Determination of the effective radiation dose for mutation breeding in purple carrot (Daucus carota L.) and possible variations formed.

BACKGROUND: Plant breeding allows altering the genetic structure of plants to meet human needs. The use of radiation technology for inducing mutations and -thereby- new phenotypic variants has become increasingly common as a tool for developing new crops. The aim of this study was to determine the effective gamma irradiation dose for inducing mutations in purple carrot. METHODS AND RESULTS: Increasing gamma radiation doses [0, 50, 100, 200, 300, 400, 500, and 600 Gy] were applied to purple carrot seeds. The irradiated seeds were sown in pots and the emergence and survival rates of the seedlings were analyzed. Considering plant emergence (%) as a response variable, the LD50 dose was 387.5 Gy. Analysis of root length, root width (shoulder diameter) and plant height in control (0 Gy) and irradiated plants (50-600 Gy) revealed an inverse association between these morphological traits and radiation dose. SRAP and ISSR markers were used to identify DNA polymorphisms in irradiated and control plants. The range of amplicons per primer set revealed by ISSR and SRAP markers was 4-10 and 2-13, respectively. In the ISSR analysis of the irradiated carrots (for the 8 doses used), we obtained range values for the average Nei's gene diversity, Shannon's information index, and polymorphism information content (PIC) of 0.13-0.25, 0.20-0.35, and 1.39-1.67, respectively, whereas in the SRAP analysis, the range values for these parameters were 0.15-0.25, 0.23-0.37, and 0.43-0.58, respectively. Cluster analysis revealed three main groups; (a) non-irradiated (control) plants, (b) plants from the 600 Gy dose, and (c) a third group with two subgroups: one with individuals from the lowest irradiation doses (50-200 Gy) and a second group with individuals from the highest irradiation doses (300-500 Gy). CONCLUSIONS: This is the first report on determining effective mutagen doses and genetic characterization of induced mutagenesis via gamma irradiation in purple carrot. ISSR and SRAP markers were successful in detecting variations among different levels of mutagen doses.

Dissecting the genetic control of root and leaf tissue-specific anthocyanin pigmentation in carrot (Daucus carota L.).

KEY MESSAGE: Inheritance, QTL mapping, phylogenetic, and transcriptome (RNA-Seq) analyses provide insight into the genetic control underlying carrot root and leaf tissue-specific anthocyanin pigmentation and identify candidate genes for root phloem pigmentation. Purple carrots can accumulate large quantities of anthocyanins in their root tissues, as well as in other plant parts. This work investigated the genetic control underlying tissue-specific anthocyanin pigmentation in the carrot root phloem and xylem, and in leaf petioles. Inheritance of anthocyanin pigmentation in these three tissues was first studied in segregating F2 and F4 populations, followed by QTL mapping of phloem and xylem anthocyanin pigments (independently) onto two genotyping by sequencing-based linkage maps, to reveal two regions in chromosome 3, namely P1 and P3, controlling pigmentation in these three tissues. Both P1 and P3 condition pigmentation in the phloem, with P3 also conditioning pigmentation in the xylem and petioles. By means of linkage mapping, phylogenetic analysis, and comparative transcriptome (RNA-Seq) analysis among carrot roots with differing purple pigmentation phenotypes, we identified candidate genes conditioning pigmentation in the phloem, the main tissue influencing total anthocyanin levels in the root. Among them, a MYB transcription factor, DcMYB7, and two cytochrome CYP450 genes with putative flavone synthase activity were identified as candidates regulating both the presence/absence of pigmentation and the concentration of anthocyanins in the root phloem. Concomitant expression patterns of DcMYB7 and eight anthocyanin structural genes were found, suggesting that DcMYB7 regulates transcription levels in the latter. Another MYB, DcMYB6, was upregulated in specific purple-rooted samples, suggesting a genotype-specific regulatory activity for this gene. These data contribute to the understanding of anthocyanin regulation in the carrot root at a tissue-specific level and maybe instrumental for improving carrot nutritional value.

Microsatellite analysis and marker development in garlic: distribution in EST sequence, genetic diversity analysis, and marker transferability across Alliaceae.

