Genetic Analysis And Positional Cloning Of The New Mutant Brown Quail-like (q-ι~b) In The Silkworm

As a kind of main patterns of biological groups, the genetic resources ofSilkworm (Bombyx mori) were abundant. That the accumulation of pigment in theskin formed many types of markings and provided a new material and researchdirection for scientific research. We had found a new novel body marking mutantbrown quail-like(q-lb) during silkworm breeding in Golden0223varieties. Comparedwith normal marking individuals, the color of its skin presented brown on theimmature stage of silkworm, so the study of the mutant to explore the methods ofpigment adjustment is of important theoretical and realistic significance.This articleconsists of the following parts:1、Classical genetic analysis of mutant q-lbThe new novel epidermal marking mutant and normal individuals were used asparent strains for the mapping panel and a single-pair cross between p50andq-lbmutant produced the F1offspring. The phenotypes of F1generation presentednormal type in reciprocal cross, and the separation ratio of normal type and q-lbmutantthat appeared in F2generation was3:1. The separation ratio of normal type andq-lbmutant that appeared in reciprocal cross was1:1,what proved thatmutant wascontrolled by a recessive gene q-lband non sex linked inheritance.The results of morphological marker tests in2th,3th,4th,5th,6th,7th,9th,12th,13th,14th,16th,18th,19th,21thand26thchromosome showed that the F1offspring were notseparated and there were no mutant appeared; the F2offspring were separated and itsseparation ratio was9:3:3:1except the7thchromosome.We used quail (q) as themorphological marker, andthe mutant brown quail-like(q-lb) was similar toquail (q),we can’t distinguish them. So there appeared two phenotypes including normalindividuals (112) and other phenotype (the mutant brown quail-like(q-lb) and quail (q)86).Their separation ratio was9:7. This phenomenon is similar to the interactionrelationship between non-allelic genes.themutant brown quail-like(q-lb)and quail(q)has the complementary relationship. In this quail (q) was similar with the mutantbrown quail-like(q-lb)on the phenotype, but they were non-allelic genes.All in all,theq-lbgene was not in these chromosomes.2、Linkage analysis and positional cloning of the q-lbgeneP1, F1and P2were used for selecting the polymorphic SSR markers, BC1Fprogeny were used for the linkage analysis, and275BC1M progeny which presented brown similar to quail on the immature stage of silkwormwere used for recombinationanalysis. This result of the linkage analysis indicated that theq-lbgene was located onthe8thchromosome. Based on this result,20polymorphic SSR markers which linkedwith theq-lbgene were developed. The result presented that a region of~230kbtightly linked to the q-lbgene between the markers S2828-30and S2828-37wasidentified. The q-lbgene linkage map of the silkworm was drawnusing Mapmaker3.0mapping software, resulting in q-lbgenetic linkage map with its genetic distancepresented to be17.7cM.3、Analysis of the candidate genesBased on the positional cloning, we found that the expression level of the18genes between the two the markers was the same using Semi-quantitative RT-PCRexcept BGIBMGA005383and BGIBMGA005418. We hadn’t designed the primersfor Semi-quantitative RT-PCR successly and the ORF of BGIBMGA005418was alsonot cloned successly. The functions of the two genes were related to the growth anddevelopment. For that reason, we need to further study the genesto find the genewhich is mutative and its mutation site.4、Differential expression of the epidermal proteins between Golden0223andq-lbmutant2-DE was used to investigate the differential expression of the epidermalproteins between Golden0223and q-lbmutant, and the result showed that there weretwo different proteins appeared. The Bmrunt protein encoded byBGIBMGA007789gene has two different spots. One of them was expressed inthenormal, but was lower expressedin theq-lbmutant, and the other was just thereverse. The Bmcryptochrome2protein encoded by BGIBMGA008906wasexpressed in the q-lbmutant, but was lower expressed in the normal.Then we clonedthe exon of the two genes respectively, and the results showed that there wereno mutation in them.To sum up, the two different proteins were not caused by the related genes in theskin of normal and q-lbmutant. The new novel epidermalmarking mutant washappened in the process of the development of silkworm, while the two differentproteins were related to the growth and development, so we forecasted that the mutanthad been related to the two different proteins.