Mapping Analysis Of The New Mutant Pale Red Egg(re~p)in The Silkworm,Bombyx Mori

The Silkworm,Bombyx mori is a complete metamorphosis insect and diapaused in egg stage,so the egg stage is the first stage of the silkworm life cycle,which shows that the study of eggs has a very special value.During the silkworm rearing,owing to the natural and human factors have an impact on it,many egg mutants have been produced on the basis of the original egg color,because of its wide variety of eggs and easy to detect and identify,it is an excellent material for the study of genetics.We had found a novel egg color mutant the pale red egg(re~p)during the breeding of silkworm.The newly laid eggs are yellowish white,then color in the following 40 hours in succession,and finally turn into pale red.Genetic analysis showed that the re~p mutant was controlled by a single recessive gene,and might be alleles with the re gene.Based on the genetic analysis of the re~p,we mapped the re~p gene and constructed the link map by the SSR Polymorphic marker and map-based cloning.The transcriptional expression analysis of the candidate genes in the locus was performed byRT-PCR?qRT-PCR and C lone sequencing,in order to find the mutation gene.The main results were as follows:.1?Classical genetic analysis of mutant(re~p)The reciprocal cross between the re~p mutant and wild type(p50)shows thatthe egg color of F1 offspring were normal.The egg color of F2 offspring appeared the separation phenomenon.There were two phenotypes of normal eggs and pale red eggs.The separation ratio was 3: 1;the backcross offspring between the wild type and F 1 were normal,while the backcross offspring between the re~p and F1 showed both phenotypes of normal and pale red eggs,the separation ratio was 1: 1,what provedthatmutant was controlled by a single autosomal recessive gene.The eggs of F1 offspring are red while the male re~p mutant hybridize with the female re mutant,and conversely the eggs of F1 offspring are pale red while the male re mutant hybridize with the female re~p mutant.The same situation appears for the eggs of F2 offspring with no phenotypic segregation.The egg color of F3,the self-cross offspring of F2 whatever reciprocal cross,appears new colors of transitional color between red and pale red except red and pale red.But the three kinds of eggs have no obvious separation.The results above indicate that the control genes of the two mutants,re mutant and re~p mutant,are alleles.For the structure proteins of egg membrane come from the matrilateral,they e xhibit a character of maternal inheritance,which cal ed as pseudo maternal inheritance.2?Linkage analysis and positional cloning of the re~p geneThe population of genetic analysis including P1?P2?F1?F2?BC1F and BC1 M were conducted.Polymorphic markers on 28 linkage groups of silkworm were found by parental P1,F1 and P2,then 11 normal and 11 re~p BC1F progenies were used to determine the linkage group on which the re~p gene was located.The results indicated that the re~p gene was located on the5 t h linkage group.Using 1087 F2 individuals and 68 re~p BC1 M individuals to performed fine mapping.The results showed that the re~p gene was located between two polymorphic markers S2674-N53 and S2674-N21.The distance between the two markers was 0.39 cM and the physical distance was about 370 kb.14 candidate genes within the particular locus.3?Analysis of candidate genesOn the basis of the mapping analysis,the expression profiles of 14 candidate genes in C1(H)and re~p at different spawning period was analyzed by qRT-PCR.The ORF sequences of some candidate genes(BGIBMGA003693? BGIBMGA003501?BGIBMGA003500?BGIBMGA003694 ?BGIBMGA003695?BGIBMGA003499)and the 3 'and 5' ends of BGIBMGA003694 and BGIBMGA003695 were cloned and sequenced.While there were no difference between in C1(H)and re~p.4?Structure analysis of MFS GeneMFS gene which coding major facilitator superfamilyand that is responsible for the red egg mutation of the silkworm.The re phenotypic is incomplete dominance opposite to the re~p phenotype through genetic analysis,which is closely related and the MFS gene is located in the localization range.So the CDS sequence of MFS gene was cloned and sequenced,the cDN A of wild type C1(H)and re~p as template.We found that a section of 59 bp sequence on MFS gene the 6 exon in re~p mutant re~placed by another section of 14 bp sequence and other sequences are not changed when compared with C1(H).When analyzing re~placed sequence,this sequence only affected the number of the re~place part of amino acidsand the other amino acid sequence close to the re~placement site has not affected,which do not change the transmembrane structure of encoding proteins.The qRT-PCR was applied to testthe expressionof MFS gene in different tissues and different egg stage among wild type C1(H),re~p and re.The results showed that the gene have a higher expression in C1(H)and re~p's head,epidermis,fat body,posterior silkgland,testis,expressed lower in trachea and ovarian.C1(H)and re~p had no significant difference,but expressed lower in re all tissues and almost no expression.The q RT-PCR of MFS gene at different egg stages also show that expression of MFS gene have no significant difference in C1(H)and re~p,and obviously very low in re mutant,which demonstrate that MFS gene is the key gene to the cause of re mutation phenotype,whether it is important for re~p mutant remains to be further study.In this study,we determined the region of the re~p mutant,where the candidate genes wre analyzed.In order to find the re~p gene,we should deeply study the candidate genes.