First Report of Bacterial Blight of Pennycress Caused by Xanthomonas campestris in Wisconsin.

In May 2023, pennycress (Thlaspi arvense, L.) lines undergoing seed production in the Walnut Street Greenhouse at the University of Wisconsin-Madison displayed symptoms of chlorosis and black necrotic leaf spots (Fig. S1-A). Lesions eventually enlarged to 1-2 cm in diameter, became necrotic, and coalesced to cover a substantial portion of leaves. Symptoms were observed in ~30% of the pennycress lines adversely affecting overall growth and reproduction. Symptomatic leaves were surface sterilized for 30 seconds in 0.75% sodium hypochlorite, rinsed in sterile deionized water, and bacteria were isolated using three-phase streaking of symptomatic tissue onto KB medium (King et al., 1954). Single colonies of three isolates (creamy white to yellow) from this initial isolation were streaked onto KB medium to obtain pure cultures. Individual colonies were transferred for growth overnight in nutrient broth (Difco) and an equal amount of the broth was added to 30% glycerol in deionized (di) water and stored at -80 °C. To validate Koch's Postulates, bacteria were grown from these stocks on Yeast Dextrose Calcium Carbonate medium (Wilson et al., 1967) and were used to inoculate 5-week-old pennycress plants in the greenhouse. The bacteria were grown for 48 hours at 26°C, suspended in 300 ml of 0.05 M PBS buffer (pH=7.2) for inoculum preparation. Plants were inoculated with three bacterial isolates (approx. 108 CFU/ml) by piercing the mid veins or hydathodes with a sterilized toothpick dipped in the suspension. Inoculated plants were then enclosed in clear plastic bags for 24-48 hours and maintained in the greenhouse at a constant temperature of 26°C with a 16-hour photoperiod. After seven days, water-soaked lesions appeared on the inoculated leaves, eventually developing into the characteristic black spots (Fig. S1-B). DNA from the original isolates was extracted, and 16S PCR and sequencing of the positive bands was done. The negative control only produced brown spots at the site of inoculation (Fig. S1-C). The primer sequences were as follows: 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT (Eden et al., 1991; Weisburg et al., 1991). A BLAST analysis showed that the isolates had an E value of 0.0 to the genus Xanthomonas as well as 100% identity. Amplification and sequencing of the bacterium using gyrB amplicons revealed a 99-100% pairwise match with Xc. To enhance taxonomy resolution and confirm the identity of these isolates, the complete genomes of three samples were sequenced using NextSeq2000 Illumina platform (NCBI bioproject ID PRJNA1040293). Average Nucleotide Identity (ANI) analysis was conducted with representative strains from the Xc species (Dubrow et al., 2022), using PanExplorer (Dereeper et al., 2020) featuring integrated FastANI module (Jain et al., 2018). The isolates genomes exhibited over 98% identity and clustered with that of Xc pv. incanae and Xc pv. barbarae (Fig S2). Further work will be required to identify the pathovar of Xc identified in this study through phenotypic host range assay. This marks the first documented case of Xc in pennycress in the Midwestern US. Given the potential use of pennycress as a cover crop in the region, further investigations are warranted to assess its economic impact on production and develop management strategies.

First Report of Tobacco Streak Virus in Cannabis sativa in New York.

