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Shinozuka H.,Australian Department of Primary Industries and Fisheries | Shinozuka H.,Molecular Plant Breeding Cooperative Research Center | Cogan N.O.I.,Australian Department of Primary Industries and Fisheries | Cogan N.O.I.,Molecular Plant Breeding Cooperative Research Center | And 6 more authors.
Plant Molecular Biology | Year: 2010

Perennial ryegrass is an obligate outbreeding pasture grass of the Poaceae family, with a two-locus (S and Z) gametophytic self-incompatibility (SI) mechanism. This system has provided a major obstacle to targeted varietal development, and enhanced knowledge is expected to support more efficient breeding strategies. Comparative genetics and physical mapping approaches have been developed to permit molecular cloning of the SI genes. SI gene-linked genetic markers based on heterologous cDNA restriction fragment length polymorphisms (RFLPs) and homologous genomic DNA-derived simple sequence repeats (SSRs) were converted to single nucleotide polymorphism (SNP) format for efficient genotyping. Genetic mapping identified the location of SI loci and demonstrated macrosynteny between related grass species. S- and Z-linked bacterial artificial chromosome (BAC) clones were sequenced using massively parallel pyrosequencing technology to provide the first physical mapping data for Poaceae SI loci. The sequence assembly process suggested a lower prevalence of middle repetitive sequences in the Z locus region and hence precedence for positional cloning strategy. In silico mapping using data from rice, Brachypodium distachyon and Sorghum revealed high sequence conservation in the vicinity of the Z locus region between SI and self-compatible (SC) grass species. Physical mapping identified a total of nine genes encoded in the Z locus region. Expression profiling and nucleotide diversity assessment identified two Z-linked genes, LpTC116908 and LpDUF247, as plausible candidates for the male and female determinants of the S-Z SI system. © 2009 Springer Science+Business Media B.V.


Dracatos P.M.,Australian Department of Primary Industries and Fisheries | Dracatos P.M.,La Trobe University | Dracatos P.M.,Molecular Plant Breeding Cooperative Research Center | Keane P.J.,La Trobe University | And 3 more authors.
Australasian Plant Pathology | Year: 2010

Puccinia coronata (crown rust) is a damaging pathogen of ryegrasses used for both forage and turf applications. Alternative sources of crown rust pathogen resistance with enhanced durability are required for perennial ryegrass breeding programs. This approach may require pyramiding of major and minor resistance gene combinations into varieties, opposing the capacity of evolving pathogen populations to overcome complex resistance mechanisms. Crown rust pathogen populations have been homogenised into distinct lineages on detached leaves through the use of molecular genotyping technology. Subsequent propagation and inoculation of such spore lineages on defined host populations and individuals requires high inoculation efficiency, given the delivery of initially small spore quantities. In this study, an inoculation protocol was developed for effective uniform infection on susceptible host plant genotypes at both seedling and adult developmental stages. A randomised complete block design was used to test the protocol on glasshouse-grown plants from 10 elite perennial ryegrass cultivars. Significant quantitative differences in pustule number and latency period were detected between cultivars, suggesting that the protocol was highly effective at providing a uniform distribution of pustules in low and high urediniospore suspension volumes. The inoculation and quantification methods provide a framework for standardised assessment of crown rust infection under controlled environment conditions, and may in principle be applied to any Pucciniahost interaction. © 2010 Australasian Plant Pathology Society.


Wang J.,Australian Department of Primary Industries and Fisheries | Wang J.,Molecular Plant Breeding Cooperative Research Center | Drayton M.C.,Australian Department of Primary Industries and Fisheries | Drayton M.C.,Molecular Plant Breeding Cooperative Research Center | And 20 more authors.
Theoretical and Applied Genetics | Year: 2010

Allotetraploid (2n = 4x = 32) white clover (Trifolium repens L.) is the most commonly cultivated legume component of temperate pastures, sown in swards with a companion grass species. Genetic control of growth performance of white clover on saline land is highly important for dairy industries, due to increasing soil salinity problems. The objective of this study was to identify quantitative trait loci (QTLs) for salinity tolerance in terms of vegetative growth under stress. Two parental genetic maps consisting of 213 and 159 marker loci and spanning 1,973.0 and 1,837.6 cM, respectively, were constructed using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers from a two-way pseudo-test cross F1 population derived from pair-crossing of the Haifa2 and LCL2 genotypes. A total of 8 unique genomic regions on 8 linkage groups (LGs) of the Haifa2 parental map and 6 unique regions on 5 LGs in the LCL2 parental map were associated with plant growth under salt stress and relative growth under stress, as compared to control conditions. The results of this study indicate that salt tolerance in white clover is controlled by multiple QTLs, some at common locations, but each of limited magnitude. Location of these QTLs provides the genetic basis and potential for pyramiding of salt tolerance genes in breeding improvement. © Her Majesty the Queen in Rights of Australia 2009.


