Genes and genome editing tools for breeding desirable phenotypes in ornamentals.

KEY MESSAGE: We review the main genes underlying commercial traits in cut flower species and critically discuss the possibility to apply genome editing approaches to produce novel variation and phenotypes. Promoting flowering and flower longevity as well as creating novelty in flower structure, colour range and fragrances are major objectives of ornamental plant breeding. The novel genome editing techniques add new possibilities to study gene function and breed new varieties. The implementation of such techniques, however, relies on detailed information about structure and function of genomes and genes. Moreover, improved protocols for efficient delivery of editing reagents are required. Recent results of the application of genome editing techniques to elite ornamental crops are discussed in this review. Enabling technologies and genomic resources are reviewed in relation to the implementation of such approaches. Availability of the main gene sequences, underlying commercial traits and in vitro transformation protocols are provided for the world's best-selling cut flowers, namely rose, lily, chrysanthemum, lisianthus, tulip, gerbera, freesia, alstroemeria, carnation and hydrangea. Results obtained so far are described and their implications for the improvement of flowering, flower architecture, colour, scent and shelf-life are discussed.

Genome-wide SNP discovery and population structure analysis in pepper (Capsicum annuum) using genotyping by sequencing.

BACKGROUND: Knowledge on population structure and genetic diversity in vegetable crops is essential for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Herein we used the GBS approach for the genome-wide identification of SNPs in a collection of Capsicum spp. accessions and for the assessment of the level of genetic diversity in a subset of 222 cultivated pepper (Capsicum annum) genotypes. RESULTS: GBS analysis generated a total of 7,568,894 master tags, of which 43.4% uniquely aligned to the reference genome CM334. A total of 108,591 SNP markers were identified, of which 105,184 were in C. annuum accessions. In order to explore the genetic diversity of C. annuum and to select a minimal core set representing most of the total genetic variation with minimum redundancy, a subset of 222 C. annuum accessions were analysed using 32,950 high quality SNPs. Based on Bayesian and Hierarchical clustering it was possible to divide the collection into three clusters. Cluster I had the majority of varieties and landraces mainly from Southern and Northern Italy, and from Eastern Europe, whereas clusters II and III comprised accessions of different geographical origins. Considering the genome-wide genetic variation among the accessions included in cluster I, a second round of Bayesian (K = 3) and Hierarchical (K = 2) clustering was performed. These analysis showed that genotypes were grouped not only based on geographical origin, but also on fruit-related features. CONCLUSIONS: GBS data has proven useful to assess the genetic diversity in a collection of C. annuum accessions. The high number of SNP markers, uniformly distributed on the 12 chromosomes, allowed the accessions to be distinguished according to geographical origin and fruit-related features. SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs.

The HIV-1 Pr55 gag polyprotein binds to plastidial membranes and leads to severe impairment of chloroplast biogenesis and seedling lethality in transplastomic tobacco plants.

Chloroplast genetic engineering has long been recognised as a powerful technology to produce recombinant proteins. To date, however, little attention has been given to the causes of pleiotropic effects reported, in some cases, as consequence of the expression of foreign proteins in transgenic plastids. In this study, we investigated the phenotypic alterations observed in transplastomic tobacco plants accumulating the Pr55(gag) polyprotein of human immunodeficiency virus (HIV-1). The expression of Pr55(gag) at high levels in the tobacco plastome leads to a lethal phenotype of seedlings grown in soil, severe impairment of plastid development and photosynthetic activity, with chloroplasts largely resembling undeveloped proplastids. These alterations are associated to the binding of Pr55(gag) to thylakoids. During particle assembly in HIV-1 infected human cells, the binding of Pr55(gag) to a specific lipid [phosphatidylinositol-(4-5) bisphosphate] in the plasma membrane is mediated by myristoylation at the amino-terminus and the so-called highly basic region (HBR). Surprisingly, the non-myristoylated Pr55(gag) expressed in tobacco plastids was likely able, through the HBR motif, to bind to nonphosphorous glycerogalactolipids or other classes of lipids present in plastidial membranes. Although secondary consequences of disturbed chloroplast biogenesis on expression of nuclear-encoded plastid proteins cannot be ruled out, results of proteomic analyses suggest that their altered accumulation could be due to retrograde control in which chloroplasts relay their status to the nucleus for fine-tuning of gene expression.

