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Lentil (Lens culinaris Medik.) is a self-pollinating, diploid, annual, cool-season, food legume crop that is cultivated throughout the world. Ascochyta blight (AB), caused by Ascochyta lentis Vassilievsky, is an economically important and widespread disease of lentil. Development of cultivars with high levels of durable resistance provides an environmentally acceptable and economically feasible method for AB control. A detailed understanding of the genetic basis of AB resistance is hence highly desirable, in order to obtain insight into the number and influence of resistance genes. Genetic linkage maps based on single nucleotide polymorphisms (SNP) and simple sequence repeat (SSR) markers have been developed from three recombinant inbred line (RIL) populations. The IH × NF map contained 460 loci across 1461.6 cM, while the IH × DIG map contained 329 loci across 1302.5 cM and the third map, NF × DIG contained 330 loci across 1914.1 cM. Data from these maps were combined with a map from a previously published study through use of bridging markers to generate a consensus linkage map containing 689 loci distributed across seven linkage groups (LGs), with a cumulative length of 2429.61 cM at an average density of one marker per 3.5 cM. Trait dissection of AB resistance was performed for the RIL populations, identifying totals of two and three quantitative trait loci (QTLs) explaining 52 and 69% of phenotypic variation for resistance to infection in the IH × DIG and IH × NF populations, respectively. Presence of common markers in the vicinity of the AB_IH1- and AB_IH2.1/AB_IH2.2-containing regions on both maps supports the inference that a common genomic region is responsible for conferring resistance and is associated with the resistant parent, Indianhead. The third QTL was derived from Northfield. Evaluation of markers associated with AB resistance across a diverse lentil germplasm panel revealed that the identity of alleles associated with AB_IH1 predicted the phenotypic responses with high levels of accuracy (~86%), and therefore have the potential to be widely adopted in lentil breeding programs. The availability of RIL-based maps, a consensus map, and validated markers linked to AB resistance provide important resources for lentil improvement.

BackgroundFor millennia, drug-type cannabis strains were extensively used for various medicinal, ritual, and inebriant applications. However, cannabis prohibition during the last century led to cultivation and breeding activities being conducted under clandestine conditions, while scientific development of the crop ceased. Recently, the potential of medicinal cannabis has been reacknowledged and the now expanding industry requires optimal and scientifically characterized varieties. However, scientific knowledge that can propel this advancement is sorely lacking. To address this issue, the current study aims to provide a better understanding of key physiological and phenological traits that can facilitate the breeding of advanced cultivars.ResultsA diverse population of 121 genotypes of high-THC or balanced THC-CBD ratio was cultivated under a controlled environment facility and 13 plant parameters were measured. No physiological association across genotypes attributed to the same vernacular classification was observed. Floral bud dry weight was found to be positively associated with plant height and stem diameter but not with days to maturation. Furthermore, the heritability of both plant height and days to maturation was relatively high, but for plant height it decreased during the vegetative growth phase. To advance breeding efficacy, a prediction equation for forecasting floral bud dry weight was generated, driven by parameters that can be detected during the vegetative growth phase solely.ConclusionsOur findings suggest that selection for taller and fast-growing genotypes is likely to lead to an increase in floral bud productivity. It was also found that the final plant height and stem diameter are determined by 5 independent factors that can be used to maximize productivity through cultivation adjustments. The proposed prediction equation can facilitate the selection of prolific genotypes without the completion of a full cultivation cycle. Future studies that will associate genome-wide variation with plants morphological traits and cannabinoid profile will enable precise and accelerated breeding through genomic selection approaches.

Advancements in high-throughput genotyping and sequencing technologies are enabling the development of a vast range of genomic tools and resources for a new revolution in plant breeding. Several genotyping-by-sequencing (GBS) methods including capture-based, genome complexity reduction and sequencing of cDNA (GBS-t) are available for application in trait dissection, association mapping, and genomic selection (GS) in crop plants. The aims of this study were to identify genomic regions conferring resistance to Ascochyta blight (AB) introgressed from the wild Cicer echinospernum into the domesticated C. arietinum, through a conventional recombinant inbred population genotyped using a variety of GBS methods. Evaluation of GBS methods revealed that capture-based approaches are robust and reproducible while GBS-t is rapid and flexible. A genetic linkage map consisting of 5886 polymorphic loci spanning 717.26 cM was generated. Using field phenotyping data from two years, a single genomic region on LG4 was identified with quantitative trait loci (QTL) mapping. Both GBS methods reported in this study are well suited for applications in genomics assisted plant breeding. Linked markers for AB resistance, identified in the current study, provide an important resource for the deployment into chickpea breeding programs for marker-assisted selection (MAS).

Potato is an important food crop worldwide and is grown in a large number of countries. As such, the crop is under disease pressures and the need for selecting disease resistance genes during breeding programs is essential. Of particular importance within Australia and other parts of the world is the potyvirus, Potato virus Y (PVY). In this paper, three commonly used PVY resistance markers, M45, RYSC3 and M6, were evaluated using existing genomic resources and phenotypic data from the Australian potato breeding program to identify a region where the PVY resistance gene, Ryadg may reside. A region of Chromosome XI was investigated, and a cluster of disease resistance genes was identified that the resistance gene Ryadg is suspected to reside within. Protein characterization was also performed on the putative resistant gene. A specific variant that had complete association with the resistance gene was identified and a single nucleotide polymorphism (SNP) assay was designed to avoid dissociation of marker and gene in future breeding programs. This SNP marker (SNP37279) was validated as a Kompetitive Allele-specific PCR (KASP) genotyping assay and was found to perform more accurately than all previously used markers for detecting Ryadg.

Blackleg disease causes yield losses in canola (Brassica napus L.). To identify resistance genes and genomic regions, genome-wide association studies (GWAS) of 585 diverse winter and spring canola accessions were performed using imputed whole-genome sequence (WGS) and transcriptome genotype-by-sequencing (GBSt). Blackleg disease phenotypes were collected across three years in six trials. GWAS were performed in several ways and their respective power was judged by the number of significant single nucleotide polymorphisms (SNP), the false discovery rate (FDR), and the percentage of SNP that validated in additional field trials in two subsequent years. WGS GWAS with 1,234,708 million SNP detected a larger number of significant SNP, achieved a lower FDR and a higher validation rate than GBSt with 64,072 SNP. A meta-analysis combining survival and average internal infection resulted in lower FDR but also lower validation rates. The meta-analysis GWAS identified 79 genomic regions (674 SNP) conferring potential resistance to L. maculans. While several GWAS signals localised in regions of known Rlm genes, fifty-three new potential resistance regions were detected. Seventeen regions had underlying genes with putative functions related to disease defence or stress response in Arabidopsis thaliana. This study provides insight into the genetic architecture and potential molecular mechanisms underlying canola L. maculans resistance.

Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9–11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Potatoes are an important human food crop, but have a number of yield limiting factors, including disease susceptibility. Potato virus Y (PVY) is found worldwide, and is one of the main virus problems for potato growers. PVY is transmitted by aphids and mechanically by machinery, tools and people, and symptoms are variable across cultivars and strains, including being symptomless in some cultivars. Therefore, breeding resistant cultivars is the best way to control this virus. This study phenotypically screened 74 of the main commercial cultivars and a few other select cultivars grown in Australia, in order to identify sources of resistance to PVY. The cultivars were screened against PVYO and PVYNTN, with 23 out of 71 resistant to PVYO and 13 out of 74 resistant to PVYNTN, and all these 13 were resistant to both strains. When the phenotypic screening was compared to the results listed on the European Cultivated Potato Database, the majority of results were found to be consistent. We then evaluated three molecular markers RYSC3, M45, and STM0003 for the extreme resistance genes Ryadg and Rysto, to validate the usefulness of the markers for marker-assisted selection (MAS) on Australian germplasm. The degree of correlation between the resistance phenotypes and the RYSC3, M45, and STM0003 markers for Ryadg and Rysto conferred PVY resistance was determined. Three cultivars amplified the RYSC3 marker, while the M45 marker amplified the same 3 and an additional 9. Of the 12 cultivars, 11 phenotyped as resistant, but 1 was susceptible. The STM0003 marker was amplified from only 2 cultivars that both had resistant phenotypes. The RYSC3, M45, and STM0003 markers were therefore able to identify all the 13 cultivars that were resistant to both strains of PVY. Therefore, these markers will enable the identification of genotypes with resistance to PVY, and enable PVY resistant parents to be used for the development of superior progeny, these genetic markers can be used for MAS in the Australian potato breeding program.

Nutritive value (NV) of forage is too time consuming and expensive to measure routinely in targeted breeding programs. Non-destructive spectroscopy has the potential to quickly and cheaply measure NV but requires an intermediate modelling step to interpret the spectral data. A novel machine learning technique for forage analysis, Cubist, was used to analyse canopy spectra to predict seven NV parameters, including dry matter (DM), acid detergent fibre (ADF), ash, neutral detergent fibre (NDF), in vivo dry matter digestibility (IVDMD), water soluble carbohydrates (WSC), and crude protein (CP). Perennial ryegrass (Lolium perenne) was used as the test crop. Independent validation of the developed models revealed prediction capabilities with R2 values and Lin’s concordance values reported between 0.49 and 0.82, and 0.68 and 0.89, respectively. Informative wavelengths for the creation of predictive models were identified for the seven NV parameters. These wavelengths included regions of the electromagnetic spectrum that are usually excluded due to high background variation, however, they contain important information and utilising them to obtain meaningful signals within the background variation is an advantage for accurate models. Non-destructive field spectroscopy along with the predictive models was deployed infield to measure NV of individual ryegrass plants. A significant reduction in labour was observed. The associated increase in speed and reduction of cost makes targeting NV in commercial breeding programs now feasible.

A conventional potato breeding strategy uses targeted outcrossing, followed by phenotypic recurrent selection over a series of generations to identify improved cultivars. This paper reviews recent research in Australia aimed at improving the efficiency of such breeding. To develop marker-assisted selection (MAS) for traits of interest, our initial targets were qualitative disease resistances for potato cyst nematode (Globodera rostochiensis Ro1), Potato virus Y and Potato virus X. We undertook a cost analysis comparison between MAS and conventional screening, confirming that MAS would be cost-effective within a breeding programme. Then, as the majority of target traits are quantitative in nature, we also looked at methods to address these traits, including progeny testing and a quantitative genetic analysis technique to develop estimated breeding values (EBVs). We found the markers were useful for detecting the disease resistance characters, while the EBVs improved the analysis of the complex traits. Using a combination of MAS, EBVs and conventional screening methods, we then designed a breeding scheme for rapid selection of cultivars with multiple desirable traits, reducing the breeding cycle from over 10 to 4 years. We then explored the factors that will affect the application of genomic selection in potato and investigated strategies to incorporate genomic selection in potato breeding, as we found that it would accelerate genetic gain as the breeding cycle can be reduced to 1 year. Improvements in computational power are also flowing on to research capabilities such as sequencing, high-throughput phenotyping and data analysis, which will accelerate germplasm improvement and breeding. High-throughput phenotyping facilities are being developed that include automated glasshouse systems equipped with imaging sensors and in-field high-throughput phenotyping systems with sensors mounted on ground- or aerial-based vehicles. Using these technological improvements in phenotypic and genotypic analysis will reduce the breeding cycle in a cost-effective manner and means that we can now breed differently.

© 2017 The Authors With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours. This study aimed to determine the ability of the MinION to metagenomically detect and characterise a virus from an infected mosquito. RNA was extracted from an Aedes notoscriptus mosquito infected with Ross River virus (RRV), converted into cDNA and sequenced on the MinION. Bioinformatic analysis of the MinION reads led to detection of full-length RRV, with reads of up to 2.5 kb contributing to the assembly. The cDNA was also sequenced on the MiSeq sequencer, and both platforms recovered the RRV genome with >98% accuracy. This proof of concept study demonstrates the metagenomic detection of an arbovirus, using the MinION, directly from a mosquito with minimal sample purification.

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.

Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next‐generation sequencing (NGS) is a high‐throughput technology that has the potential to modernize vector surveillance. When combined with DNA barcoding, it is termed ‘metabarcoding.’ The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269‐bp section of the mitochondrial cytochrome c oxidase subunit I (COI) locus. Additionally, a 67‐bp region of the RRV E2 gene was amplified from synthesized cDNA to screen for RRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using a DNA barcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species and RRV in every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species and RRV from a single mosquito. The primers used for amplification, number of PCR cycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches.

