Genome-wide Identification Of Gossypium Raimondii Dnase I Hypersensitive Sites

As a major economic crop and oil crop,cotton has always occupied an important position in agricultural production.Among them,Gossypium raimondii in wild cotton belongs to the diploid cotton species,and the smallest genome in cotton.It also has salinity,insect,drought,verticillium wilt resistance and other characteristics.The genome-wide sequencing of Gossypium raimondii has been completed,which laying the further foundation for the study of the cotton genome and the excavation of cotton’s excellent germplasm resources.The interaction between cis-regulatory elements and trans-regulatory factors controls the expression and regulation of eukaryotic genes.Usually the chromatin at the position of the cis-regulatory elements is highly sensitive to DNase I,which is the DNase I hypersensitive site referred DHSs.Studies have found that DHSs almost include all of the cis-regulatory elements,such as enhancers,repressors,insulators,and the major functional sites in the promoter.With the development of high-throughput sequencing,identification of genome-wide DHSs can be achieved through DNase I digestion and DNase-seq sequencing.This is the great significance for us to explore the specific transcription factor binding sites and construct gene expression regulation mechanisms.In this paper,we based on the existing identification methods for high sensitive sites of DNase I in rice and Arabidopsis thaliana,the optimization of DHSs constrnction methed suitable for Gossypium raimondii was completed.The young leaves and callus of Gossypium raimondii were used as materials.DHSs library in young leaves and callus of Gossypium raimondii was successfully constructed,and we sequencing of the DHSs library and correlation analysis at last.The results as follows:(1)Optimization of the library construction methods for young leaves and callus DHSs of Gossypium raimondii.For Gossypium raimondii cells contains more secondary metabolites,the cell nucleus is easily oxidized,and the nucleus extracted after addition of 2%(m/v)PVP40 in the nucleus isolation buffer is relatively pure.In addition,in order to better remove impurities such as chloroplasts in the nucleus and add0.4%(v/v)of Triton X-100 to the nuclear washing buffer,the effect of impurities such as chloroplasts can be well removed without causing degradation of the nucleus.Due to the large size and complex structure of the cotton genome,the conditions for the attachment of Adaptor 1 and Adaptor 2 were investigated during the construction of the DHSs library,and it was found that the optimal ligation ratio between Adaptor 1 and high molecular weight DNA was 1:8.Adaptor 2 has the highest effciency at 25℃ when connected.After the successful construction of the DHSs library,the library was cloned and tested.After the sequencing of the bacterial cells,it was found that about 20 bp restriction sites were obtained,which prove that the correct DHSs library has been obtained.(2)Bioinformatics analysis of young leaves and callus DHSs of Gossypium raimondii.after high-throughput sequencing,the linker sequence was removed using the bioperl software package,and read by the bowite software.Finally,DHSs were identified using F-seq.72,466,37,898 and 78,461,102,056 DHSs were obtained in the leaves and callus of Gossypium raimondii.A preliminary analysis of the approximate distribution of these DHSs on the chromosomes revealed that the DHSs in the youngleaves and callus of Gossypium raimondii were mainly concentrated at both ends of the chromosome.By further analyzing the area and proportion of these sites,it was found that DHSs were mainly distributed in the following regions: protein coding region(21%-29%),intron(3%-5%),transcription start site upstream200 bp(7%-12%),200-1000 bp upstream of the transcription initiation site(7%-9%),200 bp downstream of transcription termination site(2%-3%),200-1000 bp downstream of the transcription termination site(5%-6%),5’ UTR(12%-17%)and 3’ UTR(4%-8%),and 57% of specific DHSs exist in young leaves and callus.Finally,it was found that the abundance of DHSs in Gossypium raimondii was mainly within 500 bp downstream of the transcription initiation site,and the distribution of DHSs was mainly within 2.5 kb downstream of the transcription termination site.Although more DHSs were detected in the callus,the approximate distribution of DHSs in the young leaves and callus of Gossypium raimondii was the same.This study identified DHSs in young leaves and callus of Gossypium raimondii,providing support for subsequent studies on histone modifications.