Identification Of Mutant Gene And Quantitative Trait Locus (QTL) Mapping Of Genetic Modifiers Of Plcdl In Scant Hair Mouse Snthr^-1Bao

The scant hair mouse snthr-1Bao raised in our laboratory was a recessive mutant against DBA/2J background. The mutant gene had been located and narrowed down in a region between 117763kb and 119129kb from the centromere on the chromosome 9. What was introduced in this thesis included identification of mutant gene of scant hair mouse snthr-1Bao and mapping the modifers of mutant gene, Plcd1, through quantitative trait locus (QTL) strategy. Our jobs were divided into the following two parts:1. Identification of mutant gene of scant hair mouse snthr-1BaoOn the basis of the preliminary work that the mutant gene of Snthr-1Bao had been narrowed down to 1.3Mb region on the chromosome 9, Plcd1 gene was considered as the primary candidate gene after querying the Mouse Genome Resources database and reading relevant literatures. DBA/2J mice were designed as control group. Firstly, Plcd1 gene was identified at the level of mRNA: Primers was designed for the cDNA (2270bp) of Plcd1 gene and three fragments covering the whole length of Plcd1 cDNA respectively. Consequently, specimens of the DBA/2J mice were all amplified successfully, but the templates from snthr-1Bao mice were all amplified failfully. Secondly, Plcd1 gene was identified at level of DNA: 10 pairs of primers were designed to cover 15 exons and in-between introns of Plcd1 gene. The templates of DBA/2J mice were successfully amplified. Except exon 1 to 3, exon 4-15 of snthr-1Bao mouse were failed to be amplified. Considering the possibility of genomic deletin in the snthr-1Bao mouse,other nine pairs of primers were re-designed aimming at the genome region after exon 3 and exon 3 of the Plcd1 gene, covering the corresponding genomic region of the 3rd-15th exons and in-between introns of Plcd1 gene and the 4th-19th exon of vill gene. After adjacent primer pairs were used to run PCR amplification one by one, almost all of genomic DNA specimens from DBA/2J mice were successfully amplified. However, only the thrid exon of the Plcd1 gene and the area around the tenth exon of the vill gene from scant hair mouse snthr-1Bao were successfully amplified. Pre- and post-experimental results reminded us that the mutation should be considered as a big loss of local genome, so we matched the forward primer for the third exon of Plcd1 and the reverse primer for near the tenth exon of Vill to PCR amplify, then a clear single band around 980bp appeared in the genomic DNA samples of the snthr-1Bao mouse. After comparing genomic sequencing results of the snthr-1Bao with the mouse genomic data base, we found a big missing region in 14883bp length between 118972407bp and 118987290bp from the centromere on the chromosome 9 of the snthr-1Bao mouse, which contained the 4th-15th exons of Plcd1 gene and the 11th-19th exons of Vill gene. This missing region leads to the loss of PLCD1 and VILL protein function, and the lack of PLCD1 is most likely to be the essential cause of the hypotrichosis in snthr-1Bao mouse.2. Mapping of genetic modifiers of Plcd1 in scant hair mouse snthr-1BaoIn the process of mapping the mutant gene, a (snthr-1Bao×C57BL/6J)F1 intercross experiment was conducted, then it showed that there was a gradual change from more to less in the hair density of F2 mutant mice, which showed the typical quantitative trait. In this study, all F2 mutant mice (n=507) were raised and classified into three groups (more, intermediate and less) according to the density of hair. After extracting genomic DNA of each mouse, we chose 96 microsatellite markers to scan the genome for the QTLs acting as regulating roles of hair density. Through analysis of genotype data, the R/QTL software showed that D2Mit249,D5Mit409,D7Mit126 and D15Mit171 appeared with QTL characteristics, the LOD value of chromosome 15 was more than 7, and P value in all sites was less then 0.01, which meant a notable statistical significance. On effection of each point, D2Mit249 and D5Mit409 in the B/B-type lead to hairiness, D/D-type less hair, and the effection of D2Mit249 were stronger than D5Mit409's; D7Mit126 and D15Mit171 in the D/D-type lead to more hair, B/B-type less hair, and the two points both had strong effections. However, all four sites of QTL had no mutual antagonism or synergy. 95% confidence interval was only 12cM (42.7-54.7cM) on chromosome 15, which provided the base for furtherly searching candidated genes. This experiment firstly located 4 quantitative trait loci related to sparse hair. At the same time, the research of the interaction model by joining Plcd1 gene and regulate genes together will help to understand the molecular mechanism of hair development.