A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

Genomic distances reveal relationships of wild and cultivated beets.

Cultivated beets (Beta vulgaris ssp. vulgaris), including sugar beet, rank among the most important crops. The wild ancestor of beet crops is the sea beet Beta vulgaris ssp. maritima. Species and subspecies of wild beets are readily crossable with cultivated beets and are thus available for crop improvement. To study genomic relationships in the genus Beta, we sequence and analyse 606 beet genomes, encompassing sugar beet, sea beet, B. v. adanensis, B. macrocarpa, and B. patula. We observe two genetically distinct groups of sea beets, one from the Atlantic coast and the other from the Mediterranean area. Genomic comparisons based on k-mers identify sea beets from Greece as the closest wild relatives of sugar beet, suggesting that domestication of the ancestors of sugar beet may be traced to this area. Our work provides comprehensive insight into the phylogeny of wild and cultivated beets and establishes a framework for classification of further accessions of unknown (sub-)species assignment.

Genetic diversity among cultivated beets (Beta vulgaris) assessed via population-based whole genome sequences.

BACKGROUND: Diversification on the basis of utilization is a hallmark of Beta vulgaris (beet), as well as other crop species. Often, crop improvement and management activities are segregated by crop type, thus preserving unique genome diversity and organization. Full interfertility is typically retained in crosses between these groups and more traits may be accessible if the genetic basis of crop type lineage were known, along with available genetic markers to effect efficient transfer (e.g., via backcrossing). Beta vulgaris L. (2n =18) is a species complex composed of diverged lineages (e.g., crop types), including the familiar table, leaf (chard), fodder, and sugar beet crop types. Using population genetic and statistical methods with whole genome sequence data from pooled samples of 23 beet cultivars and breeding lines, relationships were determined between accessions based on identity-by-state metrics and shared genetic variation among lineages. RESULTS: Distribution of genetic variation within and between crop types showed extensive shared (e.g. non-unique) genetic variation. Lineage specific variation (e.g. apomorphy) within crop types supported a shared demographic history within each crop type, while principal components analysis revealed strong crop type differentiation. Relative contributions of specific chromosomes to genome wide differentiation were ascertained, with each chromosome revealing a different pattern of differentiation with respect to crop type. Inferred population size history for each crop type helped integrate selection history for each lineage, and highlighted potential genetic bottlenecks in the development of cultivated beet lineages. CONCLUSIONS: A complex evolutionary history of cultigroups in Beta vulgaris was demonstrated, involving lineage divergence as a result of selection and reproductive isolation. Clear delineation of crop types was obfuscated by historical gene flow and common ancestry (e.g. admixture and introgression, and sorting of ancestral polymorphism) which served to share genome variation between crop types and, likely, important phenotypic characters. Table beet was well differentiated as a crop type, and shared more genetic variation within than among crop types. The sugar beet group was not quite as well differentiated as the table beet group. Fodder and chard groups were intermediate between table and sugar groups, perhaps the result of less intensive selection for end use.

Determination of sucrose content in sugar beet by portable visible and near-infrared spectroscopy.

Visible and near-infrared spectra in interactance mode were acquired for intact and sliced beet samples, using two portable spectrometers for the spectral regions of 400-1100 nm and 900-1600 nm, respectively. Sucrose prediction models for intact and sliced beets were developed and then validated. The spectrometer for 400-1100 nm was able to predict the sucrose content with correlations of prediction (rp) of 0.80 and 0.88 and standard errors of prediction (SEPs) of 0.89% and 0.70%, for intact beets and beet slices, respectively. The spectrometer for 900-1600 nm had rp values of 0.74 and 0.88 and SEPs of 1.02% and 0.69% for intact beets and beet slices. These results showed the feasibility of using the portable spectrometer to predict the sucrose content of beet slices. Using simple correlation analysis, the study also identified important wavelengths that had strong correlation with the sucrose content.

The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway.

