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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.

Transient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function analysis and efficacious genome editing constructs. However, such a system has not been developed inCannabisdue to the recalcitrant nature of the plant to tissue culture as well as its illegal status for many years. In this study, young expanding leaves from asepticin vitro Cannabisexplants were used for protoplast isolation. Factorial designs were used to optimise variables in viable protoplast isolation and transient expression of GFP, with a range analyses performed to determine, and quantify, significantly impacting variables. Viable protoplast yields as high as 5.7 × 106were achieved with 2.5% (w/v) Cellulase R-10, 0.3% (w/v) Macerozyme R-10 and 0.7 M mannitol, incubated for 16 h. As indicated by the transient expression of GFP, efficiency reached 23.2% with 30 μg plasmid, 50% PEG, 1 × 106protoplasts and a transfection duration of 20 min. Application of the optimised protocol for protoplast isolation was successfully evaluated on three subsequent unrelated genotypes to highlight the robustness and broad applicability of the developed technique.

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.

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.

Genomic selection accelerates genetic progress in crop breeding through the prediction of future phenotypes of selection candidates based on only their genomic information. Here we report genetic correlations and genomic prediction accuracies in 22 agronomic, disease, and seed quality traits measured across multiple years (2015&ndash, 2017) in replicated trials under rain-fed and irrigated conditions in Victoria, Australia. Two hundred and two spring canola lines were genotyped for 62,082 Single Nucleotide Polymorphisms (SNPs) using transcriptomic genotype-by-sequencing (GBSt). Traits were evaluated in single trait and bivariate genomic best linear unbiased prediction (GBLUP) models and cross-validation. GBLUP were also expanded to include genotype-by-environment G &times, E interactions. Genomic heritability varied from 0.31to 0.66. Genetic correlations were highly positive within traits across locations and years. Oil content was positively correlated with most agronomic traits. Strong, not previously documented, negative correlations were observed between average internal infection (a measure of blackleg disease) and arachidic and stearic acids. The genetic correlations between fatty acid traits followed the expected patterns based on oil biosynthesis pathways. Genomic prediction accuracy ranged from 0.29 for emergence count to 0.69 for seed yield. The incorporation of G &times, E translates into improved prediction accuracy by up to 6%. The genomic prediction accuracies achieved indicate that genomic selection is ready for application in canola breeding.

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.

Key message Exploitation of data from a ryegrass breeding program has enabled rapid development and implementation of genomic selection for sward-based biomass yield with a twofold-to-threefold increase in genetic gain. Abstract Genomic selection, which uses genome-wide sequence polymorphism data and quantitative genetics techniques to predict plant performance, has large potential for the improvement in pasture plants. Major factors influencing the accuracy of genomic selection include the size of reference populations, trait heritability values and the genetic diversity of breeding populations. Global diversity of the important forage species perennial ryegrass is high and so would require a large reference population in order to achieve moderate accuracies of genomic selection. However, diversity of germplasm within a breeding program is likely to be lower. In addition, de novo construction and characterisation of reference populations are a logistically complex process. Consequently, historical phenotypic records for seasonal biomass yield and heading date over a 18-year period within a commercial perennial ryegrass breeding program have been accessed, and target populations have been characterised with a high-density transcriptome-based genotyping-by-sequencing assay. Ability to predict observed phenotypic performance in each successive year was assessed by using all synthetic populations from previous years as a reference population. Moderate and high accuracies were achieved for the two traits, respectively, consistent with broad-sense heritability values. The present study represents the first demonstration and validation of genomic selection for seasonal biomass yield within a diverse commercial breeding program across multiple years. These results, supported by previous simulation studies, demonstrate the ability to predict sward-based phenotypic performance early in the process of individual plant selection, so shortening the breeding cycle, increasing the rate of genetic gain and allowing rapid adoption in ryegrass improvement programs. Electronic supplementary material The online version of this article (10.1007/s00122-018-3121-7) contains supplementary material, which is available to authorized users.

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.

Summary 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.

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.

Cannabinoids are the main medicinal compounds of interest in the plant Cannabis sativa, that are primarily synthesised in the glandular trichomes; found on female floral buds. The content, composition and yield of secondary metabolites (cannabinoids and terpenoids) is influenced by the plant’s genetics and environment. Some initial gene expression experiments have been performed from strains of this plant species that contrasted in cannabinoid production, however the present knowledge about detailed trichome transcriptomics in this species is limited. An extensive transcriptome atlas was generated by RNA sequencing using root, shoot, flower and trichome tissues from a female plant strain (Cannbio-2) and was enhanced with the addition of vegetative and reproductive tissues from a male cannabis plant. Differential gene expression analysis identified genes preferentially expressed in different tissues. Detailed trichomics was performed from extractions specifically from glandular trichomes as well as female floral tissues at varying developmental stages, to identify stage-specific differentially expressed genes. Candidate genes involved in terpene and cannabinoid synthesis were identified and the majority were found to have an abundant expression in trichomes. The comprehensive transcriptome is a significant resource in cannabis for further research of functional genomics to improve the yield of specialised metabolites with high pharmacological value.