In 2022, virus-like symptoms were observed in a field of diverse hemp (Cannabis sativa L.) germplasm in Ontario County, New York. Less than 1% of plants exhibited stunting and curled leaves (Figure S1), consistent with tobacco streak virus (TSV) symptoms on other plants (Liu et al. 2022). Most typically, the plants were considerably reduced in overall size, with upwards, adaxial curling along the leaf margin with newer leaves appearing to be the most affected. Fifteen symptomatic plants representing nine accessions were tested for 12 viruses and viroids through Agdia Testing Services (Elkhart, IN). Of these, eight plants representing five accessions including: G 33204 21UO SD ('Cherry Wine S1'), G 33211 21UO SD ('Wife'), G 33225 22CL01 CL ('Candida #2'), G 33270 22UO SD ('Falkowski CBD Mix'), and G 33365 22UO SD ('Queen Dream'), were positive for TSV, a type of Ilarvirus in the Bromoviridae family. Presence of TSV was confirmed through enzyme-linked immunosorbent assay testing. TSV is a positive-sense, single-stranded RNA virus with a wide host range that can be transmitted by thrips, mechanical injury, seed, and pollen (Zambrana-Echevarría et al. 2021). To confirm the presence of TSV, two putatively TSV-infected samples were subjected to RNA-Seq analysis. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Aarhus, Denmark) per manufacturer's direction. Stranded RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant kit (San Diego, California, USA). Paired-end 2x150bp sequencing was performed on an Illumina NovaSeq6000 sequencer. RNA-Seq data was trimmed using the fastp program (Chen et al. 2018) with default parameters to remove adapter sequences and low-quality bases. After filtering, 49,696,041 and 56,126,804 paired-end reads were retained from 'Wife' and 'Falkowski CBD Mix' samples, respectively. Filtered RNA-seq reads were mapped to TSV genome accession GCF_000865505.1 using the bowtie2 (Langmead & Salzberg 2012) aligner with default parameters. From 'Wife' and 'Falkowski CBD Mix' samples, 153 and 139 reads mapped to the TSV reference genome. To further validate the presence of TSV reads, RNA-Seq data was analyzed using the Kraken2 pipeline (Wood et al. 2019). Using the Kraken2 virus database, reads associated with TSV (NCBI taxonomy ID: 12317) were identified. This analysis identified 172 and 151 TSV reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Higher numbers of reads identified using the Kraken2 analysis is due to the more permissive k-mer matching approach implemented in Kraken2. Furthermore, we identified several other virus taxa in the samples. Of note, both samples had a high number of reads associated with Amazon lily mild mottle virus with 254,493 and 116,150 reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Among other virus species belonging to Ilarviruses, Cassava Ivorian bacilliform virus and Cowpea chlorotic mottle viruses were detected from both samples. To further validate infection by TSV, samples from both ELISA-positive and ELISA-negative plants were subjected to PCR using the primers and protocol described in Zambrana-Echevarría et al. 2021. Amplification of an approximately 700 base-pair product was observed in the putatively ELISA-positive samples, but not in the ELISA-negative samples. The amplicons were further cloned into the pGEM-T Easy vector (Promega, Madison, WI, U.S.A) using the manufacturer's protocol and sequenced using M13 forward and M13 reverse primers (Functional Biosciences, Madison, WI, U.S.A). Sequencing results indicated considerable similarity to TSV genomes available in GenBank, between 88% and 99%. Raw sequence data generated from this study was deposited in NCBI under the bioproject ID PRJNA1009441. Though it cannot be ruled out that the observed symptoms were caused exclusively by TSV infection due to the high number of other viral reads, the results contribute to the literature that indicates hemp can host TSV and should be considered a potential source of TSV inoculum (Chiginsky et al. 2021). This new inoculum source could cause significant crop damage and economic loss when grown with TSV susceptible row and specialty crops.

Genetic characterization of carrot root shape and size using genome-wide association analysis and genomic-estimated breeding values.

KEY MESSAGE: The principal phenotypic determinants of market class in carrot-the size and shape of the root-are under primarily additive, but also highly polygenic, genetic control. The size and shape of carrot roots are the primary determinants not only of yield, but also market class. These quantitative phenotypes have historically been challenging to objectively evaluate, and thus subjective visual assessment of market class remains the primary method by which selection for these traits is performed. However, advancements in digital image analysis have recently made possible the high-throughput quantification of size and shape attributes. It is therefore now feasible to utilize modern methods of genetic analysis to investigate the genetic control of root morphology. To this end, this study utilized both genome wide association analysis (GWAS) and genomic-estimated breeding values (GEBVs) and demonstrated that the components of market class are highly polygenic traits, likely under the influence of many small effect QTL. Relatively large proportions of additive genetic variance for many of the component phenotypes support high predictive ability of GEBVs; average prediction ability across underlying market class traits was 0.67. GWAS identified multiple QTL for four of the phenotypes which compose market class: length, aspect ratio, maximum width, and root fill, a previously uncharacterized trait which represents the size-independent portion of carrot root shape. By combining digital image analysis with GWAS and GEBVs, this study represents a novel advance in our understanding of the genetic control of market class in carrot. The immediate practical utility and viability of genomic selection for carrot market class is also described, and concrete guidelines for the design of training populations are provided.

The influence of the Or and Carotene Hydroxylase genes on carotenoid accumulation in orange carrots [Daucus carota (L.)].

