Comparative genetic mapping between duplicated segments on maize chromosomes 3 and 8 and homoeologous regions in sorghum and sugarcane

Comparative mapping within maize, sorghum and sugarcane has previously revealed the existence of syntenic regions between the crops. In the present study, mapping on the sorghum genome of a set of probes previously located on the maize and sugarcane maps allow a detailed analysis of the relationship between maize chromosomes 3 and 8 and sorghum and sugarcane homoeologous regions. Of 49 loci revealed by 46 (4 sugarcane and 42 maize) polymorphic probes in sorghum, 42 were linked and were assigned to linkage groups G (28), E (10) and I (4). On the basis of common probes, a complete co-linearity is observed between sorghum linkage group G and the two sugarcane linkage groups II and III. The comparison between the consensus sorghum/sugarcane map (G/II/III) and the maps of maize chromosomes 3 and 8 reveals a series of linkage blocks within which gene orders are conserved. These blocks are interspersed with non-homoeologous regions corresponding to the central part of the two maize chromosomes and have been reshuffled, resulting in several inversions in maize compared to sorghum and sugarcane. The results emphasize the fact that duplication will considerably complicate precise comparative mapping at the whole genome scale between maize and other Poaceae.     

Host Plant Specialization in the Sugarcane Aphid Melanaphis sacchari

Most aphids are highly specialized on one or two related plant species and generalist species often include sympatric populations adapted to different host plants. Our aim was to test the hypothesis of the existence of host specialized lineages of the aphid Melanaphis sacchari in Reunion Island. To this end, we investigated the genetic diversity of the aphid and its association with host plants by analyzing the effect of wild sorghum Sorghum bicolor subsp. verticilliflorum or sugarcane as host plants on the genetic structuring of populations and by performing laboratory host transfer experiments to detect trade-offs in host use. Genotyping of 31 samples with 10 microsatellite loci enabled identification of 13 multilocus genotypes (MLG). Three of these, Ms11, Ms16 and Ms15, were the most frequent ones. The genetic structure of the populations was linked to the host plants. Ms11 and Ms16 were significantly more frequently observed on sugarcane, while Ms15 was almost exclusively collected in colonies on wild sorghum. Laboratory transfer experiments demonstrated the existence of fitness trade-offs. An Ms11 isofemale lineage performed better on sugarcane than on sorghum, whereas an Ms15 lineage developed very poorly on sugarcane, and two Ms16 lineages showed no significant difference in performances between both hosts. Both field and laboratory results support the existence of host plant specialization in M. sacchari in Reunion Island, despite low genetic differentiation. This study illustrates the ability of asexual aphid lineages to rapidly undergo adaptive changes including shifting from one host plant to another.     

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance.

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.     

Construction of a composite sorghum genome map and comparison with sugarcane, a related complex polyploid

 A sorghum composite linkage map was constructed with two recombinant inbred line populations using heterologous probes already mapped on maize and sugarcane. This map includes 199 loci revealed by 188 probes and distributed on 13 linkage groups. A comparison based on 84 common probes was performed between the sorghum composite map and a map of a sugarcane (Saccharum spp.) cultivar being developed and presently comprising 10 tentative linkage groups. A straight synteny was observed for 2 pairs of linkage groups; in two cases, 1 sorghum linkage group corresponded to 2 or 3 sugarcane linkage groups, respectively; in two cases 1 sugarcane link- age group corresponded to 2 separate sorghum linkage groups; for 2 sorghum linkage groups, no complete correspondance was found in the sugarcane genome. In most cases loci appeared to be colinear between homoeologous chromosomal segments in sorghum and sugarcane. These results are discussed in relation to published data on sorghum genomic maps, with specific reference to the genetic organization of sugarcane cultivars, and they, illustrate how investigations on relatively simple diploid genomes as sorghum will facilitate the mapping of related polyploid species such as sugarcane. Received: 12 August 1996 / Accepted: 30 August 1996     

Comparative analysis of QTLs affecting plant height and flowering among closely-related diploid and polyploid genomes.

