Advances in the cytogenetics of Annonaceae, the case of Annona cherimola L.

Annonaceae represent the largest extant family among the early divergent angiosperms. Despite the long-standing interest in its evolutionary and taxonomic aspects, cytogenetic studies on this family remain extremely few even on economically important species. With this study, we realized a detailed characterization of the chromosomes of Annona cherimola (2n = 14) by a combination of in situ hybridization techniques, fluorochrome banding, and karyomorphological analysis. FISH revealed that 45S and 5S rDNA sites are co-localized in correspondence to the secondary constrictions of the SAT-chromosome pair. Some hypotheses on the organization of the linked 45S and 5S rDNA repeats have been made. FISH with Arabidopsis-type telomeric arrays demonstrated that the A. cherimola telomeres are constituted by TTTAGGG sequences and that they are exclusively localized at the extremities of chromosomes. An insight into the chromosome structure of A. cherimola was obtained by the self-GISH procedure which revealed highly repeated DNA sequences localized in the centromeric regions of all chromosomes. The correspondence of s-GISH signals with DAPI banding suggests that these sequences are the principal component of the centromeric heterochromatin of this species. The karyotype of A. cherimola here described is proposed as the basic reference karyotype for successive investigations in Annonaceae.

Evaluation of Cross-Species Transferability of SSR Markers in Foeniculum vulgare.

Fennel (Foeniculum vulgare) is a species belonging to the Apiaceae family, well known for its nutritional and pharmacological properties. Despite the economic and agricultural relevance, its genomic and transcriptomic data remain poor. Microsatellites-also known as simple sequence repeats (SSRs)-are codominant markers widely used to perform cross-amplification tests starting from markers developed in related species. SSRs represent a powerful tool, especially for those species lacking genomic information. In this study, a set of primers previously designed in Daucus carota for polymorphic SSR loci was tested in commercial varieties and breeding lines of fennel in order to: (i) test their cross-genera transferability, (ii) look at their efficiency in assessing genetic diversity, and (iii) identify their usefulness for marker-assisted selection (MAS) in breeding programs. Thirty-nine SSR markers from carrot were selected and tested for their transferability score, and only 23% of them resulted suitable for fennel. The low rate of SSR transferability between the two species evidences the difficulties of the use of genomic SSR in cross-genera transferability.

Genetic Characterization of the Apple Germplasm Collection in Central Italy: The Value of Local Varieties.

In the last 50 years, intensive farming systems have been boosted by modern agricultural techniques and newly bred cultivars. The massive use of few and related cultivars has dramatically reduced the apple genetic diversity of local varieties, confined to marginal areas. In Central Italy a limited spread of intensive fruit orchards has made it possible to preserve much of the local genetic diversity, but at the same time the coexistence of both modern and ancient varieties has generated some confusion. The characterization and clarification of possible synonyms, homonyms, and/or labeling errors in old local genetic resources is an issue in the conservation and management of living collections. 175 accessions provided by 10 apple collections, mainly local varieties, some of unknown origin, and well-known modern and ancient varieties, were studied by using 19 SSRs, analyzed by STRUCTURE, Ward's clustering and parentage analysis. We were able to identify 25 duplicates, 9 synonyms, and 9 homonyms. As many as 37 unknown accession were assigned to well known local or commercial varieties. Polyploids made up 20%. Some markers were found to be significantly correlated with morphological traits and the loci associated with the fruit over color were related to QTLs for resistance to biotic stresses, aroma compounds, stiffness, and acidity. In conclusion the gene pool of Central Italy seems to be rather consistent and highly differentiated compared with other European studies (F ST = 0.147). The importance of safeguarding this diversity and the impact on the management of the germplasm living collection is discussed.

An Insight into T-DNA Integration Events in Medicago sativa.

The molecular mechanisms of transferred DNA (T-DNA) integration into the plant genome are still not completely understood. A large number of integration events have been analyzed in different species, shedding light on the molecular mechanisms involved, and on the frequent transfer of vector sequences outside the T-DNA borders, the so-called vector backbone (VB) sequences. In this work, we characterized 46 transgenic alfalfa (Medicago sativa L.) plants (events), generated in previous works, for the presence of VB tracts, and sequenced several T-DNA/genomic DNA (gDNA) junctions. We observed that about 29% of the transgenic events contained VB sequences, within the range reported in other species. Sequence analysis of the T-DNA/gDNA junctions evidenced larger deletions at LBs compared to RBs and insertions probably originated by different integration mechanisms. Overall, our findings in alfalfa are consistent with those in other plant species. This work extends the knowledge on the molecular events of T-DNA integration and can help to design better transformation protocols for alfalfa.

Characterization and Phylogenetic Analysis of Ancient Italian Landraces of Pear.

