Chromosome-scale genome sequence assemblies of the 'Autumn Bliss' and 'Malling Jewel' cultivars of the highly heterozygous red raspberry (Rubus idaeus L.) derived from long-read Oxford Nanopore sequence data.

Red raspberry (Rubus idaeus L.) is an economically valuable soft-fruit species with a relatively small (~300 Mb) but highly heterozygous diploid (2n = 2x = 14) genome. Chromosome-scale genome sequences are a vital tool in unravelling the genetic complexity controlling traits of interest in crop plants such as red raspberry, as well as for functional genomics, evolutionary studies, and pan-genomics diversity studies. In this study, we developed genome sequences of a primocane fruiting variety ('Autumn Bliss') and a floricane variety ('Malling Jewel'). The use of long-read Oxford Nanopore Technologies sequencing data yielded long read lengths that permitted well resolved genome sequences for the two cultivars to be assembled. The de novo assemblies of 'Malling Jewel' and 'Autumn Bliss' contained 79 and 136 contigs respectively, and 263.0 Mb of the 'Autumn Bliss' and 265.5 Mb of the 'Malling Jewel' assembly could be anchored unambiguously to a previously published red raspberry genome sequence of the cultivar 'Anitra'. Single copy ortholog analysis (BUSCO) revealed high levels of completeness in both genomes sequenced, with 97.4% of sequences identified in 'Autumn Bliss' and 97.7% in 'Malling Jewel'. The density of repetitive sequence contained in the 'Autumn Bliss' and 'Malling Jewel' assemblies was significantly higher than in the previously published assembly and centromeric and telomeric regions were identified in both assemblies. A total of 42,823 protein coding regions were identified in the 'Autumn Bliss' assembly, whilst 43,027 were identified in the 'Malling Jewel' assembly. These chromosome-scale genome sequences represent an excellent genomics resource for red raspberry, particularly around the highly repetitive centromeric and telomeric regions of the genome that are less complete in the previously published 'Anitra' genome sequence.

De novo sequencing and analysis of the transcriptome of two highbush blueberry (Vaccinium corymbosum L.) cultivars 'Bluecrop' and 'Legacy' at harvest and following post-harvest storage.

Fruit firmness and in particular the individual components of texture and moisture loss, are considered the key quality traits when describing blueberry fruit quality, and whilst these traits are genetically regulated, the mechanisms governing their control are not clearly understood. In this investigation, RNAseq was performed on fruits of two blueberry cultivars with very different storage properties, 'Bluecrop' and 'Legacy', at harvest, three weeks storage in a non-modified environment at 4 °C and after three weeks storage at 4 °C followed by three days at 21 °C, with the aim of understanding the transcriptional changes that occur during storage in cultivars with very different post-harvest fruit quality. De novo assemblies of the transcriptomes of the two cultivars were performed separately and a total of 39,335 and 41,896 unigenes for 'Bluecrop' and 'Legacy' respectively were resolved. Differential gene expression analyses were grouped into four cluster profiles based on changes in transcript abundance between harvest and 24 days post-harvest. A total of 290 unigenes were up-regulated in 'Legacy' only, 685 were up-regulated in 'Bluecrop', 252 were up-regulated in both cultivars and 948 were down-regulated in both cultivars between harvest and 24 days post-harvest. Unigenes showing significant differential expression between harvest and following post-harvest cold-storage were grouped into classes of biological processes including stress responses, cell wall metabolism, wax metabolism, calcium metabolism, cellular components, and biological processes. In total 21 differentially expressed unigenes with a putative role in regulating the response to post-harvest cold-storage in the two cultivars were identified from the de novo transcriptome assemblies performed. The results presented provide a stable foundation from which to perform further analyses with which to functionally validate the candidate genes identified, and to begin to understand the genetic mechanisms controlling changes in firmness in blueberry fruits post-harvest.

Major-effect candidate genes identified in cultivated strawberry (Fragaria × ananassa Duch.) for ellagic acid deoxyhexoside and pelargonidin-3-O-malonylglucoside biosynthesis, key polyphenolic compounds.