Trap-based surveillance strategies are widely used for monitoring of invasive insect species, aiming to detect newly arrived exotic taxa as well as track the population levels of established or endemic pests. Where these surveillance traps have low specificity and capture non-target endemic species in excess of the target pests, the need for extensive specimen sorting and identification creates a major diagnostic bottleneck. While the recent development of standardized molecular diagnostics has partly alleviated this requirement, the single specimen per reaction nature of these methods does not readily scale to the sheer number of insects trapped in surveillance programmes. Consequently, target lists are often restricted to a few high-priority pests, allowing unanticipated species to avoid detection and potentially establish populations.DNA metabarcoding has recently emerged as a method for conducting simultaneous, multi-species identification of complex mixed communities and may lend itself ideally to rapid diagnostics of bulk insect trap samples. Moreover, the high-throughput nature of recent sequencing platforms could enable the multiplexing of hundreds of diverse trap samples on a single flow cell, thereby providing the means to dramatically scale up insect surveillance in terms of both the quantity of traps that can be processed concurrently and number of pest species that can be targeted. In this review of the metabarcoding literature, we explore how DNA metabarcoding could be tailored to the detection of invasive insects in a surveillance context and highlight the unique technical and regulatory challenges that must be considered when implementing high-throughput sequencing technologies into sensitive diagnostic applications.

Current Australian potato cultivars suffer from a number of production and quality issues. Commercial cultivar development within Australia used a conventional potato breeding strategy, with outcrossing, and then screening of derived lines to identify improved cultivars, although we have been investigating methods to make the program more efficient. Implementation of marker-assisted selection (MAS) is highly desirable to increase efficiency, and our initial targets for MAS have been qualitative disease resistances. Two diseases of concern within the Australian potato industry are potato cyst nematode (PCN) (Globodera rostochiensis Ro1) and Potato virus Y (PVY), so we investigated markers for these diseases, including a potentially diagnostic marker, 57R, for PCN Ro1. As the majority of the target traits for the breeding program are quantitative and controlled by multiple to many genes, we have also investigated a quantitative genetic analysis technique used in livestock breeding for these complex traits to develop estimated breeding values (EBVs) for them. The breeding program has now developed a breeding scheme using a combination of MAS, EBVs and conventional screening methods for early-generation selection of cultivars with multiple desirable traits. The application of these new techniques, alongside conventional screening, will see a reduction in the breeding cycle and the cost of breeding, as well as improving genetic gain in a range of traits.

Durante el manejo postcosecha del maíz (Zea mays L.) se presentan pérdidas importantes por el ataque de plagas de almacén, particularmente por gorgojo (Sitophilus zeamais Motschulsky). El objetivo del presente estudio fue evaluar la efectividad del epazote (Dysphania ambrosioides) para controlar los daños por gorgojo en maíz. Semillas de 11 genotipos de maíz, nueve experimentales y dos híbridos comerciales, todos con humedad de 13 % y sin daño físico que indicara la presencia de insectos, fueron sometidos a tratamientos con polvo de epazote en cuatro concentraciones (0, 5, 7 y 10 g de epazote kg-1 de semilla) para controlar gorgojo. Se utilizó un diseño completamente al azar con arreglo factorial y tres repeticiones. La unidad experimental consistió en una caja de plástico acondicionada donde se colocaron 100 semillas, previamente pesadas, junto con 50 gorgojos jóvenes y la dosis correspondiente de polvo de epazote. Las variables registradas fueron pérdida de peso, granos dañados y sobrevivencia de insectos, todos expresados en porcentaje. Hubo diferencias entre genotipos de maíz solamente para pérdida de peso (P ≤ 0.05); todos los genotipos de maíz registraron pérdidas; sin embargo, el híbrido CLTHW14001 fue el menos afectado con 3.5 % de pérdida de peso. Las dosis de 7 y 10 g de epazote kg-1 de semilla fueron sobresalientes en las variables pérdida de peso, granos dañados y sobrevivencia de insectos, con 3.4, 12.2 y 3.5 % en promedio, respectivamente. El híbrido CLTHW14001 mostró el menor daño con la dosis de 7 g de epazote kg-1 de semilla con 0.94 % de pérdida de peso. El uso de polvo de epazote para el control de gorgojo es efectivo en concentraciones desde 7 g kg-1 de semilla. El genotipo de maíz influye en la efectividad del tratamiento.

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloe endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+ (gene of interest positive) and GOI− (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+ and GOI− for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI− compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI−. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte–grass associations. Endophyte–host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.

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.

Background Within the Brassicaceae, six species from the genus Brassica are widely cultivated throughout the world as oilseed, condiment, fodder or vegetable crops. The genetic relationships among the six Brassica species are described by U’s triangle model. Extensive shared traits and diverse morphotypes among Brassica species make identification and classification based on phenotypic data alone challenging and unreliable, especially when dealing with large germplasm collections. Consequently, a major issue for genebank collections is ensuring the correct identification of species. Molecular genotyping based on simple sequence repeat (SSR) marker sequencing or the Illumina Infinium Brassica napus 60K single nucleotide polymorphism (SNP) array has been used to identify species and assess genetic diversity of Brassica collections. However, these methods are technically challenging, expensive and time-consuming, making them unsuitable for routine or rapid screening of Brassica accessions for germplasm management. A cheaper, faster and simpler method for Brassica species identification is described here. Results A multiplex polymerase chain reaction (MPCR) consisting of new and existing primers specific to the Brassica A, B and C genomes was able to reliably distinguish all six Brassica species in the triangle of U with 16 control samples of known species identity. Further validation against 120 Brassica accessions previously genotyped showed that the MPCR is highly accurate and comparable to more advanced techniques such as SSR marker sequencing or the Illumina Infinium B. napus 60K SNP array. In addition, the MPCR was sensitive enough to detect seed contaminations in pooled seed samples of Brassica accessions. Conclusion A cheap and fast multiplex PCR assay for identification of Brassica species in the triangle of U was developed and validated in this study. The MPCR assay can be readily implemented in any basic molecular laboratory and should prove useful for the management of Brassica germplasm collections in genebanks.

