In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for ...In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.展开更多
DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to env...DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition, night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds. Real time qPCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.展开更多
Illegal hunting and trafficking of wildlife and their derivatives extort unprecedented population decline of relatively many species pushing them towards extinction.Notwithstanding contemporary counteracting intervent...Illegal hunting and trafficking of wildlife and their derivatives extort unprecedented population decline of relatively many species pushing them towards extinction.Notwithstanding contemporary counteracting interventions at international,regional,national and local levels,wildlife farming is advocated as an alternative approach to minimize pressure on wild populations.For wildlife farming to be an effective conservation tool,the integration of wildlife forensics is inevitable to allow distinction between captive-bred and wild-caught species.To this end,we analyzed methylation rates of skeletal muscle samples(pectoralis major,triceps brachii,gastrocnemius,biceps femoris,and neck muscles)from 60 captive-bred and 30 wild-caught Common Pheasant.A total of 13,507 differentially methylated regions were identified between five wild-caught and five captive-bred individuals through whole-genome methylation sequencing(WGBS).Based on the selected five methylation sites,LOC116231076,LOC116242223,ATAD2B,EGFL6,and HS2ST,quantitative detection technique was developed using methylation-sensitive high-resolution melting curve(MS-HRM)to measure methylation rates.The results showed significant differences in methylation rates at all differential sites between wild-caught and captive-bred individuals(|t|=0.67–33.10,P=0.000–0.042).The discrimination accuracy rate of each locus was highest in the gastrocnemius muscle and lowest in the neck muscle.The discrimination accuracy rate on LOC116231076,LOC116242223,ATAD2B,EGFL6,and HS2ST methylation sites for gastrocnemius muscle was 64.98%,100.00%,68.54%,63.79%,and 63.70%,respectively;and for neck muscle it was 67.42%,68.06%,83.61%,65.04%,and68.85%,respectively.The united discrimination accuracy rate of the five loci were 100.00%for gastrocnemius muscle,99.78%for biceps femoris muscle,97.52%for pectoralis major muscle,93.96%for triceps brachii muscle,and 91.63%for neck muscle,respectively.The panel also revealed excellent repeatability,reproducibility,sensitivity and universality to mammals and avian species.This study establishes an effective,accurate and low-cost identification technology for the identification of wild and farmed Common Pheasant,and also provides a reference for the development of identification methods for other species.展开更多
Investigation of the relationships of phenotypic and epigenetic variations might he a good way to dissect the genetic or molecular basis of phenotypic variation and plasticity in plants, Castor bean （Ricinus cornraun...Investigation of the relationships of phenotypic and epigenetic variations might he a good way to dissect the genetic or molecular basis of phenotypic variation and plasticity in plants, Castor bean （Ricinus cornraunis L）, an important non-edible oilseed crop, is a mono-species genus plant in the family Euphorbiaceae. Since it displays rich phenotypic variations with low genetic diversity, castor bean is a good model to investigate the molecular basis of phenotypic and epigenetic variations. Cytosine DNA methylation represents a major molecular mechanism of epigenetic occurrence. In this study, epigenetic diversity of sixty landrace accessions collected worldwide was investigated using the methylation- sensitive amplification polymorphism （MSAP） technique, Results showed that the epigenetic diversity （based on the polymorphism of DNA methylated loci） exhibited a medium variation （Ne = 1.395, He = 0.242, I = 0.366） at the population level though the variation was great, ranging from 3,80% to 3431% among accessions. Both population structure analysis and the phylogenetic construction （using the neighbor-joining criteria） revealed that the two main clades were identified, but they did not display a distinct geographic structure, After inspecting the location of polymorphic methylated loci on genome we identified that the polymorphic methylated loci occur widely in nuclear and organelle genomes. This study provides new data to understand phenotypic and epigenetic variations in castor bean,展开更多
DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth,development,and polyploidization.However,there is still no distinct evidence in tobacco regarding the distrib...DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth,development,and polyploidization.However,there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics.We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco,Nicotiana tabacum,using a methylation-sensitive amplified polymorphism(MSAP) technique.The results showed that methylation existed at a high level among tobacco accessions,among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic.A cluster analysis revealed distinct patterns of geography-specific groups.In addition,three polymorphic sites significantly related to tobacco mosaic virus(TMV) resistance were explored.This suggests that tobacco breeders should pay more attention to epigenetic traits.展开更多
Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this stu...Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.展开更多
AIM:To isolate and analyze the DNA sequences which are methylated differentially between gastric cancer and normal gastric mucosa. METHODS: The differentially methylated DNA sequences between gastric cancer and normal...AIM:To isolate and analyze the DNA sequences which are methylated differentially between gastric cancer and normal gastric mucosa. METHODS: The differentially methylated DNA sequences between gastric cancer and normal gastric mucosa were isolated by methylation-sensitive representational difference analysis (MS-RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with Basic Local Alignment Search Tool (BLAST). RESULTS: Three differentially methylated DNA sequences were obtained, two of which have been accepted by GenBank. The accession numbers are AY887106 and AY887107. AY887107 was highly similar to the 11th exon of LOC440683 (98%), 3' end of LOC440887 (99%), and promoter and exon regions of DRD5 (94%). AY887106 was consistent (98%) with a CpG island in ribosomal RNA isolated from colorectal cancer by Minoru Toyota in 1999. CONCLUSION: The methylation degree is different between gastric cancer and normal gastric mucosa. The differentially methylated DNA sequences can be isolated effectively by MS-RDA.展开更多
Persimmon （ Diospyros kaki Thunb. ） , a member of Ebenaceae family, is known as ＂woody grain＂ and ＂iron-stem crop＂. However, the epigenetie research has been rarely reported. In this study, tannin content in lat...Persimmon （ Diospyros kaki Thunb. ） , a member of Ebenaceae family, is known as ＂woody grain＂ and ＂iron-stem crop＂. However, the epigenetie research has been rarely reported. In this study, tannin content in late fruit development periods was determined. Moreover, the MSAP （methylation-sensitive amplified polymorphism） method was used to analyze methylation profile, and 13 primer combinations were selected. In total, 5 differential fragments were cloned and sequenced. This research would be helpful for further gene cloning, research on deastringency mechanism, as well as molecular breeding of D. kaki.展开更多
As an important wild blueberry resource,Vaccinium uliginosum has attracted more and more attention.At present,the wild resources are under destruction.The conservation of wild Vaccinium uliginosum resources is imminen...As an important wild blueberry resource,Vaccinium uliginosum has attracted more and more attention.At present,the wild resources are under destruction.The conservation of wild Vaccinium uliginosum resources is imminent.However,there are few researches on the protection and preservation of its germplasm resources.In vitro preservation is an important method for germplasm conservation.In this study,one strain of wild Vaccinium uliginosum was used as material.The effects of temperature(25℃,15℃,10℃,or 0℃),media(WPM,1/2WPM or 1/3WPM),medium supplements(sorbitol or mannose),and photoperiod(8,10,12,or 14 h•d^(-1))on the growth,survival rate and rejuvenation rate of the plantlets were studied.The physiological changes of plantlets during preservation were analyzed.Methylation-sensitive amplified polymorphism(MSAP)analysis of genomic DNA methylation of plantlets was carried out to explore the genetic stability of the plantlets after preservation.The research results provided a theoretical basis for the germplasm preservation of Vaccinium uliginosum.展开更多
Sogatella furcifera （Hovarth） is a major rice pest with sexual dimorphism. The objective of the current research was to monitor differentially cytosine methylation at CCGG sequences in male and female adults of S. f...Sogatella furcifera （Hovarth） is a major rice pest with sexual dimorphism. The objective of the current research was to monitor differentially cytosine methylation at CCGG sequences in male and female adults of S. furcifera to determine the association between gene methylation and sexual phenotypes using methylation-sensitive representa- tional difference analysis. After the second subtractive hybridization, four differentially methylated DNA bands were obtained and sequenced. Ten different fragments were found. One fragment from the positive hybridization was 120 bp, and highly similar to the tram- track genes from Nasonia vitripennis. Another fragment from the reverse hybridization was 414 bp, and homologous to the 28S rRNA gene of S. furcifera with a similarity rate as high as 99%. We also discussed how DNA methylation of tramtrack and 28S rRNA genes produced effects on sexual differentiation and development. These results provide potential evidence that DNA methylation of some genes may be related to sexual phenotype variations in S.furcifera and will facilitate future studies on the epigenetic mechanisms of insect sexual dimorphism.展开更多
基金supported by the grants from the National Natural Science Foundation of China (No. 4147 6124)the Natural Science Foundation of Zhejiang Province (No. LY17C190005)+3 种基金the Major Agriculture Program of Ningbo (No. 2017C110007)the Ningbo Science and Technology Project (No. 2016C10037)the Open Fund of Ningbo University (No. xkzsc1505)K C Wong Magana Fund in Ningbo University
文摘In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.