KEY MESSAGE: The Or and CH genes are necessary for the accumulation of high amounts of ß-carotene and other carotenoid pigments in carrot roots, in addition to the Y and Y2 genes. Carrot taproot color results from the accumulation of various carotenoid and anthocyanin pigments. Recently, the Or gene was identified as a candidate gene associated with the accumulation of ß-carotene and other provitamin A carotenoids in roots. The specific molecular mechanisms involved with this process, as well as the interactions between Or and the other genes involved in this process are not well understood. In order to better characterize the effect that Or alleles have on conditioning the accumulation of carotenoids in roots, we analyzed an F3 family fixed homozygous recessive for y and y2, derived from a cross between an orange carrot and a white wild carrot, segregating for the two known Or alleles, which we name Orc and Orw. QTL mapping across three different environments revealed that the accumulation of several carotenoids was associated with the Orc allele, with consistent patterns across environments. A second QTL on chromosome 7, harboring a carotene hydroxylase gene homologous to Lut5 in Arabidopsis, was also associated with the accumulation of several carotenoids. Two alleles for this gene, which we name CHc and CHw, were discovered to be segregating in this population. Our study provides further evidence that Or and CH are likely involved with controlling the accumulation of ß-carotene and may be involved with modulating carotenoid flux in carrot, demonstrating that both were important domestication genes in carrot.

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.

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.

Genotyping-by-sequencing provides the discriminating power to investigate the subspecies of Daucus carota (Apiaceae).

BACKGROUND: The majority of the subspecies of Daucus carota have not yet been discriminated clearly by various molecular or morphological methods and hence their phylogeny and classification remains unresolved. Recent studies using 94 nuclear orthologs and morphological characters, and studies employing other molecular approaches were unable to distinguish clearly many of the subspecies. Fertile intercrosses among traditionally recognized subspecies are well documented. We here explore the utility of single nucleotide polymorphisms (SNPs) generated by genotyping-by-sequencing (GBS) to serve as an effective molecular method to discriminate the subspecies of the D. carota complex. RESULTS: We used GBS to obtain SNPs covering all nine Daucus carota chromosomes from 162 accessions of Daucus and two related genera. To study Daucus phylogeny, we scored a total of 10,814 or 38,920 SNPs with a maximum of 10 or 30 % missing data, respectively. To investigate the subspecies of D. carota, we employed two data sets including 150 accessions: (i) rate of missing data 10 % with a total of 18,565 SNPs, and (ii) rate of missing data 30 %, totaling 43,713 SNPs. Consistent with prior results, the topology of both data sets separated species with 2n = 18 chromosome from all other species. Our results place all cultivated carrots (D. carota subsp. sativus) in a single clade. The wild members of D. carota from central Asia were on a clade with eastern members of subsp. sativus. The other subspecies of D. carota were in four clades associated with geographic groups: (1) the Balkan Peninsula and the Middle East, (2) North America and Europe, (3) North Africa exclusive of Morocco, and (4) the Iberian Peninsula and Morocco. Daucus carota subsp. maximus was discriminated, but neither it, nor subsp. gummifer (defined in a broad sense) are monophyletic. CONCLUSIONS: Our study suggests that (1) the morphotypes identified as D. carota subspecies gummifer (as currently broadly circumscribed), all confined to areas near the Atlantic Ocean and the western Mediterranean Sea, have separate origins from sympatric members of other subspecies of D. carota, (2) D. carota subsp. maximus, on two clades with some accessions of subsp. carota, can be distinguished from each other but only with poor morphological support, (3) D. carota subsp. capillifolius, well distinguished morphologically, is an apospecies relative to North African populations of D. carota subsp. carota, (4) the eastern cultivated carrots have origins closer to wild carrots from central Asia than to western cultivated carrots, and (5) large SNP data sets are suitable for species-level phylogenetic studies in Daucus.

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.

Utilizing digitized occurrence records of Midwestern feral Cannabis sativa to develop ecological niche models.

Hemp (Cannabis sativa L.) has historically played a vital role in agriculture across the globe. Feral and wild populations have served as genetic resources for breeding, conservation, and adaptation to changing environmental conditions. However, feral populations of Cannabis, specifically in the Midwestern United States, remain poorly understood. This study aims to characterize the abiotic tolerances of these populations, estimate suitable areas, identify regions at risk of abiotic suitability change, and highlight the utility of ecological niche models (ENMs) in germplasm conservation. The Maxent algorithm was used to construct a series of ENMs. Validation metrics and MOP (Mobility-oriented Parity) analysis were used to assess extrapolation risk and model performance. We also projected the final projected under current and future climate scenarios (2021-2040 and 2061-2080) to assess how abiotic suitability changes with time. Climate change scenarios indicated an expansion of suitable habitat, with priority areas for germplasm collection in Indiana, Illinois, Kansas, Missouri, and Nebraska. This study demonstrates the application of ENMs for characterizing feral Cannabis populations and highlights their value in germplasm conservation and breeding efforts. Populations of feral C. sativa in the Midwest are of high interest, and future research should focus on utilizing tools to aid the collection of materials for the characterization of genetic diversity and adaptation to a changing climate.

