Linda Milne

Construction of Reference Chromosome-Scale Pseudomolecules for Potato: Integrating the Potato Genome with Genetic and Physical Maps

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker−based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal “pseudomolecules”.

The use of genotyping by sequencing in blackcurrant ( Ribes nigrum ): developing high-resolution linkage maps in species without reference genome sequences

A genotyping by sequencing (GbS) approach is reported in blackcurrant (Ribes nigrum L.) using a de novo read assembly method developed because of the current absence of a reference genome sequence for this species. A new approach to single nucleotide polymorphism (SNP) genotype calling is described, where individual genotypes for a large number of SNPs were characterised from the GbS counts using a novel method based on a functional regression of major and minor allele read counts. The high-quality GbS SNPs were combined with SNPs and simple sequence repeats generated from other technologies to develop a linkage map with increased marker density and improved genome coverage, containing up to 204 SNPs on each linkage group. SNPs of lower quality were then located on the map using quantitative trait locus (QTL) interval mapping of the proportion of the major allele. Two QTL each for 100-berry weight and Brix scores, measured over three years, were identified using the map. The use of this approach to identify and map a significant number of novel SNPs in a woody species with hitherto limited genomic resources may have generic application to other under-resourced and minor species in the development of cost-effective and efficient high-density genetic maps.

Chromatin state analysis of the barley epigenome reveals a higher‐order structure defined by H3K27me1 and H3K27me3 abundance

Summary Combinations of histones carrying different covalent modifications are a major component of epigenetic variation. We have mapped nine modified histones in the barley seedling epigenome by chromatin immunoprecipitation next‐generation sequencing (ChIP‐seq). The chromosomal distributions of the modifications group them into four different classes, and members of a given class also tend to coincide at the local DNA level, suggesting that global distribution patterns reflect local epigenetic environments. We used this peak sharing to define 10 chromatin states representing local epigenetic environments in the barley genome. Five states map mainly to genes and five to intergenic regions. Two genic states involving H3K36me3 are preferentially associated with constitutive gene expression, while an H3K27me3‐containing genic state is associated with differentially expressed genes. The 10 states display striking distribution patterns that divide barley chromosomes into three distinct global environments. First, telomere‐proximal regions contain high densities of H3K27me3 covering both genes and intergenic DNA, together with very low levels of the repressive H3K27me1 modification. Flanking these are gene‐rich interior regions that are rich in active chromatin states and have greatly decreased levels of H3K27me3 and increasing amounts of H3K27me1 and H3K9me2. Lastly, H3K27me3‐depleted pericentromeric regions contain gene islands with active chromatin states separated by extensive retrotransposon‐rich regions that are associated with abundant H3K27me1 and H3K9me2 modifications. We propose an epigenomic framework for barley whereby intergenic H3K27me3 specifies facultative heterochromatin in the telomere‐proximal regions and H3K27me1 is diagnostic for constitutive heterochromatin elsewhere in the barley genome.

Differential expression of microRNAs and potential targets under drought stress in barley

Drought is a crucial environmental constraint limiting crop production in many parts of the world. microRNA (miRNA) based gene regulation has been shown to act in several pathways, including crop response to drought stress. Sequence based profiling and computational analysis have revealed hundreds of miRNAs and their potential targets in different plant species under various stress conditions, but few have been biologically verified. In this study, 11 candidate miRNAs were tested for their expression profiles in barley. Differences in accumulation of only four miRNAs (Ath-miR169b, Osa-miR1432, Hv-miRx5 and Hv-miR166b/c) were observed between drought-treated and well-watered barley in four genotypes. miRNA targets were predicted using degradome analysis of two, different genotypes, and genotype-specific target cleavage was observed. Inverse correlation of mature miRNA accumulation with miRNA target transcripts was also genotype dependent under drought treatment. Drought-responsive miRNAs accumulated predominantly in mesophyll tissues. Our results demonstrate genotype-specific miRNA regulation under drought stress and evidence for their role in mediating expression of target genes for abiotic stress response in barley.