Field pea (Pisum sativum L.) is a cool-season legume that is cultivated worldwide for both human consumption and stockfeed. Genetic improvement is essential for enhanced crop production and management of field pea, especially to deliver superior varieties adapted to various biotic and abiotic stresses. A detailed understanding of the genetic basis of such stress tolerances is hence desirable. Genetic linkage maps based on single nucleotide polymorphisms (SNP) and simple sequence repeat (SSR) markers have been developed from two recombinant inbred line (RIL) populations generated by crossing phenotypically divergent parental genotypes. The Kaspa × Yarrum map contained 428 loci across 1910 cM, while the Kaspa × ps1771 map contained 451 loci across 1545 cM. Data from these maps were combined through bridging markers with those from previously published studies to generate a consensus structure including 2028 loci distributed across seven linkage groups (LGs), with a cumulative length of 2387 cM at an average density of one marker per 1.2 cM. Trait dissection of powdery mildew resistance was performed for both RIL populations, identifying a single genomic region of large magnitude in the same genomic region on Ps VI, which were inferred to correspond to the er1 gene. Equivalent studies of the Kaspa × ps1771 RIL population identified a major quantitative trait locus (QTL) for boron tolerance that coincided with the disease resistance-controlling locus, permitting strategies of co-selection for these desirable traits. Resequencing of the PsMLO1 candidate gene from resistant and susceptible genotypes allowed design and validation of a putative diagnostic marker for powdery mildew resistance. The availability of a highly saturated consensus map, linked markers for key biotic and abiotic stress tolerances and a diagnostic marker for the agronomically important er1 gene provide important resources for field pea molecular breeding programs.

Large-scale SNP discovery and dense genetic mapping in a lentil intraspecific cross permitted identification of a single chromosomal region controlling tolerance to boron toxicity, an important breeding objective. Lentil (Lens culinaris Medik.) is a highly nutritious food legume crop that is cultivated world-wide. Until recently, lentil has been considered a genomic 'orphan' crop, limiting the feasibility of marker-assisted selection strategies in breeding programs. The present study reports on the identification of single-nucleotide polymorphisms (SNPs) from transcriptome sequencing data, utilisation of expressed sequence tag (EST)-derived simple sequence repeat (SSR) and SNP markers for construction of a gene-based genetic linkage map, and identification of markers in close linkage to major QTLs for tolerance to boron (B) toxicity. A total of 2,956 high-quality SNP markers were identified from a lentil EST database. Sub-sets of 546 SSRs and 768 SNPs were further used for genetic mapping of an intraspecific mapping population (Cassab × ILL2024) that exhibits segregation for B tolerance. Comparative analysis of the lentil linkage map with the sequenced genomes of Medicago truncatula Gaertn., soybean (Glycine max [L.] Merr.) and Lotus japonicus L. indicated blocks of conserved macrosynteny, as well as a number of rearrangements. A single genomic region was found to be associated with variation for B tolerance in lentil, based on evaluation performed over 2 years. Comparison of flanking markers to genome sequences of model species (M. truncatula, soybean and Arabidopsis thaliana) identified candidate genes that are functionally associated with B tolerance, and could potentially be used for diagnostic marker development in lentil.

Perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.) are important temperate forage grasses which are closely related, generating fertile interspecific hybrids. All groups are represented by multiple cultivars in the commercial pasture seeds market. Due to the close taxonomic relationship between the two species, differentiation based on morphophysiological criteria is not always readily achievable. In addition, an obligate outbreeding reproductive habit produces high levels of individual heterozygosity and intrapopulation diversity, which presents problems for discrimination between cultivars. Molecular genetic marker polymorphism provides an effective means of addressing these challenges. An iterative process of resequencing from loci distributed across the perennial ryegrass genome was used to identify single nucleotide polymorphism (SNP) markers, which were then validated and formatted in a highly multiplexed (384-plex) assay system. SNP genotyping was then performed across samples of 48–192 individuals from a total of 27 ryegrass cultivars (19 of perennial ryegrass, seven of Italian ryegrass and one hybrid cultivar). SNP markers from perennial ryegrass exhibited a high level of transfer to Italian ryegrass. Data analysis permitted quantification of intra- and inter-species diversity, as well as discrimination between cultivars within each species, including diploid and autotetraploid cultivars of perennial ryegrass. Lower levels of SNP-based diversity were detected in Italian ryegrass than in perennial ryegrass. A neighbour-joining tree based on genetic distance analysis located a hybrid cultivar to an intermediate position between the two species-specific cultivar groups. The resulting catalogue of ryegrass cultivars will provide support for the processes of cultivar accreditation and quality assurance.

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Labr., is a highly destructive disease of chickpea (Cicer arietinum L.) on a global basis, and exhibits considerable natural variation for pathogenicity. Different sources of ascochyta blight resistance are available within the cultivated species, suitable for pyramiding to improve field performance. Robust and closely linked genetic markers are desirable to facilitate this approach. A total of 4,654 simple sequence repeat (SSR) and 1,430 single nucleotide polymorphism (SNP) markers were identified from a chickpea expressed sequence tag (EST) database. Subsets of 143 EST–SSRs and 768 SNPs were further used for validation and subsequent high-density genetic mapping of two intraspecific mapping populations (Lasseter × ICC3996 and S95362 × Howzat). Comparison of the linkage maps to the genome of Medicago truncatula revealed a high degree of conserved macrosynteny. Based on field evaluation of ascochyta blight incidence performed over 2 years, two genomic regions containing resistance determinants were identified in the Lasseter × ICC3996 family. In the S95362 × Howzat population, only one quantitative trait locus (QTL) region was identified for both phenotypic evaluation trials, which on the basis of bridging markers was deduced to coincide with one of the Lasseter × ICC3996 QTLs. Of the two QTL-containing regions identified in this study, one (ab_QTL1) was predicted to be in common with QTLs identified in prior studies, while the other (ab_QTL2) may be novel. Markers in close linkage to ascochyta blight resistance genes that have been identified in this study can be further validated and effectively implemented in chickpea breeding programs.

Phalaris aquatica is a cool-season perennial grass species that is extensively cultivated in Australia, with additional usage in other areas of the world. Phalaris displays a number of desirable agronomic characteristics, although unfavourable traits include excessive seed shattering, sensitivity to aluminium toxicity, and several toxicosis syndromes. Varietal development has to date been based on traditional selection methods, but would benefit from the application of genomics-based approaches, which require the development of large-scale sequence resources. Due to a large nuclear DNA content, methods that target the expressed component of the genome and reduce the complexity of analysis are most amenable to current sequencing technologies. A reference unigene set has been developed by transcriptome sequencing of multiple tissues from a single plant belonging to the variety Landmaster. Comparisons have been made to gene complements from related species, as well as reference protein databases, and patterns of gene expression in different tissues have been evaluated. A number of candidate genes relevant to removal of undesirable attributes have been identified. The reference unigene set will provide the basis for detailed studies of differential gene expression and identification of candidate genes for potential transgenic deployment, as well as a critical resource for genotypic analysis to support future genomics-assisted breeding activities for phalaris improvement.

