Christopher A. Cullis

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.

RAPD analysis in flax: Optimization of yield and reproducibility using klenTaq 1 DNA polymerase, chelex 100, and gel purification of genomic DNA

We have developed an optimized RAPD analysis approach using the unusually heat-stable KlenTaq1 DNA polymerase. This enzyme is used in conjunction with a genomic DNA isolation method that includes a modified CTAB DNA isolation protocol, ethanol re-precipitation of resuspended nucleic acids from 2M NaCl, and Chelex 100 treatment. When needed, additional gel purification and isolation of high molecular weight DNA for use as a template in RAPD analysis is shown to remove amplification product ambiguity from within isolates of the same line as well as from between lines. This optimized RAPD analysis was used to define polymorphisms in lines of flax nearly isogenic for rust resistance at theL locus. It should also be useful for any plant species.

Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana

We have isolated a genomic clone containing Arabidopsis thaliana 5S ribosomal RNA (rRNA)-encoding genes (rDNA) by screening an A. thaliana library with a 5S rDNA probe from flax. The clone isolated contains seven repeat units of 497 bp, plus 11 kb of flanking genomic sequence at one border. Sequencing of individual subcloned repeat units shows that the sequence of the 5S rRNA coding region is very similar to that reported for other flowering plants. Four A. thaliana ecotypes were found to contain approx. 1000 copies of 5S rDNA per haploid genome. Southern-blot analysis of genomic DNA indicates that 5S rDNA occurs in long tandem arrays, and shows the presence of numerous restriction-site polymorphisms among the six ecotypes studied.

Neglecting legumes has compromised human health and sustainable food production

The United Nations declared 2016 as the International Year of Pulses (grain legumes) under the banner ‘nutritious seeds for a sustainable future’. A second green revolution is required to ensure food and nutritional security in the face of global climate change. Grain legumes provide an unparalleled solution to this problem because of their inherent capacity for symbiotic atmospheric nitrogen fixation, which provides economically sustainable advantages for farming. In addition, a legume-rich diet has health benefits for humans and livestock alike. However, grain legumes form only a minor part of most current human diets, and legume crops are greatly under-used. Food security and soil fertility could be significantly improved by greater grain legume usage and increased improvement of a range of grain legumes. The current lack of coordinated focus on grain legumes has compromised human health, nutritional security and sustainable food production.

DNA rearrangements in response to environmental stress

Publisher Summary This chapter discusses the DNA rearrangements in response to the environmental stress. Genotypic responses to changes in the environment are less documented, although there is an increasing body of evidence that the environment can influence the rate at which variation can occur. However, the definition of phenotypic and genotypic plasticity does not imply that the responses are necessarily adaptive, but there is a response to changes in the environment at both levels. The extent of phenotypic and genotypic variability is high under conditions of environmental stress for a variety of organisms. The examples of genomic changes in plants abound and appear to occur by a variety of basic mechanisms—namely, by amplification, deletion, and transposition events. The relative frequencies of these events have been examined in a number of instances. Genotypic and phenotypic variability are highest under conditions of environmental stress. The role of the environment in the generation of this variability is of central importance and the evidence is accumulating that it can have a direct role. The general conclusion from the chapter is that any shift in the environment to which an organism has become adapted should increase the variability. This conclusion needs to be tested further to define the genetic components involved in such responses and to assess the generality of the conclusion that environmental stresses can invoke genomic responses.

Molecular aspects of the environmental induction of heritable changes in flax

SummaryThe conditions for the induction and stabilisation of environmentally induced changes in flax, and the characterisation of these changes for six characters have been reviewed. A model for the induction and expression of these changes has been proposed. The environmental conditions for the successful induction of heritable changes comprise at least two components; a specific inducing component, for example a particular fertiliser treatment, which is required to induce the change, and a general inducing component, providing for vigorous healthy growth, which is necessary for the induced changes to be inherited. Stable induced changes require appropriate environments for their maintenance, for example heated greenhouses for the first 5 weeks of seedling growth and adequate pot size. Under certain growth conditions the induced changes can themselves be modified. DNA differences induced in the different genotrophs can occur at a number of sites within the genome, including the ribosomal RNA cistrons. Environmental induction causes changes in the isozyme band patterns of peroxidase, acid phosphatase and esterase isozymes. The genotrophs behave as distinct genetic types in crosses, with a complex pattern of inheritance of the hairy septa character. The association between the different induced characters is examined. All the genotrophs can be distinguished from each other on at least one of the six characters. A model for the induction of the DNA changes, involving a type of IS-element has been proposed. The expression of the induced changes, in terms of altered numbers of copies of particular sequences at specific sites within the genome, has been considered.

