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Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication.

Xiang H, Li X, Dai F, Xu X, Tan A, Chen L, Zhang G, Ding Y, Li Q, Lian J, Willden A, Guo Q, Xia Q, Wang J, Wang W - BMC Genomics (2013)

Bottom Line: We observed 2-fold more differentiated methylated cytosinces (mCs) in domesticated silkworms as compared to their wild counterparts, suggesting a trend of increasing DNA methylation during domestication.A methylation-increased gene seems to result in higher expression in domesticates and the function of its Drosophila homolog was previously found to be essential for cell volume regulation, indicating a possible correlation with the enlargement of silk glands in domesticated silkworms.Our results imply epigenetic influences at work during domestication, which gives insight into long time historical controversies regarding acquired inheritance.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiaochang Road, Kunming, Yunnan Province 650223, China. wangj@genomics.org.cn.

ABSTRACT

Background: In contrast to wild species, which have typically evolved phenotypes over long periods of natural selection, domesticates rapidly gained human-preferred agronomic traits in a relatively short-time frame via artificial selection. Under domesticated conditions, many traits can be observed that cannot only be due to environmental alteration. In the case of silkworms, aside from genetic divergence, whether epigenetic divergence played a role in domestication is an unanswered question. The silkworm is still an enigma in that it has two DNA methyltransferases (DNMT1 and DNMT2) but their functionality is unknown. Even in particular the functionality of the widely distributed DNMT1 remains unknown in insects in general.

Results: By embryonic RNA interference, we reveal that knockdown of silkworm Dnmt1 caused decreased hatchability, providing the first direct experimental evidence of functional significance of insect Dnmt1. In the light of this fact and those that DNA methylation is correlated with gene expression in silkworms and some agronomic traits in domesticated organisms are not stable, we comprehensively compare silk gland methylomes of 3 domesticated (Bombyx mori) and 4 wild (Bombyx mandarina) silkworms to identify differentially methylated genes between the two. We observed 2-fold more differentiated methylated cytosinces (mCs) in domesticated silkworms as compared to their wild counterparts, suggesting a trend of increasing DNA methylation during domestication. Further study of more domesticated and wild silkworms narrowed down the domesticates' epimutations, and we were able to identify a number of differential genes. One such gene showing demethyaltion in domesticates correspondently displays lower gene expression, and more interestingly, has experienced selective sweep. A methylation-increased gene seems to result in higher expression in domesticates and the function of its Drosophila homolog was previously found to be essential for cell volume regulation, indicating a possible correlation with the enlargement of silk glands in domesticated silkworms.

Conclusions: Our results imply epigenetic influences at work during domestication, which gives insight into long time historical controversies regarding acquired inheritance.

