CN108300797B

CN108300797B - Haplotype of upland cotton No. 25 chromosome related to fiber strength

Info

Publication number: CN108300797B
Application number: CN201711481979.9A
Authority: CN
Inventors: 商海红; 范森淼; 巩万奎; 李俊文; 刘爱英; 石玉真; 张震; 邹先炎; 葛群; 刘纪生; 范李强; 张敏; 龚举武; 袁有禄
Original assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2021-07-13
Anticipated expiration: 2037-12-29
Also published as: CN108300797A

Abstract

The invention belongs to the technical field of cotton molecular breeding, and discloses a haplotype related to the strength of upland cotton No. 25 chromosome fiber, and detection and application thereof. The haplotype is obtained by taking stable RIL population of cotton as a material through a genome re-sequencing method. The haplotypes disclosed by the invention provide a new method for molecular marker-assisted selective breeding of cotton, shorten the breeding period and have important significance in the research of cultivating excellent cotton fiber varieties.

Description

Haplotype of upland cotton No. 25 chromosome related to fiber strength

Technical Field

The invention belongs to the technical field of cotton molecular breeding, and particularly relates to screening and application of a haplotype related to the strength of upland cotton fibers.

Background

The cotton is an important strategic material related to the countryside of China, is the second crop next to the grain, relates to two industries of agriculture and textile industry, and plays an important role in national economy. With the increase of the demand of people on medium-high-grade textiles, the requirement on the quality of fibers is increasingly improved, but the traditional breeding means mainly selects through phenotypes, the breeding efficiency is low, and the requirement on quality breeding is difficult to meet. The development of molecular marker technology has made it possible to directly select quantitative trait genotypes. QTL positioning by constructing cotton genetic map can make breeder directly select genotype with quantitative characters such as fiber quality, etc. by using F₂And RIL and other mapping groups carry out QTL positioning research on fiber quality, and great results are obtained. Particularly, the development and application of the third generation marking technology SNP marker can lay a foundation for the later marker-assisted breeding.

Yuan et al utilizes an abnormal cotton high-strength fiber introgression line 7235 and a upland cotton genetic standard line TM-1 as parents to construct F₂、F_2：3Separating the population, identifying a main effect QTL which can be detected in different cotton regions in China and in a plurality of environments such as the United states and the like and can explain more than 30 percent of phenotypic variation (molecular marker screening and positioning of high-quality fiber character QTLs of Yuan and the like, genetic report 2001,28(12): 1151-; the Sunfuding takes 0-153 and the selected line sGK9708 of the cotton 41 of the insect-resistant cotton variety popularized in the yellow river basin as parents for hybridization, and the hybridization is carried out by F_2:6The single plant selection of (2) establishes a set of 196 upland cotton F_6:8Recombining inbred line group, carrying out two-year three-point four-environment (07-year Anyang, 08-year Anyang, Quzhou and Linqing) repeated test, screening multi-environment stable expression main effect QTLs, detecting 7 QTLs related to fiber strength by adopting a composite interval mapping method, and detecting interaction QTL2 pairs related to fiber strength by adopting a composite interval mapping method based on a mixed linear model(Sun Foding et al, analysis of genetic variation of fiber quality and yield traits of a recombinant inbred line population of upland cotton, Cotton science, 2010,22(4): 319-) 325) Jamseed utilizes a Recombinant Inbred Line (RIL) population to construct a genetic map, and positions 47 QTLs stable in multiple environments, wherein the QTLs exist in a form of aggregation group to control two or more traits, and the QTLs are mainly concentrated in chromosomes 4, 7,14 and 25 (Jamsed et al identification of stable trait loci (QTLs) for controlling two or more traits, wherein the QTLs are mainly concentrated in chromosomes 4, 7,14 and 25 (QTLs) for fibrous trait loci in Gossypium hi nuclear expressed in genomic traits, BMC 2016,17: 197); zhang et al constructed by two upland cotton varieties 0-153 and SGK9708 constructed a high-density genetic map with 5521 single nucleotide polymorphism markers covering a total distance of 3259.37cM by using an SLAF sequence (Zhang et al. structural of a high-density genetic map by specific Loci amplified mapping (SLAF-seq) and its mapping to a Quantitative Trap Locus (QTL) analysis for a born weight in an upper land cotton (Gossypium hi u. M. Plant Biology,2016,16: 79).

