第939回千葉医学会例会・第12回磯野外科例会

Target bed file (mandatory input file) to run primer design with MSP-HTPrimer. (TXT 74 bytes

Table_9_The Pattern and Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.XLSX

<p>Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.</p

Table_1_The Pattern, Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.XLSX

<p>Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.</p

Table_14_The Pattern, Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.XLSX

<p>Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.</p

Table_2_The Pattern and Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.XLSX

<p>Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.</p

Table_3_The Pattern, Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.XLSX

<p>Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.</p

Table_6_The Pattern, Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.XLSX

<p>Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.</p

Table_11_The Pattern and Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.XLSX

<p>Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.</p

Venn diagrams of called SNV by four variant callers using (A) BWA (B) Bowtie2 (C) GSNAP with same mapper.

<p>Result shows that 75% - 84% SNVs are common with at least two out of four variant callers. With all 3 mappers Varscan2 identified novel SNVs from 16% - 24%.</p

Identified SNVs by MutAid pipeline.

<p>SNVs were called using four variant callers (for each mapping result) with a minimum read coverage of 20, minimum variant allele coverage of 4 and a base quality of at least 20. The percentage given in brackets is the fraction of SNVs having an entry in the Single Nucleotide Polymorphism Database (dbSNP) version 137.</p