Italian ryegrass is one of the most important temperate forage grasses on a global basis. Improvement of both dry matter yield and quality of herbage have been major objectives of pasture grass breeding over the last century. F1 and F2 progeny sets derived from controlled pair-crosses between selected Italian ryegrass genotypes have been evaluated for yield and nutritive quality under field conditions. Linear regression of the performance of F1 families under sward conditions on parental genotype means in a spaced plant trial was significant for quality characteristics, but not for herbage yield. This result suggests that phenotypic selection of individual plants from spaced plant nursery is feasible for improvement of nutritive quality traits, but not for yield. The presence of significant heterosis within F1 populations was demonstrated by reduced herbage production in subsequent F2 populations (generated by one cycle of full-sib mating), an up to 22.1% total herbage yield in fresh weight, and a 30.5% survival rate at the end of the second reproductive cycle. Potential optimal crosses for exploiting such heterosis are discussed, based on construction and the inter-mating of complementary parental pools, for the implementation of a novel F1 hybrid production strategy.

With 1 figure and 1 table Abstract Potato cyst nematode (PCN) is a major pest of potato crops. Marker-assisted selection of PCN-resistant cultivars is consequently a key objective of many potato breeding programmes. The genetic marker TG689, linked to the resistance gene H1, is the current marker of choice for the prediction of PCN resistance. Polymorphism at TG689 was evaluated across 373 cultivars from Australian and Scottish breeding programmes, revealing the loss of association between the predictive allele and trait locus. Two recently identified genetic markers flanking the H1 locus were then evaluated for association with PCN-resistant phenotypes across 73 cultivars. With a single exception, presence or absence of a 452-bp amplicon for the 57R marker was predictive for the target phenotype. Further evaluation of the 57R marker on more than 300 cultivars from the Australian and Scottish potato breeding programmes provided additional evidence for robust diagnostic capability. To enable routine implementation, the 57R marker was converted into a high-throughput, fluorochrome detection–based assay and screened across a broader collection of parental cultivars and advanced breeding selections for the prediction of PCN resistance.

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 silicocomparative genomics analysis predicted colocation between a previously identified QTL for plant type (erect versus prostrate growth) and the ortholocus of the riceOsABCG5 gene (LpABCG5), as well as related QTLs in other Poaceae species. Sequencing of anLpABCG5-containing BAC clone identified presence of a paralogue (LpABCG6) in the vicinity of theLpABCG5 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 theLpABCG5 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 theLpABCG5 gene as a candidate for the plant type determinant, suggesting that manipulation of gene expression may provide valuable phenotypes for perennial ryegrass breeding.

The recent emergence of a herpes-like virus in both farmed and wild populations of abalone in Victoria, Australia, has been associated with high mortality rates in animals of all ages. Based on viral genome sequence information, a virus-specific real-time TaqMan assay was devel- oped for detection and identification of the abalone herpes-like virus (AbHV). The assay was shown to be specific as it did not detect other viruses from either the Herpesvirales or the Iridovirales orders which have genome sequence similarities. However, the TaqMan assay was able to detect DNA from the Taiwanese abalone herpes-like virus, suggesting a relationship between the Taiwanese and Aus- tralian viruses. In addition, the assay detected

The most serious foliar disease of perennial ryegrass (Lolium perenne L.) is crown rust (caused by Puccinia coronata Corda f.sp. lolii Brown). Progress in resistance breeding using recurrent selection has been slow, due to lack of genetic knowledge. Puccinia coronata is a basidiomycete fungus with a complex life-cycle involving both asexual and sexual reproductive modes. Pathotype variation may exist, but confi rmation is complicated by the outbreeding nature of the host grass and consequent intrapopulation diversity. Qualitative and quantitative resistance mechanisms may hence be due to either major resistance genes responding to mixed pathogen populations, or minor (quantitative) resistance genes. Advances in molecular biology have permitted analysis of both host and pathogen genetics. Development of pathogen-derived simple sequence repeat (SSR) genetic markers allowed detailed analysis of genetic variation, as well as generation of homogenized inoculum for detailed trait-dissection studies. Multiple genomic regions were identifi ed as containing quantitative trait loci (QTLs) for pathogen resistance, and specifi c single nucleotide polymorphism (SNP) markers for defense response (DR) and disease resistance (R) genes have been evaluated for QTL co-location. The current knowledge status of the crown rust pathogen and genetics of host resistance is reviewed, as well as future prospects for facilitated rust resistance breeding.

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.

Genetic map construction and identification of quantitative trait loci (QTLs) for blackleg resistance were performed for four mapping populations derived from five different canola source cultivars. Three of the populations were generated from crosses between single genotypes from the blackleg-resistant cultivars Caiman, Camberra and (AV)Sapphire and the blackleg-susceptible cultivar Westar(10). The fourth population was derived from a cross between genotypes from two blackleg resistant varieties (Rainbow and (AV)Sapphire). Different types of DNA-based markers were designed and characterised from a collection of 20,000 EST sequences generated from multiple Brassica species, including a new set of 445 EST-SSR markers of high value to the international community. Multiple molecular genetic marker systems were used to construct linkage maps with locus numbers varying between 219 and 468, and coverage ranging from 1173 to 1800 cM. The proportion of polymorphic markers assigned to map locations varied from 70 to 89% across the four populations. Publicly available simple sequence repeat markers were used to assign linkage groups to reference nomenclature, and a sub-set of mapped markers were also screened on the Tapidor x Ningyou (T x N) reference population to assist this process. QTL analysis was performed based on percentage survival at low and high disease pressure sites. Multiple QTLs were identified across the four mapping populations, accounting for 13-33% of phenotypic variance (V (p)). QTL-linked marker data are suitable for implementation in breeding for disease resistance in Australian canola cultivars. However, the likelihood of shifts in pathogen race structure across different geographical locations may have implications for the long-term durability of such associations.

Assignment or exclusion of an individual to specific populations or cultivars based on molecular genetic markers provides an attractive approach for varietal identification at the individual level in cross-pollinated plant species. The objectives of this study were (i) to explore the molecular diversity and relationships between Australasian perennial ryegrass (Lolium perenne L.) populations; (ii) to investigate accuracy of assignment of individuals to different types of populations including ecotypic, nonrestricted- and restricted-based cultivars; and (iii) to determine the effect of variable number of SSR loci and different statistical analysis methods on assignment accuracy. Eight forage perennial ryegrass populations comprising 48 individual plants per population were genotyped with 29 simple sequence repeat (SSR) marker loci. The number of alleles per locus ranged from 3.72 to 6.76. The mean observed heterozygosity varied from 0.419 to 0.538. Various genetic distance estimates and clustering methods obtained results consistent with breeding history. Genetic variation among (8.7%) and within populations (91.3%) was significant. Accuracy of individual assignment differed by population type, and it was higher (>90%) for restricted-based cultivars than for ecotypic and nonrestricted-based populations. These results indicate that SSR marker profiles can be effectively used to assign individuals for outbreeding populations such as perennial ryegrass. The approach used in this study may be useful for ryegrass germplasm management issues such as cultivar identification at the individual level.