Linkage Disequilibrium (LD) refers to the non-random combination of alleles at different loci. Association analysis (Association analysis) is a relatively new analysis method applied in plant quantitative trait research and plant breeding. It identifies the relationship between traits and genetic markers or candidate genes in a population based on linkage disequilibrium (Mackay I, Powell W.methods for linkage disequilibrium mapping in crops [ J ]. Trends Plant Sci, 2007, 12: 57-63). Compared with a recombinant population, the method has the obvious advantages of high flux, namely, a large number of character control gene sites or regions of germplasm resource materials with different genetic backgrounds can be effectively detected in the whole genome range; in addition to the advantage of high throughput, since whole genome association analysis is generally based on existing natural populations, it takes much less time than the typical recombinant population; meanwhile, the precision is high, and the level of a single gene can be achieved (Yangxue red and the like. plant quantitative trait association analysis research progress [ J ]. crop college, 2007, 33(4): 523-.

The decay distance (LD decay) of linkage disequilibrium determines the number and accuracy of markers required to perform genome-wide association analysis, and the level of LD in the natural population determines to some extent the resolution of genome-wide association analysis [ J ] Science, 2009, 737: 325. The magnitude of allelic frequency and recombination rates between sites can affect the level of Linkage disequilibrium, so natural mutations, recombination, sub-population structure, pressure on artificial selection, and genetic drift in a population can all affect the structure of Linkage Disequilibrium (LD) (Pitchart J K, Przewski M, Linkage disequilibrium in humans: moles and data. am J Hum Genet,2001,69: 1-14). When performing genome-wide association analysis, the genetic relationship between the structure and material of the subpopulations in the population leads to an increased degree of linkage disequilibrium across the associated population, which may provide false positive results. Therefore, it is necessary to analyze the population structure and the genetic relationship before performing the correlation analysis, and the generation of false positive markers can be effectively reduced by using the population structure and the genetic relationship as covariates (Jeffrey D W, Jonathan K P, Haplotpype blocks and linkage disequilibrium in the human genome. Nature Reviews,2003,4: 587-.

Haplotypes (haplotypes) are a set of interrelated sets of single nucleotide polymorphisms, also known as haplotypes or haplotypes, located in specific regions of a chromosome and that are predisposed to be inherited as a whole to offspring. The haplotype is the case of multiple molecular markers in linkage disequilibrium on the same chromosome. SNPs loci are not independently inherited, but tend to be inherited as a whole on a chromosome to offspring, and SNPs loci inherited in groups rarely recombine in one generation and the next. Thus, one such group of SNPs site types is also the haplotype (RW Morris, NL Kaplan. on the introduction of a polypeptide analysis in the presence of multiple discrete reactivity alloys. genetic Epidemiology,2002,23(3): 221-. Since haplotypes contain genetic information from multiple SNPs, many studies have shown that the use of haplotypes has a better statistical analysis effect than single SNPs in correlation analysis of complex traits (Hoehe M R. haplotypes and the systematic analysis of genetic variations in genes and genes. Pharmacogenomics,2003,4(5): 547-570). The fiber strength in the cotton belongs to simple quantitative traits, and the development and identification of haplotypes related to specific traits can preliminarily identify whether the cotton material is a high-strength fiber strain or not in the seedling stage.

The invention constructs 198 cotton variety association groups, performs genotyping by genome re-sequencing, performs whole genome association analysis by combining phenotypic data of fiber strength under 11 environments of multiple points in multiple years, aims to locate and screen out molecular markers related to the content of the characters, and constructs haplotypes for molecular marker-assisted selection, molecular breeding and subsequent cloning and functional verification of related genes.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: by screening the haplotype related to the strength of the upland cotton fiber and applying the haplotype to the auxiliary selection of the cotton fiber quality, the fiber quality level of the cotton variety in China can be improved as soon as possible.

The technical scheme provided by the invention is that the haplotype is linked with the cotton upland field 25 chromosome fiber strength gene, the haplotype is positioned in 3 QTLs related to the cotton upland field 25 chromosome and the fiber strength, the haplotype is qFS-chr25-2, qFS-chr25-3 and qFS-chr25-5, the number of the haplotype in each QTL is 2, the number is BLOCK1-6, and the size, the position and the polymorphic locus of the haplotype are shown in the following table:

in the above table, Chr _ D06_11946508 refers to the mutation of the nucleotide at 11946508bp of the chromosome 6, i.e., chromosome 25, of the Gossypium hirsutum D group, and the like.

