Constructing High-resolution Genetic Variation Maps And Related Studies On Silkworm Nuclear Genomes And Silkworm Mitochondrial Genomes

Silkworm is a very important economic insect. Recently, a fully-assembled reference genome was published for silkworm that dramatically stimulated the researchers to focus on molecular biology and genetic research. Silkworm has been domesticated for more than 5000 years and some important biology traits had been changed in some aspects. Although a lot of previous studies had tried to clarify the origin history of silkworm, we still have no information on which gene category change altered the biology trait in the gene level. So contructing the silkworm genetic variation map will be of help to understand these biology transformations. Moreover, the map can also provide very valuable information to the study of gene function. Here, we resequenced 41 silkworms in the whole genome wide, of which one Japanese wild silkworm was only used to mitochondrial genome analysis. Other samples consist of 29 domesticated silkworm lines and 11 wild silkworms from the various mulberry fields in China. A single-base pair resolution silkworm genetic variation map was contructed from these 40 domesticated and wild silkworms. We identified a number of single nucleotide polymorphisms, and used these dataset to perform some population genetics study, including inferring the population strcture and demographic history, as well as identification of selective signal. The major results are as follows:1. Constructing high-resolution genetic variation map on silkworm nuclear genomesWe performed whole-genome resequencing for 29 domesticated varieties and 11 wild silkworms. After aligning the data onto the fully-assembled genome, silkworm genetic variation map was created. We produced 63.25Gb raw data, which corresponds to 118.1 folds effective depth, and each sample sequenced to approximately three folds coverage, representing 99.88% of the genome. The mean genome coverage and gene coverage was 82.5% and 93.0%. respectively. We identified～16 million single-nucleotide polymorphisms,311 thousand indels and 35 thousand structural variations. The genome polymorphism level between domesticated silkworms and between wild silkworms was 1.08% and 1.3%, respectively. The difference and diversity between domesticated and wild silkworm was mainly induced by these genomic variations. We believe the high resolution SNP map can pave a way to study the functional genome of silkworm, and these molecular markers will be utilized to the positional clone in the following studies. This silkworm genetic variation map is a first successful case study among the world for the rsequencing of eukaryotes.2. Population stucture analysis on silkwormThe origin of the domesticated silkworm is a long-standing question that has not been settled by previous limited biochemical and molecular analyses. We used the SNP data to performed some population genetic analyses, and combination results of population structure, principal components analysis and neighbor-joining tree show the domesticated silkworm is tamed form the wild silkworm and there is only a unique domesticated event in the history. Addtionally, the comparison between the domesticated and wild silkworm in genome level showed that the widely used voltinism was not the dominant factor in the silkworm origin history, and we should integrate the influence of the local climate to silkworm classification. Under the simulation of demographic model, we also found the domestication event lead to a 90% reduction in effective population size during the initial bottleneck and there has not been obvious population growth since the domestication event, that the domestic lines probably have had a generally stable effective population size, and that gene-flow from the wild to domesticated varieties may not have been strong. We believed that the conclusion above is an impressive progress in the study of silkworm origin and is valuable to the silkworm community.3. Identification selection signal in Bombyx mori and related analysisSome biology traits in the domesticated silkworm have been changed during the long time human intense selection. Artificial selection has also enhanced important economic traits such as cocoon size, growth rate, and digestion efficiency. Moreover, compared to its wild ancestor Bombyx mandarina, Bombyx mori has gained some representative behavioral characteristics (such as tol-erance to human proximity and handling, as well as extensive crowding) and lost other traits (such as flight, predators, and diseases avoidance). We compared the differences in SNP frequency spectra and amounts of variability between the two groups, and identified a total of 1041 genomic regions of selective signals, covering 12.5 Mb (2.9%) of the genome, which may reflect genomic footprints left by artificial selection during domestication. Within these regions, we identified 354 protein-coding genes that represent good candidates for domestication genes. Many of them are enriched in tissues of silk gland, midgut. and testis. The genes expressed in silk gland are involved in the synthesis of silk proteins, including fibroin and sericin. Midgut-enriched genes are related to the metabolism of carbohydrates, amino acids and lipids, which play an important role in food digestion and nutrient absorption. And genes enriched in the testis are annotated as having binding, catalytic, and motor activity related to reproduction. These genes identification along with biology trait transformation provided some clues to understand the mechanism of some biology trait and the regulation pathway.4. Characterizing the silkworm mitochondrial genomesWe identified 347 single nucleotide polymorphisms (SNPs) after the resequencing data mapping to the silkworm mitochondrial genome. The polymorphism level of mitochondrial sequence in three populations declined successively as follows:Chinese wild silkworm, Japanese wild silkworm and the domesticated silkworm. This indicated that there is the largest population size in the Chinese wild population, and large reduction in the domesticated group due to inbreeding or population bottlenecking. However, the Japanese wild population, which only distributes in Japan and Korea, has a moderate effective population size compared to Chinese wild silkworm. These results are in consistence with that of the nulear genome suggested, but there is an obvious difference with human. The analysis on insect mitochondrial genome shows it has lower polymorphism level than the nuclear genome, which is may caused by rapid decay of linkage disequilibrium in the insect nuclear genome. Moreover, there is no past recombination event to have occurred in the mitochondrial evolutionary history. It is our first time to comprehensively analyze the silkworm mitochondrial genome, and we confirmed that the polymorphism level is very low, which is an obvious difference with manmal, suggesting they had exclusive genetic background, and local inbreeding had produced more effect on the domesticated species than others.5. Evolutionary study on silkworm mitochondrial genomeWe used all the SNPs identified, which produced in the chapter above, to quantify the population structure. Using the mitochondrial genome sequences of Antheraea pernyi and Eriogyna pyretorum as the outgroups, we can see clearly and conclusively that the domesticated silkworm is closer to the Chinese wild silkworm than to the Japanese wild silkworm. We also showed that the population sizes of the domesticated and Chinese wild silkworms both experience neither expansion nor contraction, which indicated there is only limit gene flow between these two groups. Similar to the analysis on the nuclear genome, through comparing the difference between synonymous and nonsynonymous among populations, we discovered that one mitochondrial gene, named cytochrome b, showed a strong signal of positive selection in the domesticated clade. This gene, which is a component of respiratory chain complexⅢand is related to electron transfer, is related to energy metabolism, and may have played an important role during silkworm domestication. Results in this chapter is coincide with that of the nuclear genome indicated, prove that the population effective size in silkworm is contant and the domestication process refined some genes to meet with the human need.In summary, we believed that the polymorphic data from the nuclear genome and mitochondrial genome are the foundation to learn the functional genome, and we will further elucidate the gene function on the silkworm genome.