Allium vegetables, such as garlic and onion, have understudied genomes and limited molecular resources, hindering advances in genetic research and breeding of these species. In this study, we characterized and compared the simple sequence repeats (SSR) landscape in the transcriptomes of garlic and related Allium (A. cepa, A. fistulosum, and A. tuberosum) and non-Allium monocot species. In addition, 110 SSR markers were developed from garlic ESTs, and they were characterized-along with 112 previously developed SSRs-at various levels, including transferability across Alliaceae species, and their usefulness for genetic diversity analysis. Among the Allium species analyzed, garlic ESTs had the highest overall SSR density, the lowest frequency of trinucleotides, and the highest of di- and tetranucleotides. When compared to more distantly related monocots, outside the Asparagales order, it was evident that ESTs of Allium species shared major commonalities with regards to SSR density, frequency distribution, sequence motifs, and GC content. A significant fraction of the SSR markers were successfully transferred across Allium species, including crops for which no SSR markers have been developed yet, such as leek, shallot, chives, and elephant garlic. Diversity analysis of garlic cultivars with selected SSRs revealed 36 alleles, with 2-5 alleles/locus, and PIC = 0.38. Cluster analysis grouped the accessions according to their flowering behavior, botanical variety, and ecophysiological characteristics. Results from this study contribute to the characterization of Allium transcriptomes. The new SSR markers developed, along with the data from the polymorphism and transferability analyses, will aid in assisting genetic research and breeding in garlic and other Allium.

A gene-derived SNP-based high resolution linkage map of carrot including the location of QTL conditioning root and leaf anthocyanin pigmentation.

BACKGROUND: Purple carrots accumulate large quantities of anthocyanins in their roots and leaves. These flavonoid pigments possess antioxidant activity and are implicated in providing health benefits. Informative, saturated linkage maps associated with well characterized populations segregating for anthocyanin pigmentation have not been developed. To investigate the genetic architecture conditioning anthocyanin pigmentation we scored root color visually, quantified root anthocyanin pigments by high performance liquid chromatography in segregating F2, F3 and F4 generations of a mapping population, mapped quantitative trait loci (QTL) onto a dense gene-derived single nucleotide polymorphism (SNP)-based linkage map, and performed comparative trait mapping with two unrelated populations. RESULTS: Root pigmentation, scored visually as presence or absence of purple coloration, segregated in a pattern consistent with a two gene model in an F2, and progeny testing of F3-F4 families confirmed the proposed genetic model. Purple petiole pigmentation was conditioned by a single dominant gene that co-segregates with one of the genes conditioning root pigmentation. Root total pigment estimate (RTPE) was scored as the percentage of the root with purple color.All five anthocyanin glycosides previously reported in carrot, as well as RTPE, varied quantitatively in the F2 population. For the purpose of QTL analysis, a high resolution gene-derived SNP-based linkage map of carrot was constructed with 894 markers covering 635.1 cM with a 1.3 cM map resolution. A total of 15 significant QTL for all anthocyanin pigments and for RTPE mapped to six chromosomes. Eight QTL with the largest phenotypic effects mapped to two regions of chromosome 3 with co-localized QTL for several anthocyanin glycosides and for RTPE. A single dominant gene conditioning anthocyanin acylation was identified and mapped.Comparative mapping with two other carrot populations segregating for purple color indicated that carrot anthocyanin pigmentation is controlled by at least three genes, in contrast to monogenic control reported previously. CONCLUSIONS: This study generated the first high resolution gene-derived SNP-based linkage map in the Apiaceae. Two regions of chromosome 3 with co-localized QTL for all anthocyanin pigments and for RTPE, largely condition anthocyanin accumulation in carrot roots and leaves. Loci controlling root and petiole anthocyanin pigmentation differ across diverse carrot genetic backgrounds.

Inheritance and mapping of Mj-2, a new source of root-knot nematode (Meloidogyne javanica) resistance in carrot.

Root-knot nematodes limit carrot production around the world by inducing taproot forking and galling deformities that render carrots unmarketable. In warmer climates, Meloidogyne javanica and Meloidogyne incognita are most prevalent. In F2 and F3 progeny from the cross between an Asian carrot resistant to M. javanica, PI 652188, and a susceptible carrot, resistance response was incompletely dominant with a relatively high heritability (H (2) = 0.78) and provided evidence for a single gene, designated Mj-2, contributing to resistance. Molecular markers linked to the previously described root-knot nematode resistance gene, Mj-1 on chromosome 8 derived from "Brasilia," demonstrated that Mj-2 does not map to that same locus but is on the same chromosome.

Expression and mapping of anthocyanin biosynthesis genes in carrot.

Anthocyanin gene expression has been extensively studied in leaves, fruits and flowers of numerous plants. Little, however, is known about anthocyanin accumulation in roots of carrots or other species. We quantified expression of six anthocyanin biosynthetic genes [phenylalanine ammonia-lyase (PAL3), chalcone synthase (CHS1), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR1), leucoanthocyanidin dioxygenase (LDOX2), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT)] in three carrot inbreds with contrasting root color: solid purple (phloem and xylem); purple outer phloem/orange xylem; and orange phloem and xylem. Transcripts for five of these genes (CHS1, DFR1, F3H, LDOX2, PAL3) accumulated at high levels in solid purple carrots, less in purple-orange carrot, and low or no transcript in orange carrots. Gene expression coincided with anthocyanin accumulation. In contrast, UFGT expression was comparable in purple and orange carrots and relatively unchanged during root development. In addition, five anthocyanin biosynthesis genes [FLS1 (flavonol synthase), F3H, LDOX2, PAL3, and UFGT] and three anthocyanin transcription factors (DcEFR1, DcMYB3 and DcMYB5) were mapped in a population segregating for the P 1 locus that conditions purple root color. P 1 mapped to chromosome 3 and of the eight anthocyanin biosynthesis genes, only F3H and FLS1 were linked to P 1. The gene expression and mapping data suggest a coordinated regulatory control of anthocyanin expression in carrot root and establish a framework for studying the anthocyanin pathway in carrots, and they also suggest that none of the genes evaluated is a candidate for P 1.

Genetic structure and domestication of carrot (Daucus carota subsp. sativus) (Apiaceae).

PREMISE OF THE STUDY: Analyses of genetic structure and phylogenetic relationships illuminate the origin and domestication of modern crops. Despite being an important worldwide vegetable, the genetic structure and domestication of carrot (Daucus carota) is poorly understood. We provide the first such study using a large data set of molecular markers and accessions that are widely dispersed around the world. • METHODS: Sequencing data from the carrot transcriptome were used to develop 4000 single nucleotide polymorphisms (SNPs). Eighty-four genotypes, including a geographically well-distributed subset of wild and cultivated carrots, were genotyped using the KASPar assay. • KEY RESULTS: Analysis of allelic diversity of SNP data revealed no reduction of genetic diversity in cultivated vs. wild accessions. Structure and phylogenetic analysis indicated a clear separation between wild and cultivated accessions as well as between eastern and western cultivated carrot. Among the wild carrots, those from Central Asia were genetically most similar to cultivated accessions. Furthermore, we found that wild carrots from North America were most closely related to European wild accessions. • CONCLUSIONS: Comparing the genetic diversity of wild and cultivated accessions suggested the absence of a genetic bottleneck during carrot domestication. In conjunction with historical documents, our results suggest an origin of domesticated carrot in Central Asia. Wild carrots from North America were likely introduced as weeds with European colonization. These results provide answers to long-debated questions of carrot evolution and domestication and inform germplasm curators and breeders on genetic substructure of carrot genetic resources.

Differential and Cultivar-Dependent Antioxidant Response of Whole and Fresh-Cut Carrots of Different Root Colors to Postharvest UV-C Radiation.

