Giovanni M Cordeiro

Characterisation of microsatellite markers from Sugarcane ( Saccharum sp.) a highly polyploid species

Cultivated sugarcane varieties (Saccharum spp) are derived from complex interspecific hybridisations between the species S. spontaneum (2n=40-128) and S. officinarum (2n=60 or 80). To analyse this complex genome, the potential of microsatellite repeats as genetic markers in sugarcane with respect to their abundance, variability and ability to detect polymorphisms was investigated. A set of microsatellite markers for genome analysis in cultivated sugarcane was identified from an enriched genomic DNA library constructed from Saccharum sp. cv Q124. Sequencing of 798 sugarcane genomic DNA clones from an enriched microsatellite library, yielded 457 inserts containing microsatellite repeat motifs. Just over 84% of the microsatellites contained dinucleotide or trinucleotide repeats averaging 15 and 13 repeat motifs, respectively. Primer sets were designed and synthesised for over 100 microsatellite sequences and tested on a set of five sugarcane cultivars. Both, heterozygosity as witnessed by the number of alleles, and length polymorphisms as seen in the differences in PCR product size for a particular allele were observed. Microsatellite markers are likely to have many applications in sugarcane genetics and breeding including germplasm analysis, cultivar identification, parent evaluation and marker assisted breeding.

Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost-effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non-trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost-effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single-locus DNA barcode for plants.

Domestication to Crop Improvement: Genetic Resources for Sorghum and Saccharum (Andropogoneae)

Background Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum) are members of the Andropogoneae tribe in the Poaceae and are each others closest relatives amongst cultivated plants. Both are relatively recent domesticates and comparatively little of the genetic potential of these taxa and their wild relatives has been captured by breeding programmes to date. This review assesses the genetic gains made by plant breeders since domestication and the progress in the characterization of genetic resources and their utilization in crop improvement for these two related species. Genetic Resources The genome of sorghum has recently been sequenced providing a great boost to our knowledge of the evolution of grass genomes and the wealth of diversity within S. bicolor taxa. Molecular analysis of the Sorghum genus has identified close relatives of S. bicolor with novel traits, endosperm structure and composition that may be used to expand the cultivated gene pool. Mutant populations (including TILLING populations) provide a useful addition to genetic resources for this species. Sugarcane is a complex polyploid with a large and variable number of copies of each gene. The wild relatives of sugarcane represent a reservoir of genetic diversity for use in sugarcane improvement. Techniques for quantitative molecular analysis of gene or allele copy number in this genetically complex crop have been developed. SNP discovery and mapping in sugarcane has been advanced by the development of high-throughput techniques for ecoTILLING in sugarcane. Genetic linkage maps of the sugarcane genome are being improved for use in breeding selection. The improvement of both sorghum and sugarcane will be accelerated by the incorporation of more diverse germplasm into the domesticated gene pools using molecular tools and the improved knowledge of these genomes.

Sugarcane microsatellites for the assessment of genetic diversity in sugarcane germplasm

Abstract The ability of microsatellite or simple sequence repeat (SSR) markers to determine the level of genetic diversity between members of the genera Saccharum (S. officinarum, S. sponfaneum, S. sinense), Old World Erianthus Michx. sect. Ripidium, North American E. giganteus (S. giganteum), Sorghum and Miscanthus were assessed. Six SSR markers were tested on 66 accessions and produced a total of 187 distinct alleles. Where available, results were compared against published data from other molecular marker systems such as RFLPs, RAPDs, AFLPs and 5S rRNA intergenic spacers. Similarity coefficient calculations and clustering revealed a genetic structure for Saccharum and Erianthus sect. Ripidium that reflected closely the relationship previously identified using other marker systems. The results indicated that SSRs will be an ideal means for the identification of the genetic constitution of modern sugarcane cultivars of interspecific origins.

EST versus Genomic Derived Microsatellite Markers for Genotyping Wild and Cultivated Barley

Genetic variation present in wild and cultivated barley populations was investigated using two sources of microsatellite also known as simple sequence repeat (SSR) markers. EST-SSRs are derived from expressed sequences and genomic SSRs are isolated from genomic DNA. Genomic SSR markers detected a higher level of polymorphism than those derived from ESTs. Polymorphism information content was higher in genomic SSRs than EST-derived SSRs. This study showed that the EST-SSR markers developed in cultivated barley are polymorphic in wild and cultivated varieties and produced high quality markers. Ten of these functional markers were polymorphic across the accessions studied. EST markers indicated clearer separation between wild and cultivated barley than genomic SSRs. The EST-SSRs are a valuable source of new polymorphic markers and should be highly applicable to barley genetic resources, providing a direct estimate of functional biodiversity.

