Resistance to frost and tuber soft rot in near-pentaploid Solanum tuberosum - S. commersonii hybrids

The objectives of the present study were to evaluate the tolerance to low temperatures and tuber soft rot in sexual near-pentaploid hybrids between incongruent 2x (1EBN) Solanum commersonii (CMM) and 4x (4EBN) S. tuberosum (TBR). For freezing resistance, killing temperatures both under non-acclimated and un- der acclimated conditions were determined using the ion leakage procedure. Values for the hybrids were dis- tributed between the wild and cultivated parental values. Some hybrids displayed an acclimation capacity close to 2.5°C, typical of hardy species. Artificial inoculation of tubers with Pectobacterium carotovorum ssp. carotovorum (formerly Erwinia carotovora ssp. carotovora) provided evidence of variability in disease response. Highly resistant hybrids were identified. After conventional phenotypic selection, wild genome content was estimated based on the presence of CMM-specific AFLP fragments. Seven primer combinations were used (Eco-AGG/Mse-CAA; Eco-ACC/Mse-CAT; Eco-ACT/Mse-CAC; Eco-ACT/Mse-CAG; Eco- ACT/Mse-CAA; Eco-ACT/Mse-CAT; Eco-AGG/Mse-CAG). The percentages of CMM-specific AFLPs ranged from 4.3% to 56.7%, with an average value of 28.1%. AFLP analysis was employed for the selection of the hybrids to be used for further breeding objectives

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding.

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids

Resistance to Ralstonia Solanacearum of sexual hybrids between Solanum commersonii and S. tuberosum

This research was carried out to study the levels of bacterial wilt resistance and genetic diversity of (near) pentaploid sexual hybrids between S. commersonii (2n = 2x = 24, 1EBN) and cultivated S. tuberosum. Following artificial inoculations with Ralstonia solanacearum, wilting degree was estimated on a scale from 0 to 4, and seven genotypes of 26 (27%) displaying a S. commersonii like behavior were identified. Latent bacterial colonizations were detected in roots of symptomless S. commersonii and hybrids, whereas no bacterial populations were detected within stems. This suggests that the movement and/or growth of the bacterium in the aerial part were strongly inhibited. A molecular study with AFLP markers clustered hybrids into nine groups and provided evidence that resistant hybrids were slightly more similar to cultivated S. tuberosum than to the wild parent. This is important in view of the re-establishment of the cultivated genetic background through backcrosses. Hybrids displayed good fertility and are being used for further breeding efforts

Combined use of molecular markers and high resolution melting (HRM) to assess chromosome dosage in potato hybrids

In plants, the most widely used cytological techniques to assess parental genome contributions are based on in situ hybridization (FISH and GISH), but they are time-consuming and need specific expertise and equipment. Recent advances in genomics and molecular biology have made PCR-based markers a straightforward, affordable technique for chromosome typing. Herein we describe the development of a molecular assay that uses single-copy conserved ortholog set II (COSII)-based SNPs and the high resolution melting (HRM) technique to assess the chromosome dosage of interspecific hybrids between a Solanum phureja-S. tuberosum diploid (2n=2x=24) hybrid and its wild relative S. commersonii. Screening and analysis of 45 COSII marker sequences allowed S. commersonii-specific SNPs to be identified for all 12 chromosomes. Combining the HRM technique with the establishment of synthetic DNA hybrids, SNP markers were successfully used to predict the expected parental chromosome ratio of five interspecific triploid hybrids. These results demonstrate the ability of this strategy to distinguish diverged genomes from each other, and to estimate chromosome dosage. The method could potentially be applied to any species as a tool to assess paternal to maternal ratios in the framework of a breeding programme or following transformation techniques

Variation of DNA methylation and phenotypic traits following unilateral sexual polyploidization in Medicago

Sexual hybridization is an important generator of biodiversity and a powerful breeding tool. Hybridization can also overcome ploidy barriers when it involves 2n gametes, as in the case of unilateral sexual polyploidization (USP) that has been utilized in several crops, among which alfalfa. This research was aimed at gaining insights into the effects of USP on genome methylation and on phenotypic traits in alfalfa, an important forage species. The Methylation-Sensi- tive Amplified Polymorphism technique was used to estimate the cytosine methylation changes occurring in a tetraploid (2n = 4x = 32) USP progeny from crosses between a diploid Medicago sativa subsp. falcata genotype that produces 2n eggs and a cultivated tetraploid Medicago sativa subsp. sativa variety. De novo methylation or demethylation in the USP progeny were observed for 13% of the detected genomic sites, indicating that methylation changes can be relevant. USP plants showed larger surface area of the leaf epidermis cells than both parents, but this did not result in larger leaf size or higher plant biomass. They displayed significant higher ovule sterility than the tetraploid parent, but normal fertility was observed in crosses with unrelated male testers. We conclude that hybridization and sexual polyploidization resulted in novel variation in terms of remodeling of the methylation landscape as well as changes in phenotypic traits in alfalfa

Fertilization fitness and offspring ploidy in 3x x 2x matings in potato.

