Genotyping-by-sequencing through transcriptomics: implementation in a range of crop species with varying reproductive habits and ploidy levels.

The application of genomics in crops has the ability to significantly improve genetic gain for agriculture. Many marker-dense tools have been developed, but few have seen broad adoption in plant genomics due to issues of significant variations of genome size, levels of ploidy, single nucleotide polymorphism (SNP) frequency and reproductive habit. When combined with limited breeding activities, small research communities and scant sequence resources, the suitability of popular systems is often suboptimal and routinely fails to effectively balance cost-effectiveness and sample throughput. Genotyping-by-sequencing (GBS) encompasses a range of protocols including resequencing of the transcriptome. This study describes a skim GBS-transcriptomics (GBS-t) approach developed to be broadly applicable, cost-effective and high-throughput while still assaying a significant number of SNP loci. A range of crop species with differing levels of ploidy and degree of inbreeding/outbreeding were chosen, including perennial ryegrass, a diploid outbreeding forage grass; phalaris, a putative segmental allotetraploid outbreeding forage grass; lentil, a diploid inbreeding grain legume; and canola, an allotetraploid partially outbreeding oilseed. GBS-t was validated as a simple and largely automated, cost-effective method which generates sufficient SNPs (from 89 738 to 231 977) with acceptable levels of missing data and even genome coverage from c. 3 million sequence reads per sample. GBS-t is therefore a broadly applicable system suitable for many crops, offering advantages over other systems. The correct choice of subsequent sequence analysis software is important, and the bioinformatics process should be iterative and tailored to the specific challenges posed by ploidy variation and extent of heterozygosity.

Reference transcriptome assembly and annotation for perennial ryegrass.

RNA-Seq methodology has been used to generate a comprehensive transcriptome sequence resource for perennial ryegrass, an important temperate pasture grass species. A total of 931 547 255 reads were obtained from libraries corresponding to 19 distinct tissue samples, including both vegetative and reproductive stages of development. Assembly of data generated a final filtered reference set of 48 713 contigs and scaffolds. The transcriptome resource will support whole genome sequence assembly, comparative genomics, implementation of genotyping-by-sequencing (GBS) methods based on transcript sampling, and identification of candidate genes for multiple biological functions.

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.

Quantitative Trait Locus (QTL) meta-analysis and comparative genomics for candidate gene prediction in perennial ryegrass (Lolium perenne L.).

BACKGROUND: In crop species, QTL analysis is commonly used for identification of factors contributing to variation of agronomically important traits. As an important pasture species, a large number of QTLs have been reported for perennial ryegrass based on analysis of biparental mapping populations. Further characterisation of those QTLs is, however, essential for utilisation in varietal improvement programs. RESULTS: A bibliographic survey of perennial ryegrass trait-dissection studies identified a total of 560 QTLs from previously published papers, of which 189, 270 and 101 were classified as morphology-, physiology- and resistance/tolerance-related loci, respectively. The collected dataset permitted a subsequent meta-QTL study and implementation of a cross-species candidate gene identification approach. A meta-QTL analysis based on use of the BioMercator software was performed to identify two consensus regions for pathogen resistance traits. Genes that are candidates for causal polymorphism underpinning perennial ryegrass QTLs were identified through in silico comparative mapping using rice databases, and 7 genes were assigned to the p150/112 reference map. Markers linked to the LpDGL1, LpPh1 and LpPIPK1 genes were located close to plant size, leaf extension time and heading date-related QTLs, respectively, suggesting that these genes may be functionally associated with important agronomic traits in perennial ryegrass. CONCLUSIONS: Functional markers are valuable for QTL meta-analysis and comparative genomics. Enrichment of such genetic markers may permit further detailed characterisation of QTLs. The outcomes of QTL meta-analysis and comparative genomics studies may be useful for accelerated development of novel perennial ryegrass cultivars with desirable traits.

Fungus-originated glucanase and monooxygenase genes in creeping bent grass (Agrostis stolonifera L.).