Fresh-cut produce have become widely popular, increasing vegetable consumption in many parts of the word. However, they are more perishable than unprocessed fresh vegetables, requiring cold storage to preserve their quality and palatability. In addition to cold storage, UV radiation has been used experimentally to try to increase nutritional quality and postharvest shelf life, revealing increased antioxidant levels in some fruits and vegetables, including orange carrots. Carrot is one of the main whole and fresh-cut vegetables worldwide. In addition to orange carrots, other root color phenotypes (e.g., purple, yellow, red) are becoming increasingly popular in some markets. The effect of the UV radiation and cold storage has not been explored in these root phenotypes. This study investigated the effect of postharvest UV-C radiation in whole and fresh-cut (sliced and shredded) roots of two purple, one yellow, and one orange-rooted cultivar, with regard to changes in concentration of total phenolics (TP) and hydroxycinnamic acids (HA), chlorogenic acid (CGA), total and individual anthocyanins, antioxidant capacity (by DPPH and ABTS), and superficial color appearance, monitoring such changes during cold storage. Results revealed that the UV-C radiation, the fresh-cut processing, and the cold storage influenced the content of antioxidant compounds and activities to varying extents, depending on the carrot cultivar, the degree of processing, and the phytochemical compound analyzed. UV-C radiation increased antioxidant capacity up to 2.1, 3.8, 2.5-folds; TP up to 2.0, 2.2, and 2.1-folds; and CGA up to 3.2, 6.6, and 2.5-folds, relative to UV-C untreated controls, for orange, yellow, and purple carrots, respectively. Anthocyanin levels were not significantly modified by the UV-C in both purple carrots evaluated. A moderate increase in tissue browning was found in some fresh-cut processed UV-C treated samples of yellow and purple but not orange roots. These data suggest variable potential for increasing functional value by UV-C radiation in different carrot root colors.

Characterization of Purple Carrot Germplasm for Antioxidant Capacity and Root Concentration of Anthocyanins, Phenolics, and Carotenoids.

The present study characterized a genetically and phenotypically diverse collection of 27 purple and two non-purple (one orange and one yellow) carrot accessions for concentration of root anthocyanins, phenolics, and carotenoids, and antioxidant capacity estimated by four different methods (ORAC, DPPH, ABTS, FRAP), in a partially replicated experimental design comprising data from two growing seasons (2018 and 2019). Broad and significant (p < 0.0001) variation was found among the accessions for all the traits. Acylated anthocyanins (AA) predominated over non-acylated anthocyanins (NAA) in all the accessions and years analyzed, with AA accounting for 55.5-100% of the total anthocyanin content (TAC). Anthocyanins acylated with ferulic acid and coumaric acid were the most abundant carrot anthocyanins. In general, black or solid purple carrots had the greatest TAC and total phenolic content (TPC), and the strongest antioxidant capacities, measured by all methods. Antioxidant capacity, estimated by all methods, was significantly, positively, and moderately-to-strongly correlated with the content of all individual anthocyanins pigments, TAC, and TPC, in both years (r = 0.59-0.90, p < 0.0001), but not with the carotenoid pigments lutein and ß-carotene; suggesting that anthocyanins and other phenolics, but not carotenoids, are major contributors of the antioxidant capacity in purple carrots. We identified accessions with high concentration of chemically stable AA, with potential value for the production of food dyes, and accessions with relatively high content of bioavailable NAA that can be selected for increased nutraceutical value (e.g., for fresh consumption).

Comparative FISH mapping of Daucus species (Apiaceae family).

The cytogenetic characterization of the carrot genome (Daucus carota L., 2n = 18) has been limited so far, partly because of its somatic chromosome morphology and scant of chromosome markers. Here, we integrate the carrot linkage groups with pachytene chromosomes by fluorescent in situ hybridization (FISH) mapping genetically anchored bacterial artificial chromosomes (BACs). We isolated a satellite repeat from the centromeric regions of the carrot chromosomes, which facilitated the study of the pachytene-based karyotype and demonstrated that heterochromatic domains were mainly confined to the pericentromeric regions of each chromosome. Chromosome-specific BACs were used to: (1) physically locate genetically unanchored DNA sequences, (2) reveal relationships between genetic and physical distances, and (3) address chromosome evolution in Daucus. Most carrot BACs generated distinct FISH signals in 22-chromosome Daucus species, providing evidence for syntenic chromosome segments and rearrangements among them. These results provide a foundation for further cytogenetic characterization and chromosome evolution studies in Daucus.

High Temperature Alters Anthocyanin Concentration and Composition in Grape Berries of Malbec, Merlot, and Pinot Noir in a Cultivar-Dependent Manner.

