QTL Mapping And Selection Of Candidate Genes Related With Blood Lipid Using "Chromosome 1 Substitution Lines Derived From Wild Mice"

Dyslipidemia is a common lipid metabolism disease,mainly manifested as the rise of plasma cholesterol and/or triglyceride,which is one of the important risk factors of cardiovascular disease.In recent years,the incidence of dyslipidemia in our country has been increasing year by year,which seriously threatens people’s health.Dyslipidemia is a complex trait disease,which is affected both by genetic factors and environmental factors at the individual level.Therefore,the identification of lipid regulation candidate genes is a worthwhile study.Because of its similarity with humans in the aspects of genetics,physiology and biochemistry,mice have a long history of genetic research as model organism and are widely used in the study of the complex trait such as dyslipidemia.In this study,F2 population was constructed using chromosome 1 substitution lines derived from wild mice B6-ChrlHZ with high blood lipid levels and recipent lines C57BL/6J(B6),which contains 215 male individuals.After F2 males were fed for 20 weeks,the blood of them was taken with fasting for biochemical determination of blood.Then the concentration of cholesterol(CHOL),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)were measured.At the same time,tail of all male F2 individuals were cut and DNA was extracted.Then 16 single nucleotide polymorphisms(SNPs)evenly distributed on chromosome 1 are selected.Specific primers were designed according to the Hi-SNP typing strategy.The PCR products were purified by electrophoresis and sent to the next-generation sequencing platform to sequencing,sequencing data was converted to typing results by bioinformatics.Combined with the phenotypic data and typing data,three quantitative trait loci(QTLs)respectively corresponding to HDL-C,TG and LDL-C were identified by R/qtl using the method of linkage analysis and standard interval mapping.Among the three QTLs,LDL-C and TG related QTLs are significant with the interval sizes of 31.0 Mb and 147.1 Mb respectively,and HDL-C related QTL is suggestive QTL with the interval size of 172.5 Mb.Due to the large range of QTLs related to TG and HDL-C,they overlap with the related QTL interval identified by Hybrid Mouse Diversity Panel(HMDP),reducing the interval to 8.0 Mb and 26.0 Mb respectively.According to the re-sequencing data in the QTL interval,genetic variations were annotated by Ensembl Variant Effect Predictor(VEP)online,and candidate genes with functional mutations were further screened.In addition,the ampicillin liver RNA was extracted and sent to the RNA-seq sequencing platform to detect the expression level,and the same differentially expressed candidate genes were also screened in the QTL interval.Through these two methods,we screened the corresponding candidate genes.There are 83 genes with functional mutations in blood lipids related QTLs.Among them,34,13,and 40 are in HDL-C,TG and LDL-C related QTLs,respectively.We also identified 20 differentially expressed genes in which 9,3,and 8 are in HDL-C,TG and LDL-C related QTLs.Finally,BioGPS platform was used to search for genes highly expressed in the liver,and two candidate genes Marcl and Aoxl were further screened out.To sum up,F2 population was constructed using B6-B6-CHrlHZ and B6 in this experiment.On the basis of obtaining phenotypic data and typing data,three QTLs related to blood lipid were identified by linkage analysis.After that,we screened candidate genes with functional mutation and differentially expressed candidate genes in QTLs.This study validates the feasibility of mapping QTL by linkage analysis of chromosome 1 substitution lines derived from wild mice.At the same time,the selection of candidate genes also provides the basis for further functional verification,which is of great significance to the final treatment of dyslipidemia.