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.

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.

CarrotOmics: a genetics and comparative genomics database for carrot (Daucus carota).

CarrotOmics (https://carrotomics.org/) is a comprehensive database for carrot (Daucus carota L.) breeding and research. CarrotOmics was developed using resources available at the MainLab Bioinformatics core (https://www.bioinfo.wsu.edu/) and is implemented using Tripal with Drupal modules. The database delivers access to download or visualize the carrot reference genome with gene predictions, gene annotations and sequence assembly. Other genomic resources include information for 11 224 genetic markers from 73 linkage maps or genotyping-by-sequencing and descriptions of 371 mapped loci. There are records for 1601 Apiales species (or subspecies) and descriptions of 9408 accessions from 11 germplasm collections representing more than 600 of these species. Additionally, 204 Apiales species have phenotypic information, totaling 28 517 observations from 10 041 biological samples. Resources on CarrotOmics are freely available, search functions are provided to find data of interest and video tutorials are available to describe the search functions and genomic tools. CarrotOmics is a timely resource for the Apiaceae research community and for carrot geneticists developing improved cultivars with novel traits addressing challenges including an expanding acreage in tropical climates, an evolving consumer interested in sustainably grown vegetables and a dynamic environment due to climate change. Data from CarrotOmics can be applied in genomic-assisted selection and genetic research to improve basic research and carrot breeding efficiency. DATABASE URL: https://carrotomics.org/.

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.

Publisher Correction: Diversity and function of terpene synthases in the production of carrot aroma and flavor compounds.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Diversity and function of terpene synthases in the production of carrot aroma and flavor compounds.

Carrot (Daucus carota L.) is an important root vegetable crop with high nutritional value, characteristic flavor, and benefits to human health. D. carota tissues produce an essential oil that is rich in volatile terpenes and plays a major role in carrot aroma and flavor. Although terpene composition represents a critical quality attribute of carrots, little is known about the biosynthesis of terpenes in this crop. Here, we functionally characterized 19 terpene synthase (TPS) genes in an orange carrot (genotype DH1) and compared tissue-specific expression profiles and in vitro products of their recombinant proteins with volatile terpene profiles from DH1 and four other colored carrot genotypes. In addition to the previously reported (E)-ß-caryophyllene synthase (DcTPS01), we biochemically characterized several TPS proteins with direct correlations to major compounds of carrot flavor and aroma including germacrene D (DcTPS7/11), γ-terpinene (DcTPS30) and α-terpinolene (DcTPS03). Random forest analysis of volatiles from colored carrot cultivars identified nine terpenes that were clearly distinct among the cultivars and likely contribute to differences in sensory quality. Correlation of TPS gene expression and terpene metabolite profiles supported the function of DcTPS01 and DcTPS03 in these cultivars. Our findings provide a roadmap for future breeding efforts to enhance carrot flavor and aroma.

An Automated Image Analysis Pipeline Enables Genetic Studies of Shoot and Root Morphology in Carrot (Daucus carota L.).

Carrot is a globally important crop, yet efficient and accurate methods for quantifying its most important agronomic traits are lacking. To address this problem, we developed an automated image analysis platform that extracts components of size and shape for carrot shoots and roots, which are necessary to advance carrot breeding and genetics. This method reliably measured variation in shoot size and shape, petiole number, petiole length, and petiole width as evidenced by high correlations with hundreds of manual measurements. Similarly, root length and biomass were accurately measured from the images. This platform also quantified shoot and root shapes in terms of principal components, which do not have traditional, manually measurable equivalents. We applied the pipeline in a study of a six-parent diallel population and an F2 mapping population consisting of 316 individuals. We found high levels of repeatability within a growing environment, with low to moderate repeatability across environments. We also observed co-localization of quantitative trait loci for shoot and root characteristics on chromosomes 1, 2, and 7, suggesting these traits are controlled by genetic linkage and/or pleiotropy. By increasing the number of individuals and phenotypes that can be reliably quantified, the development of a rapid, automated image analysis pipeline to measure carrot shoot and root morphology will expand the scope and scale of breeding and genetic studies.

