Comparative analysis of the carrot miRNAome in response to salt stress.

Soil salinity adversely affects the yield and quality of crops, including carrot. During salt stress, plant growth and development are impaired by restricted water uptake and ion cytotoxicity, leading to nutrient imbalance and oxidative burst. However, the molecular mechanisms of the carrot plant response to salt stress remain unclear. The occurrence and expression of miRNAs that are potentially involved in the regulation of carrot tolerance to salinity stress were investigated. The results of small RNA sequencing revealed that salt-sensitive (DH1) and salt-tolerant (DLBA) carrot varieties had different miRNA expression profiles. A total of 95 miRNAs were identified, including 71 novel miRNAs, of which 30 and 23 were unique to DH1 and DLBA, respectively. The comparison of NGS and qPCR results allowed identification of two conserved and five novel miRNA involved in carrot response to salt stress, and which differentiated the salt-tolerant and salt-sensitive varieties. Degradome analysis supported by in silico-based predictions and followed by expression analysis of exemplary target genes pointed at genes related to proline, glutathione, and glutamate metabolism pathways as potential miRNA targets involved in salt tolerance, and indicated that the regulation of osmoprotection and antioxidant protection, earlier identified as being more efficient in the tolerant variety, may be controlled by miRNAs. Furthermore, potential miRNA target genes involved in chloroplast protection, signal transduction and the synthesis and modification of cell wall components were indicated in plants growing in saline soil.

Population genomics identifies genetic signatures of carrot domestication and improvement and uncovers the origin of high-carotenoid orange carrots.

Here an improved carrot reference genome and resequencing of 630 carrot accessions were used to investigate carrot domestication and improvement. The study demonstrated that carrot was domesticated during the Early Middle Ages in the region spanning western Asia to central Asia, and orange carrot was selected during the Renaissance period, probably in western Europe. A progressive reduction of genetic diversity accompanied this process. Genes controlling circadian clock/flowering and carotenoid accumulation were under selection during domestication and improvement. Three recessive genes, at the REC, Or and Y2 quantitative trait loci, were essential to select for the high α- and ß-carotene orange phenotype. All three genes control high α- and ß-carotene accumulation through molecular mechanisms that regulate the interactions between the carotenoid biosynthetic pathway, the photosynthetic system and chloroplast biogenesis. Overall, this study elucidated carrot domestication and breeding history and carotenoid genetics at a molecular level.

The Retinoblastoma-related gene RBL901 can trigger drought response actions in potato.

KEY MESSAGE: This study provided new insights into response of potato to drought stress.

Transcriptome Dynamics Underlying Planticine®-Induced Defense Responses of Tomato (Solanum lycopersicum L.) to Biotic Stresses.

The induction of natural defense mechanisms in plants is considered to be one of the most important strategies used in integrated pest management (IPM). Plant immune inducers could reduce the use of chemicals for plant protection and their harmful impacts on the environment. Planticine® is a natural plant defense biostimulant based on oligomers of α(1→4)-linked D-galacturonic acids, which are biodegradable and nontoxic. The aim of this study was to define the molecular basis of Planticine's biological activity and the efficacy of its use as a natural plant resistance inducer in greenhouse conditions. Three independent experiments with foliar application of Planticine® were carried out. The first experiment in a climatic chamber (control environment, no pest pressure) subjected the leaves to RNA-seq analysis, and the second and third experiments in greenhouse conditions focused on efficacy after a pest infestation. The result was the RNA sequencing of six transcriptome libraries of tomatoes treated with Planticine® and untreated plants; a total of 3089 genes were found to be differentially expressed genes (DEGs); among them, 1760 and 1329 were up-regulated and down-regulated, respectively. DEG analysis indicated its involvement in such metabolic pathways and processes as plant-pathogen interaction, plant hormone signal transduction, MAPK signaling pathway, photosynthesis, and regulation of transcription. We detected up-regulated gene-encoded elicitor and effector recognition receptors (ELRR and ERR), mitogen-activated protein kinase (MAPKs) genes, and transcription factors (TFs), i.e., WRKY, ERF, MYB, NAC, bZIP, pathogenesis-related proteins (PRPs), and resistance-related metabolite (RRMs) genes. In the greenhouse trials, foliar application of Planticine® proved to be effective in reducing the infestation of tomato leaves by the biotrophic pathogen powdery mildew and in reducing feeding by thrips, which are insect herbivores. Prophylactic and intervention use of Planticine® at low infestation levels allows the activation of plant defense mechanisms.