Strawberries are rich in polyphenols which impart health benefits when metabolized by the gut microbiome, including anti-inflammatory, neuroprotective, and antiproliferative effects. In addition, polyphenolic anthocyanins contribute to the attractive color of strawberry fruits. However, the genetic basis of polyphenol biosynthesis has not been extensively studied in strawberry. In this investigation, ripe fruits from three cultivated strawberry populations were characterized for polyphenol content using HPLC-DAD-MSn and genotyped using the iStraw35k array. GWAS and QTL analyses identified genetic loci controlling polyphenol biosynthesis. QTL were identified on four chromosomes for pelargonidin-3-O-malonylglucoside, pelargonidin-3-O-acetylglucoside, cinnamoyl glucose, and ellagic acid deoxyhexoside biosynthesis. Presence/absence of ellagic acid deoxyhexoside and pelargonidin-3-O-malonylglucoside was found to be under the control of major gene loci on LG1X2 and LG6b, respectively, on the F. × ananassa linkage maps. Interrogation of gene predictions in the F. vesca reference genome sequence identified a single candidate gene for ellagic acid deoxyhexoside biosynthesis, while seven malonyltransferase genes were identified as candidates for pelargonidin-3-O-malonylglucoside biosynthesis. Homologous malonyltransferase genes were identified in the F. × ananassa 'Camarosa' genome sequence but the candidate for ellagic acid deoxyhexoside biosynthesis was absent from the 'Camarosa' sequence. This study demonstrated that polyphenol biosynthesis in strawberry is, in some cases, under simple genetic control, supporting previous observations of the presence or absence of these compounds in strawberry fruits. It has also shed light on the mechanisms controlling polyphenol biosynthesis and enhanced the knowledge of these biosynthesis pathways in strawberry. The above findings will facilitate breeding for strawberries enriched in compounds with beneficial health effects.

Identification of QTLs for powdery mildew (Podosphaera aphanis; syn. Sphaerotheca macularis f. sp. fragariae) susceptibility in cultivated strawberry (Fragaria ×ananassa).

Strawberry powdery mildew (Podosphaera aphanis Wallr.) is a pathogen which infects the leaves, fruit, stolon and flowers of the cultivated strawberry (Fragaria ×ananassa), causing major yield losses, primarily through unmarketable fruit. The primary commercial control of the disease is the application of fungicidal sprays. However, as the use of key active ingredients of commercial fungicides is becoming increasingly restricted, interest in developing novel strawberry cultivars exhibiting resistance to the pathogen is growing rapidly. In this study, a mapping population derived from a cross between two commercial strawberry cultivars ('Sonata' and 'Babette') was genotyped with single nucleotide polymorphism (SNP) markers from the Axiom iStraw90k genotyping array and phenotyped for powdery mildew susceptibility in both glasshouse and field environments. Three distinct, significant QTLs for powdery mildew resistance were identified across the two experiments. Through comparison with previous studies and scrutiny of the F. vesca genome sequence, candidate genes underlying the genetic control of this trait were identified.

The genome sequence and transcriptome of Potentilla micrantha and their comparison to Fragaria vesca (the woodland strawberry).

Background: The genus Potentilla is closely related to that of Fragaria, the economically important strawberry genus. Potentilla micrantha is a species that does not develop berries but shares numerous morphological and ecological characteristics with Fragaria vesca. These similarities make P. micrantha an attractive choice for comparative genomics studies with F. vesca. Findings: In this study, the P. micrantha genome was sequenced and annotated, and RNA-Seq data from the different developmental stages of flowering and fruiting were used to develop a set of gene predictions. A 327 Mbp sequence and annotation of the genome of P. micrantha, spanning 2674 sequence contigs, with an N50 size of 335,712, estimated to cover 80% of the total genome size of the species was developed. The genus Potentilla has a characteristically larger genome size than Fragaria, but the recovered sequence scaffolds were remarkably collinear at the micro-syntenic level with the genome of F. vesca, its closest sequenced relative. A total of 33,602 genes were predicted, and 95.1% of bench-marking universal single-copy orthologous genes were complete within the presented sequence. Thus, we argue that the majority of the gene-rich regions of the genome have been sequenced. Conclusions: Comparisons of RNA-Seq data from the stages of floral and fruit development revealed genes differentially expressed between P. micrantha and F. vesca.The data presented are a valuable resource for future studies of berry development in Fragaria and the Rosaceae and they also shed light on the evolution of genome size and organization in this family.

A highly specific microRNA-mediated mechanism silences LTR retrotransposons of strawberry.

Small RNAs are involved in a plethora of functions in plant genomes. In general, transcriptional gene silencing is mediated by 24-nucleotide siRNAs and is required for maintaining transposable elements in a silenced state. However, microRNAs are not commonly associated with transposon silencing. In this study, we performed small RNA transcriptome and degradome analyses of the Rosaceae model plant Fragaria vesca (the woodland strawberry) at the genome-wide level, and identified miRNA families and their targets. We report a highly specific mechanism of LTR retrotransposon silencing mediated by an abundant, ubiquitously expressed miRNA (fve-miR1511) generated from a single locus. This miRNA specifically targets LTR retroelements, silencing them post-transcriptionally by perfectly pairing to the highly conserved primer binding site for methionyl initiator tRNA that is essential for reverse transcription. We investigated the possible origins of this miRNA, and present evidence that the pre-miR1511 hairpin structure probably derived from a locus coding for tRNA(iM) (et) through a single microinversion event. Our study shows that this miRNA targets retrotransposons specifically and constitutively, and contributes to features such as genome stability, size and architecture in a far more direct way than previously thought.

