Nicole F Rice

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.

Chloroplast genome sequence confirms distinctness of Australian and Asian wild rice

Cultivated rice (Oryza sativa) is an AA genome Oryza species that was most likely domesticated from wild populations of O. rufipogon in Asia. O. rufipogon and O. meridionalis are the only AA genome species found within Australia and occur as widespread populations across northern Australia. The chloroplast genome sequence of O. rufipogon from Asia and Australia and O. meridionalis and O. australiensis (an Australian member of the genus very distant from O. sativa) was obtained by massively parallel sequencing and compared with the chloroplast genome sequence of domesticated O. sativa. Oryza australiensis differed in more than 850 sites single nucleotide polymorphism or indel from each of the other samples. The other wild rice species had only around 100 differences relative to cultivated rice. The chloroplast genomes of Australian O. rufipogon and O. meridionalis were closely related with only 32 differences. The Asian O. rufipogon chloroplast genome (with only 68 differences) was closer to O. sativa than the Australian taxa (both with more than 100 differences). The chloroplast sequences emphasize the genetic distinctness of the Australian populations and their potential as a source of novel rice germplasm. The Australian O. rufipogon may be a perennial form of O. meridionalis.

Australian Oryza: utility and conservation

Australian Oryza are an understudied and underexploited genetic resource for rice improvement. Four species are indigenous: Oryza rufipogon, Oryza meridionalis, Oryza australiensis are widespread across northern Australia, whereas Oryza officinalis is known from two localities only. Molecular analysis of these wild populations is required to better define the distinctness of the taxa and the extent of any gene flow between them and rice. Limited collections of these wild populations are held in seed and DNA banks. These species have potential for domestication in some cases but also have many traits of potential value in the improvement of domesticated rice. Stress tolerance (biotic and abiotic) and grain quality characteristics in these populations may be useful.

Genetic diversity of ICARDA's worldwide barley landrace collection

Twenty genic- and genomic SSR markers were used to study genetic diversity and geographical differentiation of barley from 29 countries through analysis of a worldwide collection of 304 ICARDA’s barley landraces. Of these, 19 loci were highly polymorphic in the material studied. Based on Nei-distance matrix, Principal Component Analysis (PCoA) and cluster analysis using UPGMA associated with AMOVA the data revealed countries’ grouping within regions. Three distinct germplasm pools were identified in the landraces. The first of these was from Eastern Africa (Eritrea and Ethiopia) and South America (Ecuador, Peru and Chile) suggesting that barley introduced to South America might have originated specifically from East Africa or that they share a common genetic basis for adaptation. The second was the Caucasus (Armenia and Georgia) and the third included the remaining regions of Central Asia, Near East, Northern Africa and Eastern Asia. Genetic diversity of barley subspecies (Six-rowed barley, Two-rowed barley, H. spontaneum C. Koch and H. agriocrithon Åberg) also discriminates them into three groups: cultivated barleys (Six-rowed barley and Two-rowed barley), wild barley H. spontaneum and subspecies H. agriocrithon. These results associated with parsimony analysis demonstrate that H. agriocrithon and H. spontaneum might be distinct and do not support a hybrid origin for H. agriocrithon suggesting further investigation of the basis of more intense sampling of the two subspecies H. spontaneum and H. agriocrithon.

Whole grain morphology of Australian rice species

The grain morphology of 17 wild rice relatives were studied by light and scanning electron microscopy and compared to two cultivated rice varieties ( Oryza sativa cv. Nipponbare and O. sativa cv. Teqing). Observations were made of the grain colour, size and shape. Grains from wild rice species exhibited a variety of colours that have potential aesthetic and nutritional value. The grains of these species exhibited a wide array of sizes and shapes, but still fell within the standard classification scale that rice breeders use for routine breeding evaluation. These results highlight the potential of these species as whole grain foods or as sources of novel alleles in conventional rice breeding programmes.

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.

Wild Oryza grain physico-chemical properties

Of the 22 species within the Oryza genus, only two, O. sativa and O. glaberrima, have been domesticated. Although food security is supported by accessing wild Oryza resources for new genes and alleles which enhance plant performance, wild Oryza grain properties have not been extensively studied. Evaluation of the grain physico-chemical properties of eight wild Oryza species found amylose content, amylopectin structure and cooking properties fell within a narrow range relative to cultivated rice. The amylopectin of the wild species had a lower proportion of short branch chains (DP 6–14) relative to cultivated rice and were all of high apparent amylose content and gelatinization temperature. The grain of the wild species did not elongate to the same extent as the cultivated rice and had lower viscosity parameters. These results highlight how significant physio-chemical changes have been made by human selection in the domestication of rice, especially japonica rice. The wild species may be useful for improving the nutritional value of rice and other cereal crops.

DNA extraction from plant tissue.

This chapter discusses the approaches that have been used in the isolation of DNA from plant tissues. A summary of approaches is presented in the appendix.

Next-generation technologies to determine plastid genome sequences

Sequencing of chloroplast genomes is a key tool for analysis of chloroplasts and the impact of manipulation of chloroplast genomes by biotechnology. Advances in genome sequencing allow the complete sequencing of the chloroplast genome and assessment of variation in the chloroplast genome sequences within a plant. Isolation of chloroplast DNA has been a traditional starting point in these analyses, but the capacity of current sequencing technologies allows effective analysis of the chloroplast genome sequence by shotgun sequencing of a preparation of total DNA from the plant. Chloroplast insertions in the nuclear genome can be distinguished by their much lower copy number. Short-read sequences are best assembled by alignment with a reference chloroplast genome.

Novel microsatellite markers for the endangered Australian rainforest tree Davidsonia jerseyana (Cunoniaceae) and cross-species amplification in the Davidsonia genus

Davidsonia jerseyana is an endangered rainforest tree endemic to far north-eastern New South Wales, Australia. The species occurs in small fragmented populations and has an edible plum-like fruit important to the Australian Native Food industry. Twenty one novel microsatellite markers were developed for D. jerseyana of which 13 were polymorphic for the species. Markers were characterised using 28 individuals, representing six populations from across the species geographic range. Species level analysis of the polymorphic markers revealed the mean number of alleles per locus was 3.154 (range 2–7) and mean expected and observed heterozygosities were 0.437 (range 0.035–0.725) and 0.044 (range 0–0.250) respectively. The heterozygote deficiency may indicate a predominantly selfing breeding system. All markers cross amplified in the other two Davidsonia species. These markers will be used to assess the genetic diversity, population structure and breeding systems in D. jerseyana and related taxa which will facilitate conservation management strategies.