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Related in: MedlinePlus

Methylation and expression of BGIMBGA003527 as well as SNPs in its upstream 2 kb region. (A) Comparison of MethylC-Seq data of the BGIMBGA003527 DMC cluster with the 454 sequencing data of traditional bisulfite PCR (BS-PCR). Methylation level of the DMCs were examined by MethylC-seq in the three domesticated silkworms Dazao, Js, C108 (Dome_MethylC-Seq) and in the four wild silkworms Wsh, Wyn, CW, XW (Wild_MethylC-Seq) and validated in the same sample sets (Dome_BS and Wild_BS). Validation and test for fixation of methylation differences in more new domesticated (L10_BS, HY_BS, 872_BS, ZZ_BS) and wild silkworms (Wgs_BS, Wzj_BS, Wjs_BS) listed in Additional file 2: Table S1, were conducted using 454 sequencing of BS-PCR amplicons. For MethylC-Seq and validation data, methylation level was calculated by dividing the total reads from each sample set covering mCG by the total reads from that sample set covering that cytosine. For fixation test, methylation level was calculated similarly except that the reads are from each individual. (B) Location of DMCs in genic regions. The gene model is at the bottom, where red blocks indicate exons and the black lines between each two blocks indicate introns. The 4 CG sites in the black ellipse are those validated and tested for fixation. Dome, domesticated silkworms, Wild, wild silkworms. (C) BGIBMGA003527 expression level changes in silk glands of each domesticated individual against wild silkworms, estimated by quantitative real-time PCR. Relative expression ratios of each domesticated to wild silkworms are normalized by logarithmic transformation. Plots shows the average relative expression ratios and error bars shows the standard errors. (D) Nucleotide frequency of the SNPs upstream 2 kb of BGIMBGA003527 surveyed from the published data (http://silkworm.swu.edu.cn/silkdb/resequencing.html) and validated for domesticated and wild silkworms listed in Additional file 2: Table S1 by Sanger sequencing.
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Figure 3: Methylation and expression of BGIMBGA003527 as well as SNPs in its upstream 2 kb region. (A) Comparison of MethylC-Seq data of the BGIMBGA003527 DMC cluster with the 454 sequencing data of traditional bisulfite PCR (BS-PCR). Methylation level of the DMCs were examined by MethylC-seq in the three domesticated silkworms Dazao, Js, C108 (Dome_MethylC-Seq) and in the four wild silkworms Wsh, Wyn, CW, XW (Wild_MethylC-Seq) and validated in the same sample sets (Dome_BS and Wild_BS). Validation and test for fixation of methylation differences in more new domesticated (L10_BS, HY_BS, 872_BS, ZZ_BS) and wild silkworms (Wgs_BS, Wzj_BS, Wjs_BS) listed in Additional file 2: Table S1, were conducted using 454 sequencing of BS-PCR amplicons. For MethylC-Seq and validation data, methylation level was calculated by dividing the total reads from each sample set covering mCG by the total reads from that sample set covering that cytosine. For fixation test, methylation level was calculated similarly except that the reads are from each individual. (B) Location of DMCs in genic regions. The gene model is at the bottom, where red blocks indicate exons and the black lines between each two blocks indicate introns. The 4 CG sites in the black ellipse are those validated and tested for fixation. Dome, domesticated silkworms, Wild, wild silkworms. (C) BGIBMGA003527 expression level changes in silk glands of each domesticated individual against wild silkworms, estimated by quantitative real-time PCR. Relative expression ratios of each domesticated to wild silkworms are normalized by logarithmic transformation. Plots shows the average relative expression ratios and error bars shows the standard errors. (D) Nucleotide frequency of the SNPs upstream 2 kb of BGIMBGA003527 surveyed from the published data (http://silkworm.swu.edu.cn/silkdb/resequencing.html) and validated for domesticated and wild silkworms listed in Additional file 2: Table S1 by Sanger sequencing.

Mentions: Due to considerable epigenetic instability, gain or loss of DNA methylation is common compared to DNA mutations, which are usually irreversible [23]. The heritable and even fixed epimutations during domestication are thus awash with prompt but reversible epigenetic changes. We therefore decided to further test more domesticated and wild samples in order to find fixed DMR. We collected four more domesticated silkworm strains (three high silk production strains, HY, L10 and 872, and one local reserved strain ZZ) and three more geographically different varieties of wild silkworms from Gansu (Wgs), Zhejiang (Wzj) and Jiangsu (Wjs) province of China (Additional file 2: Table S1, Additional file 1: Figure S2). Unfortunately, checking all the 188 DMRs with loci-specific bisulfite PCR and sequencing (BS-PCR) for these individuals is too costly, so we randomly chose 37 regions to subject to bisulfite PCR amplification followed by 454 deep sequencing, using the barcoded primers (Additional file 5: Table S4). In total, from 25 out of these 37 regions (Figures 3A &4A, Additional file 1: Figures S3 & S4), we successfully obtained effective 454 sequencing data in at least three new domesticated and two new wild samples, among which 12 regions were covered by sequencing data across all the samples. As to the rest 12 regions, although all of them were successfully amplified in domesticated silkworms and 10 out of them had effective sequencing data in at least 3 new domesticated samples, only 5 regions could be amplified in only one new wild sample, leaving us with a lack of informative sequencing data in wild silkworms for these 12 regions. Failure of sufficient amplification in wild samples may be due to mutations in primer binding regions.


Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication.