In the present invention, the QTL designation refers to the nomenclature rule of McCouch et al (1997) in Rice and is expressed in terms of the number of q + trait + linkage group + QTL (ref: McCouch SR, Cho YG, Yano M, et al report on QTL nomenclature, Rice Genet Newslett.,1997,14:11-13), for example, qFS-chr25-2 indicates the second QTL associated with fiber strength for mapping to chromosome 25.

Meanwhile, the invention also provides a method for screening the haplotype related to the strength of the upland cotton fiber, which comprises the following steps:

(1) f is constructed by using a cotton 41 selected line SGK9708 in a field-promoted upland cotton cultivation variety and an upland cotton excellent line 0-153 with an Asian cotton high-strength fiber gene as parents₂And F_2:3A population;

(2)F_2:3selfing each generation in the colony family, at F_2:6The generation carries out single plant selection once, and plants the plants for two generations till F_6:8Handle F_6:8And later generations are used as a recombinant inbred line group to carry out multi-year and multi-point experiments;

(3) extracting DNA of the recombinant inbred line population and the parents;

(4) re-sequencing a sample, developing a SLAF label by a method of clustering among reads, filtering the label according to polymorphism and integrity, carrying out genotype coding on the filtered SLAF label, dividing the SLAF label into 26 linkage groups by positioning with a reference genome, constructing a genetic map, positioning QTL according to the genetic map, positioning a haplotype aiming at the positioned QTL interval to obtain the haplotype, and determining the variation condition of the haplotype in a parent and the group.

The invention has the following beneficial effects:

according to the haplotype related to fiber strength screened by the invention, if the genotype of the sample to be tested is A, the sample is a high-fiber-strength material, if the genotype of the sample to be tested is B, the sample is a low-fiber-strength material, and if the genotype of the sample to be tested is B, the sample is a medium-strength fiber sample, or the sample to be tested is determined to have the genotype of A, B, C respectively, the fiber strength of the sample for identifying the genotype A is larger than that of the sample with the genotype of B/C, if the sample with the genotype of C is identified, the fiber strength of the sample is higher than that of the sample with the genotype of B, the identification results can be completed in the seedling stage, and the haplotype positioning is rapid and simple, the polymorphic locus molecular marker contained in the invention is obtained by a genome re-sequencing method by using software HaploView aiming at the positioned QTL interval, haplotypes are obtained and variation of the haplotypes is determined among the parents as well as the population. By using the single body type cotton breeding method, strains with high fiber strength can be quickly screened out, molecular marker-assisted breeding selection is carried out, the breeding period can be greatly shortened, and the breeding efficiency of the cotton fiber strength is improved.

Drawings

FIG. 1 is a genetic map obtained by genome re-sequencing with a total distance of 5197.17 cM.

Detailed Description

The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.

Example 1:

(1) recombinant inbred line F_6:8Obtained by

For field planting and DNA extraction between 2007 and 2008, see patent application publication No.: CN 101613761a, inventive name: patent application document of SSR markers linked to major genes of cotton fiber strength. Parents and F6:10 groups were planted in the test station of cotton institute of Chinese agricultural science institute, Quzhou test station of Chinese agricultural university and test station of Xinjiang Aksudejia science and technology, respectively in 2009. Adopting a one-line area in the circumference of Anyang and Quzhou, wherein the line length is 5 meters, the line spacing is 0.8m and (0.8+0.5) m respectively, and 20 plants are planted in each line; xinjiang adopts a 6-row area, 2 m rows long, and 15 plants in each row. Planting parents and F in the original seed field of the Hubei Gaoyao, the test station of the agricultural science institute of Anyang China in Henan province and Zhengzhou province in Henan province in 2010_6:11The group, Anyang and Zhengzhou, adopts a single-row area, the row length is 5m, and the row spacing is 0.8 m; the cognate adopts a single-row region with a row length of 4m and a wide-narrow row (0.8+0.6) m. Each test point adopts an incomplete random block design, and the planting is repeated for two times. Taking field samples in the middle and last ten days of 9 months, collecting flowers according to families, and taking fiber samples of about 12g for fiber quality determination.

