Project description:MicroRNAs (miRNAs) are emerging as essential regulators of biological processes. Somatic embryogenesis is one of the most important techniques for gymnosperm breeding programs, but there is little understanding of its underlying mechanism. To investigate the roles of miRNAs during somatic embryogenesis in larch, we constructed a small RNA library from somatic embryos. High-throughput sequencing of the library identified 83 conserved miRNAs from 35 families, 16 novel miRNAs, and 14 plausible miRNA candidates, with a high proportion specific to larch or gymnosperms. qRT-PCR analysis demonstrated that both the conserved and novel or candidate miRNAs were expressed in larch. Several miRNA precursor sequences were obtained via RACE. We predicted 110 target genes using bioinformatics, and validated nine of them by 5’ RACE. Eleven conserved miRNA families including 17 miRNAs with critical functions in plant development and six target mRNAs were detected by qRT-PCR in the larch SE. Stage-specific expression of miRNAs and their targets indicate their possible modulation on SE of larch: miR171a/b might exert function on PEMs, while miR171c acts in the induction process of larch SE; miR397 and miR398 mainly involved in modulation of PEM propagation and transition to single embryo; miR162 and miR168 exert their regulatory function during total SE process, especially during stage 5 to stage 8; miR156, miR159, miR160, miR166, miR167, and miR390 might play regulatory roles during cytoledonary embryo development. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving specific and common miRNAs operating post-transcriptionally during embryogenesis.
Project description:MicroRNAs (miRNAs) are emerging as essential regulators of biological processes. Somatic embryogenesis is one of the most important techniques for gymnosperm breeding programs, but there is little understanding of its underlying mechanism. To investigate the roles of miRNAs during somatic embryogenesis in larch, we constructed a small RNA library from somatic embryos. High-throughput sequencing of the library identified 83 conserved miRNAs from 35 families, 16 novel miRNAs, and 14 plausible miRNA candidates, with a high proportion specific to larch or gymnosperms. qRT-PCR analysis demonstrated that both the conserved and novel or candidate miRNAs were expressed in larch. Several miRNA precursor sequences were obtained via RACE. We predicted 110 target genes using bioinformatics, and validated nine of them by 5M-bM-^@M-^Y RACE. Eleven conserved miRNA families including 17 miRNAs with critical functions in plant development and six target mRNAs were detected by qRT-PCR in the larch SE. Stage-specific expression of miRNAs and their targets indicate their possible modulation on SE of larch: miR171a/b might exert function on PEMs, while miR171c acts in the induction process of larch SE; miR397 and miR398 mainly involved in modulation of PEM propagation and transition to single embryo; miR162 and miR168 exert their regulatory function during total SE process, especially during stage 5 to stage 8; miR156, miR159, miR160, miR166, miR167, and miR390 might play regulatory roles during cytoledonary embryo development. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving specific and common miRNAs operating post-transcriptionally during embryogenesis. Examination of small RNA expression profilings in Larix somatic embryos over seven developmental stages: 1) after 2 and 15 days on sub-culture; 2) 2, 6, and 11 days post-differentiation culture; and 3) somatic embryos at days 33 and 39.
Project description:Illumina HiSeq2500 technology was used to generate mRNA-sequencing expression profiles of sexual stages of the poplar rust fungus M. larici-populina: - Basidia (BSD) were collected from rust-infected dead poplar leaves; - Pycnia (PYC) -also called spermogonia- were collected from rust-infected larch needles one week after inoculation with basidiospores and corresponded to different haploid fungal cell types such as in planta infection hyphae, receptive hyphae and pycniospores; - Aecia (AEC) were collected from rust-infected larch needles two weeks after inoculation, and corresponded to freshly produced aecia and dikaryotic aeciospores and remaining pycnia. Paired-end reads of 100bp were generated for three sets of biological replicates corresponding to each of the targeted stage and aligned to the genome of M. larici-populina isolate 98AG31 (version 2.0; US Department of Energy Joint Genome Institute; http://genome.jgi.doe.gov/Mellp2_3/Mellp2_3.home.html) using CLC Genomics Workbench 12.0
Project description:Illumina HiSeq2500 technology was used to generate mRNA-sequencing expression profiles of sexual stages of the poplar rust fungus M. larici-populina: - Basidia (BSD) were collected from rust-infected dead poplar leaves; - Pycnia (PYC) were collected from rust-infected larch needles one week after inoculation with basidiospores and corresponded to different haploid fungal cell types such as in planta infection hyphae, receptive hyphae and pycniospores; - Aecia (AEC) were collected from rust-infected larch needles two weeks after inoculation, and corresponded to freshly produced aecia and dikaryotic aeciospores and remaining pycnia. Paired-end reads of 100bp were generated for three sets of biological replicates corresponding to each of the targeted stage and aligned to the genome of M. larici-populina isolate 98AG31 (version 1.0; US Department of Energy Joint Genome Institute; http://genome.jgi.doe.gov/Mellp1/Mellp1.home.html; Duplessis et al. 2011a) using CLC Genomics Workbench 8.0.1