| Rodent species of musculus are widely used in all kinds of biological research,which contains a variety of subspecies and distributed all around the word.New century,Mus musculus become a key research field of genetics and one of the most important animal model,based on the accumulation of inbred strains and genetically engineered mice.In the pos-genomic era,the study of mouse genetics from single gene research into the complex traits.Under the accumulation of mouse resources,although many quantitative trait locus(QTLs)have been mapped in the mouse genome,only small fraction of these genes have been identified.The study shows major reasons of the slow development of QTL fine mapping and functional gene cloning: 1)The disadvantages of traditional QTL mapping method are time and energy consuming,low resolution.The QTL intervals are often too large to identify causal variants(10~20c M),then would consume a few years to construct the congenic strains(accumulation of recombination events)to fine mapping QTL(1~2c M).2)The difficulties of QTL fine mapping: the existing experimental mice have a single origin and a small number of allelic genes;the lack of genetic diversity and high degree of linkage disequilibrium;only 100 years of history,and the cumulation of recombination events is low,causing the extremely difficulty of QTL fine mapping and complex trait related gene positioning cloning in classical inbred strains.In order to overcome the bottlenecks above for generating the strains which have increased mapping precision and greater genetic diversity than classical inbred strains.In this dissersion the wild mice in china has studied firstly on the following three aspects:Part one: the construction of the population of chromosome 1 substitution strains(PCSSs)from wide mouse.China is rich in resources in wild house mouse(Mus musculus),which includes two subspecies about M.m.musculus and M.m.castaneus,and have a broad subspecies hybrid zone in the Yangtze River Basin.The wild mouse is desirable to improve the performance of QTL mapping studies,because of greater genetic diversity and increased mapping precision.Nowadays,more than 1000 QTLs have been mapped on chromosome 1 in mouse,but only a small fraction of genes have been identified.In order to promote the fine mapping of these QTLs on chromosome 1,and positional cloning the functional genes.Add new members to the existing laboratory mouse for genetic study.In this dissersion,based on the 3 panels of PCR-LDR genotyping system,rapid screening the mouse that take the unrecombination events on chromosome 1 for backcross,to accelerate the construction process of PCSSs.We establish the copy number variation(CNV)detection methods based on the competitive polymerase chain reaction(c PCR),cooperative monitoring the genetic stability of chromosome one with PCR-LDR.Based on the two step self-cross method to accelerate the purification process of PCSS,and overcomes the difficulties in the large-scale feeding and genotyping work.Using whole genome microarray scanning technology,real-time monitoring the genomic background purity of chromosome engineering mice.With the increasing of backcross generation,the mouse homozygosity of genomic background is gradually increased.Finally we establish the 25 chromosome 1 substitution strains.Part two: The genetic quality evaluation of 20 chromosome 1 substitution strains.Since the turn of the century the complete genome sequence of just one mouse strain,C57BL/6J,has been available.Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed.The last decade,DNA sequencing has undergone a revolution through the development of much higher throughput sequencing technologies resulting in a significant reduction in the cost per base pair,and the Mouse Genome Project was initiated with the goal of using much higher throughput next-generation sequencing technologies to catalogue molecular variation in the 36 inbred strains.The genetic diversity of these strains,through in-depth analysis the SNPs,In Dels and structure variations(SVs),which further promoted the rapid development of mouse genetics,and evolutionary history of mice and adaptability to the environment.Based on the established PCSS,in this dissersion 1)employ the Illumina Hiseq 2500 and Illumina Hiseq X Ten platform to sequence