Ulrik P. John

Breeding Forage Plants in the Genome Era

Forage plant breeding has been largely based on phenotypic selection following sexual recombination of natural genetic variation found between and within ecotypes. Advances in plant genetic manipulation over the last 15 years have provided convincing evidence that these powerful technologies can complement and enhance plant breeding programs. Significant progress in the establishment of the methodologies required for the molecular breeding of forage plants has been made. Examples of current products and approaches for the application of these methodologies to forage grass and legume improvement are outlined. Large-scale genomic analysis of many organisms is under way with human, arabidopsis and rice genome sequences almost completed. Forage plant breeding is just now entering the genome era. The plethora of new technologies and tools now available for high-throughput gene discovery and genome-wide gene expression analysis have opened up opportunities for innovative applications in the identification, functional characterisation and use of genes of value in forage production systems and beyond. Examples of these opportunities, such as ‘molecular phenotyping’, ‘symbio-genomics’ and ‘xeno-genomics’ are introduced.

Cold acclimation induces rapid and dynamic changes in freeze tolerance mechanisms in the cryophile Deschampsia antarctica E. Desv.

The cryophilic Antarctic hair grass, Deschampsia antarctica E. Desv., one of two higher plants indigenous to Antarctica, represents a unique resource for the study of freeze tolerance mechanisms. We have previously characterized a multi-gene family in D. antarctica encoding ice recrystallization inhibition proteins (IRIPs) whose transcript levels are responsive to cold acclimation, and whose products confer ice recrystallization inhibition (RI) activity that can account for activity seen in cold acclimated plants. We used molecular and physiological analyses to investigate temporal responses of D. antarctica to cold acclimation and de-acclimation, and sub-zero acclimation. Quantitative profiling revealed that IRIP transcript levels significantly increased and decreased within hours of cold acclimation and de-acclimation, respectively, becoming up to 1000-fold more abundant in fully acclimated plants. Western analysis detected three major immuno-reactive bands whose pattern of accumulation mirrored that of transcript. These data correlated with the onset and decline of RI activity in acclimated and de-acclimated leaves. Plant survival-based testing revealed that cold acclimation enhanced freeze tolerance by 5 °C within 4 d, and that sub-zero acclimation conferred an additional 3 °C of tolerance. Thus, D. antarctica is highly responsive to temperature fluctuations, able to rapidly deploy IRIP based RI activity and enhance its freeze tolerance.

Xenogenomics: Genomic Bioprospecting in Indigenous and Exotic Plants Through EST Discovery, cDNA Microarray-Based Expression Profiling and Functional Genomics

To date, the overwhelming majority of genomics programs in plants have been directed at model or crop plant species, meaning that very little of the naturally occurring sequence diversity found in plants is available for characterization and exploitation. In contrast, ‘xenogenomics’ refers to the discovery and functional analysis of novel genes and alleles from indigenous and exotic species, permitting bioprospecting of biodiversity using high-throughput genomics experimental approaches. Such a program has been initiated to bioprospect for genetic determinants of abiotic stress tolerance in indigenous Australian flora and native Antarctic plants. Uniquely adapted Poaceae and Fabaceae species with enhanced tolerance to salt, drought, elevated soil aluminium concentration, and freezing stress have been identified, based primarily on their eco-physiology, and have been subjected to structural and functional genomics analyses. For each species, EST collections have been derived from plants subjected to appropriate abiotic stresses. Transcript profiling with spotted unigene cDNA micro-arrays has been used to identify genes that are transcriptionally modulated in response to abiotic stress. Candidate genes identified on the basis of sequence annotation or transcript profiling have been assayed in planta and other in vivo systems for their capacity to confer novel phenotypes. Comparative genomics analysis of novel genes and alleles identified in the xenogenomics target plant species has subsequently been undertaken with reference to key model and crop plants.

Targeted mining of drought stress-responsive genes from EST resources in Cleistogenes songorica.