A review of strategies used to identify transposition events in plant genomes.

Transposable elements (TEs) were initially considered redundant and dubbed 'junk DNA'. However, more recently they were recognized as an essential element of genome plasticity. In nature, they frequently become active upon exposition of the host to stress conditions. Even though most transposition events are neutral or even deleterious, occasionally they may happen to be beneficial, resulting in genetic novelty providing better fitness to the host. Hence, TE mobilization may promote adaptability and, in the long run, act as a significant evolutionary force. There are many examples of TE insertions resulting in increased tolerance to stresses or in novel features of crops which are appealing to the consumer. Possibly, TE-driven de novo variability could be utilized for crop improvement. However, in order to systematically study the mechanisms of TE/host interactions, it is necessary to have suitable tools to globally monitor any ongoing TE mobilization. With the development of novel potent technologies, new high-throughput strategies for studying TE dynamics are emerging. Here, we present currently available methods applied to monitor the activity of TEs in plants. We divide them on the basis of their operational principles, the position of target molecules in the process of transposition and their ability to capture real cases of actively transposing elements. Their possible theoretical and practical drawbacks are also discussed. Finally, conceivable strategies and combinations of methods resulting in an improved performance are proposed.

Diverse and mobile: eccDNA-based identification of carrot low-copy-number LTR retrotransposons active in callus cultures.

Long terminal repeat retrotransposons (LTR-RTs) are mobilized via an RNA intermediate using a 'copy and paste' mechanism, and account for the majority of repetitive DNA in plant genomes. As a side effect of mobilization, the formation of LTR-RT-derived extrachromosomal circular DNAs (eccDNAs) occurs. Thus, high-throughput sequencing of eccDNA can be used to identify active LTR-RTs in plant genomes. Despite the release of a reference genome assembly, carrot LTR-RTs have not yet been thoroughly characterized. LTR-RTs are abundant and diverse in the carrot genome. We identified 5976 carrot LTR-RTs, 2053 and 1660 of which were attributed to Copia and Gypsy superfamilies, respectively. They were further classified into lineages, families and subfamilies. More diverse LTR-RT lineages, i.e. lineages comprising many low-copy-number subfamilies, were more frequently associated with genic regions. Certain LTR-RT lineages have been recently active in Daucus carota. In particular, low-copy-number LTR-RT subfamilies, e.g. those belonging to the DcAle lineage, have significantly contributed to carrot genome diversity as a result of continuing activity. We utilized eccDNA sequencing to identify and characterize two DcAle subfamilies, Alex1 and Alex3, active in carrot callus. We documented 14 and 32 de novo insertions of Alex1 and Alex3, respectively, which were positioned in non-repetitive regions.

A Global Landscape of Miniature Inverted-Repeat Transposable Elements in the Carrot Genome.

Miniature inverted-repeat transposable elements (MITEs) are the most abundant group of Class II mobile elements in plant genomes. Their presence in genic regions may alter gene structure and expression, providing a new source of functional diversity. Owing to their small size and lack of coding capacity, the identification of MITEs has been demanding. However, the increasing availability of reference genomes and bioinformatic tools provides better means for the genome-wide identification and analysis of MITEs and for the elucidation of their contribution to the evolution of plant genomes. We mined MITEs in the carrot reference genome DH1 using MITE-hunter and developed a curated carrot MITE repository comprising 428 families. Of the 31,025 MITE copies spanning 10.34 Mbp of the carrot genome, 54% were positioned in genic regions. Stowaways and Tourists were frequently present in the vicinity of genes, while Mutator-like MITEs were relatively more enriched in introns. hAT-like MITEs were relatively more frequently associated with transcribed regions, including untranslated regions (UTRs). Some carrot MITE families were shared with other Apiaceae species. We showed that hAT-like MITEs were involved in the formation of new splice variants of insertion-harboring genes. Thus, carrot MITEs contributed to the accretion of new diversity by altering transcripts and possibly affecting the regulation of many genes.

