Rough Mapping Of Silkorm Mutant Flossy

Silkworm(bombyx mori), which is one of the most important economic insects, is also a good model species of genetic research. Many mutant lines of silkworm, which recorded more than 400, are preserved during the long-term evolution progresses. Classical genetic research of silkworm mutant genes, which experienced a long and fruitful period, is an important basis of sericulture.with the achievement of molecular linkage map, especially the construction of silkworm genome mapping, large-scale positional cloning of mutant genes is not only become possible but also one of the most important topics in international sericulture research.Silkworm cocoon mutant Flossy (Fl,system No.02-200)which locates located in the silkworm linkage groups 7 to 32.1,was reported by Banno et al, It a natural mutant strain which named after its wide,floating,loose cocoon. Flossy is one of few mutants related to cocoon formation and cocooning behavior, thus cloning and identification of Flossy provide great value in studying in silk production mechanism.In this study, BC1 population was built by using C108 and Fl as parents, then SNP markers were used in positonal cloning of Fl, and investigation of silk cocoon quality among different strains was performed.the results are as follows:1.Low-density linkage mapping of Fl18 markers could be used after selection and identification of the SNP markers which had already reported in the 7th chromosome.These including two fragment length polymphism markers.By using seven of these markers and 88 BC1 individuals,we constructed an low-density linkage map thought linkage analysis. The result of the map shows that the Fl-linked region was narrow to the range which is 2Mb in chromosome 7 between two SNP markers,3 and 8.The genetic distance between the two markers is 6.3cM.Meanwhile the map also indicates that 7th chromosome of the physic map is inversion to 7th linkage group in the classical genetic linkage map. 2.High-density linkage mapping of Fl79 exchanged individuals was acquired from 1248 BC1 individuals by using the marker 3 and 8 which used in the low density linkage map for screen.At the same time newly markers were developed from the data of 9 X genome sequence and map of genetic polymorphism.Using these markers and exchanged individuals to construction the high-density map of Fl.The gene of Fl is narrowed in the 470.44kb region in the nscaf2983 of 7th chromosome between the marker 31 and 69.3.Identification of candidate genes within positioning region.There exsist two annotated gene BGIBMGA010084 and BGIBMGA010084 in positioned region,More ever, no reported homology sequences was finded in all databases by BLAST. Prediction of domain shows that BGIBMGA010083 contains no 1 domain,and BGIBMGA010084 has a domain which has no functional annotation.So the two genes are newly found.The microarray of silkworm's tissue indicates that the two genes express highly in the genital gland and little in other tissue.RT-PCR result is same as the microarray date.Through analysis with the coding region of dazao,C108 and Fl,the result shows that the coding region of BGIBMGA010083 has three SNP site in the Fl:534(T→C),1158(G→A),1251(A→G),However,they are falled inte ynonymous mutation. The coding region of BGIBMGA010084 in the Fl is same as the C108,but has five SNPs compared with dazao:78(C→T),177(T→C),192(C→G),213(G→A),229(T→G)and has one base inserted between the 244 and 245.That causes two amino acids change and the protein has 112 amino acids which is even longer as before.4.Silk cocoon traits survey of Fl and other strainsThrough morphological observation of the Fl strain during the different period, the characteristics of all is same as the control except for the cocoon of Fl which is obviously flossy and contains more layer. Investigation result about characteristics of cocoon shell percentage and sericin rate of cocoons in xiafang,Fl p50 show that the cocoon shell percentage and sericin rate of cocoons of Fl are all between the xiafang and dazao. through microscopic observation after removal of sericin result shows there no differentce among the three strain in the silk.according to above analysis we specculate that the phynotype of Fl may be caused by the low adhesiveness between the cocoon layer or no adhesive layer formated between them.