Climate is determinant for grapevine geographical distribution, berry attributes, and wine quality. Due to climate change, a 2−4 °C increase in mean diurnal temperature is predicted by the end of the century for the most important Argentine viticulture region. We hypothesize that such temperature increase will affect color intensity and other quality attributes of red grapes and wines. The present study investigated the effect of high temperature (HT) on anthocyanin concentration and composition, pH, and resveratrol and solids content in berries of three major wine-producing varieties during fruit ripening in two seasons. To this end, a structure that increased mean diurnal temperature by 1.5−2.0 °C at berry sites, compared to Control (C) plants grown without such structure, was implemented in field grown vineyards of Malbec, Merlot, and Pinot Noir. Results revealed a cultivar-dependent response to HT conditions, with Malbec and Pinot Noir berries exhibiting significant decreases in total anthocyanin concentration (TAC) at veraison and harvest, respectively, while Merlot maintained an unaffected pigment content under HT. The decrease in TAC was associated with reduced levels of delphinidin, cyanidin, petunidin, peonidin, and malvidin glycosides, and increased ratios of acylated (AA)/non-acylated anthocyanins (NAA), suggesting pigment acylation as a possible stress-response mechanism for attenuating HT negative effects. Under HT, Pinot Noir, which does not produce AA, was the only cultivar with lower TAC at harvest (p < 0.05). pH, resveratrol, and solids content were not affected by HT. Our results predict high, medium, and low plasticity with regard to color quality attributes for Malbec, Merlot, and Pinot Noir, respectively, in the context of climate change.

Physicochemical properties, degradation kinetics, and antioxidant capacity of aqueous anthocyanin-based extracts from purple carrots compared to synthetic and natural food colorants.

As a means to evaluate the potential of carrot anthocyanins as food colorants and nutraceutical agents, we investigated the physicochemical stability and antioxidant capacity of purple carrot extracts under different pH (2.5-7.0) and temperature (4-40 °C) conditions, in comparison to a commercial synthetic (E131) and a natural grape-based (GRP) colorant. During incubation, the colorants were weekly-monitored for various color parameters, concentration of anthocyanins and phenolics, and antioxidant capacity. Carrot colorants were more stable than GRP; and their thermal stability was equal (at 4 °C) or higher than that of E131 (at 25-40 °C). Carrot anthocyanins had lower degradation rate at low pH and temperature, with acylated anthocyanins (AA) being significantly more stable than non-acylated anthocyanins (NAA). Anthocyanins acylated with feruloyl and coumaroyl glycosides were the most stable carrot pigments. The higher stability of carrot colorants is likely due to their richness in AA and -to a lesser extent- copigmentation with other phenolics.

De novo assembly and characterization of the carrot transcriptome reveals novel genes, new markers, and genetic diversity.

BACKGROUND: Among next generation sequence technologies, platforms such as Illumina and SOLiD produce short reads but with higher coverage and lower cost per sequenced nucleotide than 454 or Sanger. A challenge now is to develop efficient strategies to use short-read length platforms for de novo assembly and marker development. The scope of this study was to develop a de novo assembly of carrot ESTs from multiple genotypes using the Illumina platform, and to identify polymorphisms. RESULTS: A de novo assembly of transcriptome sequence from four genetic backgrounds produced 58,751 contigs and singletons. Over 50% of these assembled sequences were annotated allowing detection of transposable elements and new carrot anthocyanin genes. Presence of multiple genetic backgrounds in our assembly allowed the identification of 114 computationally polymorphic SSRs, and 20,058 SNPs at a depth of coverage of 20× or more. Polymorphisms were predominantly between inbred lines except for the cultivated x wild RIL pool which had high intra-sample polymorphism. About 90% and 88% of tested SSR and SNP primers amplified a product, of which 70% and 46%, respectively, were of the expected size. Out of verified SSR and SNP markers 84% and 82% were polymorphic. About 25% of SNPs genotyped were polymorphic in two diverse mapping populations. CONCLUSIONS: This study confirmed the potential of short read platforms for de novo EST assembly and identification of genetic polymorphisms in carrot. In addition we produced the first large-scale transcriptome of carrot, a species lacking genomic resources.

Microsatellite isolation and marker development in carrot - genomic distribution, linkage mapping, genetic diversity analysis and marker transferability across Apiaceae.