Recent studies have revealed presence of fungus-originated genes in genomes of cool-season grasses, suggesting occurrence of multiple ancestral gene transfer events between the two distant lineages. The current article describes identification of glucanase-like and monooxygenase-like genes from creeping bent grass, as lateral gene transfer candidates. An in silico analysis suggested presence of the glucanase-like gene in Agrostis, Deyeuxia, and Polypogon genera, but not in other species belonging to the clade 1 of the Poeae tribe. Similarly, the monooxygenase-like gene was confined to Agrostis and Deyeuxia genera. A consistent result was obtained from PCR-based screening. The glucanase-like gene was revealed to be ubiquitously expressed in young seedlings of creeping bent grass. Although expression of the monooxygenase-like gene was suggested in plant tissues, the levels were considerably lower than those of the glucanase-like gene. A phylogenetic analysis revealed close relationships of the two genes between the corresponding genes in fungal endophyte species of the Epichloë genus, suggesting that the genes originated from the Epichloë lineage.

Fine-scale comparative genetic and physical mapping supports map-based cloning strategies for the self-incompatibility loci of perennial ryegrass (Lolium perenne L.).

Perennial ryegrass is an obligate outbreeding pasture grass of the Poaceae family, with a two-locus (S and Z) gametophytic self-incompatibility (SI) mechanism. This system has provided a major obstacle to targeted varietal development, and enhanced knowledge is expected to support more efficient breeding strategies. Comparative genetics and physical mapping approaches have been developed to permit molecular cloning of the SI genes. SI gene-linked genetic markers based on heterologous cDNA restriction fragment length polymorphisms (RFLPs) and homologous genomic DNA-derived simple sequence repeats (SSRs) were converted to single nucleotide polymorphism (SNP) format for efficient genotyping. Genetic mapping identified the location of SI loci and demonstrated macrosynteny between related grass species. S- and Z-linked bacterial artificial chromosome (BAC) clones were sequenced using massively parallel pyrosequencing technology to provide the first physical mapping data for Poaceae SI loci. The sequence assembly process suggested a lower prevalence of middle repetitive sequences in the Z locus region and hence precedence for positional cloning strategy. In silico mapping using data from rice, Brachypodium distachyon and Sorghum revealed high sequence conservation in the vicinity of the Z locus region between SI and self-compatible (SC) grass species. Physical mapping identified a total of nine genes encoded in the Z locus region. Expression profiling and nucleotide diversity assessment identified two Z-linked genes, LpTC116908 and LpDUF247, as plausible candidates for the male and female determinants of the S-Z SI system.

Rapid and Detailed Characterization of Transgene Insertion Sites in Genetically Modified Plants via Nanopore Sequencing.

Molecular characterization of genetically modified plants can provide crucial information for the development of detection and identification methods, to comply with traceability, and labeling requirements prior to commercialization. Detailed description of the genetic modification was previously a challenging step in the safety assessment, since it required the use of laborious and time-consuming techniques. In this study an accurate, simple, and fast method was developed for molecular characterization of genetically modified (GM) plants, following a user-friendly workflow for researchers with limited bioinformatic capabilities. Three GM events from a diverse array of crop species-perennial ryegrass, white clover, and canola-were used to test the approach that exploits long-read sequencing by the MinION device, from Oxford Nanopore Technologies. The method delivered a higher degree of resolution of the transgenic events within the host genome than has previously been possible with the standard Illumina short-range sequencing strategies. The flanking sequences, copy number, and presence of backbone sequences, and overall transgene insertion structure were determined for each of the plant genomes, with the additional identification of moderate-sized secondary insertions that would have previously been missed. The proposed workflow takes only about 1 week from DNA extraction to analyzed result, and the method will complement the existing approaches for molecular characterization of GM plants, since it makes the process faster, simpler, and more cost-effective.

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.

Safety Assessment of Genetically Modified Feed: Is There Any Difference From Food?