Transgene-induced pleiotropic effects in transplastomic plants.

Since the first demonstration of stable transgene integration in the plastid genome (plastome) of higher plants, plastid transformation has been used for a wide range of purposes, including basic studies as well as biotechnological applications, showing that transplastomic plants are an effective system to produce recombinant proteins. Compared to nuclear transformation, the main advantages of this technology are the high and stable production level of proteins as well as the natural containment of transgenes. To date, more than 100 transgenes have been successfully expressed in plant chloroplasts. In some cases, however, unintended pleiotropic effects on plant growth and physiology were shown in transplastomic plants. In this paper, we review such effects and discuss some of the technologies developed to overcome them.

Mitochondrial DNA variation in cultivated and wild potato species (Solanum spp.).

The European cultivated potato, Solanum tuberosum subsp. tuberosum, has 6 related cultivated species and more than 200 wild relatives. In Solanum spp., studies of cytoplasmic organelles have been mainly confined to the plastid DNA composition of cultivated and wild species. In this study, 53 genotypes of 30 potato species belonging to the subsections Estolonifera and Potatoe, 2 tomato species, and a black nightshade genotype were examined using PCR markers to evaluate mitochondrial DNA diversity and assess whether mtDNA variability was correlated with series classification, geographical origin, ploidy, and endosperm balance number (EBN). The markers used revealed interspecific mtDNA variability in Solanum spp. and identified 13 different haplotypes. Intraspecific variability was also observed in a few species and genomic regions. Cluster analysis allowed arrangement of the 13 haplotypes into 7 subgroups, and statistical association tests showed significant relationships between mitochondrial patterns detected by molecular analysis and ploidy, EBN, and geographical origin. On the whole, the evolutionary patterns for the genomic regions analyzed reflected the species relationships established on the basis of morphological and molecular (nuclear and plastidial DNA) data. The mtDNA variability shown is also important for better characterization of genetic resources for potato breeding.

Direct gene transfer in potato: a comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts.

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression.

The rpl5- rps14 mitochondrial region: a hot spot for DNA rearrangements in Solanum spp. somatic hybrids.

Southern analysis with rpl5 and rps14 mtDNA gene probes of Solanum tuberosum, S. commersonii and a sample of somatic hybrids detected polymorphisms between parents and the appearance of a novel restriction fragment in various hybrids. In one of them, detailed mtDNA analyses revealed various configurations of the rpl5- rps14 region present at different stoichiometries. Multiple inter-parental recombination events across homologous sequences were assumed to have caused these rearrangements. Sequence similarity searches detected one sequence putatively involved in the recombination upstream of the rpl5 gene. The presence of a second recombinogenic sequence was inferred. We propose two models to explain the mechanism responsible for obtaining the different rpl5- rps14 arrangements shown after somatic hybridization. Variability in the rpl5- rps14 region observed in both the parental species and their somatic hybrids suggests this region is a hot spot for mtDNA rearrangements in Solanum spp.

Organelle DNA variation in parental Solanum spp. genotypes and nuclear-cytoplasmic interactions in Solanum tuberosum (+) S. commersonii somatic hybrid-backcross progeny.

