Project description:To determine the centromere of the maize B chromosome, we used previously published anti-CENH3-ChIP-seq data from TB-9Sb, which contain a complete functional B centromere. Distribution of centromere-specific DNA repeats, including CentC, CRM element and B-repeat, were observed in the proximal end of the assembled maize B chromosome, and this region was shown to be associated with CENH3 nucleosomes. Furthermore, six small scaffolds with sizes ranging from 10 to 174 kilobase display CENH3 enrichment, also with the distribution of these repeat sequences. These results were consistent with previously cytogenetic observation. Therefore, approximately 520 kb centromeric regions were determined in the assembled maize B chromosome.
Project description:Venomous animals have traditionally been studied from a proteomic (but also transcriptomic) perspective, often overlooking the study of venom from a genomic point of view until recently. The rise of genomics has led to an increase in the number of reference genomes for non-model organisms, including venomous taxa, enabling new questions on venom evolution from a genomic context. Although venomous snakes are the fundamental model system in venom research, the number of high-quality reference genomes in the group remains limited. In this study, we present a high-quality chromosome-level reference genome for the Arabian horned viper (Cerastes gasperettii), a highly venomous snake native to the Arabian Peninsula. Our highly-contiguous genome allowed us to explore macrochromosomal rearrangements within the Viperidae family, as well as across squamate reptile evolution. Furthermore, we identified a total of ten different toxins conforming the venom’s core, in line with our proteomic results. We also compared microsyntenic changes in the main toxin gene clusters with those of other venomous snake species, highlighting the pivotal role of gene duplication and loss in the emergence and diversification of the two main toxin families for Cerastes gasperettii. Using Illumina data, we reconstructed the demographic history and genome-wide diversity of the species, revealing how historical aridity likely drove population expansions. Finally, this study highlights the importance of using long-read sequencing as well as chromosome-level reference genomes to disentangle the origin and diversification of toxin families in venomous species.
Project description:Venomous animals have traditionally been studied from a proteomic (but also transcriptomic) perspective, often overlooking the study of venom from a genomic point of view until recently. The rise of genomics has led to an increase in the number of reference genomes for non-model organisms, including venomous taxa, enabling new questions on venom evolution from a genomic context. Although venomous snakes are the fundamental model system in venom research, the number of high-quality reference genomes in the group remains limited. In this study, we present a high-quality chromosome-level reference genome for the Arabian horned viper (Cerastes gasperettii), a highly venomous snake native to the Arabian Peninsula. Our highly-contiguous genome allowed us to explore macrochromosomal rearrangements within the Viperidae family, as well as across squamate reptile evolution. Furthermore, we identified a total of ten different toxins conforming the venom’s core, in line with our proteomic results. We also compared microsyntenic changes in the main toxin gene clusters with those of other venomous snake species, highlighting the pivotal role of gene duplication and loss in the emergence and diversification of the two main toxin families for Cerastes gasperettii. Using Illumina data, we reconstructed the demographic history and genome-wide diversity of the species, revealing how historical aridity likely drove population expansions. Finally, this study highlights the importance of using long-read sequencing as well as chromosome-level reference genomes to disentangle the origin and diversification of toxin families in venomous species.
Project description:A small fragment from maize chromosome 3 was created by irradiation by Stadler and Roman and named Duplication 3a (or Dp3a). This small chromosome does not contain any detectable CentC and CRM sequences, but when molecular features of functional centromeres such as CENH3 and CENP-C were examined, they were present. Immunolocalization analysis of phosphorylation of Ser-10 of histone H3 levels on Dp3a shows a pattern typical of a functional centromere. Meiotic analysis revealed that sister chromatids divided equationally at meiosis I as do all small chromosomes examined to date in maize. To examine the sequences associated with CENH3, chromatin immunoprecipitation (ChIP) was carried out with anti-CENH3 antibodies using material from young seedlings with and without Dp3 chromosome as the tissue source. The ChIPed DNA sample was then labeled for FISH detection and prepared for Illumina sequencing.The ChIP-Seq reads were mapped to the B73 reference genome and a significant peak was detected in the Dp3a sample that span 350 kb of the long arm of chromosome 3, which is the candidate region for association with CENH3. ChIP-bisulfite-seq results indicated that there is a slightly increased DNA methylation level after the centromere formation, approaching the level similar to normal centromere regions. Collectively, the results suggest the formation of a de novo centromere on this fragment that initially must have started at the time of X-irradiation release from the progenitor chromosome. These observations add further evidence for the epigenetic nature of centromere function in maize.
Project description:We identified a newly formed dicentric chromosome (sDic-15) in maize from intrachromosomal recombination and BFB cycles, in which only one centromere is active. The centromeres lost CentC sequences and dramatic reduced the CRM sequences, but when the molecular features of functional centromeres such as CENH3 was examined, they were present. Immunolocalization analysis of phosphorylation of H3T3, H3ser-10 and H2A levels on this new centromere shows a pattern typical of a functional centromere. Meiotic analysis revealed that this dicentric chromosome is table and transmit very well. To examine the new sequences associated with CENH3 in this centromere, chromatin immunoprecipitation (ChIP) was carried out with anti-CENH3 antibodies and material from young seedlings with or without dicentric chromosome. We mapped the ChIP-Seq reads to the reference genome and found a 723kb region from the short arm of maize chromosome 9 involve the new centromere formation. This region is gene-poor and full of TEs, but genes in this region are transcribed. The original 723kb region shows a high DNA methylation level as native centromeres but had no significant change when it involved into new centromere formation. The reactivation of this newly formed centromere indicated that centromere reactivation may not dependent on the relatively intact DNA sequences or topology of original inactive centromere.
Project description:A small fragment from maize chromosome 3 was created by irradiation by Stadler and Roman and named Duplication 3a (or Dp3a). This small chromosome does not contain any detectable CentC and CRM sequences, but when molecular features of functional centromeres such as CENH3 and CENP-C were examined, they were present. Immunolocalization analysis of phosphorylation of Ser-10 of histone H3 levels on Dp3a shows a pattern typical of a functional centromere. Meiotic analysis revealed that sister chromatids divided equationally at meiosis I as do all small chromosomes examined to date in maize. To examine the sequences associated with CENH3, chromatin immunoprecipitation (ChIP) was carried out with anti-CENH3 antibodies using material from young seedlings with and without Dp3 chromosome as the tissue source. The ChIPed DNA sample was then labeled for FISH detection and prepared for Illumina sequencing.The ChIP-Seq reads were mapped to the B73 reference genome and a significant peak was detected in the Dp3a sample that span 350 kb of the long arm of chromosome 3, which is the candidate region for association with CENH3. ChIP-bisulfite-seq results indicated that there is a slightly increased DNA methylation level after the centromere formation, approaching the level similar to normal centromere regions. Collectively, the results suggest the formation of a de novo centromere on this fragment that initially must have started at the time of X-irradiation release from the progenitor chromosome. These observations add further evidence for the epigenetic nature of centromere function in maize. ChIP-seq was carried out with anti-CENH3 antibodies using material from young seedlings with and without Dp3a chromosome. For Dp3a, some ChIPed DNA was treated with sodium bisulfite and prepared for Illumina sequencing to test its methylation level.
Project description:Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and Analysis of Sequence Counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. K-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes.
Project description:The goal of the project is to produce a standard annotation of the loci producing small RNAs in the maize genome. To achieve this goal we produced small RNA libraries from four different maize tissues, which will allow the identification of tissue-specific small RNA expression. The availability of bilogical replicates for three of the four tissues analyzed will guarantee robustness in the small RNA genes identification process.