Fungus-originated genes in the genomes of cereal and pasture grasses acquired through ancient lateral transfer.

Evidence for ancestral gene transfer between Epichloë fungal endophyte ancestors and their host grass species is described. From genomes of cool-season grasses (the Poeae tribe), two Epichloë-originated genes were identified through DNA sequence similarity analysis. The two genes showed 96% and 85% DNA sequence identities between the corresponding Epichloë genes. One of the genes was specific to the Loliinae sub-tribe. The other gene was more widely conserved in the Poeae and Triticeae tribes, including wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). The genes were independently transferred during the last 39 million years. The transferred genes were expressed in plant tissues, presumably retaining molecular functions. Multiple gene transfer events between the specific plant and fungal lineages are unique. A range of cereal crops is included in the Poeae and Triticeae tribes, and the Loliinae sub-tribe is consisted of economically important pasture and forage crops. Identification and characterisation of the 'natural' adaptation transgenes in the genomes of cereals, and pasture and forage grasses, that worldwide underpin the production of major foods, such as bread, meat, and milk, may change the 'unnatural' perception status of transgenic and gene-edited plants.

Evaluation and Recommendations for Routine Genotyping Using Skim Whole Genome Re-sequencing in Canola.

Whole genome sequencing offers genome wide, unbiased markers, and inexpensive library preparation. With the cost of sequencing decreasing rapidly, many plant genomes of modest size are amenable to skim whole genome resequencing (skim WGR). The use of skim WGR in diverse sample sets without the use of imputation was evaluated in silico in 149 canola samples representative of global diversity. Fastq files with an average of 10x coverage of the reference genome were used to generate skim samples representing 0.25x, 0.5x, 1x, 2x, 3x, 4x, and 5x sequencing coverage. Applying a pre-defined list of SNPs versus de novo SNP discovery was evaluated. As skim WGR is expected to result in some degree of insufficient allele sampling, all skim coverage levels were filtered at a range of minimum read depths from a relaxed minimum read depth of 2 to a stringent read depth of 5, resulting in 28 list-based SNP sets. As a broad recommendation, genotyping pre-defined SNPs between 1x and 2x coverage with relatively stringent depth filtering is appropriate for a diverse sample set of canola due to a balance between marker number, sufficient accuracy, and sequencing cost, but depends on the intended application. This was experimentally examined in two sample sets with different genetic backgrounds: 1x coverage of 1,590 individuals from 84 Australian spring type four-parent crosses aimed at maximizing diversity as well as one commercial F1 hybrid, and 2x coverage of 379 doubled haploids (DHs) derived from a subset of the four-parent crosses. To determine optimal coverage in a simpler genetic background, the DH sample sequence coverage was further down sampled in silico. The flexible and cost-effective nature of the protocol makes it highly applicable across a range of species and purposes.

Homology-based enzymatic DNA fragment assembly-based illumina sequencing library preparation.

The current Illumina HiSeq and MiSeq platforms can generate paired-end reads of up to 2 x 250 bp and 2 x 300 bp in length, respectively. These read lengths may be substantially longer than genomic regions of interest when a DNA sequencing library is prepared through a target enrichment-based approach. A sequencing library preparation method has been developed based on the homology-based enzymatic DNA fragment assembly scheme to allow processing of multiple PCR products within a single read. Target sequences were amplified using locus-specific PCR primers with 8 bp tags, and using the tags, homology-based enzymatic DNA assembly was performed with DNA polymerase, T7 exonuclease and T4 DNA ligase. Short PCR amplicons can hence be assembled into a single molecule, along with sequencing adapters specific to the Illumina platforms. As a proof-of-concept experiment, short PCR amplicons (57-66 bp in length) derived from genomic DNA templates of field pea and containing variable nucleotide locations were assembled and sequenced on the MiSeq platform. The results were validated with other genotyping methods. When 5 PCR amplicons were assembled, 4.3 targeted sequences (single-nucleotide polymorphisms) on average were successfully identified within each read. The utility of this for sequencing of short fragments has consequently been demonstrated.

Horizontal transfer of a ß-1,6-glucanase gene from an ancestral species of fungal endophyte to a cool-season grass host.

Molecular characterisation has convincingly demonstrated some types of horizontal gene transfer in eukaryotes, but nuclear gene transfer between distantly related eukaryotic groups appears to have been rare. For angiosperms (flowering plants), nuclear gene transfer events identified to date have been confined to genes originating from prokaryotes or other plant species. In this report, evidence for ancient horizontal transfer of a fungal nuclear gene, encoding a ß-1,6-glucanase enzyme for fungal cell wall degradation, into an angiosperm lineage is presented for the first time. The gene was identified from de novo sequencing and assembly of the genome and transcriptome of perennial ryegrass, a cool-season grass species. Molecular analysis confirmed the presence of the complete gene in the genome of perennial ryegrass. No corresponding sequence was found in other plant species, apart from members of the Poeae sub-tribes Loliinae and Dactylidinae. Evidence suggests that a common ancestor of the two sub-tribes acquired the gene from a species ancestral to contemporary grass-associated fungal endophytes around 9-13 million years ago. This first report of horizontal transfer of a nuclear gene from a taxonomically distant eukaryote to modern flowering plants provides evidence for a novel adaptation mechanism in angiosperms.

A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI.

BACKGROUND: Fragmentation at random nucleotide locations is an essential process for preparation of DNA libraries to be used on massively parallel short-read DNA sequencing platforms. Although instruments for physical shearing, such as the Covaris S2 focused-ultrasonicator system, and products for enzymatic shearing, such as the Nextera technology and NEBNext dsDNA Fragmentase kit, are commercially available, a simple and inexpensive method is desirable for high-throughput sequencing library preparation. MspJI is a recently characterised restriction enzyme which recognises the sequence motif CNNR (where R = G or A) when the first base is modified to 5-methylcytosine or 5-hydroxymethylcytosine. RESULTS: A semi-random enzymatic DNA amplicon fragmentation method was developed based on the unique cleavage properties of MspJI. In this method, random incorporation of 5-methyl-2'-deoxycytidine-5'-triphosphate is achieved through DNA amplification with DNA polymerase, followed by DNA digestion with MspJI. Due to the recognition sequence of the enzyme, DNA amplicons are fragmented in a relatively sequence-independent manner. The size range of the resulting fragments was capable of control through optimisation of 5-methyl-2'-deoxycytidine-5'-triphosphate concentration in the reaction mixture. A library suitable for sequencing using the Illumina MiSeq platform was prepared and processed using the proposed method. Alignment of generated short reads to a reference sequence demonstrated a relatively high level of random fragmentation. CONCLUSIONS: The proposed method may be performed with standard laboratory equipment. Although the uniformity of coverage was slightly inferior to the Covaris physical shearing procedure, due to efficiencies of cost and labour, the method may be more suitable than existing approaches for implementation in large-scale sequencing activities, such as bacterial artificial chromosome (BAC)-based genome sequence assembly, pan-genomic studies and locus-targeted genotyping-by-sequencing.

Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery.

BACKGROUND: Lentil (Lens culinaris Medik.) is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality. RESULTS: Tissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 106 expressed sequence tags (ESTs). De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR)-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism. CONCLUSIONS: A substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.