Catherine J Nock

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.

Population structure, effective population size and adverse effects of stocking in the endangered Australian eastern freshwater cod Maccullochella ikei

Microsatellite markers were used to examine spatio-temporal genetic variation in the endangered eastern freshwater cod Maccullochella ikei in the Clarence River system, eastern Australia. High levels of population structure were detected. A model-based clustering analysis of multilocus genotypes identified four populations that were highly differentiated by F-statistics (F(ST) = 0·09 - 0·49; P < 0·05), suggesting fragmentation and restricted dispersal particularly among upstream sites. Hatchery breeding programmes were used to re-establish locally extirpated populations and to supplement remnant populations. Bayesian and frequency-based analyses of hatchery fingerling samples provided evidence for population admixture in the hatchery, with the majority of parental stock sourced from distinct upstream sites. Comparison between historical and contemporary wild-caught samples showed a significant loss of heterozygosity (21%) and allelic richness (24%) in the Mann and Nymboida Rivers since the commencement of stocking. Fragmentation may have been a causative factor; however, temporal shifts in allele frequencies suggest swamping with hatchery-produced M. ikei has contributed to the genetic decline in the largest wild population. This study demonstrates the importance of using information on genetic variation and population structure in the management of breeding and stocking programmes, particularly for threatened species.

Application of large-scale sequencing to marker discovery in plants

Advances in DNA sequencing provide tools for efficient large-scale discovery of markers for use in plants. Discovery options include large-scale amplicon sequencing, transcriptome sequencing, gene-enriched genome sequencing and whole genome sequencing. Examples of each of these approaches and their potential to generate molecular markers for specific applications have been described. Sequencing the whole genome of parents identifies all the polymorphisms available for analysis in their progeny. Sequencing PCR amplicons of sets of candidate genes from DNA bulks can be used to define the available variation in these genes that might be exploited in a population or germplasm collection. Sequencing of the transcriptomes of genotypes varying for the trait of interest may identify genes with patterns of expression that could explain the phenotypic variation. Sequencing genomic DNA enriched for genes by hybridization with probes for all or some of the known genes simplifies sequencing and analysis of differences in gene sequences between large numbers of genotypes and genes especially when working with complex genomes. Examples of application of the above-mentioned techniques have been described.

Phylogenetics and revised taxonomy of the Australian freshwater cod genus, Maccullochella (Percichthyidae)

Determining the phylogenetic and taxonomic relationships among allopatric populations can be difficult, especially when divergence is recent and morphology is conserved. We used mitochondrial sequence data from the control region and three protein-coding genes (1253 bp in total) and genotypes determined at 13 microsatellite loci to examine the evolutionary relationships among Australia’s largest freshwater fish, the Murray cod, Maccullochella peelii peelii, from the inland Murray–Darling Basin, and its allopatric sister taxa from coastal drainages, the eastern freshwater cod, M. ikei, and Mary River cod, M. peelii mariensis. Phylogenetic analyses provided strong support for taxon-specific clades, with a clade containing both of the eastern taxa reciprocally monophyletic to M. peelii peelii, suggesting a more recent common ancestry between M. ikei and M. peelii mariensis than between the M. peelii subspecies. This finding conflicts with the existing taxonomy and suggests that ancestral Maccullochella crossed the Great Dividing Range in the Pleistocene and subsequently diverged in eastern coastal drainages. Evidence from the present study, in combination with previous morphological and allozymatic data, demonstrates that all extant taxa are genetically and morphologically distinct. The taxonomy of Maccullochella is revised, with Mary River cod now recognised as a species, Maccullochella mariensis, a sister species to eastern freshwater cod, M. ikei. As a result of the taxonomic revision, Murray cod is M. peelii.