Food security is one of major concerns for the growing global population. Modern agricultural biotechnologies, such as genetic modification, are a possible solution through enabling an increase of production, more efficient use of natural resources, and reduced environmental impacts. However, new crop varieties with altered genetic materials may be subjected to safety assessments to fulfil the regulatory requirements, prior to marketing. The aim of the assessment is to evaluate the impact of products from the new crop variety on human, animal, and the environmental health. Although, many studies on the risk assessment of genetically modified (GM) food have been published, little consideration to GM feedstuff has been given, despite that between 70 to 90% of all GM crops and their biomass are used as animal feed. In addition, in some GM plants such as forages that are only used for animal feeds, the assessment of the genetic modification may be of relevance only to livestock feeding. In this article, the regulatory framework of GM crops intended for animal feed is reviewed using the available information on GM food as the baseline. Although, the majority of techniques used for the safety assessment of GM food can be used in GM feed, many plant parts used for livestock feeding are inedible to humans. Therefore, the concentration of novel proteins in different plant tissues and level of exposure to GM feedstuff in the diet of target animals should be considered. A further development of specific methodologies for the assessment of GM crops intended for animal consumption is required, in order to provide a more accurate and standardized assessment to the GM feed safety.

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.

Development and Application of Droplet Digital PCR Tools for the Detection of Transgenes in Pastures and Pasture-Based Products.

Implementation of molecular biotechnology, such as transgenic technologies, in forage species can improve agricultural profitability through achievement of higher productivity, better use of resources such as soil nutrients, water, or light, and reduced environmental impact. Development of detection and quantification techniques for genetically modified plants are necessary to comply with traceability and labeling requirements prior to regulatory approval for release. Real-time PCR has been the standard method used for detection and quantification of genetically modified events, and droplet digital PCR is a recent alternative technology that offers a higher accuracy. Evaluation of both technologies was performed using a transgenic high-energy forage grass as a case study. Two methods for detection and quantification of the transgenic cassette, containing modified fructan biosynthesis genes, and a selectable marker gene, hygromycin B phosphotransferase used for transformation, were developed. Real-time PCR was assessed using two detection techniques, SYBR Green I and fluorescent probe-based methods. A range of different agricultural commodities were tested including fresh leaves, tillers, seeds, pollen, silage and hay, simulating a broad range of processed agricultural commodities that are relevant in the commercial use of genetically modified pastures. The real-time and droplet digital PCR methods were able to detect both exogenous constructs in all agricultural products. However, a higher sensitivity and repeatability in transgene detection was observed with the droplet digital PCR technology. Taking these results more broadly, it can be concluded that the droplet digital PCR technology provides the necessary resolution for quantitative analysis and detection, allowing absolute quantification of the target sequence at the required limits of detection across all jurisdictions globally. The information presented here provides guidance and resources for pasture-based biotechnology applications that are required to comply with traceability requirements.

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.

Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries.

Background. Multiplexed sequencing is commonly performed on massively parallel short-read sequencing platforms such as Illumina, and the efficiency of library normalisation can affect the quality of the output dataset. Although several library normalisation approaches have been established, none are ideal for highly multiplexed sequencing due to issues of cost and/or processing time. Methods. An inexpensive and high-throughput library quantification method has been developed, based on an adaptation of the melting curve assay. Sequencing libraries were subjected to the assay using the Bio-Rad Laboratories CFX Connect(TM) Real-Time PCR Detection System. The library quantity was calculated through summation of reduction of relative fluorescence units between 86 and 95 °C. Results.PCR-enriched sequencing libraries are suitable for this quantification without pre-purification of DNA. Short DNA molecules, which ideally should be eliminated from the library for subsequent processing, were differentiated from the target DNA in a mixture on the basis of differences in melting temperature. Quantification results for long sequences targeted using the melting curve assay were correlated with those from existing methods (R (2) > 0.77), and that observed from MiSeq sequencing (R (2) = 0.82). Discussion.The results of multiplexed sequencing suggested that the normalisation performance of the described method is equivalent to that of another recently reported high-throughput bead-based method, BeNUS. However, costs for the melting curve assay are considerably lower and processing times shorter than those of other existing methods, suggesting greater suitability for highly multiplexed sequencing applications.

Design of an F1 hybrid breeding strategy for ryegrasses based on selection of self-incompatibility locus-specific alleles.