A transcriptional reference map of defence hormone responses in potato

Phytohormones are involved in diverse aspects of plant life including the regulation of plant growth, development and reproduction, as well as governing biotic and abiotic stress responses. We have generated a comprehensive transcriptional reference map of the early potato responses to exogenous application of the defence hormones abscisic acid, brassinolides (applied as epibrassinolide), ethylene (applied as the ethylene precursor aminocyclopropanecarboxylic acid), salicylic acid and jasmonic acid (applied as methyl jasmonate). Of the 39000 predicted genes on the microarray, a total of 2677 and 2473 genes were significantly differentially expressed at 1 h and 6 h after hormone treatment, respectively. Specific marker genes newly identified for the early hormone responses in potato include: a homeodomain 20 transcription factor (DMG400000248) for abscisic acid; a SAUR gene (DMG400016561) induced in epibrassinolide treated plants; an osmotin gene (DMG400003057) specifically enhanced by aminocyclopropanecarboxylic acid; a gene weakly similar to AtWRKY40 (DMG402007388) that was induced by salicylic acid; and a jasmonate ZIM-domain protein 1 (DMG400002930) which was specifically activated by methyl jasmonate. An online database has been set up to query the expression patterns of potato genes represented on the microarray that can also incorporate future microarray or RNAseq-based expression studies.

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm

Gene transcript profiles during barley endosperm cellularization reveal functional modules, coexpression networks, and regulatory genes, all of which align with cell wall changes in developing grain. Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development.

An ultra-high density genetic linkage map of perennial ryegrass (Lolium perenne) using genotyping by sequencing (GBS) based on a reference shotgun genome assembly

BACKGROUND AND AIMS High density genetic linkage maps that are extensively anchored to assembled genome sequences of the organism in question are extremely useful in gene discovery. To facilitate this process in perennial ryegrass (Lolium perenne L.), a high density single nucleotide polymorphism (SNP)- and presence/absence variant (PAV)-based genetic linkage map has been developed in an F2 mapping population that has been used as a reference population in numerous studies. To provide a reference sequence to which to align genotyping by sequencing (GBS) reads, a shotgun assembly of one of the grandparents of the population, a tenth-generation inbred line, was created using Illumina-based sequencing. METHODS The assembly was based on paired-end Illumina reads, scaffolded by mate pair and long jumping distance reads in the range of 3-40 kb, with >200-fold initial genome coverage. A total of 169 individuals from an F2 mapping population were used to construct PstI-based GBS libraries tagged with unique 4-9 nucleotide barcodes, resulting in 284 million reads, with approx. 1·6 million reads per individual. A bioinformatics pipeline was employed to identify both SNPs and PAVs. A core genetic map was generated using high confidence SNPs, to which lower confidence SNPs and PAVs were subsequently fitted in a straightforward binning approach. KEY RESULTS The assembly comprises 424 750 scaffolds, covering 1·11 Gbp of the 2·5 Gbp perennial ryegrass genome, with a scaffold N50 of 25 212 bp and a contig N50 of 3790 bp. It is available for download, and access to a genome browser has been provided. Comparison of the assembly with available transcript and gene model data sets for perennial ryegrass indicates that approx. 570 Mbp of the gene-rich portion of the genome has been captured. An ultra-high density genetic linkage map with 3092 SNPs and 7260 PAVs was developed, anchoring just over 200 Mb of the reference assembly. CONCLUSIONS The combined genetic map and assembly, combined with another recently released genome assembly, represent a significant resource for the perennial ryegrass genetics community.

Towards an understanding of the control of ‘crumbly’ fruit in red raspberry

The genetic disorder known as ‘crumbly’ fruit is becoming a serious problem in the European raspberry industry. The study set out to examine the crumbly phenotype in a red raspberry mapping population under two environments (field and polytunnel) across six seasons in an effort to understand variability of the syndrome and to examine whether genetic factors were important and if so, whether QTL associated with the phenotype could be identified. This highlighted that seasonal, environmental (field or polytunnel) and genetic factors all influence the condition. Two QTL that are important for the genetic control of the condition have been located on linkage groups one and three, and an association with ripening time has been identified.

Transcriptome sequencing of an ecologically important graminivorous sawfly: a resource for marker development

Sawfly larvae (Hymenoptera: Symphyta) are an important, highly nutritious, invertebrate food source for farmland birds. Reduced numbers of farmland invertebrates are a possible factor contributing to the observed declines in many farmland bird populations associated with post-1950s intensification of agriculture. To date, studies on sawfly populations have been census-based and, therefore, the genetic factors underlying their declines are unknown. We have produced the first genetic resource for any sawfly species in the form of a de novo transcriptome assembly comprising 18,539 contiguous sequences (contigs) and 260 singletons. The assembly was sequenced using 454 pyrosequencing technology and produced 1,284 microsatellite markers for the common farmland sawfly Dolerus aeneus, which may also be useful in other closely-related species. These markers will facilitate monitoring of changes in genetic diversity and gene flow in sawfly populations therefore helping to predict extinction risk.

Contrasting genetic regulation of plant development in wild barley grown in two European environments revealed by nested association mapping

Plant development in a barley nested association mapping population is controlled by interacting genotypic (wild donor alleles) and environmental (geographical location) effects.