A new three-locus model for rootstock-induced dwarfing in apple revealed by genetic mapping of root bark percentage

Rootstock-induced dwarfing of apple scions revolutionized global apple production during the twentieth century, leading to the development of modern intensive orchards. A high root bark percentage (the percentage of the whole root area constituted by root cortex) has previously been associated with rootstock induced dwarfing in apple. In this study, the root bark percentage was measured in a full-sib family of ungrafted apple rootstocks and found to be under the control of three loci. Two QTL for root bark percentage were found to co-localise to the same genomic regions on chromosome 5 and chromosome 11 previously identified as controlling dwarfing, Dw1 and Dw2, respectively. A third QTL was identified on chromosome 13 in a region that has not been previously associated with dwarfing. The development of closely linked 3 Sequence-tagged site STS markers improved the resolution of allelic classes thereby allowing the detection of dominance and epistatic interactions between loci, with high root bark percentage only occurring in specific allelic combinations. In addition, we report a significant negative correlation between root bark percentage and stem diameter (an indicator of tree vigour), measured on a clonally propagated grafted subset of the mapping population. The demonstrated link between root bark percentage and rootstock-induced dwarfing of the scion leads us to propose a three-locus model that is able to explain levels of dwarfing from the dwarf ‘M.27’ to the semi-invigorating rootstock ‘M.116’. Moreover, we suggest that the QTL on chromosome 13 (Rb3) might be analogous to a third dwarfing QTL, Dw3 that has not previously been identified

Interpreting machine learning models to investigate circadian regulation and facilitate exploration of clock function

The circadian clock is an important adaptation to life on Earth. Here, we use machine learning to predict complex, temporal, and circadian gene expression patterns in Arabidopsis. Most significantly, we classify circadian genes using DNA sequence features generated de novo from public, genomic resources, facilitating downstream application of our methods with no experimental work or prior knowledge needed. We use local model explanation that is transcript specific to rank DNA sequence features, providing a detailed profile of the potential circadian regulatory mechanisms for each transcript. Furthermore, we can discriminate the temporal phase of transcript expression using the local, explanation-derived, and ranked DNA sequence features, revealing hidden subclasses within the circadian class. Model interpretation/explanation provides the backbone of our methodological advances, giving insight into biological processes and experimental design. Next, we use model interpretation to optimize sampling strategies when we predict circadian transcripts using reduced numbers of transcriptomic timepoints. Finally, we predict the circadian time from a single, transcriptomic timepoint, deriving marker transcripts that are most impactful for accurate prediction; this could facilitate the identification of altered clock function from existing datasets

Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening

In plants there are several classes of 21–24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf. Several conserved miRNAs showed tissue-specific expression, which, combined with target gene validation results, suggests that miRNAs may play a role in fleshy fruit development. We also identified four new nonconserved miRNAs. One of the validated targets of a novel miRNA is a member of the CTR family involved in fruit ripening. However, 62 predicted targets showing near perfect complementarity to potential new miRNAs did not validate experimentally. This suggests that target prediction of plant short RNAs could have a high false-positive rate and must therefore be validated experimentally. We also found short RNAs from a Solanaceae-specific foldback transposon, which showed a miRNA/miRNA*-like distribution, suggesting that this element may function as a miRNA gene progenitor. The other Solanaceae-specific class of short RNA was derived from an endogenous pararetrovirus sequence inserted into the tomato chromosomes. This study opens a new avenue in the field of fleshy fruit biology by raising the possibility that fruit development and ripening may be under miRNA regulation

Evidence for GC preference by monocot Dicer-like proteins

Dicot Dicer-like (DCL) enzymes operate preferably on GC rich regions when producing small interfering (si)RNA and micro (mi)RNA. This GC bias, however, is not generic in monocot miRNA productions. From wild Dactylis glomerata naturally infected by Cocksfoot streak potyvirus (CSV), CSV-siRNAs had a greater GC% than the virus genome, indicating that GC rich regions were also preferred by the grass DCLs. This supports the notion that GC preference is an ancient feature for plant DCLs, and suggests that monocot miRNA genes might have evolved to a high GC% resulting in GC bias being not detectable during mature miRNA production

Identification of novel small RNAs in tomato (Solanum lycopersicum)

To date, the majority of plant small RNAs (sRNA) have been identified in rice, poplar and Arabidopsis. To identify novel tomato sRNAs potentially involved in tomato specific processes such as fruit development and/or ripening, we cloned 4,018 sRNAs from tomato fruit tissue at the mature green stage. From this pool of sRNAs, we detected tomato homologues of nine known miRNAs, including miR482; a poplar miRNA not conserved in Arabidopsis or rice. We identified three novel putative miRNAs with flanking sequence that could be folded into a stem-loop precursor structure and which accumulated as 19-24nt RNA. One of these putative miRNAs (Put-miRNA3) exhibited significantly higher expression in fruit compared with leaf tissues, indicating a specific role in fruit development processes. We also identified nine sRNAs that accumulated as 19-24nt RNA species in tomato but genome sequence was not available for these loci. None of the nine sRNAs or three putative miRNAs possessed a homologue in Arabidopsis that had a precursor with a predicted stem-loop structure or that accumulated as a sRNA species, suggesting that the 12 sRNAs we have identified in tomato may have a species specific role in this model fruit species

Circadian regulation of the transcriptome in a complex polyploid crop.