Quantitative trait loci (QTLs) affecting plant height and flowering were studied in the two Saccharum species from which modern sugarcane cultivars are derived. Two segregating populations derived from interspecific crosses between Saccharum officinarum and Saccharum spontaneum were genotyped with 735 DNA markers. Among the 65 significant associations found between these two traits and DNA markers, 35 of the loci were linked to sugarcane genetic maps and 30 were unlinked DNA markers. Twenty-one of the 35 mapped QTLs were clustered in eight genomic regions of six sugarcane homologous groups. Some of these could be divergent alleles at homologous loci, making the actual number of genes implicated in these traits much less than 35. Four QTL clusters controlling plant height in sugarcane corresponded closely to four of the six plant-height QTLs previously mapped in sorghum. One QTL controlling flowering in sugarcane corresponded to one of three flowering QTLs mapped in sorghum. The correspondence in locations of QTLs affecting plant height and flowering in sugarcane and sorghum reinforce the notion that the simple sorghum genome is a valuable "template" for molecular dissection of the much more complex sugarcane genome.     

The production‐ecological sustainability of cassava,sugarcane and sweet sorghum cultivation for bioethanol in Mozambique

We present an approach for providing quantitative insight into the production‐ecological sustainability of biofuel feedstock production systems. The approach is based on a simple crop‐soil model and was used for assessing feedstock from current and improved production systems of cassava for bioethanol. Assessments were performed for a study area in Mozambique, a country considered promising for biomass production. Our focus is on the potential role of smallholders in the production of feedstock for biofuels. We take cassava as the crop for this purpose and compare it with feedstock production on plantations using sugarcane, sweet sorghum and cassava as benchmarks. Production‐ecological sustainability was defined by seven indicators related to resource‐use efficiency, soil quality, net energy production and greenhouse gas (GHG) emissions. Results indicate that of the assessed systems, sugarcane performed better than cassava, although it requires substantial water for irrigation. Targeted use of nutrient inputs improved sustainability of smallholder cassava. Cassava production systems on more fertile soils were more sustainable than those on less fertile soils; the latter required more external inputs for achieving the same output, affecting most indicators negatively and reducing the feasibility for smallholders. Cassava and sweet sorghum performed similarly. Cassava production requires much more labour per hectare than production of sugarcane or sweet sorghum. Production of bioethanol feedstock on cultivated lands was more sustainable and had potential for carbon sequestration, avoiding GHG emissions from clearing natural vegetation if new land is opened.     

Structure-based screening for functional non-coding RNAs in fission yeast identifies a factor repressing untimely initiation of sexual differentiation

Non-coding RNAs (ncRNAs) ubiquitously exist in normal and cancer cells. Despite their prevalent distribution, the functions of most long ncRNAs remain uncharacterized. The fission yeast Schizosaccharomyces pombe expresses >1800 ncRNAs annotated to date, but most unconventional ncRNAs (excluding tRNA, rRNA, snRNA and snoRNA) remain uncharacterized. To discover the functional ncRNAs, here we performed a combinatory screening of computational and biological tests. First, all S. pombe ncRNAs were screened in silico for those showing conservation in sequence as well as in secondary structure with ncRNAs in closely related species. Almost a half of the 151 selected conserved ncRNA genes were uncharacterized. Twelve ncRNA genes that did not overlap with protein-coding sequences were next chosen for biological screening that examines defects in growth or sexual differentiation, as well as sensitivities to drugs and stresses. Finally, we highlighted an ncRNA transcribed from SPNCRNA.1669, which inhibited untimely initiation of sexual differentiation. A domain that was predicted as conserved secondary structure by the computational operations was essential for the ncRNA to function. Thus, this study demonstrates that in silico selection focusing on conservation of the secondary structure over species is a powerful method to pinpoint novel functional ncRNAs.     

Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum

Many economically important crops have large and complex genomes that hamper their sequencing by standard methods such as whole genome shotgun (WGS). Large tracts of methylated repeats occur in plant genomes that are interspersed by hypomethylated gene‐rich regions. Gene‐enrichment strategies based on methylation profiles offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration with McrBC endonuclease digestion to enrich for euchromatic regions in the sugarcane genome. To verify the efficiency of methylation filtration and the assembly quality of sequences submitted to gene‐enrichment strategy, we have compared assemblies using methyl‐filtered (MF) and unfiltered (UF) libraries. The use of methy filtration allowed a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5× more scaffolds and 1.7× more assembled Mb in length compared with unfiltered dataset. The coverage of sorghum coding sequences (CDS) by MF scaffolds was at least 36% higher than by the use of UF scaffolds. Using MF technology, we increased by 134× the coverage of gene regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds that covered all genes of the sugarcane bacterial artificial chromosomes (BACs), 97.2% of sugarcane expressed sequence tags (ESTs), 92.7% of sugarcane RNA‐seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds from encoded enzymes of the sucrose/starch pathway discovered 291 single‐nucleotide polymorphisms (SNPs) in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes was also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and for improvement of sugarcane as a biofuel crop.     

