Evaluation of shoot-growth variation in diverse carrot (Daucus carota L.) germplasm for genetic improvement of stand establishment.

Carrot (Daucus carota L.) is a high value, nutritious, and colorful crop, but delivering carrots from seed to table can be a struggle for carrot growers. Weed competitive ability is a critical trait for crop success that carrot and its apiaceous relatives often lack owing to their characteristic slow shoot growth and erratic seedling emergence, even among genetically uniform lines. This study is the first field-based, multi-year experiment to evaluate shoot-growth trait variation over a 100-day growing season in a carrot diversity panel (N=695) that includes genetically diverse carrot accessions from the United States Department of Agriculture National Plant Germplasm System. We report phenotypic variability for shoot-growth characteristics, the first broad-sense heritability estimates for seedling emergence (0.68 < H2 < 0.80) and early-season canopy coverage ( 0.61 < H2 < 0.65), and consistent broad-sense heritability for late-season canopy height (0.76 < H2 < 0.82), indicating quantitative inheritance and potential for improvement through plant breeding. Strong correlation between emergence and canopy coverage (0.62 < r < 0.72) suggests that improvement of seedling emergence has great potential to increase yield and weed competitive ability. Accessions with high emergence and vigorous canopy growth are of immediate use to breeders targeting stand establishment, weed-tolerance, or weed-suppressant carrots, which is of particular advantage to the organic carrot production sector, reducing the costs and labor associated with herbicide application and weeding. We developed a standardized vocabulary and protocol to describe shoot-growth and facilitate collaboration and communication across carrot research groups. Our study facilitates identification and utilization of carrot genetic resources, conservation of agrobiodiversity, and development of breeding stocks for weed-competitive ability, with the long-term goal of delivering improved carrot cultivars to breeders, growers, and consumers. Accession selection can be further optimized for efficient breeding by combining shoot growth data with phenological data in this study's companion paper to identify ideotypes based on global market needs.

Advancing utilization of diverse global carrot (Daucus carota L.) germplasm with flowering habit trait ontology.

Biennial vegetable crops are challenging to breed due to long breeding cycle times. At the same time, it is important to preserve a strong biennial growth habit, avoiding premature flowering that renders the crop unmarketable. Gene banks carry important genetic variation which may be essential to improve crop resilience, but these collections are underutilized due to lack of characterization for key traits like bolting tendency for biennial vegetable crops. Due to concerns about introducing undesirable traits such as premature flowering into elite germplasm, many accessions may not be considered for other key traits that benefit growers, leaving crops more vulnerable to pests, diseases, and abiotic stresses. In this study, we develop a method for characterizing flowering to identify accessions that are predominantly biennial, which could be incorporated into biennial breeding programs without substantially increasing the risk of annual growth habits. This should increase the use of these accessions if they are also sources of other important traits such as disease resistance. We developed the CarrotOmics flowering habit trait ontology and evaluated flowering habit in the largest (N=695), and most diverse collection of cultivated carrots studied to date. Over 80% of accessions were collected from the Eurasian supercontinent, which includes the primary and secondary centers of carrot diversity. We successfully identified untapped genetic diversity in biennial carrot germplasm (n=197 with 0% plants flowering) and predominantly-biennial germplasm (n=357 with <15% plants flowering). High broad-sense heritability for flowering habit (0.81 < H2< 0.93) indicates a strong genetic component of this trait, suggesting that these carrot accessions should be consistently biennial. Breeders can select biennial plants and eliminate annual plants from a predominantly biennial population. The establishment of the predominantly biennial subcategory nearly doubles the availability of germplasm with commercial potential and accounts for 54% of the germplasm collection we evaluated. This subcollection is a useful source of genetic diversity for breeders. This method could also be applied to other biennial vegetable genetic resources and to introduce higher levels of genetic diversity into commercial cultivars, to reduce crop genetic vulnerability. We encourage breeders and researchers of biennial crops to optimize this strategy for their particular crop.

