GISH: resolving interspecific and intergeneric hybrids.

Genomic in situ hybridization (GISH) is an invaluable cytogenetic technique which enables the visualization of whole genomes in hybrids and polyploidy taxa. Total genomic DNA from one or two different species/genome is used as a probe, labeled with a fluorochrome and directly detected on mitotic chromosomes from root-tip meristems. In sugarcane we were able to characterize interspecific hybrids of two closely related species as well as intergeneric hybrids of two closely related genera.

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns.

BACKGROUND: Sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs) are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out. RESULTS: Sixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. FISH analysis resulted in the expected pattern of Gypsy/heterochromatin, Copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. Analysis of related ESTs and RT-PCR showed transcriptional variation between tissues and families. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements. CONCLUSIONS: Individual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNA's that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome.

Unprecedented enhancement of transient gene expression from minimal cassettes using a double terminator.

The potential of using vector-free minimal gene cassettes (MGCs) with a double terminator for the enhancement and stabilization of transgene expression was tested in sugarcane biolistic transformation. The MGC system used consisted of the enhanced yellow fluorescent protein (EYFP) reporter gene driven by the maize ubiquitin-1 (Ubi) promoter and a single or double terminator from nopaline synthase (Tnos) or/and Cauliflower mosaic virus 35S (35ST). Transient EYFP expression from Tnos or 35ST single terminator MGC was very low and unstable, typically peaking early (8-16 h) and diminishing rapidly (48-72 h) after bombardment. Addition of a ~260 bp vector sequence (VS) to the single MGC downstream of Tnos (Tnos + VS) or 35ST (35ST + VS) enhanced EYFP expression by 1.25- to 25-fold. However, a much more significant increase in EYFP expression was achieved when the VS in 35ST + VS was replaced by Tnos to generate a 35ST-Tnos double terminator MGC, reaching its maximum at 24 h post-bombardment. The enhanced EYFP expression from the double terminator MGC was maintained for a long period of time (168 h), resulting in an overall increase of 5- to 65-fold and 10- to 160-fold as compared to the 35ST and Tnos single terminator MGCs, respectively. The efficiency of the double terminator MGC in enhancing EYFP expression was also demonstrated in sorghum and tobacco, suggesting that the underlying mechanism is highly conserved among monocots and dicots. Our results also suggest the involvement of posttranscriptional gene silencing in the reduced and unstable transgene expression from single terminator MGCs in plants.

Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes.

BACKGROUND: Sugarcane (Saccharum spp.) has become an increasingly important crop for its leading role in biofuel production. The high sugar content species S. officinarum is an octoploid without known diploid or tetraploid progenitors. Commercial sugarcane cultivars are hybrids between S. officinarum and wild species S. spontaneum with ploidy at approximately 12x. The complex autopolyploid sugarcane genome has not been characterized at the DNA sequence level. RESULTS: The microsynteny between sugarcane and sorghum was assessed by comparing 454 pyrosequences of 20 sugarcane bacterial artificial chromosomes (BACs) with sorghum sequences. These 20 BACs were selected by hybridization of 1961 single copy sorghum overgo probes to the sugarcane BAC library with one sugarcane BAC corresponding to each of the 20 sorghum chromosome arms. The genic regions of the sugarcane BACs shared an average of 95.2% sequence identity with sorghum, and the sorghum genome was used as a template to order sequence contigs covering 78.2% of the 20 BAC sequences. About 53.1% of the sugarcane BAC sequences are aligned with sorghum sequence. The unaligned regions contain non-coding and repetitive sequences. Within the aligned sequences, 209 genes were annotated in sugarcane and 202 in sorghum. Seventeen genes appeared to be sugarcane-specific and all validated by sugarcane ESTs, while 12 appeared sorghum-specific but only one validated by sorghum ESTs. Twelve of the 17 sugarcane-specific genes have no match in the non-redundant protein database in GenBank, perhaps encoding proteins for sugarcane-specific processes. The sorghum orthologous regions appeared to have expanded relative to sugarcane, mostly by the increase of retrotransposons. CONCLUSIONS: The sugarcane and sorghum genomes are mostly collinear in the genic regions, and the sorghum genome can be used as a template for assembling much of the genic DNA of the autopolyploid sugarcane genome. The comparable gene density between sugarcane BACs and corresponding sorghum sequences defied the notion that polyploidy species might have faster pace of gene loss due to the redundancy of multiple alleles at each locus.

SacRALF1, a peptide signal from the grass sugarcane (Saccharum spp.), is potentially involved in the regulation of tissue expansion.

Rapid alkalinization factor (RALF) is part of a growing family of small peptides with hormone characteristics in plants. Initially isolated from leaves of tobacco plants, RALF peptides can be found throughout the plant kingdom and they are expressed ubiquitously in plants. We took advantage of the small gene family size of RALF genes in sugarcane and the ordered cellular growth of the grass sugarcane leaves to gain information about the function of RALF peptides in plants. Here we report the isolation of two RALF peptides from leaves of sugarcane plants using the alkalinization assay. SacRALF1 was the most abundant and, when added to culture media, inhibited growth of microcalli derived from cell suspension cultures at concentrations as low as 0.1 microM. Microcalli exposed to exogenous SacRALF1 for 5 days showed a reduced number of elongated cells. Only four copies of SacRALF genes were found in sugarcane plants. All four SacRALF genes are highly expressed in young and expanding leaves and show a low or undetectable level of expression in expanded leaves. In half-emerged leaf blades, SacRALF transcripts were found at high levels at the basal portion of the leaf and at low levels at the apical portion. Gene expression analyzes localize SacRALF genes in elongation zones of roots and leaves. Mature leaves, which are devoid of expanding cells, do not show considerable expression of SacRALF genes. Our findings are consistent with SacRALF genes playing a role in plant development potentially regulating tissue expansion.

Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis.

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and "payload" proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal beta-glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in "storage vacuoles".