De Lucas J.A.,La Trobe University | De Lucas J.A.,Molecular Plant Breeding Cooperative Research Center | Forster J.W.,La Trobe University | Forster J.W.,Molecular Plant Breeding Cooperative Research Center | And 5 more authors.
Crop and Pasture Science | Year: 2012

White clover is one of the most important pasture legumes in global temperate regions. It is an outcrossing, insect-pollinated species with gene flow occurring naturally between plants. A 2-year study was conducted to assess the relationship between gene flow and physical distance in white clover under field conditions in southern Australia. White clover plants exhibiting a red leaf mark phenotypic trait acted as pollen donors to recipient plants lacking leaf markings at distances up to 200m distant from the donor plants. Progeny were scored for the dominant red-leafed phenotype and gene flow was modelled. Paternity was confirmed using simple sequence repeat markers. A leptokurtic pattern of gene flow was observed under conditions designed to measure maximised gene flow with the majority of pollination occurring in the first 50m from the donor pollen source. The combined use of simple sequence repeat and visual markers confirmed that there was also a white clover pollen source in addition to the donor plants. This research confirms the difficulty in ensuring absolute containment of gene flow in an outcrossing species grown in an environment when endemic populations are known to exist. © CSIRO 2012.


Louie G.V.,Salk Institute for Biological Studies | Bowman M.E.,Salk Institute for Biological Studies | Tu Y.,Australian Department of Primary Industries and Fisheries | Tu Y.,Molecular Plant Breeding Cooperative Research Center | And 8 more authors.
Plant Cell | Year: 2010

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-L-homocysteine and sinapaldehyde. The product-bound complex reveals the postmethyl-transfer organization of COMT's catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde. © American Society of Plant Biologists.


Tu Y.,Australian Department of Primary Industries and Fisheries | Tu Y.,La Trobe University | Rochfort S.,Australian Department of Primary Industries and Fisheries | Liu Z.,Australian Department of Primary Industries and Fisheries | And 16 more authors.
Plant Cell | Year: 2010

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1- deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production. © 2010 American Society of Plant Biologists.


Kaur S.,La Trobe University | Francki M.G.,Food Western Australia | Francki M.G.,Molecular Plant Breeding Cooperative Research Center | Francki M.G.,Murdoch University | And 3 more authors.
Plant Biotechnology Journal | Year: 2012

An understanding of nature and extent of nucleotide sequence variation is required for programmes of discovery and characterization of single nucleotide polymorphisms (SNPs), which provide the most versatile class of molecular genetic marker. A majority of higher plant species are polyploids, and allopolyploidy, because of hybrid formation between closely related taxa, is very common. Mutational variation may arise both between allelic (homologous) sequences within individual subgenomes and between homoeologous sequences among subgenomes, in addition to paralogous variation between duplicated gene copies. Successful SNP validation in allopolyploids depends on differentiation of the sequence variation classes. A number of biological factors influence the feasibility of discrimination, including degree of gene family complexity, inbreeding or outbreeding reproductive habit, and the level of knowledge concerning progenitor diploid species. In addition, developments in high-throughput DNA sequencing and associated computational analysis provide general solutions for the genetic analysis of allopolyploids. These issues are explored in the context of experience from a range of allopolyploid species, representing grain (wheat and canola), forage (pasture legumes and grasses), and horticultural (strawberry) crop. Following SNP discovery, detection in routine genotyping applications also presents challenges for allopolyploids. Strategies based on either design of subgenome-specific SNP assays through homoeolocus-targeted polymerase chain reaction (PCR) amplification, or detection of incremental changes in nucleotide variant dosage, are described. © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.