Nearly all flowering plants produce red/violet anthocyanin pigments. Caryophyllales is the only order containing families that replace anthocyanins with unrelated red and yellow betalain pigments. Close biological correlation of pigmentation patterns suggested that betalains might be regulated by a conserved anthocyanin-regulating transcription factor complex consisting of a MYB, a bHLH and a WD repeat-containing protein (the MBW complex). Here we show that a previously uncharacterized anthocyanin MYB-like protein, Beta vulgaris MYB1 (BvMYB1), regulates the betalain pathway in beets. Silencing BvMYB1 downregulates betalain biosynthetic genes and pigmentation, and overexpressing BvMYB1 upregulates them. However, unlike anthocyanin MYBs, BvMYB1 will not interact with bHLH members of heterologous anthocyanin MBW complexes because of identified nonconserved residues. BvMYB1 resides at the historic beet pigment-patterning locus, Y, required for red-fleshed beets. We show that Y and y express different levels of BvMYB1 transcripts. The co-option of a transcription factor regulating anthocyanin biosynthesis would be an important evolutionary event allowing betalains to largely functionally replace anthocyanins.

The beet R locus encodes a new cytochrome P450 required for red betalain production.

Anthocyanins are red and violet pigments that color flowers, fruits and epidermal tissues in virtually all flowering plants. A single order, Caryophyllales, contains families in which an unrelated family of pigments, the betalains, color tissues normally pigmented by anthocyanins. Here we show that CYP76AD1 encoding a novel cytochrome P450 is required to produce the red betacyanin pigments in beets. Gene silencing of CYP76AD1 results in loss of red pigment and production of only yellow betaxanthin pigment. Yellow betalain mutants are complemented by transgenic expression of CYP76AD1, and an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation. Finally, expression of CYP76AD1 in yeast verifies its position in the betalain biosynthetic pathway. Thus, this cytochrome P450 performs the biosynthetic step that provides the cyclo-DOPA moiety of all red betacyanins. This discovery will contribute to our ability to engineer this simple, nutritionally valuable pathway into heterologous species.

Functional differentiation of the sugar beet root system as indicator of developmental phase change.

Developmental phase transitions in the plant root system have not been well characterized. In this study we compared the dynamics of sucrose accumulation with changes in gene expression analyzed with cDNA-amplified fragment length polymorphism (AFLP) in the developing tap root of sugar beet (Beta vulgaris, L.) during the first 9 weeks after emergence (WAE). Although differences between lines were evident as soon as 9 WAE, sucrose showed a marked increase in the rate of accumulation between 4 and 6 WAE and a remarkable shift in gene expression was observed between 5 and 6 WAE. These changes were evident in two unrelated genetic backgrounds and suggest that physiological and gene expression changes represent a functional differentiation of the tap root. These changes were considered as indicators of a developmental change in the sugar beet root system. To identify genes and metabolic pathways involved in this developmental shift, a root cDNA library was hybridized with probes enriched for 3- and 7-WAE transcripts and differentially expressed transcripts were analyzed by cDNA microarray. Several genes involved in the regulation of tissue development were found to be differentially regulated. Genes involved in protein metabolism, disease-related and secretory system were upregulated before the functional differentiation transition, while genes under hormonal control were upregulated after the functional differentiation transition. This developmental phase change of the root system is important to understand plant developmental regulation at the whole-plant level and will likely be useful as early selection parameter in breeding programs.

Accumulation of vitamin E in potato (Solanum tuberosum) tubers.