(2) And extracting DNA of the recombinant inbred line population and the parents.

(3) Selecting a tetraploid genome sequence of cotton provided by the research institute of Cotton of Chinese agricultural sciences as a reference genome for electronic enzyme digestion prediction (Li FG, Fan GY, Lu CR, Xiao GH, Zou CS, Kohel RJ, Ma ZY, Shang HH, Ma XF, Wu JH, et al genome sequence of conserved plan copy cotton (Gossypium hi utum TM-1) providences interactions in genome evaluation. Nature Biotechnology,2015,33(5)), finally selecting a HaeIII + SspI enzyme with an enzyme cleavage rate of 98.61%, obtaining 495.48Mreads altogether, and defining the sequence of the enzyme section in SL414 bp as an AF tag.

(4) According to the selected optimal enzyme digestion scheme, enzyme digestion experiments are carried out on the detected genome DNA of each sample, the obtained enzyme digestion fragments (SLAF labels) are processed by adding A at the 3' end, the obtained enzyme digestion fragments are connected with a Dual-index sequencing street, target fragments are selected through PCR amplification, purification, sample mixing and gel cutting, and IlluminaHiseqTM2500 is used for sequencing after the quality of the library is qualified.

(5) And identifying the original data obtained by sequencing by using the Dual-index to obtain the reads of each sample. And developing the SLAF label by a method of clustering among reads.

(6) A total of 321797 SLAF tags were obtained by bioinformatic analysis. Markers were filtered according to three criteria with polymorphism in the population, MAF >0.05, and marker integrity > 60%, to yield 20270 markers.

(7) And carrying out genotype coding on the polymorphic SLAF label, wherein the genotype coding rule is a 2-allele coding rule which is generally used in genetics, if the parental genotypes of a certain marker are aa (male parent) and bb (female parent), and the offspring genotype ab indicates that the sample is heterozygous in the coding type of the marker, wherein one genotype is from the male parent and one genotype is from the female parent.

(8) Dividing the SLAF labels into 26 linkage groups through positioning with a reference genome, calculating LOD values among high-quality molecular labels, performing linkage grouping through the LOD values, constructing a genetic map of each linkage group by adopting HighMap software, and obtaining the genetic map with the total map distance of 5197.17cM through correction.

(9) By adopting software QTL IciMapping V4.0(http:// www.isbreeding.net/software /) and software WinQTLCart 2.5, the multi-environment QTL positioning analysis of the fiber strength character is carried out by using phenotype data (shown in a table) of fiber strength characters of 11 environments (2007 Anyang, 2008 Anyang, 2009 Anxue, 2009 Xinjiang Aksu, 2010 Anyang, 2010 Gaiyi, 2010 Zheng State, 2013 Anyang) to obtain 5 QTLs related to strength, wherein more than 4 of the three environments are stable, and three of the three QTLs are positioned to a monomer type, namely qFS-chr25-2, qFS-chr25-3 and qFS-chr 25-5.

(10) And (3) utilizing software HaploView to perform haplotype positioning on the located QTL intervals qFS-chr25-2, qFS-chr25-3 and qFS-chr25-5 to obtain a haplotype, and determining the variation condition of the haplotype in the parent and the population.

(NN: sequencing data deletion)

Therefore, when the genotype of the haplotype is A, the average value of the fiber strength of the tested sample is more than or equal to 33.0cN/tex, and the fiber strength belongs to a high-strength material; the fiber strength of the samples tested ranged from 27.0cN/tex or less for the mean fiber strength when the haplotype was genotype B, to a low strength material, and approximately from 27.0 to 33.0cN/tex when the genotype was identified as C. The genotype of the haplotype can be quickly identified by means of analysis and statistics, so that the required variety can be screened better, the method is flexible and quick, the breeding period is shortened, and the breeding efficiency is improved.

Example 2:

(1) and (3) performing simplified genome re-sequencing on 306 materials by utilizing the natural cotton colony constructed by the subject, and finally selecting the cotton genome as a reference genome according to information such as the size of the cotton genome, GC content and the like. The specific information is as follows: sequencing species information: cotton, actual genome size about 2.95G, GC content 34.11%; reference species information: the cotton genome. The size of the assembled genome was 2.55G, and the GC content was 34.11%

(2) And (3) carrying out treatment of adding A to the 3' end of the obtained enzyme digestion fragment (SLAF label), connecting a Dual-index sequencing joint, carrying out PCR amplification, purifying, mixing samples, cutting gel, selecting a target fragment, and carrying out sequencing by using IlluminaHiSeq after the library quality is qualified.

