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.EEA MendozaFil: Bannoud, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carvajal, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ellison, Shelby. University of Wisconsin. Department of Horticulture; Estados Unidos.Fil: Senalik, Douglas A. United States Department of Agriculture–Agricultural Research Service. Vegetable Crops Research Unit; Estados UnidosFil: Gomez Talquenca, Gonzalo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Iorizzo, Massimo. North Carolina State University. Plants for Human Health Institute; Estados UnidosFil: Iorizzo, Massimo. North Carolina State University. Department of Horticultural Science; Estados UnidosFil: Simon, Philipp. University of Wisconsin. Department of Horticulture; Estados Unidos.Fil: Simon, Philipp. United States Department of Agriculture–Agricultural Research Service. Vegetable Crops Research Unit; Estados UnidosFil: Cavagnaro, Pablo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria La Consulta; ArgentinaFil: Cavagnaro, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cavagnaro, Pablo. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Horticultura; Argentina

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

Additional file 1 Table S1

Additional file 1: Table S1. The 162 accessions of Daucus, and two accessions of related genera characterized in this study, improvement status, locality information and new identification

Additional file 12 Figure S10

Additional file 12: Figure S10. Box plot analyses of the 23 morphological characters examined for members of Daucus carota complex (subsp. sativus not included) in this study. The box plot displays individual plant values for median, 25% and 75% percentile, range, and outliers

Data from: 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

Additional file 8 Figure S6

Additional file 8: Figure S6. Relationships among 144 accessions of Daucus carota complex and outgroup from an exhaustive quartet sampling inference using 43,713 SNPs (30% missing imputed genotypes) obtained by GBS. Numbers above branches represent bootstrap values, with only values higher than 70% shown. Names given to clades refer to the geographic origin and improvement status of the accessions of D. carota complex. ME & E refers to Middle East & Europe. Accessions designated by double stars are misplaced relative to the maximum likelihood topology of Daucus carota complex using the same number of SNPs. The outgroup taxon is D. syrticus

Additional file 7 Figure S5

Additional file 7: Figure S5. Relationships among 144 accessions of Daucus carota complex and outgroup from an exhaustive quartet sampling inference using 18,565 SNPs (10% missing imputed genotypes) obtained by GBS. Numbers above the branches represent bootstrap values, with only values higher than 70% shown. Names given to clades refer to the geographic origin and improvement status of the accessions of D. carota complex. ME & E refers to Middle East & Europe. Accessions designated by double stars are misplaced relative to the maximum likelihood topology of Daucus carota complex using the same number of SNPs. The outgroup taxon is D. syrticus

Data from: 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

Additional file 5 Figure S3

Additional file 5: Figure S3. Maximum likelihood reconstruction and structure of the genetic diversity of 144 accessions of Daucus carota complex and outgroup using 43,713 SNPs (30% missing imputed genotypes) obtained by GBS. Each accession is represented by a horizontal bar, and each color corresponds to a population (nine in total). Numbers above branches represent bootstrap values, with only values higher than 70% shown. Names given to clades refer to the geographic origin and improvement status of the accessions of D. carota complex. The outgroup taxon is D. syrticus

Additional file 2 Table S2

Additional file 2: Table S2. Summary of processing GBS data of Daucus