BACKGROUND: The Apiaceae family includes several vegetable and spice crop species among which carrot is the most economically important member, with ~21 million tons produced yearly worldwide. Despite its importance, molecular resources in this species are relatively underdeveloped. The availability of informative, polymorphic, and robust PCR-based markers, such as microsatellites (or SSRs), will facilitate genetics and breeding of carrot and other Apiaceae, including integration of linkage maps, tagging of phenotypic traits and assisting positional gene cloning. Thus, with the purpose of isolating carrot microsatellites, two different strategies were used; a hybridization-based library enrichment for SSRs, and bioinformatic mining of SSRs in BAC-end sequence and EST sequence databases. This work reports on the development of 300 carrot SSR markers and their characterization at various levels. RESULTS: Evaluation of microsatellites isolated from both DNA sources in subsets of 7 carrot F2 mapping populations revealed that SSRs from the hybridization-based method were longer, had more repeat units and were more polymorphic than SSRs isolated by sequence search. Overall, 196 SSRs (65.1%) were polymorphic in at least one mapping population, and the percentage of polymophic SSRs across F2 populations ranged from 17.8 to 24.7. Polymorphic markers in one family were evaluated in the entire F2, allowing the genetic mapping of 55 SSRs (38 codominant) onto the carrot reference map. The SSR loci were distributed throughout all 9 carrot linkage groups (LGs), with 2 to 9 SSRs/LG. In addition, SSR evaluations in carrot-related taxa indicated that a significant fraction of the carrot SSRs transfer successfully across Apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot. SSR diversity evaluated in a collection of 65 D. carota accessions revealed a high level of polymorphism for these selected loci, with an average of 19 alleles/locus and 0.84 expected heterozygosity. CONCLUSIONS: The addition of 55 SSRs to the carrot map, together with marker characterizations in six other mapping populations, will facilitate future comparative mapping studies and integration of carrot maps. The markers developed herein will be a valuable resource for assisting breeding, genetic, diversity, and genomic studies of carrot and other Apiaceae.

Genetic and Transcription Profile Analysis of Tissue-Specific Anthocyanin Pigmentation in Carrot Root Phloem.

In purple carrots, anthocyanin pigmentation can be expressed in the entire root, or it can display tissue specific-patterns. Within the phloem, purple pigmentation can be found in the outer phloem (OP) (also called the cortex) and inner phloem (IP), or it can be confined exclusively to the OP. In this work, the genetic control underlying tissue-specific anthocyanin pigmentation in the carrot root OP and IP tissues was investigated by means of linkage mapping and transcriptome (RNA-seq) and phylogenetic analyses; followed by gene expression (RT-qPCR) evaluations in two genetic backgrounds, an F2 population (3242) and the inbred B7262. Genetic mapping of 'root outer phloem anthocyanin pigmentation' (ROPAP) and inner phloem pigmentation (RIPAP) revealed colocalization of ROPAP with the P1 and P3 genomic regions previously known to condition pigmentation in different genetic stocks, whereas RIPAP co-localized with P3 only. Transcriptome analysis of purple OP (POP) vs. non-purple IP (NPIP) tissues, along with linkage and phylogenetic data, allowed an initial identification of 28 candidate genes, 19 of which were further evaluated by RT-qPCR in independent root samples of 3242 and B7262, revealing 15 genes consistently upregulated in the POP in both genetic backgrounds, and two genes upregulated in the POP in specific backgrounds. These include seven transcription factors, seven anthocyanin structural genes, and two genes involved in cellular transport. Altogether, our results point at DcMYB7, DcMYB113, and a MADS-box (DCAR_010757) as the main candidate genes conditioning ROPAP in 3242, whereas DcMYB7 and MADS-box condition RIPAP in this background. In 7262, DcMYB113 conditions ROPAP.

Genome-wide characterization of simple sequence repeats in cucumber (Cucumis sativus L.).