Molecular genetics and genomics of the Rosoideae: state of the art and future perspectives.

The Rosoideae is a subfamily of the Rosaceae that contains a number of species of economic importance, including the soft fruit species strawberry (Fragaria ×ananassa), red (Rubus idaeus) and black (Rubus occidentalis) raspberries, blackberries (Rubus spp.) and one of the most economically important cut flower genera, the roses (Rosa spp.). Molecular genetics and genomics resources for the Rosoideae have developed rapidly over the past two decades, beginning with the development and application of a number of molecular marker types including restriction fragment length polymorphisms, amplified fragment length polymorphisms and microsatellites, and culminating in the recent publication of the genome sequence of the woodland strawberry, Fragaria vesca, and the development of high throughput single nucleotide polymorphism (SNP)-genotyping resources for Fragaria, Rosa and Rubus. These tools have been used to identify genes and other functional elements that control traits of economic importance, to study the evolution of plant genome structure within the subfamily, and are beginning to facilitate genomic-assisted breeding through the development and deployment of markers linked to traits such as aspects of fruit quality, disease resistance and the timing of flowering. In this review, we report on the developments that have been made over the last 20 years in the field of molecular genetics and structural genomics within the Rosoideae, comment on how the knowledge gained will improve the efficiency of cultivar development and discuss how these advances will enhance our understanding of the biological processes determining agronomically important traits in all Rosoideae species.

Expression of Fragaria vesca PIP aquaporins in response to drought stress: PIP down-regulation correlates with the decline in substrate moisture content.

PIP aquaporin responses to drought stress can vary considerably depending on the isoform, tissue, species or level of stress; however, a general down-regulation of these genes is thought to help reduce water loss and prevent backflow of water to the drying soil. It has been suggested therefore, that it may be necessary for the plant to limit aquaporin production during drought stress, but it is unknown whether aquaporin down-regulation is gradual or triggered by a particular intensity of the stress. In this study, ten Fragaria PIP genes were identified from the woodland strawberry (Fragaria vesca L.) genome sequence and characterised at the sequence level. The water relations of F. vesca were investigated and the effect of different intensities of drought stress on the expression of four PIP genes, as well as how drought stress influences their diurnal transcription was determined. PIP down-regulation in the root corresponded to the level of drought stress. Moreover, transcript abundance of two genes highly expressed in the root (FvPIP1;1 and FvPIP2;1) was strongly correlated to the decline in substrate moisture content. The amplitude of diurnal aquaporin expression in the leaves was down-regulated by drought without altering the pattern, but showing an intensity-dependent effect. The results show that transcription of PIP aquaporins can be fine-tuned with the environment in response to declining water availability.

An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome.

BACKGROUND: Second generation sequencing has permitted detailed sequence characterisation at the whole genome level of a growing number of non-model organisms, but the data produced have short read-lengths and biased genome coverage leading to fragmented genome assemblies. The PacBio RS long-read sequencing platform offers the promise of increased read length and unbiased genome coverage and thus the potential to produce genome sequence data of a finished quality containing fewer gaps and longer contigs. However, these advantages come at a much greater cost per nucleotide and with a perceived increase in error-rate. In this investigation, we evaluated the performance of the PacBio RS sequencing platform through the sequencing and de novo assembly of the Potentilla micrantha chloroplast genome. RESULTS: Following error-correction, a total of 28,638 PacBio RS reads were recovered with a mean read length of 1,902 bp totalling 54,492,250 nucleotides and representing an average depth of coverage of 320× the chloroplast genome. The dataset covered the entire 154,959 bp of the chloroplast genome in a single contig (100% coverage) compared to seven contigs (90.59% coverage) recovered from an Illumina data, and revealed no bias in coverage of GC rich regions. Post-assembly the data were largely concordant with the Illumina data generated and allowed 187 ambiguities in the Illumina data to be resolved. The additional read length also permitted small differences in the two inverted repeat regions to be assigned unambiguously. CONCLUSIONS: This is the first report to our knowledge of a chloroplast genome assembled de novo using PacBio sequence data. The PacBio RS data generated here were assembled into a single large contig spanning the P. micrantha chloroplast genome, with a higher degree of accuracy than an Illumina dataset generated at a much greater depth of coverage, due to longer read lengths and lower GC bias in the data. The results we present suggest PacBio data will be of immense utility for the development of genome sequence assemblies containing fewer unresolved gaps and ambiguities and a significantly smaller number of contigs than could be produced using short-read sequence data alone.