Resumen Introducción. La disponibilidad de agua para la producción agrícola es esencial. La producción de maíz, sujeta al periodo de lluvias, en México supera el 70%. En las regiones agrícolas de la península de Yucatán se reportan producciones en condiciones de bajas precipitaciones o muy erráticas, que pueden provocar pérdidas totales. Una condición crítica para el establecimiento de un cultivo es contar con la humedad necesaria que permita la germinación. Objetivo. El objetivo del presente trabajo fue evaluar materiales de maíz bajo condiciones de estrés hídrico simulado por medio de soluciones osmóticas con la finalidad de identificar los que toleren estas condiciones. Materiales y métodos. El estudio se llevó a cabo en el Campo Experimental Mocochá del Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, durante el 2017; abarcó la fase de germinación y establecimiento. Se emplearon veinticinco materiales de maíz bajo un diseño completamente al azar con arreglo factorial. Se realizó una prueba de germinación bajo dos condiciones de estrés simulado por medio de PEG 8000 (-0,5 y -0,75 MPa), además se empleó un testigo (0 MPa). Se realizaron cuatro repeticiones de veinticinco semillas cada una. Las semillas se expusieron por 24 h al PEG 8000, posteriormente, se hicieron riegos con agua destilada. Se realizaron conteos de las semillas germinadas al séptimo día y se reportó como porcentaje de germinación. En una muestra de diez plántulas de cada repetición, se midió la longitud y el peso, por separado, de la parte aérea y la raíz. Resultados. De los materiales evaluados los híbridos H-563, H-565 y H-568 soportaron estrés de -0,5 MPa, y el H-520, H-567 y HEV3B presentaron germinación con -0,75 MPa y resaltaron en todas las variables evaluadas. Conclusión. Es posible identificar materiales que toleraran las condiciones de estrés durante la etapa de germinación. Abstract Introduction. The availability of water for agricultural production is essential. The production of corn, subject to the rainy season, in Mexico exceeds 70%. In the agricultural regions of the Yucatan, productions are reported in conditions of low rainfall or very erratic precipitation, which can cause total losses. A critical condition for the establishment of a crop is to have the necessary humidity that allows germination. Objective. The aim of the present work was to evaluate maize materials under simulated water stress conditions through osmotic solutions in order to identify those that can tolerate these conditions. Materials and methods. The study was carried out in the Campo Experimental Mococha of the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, during 2017; including the germination and establishment phase. Twenty-five maize materials were used under a completely random design with a factorial arrangement. A germination test was performed under simulated two stress conditions by means of the PEG 8000 (-0.50 and -0,75 MPa), also a control (0 MPa) was used. Four replicates of twenty-five seeds were done. The seeds were exposed for 24 h to the PEG 8000, subsequent, irrigations were done with distilled water. The seeds were exposed for 24 h to PEG 8000, later, the seeds were irrigated with distilled water. The germinated seeds were counted on the seventh day and reported as germination percentage. In a sample, of ten seedlings of each repetition, length and weight were measured, separately, from the aerial part and the root. Results. From the evaluated materials, the hybrids H-563, H-565 and H-568 endured a stress of -0.5 MPa, and the H-520, H-567 and HEV3B hybrids presented germination with -0.75 MPa and stood out in all the variables evaluated. Conclusion. It is possible to identify materials that will tolerate stress conditions during the germination stage.

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.

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.

Objective Through colonoscopy, polyps can be identified and removed to reduce colorectal cancer incidence and mortality. Appropriate use of surveillance colonoscopy, post polypectomy, is a focus of healthcare reform. Materials and Methods The authors developed and implemented the first electronic medical record–based colonoscopy reporting system (CoRS) that matches endoscopic findings with guideline-consistent surveillance recommendations and generates tailored results and recommendation letters for patients and providers. Results In its first year, CoRS was used in 98.6% of indicated cases. Via a survey, colonoscopists agreed/strongly agreed it is easy to use (83%), provides guideline-based recommendations (89%), improves quality of Spanish letters (94%), they would recommend it for other institutions (78%), and it made their work easier (61%), and led to improved practice (56%). Discussion CoRS’ widespread adoption and acceptance likely resulted from stakeholder engagement throughout the development and implementation process. Conclusion CoRS is well-accepted by clinicians and provides guideline-based recommendations and results communications to patients and providers.

