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Updated: May 3, 2026
Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development
Published on: March 5, 2017
Gene discovery and molecular marker development, based on high-throughput transcript sequencing of Paspalum dilatatum
Andrea Giordano1, Noel O I Cogan2, Sukhjiwan Kaur3
1Department of Environment and Primary Industries, AgriBio, Centre for AgriBioscience, Bundoora, Victoria, Australia ; Molecular Plant Breeding Cooperative Research Centre, Bundoora, Victoria, Australia ; Dairy Futures Cooperative Research Centre, Bundoora, Victoria, Australia ; La Trobe University, Bundoora, Victoria, Australia.
Background:
Paspalum dilatatum Poir. (common name dallisgrass) is a native grass species of South America, with special relevance to dairy and red meat production. P. dilatatum exhibits higher forage quality than other C4 forage grasses and is tolerant to frost and water stress. This species is predominantly cultivated in an apomictic monoculture, with an inherent high risk that biotic and abiotic stresses could potentially devastate productivity. Therefore, advanced breeding strategies that characterise and use available genetic diversity, or assess germplasm collections effectively are required to deliver advanced cultivars for production systems. However, there are limited genomic resources available for this forage grass species.
Results:
Transcriptome sequencing using second-generation sequencing platforms has been employed using pooled RNA from different tissues (stems, roots, leaves and inflorescences) at the final reproductive stage of P. dilatatum cultivar Primo. A total of 324,695 sequence reads were obtained, corresponding to c. 102 Mbp. The sequences were assembled, generating 20,169 contigs of a combined length of 9,336,138 nucleotides. The contigs were BLAST analysed against the fully sequenced grass species of Oryza sativa subsp. japonica, Brachypodium distachyon, the closely related Sorghum bicolor and foxtail millet (Setaria italica) genomes as well as against the UniRef 90 protein database allowing a comprehensive gene ontology analysis to be performed. The contigs generated from the transcript sequencing were also analysed for the presence of simple sequence repeats (SSRs). A total of 2,339 SSR motifs were identified within 1,989 contigs and corresponding primer pairs were designed. Empirical validation of a cohort of 96 SSRs was performed, with 34% being polymorphic between sexual and apomictic biotypes.
Conclusions:
The development of genetic and genomic resources for P. dilatatum will contribute to gene discovery and expression studies. Association of gene function with agronomic traits will significantly enable molecular breeding and advance germplasm enhancement.
