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Chagne D.,The New Zealand Institute for Plant and Food Research Ltd | Crowhurst R.N.,Mount Albert Research Center | Pindo M.,Instituto Agrario San Michele allAdige Research and Innovation Center | Thrimawithana A.,Mount Albert Research Center | And 39 more authors.
PLoS ONE | Year: 2014

We present a draft assembly of the genome of European pear (Pyrus communis) 'Bartlett'. Our assembly was developed employing second generation sequencing technology (Roche 454), from single-end, 2 kb, and 7 kb insert paired-end reads using Newbler (version 2.7). It contains 142,083 scaffolds greater than 499 bases (maximum scaffold length of 1.2 Mb) and covers a total of 577.3 Mb, representing most of the expected 600 Mb Pyrus genome. A total of 829,823 putative single nucleotide polymorphisms (SNPs) were detected using re-sequencing of 'Louise Bonne de Jersey' and 'Old Home'. A total of 2,279 genetically mapped SNP markers anchor 171 Mb of the assembled genome. Ab initio gene prediction combined with prediction based on homology searching detected 43,419 putative gene models. Of these, 1219 proteins (556 clusters) are unique to European pear compared to 12 other sequenced plant genomes. Analysis of the expansin gene family provided an example of the quality of the gene prediction and an insight into the relationships among one class of cell wall related genes that control fruit softening in both European pear and apple (Malusxdomestica). The 'Bartlett' genome assembly v1.0 (http://www.rosaceae.org/species/pyrus/ pyrus-communis/genome-v1.0) is an invaluable tool for identifying the genetic control of key horticultural traits in pear and will enable the wide application of marker-assisted and genomic selection that will enhance the speed and efficiency of pear cultivar development. © 2014 Chagné et al.


Knabel M.,The New Zealand Institute for Plant and Food Research Ltd | Knabel M.,University of Auckland | Friend A.P.,Motueka Research Center | Palmer J.W.,Motueka Research Center | And 10 more authors.
BMC Plant Biology | Year: 2015

Background: The vigour and precocity of trees highly influences their efficiency in commercial production. In apple, dwarfing rootstocks allow high-density plantings while their precocious flowering enables earlier fruit production. Currently, there is a lack of pear (Pyrus communis L.) rootstocks that are equivalent to the high yielding apple rootstock 'M9'. For the efficient breeding of new Pyrus rootstocks it is crucial to understand the genetic determinants of vigour control and precocity. In this study we used quantitative trait loci (QTLs) analysis to identify genetic loci associated with the desired traits, using a segregating population of 405 F1 P. communis seedlings from a cross between 'Old Home' and 'Louise Bonne de Jersey' (OHxLBJ). The seedlings were grafted as rootstocks with 'Doyenne du Comice' scions and comprehensively phenotyped over four growing seasons for traits related to tree architecture and flowering, in order to describe the growth of the scions. Results: A high density single nucleotide polymorphism (SNP)-based genetic map comprising 597 polymorphic pear and 113 apple markers enabled the detection of QTLs influencing expression of scion vigour and precocity located on linkage groups (LG)5 and LG6 of 'Old Home'. The LG5 QTL maps to a position that is syntenic to the apple 'Malling 9' ('M9') Dw1 locus at the upper end of LG5. An allele of a simple sequence repeat (SSR) associated with apple Dw1 segregated with dwarfing and precocity in pear and was identified in other pear germplasm accessions. The orthology of the vigour-controlling LG5 QTL between apple and pear raises the possibility that the dwarfing locus Dw1 arose before the divergence of apple and pear, and might therefore be present in other Rosaceae species. Conclusion: We report the first QTLs associated with vigour control and flowering traits in pear rootstocks. Orthologous loci were found to control scion growth and precocity in apple and pear rootstocks. The application of our results may assist in the breeding process of a pear rootstock that confers both vigour control and precocity to the grafted scion cultivar. © 2015 Knäbel et al.