A genome-wide BAC end-sequence survey of sugarcane elucidates genome composition, and identifies BACs covering much of the euchromatin

BAC-end sequences (BESs) of hybrid sugarcane cultivar R570 are presented. A total of 66,990 informative BESs were obtained from 43,874 BAC clones. Similarity search using a variety of public databases revealed that 13.5 and 42.8 % of BESs match known gene-coding and repeat regions, respectively. That 11.7 % of BESs are still unmatched to any nucleotide sequences in the current public databases despite the fact that a close relative, sorghum, is fully sequenced, indicates that there may be many sugarcane-specific or lineage-specific sequences. We found 1,742 simple sequence repeat motifs in 1,585 BESs, spanning 27,383 bp in length. As simple sequence repeat markers derived from BESs have some advantages over randomly generated markers, these may be particularly useful for comparing BAC-based physical maps with genetic maps. BES and overgo hybridization information was used for anchoring sugarcane BAC clones to the sorghum genome sequence. While sorghum and sugarcane have extensive similarity in terms of genomic structure, only 2,789 BACs (6.4 %) could be confidently anchored to the sorghum genome at the stringent threshold of having both-end information (BESs or overgos) within 300 Kb. This relatively low rate of anchoring may have been caused in part by small- or large-scale genomic rearrangements in the Saccharum genus after two rounds of whole genome duplication since its divergence from the sorghum lineage about 7.8 million years ago. Limiting consideration to only low-copy matches, 1,245 BACs were placed to 1,503 locations, covering ~198 Mb of the sorghum genome or about 78 % of the estimated 252 Mb of euchromatin. BESs and their analyses presented here may provide an early profile of the sugarcane genome as well as a basis for BAC-by-BAC sequencing of much of the basic gene set of sugarcane.     

Ethanol production potential of sweet sorghum assessed using forage fiber analysis procedures

Sweet sorghum (Sorghum bicolor (L.) Moench) is widely recognized as a highly promising biomass energy crop with particular potential to complement sugarcane production in diversified cropping systems. Agronomic assessments have led to identification of four cultivars well suited for such sugarcane‐based production systems in southern Louisiana. Sweet sorghum biofuel production systems are currently being developed, and research producing large sample numbers requiring ethanol yield assessment is anticipated. Fiber analysis approaches developed for forage evaluation appear to be useful for screening such large numbers of samples for relative ethanol yield. Chemical composition, forage fiber characteristics, digestibility, and ethanol production of sweet sorghum bagasse from the four cultivars were assessed. Measures of detergent fiber, lignin, and digestibility were highly correlated with ethanol production (P < 0.01). The best linear regression models accounted for about 80% of the variation among cultivars in ethanol production. Bagasse from the cultivar Dale produced more ethanol per gram of material than any of the other cultivars. This superior ethanol production was apparently associated with less lignin in stems of Dale. Forage evaluation measures including detergent fiber analyses, in vitro digestibility, and an in vitro gas production technique successfully identified the cultivar superior in ethanol yield indicating their usefulness for screening sweet sorghum samples for potential ethanol production in research programs generating large sample numbers from evaluations of germ plasm or agronomic treatments. These screening procedures reduce time and expense of alternatives such as hexose sugar assessment for calculating theoretical ethanol yield.     