Whole genome shotgun sequences for microsatellite discovery and application in cultivated and wild Macadamia (Proteaceae)

Premise of the study: Next-generation sequencing (NGS) data are widely used for single-nucleotide polymorphism discovery and genetic marker development in species with limited available genome information. We developed microsatellite primers for the Proteaceae nut crop species Macadamia integrifolia and assessed cross-species transferability in all congeners to investigate genetic identification of cultivars and gene flow. Methods and Results: Primers were designed from both raw and assembled Illumina NGS paired-end reads. The final 12 microsatellite markers selected were polymorphic among wild individuals of all four Macadamia species—M. integrifolia, M. tetraphylla, M. ternifolia, and M. jansenii—and in commercial macadamia cultivars including hybrids. Conclusions: We demonstrate the utility of raw and assembled Illumina NGS reads from total genomic DNA for the rapid development of microsatellites in Macadamia. These primers will facilitate future studies of population structure, hybridization, parentage, and cultivar identification in cultivated and wild Macadamia populations.

Genetic differentiation among and within three red mahoganies (series Annulares), Eucalyptus pellita, E. resinifera and E. scias (Myrtaceae)

Theredmahoganygroup(Eucalyptusser.AnnularesBlakely)includessomeofthemostimportantcommercial species (i.e. Eucalyptus urophyllaS.T.Blake) worldwide for forestry in the subtropics and tropics. However, the taxonomic status of some species in this group is unclear and the relationship among and genetic structuring within some species is unresolved. The present study examined genetic variation at 13 microsatellite loci in E. pellitaF.Muell.,E. resiniferaSmith and E. scias L.Johnson & K.Hill. Despite close geographical proximity and natural hybridisation in northern Queensland, E.resiniferaandE.pellitaremaingeneticallydistinctastaxa.WithinE.pellita,twogeneticgroupswereclearlyresolved,one from New Guinea and one from Queensland (Cape York Peninsula populations were not sampled). Geographic structuring wasalsoevidentinE.resinifera,withnorthernQueenslandpopulationsseparatingfromthosefromFraserIslandsouthwards. Ecologicalfactorsandspeciesdisjunctionswereimplicatedinthegeneticsubstructuringofthesetwotaxabecausepatternsof geographicvariationalignedwithbiogeographicalregions.E.sciaswasindistinguishablefromsouthernE.resiniferaandits three subspecies could not be resolved.

Polymorphic microsatellite loci for species of Australian freshwater cod, Maccullochella

The Australian freshwater cod genus, Maccullochella is represented by three species: Murray cod, M. peeliipeelii, eastern freshwater cod, M. ikei, and trout cod, M.macquariensis. Seven novel microsatellite loci from M. ikei and six previously published loci from M. peelii peelii were tested on wild populations of Murray, eastern and trout cod. Levels of polymorphism varied between species with 13 loci polymorphic in Murray cod, 9 in trout cod and 7 in eastern cod. Observed heterozygosities ranged from 0.053 to 0.842. This suite of microsatellite loci will facilitate future studies of the genetic status of wild and hatchery bred populations of Maccullochella.

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.

Efficient Diagnosis of Ratoon Stunting Disease of Sugarcane by Quantitative PCR on Pooled Leaf Sheath Biopsies

Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli, is arguably one of the most devastating diseases of sugarcane. Four diagnostic techniques were compared for 100 fields of sugarcane (Saccharum interspecific hybrids) of unknown infection status. These were quantitative polymerase chain reaction on pooled leaf sheath biopsies (LSB-qPCR), conventional PCR on the same templates (LSB-PCR), evaporative-binding enzyme immunoassay (EB-EIA) coupled with phase contrast microscopy (PCM) on expressed xylem sap from the same fields, and conventional PCR on the same xylem sap samples. LSB-qPCR and LSB-PCR detected the causal agent in 27 and 18 fields, respectively, whereas, from samples of expressed xylem sap from the same fields, conventional PCR identified 12 infections and EB-EIA/PCM detected L. xyli subsp. xyli in 3 fields. The sensitivities of qPCR and PCR were approximately 103 and 104 CFU ml-1, respectively, determined from plate counts of a dilution series. Tests were conducted on a further 139 LSB samples from across the Australian industry, with qPCR and PCR diagnosing RSD in 31 and 25 fields, respectively. Using qPCR and PCR on LSB samples, RSD was diagnosed in a range of cultivars throughout the year, and qPCR and PCR could detect L. xyli subsp. xyli in sugarcane ranging from 3 months to greater than 1 year old.