker in early prediction and screening of the length of upland cotton fibers. The method specifically comprises the following steps:
(1) Extracting genome DNA of a sample to be detected;
(2) Designing primers according to SNP molecular markers by taking the extracted DNA as a template, and respectively carrying out PCR amplification;
(3) Based on the PCR amplification product, the upland cotton fiber length was analyzed.
A primer or reagent for detecting the SNP molecular marker.
A kit for detecting the SNP molecular marker.
A gene chip containing the SNP molecular marker.
The invention has the beneficial effects that:
the invention plants 1812 parts of cotton materials in 2 years, 5 places and 10 natural environments, and detects and analyzes the fiber length of the varieties. Genome re-sequencing is carried out on 1812 cotton varieties through an IlluminaHiseq sequencing platform, so that high-quality clean data is obtained, the data size is 20.47Tb, the parent average sequencing depth is 35X, and the offspring average sequencing depth is more than 4X. The total of 21 calculated values (10 environments in total, 5 test points in 2 years, 1 BLUP value of all 10 environments, 10 breeding values in total, 20 breeding values in 5 test points in 2 years, 21 calculated values in total) were accumulated by GWAS analysis, and 16 SNP molecular markers associated with the length of upland cotton fibers, which appeared stably in at least three or more environments, were obtained.
The SNP molecular marker related to the length of the upland cotton fiber can be used for early prediction and screening of the length character of the cotton fiber and can also be used for breeding of long fiber cotton varieties. The method is directly expressed in the form of DNA, can be detected in all tissues and all development stages of cotton, and is not limited by seasons and environment, and the problems of no expression and the like are avoided; the expression is neutral, and the expression of the target character is not influenced; SNPs are suitable for rapid, large-scale screening. SNPs in genome screening are often analyzed only +/-, and the length of fragments is not needed, so that the development of an automatic technology for screening or detecting SNPs is facilitated.
Detailed Description
The following examples are given to illustrate the present invention in further detail, but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
Example 1: acquisition of SNP molecular markers
(1) Fiber length measurement:
the population was tested 5-point 2-replicates (part of sites 1-replicates) in 2017, 2018. The 1799 progeny and 13 parents total 1812 material were randomly arranged within and between the sub-populations. The parents of the sub-population are randomly added to the sub-population. The population is provided with 3 controls, namely parent ZZM3, lu cotton grinding 28 and Jinke 178 of the population. The 3 controls appeared in sequence every 15 materials in the population and eventually covered evenly throughout the population. The 5 test points are: henan Anyang (AY), anhui Anqing (AQ), hebei Chengtai (XT), xinjiang Stone (SHZ), xinjiang Alar (ALE). Each test point is planted according to a single row area (except for Xinjiang Alar which is double rows) and 2m rows, the number of plants in each row is between 10 and 30 plants (specifically according to a local cultivation mode), the sampling time is different from 9 months to 20 days to 10 months and 20 days (specifically according to a local frost period and a cultivation mode), the middle parts of the rest plants are close to main stem bell in each cell except for 2 plants at two ends, 1 to 2 rings are not equal, and 20 rings are taken in total. In order to reduce errors, the unique bar code numbering system is adopted, the number plates are placed in bell weight bags, in order to reduce errors, all land bell weight materials are ginned on a Henan Anyang farm of China national academy of agricultural sciences, a new county MPSY-20A skin gin is adopted to gin the harvested 20 bell samples of each district, ginned cotton samples are obtained, and 2.5% span length (fiber length) measurement is carried out according to the specification by using an agricultural rural cotton fiber quality supervision and inspection center (Anyang) HFT9000 (HVICC calibration level) instrument. Using the R software package lme (https:// gitsub.com/lme 4/lme 4), the optimal linear unbiased prediction of fiber length (mm) was estimated (two replicates in 2 years).