(3) Finally, determining that the digestion is carried out by HaeIII + SspI-HF, the digestion efficiency is 95.70%, the sequence of the fragment length at 364-414 is defined as the SLAF label, and sequencing to obtain 1,970.53M reads. By bioinformatic analysis, 1,577,828 SLAF tags were obtained with an average sequencing depth of 13.19 x.

(4) And (3) comparing sequencing reads to a reference genome by using BWA, developing SNP by using two methods of GATK and samtools, and obtaining 3,464,561 group SNPs by using SNP marker intersection obtained by the two methods as a final reliable SNP marker data set. A total of 133,846 high-identity population SNPs were obtained by filtration with a completeness >0.6, MAF >0.05, with 889,412 polymorphic SLAF tags, yielding 3,464,561 population SNPs.

(5) Using the SNP marker of BLOCK2 mentioned above as an example, statistics were made with the SNP results of example 1, and 10 to 20 portions of the material were randomly sampled. The results are shown in the following table:

(6) the field data of the 20 parts of materials and the fiber strength values in the fiber inspection results are checked, three environments are selected, namely 2016 angyang, 2015 angyang and 2015 alaer respectively, and the statistical results are shown in the following table:

in conclusion, when the genotype of the haplotype is A, the average fiber strength value of the tested sample is more than or equal to 33.0cN/tex, and the sample belongs to a high-strength material; when the genotype of the haplotype is B, the fiber strength mean value range of the tested sample is less than or equal to 27.0cN/tex and belongs to low-strength material, and when the genotype is identified as C, the fiber strength range is 27.0-33.0cN/tex, therefore, the marker can be used for quickly determining whether the tested sample is high-strength material, the detection method is quick and effective, the breeding period can be greatly shortened, and the breeding efficiency is improved.

Claims

1. The haplotype is located in 3 QTLs related to the fiber strength of the upland cotton No. 25 chromosome, wherein the haplotype is qFS-chr25-2, qFS-chr25-3 and qFS-chr25-5, the number of the haplotypes in each QTL is 2, and the haplotypes are numbered as BLOCK 1-6;

these 3 QTLs were stably detectable in multiple environments (. gtoreq.3) and the polymorphic sites of the haplotype are shown in the following table:

and the genotype polymorphism sites corresponding to each haplotype are the following a), b) and C) in the following order, wherein the corresponding genotypes are A, B and C, which are shown in the following table:

wherein, when the genotype is A, the fiber strength mean value of the cotton is more than or equal to 33.0cN/tex, and the cotton belongs to a high-strength material; when the genotype of the haplotype is B, the fiber strength mean value range of the cotton is less than or equal to 27.0cN/tex, and the fiber strength mean value range belongs to low-strength material, and when the genotype is identified as C, the fiber strength range of the cotton is approximately 27.0-33.0 cN/tex.

2. A method for detecting the strength of upland cotton fibers is characterized by comprising the following steps: the haplotype genotype and SLAF sequencing result of claim 1, wherein the fiber strength of the cotton sample can be rapidly detected, and the fiber strength of the cotton sample is high when the genotype of the cotton sample is A, low fiber strength material when the genotype of the cotton sample is B, and medium fiber strength material in other cases, or the fiber strength of the cotton sample is A, B, C when the genotype of the cotton sample is determined to be B, wherein the fiber strength of the sample with genotype A is higher than that of the sample with genotype B/C, and the fiber strength of the sample with genotype C is higher than that of the sample with genotype B when the genotype of the cotton sample is determined to be C.

3. The method of claim 2, wherein: when the genotype of the haplotype is A, the average value of the fiber strength of the tested sample is more than or equal to 33.0cN/tex, and the fiber strength belongs to a high-strength material; when the genotype of the haplotype is B, the fiber strength of the tested sample is in the average range of less than or equal to 27.0cN/tex, and belongs to a low-strength material, and when the genotype is identified as C, the fiber strength is approximately in the range of 27.0-33.0 cN/tex.