The first two chromosome-scale genome assemblies of American hazelnut enable comparative genomic analysis of the genus Corylus.

The native, perennial shrub American hazelnut (Corylus americana) is cultivated in the Midwestern United States for its significant ecological benefits, as well as its high-value nut crop. Implementation of modern breeding methods and quantitative genetic analyses of C. americana requires high-quality reference genomes, a resource that is currently lacking. We therefore developed the first chromosome-scale assemblies for this species using the accessions 'Rush' and 'Winkler'. Genomes were assembled using HiFi PacBio reads and Arima Hi-C data, and Oxford Nanopore reads and a high-density genetic map were used to perform error correction. N50 scores are 31.9 Mb and 35.3 Mb, with 90.2% and 97.1% of the total genome assembled into the 11 pseudomolecules, for 'Rush' and 'Winkler', respectively. Gene prediction was performed using custom RNAseq libraries and protein homology data. 'Rush' has a BUSCO score of 99.0 for its assembly and 99.0 for its annotation, while 'Winkler' had corresponding scores of 96.9 and 96.5, indicating high-quality assemblies. These two independent assemblies enable unbiased assessment of structural variation within C. americana, as well as patterns of syntenic relationships across the Corylus genus. Furthermore, we identified high-density SNP marker sets from genotyping-by-sequencing data using 1343 C. americana, C. avellana and C. americana × C. avellana hybrids, in order to assess population structure in natural and breeding populations. Finally, the transcriptomes of these assemblies, as well as several other recently published Corylus genomes, were utilized to perform phylogenetic analysis of sporophytic self-incompatibility (SSI) in hazelnut, providing evidence of unique molecular pathways governing self-incompatibility in Corylus.

Developing high-quality value-added cereals for organic systems in the US Upper Midwest: hard red winter wheat (Triticum aestivum L.) breeding.

There is an increased demand for food-grade grains grown sustainably. Hard red winter wheat has comparative advantages for organic farm rotations due to fall soil cover, weed competition, and grain yields. However, limitations of currently available cultivars such as poor disease resistance, winter hardiness, and baking quality, challenges its adoption and use. Our goal was to develop a participatory hard red winter wheat breeding program for the US Upper Midwest involving farmers, millers, and bakers. Specifically, our goals include (1) an evaluation of genotype-by-environment interaction (GEI) and genotypic stability for both agronomic and quality traits, and (2) the development of on-farm trials as well as baking and sensory evaluations of genotypes to include farmers, millers, and bakers' perspectives in the breeding process. Selection in early generations for diseases and protein content was followed by multi-environment evaluations for agronomic, disease, and quality traits in three locations during five years, on-farm evaluations, baking trials, and sensory evaluations. GEI was substantial for most traits, but no repeatable environmental conditions were significant contributors to GEI making selection for stability a critical trait. Breeding lines had similar performance in on-station and on-farm trials compared to commercial checks, but some breeding lines were more stable than the checks for agronomic, quality traits, and baking performance. These results suggest that stable lines can be developed using a participatory breeding approach under organic management. Crop improvement explicitly targeting sustainable agriculture practices for selection with farm to table participatory perspectives are critical to achieve long-term sustainable crop production. KEY MESSAGE: We describe a hard red winter wheat breeding program focused on developing genotypes adapted to organic systems in the US Upper Midwest for high-end artisan baking quality using participatory approaches.

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.

Beta vulgaris ssp. vulgaris chromosome 8 shows significant association with geosmin concentration in table beet.

