Identification, utilisation and mapping of novel transcriptome-based markers from blackcurrant (Ribes nigrum)

<p>Abstract</p> <p>Background</p> <p>Deep-level second generation sequencing (2GS) technologies are now being applied to non-model species as a viable and favourable alternative to Sanger sequencing. Large-scale SNP discovery was undertaken in blackcurrant (<it>Ribes nigrum </it>L.) using transcriptome-based 2GS 454 sequencing on the parental genotypes of a reference mapping population, to generate large numbers of novel markers for the construction of a high-density linkage map.</p> <p>Results</p> <p>Over 700,000 reads were produced, from which a total of 7,000 SNPs were found. A subset of polymorphic SNPs was selected to develop a 384-SNP OPA assay using the Illumina BeadXpress platform. Additionally, the data enabled identification of 3,000 novel EST-SSRs. The selected SNPs and SSRs were validated across diverse <it>Ribes </it>germplasm, including mapping populations and other selected <it>Ribes </it>species.</p> <p>SNP-based maps were developed from two blackcurrant mapping populations, incorporating 48% and 27% of assayed SNPs respectively. A relatively high proportion of visually monomorphic SNPs were investigated further by quantitative trait mapping of theta score outputs from BeadStudio analysis, and this enabled additional SNPs to be placed on the two maps.</p> <p>Conclusions</p> <p>The use of 2GS technology for the development of markers is superior to previously described methods, in both numbers of markers and biological informativeness of those markers. Whilst the numbers of reads and assembled contigs were comparable to similar sized studies of other non-model species, here a high proportion of novel genes were discovered across a wide range of putative function and localisation. The potential utility of markers developed using the 2GS approach in downstream breeding applications is discussed.</p

Modelling life cycle related and individual shape variation in biological specimens

The main purpose of this research is to develop methods for automatic identification of biological specimens in digital photographs and drawings held in a database. Incorporation of taxonomic drawings into a visual indexing system has not been attempted to date. Diatoms are a single cell microscopic algae that provide a particularly suitable case study. Identification of diatoms is a challenging task due to the huge number of the species, blurred boundaries between species, and life cycle related shape changes. A novel model based on principal curves representing the life cycle related shape variation of a number of diatom species has been developed. Our model is suitable for reconstruction purposes, allowing us to produce drawings of a variety of diatom shapes, thus providing a link between the photographs and drawings. We present the classification results of photographed and drawn specimens based on the model and compare our results to another recent system for diatom identification. Finally, given a diatom specimen, we are able not only to identify the species it belongs to but also to pinpoint the stage in the life cycle it represents

SMRT-AgRenSeq-d in potato (Solanum tuberosum) as a method to identify candidates for the nematode resistance Gpa5

Potato is the third most important food crop in the world. Diverse pathogens threaten sustainable crop production but can be controlled, in many cases, through the deployment of disease resistance genes belonging to the family of nucleotide-binding, leucine-rich-repeat (NLR) genes. To identify effective disease resistance genes in established varieties, we have successfully established SMRT-AgRenSeq in tetraploid potatoes and have further enhanced the methodology by including dRenSeq in an approach that we term SMR-AgRenSeq-d. The inclusion of dRenSeq enables the filtering of candidates after the association analysis by establishing a presence/absence matrix across resistant and susceptible varieties that is translated into an F1 score. Using a SMRT-RenSeq-based sequence representation of the NLRome from the cultivar Innovator, SMRT-AgRenSeq-d analyses reliably identified the late blight resistance benchmark genes Rpi-R1, Rpi-R2-like, Rpi-R3a, and Rpi-R3b in a panel of 117 varieties with variable phenotype penetrations. All benchmark genes were identified with an F1 score of 1, which indicates absolute linkage in the panel. This method also identified nine strong candidates for Gpa5 that controls the potato cyst nematode (PCN) species Globodera pallida (pathotypes Pa2/3). Assuming that NLRs are involved in controlling many types of resistances, SMRT-AgRenSeq-d can readily be applied to diverse crops and pathogen systems.</p

SMRT-AgRenSeq-d in potato (Solanum tuberosum) as a method to identify candidates for the nematode resistance Gpa5

