Single Primer Enrichment Technology (SPET) for High-Throughput Genotyping in Tomato and Eggplant Germplasm

[EN] Single primer enrichment technology (SPET) is a new, robust, and customizable solution for targeted genotyping. Unlike genotyping by sequencing (GBS), and like DNA chips, SPET is a targeted genotyping technology, relying on the sequencing of a region flanking a primer. Its reliance on single primers, rather than on primer pairs, greatly simplifies panel design, and allows higher levels of multiplexing than PCR-based genotyping. Thanks to the sequencing of the regions surrounding the target SNP, SPET allows the discovery of thousands of closely linked, novel SNPs. In order to assess the potential of SPET for high-throughput genotyping in plants, a panel comprising 5k target SNPs, designed both on coding regions and introns/UTRs, was developed for tomato and eggplant. Genotyping of two panels composed of 400 tomato and 422 eggplant accessions, comprising both domesticated material and wild relatives, generated a total of 12,002 and 30,731 high confidence SNPs, respectively, which comprised both target and novel SNPs in an approximate ratio of 1:1.6, and 1:5.5 in tomato and eggplant, respectively. The vast majority of the markers was transferrable to related species that diverged up to 3.4 million years ago (Solanum pennellii for tomato and S. macrocarpon for eggplant). Maximum Likelihood phylogenetic trees and PCA outputs obtained from the whole dataset highlighted genetic relationships among accessions and species which were congruent with what was previously reported in literature. Better discrimination among domesticated accessions was achieved by using the target SNPs, while better discrimination among wild species was achieved using the whole SNP dataset. Our results reveal that SPET genotyping is a robust, high-throughput technology for genetic fingerprinting, with a high degree of cross-transferability between crops and their cultivated and wild relatives, and allows identification of duplicates and mislabeled accessions in genebanks.This work has been funded by the European Union's Horizon 2020 Research and Innovation Programme under the grant agreement number 677379 (G2P-SOL project: Linking genetic resources, genomes, and phenotypes of solanaceous crops).Barchi, L.; Acquadro, A.; Alonso-Martín, D.; Aprea, G.; Bassolino, L.; Demurtas, O.; Ferrante, P.... (2019). Single Primer Enrichment Technology (SPET) for High-Throughput Genotyping in Tomato and Eggplant Germplasm. Frontiers in Plant Science. 10:1-17. https://doi.org/10.3389/fpls.2019.01005S11710Acquadro, A., Barchi, L., Gramazio, P., Portis, E., Vilanova, S., Comino, C., … Lanteri, S. (2017). 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Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study

Abstract Background Chlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time. Results We constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci. Conclusions Due to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed

Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates

Heterologous expression of Bixa orellana cleavage dioxygenase 4–3 drives crocin but not bixin biosynthesis

Annatto (Bixa orellana) is a perennial shrub native to the Americas, and bixin, derived from its seeds, is a methoxylated apocarotenoid used as a food and cosmetic colorant. Two previous reports claimed to have isolated the carotenoid cleavage dioxygenase (CCD) responsible for the production of the putative precursor of bixin, the C24 apocarotenal bixin dialdehyde. We re-assessed the activity of six Bixa CCDs and found that none of them produced substantial amounts of bixin dialdehyde in Escherichia coli. Unexpectedly, BoCCD4-3 cleaved different carotenoids (lycopene, β-carotene, and zeaxanthin) to yield the C20 apocarotenal crocetin dialdehyde, the known precursor of crocins, which are glycosylated apocarotenoids accumulated in saffron stigmas. BoCCD4-3 lacks a recognizable transit peptide but localized to plastids, the main site of carotenoid accumulation in plant cells. Expression of BoCCD4-3 in Nicotiana benthamiana leaves (transient expression), tobacco (Nicotiana tabacum) leaves (chloroplast transformation, under the control of a synthetic riboswitch), and in conjunction with a saffron crocetin glycosyl transferase, in tomato (Solanum lycopersicum) fruits (nuclear transformation) led to high levels of crocin accumulation, reaching the highest levels (>100 µg/g dry weight) in tomato fruits, which also showed a crocin profile similar to that found in saffron, with highly glycosylated crocins as major compounds. Thus, while the bixin biosynthesis pathway remains unresolved, BoCCD4-3 can be used for the metabolic engineering of crocins in a wide range of different plant tissues

Additional file 7: of Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study

Table S1. Oligonucleotides used to screen Chlamydomonas transformants. Table S2. Oligonucleotides used to study the T-DNA deletion pattern. Table S3. Oligonucleotides used to study the influence of R and L borders on T-DNA rearrangements. Table S4. Luciferase activity data at the 3rd and 20th subcultures. Table S5. NGS library mapping statistics. (DOCX 75 kb

Additional file 5: of Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study

Figure S5. Deletion pattern on the T-DNA in the pAgroLucR transformants obtained though co-cultivation of Chlamydomonas with Agrobacterium cells transformed with the pAgroLucR plasmid. The figure shows respectively a PCR analysis of a set of 29 independent cw15 transformants obtained with C58C1 Agrobacterium cells carrying the pAgroLucR vector. Wt: cw15, P: pAgroLucR plasmid; C-: negative control. Oligonucleotide sequences are reported in Additional file 7: Table S3. (PPTX 1196 kb

Additional file 4: of Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study

Figure S4. PCR analysis on a set of nine independent transformants obtained through electroporation to study the deletion pattern along the T-DNA. Chlamydomonas cw15 cells were electroporated with the pAgroLucR plasmid. Six PCR reactions were performed on extracted DNA with nested pairs of oligonucleotides annealing in the T-DNA from the LB to the RB (Panel A). The results (Panel B) show that there is a gradient of deletions from the LB to the RB. β-tubulin was used as positive control for DNA extraction. M: 1 Kb Plus DNA Ladder (Life Technologies); wt: cw15 strain; P: pAgroLucR; −: negative control. Oligonucleotide sequences are reported in Additional file 7: Table S2. (PPTX 128 kb