Pear is one of the oldest fruit tree crops and the third most important temperate fruit species. Its domestication took place independently in the Far East (China) and in the Caucasus region. While the origin of Eastern Asian cultivars is clear, that of European cultivars is still in doubt. Italy has a wealth of local varieties and genetic resources safeguarded by several public and private collections to face the erosion caused by the introduction of improved varieties in specialized orchards. The objectives of the present study were: (i) to characterize the existing germplasm through nuclear (SSR) and (ii) to clarify the genetic divergence between local and cultivated populations through chloroplast DNA (cpDNA) markers in order to provide insights into phylogenetic relationships of Pyrus spp. For this reason, 95 entries from five different germplasm collections, including nine European, Mediterranean and Eastern Asian species, were analyzed, and the intergenic accD-psaI sequences were compared to the worldwide distributed dataset encompassing a total of 298 sequences from 26 different Pyrus species. The nine nuclear SSRs were able to identify a total of 179 alleles, with a loci polymorphism P = 0.89. Most of the variation (97%) was found within groups. Five accessions from different sources were confirmed to be the same. Eight out of 20 accessions of unknown origin were identified, and six synonyms were detected. Locus NH030a was found to be monomorphic in all the cultivated accessions and in reference species interfertile with P. communis, leading to hypothesize selection pressures for adaptation to cultivation. The cpDNA sequences of the 95 accessions were represented by 14 haplotypes, six of which (derived from P. communis, P. cossonii and P. ussuriensis) are recorded here for the first time and may suggest the ancient origin of some local varieties. The network analysis of the 298 cpDNA sequences allowed two different haplogroups, Eastern and Western Eurasia, to be defined, supporting recent views of a clear division between Occidental and Oriental species. By combining the results from nuclear and uniparental markers, it was possible to better define many unknown accessions.

Sexual Polyploidization in Medicago sativa L.: Impact on the Phenotype, Gene Transcription, and Genome Methylation.

Polyploidization as the consequence of 2n gamete formation is a prominent mechanism in plant evolution. Studying its effects on the genome, and on genome expression, has both basic and applied interest. We crossed two diploid (2n = 2x = 16) Medicago sativa plants, a subsp. falcata seed parent, and a coerulea × falcata pollen parent that form a mixture of n and 2n eggs and pollen, respectively. Such a cross produced full-sib diploid and tetraploid (2n = 4x = 32) hybrids, the latter being the result of bilateral sexual polyploidization (BSP). These unique materials allowed us to investigate the effects of BSP, and to separate the effect of intraspecific hybridization from those of polyploidization by comparing 2x with 4x full sib progeny plants. Simple sequence repeat marker segregation demonstrated tetrasomic inheritance for all chromosomes but one, demonstrating that these neotetraploids are true autotetraploids. BSP brought about increased biomass, earlier flowering, higher seed set and weight, and larger leaves with larger cells. Microarray analyses with M. truncatula gene chips showed that several hundred genes, related to diverse metabolic functions, changed their expression level as a consequence of polyploidization. In addition, cytosine methylation increased in 2x, but not in 4x, hybrids. Our results indicate that sexual polyploidization induces significant transcriptional novelty, possibly mediated in part by DNA methylation, and phenotypic novelty that could underpin improved adaptation and reproductive success of tetraploid M. sativa with respect to its diploid progenitor. These polyploidy-induced changes may have promoted the adoption of tetraploid alfalfa in agriculture.

Efficient, Antibiotic Marker-Free Transformation of a Dicot and a Monocot Crop with Glutamate 1-Semialdehyde Aminotransferase Selectable Marker Genes.

Antibiotic-free, efficient in vitro selection in plant genetic engineering can improve risk perception and speed up pre-market scrutiny of genetically modified crops. We provide a protocol for genetic transformation of two important crops, durum wheat and alfalfa, using a bacterial and a plant-derived selectable marker gene encoding mutated, gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA) enzymes. These methods can potentially be applied, with minor adaptations, to many other monocot and dicot crop plants.

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids.

KEY MESSAGE: A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.

Assessment of heat shock protein 70 induction by heat in alfalfa varieties and constitutive overexpression in transgenic plants.

Heat shock proteins (HSPs) are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock.

Occurrence of a number of enzymes involved in either gluconeogenesis or other processes in the pericarp of three cultivars of grape (Vitis vinifera L.) during development.