Carotenoid Presence Is Associated with the Or Gene in Domesticated Carrot.

Carrots are among the richest sources of provitamin A carotenes in the human diet, but genetic variation in the carotenoid pathway does not fully explain the high levels of carotenoids in carrot roots. Using a diverse collection of modern and historic domesticated varieties, and wild carrot accessions, an association analysis for orange pigmentation revealed a significant genomic region that contains the Or gene, advancing it as a candidate for carotenoid presence in carrot. Analysis of sequence variation at the Or locus revealed a nonsynonymous mutation cosegregating with carotenoid content. This mutation was absent in all wild carrot samples and nearly fixed in all orange domesticated samples. Or has been found to control carotenoid presence in other crops but has not previously been described in carrot. Our analysis also allowed us to more completely characterize the genetic structure of carrot, showing that the Western domesticated carrot largely forms one genetic group, despite dramatic phenotypic differences among market classes. Eastern domesticated and wild accessions form a second group, which reflects the recent cultivation history of carrots in Central Asia. Other wild accessions form distinct geographic groups, particularly on the Iberian peninsula and in Northern Africa. Using genome-wide Fst , nucleotide diversity, and the cross-population composite likelihood ratio, we analyzed the genome for regions putatively under selection during domestication and identified 12 regions that were significant for all three methods of detection, one of which includes the Or gene. The Or domestication allele appears to have been selected after the initial domestication of yellow carrots in the East, near the proposed center of domestication in Central Asia. The rapid fixation of the Or domestication allele in almost all orange and nonorange carrots in the West may explain why it has not been found with less genetically diverse mapping populations.

Fine Mapping, Transcriptome Analysis, and Marker Development for Y2 , the Gene That Conditions ß-Carotene Accumulation in Carrot (Daucus carota L.).

Domesticated carrots, Daucus carota subsp. sativus, are the richest source of ß-carotene in the US diet, which, when consumed, is converted into vitamin A, an essential component of eye health and immunity. The Y2 locus plays a significant role in beta-carotene accumulation in carrot roots, but a candidate gene has not been identified. To advance our understanding of this locus, the genetic basis of ß-carotene accumulation was explored by utilizing an advanced mapping population, transcriptome analysis, and nucleotide diversity in diverse carrot accessions with varying levels of ß-carotene. A single large effect Quantitative Trait Locus (QTL) on the distal arm of chromosome 7 overlapped with the previously identified ß-carotene accumulation QTL, Y2 Fine mapping efforts reduced the genomic region of interest to 650 kb including 72 genes. Transcriptome analysis within this fine mapped region identified four genes differentially expressed at two developmental time points, and 13 genes differentially expressed at one time point. These differentially expressed genes included transcription factors and genes involved in light signaling and carotenoid flux, including a member of the Di19 gene family involved in Arabidopsis photomorphogenesis, and a homolog of the bHLH36 transcription factor involved in maize carotenoid metabolism. Analysis of nucleotide diversity in 25 resequenced carrot accessions revealed a drastic decrease in diversity of this fine-mapped region in orange cultivated accessions as compared to white and yellow cultivated and to white wild samples. The results presented in this study provide a foundation to identify and characterize the gene underlying ß-carotene accumulation in carrot.

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.

Application of genomics-assisted breeding for generation of climate resilient crops: progress and prospects.

Climate change affects agricultural productivity worldwide. Increased prices of food commodities are the initial indication of drastic edible yield loss, which is expected to increase further due to global warming. This situation has compelled plant scientists to develop climate change-resilient crops, which can withstand broad-spectrum stresses such as drought, heat, cold, salinity, flood, submergence and pests, thus helping to deliver increased productivity. Genomics appears to be a promising tool for deciphering the stress responsiveness of crop species with adaptation traits or in wild relatives toward identifying underlying genes, alleles or quantitative trait loci. Molecular breeding approaches have proven helpful in enhancing the stress adaptation of crop plants, and recent advances in high-throughput sequencing and phenotyping platforms have transformed molecular breeding to genomics-assisted breeding (GAB). In view of this, the present review elaborates the progress and prospects of GAB for improving climate change resilience in crops, which is likely to play an ever increasing role in the effort to ensure global food security.