Nuclear-cytoplasmic interactions can influence fertility and agronomic performance of interspecific hybrids in potato as well as other species. With the aim of assessing the potential value of a novel recombinant cytoplasm derived by interspecific somatic hybridization, backcross progeny were produced by crossing a somatic hybrid between Solanum tuberosum (tbr) and the wild incongruous species S. commersonii (cmm) with various potato clones. BC1 clones were evaluated for male fertility and other agronomic traits. Male fertility clearly depended on the cross direction and the cytoplasm source. Genotypes with cytoplasms sensitive to nuclear genes derived from Solanum commersonii and inducing male sterility showed identical mtDNA composition, as based on mtDNA analyses with various PCR-based and RFLP markers. On the other hand, genotypes with cytoplasms not inducing male sterility in the presence of the cmm nuclear genes showed a different mtDNA organisation. Analysis of cpDNA confirmed similarity of cytoplasmic composition in CMS-inducing genotypes and clear differences with the others. Genotypes with recombinant cytoplasm induced by somatic hybridization generally showed similar agronomic performances in reciprocal hybrids with tbr cytoplasm, suggesting that the novel cytoplasm can be used in potato breeding.

Evidence for tetrasomic inheritance in a tetraploid Solanum commersonii (+) S. tuberosum somatic hybrid through the use of molecular markers.

In order to assess the potential for interspecific recombination between the cultivated Solanum tuberosum (tbr) and the sexually isolated wild species Solanum commersonii(cmm), genetic analysis of a F(2) progeny obtained by selfing one tetraploid cmm (+) tbr somatic hybrid was performed through molecular markers. For this purpose, the extent of disomic and/or tetrasomic inheritance of species-specific RAPD and AFLP markers was determined by following their segregation in a 90-genotype progeny, and testing all the possible segregation ratios in a selfed tetraploid progeny. The RAPD analysis performed using 16 primers revealed that the cmm-specific RAPDs were mainly (93.7%) duplex markers and were equally distributed between loci with a disomic (46.7%) and tetrasomic (53.3%) inheritance. The AFLP analysis led to the identification of 272 (58%) informative AFLPs, which were either cmm- or tbr-specific markers. About 63% of cmm-specific AFLPs were duplex loci, most of which (92.6%) were inherited as tetrasomic loci. As regards the tbr-specific AFLPs, the percentage of simplex loci (52.9%) was higher than that of duplex loci (32.6%), and among the latter most (88.5%) were inherited as tetrasomic loci. Overall, 130 duplex markers were found, of which 53.1% were cmm-specific and 46.9% were tbr-specific. Out of 130 markers, 18 (13.8%) were inherited as disomic, and 112 (86.2%) as tetrasomic, loci. This implies that the majority of duplex markers were located on chromosomes which at meiosis tend to randomly pair as bivalents or to form tetravalents. The total number of simplex loci was 119, and most of them (82.3%) were tbr-specific loci. In some cases the observed segregation ratios even allowed us to clearly determine whether a random chromosome or chromatid segregation was detected. This was the case of three cmm-specific RAPDs, 19 cmm- and 25 tbr-specific AFLPs, which fit a 20.8:1 or 2.5:1 ratio, both cases for which a clear random chromatid segregation can be assumed, since they represent the limit of segregation expected when the distance between the locus and the centromere always leads to a cross-over event. The percentage of ascertained crossing-over events was around 37% out of the tetrasomically inherited loci clearly identified (128 loci), a value indicating that the flow of genes from the sexually isolated S. commersonii to the cultivated potato is possible, for at least a large proportion of genes.

Characterization and expression of cold-induced glutathione S-transferase in freezing tolerant Solanum commersonii, sensitive S. tuberosum and their interspecific somatic hybrids.