Genetic diversity structure of western-type carrots.

BACKGROUND: Carrot is a crop with a wide range of phenotypic and molecular diversity. Within cultivated carrots, the western gene pool comprises types characterized by different storage root morphology. First western carrot cultivars originated from broad-based populations. It was followed by intercrosses among plants representing early open-pollinated cultivars, combined with mass phenotypic selection for traits of interest. Selective breeding improved root uniformity and led to the development of a range of cultivars differing in root shape and size. Based on the root shape and the market use of cultivars, a dozen of market types have been distinguished. Despite their apparent phenotypic variability, several studies have suggested that western cultivated carrot germplasm was genetically non-structured. RESULTS: Ninety-three DcS-ILP markers and 2354 SNP markers were used to evaluate the structure of genetic diversity in the collection of 78 western type open-pollinated carrot cultivars, each represented by five plants. The mean percentage of polymorphic loci segregating within a cultivar varied from 31.18 to 89.25% for DcS-ILP markers and from 45.11 to 91.29% for SNP markers, revealing high levels of intra-cultivar heterogeneity, in contrast to its apparent phenotypic stability. Average inbreeding coefficient for all cultivars was negative for both DcS-ILP and SNP, whereas the overall genetic differentiation across all market classes, as measured by FST, was comparable for both marker systems. For DcS-ILPs 90-92% of total genetic variation could be attributed to the differences within the inferred clusters, whereas for SNPs the values ranged between 91 to 93%. Discriminant Analysis of Principal Components enabled the separation of eight groups cultivars depending mostly on their market type affiliation. Three groups of cultivars, i.e. Amsterdam, Chantenay and Imperator, were characterized by high homogeneity regardless of the marker system used for genotyping. CONCLUSIONS: Both marker systems used in the study enabled detection of substantial variation among carrot plants of different market types, therefore can be used in germplasm characterization and analysis of genome relationships. The presented results likely reveal the actual genetic diversity structure within the western carrot gene pool and point at possible discrepancies within the cultivars' passport data.

Mining for Candidate Genes Controlling Secondary Growth of the Carrot Storage Root.

BACKGROUND: Diverse groups of carrot cultivars have been developed to meet consumer demands and industry needs. Varietal groups of the cultivated carrot are defined based on the shape of roots. However, little is known about the genetic basis of root shape determination. METHODS: Here, we used 307 carrot plants from 103 open-pollinated cultivars for a genome wide association study to identify genomic regions associated with the storage root morphology. RESULTS: A 180 kb-long region on carrot chromosome 1 explained 10% of the total observed phenotypic variance in the shoulder diameter. Within that region, DcDCAF1 and DcBTAF1 genes were proposed as candidates controlling secondary growth of the carrot storage root. Their expression profiles differed between the cultivated and the wild carrots, likely indicating that their elevated expression was required for the development of edible roots. They also showed higher expression at the secondary root growth stage in cultivars producing thick roots, as compared to those developing thin roots. CONCLUSIONS: We provided evidence for a likely involvement of DcDCAF1 and/or DcBTAF1 in the development of the carrot storage root and developed a genotyping assay facilitating the identification of variants in the region on carrot chromosome 1 associated with secondary growth of the carrot root.

Stowaway miniature inverted repeat transposable elements are important agents driving recent genomic diversity in wild and cultivated carrot.