Potato is the third most important food crop in the world. Diverse pathogens threaten sustainable crop production but can be controlled, in many cases, through the deployment of disease resistance genes belonging to the family of nucleotide-binding, leucine-rich-repeat (NLR) genes. To identify effective disease resistance genes in established varieties, we have successfully established SMRT-AgRenSeq in tetraploid potatoes and have further enhanced the methodology by including dRenSeq in an approach that we term SMR-AgRenSeq-d. The inclusion of dRenSeq enables the filtering of candidates after the association analysis by establishing a presence/absence matrix across resistant and susceptible varieties that is translated into an F1 score. Using a SMRT-RenSeq-based sequence representation of the NLRome from the cultivar Innovator, SMRT-AgRenSeq-d analyses reliably identified the late blight resistance benchmark genes Rpi-R1, Rpi-R2-like, Rpi-R3a, and Rpi-R3b in a panel of 117 varieties with variable phenotype penetrations. All benchmark genes were identified with an F1 score of 1, which indicates absolute linkage in the panel. This method also identified nine strong candidates for Gpa5 that controls the potato cyst nematode (PCN) species Globodera pallida (pathotypes Pa2/3). Assuming that NLRs are involved in controlling many types of resistances, SMRT-AgRenSeq-d can readily be applied to diverse crops and pathogen systems.</p

The evolutionary patterns of barley pericentromeric chromosome regions, as shaped by linkage disequilibrium and domestication

The distribution of recombination events along large cereal chromosomes is uneven and is generally restricted to gene-rich telomeric ends. To understand how the lack of recombination affects diversity in the large pericentromeric regions, we analysed deep exome capture data from a final panel of 815 Hordeum vulgare (barley) cultivars, landraces and wild barleys, sampled from across their eco-geographical ranges. We defined and compared variant data across the pericentromeric and non-pericentromeric regions, observing a clear partitioning of diversity both within and between chromosomes and germplasm groups. Dramatically reduced diversity was found in the pericentromeres of both cultivars and landraces when compared with wild barley. We observed a mixture of completely and partially differentiated single-nucleotide polymorphisms (SNPs) between domesticated and wild gene pools, suggesting that domesticated gene pools were derived from multiple wild ancestors. Patterns of genome-wide linkage disequilibrium, haplotype block size and number, and variant frequency within blocks showed clear contrasts among individual chromosomes and between cultivars and wild barleys. Although most cultivar chromosomes shared a single major pericentromeric haplotype, chromosome 7H clearly differentiated the two-row and six-row types associated with different geographical origins. Within the pericentromeric regions we identified 22 387 non-synonymous SNPs, 92 of which were fixed for alternative alleles in cultivar versus wild accessions. Surprisingly, only 29 SNPs found exclusively in the cultivars were predicted to be 'highly deleterious'. Overall, our data reveal an unconventional pericentromeric genetic landscape among distinct barley gene pools, with different evolutionary processes driving domestication and diversification.</p

Conserved signalling components coordinate epidermal patterning and cuticle deposition in barley

Faced with terrestrial threats, land plants seal their aerial surfaces with a lipid-rich cuticle. To breathe, plants interrupt their cuticles with adjustable epidermal pores, called stomata, that regulate gas exchange, and develop other specialised epidermal cells such as defensive hairs. Mechanisms coordinating epidermal features remain poorly understood. Addressing this, we studied two loci whose allelic variation causes both cuticular wax-deficiency and misarranged stomata in barley, identifying the underlying genes, Cer-g/ HvYDA1, encoding a YODA-like (YDA) MAPKKK, and Cer-s/ HvBRX-Solo, encoding a single BREVIS-RADIX (BRX) domain protein. Both genes control cuticular integrity, the spacing and identity of epidermal cells, and barley’s distinctive epicuticular wax blooms, as well as stomatal patterning in elevated CO(2) conditions. Genetic analyses revealed epistatic and modifying relationships between HvYDA1 and HvBRX-Solo, intimating that their products participate in interacting pathway(s) linking epidermal patterning with cuticular properties in barley. This may represent a mechanism for coordinating multiple adaptive features of the land plant epidermis in a cultivated cereal

Characterisation of barley resistance to rhynchosporium on chromosome 6HS

Key Message: Major resistance gene to rhynchosporium, Rrs18, maps close to the telomere on the short arm of chromosome 6H in barley. Rhynchosporium or barley scald caused by a fungal pathogen Rhynchosporium commune is one of the most destructive and economically important diseases of barley in the world. Testing of Steptoe × Morex and CIho 3515 × Alexis doubled haploid populations has revealed a large effect QTL for resistance to R. commune close to the telomere on the short arm of chromosome 6H, present in both populations. Mapping markers flanking the QTL from both populations onto the 2017 Morex genome assembly revealed a rhynchosporium resistance locus independent of Rrs13 that we named Rrs18. The causal gene was fine mapped to an interval of 660 Kb using Steptoe × Morex backcross 1 S₂ and S₃ lines with molecular markers developed from Steptoe exome capture variant calling. Sequencing RNA from CIho 3515 and Alexis revealed that only 4 genes within the Rrs18 interval were transcribed in leaf tissue with a serine/threonine protein kinase being the most likely candidate for Rrs18.Max Coulter, Bianca Büttner, Kerstin Hofmann, Micha Bayer, Luke Ramsay, Günther Schweizer, Robbie Waugh, Mark E. Looseley, Anna Avrov

Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world's oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations