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Flow cytometric characterisation of the complex polyploid genome of Saccharum officinarum and modern sugarcane cultivars.

ABSTRACT: Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.

SUBMITTER: Metcalfe CJ

PROVIDER: S-EPMC6920420 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Flow cytometric characterisation of the complex polyploid genome of Saccharum officinarum and modern sugarcane cultivars.

Metcalfe Cushla J CJ Li Jingchuan J Giorgi Debora D Doležel Jaroslav J Piperidis Nathalie N Aitken Karen S KS

Scientific reports 20191218 1

Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were ...[more]

PMID: 31852940

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Project description:BackgroundSugarcane (Saccharum officinarum L.) is an economically important crop, mainly due to the production of sugar and biofuel (Azevedo RA, Carvalho RF, Cia MC, & Gratão PL, Trop Plant Biol 4:42-51, 2011). Grown mainly in tropical and subtropical countries, sugarcane is a highly polyploid plant with up to ten copies of each chromosome, which increases the difficulties of genome assembly and genetic, physiologic and biochemical analyses. The increasing demands of sugar and the increasing cost of sugarcane harvest require sugarcane varieties which can shed their leaves during the maturity time, so it is important to study the mechanism of leaf abscission in sugarcane.ResultsTo improve the understanding of miRNA roles in sugarcane leaf abscission, we reported the genome-wide characterization of miRNAs and their putative targets in sugarcane using deep sequencing for six small RNA libraries. In total, 93 conserved miRNAs and 454 novel miRNAs were identified in sugarcane using previously reported transcriptome as reference. Among them, 25 up-regulated and 13 down-regulated miRNAs were identified in leaf abscission sugarcane plants (LASP) compared to leaf packaging sugarcane plants (LPSP). Target prediction revealed several miRNA-mRNA modules including miR156-SPL, miR319-TPR2, miR396-GRF and miR408-LAC3 might be involved in the sugarcane leaf abscission. KEGG pathway enrichment analysis showed differentially expressed miRNAs may regulate pathways like "plant hormone signal transduction" and "plant-pathogen interaction", which is consistent with previous transcriptome study. In addition, we identified 96 variant miRNAs with 135 single nucleotide polymorphisms (SNPs). The expression of sugarcane miRNAs and variant miRNAs were confirmed by qRT-PCR. We identified a possible poaceae specific miRNA called miR5384 for the first time in sugarcane.ConclusionsWe not only reported miR5384, a possible poaceae specific miRNA, for the first time in sugarcane but also presented some miRNA-mRNA modules including miR156-SPL, miR319-TPR2, miR396-GRF and miR408-LAC in sugarcane. These modules might be involved in the regulation of sugarcane leaf abscission during the maturity time. All of these findings may lay ground work for future application of sugarcane breeding program and benefit research studies of sugarcane miRNAs.

Project description:BackgroundSugarcane (Saccharum officinarum L.) is an important sugar crop which belongs to the grass family and can be used for fuel ethanol production. The growing demands for sugar and biofuel is asking for breeding a sugarcane variety that can shed their leaves during the maturity time due to the increasing cost on sugarcane harvest.ResultsTo determine leaf abscission related genes in sugarcane, we generated 524,328,950 paired reads with RNA-Seq and profiled the transcriptome of new born leaves of leaf abscission sugarcane varieties (Q1 and T) and leaf packaging sugarcane varieties (Q2 and B). Initially, 275,018 transcripts were assembled with N50 of 1,177 bp. Next, the transcriptome was annotated by mapping them to NR, UniProtKB/Swiss-Prot, Gene Ontology and KEGG pathway databases. Further, we used TransDecoder and Trinotate to obtain the likely proteins and annotate them in terms of known proteins, protein domains, signal peptides, transmembrane regions and rRNA transcripts. Different expression analysis showed 1,202 transcripts were up regulated in leaf abscission sugarcane varieties, relatively to the leaf packaging sugarcane varieties. Functional analysis told us 62, 38 and 10 upregulated transcripts were involved in plant-pathogen interaction, response to stress and abscisic acid associated pathways, respectively. The upregulation of transcripts encoding 4 disease resistance proteins (RPM1, RPP13, RGA2, and RGA4), 6 ABC transporter G family members and 16 transcription factors including WRK33 and heat stress transcription factors indicate they may be used as candidate genes for sugarcane breeding. The expression levels of transcripts were validated by qRT-PCR. In addition, we characterized 3,722 SNPs between leaf abscission and leaf packaging sugarcane plants.ConclusionOur results showed leaf abscission associated genes in sugarcane during the maturity period. The output of this study provides a valuable resource for future genetic and genomic studies in sugarcane.

Project description:Sugarcane Bacilliform Guadeloupe A Virus (SCBGAV, genus Badnavirus, family Caulimoviridae) is an emerging, deleterious pathogen of sugarcane which presents a substantial barrier to producing high sugarcane earnings. Sugarcane bacilliform viruses (SCBVs) are one of the main species that infect sugarcane. During the last 30 years, significant genetic changes in SCBV strains have been observed with a high risk of disease incidence associated with crop damage. SCBV infection may lead to significant losses in biomass production in susceptible sugarcane cultivars. The circular, double-stranded (ds) DNA genome of SCBGAV (7.4 Kb) is composed of three open reading frames (ORFs) on the positive strand that replicate by a reverse transcriptase. SCBGAV can infect sugarcane in a semipersistent manner via the insect vectors sugarcane mealybug species. In the current study, we used miRNA target prediction algorithms to identify and comprehensively analyze the genome-wide sugarcane (Saccharum officinarum L.)-encoded microRNA (miRNA) targets against the SCBGAV. Mature miRNA target sequences were retrieved from the miRBase (miRNA database) and were further analyzed for hybridization to the SCBGAV genome. Multiple computational approaches-including miRNA-target seed pairing, multiple target positions, minimum free energy, target site accessibility, maximum complementarity, pattern recognition and minimum folding energy for attachments-were considered by all algorithms. Among them, sof-miR396 was identified as the top effective candidate, capable of targeting the vital ORF3 of the SCBGAV genome. miRanda, RNA22 and RNAhybrid algorithms predicted hybridization of sof-miR396 at common locus position 3394. The predicted sugarcane miRNAs against viral mRNA targets possess antiviral activities, leading to translational inhibition by mRNA cleavage. Interaction network of sugarcane-encoded miRNAs with SCBGAV genes, created using Circos, allow analyze new targets. The finding of the present study acts as a first step towards the creation of SCBGAV-resistant sugarcane through the expression of the identified miRNAs.

Dataset Information

Flow cytometric characterisation of the complex polyploid genome of Saccharum officinarum and modern sugarcane cultivars.

Publications

Flow cytometric characterisation of the complex polyploid genome of Saccharum officinarum and modern sugarcane cultivars.

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