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)-SSR markers from Brassica napus and their cross-amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST-SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 16 expressed sequence tags (EST)-SSR markers from Brassica juncea and their cross-amplification across Brassica species. Sixteen primer pairs were assessed for polymorphism in all genomes of the diploid and amphidiploid Brassica species. The markers show reliable amplification, considerable polymorphism and high transferability across species, demonstrating the utility of EST-SSRs for genetic analysis of brassicas.

Field pea (Pisum sativum L.) is an important grain legume consumed both as human food and animal feed. However, productivity in low rainfall regions can be significantly reduced by inferior soils containing high levels of boron and/or salinity. Furthermore, powdery mildew (Erysiphe pisi) disease also causes significant yield loss in warmer regions. Breeding for tolerance to these abiotic and biotic stresses are major aims for pea breeding programs and the application of molecular markers for these traits could greatly assist in developing improved germplasm at a faster rate. The current study reports the evaluation of a near diagnostic marker, PsMlo, associated with powdery mildew (PM) resistance and boron (B) tolerance as well as linked markers associated with salinity tolerance across a diverse set of pea germplasm. The PsMlo1 marker predicted the PM and B phenotypic responses with high levels of accuracy (>80%) across a wide range of field pea genotypes, hence offers the potential to be widely adapted in pea breeding programs. In contrast, linked markers for salinity tolerance were population specific, therefore, application of these markers would be suitable to relevant crosses within the program. Our results also suggest that there are possible new sources of salt tolerance present in field pea germplasm that could be further exploited.

A targeted amplicon-based genotyping-by-sequencing approach has permitted cost-effective and accurate discrimination between ryegrass species (perennial, Italian and inter-species hybrid), and identification of cultivars based on bulked samples. Perennial ryegrass and Italian ryegrass are the most important temperate forage species for global agriculture, and are represented in the commercial pasture seed market by numerous cultivars each composed of multiple highly heterozygous individuals. Previous studies have identified difficulties in the use of morphophysiological criteria to discriminate between these two closely related taxa. Recently, a highly multiplexed single nucleotide polymorphism (SNP)-based genotyping assay has been developed that permits accurate differentiation between both species and cultivars of ryegrasses at the genetic level. This assay has since been further developed into an amplicon-based genotyping-by-sequencing (GBS) approach implemented on a second-generation sequencing platform, allowing accelerated throughput and ca. sixfold reduction in cost. Using the GBS approach, 63 cultivars of perennial, Italian and interspecific hybrid ryegrasses, as well as intergeneric Festulolium hybrids, were genotyped. The genetic relationships between cultivars were interpreted in terms of known breeding histories and indistinct species boundaries within the Lolium genus, as well as suitability of current cultivar registration methodologies. An example of applicability to quality assurance and control (QA/QC) of seed purity is also described. Rapid, low-cost genotypic assays provide new opportunities for breeders to more fully explore genetic diversity within breeding programs, allowing the combination of novel unique genetic backgrounds. Such tools also offer the potential to more accurately define cultivar identities, allowing protection of varieties in the commercial market and supporting processes of cultivar accreditation and quality assurance.

Field pea (Pisum sativum L.) is a grain legume crop that is cultivated for either human or livestock consumption. Development of varieties adapted to damaging abiotic and biotic stresses is a major objective for field pea breeding. Bacterial blight is a serious disease caused by the pathogenic agents Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. pisi. A recombinant inbred line (RIL) genetic mapping population was generated by crossing the susceptible genotype Kaspa to the resistant genotype PBA Oura. Previously described sets of single-nucleotide polymorphism and simple sequence repeat markers were assigned to a genetic linkage map of the Kaspa × PBA Oura population, which contained 358 markers spanning 1070 cM with an average marker density of 1 locus per 3.0 cM. Combination with multiple previously published maps (including that of Kaspa × Parafield) subsequently generated an integrated structure with much higher marker density of 1 locus per 0.85 cM. The Kaspa × PBA Oura and Kaspa × Parafield RILs were screened at the seedling stage for resistance to both pathovars. Totals of four and two QTLs for resistance to infection by P. syringae pv. syringae were detected in the Kaspa × Parafield and Kaspa × PBA Oura populations, respectively. A single common genomic region associated with P. syringae pv. pisi resistance was identified in both mapping populations. To integrate information on bacterial blight resistance from various QTL mapping studies, the relevant regions were extrapolated on to the integrated map through use of common flanking markers. The resources generated in this study will support map enhancement, identification of marker-trait associations, genomics-assisted breeding, map-based gene isolation and comparative genetics.

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 vigour 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.

Background Field pea (Pisum sativum L.) is a cool-season grain legume that is cultivated world-wide for both human consumption and stock-feed purposes. Enhancement of genetic and genomic resources for field pea will permit improved understanding of the control of traits relevant to crop productivity and quality. Advances in second-generation sequencing and associated bioinformatics analysis now provide unprecedented opportunities for the development of such resources. The objective of this study was to perform transcriptome sequencing and characterisation from two genotypes of field pea that differ in terms of seed and plant morphological characteristics. Results Transcriptome sequencing was performed with RNA templates from multiple tissues of the field pea genotypes Kaspa and Parafield. Tissue samples were collected at various growth stages, and a total of 23 cDNA libraries were sequenced using Illumina high-throughput sequencing platforms. A total of 407 and 352 million paired-end reads from the Kaspa and Parafield transcriptomes, respectively were assembled into 129,282 and 149,272 contigs, which were filtered on the basis of known gene annotations, presence of open reading frames (ORFs), reciprocal matches and degree of coverage. Totals of 126,335 contigs from Kaspa and 145,730 from Parafield were subsequently selected as the reference set. Reciprocal sequence analysis revealed that c. 87 % of contigs were expressed in both cultivars, while a small proportion were unique to each genotype. Reads from different libraries were aligned to the genotype-specific assemblies in order to identify and characterise expression of contigs on a tissue-specific basis, of which 87 % were expressed in more than one tissue, while others showed distinct expression patterns in specific tissues, providing unique transcriptome signatures. Conclusion This study provided a comprehensive assembled and annotated transcriptome set for field pea that can be used for development of genetic markers, in order to assess genetic diversity, construct linkage maps, perform trait-dissection and implement whole-genome selection strategies in varietal improvement programs, as well to identify target genes for genetic modification approaches on the basis of annotation and expression analysis. In addition, the reference field pea transcriptome will prove highly valuable for comparative genomics studies and construction of a finalised genome sequence. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1815-7) contains supplementary material, which is available to authorized users.