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.

Potatoes are highly heterozygous and the conventional breeding of superior germplasm is challenging, but use of a combination of MAS and EBVs can accelerate genetic gain. Cultivated potatoes are highly heterozygous due to their outbreeding nature, and suffer acute inbreeding depression. Modern potato cultivars also exhibit tetrasomic inheritance. Due to this genetic heterogeneity, the large number of target traits and the specific requirements of commercial cultivars, potato breeding is challenging. A conventional breeding strategy applies phenotypic recurrent selection over a number of generations, a process which can take over 10 years. Recently, major advances in genetics and molecular biology have provided breeders with molecular tools to accelerate gains for some traits. Marker-assisted selection (MAS) can be effectively used for the identification of major genes and quantitative trait loci that exhibit large effects. There are also a number of complex traits of interest, such as yield, that are influenced by a large number of genes of individual small effect where MAS will be difficult to deploy. Progeny testing and the use of pedigree in the analysis can provide effective identification of the superior genetic factors that underpin these complex traits. Recently, it has been shown that estimated breeding values (EBVs) can be developed for complex potato traits. Using a combination of MAS and EBVs for simple and complex traits can lead to a significant reduction in the length of the breeding cycle for the identification of superior germplasm.

Detection of genetic diversity is important for characterisation of crop plant collections in order to detect the presence of valuable trait variation for use in breeding programs. A collection of faba bean (Vicia faba L.) genotypes was evaluated for intra- and inter-population diversity using a set of 768 genome-wide distributed single nucleotide polymorphism (SNP) markers, of which 657 obtained successful amplification and detected polymorphisms. Gene diversity and polymorphism information content (PIC) values varied between 0.022–0.500 and 0.023–1.00, with averages of 0.363 and 0.287, respectively. The genetic structure of the germplasm collection was analysed and a neighbour-joining (NJ) dendrogram was constructed. The faba bean accessions grouped into two major groups, with several additional smaller sub-groups, predominantly on the basis of geographical origin. These results were further supported by principal co-ordinate analysis (PCoA), deriving two major groupings which were differentiated on the basis of site of origin and pedigree relationships. In general, high levels of heterozygosity were observed, presumably due to the partially allogamous nature of the species. The results will facilitate targeted crossing strategies in future faba bean breeding programs in order to achieve genetic gain.

Best linear unbiased prediction (BLUP), which uses pedigree to estimate breeding values, can result in increased genetic gains for low heritability traits in autotetraploid potato. Conventional potato breeding strategies, based on outcrossing followed by phenotypic recurrent selection over a number of generations, can result in slow but steady improvements of traits with moderate to high heritability. However, faster gains, particularly for low heritability traits, could be made by selection on estimated breeding values (EBVs) calculated using more complete pedigree information in best linear unbiased prediction (BLUP) analysis. One complication in applying BLUP predictions of breeding value to potato breeding programs is the autotetraploid inheritance pattern of this species. Here we have used a large pedigree, dating back to 1908, to estimate heritability for nine key traits for potato breeding, modelling autotetraploid inheritance. We estimate the proportion of double reduction in potatoes from our data, and across traits, to be in the order of 10 %. Estimates of heritability ranged from 0.21 for breeder's visual preference, 0.58 for tuber yield, to 0.83 for plant maturity. Using the accuracies of the EBVs determined by cross generational validation, we model the genetic gain that could be achieved by selection of genotypes for breeding on BLUP EBVs and demonstrate that gains can be greater than in conventional schemes.

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.

The primary aim of plant breeders is to develop new cultivars in order to improve productivity and to combat threats from pests and diseases. Recent advances in genomics have been recognised as providing important tools for plant breeders in the form of molecular genetic markers that can be used to tag genes of interest. However, the cost-effective use of marker technology is dependent on the nature and timing of the use of such markers. A conventional potato breeding programme typically creates a large breeding population and then employs phenotypic recurrent selection over a number of generations to identify superior genotypes. Marker-assisted selection (MAS) provides the advantage of being applicable at the seedling or an early generation stage. We have analysed the cost of MAS and compared it to conventional screening, then modelled the respective costs against the breeding population size of the generation in which they would be applied to determine whether MAS in the early generations of a potato breeding programme would be cost-effective. As various potato breeding programmes employ different selection rates in early generations, these rates have also been modelled to determine the effect. Our results indicate that MAS could be applied cost-effectively in the second clonal generation for all models currently employed in potato breeding.