(2) Detection of SNP:
samples 1812 parts of upland cotton were collected together for genome re-sequencing, with 13 parts of the parent, and 1799 parts of the recombinant inbred line RIL (Recombinant Inbred Lines). When a sample is collected, seeds of each strain are sown in an incubator, and young leaves of cotton plants are collected. All samples were extracted with 5 μg of high quality cotton genomic DNA by CTAB method. The extracted genome DNA is sent to Shenzhen Dacron Gene technology Co., ltd for genome resequencing. Sequencing to obtain high-quality clean data volume of 20.47Tb, parent average sequencing depth of 35X and offspring average sequencing depth of more than 4X. The genome of high-quality tetraploid cotton (G. Hirsutum 'Texas Marker 1') is used as a reference genome for sequence positioning. All unassembled contigs were ligated to one pseudochromosome (named "ChrUN") prior to mapping. Short sequences of 1812 samples were mapped to the reference genome using BWA (v.0.7.12) software, rejecting all unaligned reads and reads of low quality (mapping quality less than 20), respectively. Then, mutation identification was performed on each sample using GATK UnifiedGenotyper (v.3.8.0), and the mutation files of all samples (n=1812) were combined into one total VCF file. Finally, 11,856,129 and 4,543,742 high quality SNPs and indels, respectively, were identified. Based on the minor allele frequency being greater than 0.05 and the deletion rate being less than 0.2, VCFtools were used to further filter the mutation sites to screen out high quality SNPs and indels 1,855,955 and l,309,084, respectively, for subsequent whole genome association analysis. The effects of all variations are noted by ANNOVAR.
(3) Whole genome association analysis of upland cotton fiber length traits:
upland cotton fiber length trait whole genome scanning (GWAS) positioning, and carrying out statistical analysis on the upland cotton fiber length trait result obtained in the step (1) and genotype data obtained in the step (2) by adopting a mixed linear model of (effective mixed-model association Expedited) (EMMA) statistical analysis software, wherein specific reference can be made to: http:// csg.sph.umichi.edu/kang/emmax/download/index. The statistical model is as follows:
y＝Xα+Zβ+Wμ+e
y is a phenotypic character, X is an indication matrix of a fixed effect, and alpha is an estimated parameter of the fixed effect; z is an indication matrix of SNP, and beta is the effect of SNP; w is an indication matrix of random effects, mu is a predicted random individual, e is a random residual, obeying e to (0, delta) e 2 ). In this model, population analysis was corrected by adding the affinity matrix to μ. Analysis shows that 16 SNPs are obviously related to the length characters of the upland cotton fibers, and the allele locus information of the SNP markers is shown in table 1. The reference sequence is upland cotton cultivar TM-1, reference genome version number G.hirsutum_TM-1_ICR: (http:// grand. Crycas. Com. Cn/page/download/download). The nucleotide sequence of 50bp upstream and downstream of the SNP loci is shown as SEQ ID NO.1-SEQ ID NO. 16.
TABLE 1 SNP molecular markers associated with upland cotton fiber length
(4) And (3) verification: the effect of the SNP is verified by using the fiber length BLUP value (optimal linear unbiased predicted value) of 1812 cotton multi-parent group under 10 environments in total of 2 years and 5 points, and the result shows that 100% of the SNP shows that the variation of the fiber length character of upland cotton has obvious influence.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. The SNP molecular marker related to the length of the upland cotton fiber is characterized in that the SNP molecular marker is at least one of nucleotide sequences shown as SEQ ID NO.1-SEQ ID NO. 16.
2. The SNP molecular marker according to claim 1, wherein the SNP molecular site is mutated at the 51bp of the sequence, and the mutation form of the SNP molecular marker is as follows:
3. use of the SNP molecular marker of claim 1 in early prediction and screening of upland cotton fiber length.
4. The use according to claim 3, characterized by the following steps:
(1) Extracting genome DNA of a sample to be detected;
(2) Designing primers according to SNP molecular markers by taking the extracted DNA as a template, and respectively carrying out PCR amplification;
(3) Based on the PCR amplification product, the upland cotton fiber length was analyzed.
5. A primer or reagent for detecting the SNP molecular marker according to claim 1.
6. A kit for detecting the SNP molecular marker according to claim 1.
7. A gene chip comprising the SNP molecular marker according to claim 1.
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