BACKGROUND: Miniature inverted repeat transposable elements (MITEs) are small non-autonomous DNA transposons that are ubiquitous in plant genomes, and are mobilised by their autonomous relatives. Stowaway MITEs are derived from and mobilised by elements from the mariner superfamily. Those elements constitute a significant portion of the carrot genome; however the variation caused by Daucus carota Stowaway MITEs (DcStos), their association with genes and their putative impact on genome evolution has not been comprehensively analysed. RESULTS: Fourteen families of Stowaway elements DcStos occupy about 0.5% of the carrot genome. We systematically analysed 31 genomes of wild and cultivated Daucus carota, yielding 18.5 thousand copies of these elements, showing remarkable insertion site polymorphism. DcSto element demography differed based on the origin of the host populations, and corresponded with the four major groups of D. carota, wild European, wild Asian, eastern cultivated and western cultivated. The DcStos elements were associated with genes, and most frequently occurred in 5' and 3' untranslated regions (UTRs). Individual families differed in their propensity to reside in particular segments of genes. Most importantly, DcSto copies in the 2 kb regions up- and downstream of genes were more frequently associated with open reading frames encoding transcription factors, suggesting their possible functional impact. More than 1.5% of all DcSto insertion sites in different host genomes contained different copies in exactly the same position, indicating the existence of insertional hotspots. The DcSto7b family was much more polymorphic than the other families in cultivated carrot. A line of evidence pointed at its activity in the course of carrot domestication, and identified Dcmar1 as an active carrot mariner element and a possible source of the transposition machinery for DcSto7b. CONCLUSION: Stowaway MITEs have made a substantial contribution to the structural and functional variability of the carrot genome.

Comparative Transcriptomics of Root Development in Wild and Cultivated Carrots.

The carrot is the most popular root vegetable worldwide. The genetic makeup underlying the development of the edible storage root are fragmentary. Here, we report the first comparative transcriptome analysis between wild and cultivated carrot roots at multiple developmental stages. Overall, 3285, 4637, and 570 genes were differentially expressed in the cultivated carrot in comparisons made for young plants versus developing roots, young plants versus mature roots, and developing roots versus mature roots, respectively. Of those, 1916, 2645, and 475, respectively, were retained after filtering out genes showing similar profiles of expression in the wild carrot. They were assumed to be of special interest with respect to the development of the storage root. Among them, transcription factors and genes encoding proteins involved in post-translational modifications (signal transduction and ubiquitination) were mostly upregulated, while those involved in redox signaling were mostly downregulated. Also, genes encoding proteins regulating cell cycle, involved in cell divisions, development of vascular tissue, water transport, and sugar metabolism were enriched in the upregulated clusters. Genes encoding components of photosystem I and II, together with genes involved in carotenoid biosynthesis, were upregulated in the cultivated roots, as opposed to the wild roots; however, they were largely downregulated in the mature storage root, as compared with the young and developing root. The experiment produced robust resources for future investigations on the regulation of storage root formation in carrot and Apiaceae.

Miniature Inverted Repeat Transposable Element Insertions Provide a Source of Intron Length Polymorphism Markers in the Carrot (Daucus carota L.).

The prevalence of non-autonomous class II transposable elements (TEs) in plant genomes may serve as a tool for relatively rapid and low-cost development of gene-associated molecular markers. Miniature inverted-repeat transposable element (MITE) copies inserted within introns can be exploited as potential intron length polymorphism (ILP) markers. ILPs can be detected by PCR with primers anchored in exon sequences flanking the target introns. Here, we designed primers for 209 DcSto (Daucus carota Stowaway-like) MITE insertion sites within introns along the carrot genome and validated them as candidate ILP markers in order to develop a set of markers for genotyping the carrot. As a proof of concept, 90 biallelic DcS-ILP markers were selected and used to assess genetic diversity of 27 accessions comprising wild Daucus carota and cultivated carrot of different root shape. The number of effective alleles was 1.56, mean polymorphism informative content was 0.27, while the average observed and expected heterozygosity was 0.24 and 0.34, respectively. Sixty-seven loci showed positive values of Wright's fixation index. Using Bayesian approach, two clusters comprising four wild and 23 cultivated accessions, respectively, were distinguished. Within the cultivated carrot gene pool, four subclusters representing accessions from Chantenay, Danvers, Imperator, and Paris Market types were revealed. It is the first molecular evidence for root-type associated diversity structure in western cultivated carrot. DcS-ILPs detected substantial genetic diversity among the studied accessions and, showing considerable discrimination power, may be exploited as a tool for germplasm characterization and analysis of genome relationships. The developed set of DcS-ILP markers is an easily accessible molecular marker genotyping system based on TE insertion polymorphism.