Xiang H, Li X, Dai F, Xu X, Tan A, Chen L, Zhang G, Ding Y, Li Q, Lian J, Willden A, Guo Q, Xia Q, Wang J, Wang W - BMC Genomics (2013)

Methylation and expression of BGIMBGA003527 as well as SNPs in its upstream 2 kb region. (A) Comparison of MethylC-Seq data of the BGIMBGA003527 DMC cluster with the 454 sequencing data of traditional bisulfite PCR (BS-PCR). Methylation level of the DMCs were examined by MethylC-seq in the three domesticated silkworms Dazao, Js, C108 (Dome_MethylC-Seq) and in the four wild silkworms Wsh, Wyn, CW, XW (Wild_MethylC-Seq) and validated in the same sample sets (Dome_BS and Wild_BS). Validation and test for fixation of methylation differences in more new domesticated (L10_BS, HY_BS, 872_BS, ZZ_BS) and wild silkworms (Wgs_BS, Wzj_BS, Wjs_BS) listed in Additional file 2: Table S1, were conducted using 454 sequencing of BS-PCR amplicons. For MethylC-Seq and validation data, methylation level was calculated by dividing the total reads from each sample set covering mCG by the total reads from that sample set covering that cytosine. For fixation test, methylation level was calculated similarly except that the reads are from each individual. (B) Location of DMCs in genic regions. The gene model is at the bottom, where red blocks indicate exons and the black lines between each two blocks indicate introns. The 4 CG sites in the black ellipse are those validated and tested for fixation. Dome, domesticated silkworms, Wild, wild silkworms. (C) BGIBMGA003527 expression level changes in silk glands of each domesticated individual against wild silkworms, estimated by quantitative real-time PCR. Relative expression ratios of each domesticated to wild silkworms are normalized by logarithmic transformation. Plots shows the average relative expression ratios and error bars shows the standard errors. (D) Nucleotide frequency of the SNPs upstream 2 kb of BGIMBGA003527 surveyed from the published data (http://silkworm.swu.edu.cn/silkdb/resequencing.html) and validated for domesticated and wild silkworms listed in Additional file 2: Table S1 by Sanger sequencing.
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Related In: Results  -  Collection

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Figure 3: Methylation and expression of BGIMBGA003527 as well as SNPs in its upstream 2 kb region. (A) Comparison of MethylC-Seq data of the BGIMBGA003527 DMC cluster with the 454 sequencing data of traditional bisulfite PCR (BS-PCR). Methylation level of the DMCs were examined by MethylC-seq in the three domesticated silkworms Dazao, Js, C108 (Dome_MethylC-Seq) and in the four wild silkworms Wsh, Wyn, CW, XW (Wild_MethylC-Seq) and validated in the same sample sets (Dome_BS and Wild_BS). Validation and test for fixation of methylation differences in more new domesticated (L10_BS, HY_BS, 872_BS, ZZ_BS) and wild silkworms (Wgs_BS, Wzj_BS, Wjs_BS) listed in Additional file 2: Table S1, were conducted using 454 sequencing of BS-PCR amplicons. For MethylC-Seq and validation data, methylation level was calculated by dividing the total reads from each sample set covering mCG by the total reads from that sample set covering that cytosine. For fixation test, methylation level was calculated similarly except that the reads are from each individual. (B) Location of DMCs in genic regions. The gene model is at the bottom, where red blocks indicate exons and the black lines between each two blocks indicate introns. The 4 CG sites in the black ellipse are those validated and tested for fixation. Dome, domesticated silkworms, Wild, wild silkworms. (C) BGIBMGA003527 expression level changes in silk glands of each domesticated individual against wild silkworms, estimated by quantitative real-time PCR. Relative expression ratios of each domesticated to wild silkworms are normalized by logarithmic transformation. Plots shows the average relative expression ratios and error bars shows the standard errors. (D) Nucleotide frequency of the SNPs upstream 2 kb of BGIMBGA003527 surveyed from the published data (http://silkworm.swu.edu.cn/silkdb/resequencing.html) and validated for domesticated and wild silkworms listed in Additional file 2: Table S1 by Sanger sequencing.
Mentions: Due to considerable epigenetic instability, gain or loss of DNA methylation is common compared to DNA mutations, which are usually irreversible [23]. The heritable and even fixed epimutations during domestication are thus awash with prompt but reversible epigenetic changes. We therefore decided to further test more domesticated and wild samples in order to find fixed DMR. We collected four more domesticated silkworm strains (three high silk production strains, HY, L10 and 872, and one local reserved strain ZZ) and three more geographically different varieties of wild silkworms from Gansu (Wgs), Zhejiang (Wzj) and Jiangsu (Wjs) province of China (Additional file 2: Table S1, Additional file 1: Figure S2). Unfortunately, checking all the 188 DMRs with loci-specific bisulfite PCR and sequencing (BS-PCR) for these individuals is too costly, so we randomly chose 37 regions to subject to bisulfite PCR amplification followed by 454 deep sequencing, using the barcoded primers (Additional file 5: Table S4). In total, from 25 out of these 37 regions (Figures 3A &4A, Additional file 1: Figures S3 & S4), we successfully obtained effective 454 sequencing data in at least three new domesticated and two new wild samples, among which 12 regions were covered by sequencing data across all the samples. As to the rest 12 regions, although all of them were successfully amplified in domesticated silkworms and 10 out of them had effective sequencing data in at least 3 new domesticated samples, only 5 regions could be amplified in only one new wild sample, leaving us with a lack of informative sequencing data in wild silkworms for these 12 regions. Failure of sufficient amplification in wild samples may be due to mutations in primer binding regions.