基金supported by National Nonprofit Institute Research Grant of Chinese Academy of Forestry(TGB2013010)
文摘DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition, night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds. Real time qPCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.
基金supported by the Fundamental Research Funds for the Central Universities(2572020DR10)Project on the Investigation,Supervision and Industry Regulation of Rare and Endangered Species(2024)。
文摘Illegal hunting and trafficking of wildlife and their derivatives extort unprecedented population decline of relatively many species pushing them towards extinction.Notwithstanding contemporary counteracting interventions at international,regional,national and local levels,wildlife farming is advocated as an alternative approach to minimize pressure on wild populations.For wildlife farming to be an effective conservation tool,the integration of wildlife forensics is inevitable to allow distinction between captive-bred and wild-caught species.To this end,we analyzed methylation rates of skeletal muscle samples(pectoralis major,triceps brachii,gastrocnemius,biceps femoris,and neck muscles)from 60 captive-bred and 30 wild-caught Common Pheasant.A total of 13,507 differentially methylated regions were identified between five wild-caught and five captive-bred individuals through whole-genome methylation sequencing(WGBS).Based on the selected five methylation sites,LOC116231076,LOC116242223,ATAD2B,EGFL6,and HS2ST,quantitative detection technique was developed using methylation-sensitive high-resolution melting curve(MS-HRM)to measure methylation rates.The results showed significant differences in methylation rates at all differential sites between wild-caught and captive-bred individuals(|t|=0.67–33.10,P=0.000–0.042).The discrimination accuracy rate of each locus was highest in the gastrocnemius muscle and lowest in the neck muscle.The discrimination accuracy rate on LOC116231076,LOC116242223,ATAD2B,EGFL6,and HS2ST methylation sites for gastrocnemius muscle was 64.98%,100.00%,68.54%,63.79%,and 63.70%,respectively;and for neck muscle it was 67.42%,68.06%,83.61%,65.04%,and68.85%,respectively.The united discrimination accuracy rate of the five loci were 100.00%for gastrocnemius muscle,99.78%for biceps femoris muscle,97.52%for pectoralis major muscle,93.96%for triceps brachii muscle,and 91.63%for neck muscle,respectively.The panel also revealed excellent repeatability,reproducibility,sensitivity and universality to mammals and avian species.This study establishes an effective,accurate and low-cost identification technology for the identification of wild and farmed Common Pheasant,and also provides a reference for the development of identification methods for other species.
基金jointly supported by Chinese National Key Technology R & D Program (2015BAD15B02)National Natural Science Foundation of China (31661143002 and 31501034)
文摘Investigation of the relationships of phenotypic and epigenetic variations might he a good way to dissect the genetic or molecular basis of phenotypic variation and plasticity in plants, Castor bean （Ricinus cornraunis L）, an important non-edible oilseed crop, is a mono-species genus plant in the family Euphorbiaceae. Since it displays rich phenotypic variations with low genetic diversity, castor bean is a good model to investigate the molecular basis of phenotypic and epigenetic variations. Cytosine DNA methylation represents a major molecular mechanism of epigenetic occurrence. In this study, epigenetic diversity of sixty landrace accessions collected worldwide was investigated using the methylation- sensitive amplification polymorphism （MSAP） technique, Results showed that the epigenetic diversity （based on the polymorphism of DNA methylated loci） exhibited a medium variation （Ne = 1.395, He = 0.242, I = 0.366） at the population level though the variation was great, ranging from 3,80% to 3431% among accessions. Both population structure analysis and the phylogenetic construction （using the neighbor-joining criteria） revealed that the two main clades were identified, but they did not display a distinct geographic structure, After inspecting the location of polymorphic methylated loci on genome we identified that the polymorphic methylated loci occur widely in nuclear and organelle genomes. This study provides new data to understand phenotypic and epigenetic variations in castor bean,
基金supported by the Program for High-Quality Tobacco Development of China (No. [2010]221)the Foundation of Science and Technology of Guizhou Province (No. J[2010]2251)the Program for Guizhou Tobacco Science of China (No. 200910)
文摘DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth,development,and polyploidization.However,there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics.We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco,Nicotiana tabacum,using a methylation-sensitive amplified polymorphism(MSAP) technique.The results showed that methylation existed at a high level among tobacco accessions,among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic.A cluster analysis revealed distinct patterns of geography-specific groups.In addition,three polymorphic sites significantly related to tobacco mosaic virus(TMV) resistance were explored.This suggests that tobacco breeders should pay more attention to epigenetic traits.