Characterization of a Genomic Region under Selection in Cultivated Carrot (Daucus carota subsp. sativus) Reveals a Candidate Domestication Gene.

Carrot is one of the most important vegetables worldwide, owing to its capability to develop fleshy, highly nutritious storage roots. It was domesticated ca. 1,100 years ago in Central Asia. No systematic knowledge about the molecular mechanisms involved in the domestication syndrome in carrot are available, however, the ability to form a storage root is undoubtedly the essential transition from the wild Daucus carota to the cultivated carrot. Here, we expand on the results of a previous study which identified a polymorphism showing a significant signature for selection upon domestication. We mapped the region under selection to the distal portion of the long arm of carrot chromosome 2, confirmed that it had been selected, as reflected in both the lower nucleotide diversity in the cultivated gene pool, as compared to the wild (πw/πc = 7.4 vs. 1.06 for the whole genome), and the high FST (0.52 vs. 0.12 for the whole genome). We delimited the region to ca. 37 kb in length and identified a candidate domestication syndrome gene carrying three non-synonymous single nucleotide polymorphisms and one indel systematically differentiating the wild and the cultivated accessions. This gene, DcAHLc1, belongs to the AT-hook motif nuclear localized (AHL) family of plant regulatory genes which are involved in the regulation of organ development, including root tissue patterning. AHL genes work through direct interactions with other AHL family proteins and a range of other proteins that require intercellular protein movement. Based on QTL data on root thickening we speculate that DcAHLc1 might be involved in the development of the carrot storage root, as the localization of the gene overlapped with one of the QTLs. According to haplotype information we propose that the 'cultivated' variant of DcAHLc1 has been selected from wild Central Asian carrot populations upon domestication and it is highly predominant in the western cultivated carrot gene pool. However, some primitive eastern landraces and the derived B7262 purple inbred line still carry the 'wild' variant, reflecting a likely complexity of the genetic determination of the formation of carrot storage roots.

A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution.

We report a high-quality chromosome-scale assembly and analysis of the carrot (Daucus carota) genome, the first sequenced genome to include a comparative evolutionary analysis among members of the euasterid II clade. We characterized two new polyploidization events, both occurring after the divergence of carrot from members of the Asterales order, clarifying the evolutionary scenario before and after radiation of the two main asterid clades. Large- and small-scale lineage-specific duplications have contributed to the expansion of gene families, including those with roles in flowering time, defense response, flavor, and pigment accumulation. We identified a candidate gene, DCAR_032551, that conditions carotenoid accumulation (Y) in carrot taproot and is coexpressed with several isoprenoid biosynthetic genes. The primary mechanism regulating carotenoid accumulation in carrot taproot is not at the biosynthetic level. We hypothesize that DCAR_032551 regulates upstream photosystem development and functional processes, including photomorphogenesis and root de-etiolation.

Conversion of a diversity arrays technology marker differentiating wild and cultivated carrots to a co-dominant cleaved amplified polymorphic site marker.

Cultivated carrot and its wild ancestor co-occur in most temperate regions of the world and can easily hybridize. The genetic basis of the process of domestication in carrot is not well understood. Recent results of an investigation on genetic diversity structure of cultivated and wild carrot and signatures for domestication using Diversity Arrays Technology (DArT) allowed identification of polymorphisms differentiating wild and cultivated accessions. We selected one of these polymorphisms, showing the strongest evidence for directional selection in the course of domestication, and converted it into a co-dominant cleaved amplified polymorphic site (CAPS) marker named cult. To achieve that, we designed site-specific primers anchored in sequences flanking the original DArT clone, amplified and sequenced the PCR products derived from cultivated and wild carrot. A PstI restriction site present in the 'cultivated' variant and absent in the 'wild' was subsequently used for routine differentiation the two variants. We validated the cult marker on 88 accessions of cultivated and wild carrot, each represented by five individuals. The allelic variant associated with the wild phenotype was only rarely observed in cultivated carrot, mostly in purple-rooted accessions originating Turkey and Iran, possibly indicating that the physical association between the diagnostic polymorphism and the putative 'domestication gene' has been broken in a group of Eastern carrots.