Quantitative trait loci (QTLs) for a number of agronomically important traits of perennial ryegrass (Lolium perenne L.) were identified by means of a reference molecular marker-based genetic map. Replicated phenotypic data was obtained for a number of field-assessed morphological and developmental traits as well as the winter hardiness-associated characters of winter survival and electrical conductivity. Marker-trait association analysis was performed by a number of methods, and a high degree of congruence was observed between the respective results. QTLs were detected for morphological traits such as plant height, tiller size, leaf length, leaf width, fresh weight at harvest, plant type, spikelet number per spike and spike length, as well as the developmental traits of heading date and degree of aftermath heading. A number of traits were significantly correlated, and coincident QTL locations were identified. No significant QTLs for winter survival in the field were identified. However, a QTL for electrical conductivity corresponding to frost tolerance was located close to a heading date QTL in a region that may show conserved synteny with chromosomal regions associated with both winter hardiness and flowering time variation in cereals. The QTL analysis of multiple phenotypic traits provides the basis for marker assisted selection (MAS) of important agronomic characters, allowing genetic improvement of yield, quality and adaptation in perennial ryegrass breeding.

We have identified quantitative trait loci (QTL) for transgenic and adventitious root production using an Agrobacterium rhizogenes-mediated co-transformation system in conjunction with a Brassica oleracea double haploid (DH) mapping population. Three QTL for green fluorescent protein (GFP)-fluorescent root production and four QTL for adventitious root production were identified as accounting for 26% and 32% of the genetic variation in the population, respectively. Two of the QTL regions identified were common to both transgenic and adventitious root production. Two different methods of QTL analysis were employed (marker regression and interval mapping) and with the exception of one region on linkage group O7 for transgenic root production, both techniques detected the same regions of the genome. The regions we identified to be associated with the control of transgenic root production following A. rhizogenes-mediated transformation are the first to be detected using a QTL mapping approach. In addition, this is the first study to identify genetic regions that co-regulate both transgenic and adventitious root production within the constraints of an A. rhizogenes-mediated transformation process. We have identified plant genotypes that do not produce any transgenic roots that may be deficient for T-DNA integration via illegitimate recombination and that may also be potentially important for the development of homologous recombination protocols. Conversely, we have also identified plant genotypes with high rates of transgenic root production that will be critical in the development of high throughput transformation systems.

The production of transgenic roots was scored for eight Brassica oleracea cultivars from broccoli, cabbage, cauliflower and kale following inoculation with an Agrobacterium rhizogenes cell line carrying a binary plasmid bearing the green fluorescence protein (gfp) gene in the T-DNA. Significant differences in the numbers of explants producing transgenic roots were observed between cultivars, ranging from 1.4% for Marathon F1 to 57.8% for the Green Duke F1. Three F1 cultivars were subjected to anther culture, and doubled-haploid (DH) lines were used for transformation. The DH lines produced showed considerable variation for transgenic root production with some lines showing increased efficiency compared to the parental F1 cultivar. Grouping of the DH lines into response classes with respect to transgenic root production allowed the development of potential genetic models to explain the variation in performance released from each F1 cultivar. No apparent segregation distortion for transgenic root production was observed in the DH lines following anther culture.

A sample of 22 accessions of Brassica oleracea were screened for their responses at the seedling stage to twelve isolates of the fungal pathogen Peronospora parasitica (downy mildew), and 14 accessions were screened for their responses at the seedling stage to nine isolates of Albugo candida (white blister). The results suggested that classic gene-for-gene interactions operate in the P. parasitica pathosystem whereas in the A. candida pathosystem the results could be explained either by a gene-for-gene model or an additive gene model. Interpretation of the results for both pathogens was complicated by the heterogeneous nature of the host plant accessions and differential lines are therefore being developed in a homogeneous genetic background to allow further investigation of these pathosystems to be carried out.

Background Field pea (Pisum sativum L.) is a self-pollinating, diploid, cool-season food legume. Crop production is constrained by multiple biotic and abiotic stress factors, including salinity, that cause reduced growth and yield. Recent advances in genomics have permitted the development of low-cost high-throughput genotyping systems, allowing the construction of saturated genetic linkage maps for identification of quantitative trait loci (QTLs) associated with traits of interest. Genetic markers in close linkage with the relevant genomic regions may then be implemented in varietal improvement programs. Results In this study, single nucleotide polymorphism (SNP) markers associated with expressed sequence tags (ESTs) were developed and used to generate comprehensive linkage maps for field pea. From a set of 36,188 variant nucleotide positions detected through in silico analysis, 768 were selected for genotyping of a recombinant inbred line (RIL) population. A total of 705 SNPs (91.7%) successfully detected segregating polymorphisms. In addition to SNPs, genomic and EST-derived simple sequence repeats (SSRs) were assigned to the genetic map in order to obtain an evenly distributed genome-wide coverage. Sequences associated with the mapped molecular markers were used for comparative genomic analysis with other legume species. Higher levels of conserved synteny were observed with the genomes of Medicago truncatula Gaertn. and chickpea (Cicer arietinum L.) than with soybean (Glycine max [L.] Merr.), Lotus japonicus L. and pigeon pea (Cajanus cajan [L.] Millsp.). Parents and RIL progeny were screened at the seedling growth stage for responses to salinity stress, imposed by addition of NaCl in the watering solution at a concentration of 18 dS m-1. Salinity-induced symptoms showed normal distribution, and the severity of the symptoms increased over time. QTLs for salinity tolerance were identified on linkage groups Ps III and VII, with flanking SNP markers suitable for selection of resistant cultivars. Comparison of sequences underpinning these SNP markers to the M. truncatula genome defined genomic regions containing candidate genes associated with saline stress tolerance. Conclusion The SNP assays and associated genetic linkage maps developed in this study permitted identification of salinity tolerance QTLs and candidate genes. This constitutes an important set of tools for marker-assisted selection (MAS) programs aimed at performance enhancement of field pea cultivars.