the 20 PCSS strains(the average depth > 30×).Under the quality control,which filtering the low quality data and adaptor,leave over the adequate,effective,high quality sequencing data are all suited for subsequent biostatistics analysis.2)Then the Speedseq software is used to excavate the genetic diversity of the 20 PCSS strains,and find 4.1×107 genetic variants.The B6-Chr1 KM have the least genetic marker(7.62×105),but 19 chromosome 1 from wild mice contain substantial genetic diversity,which is far more than the common experimental mice.The average density of SNP on chromosome 1 from wild mice is 7.06 SNP/Kb,which is much higher than the laboratory mice(1.25 SNP/Kb).Using the Effect Predictor Variant software we annotate a total of 2684 genes.3)According to the SNP data of 20 strains: the homozygosity on chromosome 1 of 16 strains are more than 98%,but the B6-Chr1 HZ,B6-Chr1 TZ,B6-Chr1 TZ and B6-Chr1 TZ respectively is 97.19%,91.90%,80.00% and 75.96%,and all these strains are suitable for the study of complex traits.4)On the genomic background of the 20 strains,traces of genetic information come from the donor strains(named "Islands")and the lowest heterozygosity is 0.27%,the B6-Chr1 YX is 6.46%.5).We analysis the genetic structure of 20 PCSS stains,which shows that Mus musculus mainly distributed two subspecies M.m.castaneus and M.m.musculus in China.With the increase of human activities and frequent flow,there is mutual penetration between the north and south Mus musculus,which formed a larger hybrid zone.The whole genome DNA sequencing data and preliminary analysis results show a useful complement to the mouse genetics studies.Filled the blank of wild mice DNA sequence information in china,and laid a solid foundation for QTL fine mapping and positional cloning.Part three: The phenotype detection and statistics analysis of PCSS strains.The mouse is an important mammalian model of human diseases,and widely used in physiological and pathological study.The genetic mapping studies in the mouse and other model organisms are used to search for genes underlying complex phenotypes.Phenotyping is the important content of genetic studies in mice.In the post-genome era,single and simple phenotypic detection is unsuitable for the study of complex traits,so carry out extensive phenotypic detection of complex traits has been widely concerned.In this dissersion,we access the B6-Chr1 SJ,B6-Chr1ZZ2,B6-Chr1 ZC,B6-Chr1 TW,B6-Chr1 SMX,B6-Chr1ZZ1,B6-Chr1 CM,B6-Chr1 HZ and B6-Chr1 KM strains and study the growth,physiological and biochemical phenotype,meanwhile establish a databank.Based on the results of statistical analysis,we identify the significant differences between the PCSS strains and B6.For example,The B6-Chr1KM(P=0.000),B6-Chr1ZZ1(P=0.014)and B6-Chr1TW(P=0.031)weight have significant differences with B6 female,but there is no significant difference with other strains.The Vaginal opening(VO)time of B6-Chr1ZZ1,B6-Chr1 TW,B6-Chr1 SJ,B6-Chr1 SMX and B6-Chr1ZZ2 are significant higher than B6(P?0.05).The Wrapping off time of B6-Chr1CM?B6-Chr1SJ?B6-Chr1 SM and B6-Chr1ZZ2 are significant higher than B6(P?0.05).The ALP of B6-Chr1 HZ female is significant higher than B6(P=0.001),and the B6-Chr1 KM male is significant lower than B6(P=0.031).The ALT of B6-Chr1 CM female is significant higher than B6(P=0.011).The TB of B6-Chr1CM(P=0.000),B6-Chr1SMX(P=0.000),B6-Chr1HZ(P=0.041)male are significant higher than B6.The TG of B6-Chr1 SMX female is significant higher than B6(P=0.044)The TC of B6-Chr1 TW male is significant higher than B6(P=0.005)and so on.By the induction of phenotypic data and input phenotype database,that lay a good foundation for study the phenotype of PCSS and QTL mining.In this dissersion,under the view of the difficulties in study mouse complex traits,we established the PCSS from wild mice,achieved the preliminary analysis of whole genome information and genetic diversity about the 20 strains,and further collected the basic and blood biochemical phenotype data of 9 strains,then established the basic database.After all these data lay a solid foundation for subsequent QTL fine mapping and positioning cloning of complex traits,and provide high-quality genetic resources for mouse genetic studies. |
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