Potato ( L.) breeders consider a large number of traits during cultivar development and progress in conventional breeding can be slow. There is accumulating evidence that some of these traits, such as yield, are affected by a large number of genes with small individual effects. Recently, significant efforts have been applied to the development of genomic resources to improve potato breeding, culminating in a draft genome sequence and the identification of a large number of single nucleotide polymorphisms (SNPs). The availability of these genome-wide SNPs is a prerequisite for implementing genomic selection for improvement of polygenic traits such as yield. In this review, we investigate opportunities for the application of genomic selection to potato, including novel breeding program designs. We have considered a number of factors that will influence this process, including the autotetraploid and heterozygous genetic nature of potato, the rate of decay of linkage disequilibrium, the number of required markers, the design of a reference population, and trait heritability. Based on estimates of the effective population size derived from a potato breeding program, we have calculated the expected accuracy of genomic selection for four key traits of varying heritability and propose that it will be reasonably accurate. We compared the expected genetic gain from genomic selection with the expected gain from phenotypic and pedigree selection, and found that genetic gain can be substantially improved by using genomic selection.

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 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.

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 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.

Crown rust fungus, Puccinia coronata f.sp. lolii is an obligate biotrophic pathogen of ryegrasses which causes significant reductions of herbage yield, palatability and digestibility. Genetic diversity in virulence has been reported in all the major temperate regions of the world and is therefore a major problem for pasture and turf breeders developing varieties with durable resistance for crown rust. Knowledge of the genetic variation present both within and between Australasian crown rust populations is essential for the efficient production of resistant varieties. A total of 11 efficient simple sequence repeat (SSR) markers developed from a urediniospore-derived expressed sequence tag (EST) resource have been used for Australasian intraspecific genetic diversity analysis. Seventy-two single pustule samples comprising three main populations from both North and South Islands of New Zealand and from south-eastern Australia were genotyped. The analysis identified 59 distinct genotypes, high levels of genetic diversity being detected both within and between populations. All methods of analysis detected no significant difference between isolates from the North and South Islands of New Zealand (p > 0.05), and high intrapopulation diversity between Victorian isolates. However high population differentiation (p < 0.001) was detected between Victorian isolates and those from the South Island (PhiPT [estimate of genetic variability = 0.101) and especially the North Islands (PhiPT = 0.162) of New Zealand. Genetic dissection of crown rust population structure within Australasia will inform the magnitude of gene pyramiding required for the development of varieties with durable resistance.

The key components for implementation of functionally associated genetic markers in perennial ryegrass and white clover breeding have been established. They include: generation of substantial genomic sequence resources; methods for functional analysis and verification of candidate gene status, including transcriptomics and transgenesis; efficient methods for in silico and in vitro discovery and validation of gene-associated SNP loci; evaluation of intra- and inter-population diversity; estimation of SNP haplotype complexity and LD; development of robust protocols for phenotypic analysis of key agronomic traits; design and implementation of experiments for haplotype-phenotype correlation; and development of efficient strategies for gene-based marker deployment in existing germplasm improvement programmes. These processes are mature for perennial ryegrass, and despite the added complexities of genetic architecture in white clover, optimized methods for SNP discovery should be equally effective for the latter species. The strategies are also suitable for other agronomically important temperate forage species, such as the grasses Italian ryegrass, tall fescue (Festuca arundinacea), meadow fescue (Festuca pratensis), cocksfoot (Dactylis glomerata) and timothy (Phleum pratense), as well as the legumes red clover (Trifolium pratense) and lucerne (Medicago sativa).

Susceptibility to foliar pathogens commonly causes significant reductions in productivity of the important temperate forage perennial ryegrass. Breeding for durable disease resistance involves not only the deployment of major genes but also the additive effects of minor genes. An approach based on in vitro single nucleotide polymorphism (SNP) discovery in candidate defence response (DR) genes has been used to develop potential diagnostic genetic markers. SNPs were predicted, validated and mapped for representatives of the pathogenesis-related (PR) protein-encoding and reactive oxygen species (ROS)-generating gene classes. The F(1)(NA(6) x AU(6)) two-way pseudo-test cross population was used for SNP genetic mapping and detection of quantitative trait loci (QTLs) in response to a crown rust field infection. Novel resistance QTLs were coincident with mapped DR gene SNPs. QTLs on LG3 and LG7 also coincided with both herbage quality QTLs and candidate genes for lignin biosynthesis. Multiple DR gene SNP loci additionally co-located with QTLs for grey leaf spot, bacterial wilt and crown rust resistance from other published studies. Further functional validation of DR gene SNP loci using methods such as fine-mapping and association genetics will improve the efficiency of parental selection based on superior allele content.