Identification of microsatellite markers for fragrance in rice by analysis of the rice genome sequence

Several chemical constituents are important to the fragrance of cooked rice. However, the chemical compound 2-acetyl-1-pyrroline (AP) is regarded as the most important component of fragrance in the basmati- and jasmine-style fragrant rices. AP is found in all parts of the plant except the roots. It is believed that a single recessive gene is responsible for the production of fragrance in most rice plants. The detection of fragrance can be carried out via sensory or chemical methods, although each has their disadvantages. To overcome these difficulties, we have identified an (AT)40 repeat microsatellite or simple sequence repeat (SSR) marker for fragrant and non-fragrant alleles of the fgr gene. Identification of this marker was facilitated through use of both the publicly available and restricted access sequence information of the Monsanto rice sequence databases. Fifty F2 individuals from a mapping population were genotyped for the polymorphic marker. This marker has a high polymorphism information content (PIC = 0.9). Other SSR markers linked to fragrance could be identified in the same way of use in other populations. This study demonstrates that analysis of the rice genome sequence is an effective option for identification of markers for use in rice improvement.

A single nucleotide polymorphism (SNP) marker linked to the fragrance gene in rice (Oryza sativa L.)

The whole rice genome sequence was used to assist in the identification of a single nucleotide polymorphism (SNP) marker linked to the fragrance gene (fgr) in rice. Genes flanked by restriction fragment length polymorphism and microsatellite markers known to be linked to the fragrance gene were identified by DNA sequence alignment of EST sequences against BAC clones covering this region of chromosome eight. Re-sequencing and comparison of parts of these genes derived from a fragrant and a non-fragrant cultivar revealed only one SNP (a C/T transition) in more than 6 kbp of sequence from 14 genes. Ten of eleven fragrant genotypes and six of 14 non-fragrant genotypes tested carried the C allele. This approach indicated a generally low level of SNP polymorphism in cultivated rice suggesting that association of SNP with phenotypes should be an efficient path to gene discovery in cultivated rice.

Optimisation of a microsatellite enrichment technique in Saccharum spp.

The creation of enriched microsatellite libraries can ensure an abundant supply of microsatellite sequences at a considerably reduced cost. We report here the modification to an existing enrichment protocol and the optimisation of this technique for use on specific plant species, in this case, sugarcane. Using this optimised protocol, we achieved a 40% increase in the level of enrichment.

DNA Banks and their role in facilitating the application of genomics to plant germplasm

Advances in genomics have provided technologies for high throughput analysis of plant genomes with potential for use in gene discovery in germplasm collections. The establishment of DNA banks facilitates this screening by making DNA from large numbers of plant accessions widely available. DNA banks require the development of appropriate policies for access and benefit sharing. Tools for automating sample and data handling are essential. Standard molecular methods for fingerprinting DNA accessions for international comparisons need to be determined. New screening technologies are required to take advantage of the emerging availability of large DNA collections. The Australian Plant DNA Bank aims to collect DNA from all Australian plant species and to sample the diversity within each species. DNA from all individuals of the species is being stored for rare species. Domesticated or economically important species from all countries are also being collected and stored. International networking of DNA banks will be a key step in linking genomics tools to global plant diversity.

New Saccharum hybrids in S. spontaneum cytoplasm developed through a combination of conventional and molecular breeding approaches

Identification of sugarcane F 1 hybrids is difficult when selections are based solely on morphological traits. Our objective was to combine morphological traits and molecular marker analysis to select F 1 hybrids from two separate crosses between Djatiroto, a clone of Saccha rum spontaneum, and elite sugarcane clones, LCP 85-384 (Cross 97-3144) and CP 62-258 (Cross 97-3146). The maternal inflorescences of Djatiroto were emasculated by submersion in a circulating 45°C hot-water tank for 10 min to minimize self-fertilization. Cross 97-3144 produced 4.7 g of seeds with 338 viable seeds per gram and Cross 97-3146 produced 2.4 g of seeds with 166 viable seeds per gram. After greenhouse germination, 96 progeny from each cross were evaluated in a field plot. Evaluations were conducted on the ratoon crops for stalk diameter (mm), juice Brix (percentage soluble solids), and a randomly amplified polymorphic DNA (RAPD) marker OPA-1 1-366 that was reproducibly amplified through PCR from the elite clones, but not the maternal S. spontaneum clone. Fifty progeny (52.1%) from Cross 97-3144 and 36 progeny (37.5%) from Cross 97-3146 inherited the RAPI) marker. Five putative F1 progeny were selected from each cross, namely US 99-43, US 99-44, US 99-45, US 99-46 and US 99-47 from Cross 97-3144, and US 99-48, US 99-49, US 99-50, US 99-51 and US 99-52 from Cross 97-3146, based on their relatively larger stalk diameter, higher Brix and inheritance of the RAP ID marker. The hybrid nature of these selected progeny was verified with sugarcane microsatellite markers. This is the first report of the development of Saccharum hybrids with the cytoplasm of S. spontaneum for breeding purpose through a combination of conventional and molecular breeding approaches. Availability of these F1 hybrids could enhance the genetic diversity of Saccharum germplasm and has enabled sugarcane geneticists and breeders to explore the possible contribution of S. spontaneum cytoplasm in the development of new sugarcane cultivars.