BACKGROUND: Cucumber, Cucumis sativus L. is an important vegetable crop worldwide. Until very recently, cucumber genetic and genomic resources, especially molecular markers, have been very limited, impeding progress of cucumber breeding efforts. Microsatellites are short tandemly repeated DNA sequences, which are frequently favored as genetic markers due to their high level of polymorphism and codominant inheritance. Data from previously characterized genomes has shown that these repeats vary in frequency, motif sequence, and genomic location across taxa. During the last year, the genomes of two cucumber genotypes were sequenced including the Chinese fresh market type inbred line '9930' and the North American pickling type inbred line 'Gy14'. These sequences provide a powerful tool for developing markers in a large scale. In this study, we surveyed and characterized the distribution and frequency of perfect microsatellites in 203 Mbp assembled Gy14 DNA sequences, representing 55% of its nuclear genome, and in cucumber EST sequences. Similar analyses were performed in genomic and EST data from seven other plant species, and the results were compared with those of cucumber. RESULTS: A total of 112,073 perfect repeats were detected in the Gy14 cucumber genome sequence, accounting for 0.9% of the assembled Gy14 genome, with an overall density of 551.9 SSRs/Mbp. While tetranucleotides were the most frequent microsatellites in genomic DNA sequence, dinucleotide repeats, which had more repeat units than any other SSR type, had the highest cumulative sequence length. Coding regions (ESTs) of the cucumber genome had fewer microsatellites compared to its genomic sequence, with trinucleotides predominating in EST sequences. AAG was the most frequent repeat in cucumber ESTs. Overall, AT-rich motifs prevailed in both genomic and EST data. Compared to the other species examined, cucumber genomic sequence had the highest density of SSRs (although comparable to the density of poplar, grapevine and rice), and was richest in AT dinucleotides. Using an electronic PCR strategy, we investigated the polymorphism between 9930 and Gy14 at 1,006 SSR loci, and found unexpectedly high degree of polymorphism (48.3%) between the two genotypes. The level of polymorphism seems to be positively associated with the number of repeat units in the microsatellite. The in silico PCR results were validated empirically in 660 of the 1,006 SSR loci. In addition, primer sequences for more than 83,000 newly-discovered cucumber microsatellites, and their exact positions in the Gy14 genome assembly were made publicly available. CONCLUSIONS: The cucumber genome is rich in microsatellites; AT and AAG are the most abundant repeat motifs in genomic and EST sequences of cucumber, respectively. Considering all the species investigated, some commonalities were noted, especially within the monocot and dicot groups, although the distribution of motifs and the frequency of certain repeats were characteristic of the species examined. The large number of SSR markers developed from this study should be a significant contribution to the cucurbit research community.

Carrot Anthocyanins Genetics and Genomics: Status and Perspectives to Improve Its Application for the Food Colorant Industry.

Purple or black carrots (Daucus carota ssp. sativus var. atrorubens Alef) are characterized by their dark purple- to black-colored roots, owing their appearance to high anthocyanin concentrations. In recent years, there has been increasing interest in the use of black carrot anthocyanins as natural food dyes. Black carrot roots contain large quantities of mono-acylated anthocyanins, which impart a measure of heat-, light- and pH-stability, enhancing the color-stability of food products over their shelf-life. The genetic pathway controlling anthocyanin biosynthesis appears well conserved among land plants; however, different variants of anthocyanin-related genes between cultivars results in tissue-specific accumulations of purple pigments. Thus, broad genetic variations of anthocyanin profile, and tissue-specific distributions in carrot tissues and organs, can be observed, and the ratio of acylated to non-acylated anthocyanins varies significantly in the purple carrot germplasm. Additionally, anthocyanins synthesis can also be influenced by a wide range of external factors, such as abiotic stressors and/or chemical elicitors, directly affecting the anthocyanin yield and stability potential in food and beverage applications. In this study, we critically review and discuss the current knowledge on anthocyanin diversity, genetics and the molecular mechanisms controlling anthocyanin accumulation in carrots. We also provide a view of the current knowledge gaps and advancement needs as regards developing and applying innovative molecular tools to improve the yield, product performance and stability of carrot anthocyanin for use as a natural food colorant.

Characterization of a deep-coverage carrot (Daucus carota L.) BAC library and initial analysis of BAC-end sequences.

Carrot is the most economically important member of the Apiaceae family and a major source of provitamin A carotenoids in the human diet. However, carrot molecular resources are relatively underdeveloped, hampering a number of genetic studies. Here, we report on the synthesis and characterization of a bacterial artificial chromosome (BAC) library of carrot. The library is 17.3-fold redundant and consists of 92,160 clones with an average insert size of 121 kb. To provide an overview of the composition and organization of the carrot nuclear genome we generated and analyzed 2,696 BAC-end sequences (BES) from nearly 2,000 BACs, totaling 1.74 Mb of BES. This analysis revealed that 14% of the BES consists of known repetitive elements, with transposable elements representing more than 80% of this fraction. Eleven novel carrot repetitive elements were identified, covering 8.5% of the BES. Analysis of microsatellites showed a comparably low frequency for these elements in the carrot BES. Comparisons of the translated BES with protein databases indicated that approximately 10% of the carrot genome represents coding sequences. Moreover, among eight dicot species used for comparison purposes, carrot BES had highest homology to protein-coding sequences from tomato. This deep-coverage library will aid carrot breeding and genetics.