Vitamin E (tocopherol) is a powerful antioxidant essential for human health and synthesized only by photosynthetic organisms. The effects of over-expression of tocopherol biosynthetic enzymes have been studied in leaves and seeds, but not in a non-photosynthetic, below-ground plant organ. Genetic and molecular approaches were used to determine if increased levels of tocopherols can be accumulated in potato (Solanum tuberosum L.) tubers through metabolic engineering. Two transgenes were constitutively over-expressed in potato: Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana homogentisate phytyltransferase (At-HPT). alpha-Tocopherol levels in the transgenic plants were determined by high-performance liquid chromatography. In potato tubers, over-expression of At-HPPD resulted in a maximum 266% increase in alpha-tocopherol, and over-expression of At-HPT yielded a 106% increase. However, tubers from transgenic plants still accumulated approximately 10- and 100-fold less alpha-tocopherol than leaves or seeds, respectively. The results indicate that physiological and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato tubers. Studying regulation and induction of tocopherol biosynthesis should reveal approaches to more effectively engineer crops with enhanced tocopherol content.

Sugarbeet sucrose synthase genes differ in organ-specific and developmental expression.

A full-length sucrose synthase (SBSS2) cDNA clone was isolated from sugarbeet. Comparison of its composition and organ-specific and developmental expression with a previously isolated sugarbeet sucrose synthase gene (SBSS1) revealed distinct differences between the two genes. The two genes share 80% similarity in deduced amino acid sequence but belong to different sucrose synthase subclasses based on phylogenic analysis. Both sucrose synthases were highly expressed in roots, and had low levels of expression in leaf tissue. Transcript abundance of SBSS2, relative to SBSS1, was greater in young vegetative and floral tissues, and reduced in mature vegetative tissues. The organ-specific and developmental expression of SBSS1 and SBSS2 proteins was similar to SBSS1 and SBSS2 transcript levels, although developmental changes in protein abundance lagged transcriptional changes by many weeks. The similarities and differences in transcript and protein abundance suggest that both transcriptional and post-transcriptional regulatory mechanisms are likely to contribute to sucrose synthase expression in sugarbeet.

Fluorometric sucrose evaluation for sugar beet.

Sucrose is the economic product from sugar beet. Disease resistance is often available in low-sucrose genotypes and, prior to the deployment of such novel genes as available into the cultivated spectrum, selection for increased sucrose content is required during introgression. The objective of this work was to evaluate a relatively rapid and inexpensive enzymatic-fluorometric microtiter plate assay for sucrose quantification in sugar beet root dry matter, both for progeny testing in the greenhouse and for evaluation of field-grown mother roots. As determined using HPLC, sucrose content in diverse populations of sugar and table beet assayed over various developmental stages ranged from 0.213 to 2.416 mmol g(-1) of dry matter, and these values were used as references for both refractometry and enzymatic-fluorometric assay. As expected, refractometric analysis generally overestimated sucrose content. Enzymatic-fluorometric analyses were reasonably well correlated with HPLC results for young greenhouse-grown root tissues (R2 = 0.976), and less so with older field-grown roots (R2 = 0.605), for unknown reasons. Enzymatic-fluorometric assays may be best deployed for progeny testing of young seedlings.

Cultivar-specific seedling vigor and expression of a putative oxalate oxidase germin-like protein in sugar beet (Beta vulgaris L.).

For genetic screening and breeding purposes, an in vitro germination system that reflects relative field emergence potential was used to screen for germination-enhancing and stress-induced genes from germinating seedlings from two varieties of sugar beet. Three full-length germin-like protein (GLP) gene classes were recovered from stress-germinated seedlings of a superior emerging variety. GLP gene expression, oxalate oxidase protein activity, the H(2)O(2) content of stressed seedlings, but not catalase activity, were induced by stress germination conditions (e.g. excess water, NaCl, mannitol, or oxalate) in a good emerging hybrid and were not induced in a poor emerging variety. Only one of the three germin-like protein genes ( BvGer165) was differentially regulated, and was induced only in the good emerger. Hydrogen peroxide promoted germination and partially compensated solute-depressed germination percentages. Unlike other solute recovery by hydrogen peroxide regimes, recovery in oxalic acid plus H(2)O(2) was cultivar-independent. A block in oxalate metabolism is postulated to contribute to lower germination under stress in the lower emerging variety. Selection for stress-induced germin expression, or for down-stream targets, presents the first direct target to enable breeding for improved field emergence of sugar beet.