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.

Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation.

KEY MESSAGE: We have developed and validated an efficient protocol for producing gene-edited carrot plants that do not result in the stable incorporation of foreign DNA in the edited plant's genome. We report here a method for producing transgene-free, gene-edited carrot (Daucus carota subs. sativus) plants. With this approach, PEG-mediated transformation is used to transiently express a cytosine base editor and a guide RNA in protoplasts to induce targeted mutations in the carrot genome. These protoplasts are then cultured under conditions that lead to the production of somatic embryos which subsequently develop into carrot plants. For this study, we used the Centromere-Specific Histone H3 (CENH3) gene as a target for evaluating the efficiency with which regenerated, edited plants could be produced. After validating sgRNA performance and protoplast transformation efficiency using transient assays, we performed two independent editing experiments using sgRNAs targeting different locations within CENH3. In the first experiment, we analyzed 184 regenerated plants and found that 22 of them (11.9%) carried targeted mutations within CENH3, while in the second experiment, 28 out of 190 (14.7%) plants had mutations in CENH3. Of the 50 edited carrot lines that we analyzed, 43 were homozygous or bi-allelic for mutations in CENH3. No evidence of the base editor expression plasmid was found in the edited lines tested, indicating that this approach is able to produce transgene-free, gene-edited lines. The protocol that we describe provides an efficient method for easily generating large numbers of transgene-free, gene-edited carrot plants.

Freeing Crop Genetics through the Open Source Seed Initiative.

For millennia, seeds have been freely available to use for farming and plant breeding without restriction. Within the past century, however, intellectual property rights (IPRs) have threatened this tradition. In response, a movement has emerged to counter the trend toward increasing consolidation of control and ownership of plant germplasm. One effort, the Open Source Seed Initiative (OSSI, www.osseeds.org), aims to ensure access to crop genetic resources by embracing an open source mechanism that fosters exchange and innovation among farmers, plant breeders, and seed companies. Plant breeders across many sectors have taken the OSSI Pledge to create a protected commons of plant germplasm for future generations.

Gender differences in the order of appearance of elbow ossification centres.

BACKGROUND: The predictable order of appearance of elbow secondary ossification centres in children is important in interpreting elbow radiographs, most commonly in the context of trauma. The usual sequence of appearance of these ossification centres is given by the acronym CRITOL, but exceptions have been described and are recognised as normal variants. AIM: To investigate the extent to which the appearance of the elbow secondary ossification centres follows a predictable sequence, and to establish the incidence of exceptions to the usual CRITOL order. METHODS: Two-stage retrospective review of 421 elbow radiographs in children at our centre. RESULTS: Of 64 children with two visible ossification centres, there was a 23% overall variant incidence (49 CR, 15 CI). Twenty-eight children had four visible ossification centres, with an overall variant incidence of 32% (19 CRIT, 9 CRIO). The variants were significantly more common in girls than in boys ( p < 0.001). CONCLUSION: Whilst the majority of radiographs remained consistent with the described CRITOL sequence, variance in this order was seen in a quarter of children with two visible ossification centres, and a third of children with four visible ossification centres. Incidence of variance was much more common in girls, reaching statistical significance. Awareness of these normal variations, particularly in girls, should help to avoid misinterpretation.

Subtyping cognitive profiles in Autism Spectrum Disorder using a Functional Random Forest algorithm.

DSM-5 Autism Spectrum Disorder (ASD) comprises a set of neurodevelopmental disorders characterized by deficits in social communication and interaction and repetitive behaviors or restricted interests, and may both affect and be affected by multiple cognitive mechanisms. This study attempts to identify and characterize cognitive subtypes within the ASD population using our Functional Random Forest (FRF) machine learning classification model. This model trained a traditional random forest model on measures from seven tasks that reflect multiple levels of information processing. 47 ASD diagnosed and 58 typically developing (TD) children between the ages of 9 and 13 participated in this study. Our RF model was 72.7% accurate, with 80.7% specificity and 63.1% sensitivity. Using the random forest model, the FRF then measures the proximity of each subject to every other subject, generating a distance matrix between participants. This matrix is then used in a community detection algorithm to identify subgroups within the ASD and TD groups, and revealed 3 ASD and 4 TD putative subgroups with unique behavioral profiles. We then examined differences in functional brain systems between diagnostic groups and putative subgroups using resting-state functional connectivity magnetic resonance imaging (rsfcMRI). Chi-square tests revealed a significantly greater number of between group differences (p < .05) within the cingulo-opercular, visual, and default systems as well as differences in inter-system connections in the somato-motor, dorsal attention, and subcortical systems. Many of these differences were primarily driven by specific subgroups suggesting that our method could potentially parse the variation in brain mechanisms affected by ASD.