Geosmin, a degraded sesquiterpene molecule with earthy and musty odor, imbues table beet with its characteristic aroma. Geosmin is heritable and endogenously produced in table beet; its earthy aroma is sought by some consumers but deters others. Geosmin biosynthesis is catalyzed by a bifunctional geosmin synthase enzyme in diverse bacteria and fungi, but a mechanism for geosmin biosynthesis in plants has not been reported. This work employed association analysis and selective genotyping of a segregating F2:3 mapping population to seek QTL associated with geosmin concentration in table beet. GBS reads were aligned to sugar beet reference genome EL10.2, and association analysis revealed two QTL for geosmin concentration on Beta vulgaris ssp. vulgaris chromosome 8. QTL at EL10.2 positions 28,017,624 and 38,488,687 each show effect size 8.7 µg·kg-1 geosmin and explain 8.5% and 6.4% of total variation in geosmin concentration, respectively. Resolution was low due to large recombination bin size and imperfect alignment between the reference genome and mapping population, but population size and selection proportion were sufficient to detect moderate to large effect QTL. This study, the first molecular genetic mapping experiment in table beet, succeeded in finding QTL for geosmin concentration in table beet, and it provides the basis for fine mapping or candidate gene investigation of functional loci for this distinctive sensory trait.

A Digital Image-Based Phenotyping Platform for Analyzing Root Shape Attributes in Carrot.

Root shape in carrot (Daucus carota subsp. sativus), which ranges from long and tapered to short and blunt, has been used for at least several centuries to classify carrot cultivars. The subjectivity involved in determining market class hinders the establishment of metric-based standards and is ill-suited to dissecting the genetic basis of such quantitative phenotypes. Advances in digital image acquisition and analysis has enabled new methods for quantifying sizes of plant structures and shapes, but in order to dissect the genetic control of the shape features that define market class in carrot, a tool is required that quantifies the specific shape features used by humans in distinguishing between classes. This study reports the construction and demonstration of the first such platform, which facilitates rapid phenotyping of traits that are measurable by hand, such as length and width, as well as principal component analysis (PCA) of the root contour and its curvature. This latter approach is of particular interest, as it enabled the detection of a novel and significant quantitative trait, defined here as root fill, which accounts for 85% of the variation in root shape. Curvature analysis was demonstrated to be an effective method for precise measurement of the broadness of the carrot shoulder, and degree of tip fill; the first principal component of the respective curvature profiles captured 87% and 84% of the total variance. This platform's performance was validated in two experimental panels. First, a diverse, global collection of germplasm was used to assess its capacity to identify market classes through clustering analysis. Second, a diallel mating design between inbred breeding lines of differing market classes was used to estimate the heritability of the key phenotypes that define market class, which revealed significant variation in the narrow-sense heritability of size and shape traits, ranging from 0.14 for total root size, to 0.84 for aspect ratio. These results demonstrate the value of high-throughput digital phenotyping in characterizing the genetic control of complex quantitative phenotypes.

SHiNeMaS: a web tool dedicated to seed lots history, phenotyping and cultural practices.

MOTIVATION: In 2005, researchers from the French National Research Institute for Agriculture, Food and Environment (Institut national de recherche pour l'agriculture, l'alimentation et l'environnement, INRAE) started a collaboration with the French farmers' seed network Réseau Semences Paysannes (RSP) on bread wheat participatory breeding (PPB). The aims were: (1) to study on-farm management of crop diversity, (2) to develop population-varieties adapted to organic and low-inputs agriculture, (3) to co-develop tools and methods adapted to on-farm experiments. In this project, researchers and farmers' organizations needed to map the history and life cycle of the population-varieties using network formalism to represent relationships between seed lots. All this information had to be centralized and stored in a database. RESULTS: We describe here SHiNeMaS (Seeds History and Network Management System) a web tool database. SHiNeMaS aims to provide useful interfaces to track seed lot history and related data (phenotyping, environment, cultural practices). Although SHiNeMaS has been developed in the context of a bread wheat participatory breeding program, the database has been designed to manage any kind and even multiple cultivated plant species. SHiNeMaS is available under Affero GPL licence and uses free technologies such as the Python language, Django framework or PostgreSQL database management system (DBMS). CONCLUSION: We developed SHiNeMaS, a web tool database, dedicated to the management of the history of seed lots and related data like phenotyping, environmental information and cultural practices. SHiNeMaS has been used in production in our laboratory for 5 years and farmers' organizations facilitators manage their own information in the system.