It is uncertain whether the enzymes pyruvate orthophosphate dikinase (PPDK) or isocitrate lyase (ICL) are present in the pericarp of grape, in which they could function in gluconeogenesis. The occurrence of these and other enzymes was investigated in the pericarp of three cultivars of grape (Vitis vinifera L.). In particular, the abundance of the enzymes aldolase, glutamine synthase (GS), acid invertase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC), PPDK and ICL were determined during the development of the pericarp of the cultivars Cabernet Sauvignon, Chardonnay and Zibibbo. PPDK and ICL were not detected at any stage of development. Each of the other enzymes showed different changes in abundance during development. However, for a given enzyme its changes in abundance were similar in each cultivar. In the ripe pericarp of Cabernet Sauvignon, PEPC, cytosolic GS and aldolase were equally distributed between the vasculature and parenchyma cells of the flesh and skin. The absence or very low abundance of PPDK provides strong evidence that any gluconeogenesis from malate utilises phosphoenolpyruvate carboxykinase (PEPCK). The absence or very low abundance of ICL in the pericarp precludes any gluconeogenesis from ethanol.

Population structure of barley landrace populations and gene-flow with modern varieties.

Landraces are heterogeneous plant varieties that are reproduced by farmers as populations that are subject to both artificial and natural selection. Landraces are distinguished by farmers due to their specific traits, and different farmers often grow different populations of the same landrace. We used simple sequence repeats (SSRs) to analyse 12 barley landrace populations from Sardinia from two collections spanning 10 years. We analysed the population structure, and compared the population diversity of the landraces that were collected at field level (population). We used a representative pool of barley varieties for diversity comparisons and to analyse the effects of gene flow from modern varieties. We found that the Sardinian landraces are a distinct gene pool from those of both two-row and six-row barley varieties. There is also a low, but significant, mean level and population-dependent level of introgression from the modern varieties into the Sardinian landraces. Moreover, we show that the Sardinian landraces have the same level of gene diversity as the representative sample of modern commercial varieties grown in Italy in the last decades, even within population level. Thus, these populations represent crucial sources of germplasm that will be useful for crop improvement and for population genomics studies and association mapping, to identify genes, loci and genome regions responsible for adaptive variations. Our data also suggest that landraces are a source of valuable germplasm for sustainable agriculture in the context of future climate change, and that in-situ conservation strategies based on farmer use can preserve the genetic identity of landraces while allowing adaptation to local environments.

Assessment of simple marker-free genetic transformation techniques in alfalfa.

Methods to avoid the presence of selectable marker genes (SMG) in transgenic plants are available but not implemented in many crop species. We assessed the efficiency of simple marker-free Agrobacterium-mediated transformation techniques in alfalfa: regeneration without selection, or marker-less, and co-transformation with two vectors, one containing the SMG and one containing a non-selected gene. To easily estimate the efficiency of marker-less transformation, the nptII and the GUS markers were used as non-selected genes. After Agrobacterium treatment, somatic embryos were regenerated without selection. The percentage of transgenic embryos was determined by a second cycle of regeneration using the embryos as starting material, in the presence of kanamycin, by PCR screening of T1 progenies, and by the GUS test. In two experiments, from 0 to 1.7% of the somatic embryos were transgenic. Co-transformation was performed with two vectors, one with the hemL SMG and one with the unselected nptII gene, each carried by a different culture of Agrobacterium. Only 15 putative co-transformed plants were regenerated from two experiments, with an average co-transformation percentage of 3.7. Southern blot hybridizations and/or T(1) progeny segregation were used to confirm transgene integration, and qPCR was also used to estimate the T-DNA copy number. In the T(1) progenies obtained by crossing with a non-transgenic pollinator, marker-free segregants were obtained. Both marker-free approaches showed very low efficiency.

Non-antibiotic, efficient selection for alfalfa genetic engineering.

A selectable marker gene (SMG), usually conferring resistance to an antibiotic or herbicide, is generally introduced into the plant cells with the gene(s) for the trait of interest to allow only the cells that have integrated and express the foreign sequences to regenerate into a plant. The availability of several SMGs for each plant species is useful for both basic and applied research to combine several genes of interest in the same plant. A selection system based on gabaculine (3-amino-2,3-dihydrobenzoic acid) as the selective substance and the bacterial hemL gene [encoding a mutant for of the enzyme glutamate 1-semialdehyde aminotransferase (GSA-AT)] as the SMG was previously used for genetic transformation of tobacco. The hemL gene is a good candidate for a safe SMG, because GSA-AT is present in all plants and is likely involved in one metabolic step only, so that unintended effects of its overexpression in plants are not probable. In this work, we have compared this new selection system with the conventional, kanamycin-based system for alfalfa Agrobacterium-mediated transformation. The hemL and NptII genes were placed together into a T-DNA under the control of identical promoters and terminators. We show that the gabaculine-based system is more efficient than the conventional, kanamycin-based system. The inheritance of hemL was Mendelian, and no obvious phenotypic effect of its expression was observed.