Linkage mapping of root shape traits in two carrot populations.

This study investigated the genetic basis of carrot root shape traits using composite interval mapping in two biparental populations (n = 119 and n = 128). The roots of carrot F2:3 progenies were grown over 2 years and analyzed using a digital imaging pipeline to extract root phenotypes that compose market class. Broad-sense heritability on an entry-mean basis ranged from 0.46 to 0.80 for root traits. Reproducible quantitative trait loci (QTL) were identified on chromosomes 2 and 6 on both populations. Colocalization of QTLs for phenotypically correlated root traits was also observed and coincided with previously identified QTLs in published association and linkage mapping studies. Individual QTLs explained between 14 and 27% of total phenotypic variance across traits, while four QTLs for length-to-width ratio collectively accounted for up to 73% of variation. Predicted genes associated with the OFP-TRM (OVATE Family Proteins-TONNEAU1 Recruiting Motif) and IQD (IQ67 domain) pathway were identified within QTL support intervals. This observation raises the possibility of extending the current regulon model of fruit shape to include carrot storage roots. Nevertheless, the precise molecular mechanisms through which this pathway operates in roots characterized by secondary growth originating from cambium layers remain unknown.

Chromosome-level changes and genome elimination by manipulation of CENH3 in carrot (Daucus carota).

Hybrid cultivars are valuable in many crop species due to their high yield, uniformity, and other desirable traits. Doubled haploids, which have two identical sets of chromosomes, are valuable for hybrid breeding because they can be produced in one generation, in comparison to the multigenerational process typically used to produce inbred parents for hybrid production. One method to produce haploid plants is manipulation of centromeric histone H3 (CENH3). This method of producing haploids has so far been successful in Arabidopsis, maize (Zea mays), and wheat (Triticum aestivum). Here we describe modification of CENH3 in carrot (Daucus carota) to test for the ability of these modifications to induce uniparental genome elimination, which is the basis for haploid induction. Base editing was used to make cenh3 mutant plants with amino acid substitutions in the region of CENH3 encoding the histone fold domain. These cenh3 mutant plants were then outcrossed with CENH3 wild-type plants. Using PCR-based genotyping assays, we identified two candidates for genome elimination. One candidate was classified as a putative aneuploid plant in which chromosome 7 is in a single copy state. The other candidate was characterized as a putative tetraploid that was likely haploid during its genesis. Our results suggest that this putative tetraploid inherited all of its chromosomes from the CENH3 wild-type parent and that the genome of the cenh3 mutant plant was lost. This study provides evidence that modification of CENH3 in carrot has the potential to induce genome elimination and ploidy changes in carrot.

Form and contour: breeding and genetics of organ shape from wild relatives to modern vegetable crops.

Shape is a primary determinant of consumer preference for many horticultural crops and it is also associated with many aspects of marketing, harvest mechanics, and postharvest handling. Perceptions of quality and preference often map to specific shapes of fruits, tubers, leaves, flowers, roots, and other plant organs. As a result, humans have greatly expanded the palette of shapes available for horticultural crops, in many cases creating a series of market classes where particular shapes predominate. Crop wild relatives possess organs shaped by natural selection, while domesticated species possess organs shaped by human desires. Selection for visually-pleasing shapes in vegetable crops resulted from a number of opportunistic factors, including modification of supernumerary cambia, allelic variation at loci that control fundamental processes such as cell division, cell elongation, transposon-mediated variation, and partitioning of photosynthate. Genes that control cell division patterning may be universal shape regulators in horticultural crops, influencing the form of fruits, tubers, and grains in disparate species. Crop wild relatives are often considered less relevant for modern breeding efforts when it comes to characteristics such as shape, however this view may be unnecessarily limiting. Useful allelic variation in wild species may not have been examined or exploited with respect to shape modifications, and newly emergent information on key genes and proteins may provide additional opportunities to regulate the form and contour of vegetable crops.

Steam-cooking rapidly destroys and reverses onion-induced antiplatelet activity.

