Upland cotton No. 4 chromosome and SNP molecular markers associated with fiber strength

[0022] The present invention will be further clarified through the detailed description of the specific embodiments below, but it is not a limitation to the present invention, but only an example is given.

[0023] (1) Recombinant inbred line F 6:8 Gain

[0024] For field planting and DNA extraction from 2007 to 2008, please refer to Patent Application Publication Number: CN 101613761A, Title of Invention: Patent application documents for SSR markers linked to the main genes of cotton fiber strength. In 2009, they were planted at the experimental station of Cotton Research Institute of Chinese Academy of Agricultural Sciences in Anyang, Henan, the Quzhou experimental station of China Agricultural University and the experimental station of Xinjiang Aksu Dejia Science and Technology Seed Industry Co., Ltd. 6:10 group. Anyang and Quzhou use single-row areas, with a row length of 5 meters and a row spacing of 0.8m and (0.8+0.5)m, respectively, with 20 plants per row; Xinjiang uses a 6-row area with a row length of 2 meters and 15 plants per row. In 2010, they were planted at the original seed field in Gaoyi, Hebei, the experimental station of the Chinese Academy of Agricultural Sciences in Anyang, Henan, and Zhengzhou, Henan. 6:11 For groups, Anyang and Zhengzhou adopt single-row areas with a row length of 5m and row spacing of 0.8m; Gaoyi adopts a single-row area with rows of 4m in length and (0.8+0.6)m wide and narrow rows. Each of the above pilots adopted an incomplete random block design, planting two replicates. Field sampling was carried out in mid-to-late September, flowers were harvested according to family, and about 12g fiber samples were taken to determine fiber quality.

[0025] (2) Extract the DNA of recombinant inbred line populations and parents. Refer to the literature for specific methods (Song Guoli, Improved CTAB Method for Rapid Extraction of Cotton DNA, Acta Cotton: 1998, 10(5) 273-275).

[0026] (3) Choose to use the cotton tetraploid genome sequence provided by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences as the reference genome for electronic restriction 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 cultivated Upland cotton( Gossypium hirsutum TM-1) provides insights into genome evolution. NatureBiotechnology, 2015, 33(5)), and finally choose HaeIII+SspI enzyme, the digestion rate is 98.61%, and a total of 495.48 Mreads are obtained, and the digestion fragment length is 364-414bp. Defined as SLAF label.

[0027] (4) According to the selected most suitable restriction enzyme digestion plan, the genomic DNA of each sample to be tested is subjected to the restriction enzyme digestion experiment, and the 3'end of the digested fragment (SLAF label) is treated with A, and connected to the Dual-index sequencing street. PCR amplification, purification, sample mixing, gel cutting to select the target fragments, library quality inspection after passing the IlluminaHiseqTM2500 for sequencing.

[0028] (5) Use Dual-index to identify the original data obtained by sequencing, and obtain the reads of each sample. By clustering between reads, SLAF tags are developed in parents and offspring.

[0029] (6) Through bioinformatics analysis, a total of 321,797 SLAF tags were obtained, of which 35,300 were polymorphic SLAF tags.

[0030] (7) Perform genotype coding for the polymorphic SLAF tag. The genotype coding rule is the 2 allelic coding rule commonly used in genetics. For example, the parental genotype of a marker is aa (male parent) and bb (female parent). The offspring genotype ab indicates that the coding type of the marker in the sample is heterozygous, in which one genotype is from the male parent and one genotype is from the female parent.

[0031] (8) In order to ensure the quality of the genetic map, the SLAF tag should be sequenced according to the parent’s depth of less than 10x, completeness less than 30%, and severe partial separation (p-value). <0.05), the parents are heterozygous, and the conditions of simultaneous comparison to two sets of genomes were filtered, and a total of 7958 SLAF tags were screened.

[0032] (9) Divide the SLAF tags into 26 linkage groups by positioning with the reference genome, calculate the LOD value between high-quality molecular tags, use the LOD value for linkage grouping, and use HighMap software to construct a genetic map for each linkage group. Through correction, a genetic map with a total map distance of 5197.17cM (such as figure 1 Shown). Among them, the HighMap software is independently developed by Beijing Biomark Biotechnology Company.

[0033] (10) Based on the sequencing data of SLAF, comparing BWA with the reference genomes of two diploid cottons, 44583 SNPs with polymorphisms between the parents were obtained. After quality filtering, 10440 were located on the map. SNP markers.

[0034] (11) Using software QTL IciMapping V4.0 (http://www.isbreeding.net/software/) and software WinQTLCart 2.5, through 11 environments (Anyang in 2007, Anyang in 2008, Linqing in 2008, Song in 2008 Zhou, Anyang in 2009, Qu Zhou in 2009, Aksu in 2009, Anyang in 2010, Gaoyi in 2010, Zhengzhou in 2010, Anyang in 2013) phenotypic data and genotypic data for fiber strength traits, and more fiber strength traits Environmental QTL mapping analysis showed that a total of 6 QTLs related to fiber strength were obtained, of which 4 were stable in multiple environments. The obtained QTLs were correlated with SNP markers, and finally SNPs that were clearly classified with fiber strength were screened. Marker (the position of QTL on the chromosome such as figure 2 Shown), where and qFS-chr04-2 The linked SNP markers are CRI-SNP-198739, CRI-SNP-198740, CRI-SNP-198741, CRI-SNP-198742; and qFS-chr04-3 The linked SNP markers are CRI-SNP-198743, CRI-SNP-198744, CRI-SNP-198745, CRI-SNP-198746, CRI-SNP-198747, CRI-SNP-198748, CRI-SNP-198749, CRI-SNP -198750, CRI-SNP-198751, CRI-SNP-198752, CRI-SNP-198753, CRI-SNP-198754, CRI-SNP-198755, CRI-SNP-198756, CRI-SNP-198757, CRI-SNP-198758 , CRI-SNP-198759, CRI-SNP-198760, CRI-SNP-198761, CRI-SNP-198762, CRI-SNP-198763, CRI-SNP-198764, CRI-SNP-198765, CRI-SNP-198766, CRI -SNP-198767, CRI-SNP-198768, CRI-SNP-198769, CRI-SNP-198770; and qFS-chr04-5 The linked SNP markers are CRI-SNP-198771 and CRI-SNP-198772.