Environmentally induced DNA changes in plants

The environment has been shown to induce heritable changes in three plant systems, namely flax, Nicotiana rustica, and soybean cell suspension cultures. Changes in nuclear DNA sequences associated with the induction phenomenon have been demonstrated in both flax and soybean. In flax, which has been most extensively studied, the overall variation in the nuclear DNA is described together with a number of transcribed and nontranscribed sequences which have been shown to vary. Possible mechanisms by which the observed DNA changes could arise are discussed and the phenomenon of environmentally induced DNA variation compared with examples of gene amplification or deletion in other systems.

Is photosynthetic transcriptional regulation in Triticum aestivum L. cv. ‘TugelaDN’ a contributing factor for tolerance to Diuraphis noxia (Homoptera: Aphididae)?

Diuraphis noxia (Russian wheat aphid, RWA) is a major pest on wheat in South Africa and most other wheat growing countries. Being a probing-sucking insect, RWAs insert their stylets into the phloem sieve elements and feed on the phloem sap. This feeding causes necrotic lesions in resistant varieties, or decoloration of leaves and death in susceptible varieties. In an effort to broaden our understanding on the response of the plant to RWA feeding, we synthesized and analyzed expressed sequence tags (ESTs) from suppression subtractive hybridization (SSH) libraries. These libraries were constructed using near isogenic wheat lines susceptible ‘Tugela’ and resistant ‘TugelaDN’(Dn1) to RWA, as well as accession lines PI137739 (Dn1) and PI294994 (Dn5). Analysis of 200 ESTs from the libraries revealed the involvement of transcripts encoding genes involved in cell maintenance, growth and regulation, plant defense and signaling, photosynthesis and energy production, and of unknown function. A selection of these ESTs, in combination with clones obtained from other sources, were used on a custom array to study the expression profiles of 256 candidate wheat sequences putatively involved in plant defense against RWA. The selected sequences included wheat genomic clones with putative nucleotide binding site (NBS) motifs, rapid amplification of cDNA ends PCR (RACE-PCR), and cDNA clones from RWA induced libraries. Genomic banana and flax clones that were obtained using representative difference analysis (RDA), and suspected to be involved in abiotic stress responses, were also spotted onto the microarray slides. The spotted custom arrays were then hybridized against cDNA isolated from a resistant cultivar ‘Tugela DN’ on 0, 2, 5, and 8 days after infestation, post-labeled with Cy3- or Cy5-fluorescent dyes. The subsequent expression profiling using DNA microarray, RT-PCR, and Northern Blot analysis identified 29 transcripts associated with the feeding response. These transcripts encoded proteins functioning in direct defense and signaling, oxidative burst, cell wall degradation, cell maintenance, photosynthesis, and energy production. Results indicate that plants co-ordinately regulate gene expression when attacked by RWA. It is hypothesized that the NBS-LRR proteins are important in receptor recognition and signaling, which enable the plant to overcome the stresses inflicted by RWA feeding. It is further suggested that the ability to maintain photosynthetic function with resultant energy production is one of the determining factors ensuring the survival of the resistant varieties when coping with the RWA feeding.

The Generation of Somatic and Heritable Variation in Response to Stress

In many organisms, including all higher plants, the life cycle is such that somatic mutations have the potential to be inherited. The rate at which somatic mutations arise in higher plants can be affected by a number of factors, including the environment in which the plants grow. One well-documented case in which this occurs is the environmental induction of heritable changes in flax. In this system, phenotypic and genomic changes can arise as a consequence of the particular environment under which plants are allowed to develop. These changes, which occur in a single generation, can be stably inherited for a large number of generations. The majority of the genomic alterations occur in the highly repeated sequences, although not all the sequence families alter to the same extent. Even within a family there appear to be specific subsets that are especially labile, although the characteristics differentiating the labile subset from the rest of the family of similar (or identical) sequences is not known. Genomic changes have also been demonstrated when flax cells have been taken through a cycle of tissue culture and regeneration. In this case, the genomic changes were similar to those previously observed in response to particular environments. A possible role for these types of changes is the generation of bursts of variation in higher plants as a response to stress.

Phenotypic consequences of environmentally induced changes in plant DNA

Abstract Rapid genomic changes accompanied by heritable phenotypic changes can be induced in flax by environmental stress. The DNA changes are confined to a specific subset of the genome and may affect the phenotype by virtue of their position in the genome rather than by their specific nucleotide sequence.