CRISPR/Cas9 Editing Sites Identification and Multi-Elements Association Analysis in Camellia sinensis.

CRISPR/Cas9 is an efficient genome-editing tool, and the identification of editing sites and potential influences in the Camellia sinensis genome have not been investigated. In this study, bioinformatics methods were used to characterise the Camellia sinensis genome including editing sites, simple sequence repeats (SSRs), G-quadruplexes (GQ), gene density, and their relationships. A total of 248,134,838 potential editing sites were identified in the genome, and five PAM types, AGG, TGG, CGG, GGG, and NGG, were observed, of which 66,665,912 were found to be specific, and they were present in all structural elements of the genes. The characteristic region of high GC content, GQ density, and PAM density in contrast to low gene density and SSR density was identified in the chromosomes in the joint analysis, and it was associated with secondary metabolites and amino acid biosynthesis pathways. CRISPR/Cas9, as a technology to drive crop improvement, with the identified editing sites and effector elements, provides valuable tools for functional studies and molecular breeding in Camellia sinensis.

Stoichiometry versus ecology: the relationships between genome size and guanine-cytosine content, and tissue nitrogen and phosphorus in grassland herbs.

BACKGROUND AND AIMS: Plant tissue nitrogen (N) and phosphorus (P) and genome traits, such as genome size and guanine-cytosine (GC) content, scale with growth or metabolic rates and are linked to plant ecological strategy spectra. Tissue NP stoichiometry and genome traits are reported to affect plant growth, metabolic rates or ecological strategies in contrasting ways, although the elemental costs for building and maintaining DNA are typically overlooked. METHODS: We formulated stoichiometry- and ecology-based predictions on the relationship between genome size and GC content to tissue N, P and N : P and tested them on a set of 130 herbaceous species from a temperate grassland using ordinary, phylogenetic and quantile regression. KEY RESULTS: Genome size was only negatively linked to plant N and N : P in species with very small genomes. We found no link between genome size and plant P. GC content was negatively linked to plant N and P but we found these significant links consistently in both GC-rich and GC-poor species. Finally, GC content correlated positively with plant N : P but only in species with GC-rich genomes. CONCLUSIONS: Our results provide stronger support for the ecology-based predictions than the stoichiometry-based predictions, and for the links between GC content and plant N and P stoichiometry than for genome size. We argue that the theories of plant metabolic rates and ecological strategies (resource-acquisitive vs. conservative or ruderal vs. stress-tolerator spectra) better explain interspecific genome-NP stoichiometry relationships at the tissue level (although relatively weakly) than the stoichiometric theory based on the elemental costs for building and maintaining DNA.

Contributions to cytogenetics of Plectranthusbarbatus Andr. (Lamiaceae): a medicinal plant.

Accessions of Plectranthusbarbatus (Lamiaceae), a medicinal plant, were investigated using a cytogenetic approach and flow cytometry (FCM). Here, we describe for the first time details of the karyotype including chromosome morphology, physical mapping of GC rich bands (CMA3 banding), as well as the mapping of 45S and 5S rDNA sites. All accessions studied showed karyotypes with 2n = 30 small metacentric and submetacentric chromosomes. The CMA3 banding and fluorescent in situ hybridization techniques revealed coincidence between CMA3 bands and 45S rDNA sites (6 terminal marks) while for the 5S rDNA were observed 4 subterminal marks no coincident with CMA3 marks. For nuclear genome size measurement, the FCM procedure provided histograms with G0/G1 peaks exhibiting CV between 2.0-4.9 and the mean values obtained for the species was 2C = 2.78 pg, with AT% = 61.08 and GC% = 38.92. The cytogenetic data obtained here present new and important information which enables the characterization of Plectranthusbarbatus.

Novel polymorphisms in UTR and coding region of inducible heat shock protein 70.1 gene in tropically adapted Indian zebu cattle (Bos indicus) and riverine buffalo (Bubalus bubalis).

Due to evolutionary divergence, cattle (taurine, and indicine) and buffalo are speculated to have different responses to heat stress condition. Variation in candidate genes associated with a heat-shock response may provide an insight into the dissimilarity and suggest targets for intervention. The present work was undertaken to characterize one of the inducible heat shock protein genes promoter and coding regions in diverse breeds of Indian zebu cattle and buffaloes. The genomic DNA from a panel of 117 unrelated animals representing 14 diversified native cattle breeds and 6 buffalo breeds were utilized to determine the complete sequence and gene diversity of HSP70.1 gene. The coding region of HSP70.1 gene in Indian zebu cattle, Bos taurus and buffalo was similar in length (1,926 bp) encoding a HSP70 protein of 641 amino acids with a calculated molecular weight (Mw) of 70.26 kDa. However buffalo had a longer 5' and 3' untranslated region (UTR) of 204 and 293 nucleotides respectively, in comparison to Indian zebu cattle and Bos taurus wherein length of 5' and 3'-UTR was 172 and 286 nucleotides, respectively. The increased length of buffalo HSP70.1 gene compared to indicine and taurine gene was due to two insertions each in 5' and 3'-UTR. Comparative sequence analysis of cattle (taurine and indicine) and buffalo HSP70.1 gene revealed a total of 54 gene variations (50 SNPs and 4 INDELs) among the three species in the HSP70.1 gene. The minor allele frequencies of these nucleotide variations varied from 0.03 to 0.5 with an average of 0.26. Among the 14 B. indicus cattle breeds studied, a total of 19 polymorphic sites were identified: 4 in the 5'-UTR and 15 in the coding region (of these 2 were non-synonymous). Analysis among buffalo breeds revealed 15 SNPs throughout the gene: 6 at the 5' flanking region and 9 in the coding region. In bubaline 5'-UTR, 2 additional putative transcription factor binding sites (Elk-1 and C-Re1) were identified, other than three common sites (CP2, HSE and Pax-4) observed across all the analyzed animals. No polymorphism was found within the 3'-UTR of Indian cattle or buffalo as it was found to be monomorphic. The promoter sequences generated in 117 individuals showed a rich array of sequence elements known to be involved in transcription regulation. A total of 11 nucleotide changes were observed in the promoter sequence across the analyzed species, 3 of these changes were located within the potential transcription factor binding domains. We also identified 4 microsatellite markers within the buffalo HSP70.1 gene and 3 microsatellites within bovine HSP70.1. The present study identified several distinct changes across indicine, taurine and bubaline HSP70.1 genes that could further be evaluated as molecular markers for thermotolerance.