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.

Chloroplast genome sequences are of broad significance in plant biology, due to frequent use in molecular phylogenetics, comparative genomics, population genetics, and genetic modification studies. The present study used a second-generation sequencing approach to determine and assemble the plastid genomes (plastomes) of four representatives from the agriculturally important Lolium-Festuca species complex of pasture grasses (Lolium multiflorum, Festuca pratensis, Festuca altissima, and Festuca ovina). Total cellular DNA was extracted from either roots or leaves, was sequenced, and the output was filtered for plastome-related reads. A comparison between sources revealed fewer plastome-related reads from root-derived template but an increase in incidental bacterium-derived sequences. Plastome assembly and annotation indicated high levels of sequence identity and a conserved organization and gene content between species. However, frequent deletions within the F. ovina plastome appeared to contribute to a smaller plastid genome size. Comparative analysis with complete plastome sequences from other members of the Poaceae confirmed conservation of most grass-specific features. Detailed analysis of the rbcL–psaI intergenic region, however, revealed a “hot-spot” of variation characterized by independent deletion events. The evolutionary implications of this observation are discussed. The complete plastome sequences are anticipated to provide the basis for potential organelle-specific genetic modification of pasture grasses.

Background Single nucleotide polymorphisms (SNPs) provide essential tools for the advancement of research in plant genomics, and the development of SNP resources for many species has been accelerated by the capabilities of second-generation sequencing technologies. The current study aimed to develop and use a novel bioinformatic pipeline to generate a comprehensive collection of SNP markers within the agriculturally important pasture grass tall fescue; an outbreeding allopolyploid species displaying three distinct morphotypes: Continental, Mediterranean and rhizomatous. Results A bioinformatic pipeline was developed that successfully identified SNPs within genotypes from distinct tall fescue morphotypes, following the sequencing of 414 polymerase chain reaction (PCR) – generated amplicons using 454 GS FLX technology. Equivalent amplicon sets were derived from representative genotypes of each morphotype, including six Continental, five Mediterranean and one rhizomatous. A total of 8,584 and 2,292 SNPs were identified with high confidence within the Continental and Mediterranean morphotypes respectively. The success of the bioinformatic approach was demonstrated through validation (at a rate of 70%) of a subset of 141 SNPs using both SNaPshot™ and GoldenGate™ assay chemistries. Furthermore, the quantitative genotyping capability of the GoldenGate™ assay revealed that approximately 30% of the putative SNPs were accessible to co-dominant scoring, despite the hexaploid genome structure. The sub-genome-specific origin of each SNP validated from Continental tall fescue was predicted using a phylogenetic approach based on comparison with orthologous sequences from predicted progenitor species. Conclusions Using the appropriate bioinformatic approach, amplicon resequencing based on 454 GS FLX technology is an effective method for the identification of polymorphic SNPs within the genomes of Continental and Mediterranean tall fescue. The GoldenGate™ assay is capable of high-throughput co-dominant SNP allele detection, and minimises the problems associated with SNP genotyping in a polyploid by effectively reducing the complexity to a diploid system. This SNP collection may now be refined and used in applications such as cultivar identification, genetic linkage map construction, genome-wide association studies and genomic selection in tall fescue. The bioinformatic pipeline described here represents an effective general method for SNP discovery within outbreeding allopolyploid species.

Perennial ryegrass is a globally cultivated obligate outbreeding diploid species (2n = 2x = 14) which is subjected to periods of waterlogging stress due to flood irrigation during winter and the lead-up to summer. Reduction of oxygen supply to root systems due to waterlogging produces consequent deleterious effects on plant performance. Framework genetic maps for a large-scale genetic mapping family [F 1(NA x × AU6)] were constructed containing 91 simple sequence repeat and 24 single nucleotide polymorphism genetic markers. Genetic trait dissection using both control and waterlogging treatments was performed in the glasshouse, a total of 143 maximally recombinant genotypes being selected from the overall sib-ship and replicated threefold in the trial. Analysis was performed for nine quantitative morphological traits measured 8 weeks after stress treatments were applied. A total of 37 quantitative trait loci (QTLs) were identified; 19 on the NA x parental genetic map, and 18 on the AU6 parental genetic map. Regions of particular interest were identified on linkage groups (LGs) 4 and 3 of the respective maps, which have been targeted for further analysis by selection of critical recombinants. This first study of genetic control of waterlogging tolerance in ryegrasses has important implications for breeding improvement of abiotic stress adaptation.

Background White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised. Results A total of four paired homoeologous BACs were selected and sequenced to generate 173 kb of overlapping sequence between the O and P' sub-genomes. Equivalent gene content was generally observed, apart from small-scale deletions, in contrast to conservation of intergenic sequences, which varied between the four selected regions. Measurement of the number of synonymous substitutions between homoeologous genes led to estimation of a 4.2 million year divergence time between the two sub-genomes. Microsynteny was observed between the genomes of white clover and L. japonicus for all four targeted regions, but corresponding M. truncatula genomic regions were only identified for two BAC pairs. Conclusions This study describes the first analysis of sub-genome structural conservation across selected genomic regions in white clover. Although the high levels of sequence conservation between the O and P' sub-genomes would complicate efforts for whole genome sequence assembly, the conserved microsynteny with model legume genomes, especially that of L. japonicus, will be highly valuable for the future of white clover genomics and molecular breeding.

Allotetraploid (2n = 4x = 32) white clover (Trifolium repens L.) is the most commonly cultivated legume component of temperate pastures, sown in swards with a companion grass species. Genetic control of growth performance of white clover on saline land is highly important for dairy industries, due to increasing soil salinity problems. The objective of this study was to identify quantitative trait loci (QTLs) for salinity tolerance in terms of vegetative growth under stress. Two parental genetic maps consisting of 213 and 159 marker loci and spanning 1,973.0 and 1,837.6 cM, respectively, were constructed using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers from a two-way pseudo-test cross F(1) population derived from pair-crossing of the Haifa(2) and LCL(2) genotypes. A total of 8 unique genomic regions on 8 linkage groups (LGs) of the Haifa(2) parental map and 6 unique regions on 5 LGs in the LCL(2) parental map were associated with plant growth under salt stress and relative growth under stress, as compared to control conditions. The results of this study indicate that salt tolerance in white clover is controlled by multiple QTLs, some at common locations, but each of limited magnitude. Location of these QTLs provides the genetic basis and potential for pyramiding of salt tolerance genes in breeding improvement.