Development of microsatellite markers in autopolyploid sugarcane and comparative analysis of conserved microsatellites in sorghum and sugarcane

Sugarcane has become an increasingly important first-generation biofuel crop in tropical and subtropical regions. It has a large, complex, polyploid genome that has hindered the progress of genomic research and marker-assisted selection. Genetic mapping and ultimately genome sequence assembly require a large number of DNA markers. Simple sequence repeats (SSRs) are widely used in genetic mapping because of their abundance, high rates of polymorphism, and ease of use. The objectives of this study were to develop SSR markers for construction of a saturated genetic map and to characterize the frequency and distribution of SSRs in a polyploid genome. SSR markers were mined from expressed sequence tag (EST), reduced representation library genomic sequences, and bacterial artificial chromosome (BAC) sequences. A total of 5,675 SSR markers were surveyed in a segregating population. The overall successful amplification and polymorphic rates were 87.9 and 16.4%, respectively. The trinucleotide repeat motifs were most abundant, with tri- and hexanucleotide motifs being the most abundant for the ESTs. BAC and genomic SSRs were mostly AT-rich while the ESTs were relatively GC-rich due to codon bias. These markers were also aligned to the sorghum genome, resulting in 1,203 markers mapped in the sorghum genome. This set of SSRs conserved in sugarcane and sorghum would be the most informative for mapping quantitative trait loci in sugarcane and for comparative genomic analyses. This large collection of SSR markers is a valuable resource for sugarcane genomic research and crop improvement.     

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.     

An EST survey of the sugarcane transcriptome

Its large genome and high polyploidy makes sugarcane (Saccharum spp.) a singularly challenging crop to study and improve using genetic approaches. To provide large numbers of functionally characterized candidate genes that might be tested for direct association (rather than distant linkage) with economically important traits, we sequenced the 5' ends of 9,216 clones from three cDNA libraries (apex, leaf and mature internode), representing 3,401 non-redundant sequences. About 57% of these sequences could be assigned a tentative function based on statistically significant similarity to previously characterized proteins or DNA sequences. Another 28% corresponded to previously identified, but uncharacterized, sequences. Some of the remaining unidentified sequences were predicted to be genes which could potentially be new to plants or unique to sugarcane. Comparisons of the sugarcane ESTs to a large sorghum EST database revealed similar compositions of expressed genes between some different tissues. Comparison to a detailed Arabidopsis protein database showed some highly conserved sequences, which might be useful DNA markers for pan-angiosperm comparative mapping. These EST sequences provide a foundation for many new studies to accelerate isolation of agronomically important genes from the cumbersome sugarcane genome.     

Genomic organization of the ribosomal DNA of sorghum and its close relatives

Summary The structure and organization of the ribosomal DNA (rDNA) of sorghum (Sorghum bicolor) and several closely related grasses were determined by gel blot hybridization to cloned maize rDNA. Monocots of the genus Sorghum (sorghum, shattercane, Sudangrass, and Johnsongrass) and the genus Saccharum (sugarcane species) were observed to organize their rDNA as direct tandem repeats of several thousand rDNA monomer units. For the eight restriction enzymes and 14 cleavage sites examined, no variations were seen within all of the S. bicolor races and other Sorghum species investigated. Sorghum, maize, and sugarcane were observed to have very similar rDNA monomer sizes and restriction maps, befitting their close common ancestry. The restriction site variability seen between these three genera demonstrated that sorghum and sugarcane are more closely related to each other than either is to maize. Variation in rDNA monomer lengths were observed frequently within the Sorghum genus. These size variations were localized to the intergenic spacer region of the rDNA monomer. Unlike many maize inbreds, all inbred Sorghum diploids were found to contain only one rDNA monomer size in an individual plant. These results are discussed in light of the comparative timing, rates, and modes of evolutionary events in Sorghum and other grasses. Spacer size variation was found to provide a highly sensitive assay for the genetic contribution of different S. bicolor races and other Sorghum species to a Sorghum population.     

Multivariate analysis of sorghum volatiles for the fast screening of sugarcane aphid infestation

The sugarcane aphid, Melanaphis sacchari, has been a severe pest throughout the sorghum field in Texas, which can worse the sorghum yield economically. For this purpose of early detection, the mechanism of herbivore-induced plant volatiles (HIPVs) needs to be utilized in the detection method. In this study, the HayeSep Q adsorbent combined gas chromatography mass spectrometry (GC/MS) was tested to analyze the volatile organic compounds (VOCs) that sorghum can emit when they are in good shape as well as they are infested by the sugarcane aphids, and multivariate techniques were performed for the fast screening of the infestation. Several VOCs identified from Student’s t-test with p < 0.05 were finally chosen as variables for multivariate analysis, and both unsupervised learning of principal component analysis (PCA) and clustering analysis (CA) and supervised learning of linear discriminant analysis (LDA) were done, showing good performance on discrimination between healthy and infested sorghum.