Dracatos P.M.,La Trobe University | Dracatos P.M.,Molecular Plant Breeding Cooperative Research Center | Cogan N.O.I.,La Trobe University | Keane P.J.,Molecular Plant Breeding Cooperative Research Center | And 6 more authors.
Crop Science | Year: 2010

The most serious foliar disease of perennial ryegrass (Lolium perenne L.) is crown rust (caused by Puccinia coronata Corda f.sp. lolii Brown). Progress in resistance breeding using recurrent selection has been slow, due to lack of genetic knowledge. Puccinia coronata is a basidiomycete fungus with a complex life-cycle involving both asexual and sexual reproductive modes. Pathotype variation may exist, but confi rmation is complicated by the outbreeding nature of the host grass and consequent intrapopulation diversity. Qualitative and quantitative resistance mechanisms may hence be due to either major resistance genes responding to mixed pathogen populations, or minor (quantitative) resistance genes. Advances in molecular biology have permitted analysis of both host and pathogen genetics. Development of pathogen-derived simple sequence repeat (SSR) genetic markers allowed detailed analysis of genetic variation, as well as generation of homogenized inoculum for detailed trait-dissection studies. Multiple genomic regions were identifi ed as containing quantitative trait loci (QTLs) for pathogen resistance, and specifi c single nucleotide polymorphism (SNP) markers for defense response (DR) and disease resistance (R) genes have been evaluated for QTL co-location. The current knowledge status of the crown rust pathogen and genetics of host resistance is reviewed, as well as future prospects for facilitated rust resistance breeding. © Crop Science Society of America.


Shinozuka H.,La Trobe University | Shinozuka H.,Molecular Plant Breeding Cooperative Research Center | Shinozuka H.,Dairy Futures Cooperative Research Center | Cogan N.O.I.,La Trobe University | And 8 more authors.
International Journal of Plant Genomics | Year: 2011

Perennial ryegrass is an important pasture grass in temperate regions. As a forage biomass-generating species, plant architecture-related characters provide key objectives for breeding improvement. In silico comparative genomics analysis predicted colocation between a previously identified QTL for plant type (erect versus prostrate growth) and the ortholocus of the rice OsABCG5 gene (LpABCG5), as well as related QTLs in other Poaceae species. Sequencing of an LpABCG5-containing BAC clone identified presence of a paralogue (LpABCG6) in the vicinity of the LpABCG5 locus, in addition to three other gene-like sequences. Comparative genomics involving five other 5 grass species (rice, Brachypodium, sorghum, maize, and foxtail millet) revealed conserved microsynteny in the ABCG5 ortholocus-flanking region. Gene expression profiling and phylogenetic analysis suggested that the two paralogues are functionally distinct. Fourteen additional ABCG5 gene family members, which may interact with the LpABCG5 gene, were identified through sequencing of transcriptomes from perennial ryegrass leaf, anther, and pistils. A larger-scale phylogenetic analysis of the ABCG gene family suggested conservation between major branches of the Poaceae family. This study identified the LpABCG5 gene as a candidate for the plant type determinant, suggesting that manipulation of gene expression may provide valuable phenotypes for perennial ryegrass breeding. © 2011 Hiroshi Shinozuka et al.


Hand M.L.,La Trobe University | Hand M.L.,Molecular Plant Breeding Cooperative Research Center | Cogan N.O.I.,La Trobe University | Cogan N.O.I.,Molecular Plant Breeding Cooperative Research Center | And 6 more authors.
BMC Plant Biology | Year: 2010

Background: White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised.Results: A total of four paired homoeologous BACs were selected and sequenced to generate 173 kb of overlapping sequence between the O and P' sub-genomes. Equivalent gene content was generally observed, apart from small-scale deletions, in contrast to conservation of intergenic sequences, which varied between the four selected regions. Measurement of the number of synonymous substitutions between homoeologous genes led to estimation of a 4.2 million year divergence time between the two sub-genomes. Microsynteny was observed between the genomes of white clover and L. japonicus for all four targeted regions, but corresponding M. truncatula genomic regions were only identified for two BAC pairs.Conclusions: This study describes the first analysis of sub-genome structural conservation across selected genomic regions in white clover. Although the high levels of sequence conservation between the O and P' sub-genomes would complicate efforts for whole genome sequence assembly, the conserved microsynteny with model legume genomes, especially that of L. japonicus, will be highly valuable for the future of white clover genomics and molecular breeding. © 2010 Hand et al; licensee BioMed Central Ltd.

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