基金supported by the National Natural Science Foundation of China (30860149 and 31360364)the Joint Support Program from Tropical Crop Breeding Engineering Center of Ministry of Education of China+1 种基金the Crop Science National Key Disciplines of China (lhxm-2012-2)the Key Scientific Research Program from Hainan Province,China (ZDZX2013023)
文摘Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.
基金The Key Project Fund of the Science and Technology Program of Hunan Province, No. 04SK1004the Scientific Research Fund of Educational Department, Hunan Province, No. 99C195
文摘AIM:To isolate and analyze the DNA sequences which are methylated differentially between gastric cancer and normal gastric mucosa. METHODS: The differentially methylated DNA sequences between gastric cancer and normal gastric mucosa were isolated by methylation-sensitive representational difference analysis (MS-RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with Basic Local Alignment Search Tool (BLAST). RESULTS: Three differentially methylated DNA sequences were obtained, two of which have been accepted by GenBank. The accession numbers are AY887106 and AY887107. AY887107 was highly similar to the 11th exon of LOC440683 (98%), 3' end of LOC440887 (99%), and promoter and exon regions of DRD5 (94%). AY887106 was consistent (98%) with a CpG island in ribosomal RNA isolated from colorectal cancer by Minoru Toyota in 1999. CONCLUSION: The methylation degree is different between gastric cancer and normal gastric mucosa. The differentially methylated DNA sequences can be isolated effectively by MS-RDA.
基金Supported by College Students’Innovation and Entrepreneurship Training Program of Huanggang Normal University(zx201425)Fund of Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains(2015TD07)
文摘Persimmon （ Diospyros kaki Thunb. ） , a member of Ebenaceae family, is known as ＂woody grain＂ and ＂iron-stem crop＂. However, the epigenetie research has been rarely reported. In this study, tannin content in late fruit development periods was determined. Moreover, the MSAP （methylation-sensitive amplified polymorphism） method was used to analyze methylation profile, and 13 primer combinations were selected. In total, 5 differential fragments were cloned and sequenced. This research would be helpful for further gene cloning, research on deastringency mechanism, as well as molecular breeding of D. kaki.
基金Supported by the National Natural Science Foundation of China(32172521)。
文摘As an important wild blueberry resource,Vaccinium uliginosum has attracted more and more attention.At present,the wild resources are under destruction.The conservation of wild Vaccinium uliginosum resources is imminent.However,there are few researches on the protection and preservation of its germplasm resources.In vitro preservation is an important method for germplasm conservation.In this study,one strain of wild Vaccinium uliginosum was used as material.The effects of temperature(25℃,15℃,10℃,or 0℃),media(WPM,1/2WPM or 1/3WPM),medium supplements(sorbitol or mannose),and photoperiod(8,10,12,or 14 h•d^(-1))on the growth,survival rate and rejuvenation rate of the plantlets were studied.The physiological changes of plantlets during preservation were analyzed.Methylation-sensitive amplified polymorphism(MSAP)analysis of genomic DNA methylation of plantlets was carried out to explore the genetic stability of the plantlets after preservation.The research results provided a theoretical basis for the germplasm preservation of Vaccinium uliginosum.
基金This research was supported by the National Nat- ural Science Foundation of China （31171844） and Natural Science Foundation of Guangdong Province （$2011010001353）. We are very grateful to Dr. Min Zhang and Mrs. Wen-Jing Wu for technical assistance.
文摘Sogatella furcifera （Hovarth） is a major rice pest with sexual dimorphism. The objective of the current research was to monitor differentially cytosine methylation at CCGG sequences in male and female adults of S. furcifera to determine the association between gene methylation and sexual phenotypes using methylation-sensitive representa- tional difference analysis. After the second subtractive hybridization, four differentially methylated DNA bands were obtained and sequenced. Ten different fragments were found. One fragment from the positive hybridization was 120 bp, and highly similar to the tram- track genes from Nasonia vitripennis. Another fragment from the reverse hybridization was 414 bp, and homologous to the 28S rRNA gene of S. furcifera with a similarity rate as high as 99%. We also discussed how DNA methylation of tramtrack and 28S rRNA genes produced effects on sexual differentiation and development. These results provide potential evidence that DNA methylation of some genes may be related to sexual phenotype variations in S.furcifera and will facilitate future studies on the epigenetic mechanisms of insect sexual dimorphism.