Nakasugi K.,University of Sydney | Crowhurst R.,Mount Albert Research Center | Bally J.,University of Sydney | Waterhouse P.,University of Sydney | Waterhouse P.,Queensland University of Technology
PLoS ONE | Year: 2014

Background: Nicotiana benthamiana is an allo-tetraploid plant, which can be challenging for de novo transcriptome assemblies due to homeologous and duplicated gene copies. Transcripts generated from such genes can be distinct yet highly similar in sequence, with markedly differing expression levels. This can lead to unassembled, partially assembled or mis-assembled contigs. Due to the different properties of de novo assemblers, no one assembler with any one given parameter space can re-assemble all possible transcripts from a transcriptome. Results:In an effort to maximise the diversity and completeness of de novo assembled transcripts, we utilised four de novo transcriptome assemblers, TransAbyss, Trinity, SOAPdenovo-Trans, and Oases, using a range of k-mer sizes and different input RNA-seq read counts. We complemented the parameter space biologically by using RNA from 10 plant tissues. We then combined the output of all assemblies into a large super-set of sequences. Using a method from the EvidentialGene pipeline, the combined assembly was reduced from 9.9 million de novo assembled transcripts to about 235,000 of which about 50,000 were classified as primary. Metrics such as average bit-scores, feature response curves and the ability to distinguish paralogous or homeologous transcripts, indicated that the EvidentialGene processed assembly was of high quality. Of 35 RNA silencing gene transcripts, 34 were identified as assembled to full length, whereas in a previous assembly using only one assembler, 9 of these were partially assembled. Conclusions:To achieve a high quality transcriptome, it is advantageous to implement and combine the output from as many different de novo assemblers as possible. We have in essence taking the 'best' output from each assembler while minimising sequence redundancy. We have also shown that simultaneous assessment of a variety of metrics, not just focused on contig length, is necessary to gauge the quality of assemblies. © 2014 Nakasugi et al.


Bally J.,Queensland University of Technology | Bally J.,University of Sydney | Nakasugi K.,University of Sydney | Jia F.,University of Sydney | And 14 more authors.
Nature Plants | Year: 2015

A single lineage of Nicotiana benthamiana is widely used as a model plant 1 and has been instrumental in making revolutionary discoveries about RNA interference (RNAi), viral defence and vaccine production. It is peerless in its susceptibility to viruses and its amenability in transiently expressing transgenes 2,3. These unparalleled characteristics have been associated both positively and negatively with a disruptive insertion in the RNA-dependent RNA polymerase 1 gene, Rdr1 4-6. For a plant so routinely used in research, the origin, diversity and evolution of the species, and the basis of its unusual abilities, have been relatively unexplored. Here, by comparison with wild accessions from across the spectrum of the speciesa € natural distribution, we show that the laboratory strain of N. benthamiana is an extremophile originating from a population that has retained a mutation in Rdr1 for a 1/40.8 Myr and thereby traded its defence capacity for early vigour and survival in the extreme habitat of central Australia. Reconstituting Rdr1 activity in this isolate provided protection. Silencing the functional allele in a wild strain rendered it hypersusceptible and was associated with a doubling of seed size and enhanced early growth rate. These findings open the way to a deeper understanding of the delicate balance between protection and vigour. © 2015 Macmillan Publishers Limited. All rights reserved.


PubMed | University of New South Wales, Mount Albert Research Center, Queensland University of Technology and CSIRO
Type: | Journal: Nature plants | Year: 2016

A single lineage of Nicotiana benthamiana is widely used as a model plant(1) and has been instrumental in making revolutionary discoveries about RNA interference (RNAi), viral defence and vaccine production. It is peerless in its susceptibility to viruses and its amenability in transiently expressing transgenes(2,3). These unparalleled characteristics have been associated both positively and negatively with a disruptive insertion in the RNA-dependent RNA polymerase 1 gene, Rdr1(4-6). For a plant so routinely used in research, the origin, diversity and evolution of the species, and the basis of its unusual abilities, have been relatively unexplored. Here, by comparison with wild accessions from across the spectrum of the species natural distribution, we show that the laboratory strain of N.benthamiana is an extremophile originating from a population that has retained a mutation in Rdr1 for 0.8 Myr and thereby traded its defence capacity for early vigour and survival in the extreme habitat of central Australia. Reconstituting Rdr1 activity in this isolate provided protection. Silencing the functional allele in a wild strain rendered it hypersusceptible and was associated with a doubling of seed size and enhanced early growth rate. These findings open the way to a deeper understanding of the delicate balance between protection and vigour.


Wee S.-L.,National University of Malaysia | Wee S.-L.,The New Zealand Institute for Plant and Food Research Ltd | Suckling D.M.,The New Zealand Institute for Plant and Food Research Ltd | Barrington A.M.,Mount Albert Research Center
Australian Journal of Entomology | Year: 2011

