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.

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.

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.

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.