Development of Protocols for Regeneration and Transformation of Apomitic and Sexual Forms of Dallisgrass (Paspalum dilatatum Poir.).

Paspalum dilatatum (common name dallisgrass), a productive C4 grass native to South America, is an important pasture grass found throughout the temperate warm regions of the world. It is characterized by its tolerance to frost and water stress and a higher forage quality than other C4 forage grasses. P. dilatatum includes tetraploid (2n = 40), sexual, and pentaploid (2n = 50) apomictic forms, but is predominantly cultivated in an apomictic monoculture, which implies a high risk that biotic and abiotic stresses could seriously affect the grass productivity. The obtention of reproducible and efficient protocols of regeneration and transformation are valuable tools to obtain genetic modified grasses with improved agronomics traits. In this review, we present the current regeneration and transformation methods of both apomictic and sexual cultivars of P. dilatatum, discuss their strengths and limitations, and focus on the perspectives of genetic modification for producing new generation of forages. The advances in this area of research lead us to consider Paspalum dilatatum as a model species for the molecular improvement of C4 perennial forage species.

Gene discovery and molecular marker development, based on high-throughput transcript sequencing of Paspalum dilatatum Poir.

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.

Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv.

Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.

Use of AFLP and RAPD molecular genetic markers and cytogenetic analysis to explore relationships among taxa of the Patagonian Bromus setifolius complex.

Bromus setifolius var. pictus (Hook) Skottsb., B. setifolius var. setifolius Presl. and B.setifolius var. brevifolius Ness are three native Patagonian taxa in the section Pnigma Dumort of the genus Bromus L. AFLP and RAPD analysis, in conjunction with genetic distance measurements and statistical techniques, revealed variation within this group and indicated that B. setifolius var. brevifolius was closely related to B. setifolius var. pictus, with both taxa being more distantly related to B. setifolius var. setifolius. Cytogenetic analysis confirmed the chromosomal number of B. setifolius var. pictus (2n = 70) and B. setifolius var. setifolius (2n = 28) and showed for the first time that B. setifolius var. brevifolius had 2n = 70. The combination of molecular genetic and cytogenetic evidence supported a species status for two of the three taxa and suggested hypotheses for the evolutionary origin of these complex taxa. Species status was also indicated for B. setifolius var. setifolius. Based on these findings, we suggest that B. setifolius var. pictus be referred to as B. pictus Hook var. pictus, and B. setifolius var brevifolius as B. pictus Hook var brevifolius. The correlation between AFLP diversity and variation in ecological parameters suggested that this marker system could be used to assess breeding progress and to monitor the domestication of Patagonian Bromus species for agronomic use.

Use of AFLP and RAPD molecular genetic markers and cytogenetic analysis to explore relationships among taxa of the Patagonian Bromus setifolius complex

Bromus setifolius var. pictus (Hook) Skottsb., B. setifolius var. setifolius Presl. and B. setifolius var. brevifolius Ness are three native Patagonian taxa in the section Pnigma Dumort of the genus Bromus L. AFLP and RAPD analysis, in conjunction with genetic distance measurements and statistical techniques, revealed variation within this group and indicated that B. setifolius var. brevifolius was closely related to B. setifolius var. pictus, with both taxa being more distantly related to B. setifolius var. setifolius. Cytogenetic analysis confirmed the chromosomal number of B. setifolius var. pictus (2n = 70) and B. setifolius var. setifolius (2n = 28) and showed for the first time that B. setifolius var. brevifolius had 2n = 70. The combination of molecular genetic and cytogenetic evidence supported a species status for two of the three taxa and suggested hypotheses for the evolutionary origin of these complex taxa. Species status was also indicated for B. setifolius var. setifolius. Based on these findings, we suggest that B. setifolius var. pictus be referred to as B. pictus Hook var. pictus, and B. setifolius var brevifolius as B. pictus Hook var brevifolius. The correlation between AFLP diversity and variation in ecological parameters suggested that this marker system could be used to assess breeding progress and to monitor the domestication of Patagonian Bromus species for agronomic use.