Investigating the genetic components of tuber bruising in a breeding population of tetraploid potatoes.

BACKGROUND: Tuber bruising in tetraploid potatoes (Solanum tuberosum) is a trait of economic importance, as it affects tubers' fitness for sale. Understanding the genetic components affecting tuber bruising is a key step in developing potato lines with increased resistance to bruising. As the tetraploid setting renders genetic analyses more complex, there is still much to learn about this complex phenotype. Here, we used capture sequencing data on a panel of half-sibling populations from a breeding programme to perform a genome-wide association analysis (GWAS) for tuber bruising. In addition, we collected transcriptomic data to enrich the GWAS results. However, there is currently no satisfactory method to represent both GWAS and transcriptomics analysis results in a single visualisation and to compare them with existing knowledge about the biological system under study. RESULTS: When investigating population structure, we found that the STRUCTURE algorithm yielded greater insights than discriminant analysis of principal components (DAPC). Importantly, we found that markers with the highest (though non-significant) association scores were consistent with previous findings on tuber bruising. In addition, new genomic regions were found to be associated with tuber bruising. The GWAS results were backed by the transcriptomics differential expression analysis. The differential expression notably highlighted for the first time the role of two genes involved in cellular strength and mechanical force sensing in tuber resistance to bruising. We proposed a new visualisation, the HIDECAN plot, to integrate the results from the genomics and transcriptomics analyses, along with previous knowledge about genomic regions and candidate genes associated with the trait. CONCLUSION: This study offers a unique genome-wide exploration of the genetic components of tuber bruising. The role of genetic components affecting cellular strength and resistance to physical force, as well as mechanosensing mechanisms, was highlighted for the first time in the context of tuber bruising. We showcase the usefulness of genomic data from breeding programmes in identifying genomic regions whose association with the trait of interest merit further investigation. We demonstrate how confidence in these discoveries and their biological relevance can be increased by integrating results from transcriptomics analyses. The newly proposed visualisation provides a clear framework to summarise of both genomics and transcriptomics analyses, and places them in the context of previous knowledge on the trait of interest.

In vivo single-molecule analysis reveals COOLAIR RNA structural diversity.

Cellular RNAs are heterogeneous with respect to their alternative processing and secondary structures, but the functional importance of this complexity is still poorly understood. A set of alternatively processed antisense non-coding transcripts, which are collectively called COOLAIR, are generated at the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC)1. Different isoforms of COOLAIR influence FLC transcriptional output in warm and cold conditions2-7. Here, to further investigate the function of COOLAIR, we developed an RNA structure-profiling method to determine the in vivo structure of single RNA molecules rather than the RNA population average. This revealed that individual isoforms of the COOLAIR transcript adopt multiple structures with different conformational dynamics. The major distally polyadenylated COOLAIR isoform in warm conditions adopts three predominant structural conformations, the proportions and conformations of which change after cold exposure. An alternatively spliced, strongly cold-upregulated distal COOLAIR isoform6 shows high structural diversity, in contrast to proximally polyadenylated COOLAIR. A hyper-variable COOLAIR structural element was identified that was complementary to the FLC transcription start site. Mutations altering the structure of this region changed FLC expression and flowering time, consistent with an important regulatory role of the COOLAIR structure in FLC transcription. Our work demonstrates that isoforms of non-coding RNA transcripts adopt multiple distinct and functionally relevant structural conformations, which change in abundance and shape in response to external conditions.

Natural temperature fluctuations promote COOLAIR regulation of FLC.

Plants monitor many aspects of their fluctuating environments to help align their development with seasons. Molecular understanding of how noisy temperature cues are registered has emerged from dissection of vernalization in Arabidopsis, which involves a multiphase cold-dependent silencing of the floral repressor locus FLOWERING LOCUS C (FLC). Cold-induced transcriptional silencing precedes a low probability PRC2 epigenetic switching mechanism. The epigenetic switch requires the absence of warm temperatures as well as long-term cold exposure. However, the natural temperature inputs into the earlier transcriptional silencing phase are less well understood. Here, through investigation of Arabidopsis accessions in natural and climatically distinct field sites, we show that the first seasonal frost strongly induces expression of COOLAIR, the antisense transcripts at FLC Chamber experiments delivering a constant mean temperature with different fluctuations showed the freezing induction of COOLAIR correlates with stronger repression of FLC mRNA. Identification of a mutant that ectopically activates COOLAIR revealed how COOLAIR up-regulation can directly reduce FLC expression. Consistent with this, transgenes designed to knockout COOLAIR perturbed the early phase of FLC silencing. However, all transgenes designed to remove COOLAIR resulted in increased production of novel convergent FLC antisense transcripts. Our study reveals how natural temperature fluctuations promote COOLAIR regulation of FLC, with the first autumn frost acting as a key indicator of autumn/winter arrival.

The Arabidopsis epigenetic regulator ICU11 as an accessory protein of Polycomb Repressive Complex 2.

Molecular mechanisms enabling the switching and maintenance of epigenetic states are not fully understood. Distinct histone modifications are often associated with ON/OFF epigenetic states, but how these states are stably maintained through DNA replication, yet in certain situations switch from one to another remains unclear. Here, we address this problem through identification of Arabidopsis INCURVATA11 (ICU11) as a Polycomb Repressive Complex 2 accessory protein. ICU11 robustly immunoprecipitated in vivo with PRC2 core components and the accessory proteins, EMBRYONIC FLOWER 1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1), and TELOMERE_REPEAT_BINDING FACTORS (TRBs). ICU11 encodes a 2-oxoglutarate-dependent dioxygenase, an activity associated with histone demethylation in other organisms, and mutant plants show defects in multiple aspects of the Arabidopsis epigenome. To investigate its primary molecular function we identified the Arabidopsis FLOWERING LOCUS C (FLC) as a direct target and found icu11 disrupted the cold-induced, Polycomb-mediated silencing underlying vernalization. icu11 prevented reduction in H3K36me3 levels normally seen during the early cold phase, supporting a role for ICU11 in H3K36me3 demethylation. This was coincident with an attenuation of H3K27me3 at the internal nucleation site in FLC, and reduction in H3K27me3 levels across the body of the gene after plants were returned to the warm. Thus, ICU11 is required for the cold-induced epigenetic switching between the mutually exclusive chromatin states at FLC, from the active H3K36me3 state to the silenced H3K27me3 state. These data support the importance of physical coupling of histone modification activities to promote epigenetic switching between opposing chromatin states.

Absence of warmth permits epigenetic memory of winter in Arabidopsis.

Plants integrate widely fluctuating temperatures to monitor seasonal progression. Here, we investigate the temperature signals in field conditions that result in vernalisation, the mechanism by which flowering is aligned with spring. We find that multiple, distinct aspects of the temperature profile contribute to vernalisation. In autumn, transient cold temperatures promote transcriptional shutdown of Arabidopsis FLOWERING LOCUS C (FLC), independently of factors conferring epigenetic memory. As winter continues, expression of VERNALIZATION INSENSITIVE3 (VIN3), a factor needed for epigenetic silencing, is upregulated by at least two independent thermosensory processes. One integrates long-term cold temperatures, while the other requires the absence of daily temperatures above 15 °C. The lack of spikes of high temperature, not just prolonged cold, is thus the major driver for vernalisation. Monitoring of peak daily temperature is an effective mechanism to judge seasonal progression, but is likely to have deleterious consequences for vernalisation as the climate becomes more variable.

The genetic architecture of constitutive and induced trichome density in two new recombinant inbred line populations of Arabidopsis thaliana: phenotypic plasticity, epistasis, and bidirectional leaf damage response.

BACKGROUND: Herbivory imposes an important selective pressure on plants. In Arabidopsis thaliana leaf trichomes provide a key defense against insect herbivory; however, trichome production incurs a fitness cost in the absence of herbivory. Previous work on A. thaliana has shown an increase in trichome density in response to leaf damage, suggesting a mechanism by which the cost associated with constitutively high trichome density might be mitigated; however, the genetic basis of trichome density induction has not been studied. RESULTS: Here, we describe the mapping of quantitative trait loci (QTL) for constitutive and damage induced trichome density in two new recombinant inbred line populations of A. thaliana; mapping for constitutive and induced trichome density also allowed for the investigation of damage response (plasticity) QTL. Both novel and previously identified QTL for constitutive trichome density and the first QTL for induced trichome density and response are identified. Interestingly, two of the four parental accessions and multiple RILs in each population exhibited lower trichome density following leaf damage, a response not previously described in A. thaliana. Importantly, a single QTL was mapped for the response phenotype and allelic variation at this locus appears to determine response trajectory in RILs. The data also show that epistatic interactions are a significant component of the genetic architecture of trichome density. CONCLUSIONS: Together, our results provide further insights into the genetic architecture of constitutive trichome density and new insights into induced trichome density in A. thaliana specifically and to our understanding of the genetic underpinnings of natural variation generally.