The circadian clock is a finely balanced timekeeping mechanism that coordinates programmes of gene expression. It is currently unknown how the clock regulates expression of homoeologous genes in polyploids. Here, we generate a high-resolution time-course dataset to investigate the circadian balance between sets of 3 homoeologous genes (triads) from hexaploid bread wheat. We find a large proportion of circadian triads exhibit imbalanced rhythmic expression patterns, with no specific subgenome favoured. In wheat, period lengths of rhythmic transcripts are found to be longer and have a higher level of variance than in other plant species. Expression of transcripts associated with circadian controlled biological processes is largely conserved between wheat and ; however, striking differences are seen in agriculturally critical processes such as starch metabolism. Together, this work highlights the ongoing selection for balance versus diversification in circadian homoeologs and identifies clock-controlled pathways that might provide important targets for future wheat breeding

Hidden variation in polyploid wheat drives local adaptation.

Wheat has been domesticated into a large number of agricultural environments and has the ability to adapt to diverse environments. To understand this process, we survey genotype, repeat content, and DNA methylation across a bread wheat landrace collection representing global genetic diversity. We identify independent variation in methylation, genotype, and transposon copy number. We show that these, so far unexploited, sources of variation have had a significant impact on the wheat genome and that ancestral methylation states become preferentially "hard coded" as single nucleotide polymorphisms (SNPs) via 5-methylcytosine deamination. These mechanisms also drive local adaption, impacting important traits such as heading date and salt tolerance. Methylation and transposon diversity could therefore be used alongside SNP-based markers for breeding

Proposed pre-selection method for identification of dwarfing peach rootstocks based on rapid shoot xylem vessel analysis

The association between a genotype's dwarfing ability and a genetic, physiological, or anatomical trait would be of great value for selecting dwarf rootstocks at an early stage of development. Dwarfing peach rootstocks have been associated with decreased hydraulic conductance due to smaller xylem vessel diameters compared to invigorating rootstock genotypes. We evaluated tentative anatomical criteria for selecting dwarfing peach rootstocks based on measurements of the diameter of the largest xylem vessels and the number of xylem vessels surrounding the largest vessels in cross sections of shoots. Epicormic and proleptic shoots were collected from trees of the standard, invigorating ‘Nemaguard’ peach rootstock and a series of dwarfing rootstocks: ‘Controller 9.5’ ™ (HBOK 50), ‘Controller 9’ ™ (P30-135), ‘Controller 8’ ™ (HBOK 10), ‘Controller 7’ ™ (HBOK 32), ‘Controller 6’ ™ (HBOK 27) and ‘Controller 5’ ™ (K146-43). Transverse sections were cut from 2, 3, and 4 mm diameter stems of proleptic shoots and from 4, 5, and 6 mm diameter stems of epicormic shoots. The samples were fresh sectioned to a thickness of about 150 μm, stained with Toluidin-Blue-O and photographed on a light microscope. The largest xylem vessel was visually identified in each microscopic view field, and the surrounding circle of vessels with a diameter of ∼164 micrometers were counted. Rootstock genotype, shoot diameter, and the interactions between rootstock × shoot diameter significantly affected the diameters of the largest vessels of both proleptic and epicormic shoots. Most of the evaluations of the largest vessel diameter were able to distinguish the lowest vigor rootstock from the vigorous, but the intermediate vigor rootstocks were not clearly distinguished by this criterion. The less vigorous rootstocks showed a tendency for having more vessels. The larger vessel diameters in more vigorous rootstocks appeared to be related with fewer vessels. For rootstock screening purposes epicormic shoots appeared to be better for evaluation than proleptic shoots. Young, vigorous seedlings are in the vegetative phase of plant growth, and probably more similar to epicormic shoots than to proleptic onesshoots that are physiologically more related to fruit bearing than to rapid vegetative growth. We propose that the evaluation of the largest vessel of a microscopic view field associated with the number of vessels around the largest vessel, measured on 5 mm vigorous shoots would be useful as a criterion for selecting dwarfing peach rootstock genotypes

Structural and Functional Analysis of Viral siRNAs

A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase