Mutation Rate And Spectrum Of The Silkworm In Normal And Stress Conditions

Mutation rate is an important parameter of evolutionary genetics and also a necessary parameter for calculating the time of species differentiation.The classic theory,"molecular clock",holds that the mutation rate of the genome is constant in different species and environments,and a recent study in insects have also shown that the mutation rate of the insect genome is almost the same,but this inference is based on the known mutation rates of the four insect species（Drosophila melanogaster,Heliconius melpomene,Apis mellifera,and Bumblebe）.Whether the insect mutation rates are constant or not remains to be estimated for more insect mutation rates.The silkworm（Bombyx mori）is an important economic insect.The silkworm industry has a long history in China and is a representative of Chinese civilization.The ancient Silk Road derived from it has played an important role in economic and cultural exchanges between China and the West.Therefore,the history of silkworm domestication has become a long-term focus of silkworm geneticists.A large number of existing studies indicate that the domestic silkworm domesticated from Chinese wild silkworm,Bombyx manderina,at least 4100 years ago,but these estimates are based on the genomic mutation rate of the Drosophila or the Heliconius.Whether the mutation rate is consistent remains to be studied.In addition,the classical"molecular clock"hypothesis holds that the genomic mutation rate in different species and different environments is almost constant,but whether different environments affect the genomic mutation rate in multicellular organisms is still unknown.Therefore,in this study,silkworm was used as the research object,and the parents-offspring illumina sequencing method was used to study the mutations of silkworm in normal environment and four stress conditions.The main results of this study were showed as followed:1.Spontaneous mutation rate of the silkworm genome is higher than that of the other four insect speciesIn this study,genome sequencing and SNP identification of two silkworm parents and their 30 offspring were performed,and a total of 106 new spontaneous mutations were identified in the 30 offspring.After PCR detection and artificial simulated mutations to remove the false positive rate and false negative rate,the spontaneous mutation rate of the silkworm genome under normal conditions was0.41×10^-8(95%CI=0.34×10^-8～0.49×10^-8),which is higher（differ by 1.5-fold）than other four previously investigated insects.It was also found that the mutation rate of insect genome was significantly positively correlated with the genome size.Based on the spontaneous mutation rate of the silkworm obtained from this study,the silkworm domestication history was re-estimated.It was found that the start time of the silkworm domestication can be traced back to 9700 years ago.2.The mutation rate of the silkworm genome changes with environmental changesThe fertilized eggs of silkworm were subjected to low temperature（0℃,10 hours）,high temperature（32℃,10 hours）,ultraviolet ray（7800μW/cm2,3 minutes）and gamma ray（0.35Gy/min,7 minutes）.After paternity sequencing and SNP identification,it was found that the mutation rates in the low temperature,high temperature,ultraviolet irradiation,and gamma radiation treatment groups were 0.54×10^-8,0.56×10^-8,1.13×10^-8,2.25×10^-8,respectively.The mutation rate of the silkworm in four stress conditions was higher than that in the normal environment,and the highest rate was 5.5times.Therefore,the mutation rate of the genome in different environments is not constant,which implies that the related evolutionary model based on the constant mutation rate may need to be revised.3.Mutation rates in gene regions are relatively constant under mild stress conditionsAfter comparing the mutation rates of different genomic regions in different environments,we found that the mutation rates of the gene region in the normal and mild str ess conditions are 1.50-1.55 times that of the normal group.However,the mutation rates of the gene region in the normal and mild stress conditions are onl y 1.07 times that of the normal group,so the mutation rate in the gene region is relativel y constant.With the development of whole genome sequencing technology and the reduction of the costs,more and more studies use whole genome sequences for evolutionary anal ysis.The results of this study imply that it may be more accurate to use the single-base mutation rate of gene region s as molecular clock to estimate species differentiation time or perform phylogenetic anal ysis.4.G→A/C→T mutation frequency is significantly higher than other mutation typesAfter counting the mutation frequencies of different single base mutation types in different environments,it was found that the frequency of G→A/C→T mutations,which were higher than 31%,was significantly higher than other mutation types.Further analysis and comparison of the mutation frequencies of different base mutation types of other species It was found that the frequency of G→A/C→T mutations in other species was generally higher than other mutation types,but the cause of this phenomenon remains to be studied.In conclusion,this study revealed the spontaneous mutation rate of the silkworm genome,which has important reference value for the domestication of silkworm and the historical deduction of the ancient Silk Road.And our research results show that there are significant differences in the genome mutation rate in different species and different environments,and the mutation rate in the gene region is relatively constant,which has important guidance for the improvement of a large number of evolutionary models based on the"molecular clock"hypothesis and phylogenetic analysis significance.