The advantage of inherited sterility over complete sterility in lepidopteran sterile insect technique programs results from the improvement of mating fitness of male-only releases with wild females, and the resulting large multiplier effect from viable but sterile progeny from every mating. The deleterious effects induced by irradiation are inherited by the F 1 generation, but it is very difficult to measure population introgression at that stage, and the alternative has been to await population suppression at the F 2 generation. This work, conducted in support of the successful elimination of painted apple moth (Teia anartoides) in New Zealand, aimed to determine the feasibility of a cytological assessment on the F 1 sperm bundles of this species, as a new forensic biosecurity tool providing information for decision support. The technique successfully distinguished the homogeneous nuclei clusters of eupyrene bundles of the normal fertile males from the heterogeneously stained nuclei clusters of the F 1 progeny. However, the challenge for the technique involved obtaining good specimens for cytological diagnosis. The percentage of positive staining results was correlated strongly with survival, which was <5 days. Moths that had spent 24h on a sticky base in a monitoring trap were equivalent to freshly killed specimens, but the efficacy of the technique decreased after that. Some specimens that were 'dead' to the naked eye but potentially alive internally produced reliable results. The technique may be potentially useful as a forensic biosecurity tool in the future when a communicating trap is deployed, ensuring that fresh specimens could be used to monitor the success of inherited sterility in population suppression or eradication. © 2011 The Authors. Journal compilation © 2011 Australian Entomological Society.


Nakasugi K.,University of Sydney | Crowhurst R.N.,Mount Albert Research Center | Bally J.,University of Sydney | Wood C.C.,CSIRO | And 2 more authors.
PLoS ONE | Year: 2013

Background: Nicotiana benthamiana has been widely used for transient gene expression assays and as a model plant in the study of plant-microbe interactions, lipid engineering and RNA silencing pathways. Assembling the sequence of its transcriptome provides information that, in conjunction with the genome sequence, will facilitate gaining insight into the plant's capacity for high-level transient transgene expression, generation of mobile gene silencing signals, and hyper-susceptibility to viral infection. Methodology/Results: RNA-seq libraries from 9 different tissues were deep sequenced and assembled, de novo, into a representation of the transcriptome. The assembly, of16GB of sequence, yielded 237,340 contigs, clustering into 119,014 transcripts (unigenes). Between 80 and 85% of reads from all tissues could be mapped back to the full transcriptome. Approximately 63% of the unigenes exhibited a match to the Solgenomics tomato predicted proteins database. Approximately 94% of the Solgenomics N. benthamiana unigene set (16,024 sequences) matched our unigene set (119,014 sequences). Using homology searches we identified 31 homologues that are involved in RNAi-associated pathways in Arabidopsis thaliana, and show that they possess the domains characteristic of these proteins. Of these genes, the RNA dependent RNA polymerase gene, Rdr1, is transcribed but has a 72 nt insertion in exon1 that would cause premature termination of translation. Dicer-like 3 (DCL3) appears to lack both the DEAD helicase motif and second dsRNA binding motif, and DCL2 and AGO4b have unexpectedly high levels of transcription. Conclusions: The assembled and annotated representation of the transcriptome and list of RNAi-associated sequences are accessible at www.benthgenome.com alongside a draft genome assembly. These genomic resources will be very useful for further study of the developmental, metabolic and defense pathways of N. benthamiana and in understanding the mechanisms behind the features which have made it such a well-used model plant. © 2013 Nakasugi et al.


Kumar S.,The New Zealand Institute for Plant and Food Research Ltd | Rowan D.,Palmerston North Research Center | Hunt M.,Palmerston North Research Center | Chagne D.,Palmerston North Research Center | And 2 more authors.
Molecular Breeding | Year: 2015

Flavour is an important food trait, yet little is known about the genetic architecture and mode of inheritance of apple flavour compounds. The objectives of this study were to: understand the inheritance of flavour volatiles in a clonally replicated germplasm population; unravel correlation networks of volatiles; and to use genome-wide single nucleotide polymorphism (SNP) markers to identify genomic regions that play a role in the expression of flavour volatiles. This analysis revealed that more than half of the 37 volatiles (measured by gas chromatography–mass spectrometry) showed high heritability (h2 > 0.4), with only a small number (3 of the 37) displaying low heritability (h2 < 0.2). Majority (~85 %) of the significant SNP loci displayed the additive mode of inheritance. Our results supported the roles of MdAAT, MdCXE and MdLOX genes in the expression of apple flavour volatiles. Effect sizes of SNP loci, some of which are associated with multiple compounds, were small (<10 %), which is consistent with a polygenic quantitative inheritance model. New genomic locations associated with multiple flavour compounds were found, and some SNPs were associated with both sensory flavour and some flavour volatiles. Simultaneous genome-wide association study for sensory flavour and flavour compounds is recommended for unravelling genetic mechanisms to facilitate marker-assisted breeding for targeted flavours. © 2015, Springer Science+Business Media Dordrecht.

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