Identification of an SCPL Gene Controlling Anthocyanin Acylation in Carrot (Daucus carota L.) Root.

Anthocyanins are natural health promoting pigments that can be produced in large quantities in some purple carrot cultivars. Decoration patterns of anthocyanins, such as acylation, can greatly influence their stability and biological properties and use in the food industry as nutraceuticals and natural colorants. Despite recent advances made toward understanding the genetic control of anthocyanin accumulation in purple carrot, the genetic mechanism controlling acylation of anthocyanin in carrot root have not been studied yet. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify the genetic factor conditioning the accumulation of non-acylated (Cy3XGG) versus acylated (Cy3XFGG and Cy3XSGG) cyanidin derivatives, in three carrot populations. Segregation and mapping analysis pointed to a single gene with dominant effect controlling anthocyanin acylation in the root, located in a 576kb region containing 29 predicted genes. Orthologous and phylogenetic analyses enabled the identification of a cluster of three SCPL-acyltransferases coding genes within this region. Comparative transcriptome analysis indicated that only one of these three genes, DcSCPL1, was always expressed in association with anthocyanin pigmentation in the root and was co-expressed with DcMYB7, a gene known to activate anthocyanin biosynthetic genes in carrot. DcSCPL1 sequence analysis, in root tissue containing a low level of acylated anthocyanins, demonstrated the presence of an insertion causing an abnormal splicing of the 3rd exon during mRNA editing, likely resulting in the production of a non-functional acyltransferase and explaining the reduced acylation phenotype. This study provides strong linkage-mapping and functional evidences for the candidacy of DcSCPL1 as a primary regulator of anthocyanin acylation in carrot storage root.

A Cluster of MYB Transcription Factors Regulates Anthocyanin Biosynthesis in Carrot (Daucus carota L.) Root and Petiole.

Purple carrots can accumulate large quantities of anthocyanins in their roots and -in some genetic backgrounds- petioles, and therefore they represent an excellent dietary source of antioxidant phytonutrients. In a previous study, using linkage analysis in a carrot F2 mapping population segregating for root and petiole anthocyanin pigmentation, we identified a region in chromosome 3 with co-localized QTL for all anthocyanin pigments of the carrot root, whereas petiole pigmentation segregated as a single dominant gene and mapped to one of these "root pigmentation" regions conditioning anthocyanin biosynthesis. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify candidate genes controlling anthocyanin pigmentation in the carrot root and petiole. Fine mapping was performed in four carrot populations with different genetic backgrounds and patterns of pigmentation. The regions controlling root and petiole pigmentation in chromosome 3 were delimited to 541 and 535 kb, respectively. Genome wide prediction of transcription factor families known to regulate the anthocyanin biosynthetic pathway coupled with orthologous and phylogenetic analyses enabled the identification of a cluster of six MYB transcription factors, denominated DcMYB6 to DcMYB11, associated with the regulation of anthocyanin biosynthesis. No anthocyanin biosynthetic genes were present in this region. Comparative transcriptome analysis indicated that upregulation of DcMYB7 was always associated with anthocyanin pigmentation in both root and petiole tissues, whereas DcMYB11 was only upregulated with pigmentation in petioles. In the petiole, the level of expression of DcMYB11 was higher than DcMYB7. DcMYB6, a gene previously suggested as a key regulator of carrot anthocyanin biosynthesis, was not consistently associated with pigmentation in either tissue. These results strongly suggest that DcMYB7 is a candidate gene for root anthocyanin pigmentation in all the genetic backgrounds included in this study. DcMYB11 is a candidate gene for petiole pigmentation in all the purple carrot sources in this study. Since DcMYB7 is co-expressed with DcMYB11 in purple petioles, the latter gene may act also as a co-regulator of anthocyanin pigmentation in the petioles. This study provides linkage-mapping and functional evidence for the candidacy of these genes for the regulation of carrot anthocyanin biosynthesis.