Cleistogenes songorica is an important perennial grass found in the pastoral steppe of Inner Mongolia. C. songorica flourishes in drought prone environments, and therefore provides an ideal candidate plant system for the identification of drought-tolerance conferring genes. We constructed cDNA libraries from leaves and roots of drought-stressed C. songorica seedlings. Expressed sequence tag (EST) sequencing of 5664 random cDNA clones produced 3579 high quality, trimmed sequences. The average read length of trimmed ESTs was 613bp. Clustering and assembly identified a non-redundant set of 1499 contigs, including 805 singleton unigenes and 694 multi-member unigenes. The resulting unigenes were functionally categorized according to the Gene Ontology (GO) hierarchy using the in house Bioinformatic Advanced Scientific Computing (BASC) annotation pipeline. Among the total 2.2Mbp of EST sequence data, 161 putative SSRs were found, a frequency similar to that previously observed in oat and Arabidopsis ESTs. Sixty-three unigenes were functionally annotated as being stress responsive, of which 22 were similar to genes implicated in drought stress response. Using quantitative real time RT-PCR, transcripts of 13 of these 22 genes were shown to be at least three fold more, or less abundant in drought-stressed leaves or roots, with 8 increased and 5 decreased in relative transcript abundance. The C. songorica EST and cDNA collections generated in this study are a valuable resource for microarray-based expression profiling, and functional genomics in order to elucidate their role, and to understand the underlying mechanisms of drought-tolerance in C. songorica.

Transgenesis and Genomics in Molecular Breeding of Temperate Pasture Grasses and Legumes

Significant advances in the establishment of the methodologies required for the molecular breeding of temperate forage grasses (Lolium and Festuca species) and legumes (Trifolium and Medicago species) are reviewed. Examples of current products and approaches for the application of these methodologies to forage grass and legume improvement are outlined. The plethora of new technologies and tools now available for high-throughput gene discovery and genome-wide expression analysis have opened up opportunities for innovative applications in the identification, functional characterisation and use of genes of value in forage production systems and beyond. Selected examples of our current work in pasture plant genomics, xenogenomics, symbiogenomics and microarray-based molecular phenotyping are discussed.

Xenogenomics: genomic bioprospecting in indigenous and exotic plants through EST discovery, cDNA microarray-based expression profiling and functional genomics: Conference Papers

The bovine RPCI-42 BAC library was screened to construct a sequence-ready ∼4 Mb single contig of 92 BAC clones on BTA 1q12. The contig covers the region between the genes KRTAP8P1 and CLIC6. This genomic segment in cattle is of special interest as it contains the dominant gene responsible for the hornless or polled phenotype in cattle. The construction of the BAC contig was initiated by screening the bovine BAC library with heterologous cDNA probes derived from 12 human genes of the syntenic region on HSA 21q22. Contig building was facilitated by BAC end sequencing and chromosome walking. During the construction of the contig, 165 BAC end sequences and 109 single-copy STS markers were generated. For comparative mapping of 25 HSA 21q22 genes, genomic PCR primers were designed from bovine EST sequences and the gene-associated STSs mapped on the contig. Furthermore, bovine BAC end sequence comparisons against the human genome sequence revealed significant matches to HSA 21q22 and allowed the in silico mapping of two new genes in cattle. In total, 31 orthologues of human genes located on HSA 21q22 were directly mapped within the bovine BAC contig, of which 16 genes have been cloned and mapped for the first time in cattle. In contrast to the existing comparative bovine–human RH maps of this region, these results provide a better alignment and reveal a completely conserved gene order in this 4 Mb segment between cattle, human and mouse. The mapping of known polled linked BTA 1q12 microsatellite markers allowed the integration of the physical contig map with existing linkage maps of this region and also determined the exact order of these markers for the first time. Our physical map and transcript map may be useful for positional cloning of the putative polled gene in cattle. The nucleotide sequence data reported in this paper have been submitted to EMBL and have been assigned Accession Numbers AJ698510–AJ698674. Copyright