Relatively modest levels of genetic gain have been achieved in conventional ryegrass breeding when compared to cereal crops such as maize, current estimates indicating an annual improvement of 0.25-0.6% in dry matter production. This property is partially due to an inability to effectively exploit heterosis through the formation of F1 hybrids. Controlled crossing of ryegrass lines from geographically distant origins has demonstrated the occurrence of heterosis, which can result in increases of dry matter production in the order of 25%. Although capture of hybrid vigor offers obvious advantages for ryegrass cultivar production, to date there have been no effective and commercially suitable methods for obtaining high proportions of F1 hybrid seed. Continued advances in fine-scale genetic and physical mapping of the gametophytic self-incompatibility (SI) loci (S and Z) of ryegrasses are likely in the near future to permit the identification of closely linked genetic markers that define locus-specific haplotypes, allowing prediction of allelic variants and hence compatibility between different plant genotypes. Given the availability of such information, a strategy for efficient generation of ryegrass cultivars with a high proportion of F1 hybrid individuals has been simulated, which is suitable for commercial implementation. Through development of two parental pools with restricted diversity at the SI loci, relative crossing compatibility between pools is increased. Based on simulation of various levels of SI allele diversity restriction, the most effective scheme will generate 83.33% F1 hybrids. Results from the study, including the impact of varying flowering time, are discussed along with a proposed breeding design for commercial application.

Nucleotide diversity of vernalization and flowering-time-related genes in a germplasm collection of meadow fescue (Festuca pratensis Huds. syn. Lolium pratense (Huds.) Darbysh.).

In plant species, control of flowering time is an important factor for adaptation to local natural environments. The Vrn1 , CO , FT1 and CK2α genes are key components in the flowering-specific signaling pathway of grass species. Meadow fescue is an agronomically important forage grass species, which is naturally distributed across Europe and Western Asia. In this study, meadow fescue flowering-time-related genes were resequenced to assess nucleotide diversity in European and Western Asian subpopulations. Identified sequence polymorphisms were then converted into PCR-based molecular genetic markers, and a meadow fescue germplasm collection was genotyped to investigate global allelic variation. Lower nucleotide diversities were observed for the Vrn1 and CO orthologs, while relatively higher values were observed for the FT1 and casein kinase II α-subunit (CK2α) orthologs. The nucleotide diversity for FT1 orthologs in the Western Asian subpopulation was significantly higher than those of the European subpopulation. Similarly, significant differences in nucleotide diversity for the remaining genes were observed between several combinations of subpopulation. The global allele distribution pattern was consistent with observed level of nucleotide diversity. These results suggested that the degree of purifying selection acting on the genes differs according to geographical location. As previously shown for model plant species, functional specificities of flowering-time-related genes may also vary according to environmental conditions.

Comparative Genomics in Perennial Ryegrass (Lolium perenne L.): Identification and Characterisation of an Orthologue for the Rice Plant Architecture-Controlling Gene OsABCG5.

Perennial ryegrass is an important pasture grass in temperate regions. As a forage biomass-generating species, plant architecture-related characters provide key objectives for breeding improvement. In silico comparative genomics analysis predicted colocation between a previously identified QTL for plant type (erect versus prostrate growth) and the ortholocus of the rice OsABCG5 gene (LpABCG5), as well as related QTLs in other Poaceae species. Sequencing of an LpABCG5-containing BAC clone identified presence of a paralogue (LpABCG6) in the vicinity of the LpABCG5 locus, in addition to three other gene-like sequences. Comparative genomics involving five other 5 grass species (rice, Brachypodium, sorghum, maize, and foxtail millet) revealed conserved microsynteny in the ABCG5 ortholocus-flanking region. Gene expression profiling and phylogenetic analysis suggested that the two paralogues are functionally distinct. Fourteen additional ABCG5 gene family members, which may interact with the LpABCG5 gene, were identified through sequencing of transcriptomes from perennial ryegrass leaf, anther, and pistils. A larger-scale phylogenetic analysis of the ABCG gene family suggested conservation between major branches of the Poaceae family. This study identified the LpABCG5 gene as a candidate for the plant type determinant, suggesting that manipulation of gene expression may provide valuable phenotypes for perennial ryegrass breeding.