Bottom Line: We observed 2-fold more differentiated methylated cytosinces (mCs) in domesticated silkworms as compared to their wild counterparts, suggesting a trend of increasing DNA methylation during domestication.A methylation-increased gene seems to result in higher expression in domesticates and the function of its Drosophila homolog was previously found to be essential for cell volume regulation, indicating a possible correlation with the enlargement of silk glands in domesticated silkworms.Our results imply epigenetic influences at work during domestication, which gives insight into long time historical controversies regarding acquired inheritance.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiaochang Road, Kunming, Yunnan Province 650223, China. wangj@genomics.org.cn.

ABSTRACT

Background: In contrast to wild species, which have typically evolved phenotypes over long periods of natural selection, domesticates rapidly gained human-preferred agronomic traits in a relatively short-time frame via artificial selection. Under domesticated conditions, many traits can be observed that cannot only be due to environmental alteration. In the case of silkworms, aside from genetic divergence, whether epigenetic divergence played a role in domestication is an unanswered question. The silkworm is still an enigma in that it has two DNA methyltransferases (DNMT1 and DNMT2) but their functionality is unknown. Even in particular the functionality of the widely distributed DNMT1 remains unknown in insects in general.

Results: By embryonic RNA interference, we reveal that knockdown of silkworm Dnmt1 caused decreased hatchability, providing the first direct experimental evidence of functional significance of insect Dnmt1. In the light of this fact and those that DNA methylation is correlated with gene expression in silkworms and some agronomic traits in domesticated organisms are not stable, we comprehensively compare silk gland methylomes of 3 domesticated (Bombyx mori) and 4 wild (Bombyx mandarina) silkworms to identify differentially methylated genes between the two. We observed 2-fold more differentiated methylated cytosinces (mCs) in domesticated silkworms as compared to their wild counterparts, suggesting a trend of increasing DNA methylation during domestication. Further study of more domesticated and wild silkworms narrowed down the domesticates' epimutations, and we were able to identify a number of differential genes. One such gene showing demethyaltion in domesticates correspondently displays lower gene expression, and more interestingly, has experienced selective sweep. A methylation-increased gene seems to result in higher expression in domesticates and the function of its Drosophila homolog was previously found to be essential for cell volume regulation, indicating a possible correlation with the enlargement of silk glands in domesticated silkworms.

Conclusions: Our results imply epigenetic influences at work during domestication, which gives insight into long time historical controversies regarding acquired inheritance.

Show MeSH
Related in: MedlinePlus