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.

Assessment and Accessibility of Phenotypic and Genotypic Diversity of Carrot (Daucus carota L. var. sativus) Cultivars Commercially Available in the United States.

Increased use of intellectual property rights over plant germplasm has led to a complicated landscape for exchange among plant breeders. Our goal was to examine phenotypic and genotypic diversity present in commercially available carrot (Daucus carota L. var. sativus) germplasm in relation to the freedom to operate-the ability for plant breeders to access and use crop genetic diversity. A collection of 140 commercially available carrot cultivars were grown in replicated field trials in the Madison, WI area in 2013 and 2014. Phenotypic measurements were recorded for leaf and root characteristics. Illumina sequencing was used to conduct genotyping by sequencing analysis on all cultivars to understand the range of genetic diversity present. Additionally, the intellectual property rights associated with each cultivar was noted to determine the freedom to operate. We found that although one-third of the commercially available US carrot cultivars in our study are restricted through some form of intellectual property rights, the genetic and phenotypic variability of the protected cultivars does not represent a completely separate group from the available material. Phenotypic analyses including ANOVA and principal components analysis, suggest that many of the traits differed significantly based on market class, but not by whether the cultivar had freedom to operate. The principal components and Fst analyses on the genotyping by sequencing data revealed that carrot market classes (Fst = 0.065) and freedom to operate classes (Fst = 0.023) were not genetically distinct, and that principle components 1 and 2 account for only 10.1% of the total genotypic variation, implying that cultivated carrot germplasm in the US forms an unstructured population. Our findings suggest that the genetic diversity present in carrot cultivars that have freedom to operate is potentially large enough to support carrot breeding efforts in most market classes given present levels of intellectual property protection.

On-farm dynamic management of genetic diversity: the impact of seed diffusions and seed saving practices on a population-variety of bread wheat.

Since the domestication of crop species, humans have derived specific varieties for particular uses and shaped the genetic diversity of these varieties. Here, using an interdisciplinary approach combining ethnobotany and population genetics, we document the within-variety genetic structure of a population-variety of bread wheat (Triticum aestivum L.) in relation to farmers' practices to decipher their contribution to crop species evolution. Using 19 microsatellites markers, we conducted two complementary graph theory-based methods to analyze population structure and gene flow among 19 sub-populations of a single population-variety [Rouge de Bordeaux (RDB)]. The ethnobotany approach allowed us to determine the RDB history including diffusion and reproduction events. We found that the complex genetic structure among the RDB sub-populations is highly consistent with the structure of the seed diffusion and reproduction network drawn based on the ethnobotanical study. This structure highlighted the key role of the farmer-led seed diffusion through founder effects, selection and genetic drift because of human practices. An important result is that the genetic diversity conserved on farm is complementary to that found in the genebank indicating that both systems are required for a more efficient crop diversity conservation.

Dynamic management of crop diversity: From an experimental approach to on-farm conservation.

In agricultural systems, biodiversity includes diversity within species and among species and provides many benefits for production, resilience and conservation. This article addresses the effects of a strategy of in situ conservation called dynamic management (DM) on population evolution, adaptation and diversity. Two French DM initiatives are considered, the first one corresponding to an experimental context, the second to an on-farm management. Results from a study over 26 years of experimental DM of bread wheat (Triticum aestivum L.) are first presented, including the evolution of agronomic traits and genetic diversity at neutral and fitness related loci. While this experiment greatly increased scientific knowledge of the effects of natural selection on cultivated populations, it also showed that population conservation cannot rely only on a network of experimental stations. In collaboration with a farmers' network in France, researchers have begun studying the effects of on-farm DM (conservation and selection) on diversity and adaptation. Results from these studies show that on-farm DM is a key element for the long-term conservation and use of agricultural biodiversity. This method of in situ conservation deserves more attention in industrialised countries.