Glutathione S-transferases (GST) form a large family of non-photosynthetic enzymes known to function in detoxification of xenobiotics. We have cloned and characterized a novel, low temperature regulated GST, Solanum commersonii glutathione S-transferase (Scgst1), from a cold acclimated wild potato species S. commersonii and studied the level of its transcription in freezing tolerant and sensitive Solanum genotypes. Active oxygen species (AOS) were associated with the early steps of Scgst1 regulation since a strong mRNA signal was detected in hydrogen peroxide and salicylic acid treated plants. In experimental conditions where the formation of AOS is known to accelerate, such as excessive light at low temperature, significant accumulation of the transcript was observed in S. commersonii. Under similar experimental conditions, Scgst1 transcript did not accumulate in freezing sensitive S. tuberosum eventhough a single copy of the Scgst1 sequence was present in both species. Thus, Scgst1 in the S. tuberosum genome did not exhibit the same cold-induction properties as in S. commersonii. In comparison with the parental lines, the somatic hybrid SH9A (S. commersonii (+) S. tuberosum) had an interparental level of Scgst1 accumulation as well as freezing tolerance. The abundance of Scgst1 transcript thus correlated well with the freezing tolerance of the parental lines and the somatic hybrid SH9A. Increased GST enzyme activity was observed in S. commersonii and SH9A after 2 days of cold acclimation whereas the activity declined in S. tuberosum during the same period. Further studies of potato lines (S1) that were derived by selfing the somatic hybrid revealed a more complex relationship between freezing tolerance and Scgst1 expression level. In the S1 genotypes, the regulation of Scgst1 transcription resembled more that of S. tuberosum and was not directly related to their freezing tolerance. This could be due to the interaction of the two genomes in S1 genotypes as well as chromosomal rearrangements during meiosis.

Endosperm balance number manipulation for direct in vivo germplasm introgression to potato from a sexually isolated relative (Solanum commersonii Dun.).

Diploid (2n = 2x = 24) Solanum species with endosperm balance number (EBN) = 1 are sexually isolated from diploid 2EBN species and both tetraploid (2n = 4x = 48, 4EBN) and haploid (2n = 2x = 24, 2EBN) S. tuberosum Group Tuberosum. To sexually overcome these crossing barriers in the diploid species S. commersonii (1EBN), the manipulation of the EBN was accomplished by scaling up and down ploidy levels. Triploid F1 hybrids between an in vitro-doubled clone of S. commersonii (2n = 4x = 48, 2EBN) and diploid 2EBN clones were successfully used in 3x x 4x crosses with S. tuberosum Group Tuberosum, resulting in pentaploid/near pentaploid BC1 progenies. This provided evidence of 2n (3x) egg formation in the triploid female parents. Two selected BC1 pentaploid hybrids were successfully backcrossed both as male and as female parents with S. tuberosum Group Tuberosum. The somatic chromosome number varied greatly among the resulting BC2 progenies, which included hyperaneuploids, but also a number (4.8%) of 48-chromosome plants. The introgression of S. commersonii genomes was confirmed by the presence of S. commersonii-specific randomly amplified polymorphic DNA markers in the BC2 population analyzed. The results clearly demonstrate the feasibility of germplasm introgression from sexually isolated diploid 1EBN species into the 4x (4EBN) gene pool of the cultivated potato using sexual hybridization. Based on the amount and type of genetic variation generated, cumbersomeness, general applicability, costs, and other factors, it would be interesting to compare the approach reported here with other in vitro or in vivo, direct or indirect, approaches previously reported.

Cremart: A new chemical for efficient induction of micronuclei in cells and protoplasts for partial genome transfer.

Results on efficient induction of micronuclei by Cremart in suspension cells and protoplasts of potato are reported. Cremart is a highly effective phosphoric amide herbicide, which acts on the mitotic spindle, and induces micronuclei through modification of mitosis. After treatment with Cremart, metaphase chromosomes changed directly into micronuclei without centromere division and chromatid separation. When suspension cells were treated with Cremart (3.7-15.0 µM) for 48h, and subsequently incubated in a mixture of cell wall-digesting enzymes in the presence of cytochalasin-B and Cremart for 18h, the frequency of micronucleation in the cell/protoplast mixture increased significantly, as compared to that obtained after treatment of suspension cells with Cremart (3.7-15.0 µM) for 48 h. Sieving after enzyme incubation resulted in the recovery of protoplasts, showing mass induction of micronuclei. Also synchronized suspension cells of Nicotiana plumbaginifolia responded with high frequency of micronucleation after Cremart (3.7 µM) treatment. The application of this procedure for partial genome transfer is discussed.

Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants.

Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic esearch on frost tolerance and cold-hardening is suggested.