BACKGROUND: Foods in the diet that can aid in the prevention of diseases are of major interest. Onions are key ingredients in many cuisines around the world and moreover, onion demand has trended higher over the past three decades. An important pharmacological aspect of onion is the ability to inhibit platelet aggregation. Raw onions inhibit platelet aggregation; however, when onions are boiled or heated, antiplatelet activity may be abolished. METHODS: Onion quarters were steamed for 0, 1, 3, 6, 10, and 15 min. The in vitro antiplatelet activity of a yellow hybrid storage onion was examined at these times on the blood of 12 human subjects using in vitro whole blood aggregometry. RESULTS: Contrary to findings reported for boiling, antiplatelet activity was destroyed between 3 and 6 min of steaming, and at 10 min of steaming, cooked onions stimulated platelet activity. Extracts from cooked onion had the potential to reverse the inhibitory effect on blood platelets by 25%. Responses were consistent across all donors. Total polyphenolic concentration and soluble solids were not affected by steaming time. CONCLUSIONS: The potential value of cooked onion preparations may result in destruction or reversal of antiplatelet activity, without affecting the polyphenolic concentration.

Importance of mitochondrial haplotypes and maternal lineage in sprint performance among individuals of West African ancestry.

Mitochondrial DNA (mtDNA) is inherited solely along the matriline, giving insight into both ancestry and prehistory. Individuals of sub-Saharan ancestry are overrepresented in sprint athletics, suggesting a genetic advantage. The purpose of this study was to compare the mtDNA haplogroup data of elite groups of Jamaican and African-American sprinters against respective controls to assess any differences in maternal lineage. The first hypervariable region of mtDNA was haplogrouped in elite Jamaican athletes (N=107) and Jamaican controls (N=293), and elite African-American athletes (N=119) and African-American controls (N=1148). Exact tests of total population differentiation were performed on total haplogroup frequencies. The frequency of non-sub-Saharan haplogroups in Jamaican athletes and Jamaican controls was similar (1.87% and 1.71%, respectively) and lower than that of African-American athletes and African-American controls (21.01% and 8.19%, respectively). There was no significant difference in total haplogroup frequencies between Jamaican athletes and Jamaican controls (P=0.551 ± 0.005); however, there was a highly significant difference between African-American athletes and African-American controls (P<0.001). The finding of statistically similar mtDNA haplogroup distributions in Jamaican athletes and Jamaican controls suggests that elite Jamaican sprinters are derived from the same source population and there is neither population stratification nor isolation for sprint performance. The significant difference between African-American sprinters and African-American controls suggests that the maternal admixture may play a role in sprint performance.

Proposed Risk Stratification and Patterns of Radioactive Iodine Therapy in Malignant Struma Ovarii.

Introduction: Malignant struma ovarii (MSO) is a rare thyroid cancer arising within an ovarian teratoma. While surgical excision of the primary tumor is widely accepted as standard of care, recommendations for adjuvant treatment of MSO-whether or not to administer radioactive iodine (RAI)-are based largely on case reports and remain debated. In this study, we aimed to propose a risk stratification and analyze RAI utilization patterns in MSO cases. Methods: The National Cancer Database (NCDB) was queried for patients with MSO between 2004 and 2016. Demographic, oncological, and clinicopathologic data were compared between groups using Fisher's exact test. Kaplan-Meier curves were used to estimate overall survival (OS), and variables associated with OS were assessed via univariate Cox regression. We adapted the 2015 American Thyroid Association risk guidelines for MSO patients. We stratified patients into low-, intermediate-, and high-risk groups using metastasis, extraovarian extension, lymphovascular invasion, lymph node status, surgical margins, tumor size, and grade. Risk stratification, demographic, oncological, and clinicopathologic data were compared between the groups receiving and not receiving RAI therapy. We then queried the Surveillance, Epidemiology, and End Results (SEER) 18 registry for patients with MSO between 2000 and 2018 to confirm our risk stratification analysis. Results: In the NCDB analysis, a total of 158 patients were identified, and 19 received RAI. RAI therapy was associated with distant metastasis (p = 0.005) and lymph node status (p = 0.012). Twenty-one NCDB patients were stratified as high risk, and 30% of high-risk patients received RAI. High-risk stratification was associated with decreased OS via univariate Cox regression (hazard ratio = 4.0 [95% confidence interval 1.11-14.26], p = 0.034). In our subsequent analysis using the SEER registry, there were 95 MSO patients, and 18 received RAI. Again, the majority of high-risk patients did not receive RAI, with only 41% of high-risk patients receiving RAI. Conclusions: MSO is a rare malignancy with apparently variable and inconsistent patterns of postoperative RAI administration. The risk stratification described here provides a framework to identify patients potentially at risk for mortality, and utilization of RAI in this group should be studied further.

Evolution of Root Morphology in Table Beet: Historical and Iconographic.

