Clizia Villano

Genetic diversity among potato species as revealed by phenotypic resistances and SSR markers

The evolutionary diversity of wild potato species makes them excellent materials for improving the narrow genetic basis of the cultivated potato Solanum tuberosum. Understanding their genetic diversity is important not only to choose the best parents for breeding, but also to design proper crossing schemes and selection strategies. The objectives of this study were to determine the resistance response to Ralstonia solanacearum, Potato virus Y and low temperatures of 21 clones of 12 potato species, and to determine their genetic diversity through simple sequence repeat (SSR) markers. Sources of resistance have been found for all the investigated traits, with high resistance variability not only between but also within species. Combined resistances were also identified, with positive implications for efficient breeding. SSR analysis allowed the detection of 12 loci and 46 alleles across all genotypes, with an average value of 3.8 alleles per locus. Both unique and rare alleles useful for marker-assisted selection were found. SSR-based cluster analysis revealed that resistant genotypes were distributed among all clusters, suggesting that genetically different resistant genotypes were identified. The information obtained in this study is discussed from a breeding perspective.

Use of SSR and Retrotransposon-Based Markers to Interpret the Population Structure of Native Grapevines from Southern Italy

Native grapevines are the quintessential elements of Southern Italy winemaking, and genomic characterization plays a role of primary importance for preservation and sustainable use of these unexploited genetic resources. Among the various molecular techniques available, SSR and retrotransposons-based markers result to be the most valuable for cultivars and biotypes distinctiveness. A total of 62 accessions including 38 local grape cultivars were analyzed with 30 SSR, four REMAP and one IRAP markers to assess their genetic diversity and obtain a complete genomic profiling. The use of VrZAG79, VrZAG112, VVS2, VVMD25 and VVMD5 combined with retrotransposon-based markers proved to be the most discriminating and polymorphic markers for the rapid and unambiguous identification of minority grapevines from Campania region, which is considered one of the most appreciated Italian districts for wine production. Results revealed 58 SSR marker-specific alleles, 22 genotype-specific SSR alleles, and four REMAP and IRAP private bands. Cases of synonymy and homonymy were discovered. In conclusion, we provided evidences that the integrating SSR and retrotransposon-based markers is an effective strategy to assess the genetic diversity of autochthonous grapes, allowing their easy identification.

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. Here, we describe the development of a molecular assay that uses single-copy conserved ortholog set II (COSII)-based single nucleotide polymorphisms (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 5 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 program or following transformation techniques.

Metabolic and RNA profiling elucidates proanthocyanidins accumulation in Aglianico grape

Aglianico grapes are known for their high content of proanthocyanidins (PAs), which are responsible for the astringency of wines derived from this cultivar. However, the accumulation of PAs and their genetic control during berry development remain largely unexplored. This work aimed to monitor astringency-causing PAs in Aglianico berries and correlate them with the expression of 14 key genes. Berries were collected during ripening and dissected in skins and seeds. PAs were fractionated and the content of total phenolics, flavans, anthocyanins, tannins reactive towards salivary proteins and tannin structural composition were evaluated. The results provided evidence that PAs were more abundant in seeds than in skins as expected, with differences in the structural composition between tissues, which did not varied during ripening. Expression analysis showed that Aglianico is able to accumulate polyphenols due to its ability to modulate key genes in a tissue-specific manner.

Dicer-like and RNA-dependent RNA polymerase gene family identification and annotation in the cultivated Solanum tuberosum and its wild relative S. commersonii

Main conclusionWe provide advances in DCL and RDR gene diversity in Solanaceae. We also shed light on DCL and RDR gene expression in response to cold stress.DICER-like (DCL) and RNA-dependent RNA polymerase (RDR) genes form the core components to trigger small non-coding RNA (ncRNA) production. In spite of this, little is known about the two gene families in non-model plant species. As their genome sequences are now available, the cultivated potato (Solanum tuberosum) and its cold-tolerant wild relative Solanum commersonii offer a valuable opportunity to advance our understanding of the above genes. To determine the extent of diversification and evolution of DCLs and RDRs in these species, we performed a comparative analysis. Seven DCLs were identified in the two species, whereas seven and six RDR genes were found in S. tuberosum and S. commersonii, respectively. Based on phylogenetic analysis with DCLs and RDRs from several species, we provide evidence for an increase in their number in both potato species. We also disclosed that tandem duplications played a major role in the evolution of these gene families in Solanaceae. DCL and RDR expression was investigated in different tissues and under cold and virus stresses, with divergent profiles of the tandem duplicated genes being found in different tissues. DCL paralogs showed a contrasting expression in S. tuberosum and S. commersonii following cold stress and virus infection. By contrast, no change in RDR transcript activity was detected following both stresses. Overall, this study provides the first comparative genomic analysis of the core components of the RNAi machinery in Solanaceae and offers a scaffold for future functional analysis of these gene families.

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity

Summary Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.

The Wild Side of Potato: Insights into the Genome Sequence of the Stress-Tolerant S. commersonii

Solanum commersonii is a potato species native to Central and South America. Despite being genetically isolated from cultivated potato, in the past few years it has garnered significant research interest because it exhibits high tolerance to both biotic and abiotic stresses. Among the abiotic stresses, particularly interesting are its freezing tolerance and capacity to cold acclimatize. Little is understood of the genetic determinants and mechanisms beyond its resistance traits. This is partially due to the lack of genomic resources for potato germplasm. The group at the University of Naples has recently decoded, for the first time, the genome of S. commersonii, ushering in a new era of whole-genome sequencing of wild potato relatives. After illustrating the genome structure and organization of this species and its intriguing evolutionary roots, this chapter describes findings relative to the identification of the candidate genes for cold stress tolerance. The genome sequence of S. commersonii will pave the way to an understanding of the molecular dynamics that have given this species so many adaptive characteristics.

Chipping ability, specific gravity and resistance to Pectobacterium carotovorum in advanced potato selections

ABSTRACT The availability of potato varieties combining good tuber quality and resistance traits is very important for both processors and supermarkets. In the present study, post-harvest quality traits as well as resistance to tuber bacterial soft rot were evaluated in 42 advanced potato clones belonging to seven hybrid families. Differences in specific gravity were found among families and among clones, with about 30% of clones showing a high specific gravity (>1.080). Clones and varieties were assayed also for a comprehensive chipping profile. Overall, 43% of clones presented an acceptable chipping score (≤4.5) at harvest. In addition, following cold storage with and without reconditioning, various clones performed acceptably. Five clones from three families (Bolestra X MC 329, Spunta X Victoria and Majestic X Alcmaria) were classified as resistant to bacterial soft rot. An evaluation index is proposed to help identify clones with interesting combinations of traits.