Annalisa Marchese

Morphological characteristics, microsatellite fingerprinting and determination of incompatibility genotypes of Sicilian sweet cherry cultivars

Summary Sicily has extensive germplasm of diploid sweet cherry (Prunus avium L.) that has not been well studied. In this investigation, 39 cherry accessions, selected from collections and farms, were analysed using molecular markers and characterised for various morphological and other agronomic characters such as flesh colour, fruit size, quality and, in some cases, ripening periods. Thirteen Simple Sequence Repeat (SSR) primer pairs, as well as two primer pairs for the incompatibility (S) locus, which amplified across the first intron of the S-RNase gene and across the intron of the SFB gene, were used in three multiplexed reactions to analyse the accessions. The number of alleles per SSR locus ranged from four to 11 (mean 7.2). Twelve S-alleles were found. Allele S4, which has been reported to be common in sweet cherry, was absent from the Sicilian germplasm and alleles S16 and S22 were frequent, although previously reported largely in wild populations. The accessions were assigned to their incompatibility groups. A UPGMA dendrogram was constructed and revealed possible synonyms. The set of SSR primers amplified unique fingerprints for 27 accessions, while twelve fell into six non-distinguishable groups. This approach could be used for comparing cultivars and wild accessions from other regions, and for establishing a database of this important species for breeding and conservation purposes. Several accessions useful for breeding are reported.

Genetic relationships, structure and parentage simulation among the olive tree (Olea europaea L. subsp. europaea) cultivated in Southern Italy revealed by SSR markers

In this work, we assess both the morphological and genetic diversity of 68 important olive cultivars from three Southern Italian regions: Calabria, Campania and Sicily. Twenty-five phenotypic traits were evaluated and 12 simple sequence repeat (SSR) markers were analysed. All SSR primers were polymorphic and reliable. The total number of alleles per locus varied from 5 to 19 with an average number of 13.1 and a mean polymorphic information content (PIC) of 0.81. These results suggested high genetic diversity within these three olive germplasm collections. Morphological traits also showed significant variability amongst cultivars. Two cases of identity were found and ten statistically significant cases of putative parent/sibling were discovered by performing a SSR-based parentage simulation analysis with CERVUS. The Mantel test indicated low but significant correlations between the morphological data and SSR allelic frequency, origin and SSR allelic frequency, and origin and morphology. Structure software allowed inference of relationships between the three olive germplasm collections and allowed us to obtain the most consistent grouping and to identify putative admixed or exchanged cultivars. Cluster and multivariate analysis, based on morphological traits, revealed geographic grouping in agreement with UPGMA dendrogram and structure analysis using SSRs. Sicilian cultivars showed a more homogenous genetic makeup, probably due to geographical isolation, whilst Calabrian and Campanian cultivars seemed to have a less distinct genetic structure, with a greater degree of intermixing. A correlation between the presence of certain SSR alleles and fruit size was also found.

Simple Sequence Repeat Marker Development and Mapping Targeted to Previously Unmapped Regions of the Strawberry Genome Sequence

The genome sequence of the woodland strawberry (Fragaria vesca L.) is an important resource providing a reference for comparative genomics studies and future sequenced rosaceous species and has great utility as a model for the development of markers for mapping in the cultivated strawberry Fragaria ×ananassa Duchesne ex Rozier. A set of 152 microsatellite simple sequence repeat (SSR) primer pairs was developed and mapped, along with 42 previously published but unmapped SSRs, permitting the precise assignment of 28.2 Mbp of previously unanchored genome sequence scaffolds (13% of the F. vesca genome sequence). The original ordering of F. vesca sequence scaffolds was performed without a physical map, using predominantly SSR markers to order scaffolds via anchoring to a comprehensive linkage map. This report complements and expands resolution of the Fragaria spp. reference map and refines the scaffold ordering of the F. vesca genome sequence using newly devised tools. The results of this study provide two significant resources: (i) the concurrent validation of a substantial set of SSRs associated with these previously unmapped regions of the Fragaria spp. genome and (ii) the precise placement of previously orphaned genomic sequence. Together, these resources improve the resolution and completeness of the strawberry genome sequence, making it a better resource for downstream studies in Fragaria spp. and the family Rosaceae.

Microspore embryogenesis induced through in vitro anther culture of almond (Prunus dulcis Mill.)

Anther culture is one of the most widely used methods to induce gametic embryogenesis. The aim of this investigation was to induce microspore embryogenesis in almond (Prunus dulcis Mill.), through this technique. Anthers were cultured at the vacuolated developmental stage, and seven cultivars, two culture media and two temperature treatments were assessed. Although evidence of the microspore induction was observed in all the genotypes and treatments tested (symmetrical nucleus division and multinucleated structures), calli were produced merely by anthers cultured in the medium P and the regeneration of embryos was detected only in anthers of the cultivars Filippo Ceo, Lauranne and Genco, placed on medium P and subjected to the Control treatment (direct culture at 25 ± 1 °C, without the hot thermal shock at 35 ± 1 °C for 7 days). Characterization by SSR marker analysis of the embryo genotypes revealed that the regenerants had a single allele for each locus whereas the parent cultivar was heterozygous, indicating their development from haploid microspores. This study reports the evidence of gametic embryogenesis and, particularly, of microspore embryogenesis through in vitro anther culture, in almond, and, for the first time to our knowledge, the production of homozygous embryos.

S-genotype identification, genetic diversity and structure analysis of Italian sweet cherry germplasm

In this study, 186 local sweet cherry accessions from 12 Italian regions, plus eight reference accessions, were analysed for the first time, using 13 microsatellite markers. Moreover, their S-incompatibility genotypes were identified with consensus primers for the S-RNase and SFB genes. A total of 161 unique genotypes were found; 18 groups of synonyms, along with the discovery of cases of misidentification. The average number of alleles per locus was 9.7, the mean expected heterozygosity (He) was 0.63, the mean observed heterozygosity (Ho) was 0.65 and the mean polymorphic information content (PIC) was 0.58. The structure analysis revealed the presence of six populations, which reflected in some cases geographical areas, the exchange of material among regions and introduction of material from abroad. A total of 17 different S-alleles were found, combined in 24 incompatibility groups of the 47 reported so far. Furthermore, 10 new incompatibility groups, from XLVII to LVI, were identified. Seven genotypes with unique S-allele combinations were included in the pollen donor group 0. The mutant allele of the pollen SFB5′ was found in early ripening genotypes from Sicily and Sardinia. The variability of SSRs present in both introns of the allele S13 was also explored; new combinations of variants were found and some accessions presented SSR variants typical of wild cherry. It is evident that the Italian sweet cherry germplasm collection represents a relevant source of genetic diversity that needs to be preserved for future breeding programmes.