Diversity, genetic mapping, and signatures of domestication in the carrot (Daucus carota L.) genome, as revealed by Diversity Arrays Technology (DArT) markers.

Carrot is one of the most economically important vegetables worldwide, but genetic and genomic resources supporting carrot breeding remain limited. We developed a Diversity Arrays Technology (DArT) platform for wild and cultivated carrot and used it to investigate genetic diversity and to develop a saturated genetic linkage map of carrot. We analyzed a set of 900 DArT markers in a collection of plant materials comprising 94 cultivated and 65 wild carrot accessions. The accessions were attributed to three separate groups: wild, Eastern cultivated and Western cultivated. Twenty-seven markers showing signatures for selection were identified. They showed a directional shift in frequency from the wild to the cultivated, likely reflecting diversifying selection imposed in the course of domestication. A genetic linkage map constructed using 188 F2 plants comprised 431 markers with an average distance of 1.1 cM, divided into nine linkage groups. Using previously anchored single nucleotide polymorphisms, the linkage groups were physically attributed to the nine carrot chromosomes. A cluster of markers mapping to chromosome 8 showed significant segregation distortion. Two of the 27 DArT markers with signatures for selection were segregating in the mapping population and were localized on chromosomes 2 and 6. Chromosome 2 was previously shown to carry the Vrn1 gene governing the biennial growth habit essential for cultivated carrot. The results reported here provide background for further research on the history of carrot domestication and identify genomic regions potentially important for modern carrot breeding.

DcSto: carrot Stowaway-like elements are abundant, diverse, and polymorphic.

We investigated nine families of Stowaway-like miniature inverted-repeat transposable elements (MITEs) in the carrot genome, named DcSto1 to DcSto9. All of them were AT-rich and shared a highly conserved 6 bp-long TIR typical for Stowaways. The copy number of DcSto1 elements was estimated as ca. 5,000 per diploid genome. We observed preference for clustered insertions of DcSto and other MITEs. Distribution of DcSto1 hybridization signals revealed presence of DcSto1 clusters within euchromatic regions along all chromosomes. An arrangement of eight regions encompassing DcSto insertion sites, studied in detail, was highly variable among plants representing different populations of Daucus carota. All of these insertions were polymorphic which most likely suggests a very recent mobilization of those elements. Insertions of DcSto near carrot genes and presence of putative promoters, regulatory motifs, and polyA signals within their sequences might suggest a possible involvement of DcSto in the regulation of gene expression.

Population dynamics of miniature inverted-repeat transposable elements (MITEs) in Medicago truncatula.

Miniature inverted-repeat transposable elements (MITEs) are small and high copy number transposons, related to and mobilized by some class II autonomous elements. New MITE families can be identified by computer-based mining of sequenced genomes. We describe four MITE families related to MtPH transposons mined de novo in the genome of Medicago truncatula, together with one previously described family MITRAV. Different levels of their intra-family sequence diversity and insertion polymorphism indicate that they were active at different evolutionary periods. MetMIT1 and MITRAV families were uniform in sequence and produced highly polymorphic insertion sites in 26 ecotypes representing a M. truncatula core collection. A subset of insertions was present only in the reference genome of A17 'Jemalong', suggesting that the two families might have been active in the course of domestication. In contrast, all investigated insertions of the MetMIT2 family were fixed, showing that it was not active after M. truncatula speciation. MetMIT1 elements were divided into three clusters, i.e. (I) relatively heterogenous copies fixed in the genome of M. truncatula, (II) uniform but also mostly fixed, and (III) uniform and polymorphic among the investigated accessions. It might reflect the evolutionary history of the MetMIT1 family, showing multiple bursts of activity. A number of MetMIT1 and MITRAV insertions were present within 1 kb upstream or downstream the ORF. A high proportion of insertions proximal to coding regions was unique to A17 'Jemalong'.