The main objective of the current research was to study the reproductive behaviour of artificial triploid potato hybrids between wild Solanum commersonii and the cultivated potato Solanum tuberosum. When used in 3x 6 2x crosses, triploids gave aneuploid progenies with somatic chromosome number ranging from 29 to 36. Fertilization fitness data suggested that the survival rate of gametes produced by the triploid parents may be related to their chromosome number. In addition, consistent with molecular data, our results indicated that fitness of gametes and chromosome number of progenies are influenced by the genome dosage of interspecific triploids. Since a main route to polyploidy formation is via 2n gametes and triploids, our study may contribute to a better understanding of polyploid plant reproduction, evolution and breeding

DNA-based technologies for grapevine biodiversity exploitation: state of the art and future perspectives

The cultivated grapevine, Vitis vinifera subsp. vinifera L., is represented by an enormous population of varieties and clones. They arise from the accumulation of gametic and somatic mutations during centuries of sexual and asexual propagation. These varieties represent a vast reservoir of traits/alleles that could be useful in improving the berry quality as well as against environmental stresses. However, most of them are still unexploited. For this reason, an efficient characterization system is essential to define the varietal identity, avoid cases of synonymy (identical genotypes but different names) and homonymy (same names but different genotypes) and deepen our understanding of the existing diversity within the grape germplasm. The plethora of DNA-based high-throughput technologies currently available provides promising tools for the analysis of diversity, overcoming many of the limitations of phenotypic-based diversity analyses. However, the analysis of intra-varietal diversity remains challenging. In this scenario, after summarizing the causes and consequences of grapevine genetic inter- and intra-varietal diversity, we review the DNA-based technologies used for varietal genotyping, emphasizing those able to distinguish clones within a variety. This review provides an update on the technologies used to explore grapevine diversity, the knowledge of which is necessary for an efficient exploitation and conservation of the grapevine germplasm

Genome-wide identification and analysis of candidate genes for disease resistance in tomato

Tomato (Solanum lycopersicum L.) has served as an important model system for studying the genetics and molecular basis of resistance mecha- nisms in plants. An unprecedented challenge is now to capitalize on the genetic and genomic achievements obtained in this species. In this study, we show that information on the tomato genome can be used predictively to link resistance function with specific sequences. An integrated genomic approach for identifying new resistance (R) gene candidates was developed. An R gene functional map was created by co-localization of candidate pathogen recognition genes and anchoring molecular markers associated with resistance phenotypes. In-depth characterization of the identified pathogen recognition genes was performed. Finally, in order to highlight expressed pathogen recognition genes and to provide a first step in validation, the tomato transcriptome was explored and basic molecular analyses were conducted. Such methodology can help to better direct positional cloning, reducing the amount of effort required to identify a functional gene. The resulting candidate loci selected are available for exploiting their specific function

Utilizzazione di marcatori molecolari SSR e AFLP per l'identificazione varietale in patata

Obiettivo. La tracciabilità dei prodotti alimentari tra- mite la caratterizzazione varietale delle produzioni agricole è uno degli aspetti di maggior rilievo nel cam- po della valorizzazione del patrimonio agroalimentare italiano e della tutela del consumatore. Le moderne tecniche di biologia molecolare offrono strumenti ana- litici di grande ef cacia nell’identi cazione varietale. Tra i prodotti caratteristici dell’agricoltura italiana, la patata precoce, coltivata tipicamente in Campania, Pu- glia, Sicilia e Sardegna, riveste un ruolo di primaria importanza, ma è oggetto di frodi alimentari tramite il supplemento di materiale proveniente dall’Africa set- tentrionale o da Cipro. L’obiettivo di questa ricerca è stato l’ottenimento di un ngerprinting molecolare di varietà di patata comunemente utilizzate per la produ- zione extrastagionale. Metodi. Il materiale genomico è stato estratto dai tu- beri di 22 varietà, raccolte nelle zone di origine, e ana- lizzato con otto microsatelliti e cinque combinazioni di primer AFLP. Risultati. Dal confronto dei pro li allelici è risultato che il numero minimo di loci SSR necessario per di- stinguere le varietà analizzate è stato cinque (STI0032, STG0001, STI0012, STM5127 e STM1106). L’analisi AFLP, invece, ha permesso di individuare 83 frammen- ti speci ci per le quattro varietà maggiormente colti- vate nei cicli extrastagionali e, in particolare, 34 per Sieglinde, 23 per Spunta, 15 per Elvira e 11 per Agria. Conclusione. In conclusione, è stato possibile svilup- pare nuovi marcatori molecolari speci ci di varietà di patata precoce, utili per la tracciabilità molecolare e per garantire la veridicità delle indicazioni presenti sulle etichette dei prodotti

AFLP analysis to assess genomic stability in Solanum regenerants derived from wild and cultivated species

The cultivated potato as well as its tuber-bearing relatives are considered model plants for cell and tissue culture, and therefore for exploiting the genetic variation induced by in vitro culture. The association between molecular stability and tissue culture in different genetic backgrounds and ploidy levels has already been explored. However, it still remains to be ascertained whether somaclonal variation differs between callus-derived chromosome- doubled and undoubled regenerants. Our research aimed at investigating, through amplified fragment length polymorphism (AFLP) markers, the genetic changes in marker- banding patterns of diploid and tetraploid regenerants obtained from one clone each of Solanum bulbocastanum Dunal and S. cardiophyllum Lindl (both 2n = 2x = 24) and tetraploids from cultivated S. tuberosum L. (2n = 4x = 48). Pairwise comparisons between the banding patterns of regenerants and parents allowed detecting considerable changes associated to in vitro culture both at diploid and tetraploid level. The percentages of polymorphic bands between diploid and tetraploid regenerants were, respectively, 57 and 69% in S. bulbocastanum and 58 and 63% in S. cardiophyllum. On average, the frequencies of lost parental fragments in regenerants were significantly higher than novel bands both in S. bulbocastanum (48 vs. 22%) and S. tuberosum (36 vs. 18%) regenerants. By contrast, in S. cardiophyllum, a similar incidence of the two events was detected (32 vs. 29%). Our results revealed that structural changes after tissue culture process strongly affected the genome of the species studied, but diploid and tetraploids regenerated plants responded equally