Evaluation of SNP Data from the Malus Infinium Array Identifies Challenges for Genetic Analysis of Complex Genomes of Polyploid Origin.

High throughput arrays for the simultaneous genotyping of thousands of single-nucleotide polymorphisms (SNPs) have made the rapid genetic characterisation of plant genomes and the development of saturated linkage maps a realistic prospect for many plant species of agronomic importance. However, the correct calling of SNP genotypes in divergent polyploid genomes using array technology can be problematic due to paralogy, and to divergence in probe sequences causing changes in probe binding efficiencies. An Illumina Infinium II whole-genome genotyping array was recently developed for the cultivated apple and used to develop a molecular linkage map for an apple rootstock progeny (M432), but a large proportion of segregating SNPs were not mapped in the progeny, due to unexpected genotype clustering patterns. To investigate the causes of this unexpected clustering we performed BLAST analysis of all probe sequences against the 'Golden Delicious' genome sequence and discovered evidence for paralogous annealing sites and probe sequence divergence for a high proportion of probes contained on the array. Following visual re-evaluation of the genotyping data generated for 8,788 SNPs for the M432 progeny using the array, we manually re-scored genotypes at 818 loci and mapped a further 797 markers to the M432 linkage map. The newly mapped markers included the majority of those that could not be mapped previously, as well as loci that were previously scored as monomorphic, but which segregated due to divergence leading to heterozygosity in probe annealing sites. An evaluation of the 8,788 probes in a diverse collection of Malus germplasm showed that more than half the probes returned genotype clustering patterns that were difficult or impossible to interpret reliably, highlighting implications for the use of the array in genome-wide association studies.

Basic RNases of wild almond (Prunus webbii): cloning and characterization of six new S-RNase and one "non-S RNase" genes.

In order to investigate the S-RNase allele structure of a Prunus webbii population from the Montenegrin region of the Balkans, we analyzed 10 Prunus webbii accessions. We detected 10 different S-RNase allelic variants and obtained the nucleotide sequences for six S-RNases. The BLAST analysis showed that these six sequences were new Prunus webbii S-RNase alleles. It also revealed that one of sequenced alleles, S(9)-RNase, coded for an amino acid sequence identical to that for Prunus dulcis S(14)-RNase, except for a single conservative amino acid replacement in the signal peptide region. Another, S(3)-RNase, was shown to differ by only three amino acid residues from Prunus salicina Se-RNase. The allele S(7)-RNase was found to be inactive by stylar protein isoelectric focusing followed by RNase-specific staining, but the reason for the inactivity was not at the coding sequence level. Further, in five of the 10 analyzed accessions, we detected the presence of one active basic RNase (marked PW(1)) that did not amplify with S-RNase-specific DNA primers. However, it was amplified with primers designed from the PA1 RNase nucleotide sequence (basic "non-S RNase" of Prunus avium) and the obtained sequence showed high homology (80%) with the PA1 allele. Although homologs of PA1 "non-S RNases" have been reported in four other Prunus species, this is the first recorded homolog in Prunus webbii. The evolutionary implications of the data are discussed.

Self-incompatibility of Prunus tenella and evidence that reproductively isolated species of Prunus have different SFB alleles coupled with an identical S-RNase allele.

Many species of Prunus display an S-RNase-based gametophytic self-incompatibility (SI), controlled by a single highly polymorphic multigene complex termed the S-locus. This comprises tightly linked stylar- and pollen-expressed genes that determine the specificity of the SI response. We investigated SI of Prunus tenella, a wild species found in small, isolated populations on the Balkan peninsula, initially by pollination experiments and identifying stylar-expressed RNase alleles. Nine P. tenella S-RNase alleles (S(1)-S(9)) were cloned; their sequence analysis showed a very high ratio of non-synonymous to synonymous nucleotide substitutions (K(a)/K(s)) and revealed that S-RNase alleles of P. tenella, unlike those of Prunus dulcis, show positive selection in all regions except the conserved regions and that between C2 and RHV. Remarkably, S(8)-RNase, was found to be identical to S(1)-RNase from Prunus avium, a species that does not interbreed with P. tenella and, except for just one amino acid, to S(11) of P. dulcis. However, the corresponding introns and S-RNase-SFB intergenic regions showed considerable differences. Moreover, protein sequences of the pollen-expressed SFB alleles were not identical, harbouring 12 amino-acid replacements between those of P. tenella SFB(8) and P. avium SFB(1). Implications of this finding for hypotheses about the evolution of new S-specificities are discussed.