The biological feasibility and social context of gene-edited, caffeine-free coffee.

Coffee, especially the species Coffea arabica and Coffea canephora, is one of the world's most consumed beverages. The consumer demand for caffeine-free coffee is currently being met through chemical decaffeination processes. However, this method leads to loss of beverage quality. In this review, the feasibility of using gene editing to produce caffeine-free coffee plants is reviewed. The genes XMT (7-methylxanthosine methyltransferase) and DXMT (3,7-dimethylxanthine methyltransferase) were identified as candidate target genes for knocking out caffeine production in coffee plants. The possible effect of the knock-out of the candidate genes was assessed. Using Agrobacterium tumefaciens-mediated introduction of the CRISPR-Cas system to Knock out XMT or DXMT would lead to blocking caffeine biosynthesis. The use of CRISPR-Cas to genetically edit consumer products is not yet widely accepted, which may lead to societal hurdles for introducing gene-edited caffeine-free coffee cultivars onto the market. However, increased acceptance of CRISPR-Cas/gene editing on products with a clear benefit for consumers offers better prospects for gene editing efforts for caffeine-free coffee.

Genome editing of polyploid crops: prospects, achievements and bottlenecks.

Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.

Exploring the alpha-gliadin locus: the 33-mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii.

Most alpha-gliadin genes of the Gli-D2 locus on the D genome of hexaploid bread wheat (Triticum aestivum) encode for proteins with epitopes that can trigger coeliac disease (CD), and several contain a 33-mer peptide with six partly overlapping copies of three epitopes, which is regarded as a remarkably potent T-cell stimulator. To increase genetic diversity in the D genome, synthetic hexaploid wheat lines are being made by hybridising accessions of Triticum turgidum (AB genome) and Aegilops tauschii (the progenitor of the D genome). The diversity of alpha-gliadins in A. tauschii has not been studied extensively. We analysed the alpha-gliadin transcriptome of 51 A. tauschii accessions representative of the diversity in A. tauschii. We extracted RNA from developing seeds and performed 454 amplicon sequencing of the first part of the alpha-gliadin genes. The expression profile of allelic variants of the alpha-gliadins was different between accessions, and also between accessions of the Western and Eastern clades of A. tauschii. Generally, both clades expressed many allelic variants not found in bread wheat. In contrast to earlier studies, we detected the 33-mer peptide in some A. tauschii accessions, indicating that it was introduced along with the D genome into bread wheat. In these accessions, transcripts with the 33-mer peptide were present at lower frequencies than in bread wheat varieties. In most A. tauschii accessions, however, the alpha-gliadins do not contain the epitope, and this may be exploited, through synthetic hexaploid wheats, to breed bread wheat varieties with fewer or no coeliac disease epitopes.

CRISPR/Cas9 Gene Editing of Gluten in Wheat to Reduce Gluten Content and Exposure-Reviewing Methods to Screen for Coeliac Safety.

Ingestion of gluten proteins (gliadins and glutenins) from wheat, barley and rye can cause coeliac disease (CD) in genetically predisposed individuals. The only remedy is a strict and lifelong gluten-free diet. There is a growing desire for coeliac-safe, whole-grain wheat-based products, as consumption of whole-grain foods reduces the risk of chronic diseases. However, due to the large number of gluten genes and the complexity of the wheat genome, wheat that is coeliac-safe but retains baking quality cannot be produced by conventional breeding alone. CD is triggered by immunogenic epitopes, notably those present in α-, γ-, and ω-gliadins. RNA interference (RNAi) silencing has been used to down-regulate gliadin families. Recently, targeted gene editing using CRISPR/Cas9 has been applied to gliadins. These methods produce offspring with silenced, deleted, and/or edited gliadins, that overall may reduce the exposure of patients to CD epitopes. Here we review methods to efficiently screen and select the lines from gliadin gene editing programs for CD epitopes at the DNA and protein level, for baking quality, and ultimately in clinical trials. The application of gene editing for the production of coeliac-safe wheat is further considered within the context of food production and in view of current national and international regulatory frameworks.

Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families.

BACKGROUND: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. RESULTS: In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. CONCLUSIONS: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.

Food processing and breeding strategies for coeliac-safe and healthy wheat products.

A strict gluten-free diet is currently the only treatment for the 1-2% of the world population who suffer from coeliac disease (CD). However, due to the presence of wheat and wheat derivatives in many food products, avoiding gluten consumption is difficult. Gluten-free products, made without wheat, barley or rye, typically require the inclusion of numerous additives, resulting in products that are often less healthy than gluten-based equivalents. Here, we present and discuss two broad approaches to decrease wheat gluten immunogenicity for CD patients. The first approach is based on food processing strategies, which aim to remove gliadins or all gluten from edible products. We find that several of the candidate food processing techniques to produce low gluten-immunogenic products from wheat already exist. The second approach focuses on wheat breeding strategies to remove immunogenic epitopes from the gluten proteins, while maintaining their food-processing properties. A combination of breeding strategies, including mutation breeding and possibly genome editing, will be necessary to produce coeliac-safe wheat. Individuals suffering from CD and people genetically susceptible who may develop CD after prolonged gluten consumption would benefit from reduced CD-immunogenic wheat. Although the production of healthy and less CD-toxic wheat varieties and food products will be challenging, increasing global demand may require these issues to be addressed in the near future by food processing and cereal breeding companies.

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

KEY MESSAGE: Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

Opportunities for Products of New Plant Breeding Techniques.

Various new plant breeding techniques (NPBT) have a similar aim, namely to produce improved crop varieties that are difficult to obtain through traditional breeding methods. Here, we review the opportunities for products created using NPBTs. We categorize products of these NPBTs into three product classes with a different degree of genetic modification. For each product class, recent examples are described to illustrate the potential for breeding new crops with improved traits. Finally, we touch upon the future applications of these methods, such as cisgenic potato genotypes in which specific combinations of Phytophthora infestans resistance genes have been stacked for use in durable cultivation, or the creation of new disease resistances by knocking out or removing S-genes using genome-editing techniques.

Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

Cisgenic apple trees; development, characterization, and performance.

Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.

Regeneration and transformation of Crambe abyssinica.

BACKGROUND: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. RESULTS: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L(-1) were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L(-1) kanamycin, 25 mg · L(-1), then back to 10 mg · L(-1)) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L(-1)) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion. CONCLUSIONS: Present research revealed the potential of using crambe meristematic tissue for genetic transformation and in vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.

Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (Fragaria × ananassa) fruits.

Strawberry (Fragaria × ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein). A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators. Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3∆). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts. Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.

One-step Agrobacterium mediated transformation of eight genes essential for rhizobium symbiotic signaling using the novel binary vector system pHUGE.

Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.

The mQTL hotspot on linkage group 16 for phenolic compounds in apple fruits is probably the result of a leucoanthocyanidin reductase gene at that locus.

BACKGROUND: Our previous study on ripe apples from a progeny of a cross between the apple cultivars 'Prima' and 'Fiesta' showed a hotspot of mQTLs for phenolic compounds at the top of LG16, both in peel and in flesh tissues. In order to find the underlying gene(s) of this mQTL hotspot, we investigated the expression profiles of structural and putative transcription factor genes of the phenylpropanoid and flavonoid pathways during different stages of fruit development in progeny genotypes. RESULTS: Only the structural gene leucoanthocyanidin reductase (MdLAR1) showed a significant correlation between transcript abundance and content of metabolites that mapped on the mQTL hotspot. This gene is located on LG16 in the mQTL hotspot. Progeny that had inherited one or two copies of the dominant MdLAR1 alleles (Mm, MM) showed a 4.4- and 11.8-fold higher expression level of MdLAR1 respectively, compared to the progeny that had inherited the recessive alleles (mm). This higher expression was associated with a four-fold increase of procyanidin dimer II as one representative metabolite that mapped in the mQTL hotspot. Although expression level of several structural genes were correlated with expression of other structural genes and with some MYB and bHLH transcription factor genes, only expression of MdLAR1 was correlated with metabolites that mapped at the mQTL hotspot. MdLAR1 is the only candidate gene that can explain the mQTL for procyanidins and flavan-3-ols. However, mQTLs for other phenylpropanoids such as phenolic esters, dihydrochalcones and flavonols, that appear to map at the same locus, have so far not been considered to be dependent on LAR, as their biosynthesis does not involve LAR activity. An explanation for this phenomenon is discussed. CONCLUSIONS: Transcript abundances and genomic positions indicate that the mQTL hotspot for phenolic compounds at the top of LG16 is controlled by the MdLAR1 gene. The dominant allele of the MdLAR1 gene, causing increased content of metabolites that are potentially health beneficial, could be used in marker assisted selection of current apple breeding programs and for cisgenesis.

Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples.

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (P(MdRbc)) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar 'Gala' was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. 'Santana'.

Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines.

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. 'Elstar' and 'Gala' in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable.

Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria x ananassa).

An octaploid (Fragaria x ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria x ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens. Out of 25 transgenic lines, nine lines showed a reduction in CHS mRNA accumulation of more than 50% as compared to the untransformed cv. Calypso control. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from one to four. The pigmentation of the fruit was only affected when less than 5% of the control CHS expression level was detected. A stable antisense phenotype over a period of 4 years was obtained in the primary transgenic lines at a rate of 1:20. As a consequence of the reduced activity of CHS, the levels of anthocyanins, flavonols, and proanthocyanidins were downregulated and precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway leading to highly increased levels of cinnamoyl glucose (520% of control), caffeoyl glucose (816% of control), and feruloyl glucose (1092% of control) as well as p-coumaryl alcohol (363% of control) and p-coumaryl-1-acetate (1079% of control), which occur only as trace components in untransformed control fruits. These results demonstrate that the introduction of an antisense CHS construct in strawberry results in an unpredictable biochemical phenotype, thereby confirming that CHS function is an important regulatory point of substrate flow between the flavonoid and the phenylpropanoid pathways.

Quantification of allele-specific expression of a gene encoding strawberry polygalacturonase-inhibiting protein (PGIP) using Pyrosequencing.

Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated the transcript abundance of six individual, highly homologous alleles of a polygalacturonase-inhibiting protein gene (FaPGIP) from octoploid strawberry (Fragaria x ananassa). We applied the highly quantitative Pyrosequencing method which, for the gene under study, detected allele frequency differences as small as 4.0 +/- 2.8%. Pyrosequencing of RT-PCR products showed that one FaPGIP allele was preferentially expressed in leaf tissue, while two other alleles were expressed in a fruit-specific way. For fruits that were inoculated with Botrytis cinerea a strong increase in overall FaPGIP gene expression was observed. This upregulation was accompanied by a significant change in FaPGIP allele frequencies when compared with non-treated fruits. Remarkably, in the five cultivars tested, the allele frequency in cDNA from the inoculated fruits was similar to that in genomic DNA, suggesting uniform upregulation of all FaPGIP alleles present as a result of pathogenesis-related stress. The results demonstrate that when Pyrosequencing of RT-PCR products is performed, novel allele-specific gene regulation can be detected and quantified.

Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene.

Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. We describe the development of an effective transformation system for generating such marker-free transgenic plants, without the need for repeated transformation or sexual crossing. This system combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. The described system can be generally applied to existing transformation protocols, and was tested in strawberry using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events. Fully marker-free transgenic strawberry plants were obtained following two different selection/regeneration strategies.