The Beta vulgaris complex includes sugar beet, mangel wurzel, Swiss chard, fodder beet, and table beet. Mangel wurzel and fodder beet are considered to be the same general crop type, with the former possessing lower dry matter content (<13%) than the latter. Mangel is likely derived from crosses between table beet and chard, while fodder beet may have a more recent origin, arising from crosses between mangel and sugarbeet. The table beet was derived from the wild sea beet, B. vulgaris (L.) subsp. maritima (L.) Arcang, with small non-spherical roots. Table beet is presently a popular vegetable cultivated for its pigmented roots, typically red but also yellow and other colors. Wild forms were consumed in antiquity mainly for their leaves with roots used medicinally. Beet is referred to in the Septuagint, a Greek translation of the first five books of the Hebrew bible, made in Ptolomeic Egypt in the third century BCE. A beet identified as Beta maritima is included in De Material Medicus of Pedanius Dioscorides written in the first century CE, and the first illustrated version of 512, known as the Juliana Anicia Codex, includes an image with non-spherical root. Beet is mentioned in several tractates of the Talmud, a sixth century collection of history and civil law written in Babylonia. Beta maritima possesses supernumerary root cambia, which facilitated selection of swollen rooted forms. The first colored illustration of swollen rooted table beet, B. vulgaris, can be found in the 1515-1517 frescos of Raphael Sanzio and Giovanni Martina da Udine in the Villa Farnesina in Rome. Swollen roots in Roman beet are illustrated and described in the 1587 French herbal Historia Generalis Plantarum of Jacques Dalechamps. Conically shaped beet roots are found in the market painting of Franz Snijders in the 17th century. Various spherical forms of beet root are found in the work of American painter James Peale in 1826. A complete array of beet root types is found in the Benary catalog of 1876. Modern, spherical beet roots were depicted in 1936 by the Russian painter Zinaida Serebriankov, 1936. Artistic and historical representations of table beet suggest that swollen rooted forms have existed during the past five centuries, but conically shaped roots were gradually replaced by spherically shaped roots during this period.

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.

Human chorionic gonadotrophin producing epithelioid sarcoma metastatic to the placenta.

Pregnancy complicated by sarcoma is a rare event, and metastasis to the placenta is exceptionally rare. Of reported malignancies involving the products of conception, only 8% are sarcomas. We report the first case of placental metastasis of an epithelioid sarcoma, an uncommon malignancy which most frequently affects young adults. In addition to the typical histological features and immunophenotypic profile expected of epithelioid sarcoma, this tumor also expressed human chorionic gonadotrophin (HCG). Although uncharacteristic, the production of HCG has been previously reported in other nontrophoblastic tumors. It may have contributed to the pathogenesis of the tumor metastatic to the placenta in this case.

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.

Genetic variation and selection response in model breeding populations of Brassica rapa following a diversity bottleneck.

Domestication and breeding share a common feature of population bottlenecks followed by significant genetic gain. To date, no crop models for investigating the evolution of genetic variance, selection response, and population diversity following bottlenecks have been developed. We developed a model artificial selection system in the laboratory using rapid-cycling Brassica rapa. Responses to 10 cycles of recurrent selection for cotyledon size were compared across a broad population founded with 200 individuals, three bottleneck populations initiated with two individuals each, and unselected controls. Additive genetic variance and heritability were significantly larger in the bottleneck populations prior to selection and this corresponded to a heightened response of bottleneck populations during the first three cycles. However, the overall response was ultimately greater and more sustained in the broad population. AFLP marker analyses revealed the pattern and extent of population subdivision were unaffected by a bottleneck even though the diversity retained in a selection population was significantly limited. Rapid gain in genetically more uniform bottlenecked populations, particularly in the short term, may offer an explanation for why domesticators and breeders have realized significant selection progress over relatively short time periods.

William Friedman, Geneticist Turned Cryptographer.

William Friedman (1891-1969), trained as a plant geneticist at Cornell University, was employed at Riverbank Laboratories by the eccentric millionaire George Fabyan to work on wheat breeding. Friedman, however, soon became intrigued by and started working on a pet project of Fabyan's involving the conjecture that Francis Bacon, a polymath known for the study of ciphers, was the real author of Shakespeare's plays. Thus, beginning in ∼1916, Friedman turned his attention to the so called "Baconian cipher," and developed decryption techniques that bore similarity to approaches for solving problems in population genetics. His most significant, indeed pathbreaking, work used ideas from genetics and statistics, focusing on analysis of the frequencies of letters in language use. Although he had transitioned from being a geneticist to a cryptographer, his earlier work had resonance in his later pursuits. He soon began working directly for the United States government and produced solutions used to solve complex military ciphers, in particular to break the Japanese Purple code during World War II. Another important legacy of his work was the establishment of the Signal Intelligence Service and eventually the National Security Agency.