Comparison of DNA extraction methods on different sample matrices within the same terrestrial ecosystem.

Metataxonomic studies of ecosystem microbiotas require the simultaneous processing of samples with contrasting physical and biochemical traits. However, there are no published studies of comparisons of different DNA extraction kits to characterize the microbiotas of the main components of terrestrial ecosystems. Here, and to our knowledge for the first time, five DNA extraction kits were used to investigate the composition and diversity of the microbiota of a subset of samples typically studied in terrestrial ecosystems such as bulk soil, rhizosphere soil, invertebrate taxa and mammalian feces. DNA extraction kit was associated with changes in the relative abundance of hundreds of ASVs, in the same samples, resulting in significant differences in alpha and beta diversity estimates of their microbiotas. Importantly, the impact of DNA extraction kit on sample diversity varies according to sample type, with mammalian feces and soil samples showing the most and least consistent diversity estimates across DNA extraction kits, respectively. We show that the MACHEREY-NAGEL NucleoSpin® Soil kit was associated with the highest alpha diversity estimates, providing the highest contribution to the overall sample diversity, as indicated by comparisons with computationally assembled reference communities, and is recommended to be used for any large-scale microbiota study of terrestrial ecosystems.

Gut microbiota variations in wild yellow baboons (Papio cynocephalus) are associated with sex and habitat disturbance.

Although male and female mammals differ in biological traits and functional needs, the contribution of this sexual dimorphism to variations in gut bacteria and fungi (gut microbiota) in relation to habitat type has not been fully examined. To understand whether the combination of sex and habitat affects gut microbiota variation, we analyzed 40 fecal samples of wild yellow baboons (Papio cynocephalus) living in contrasting habitat types (intact, well-protected vs. fragmented, less protected forests) in the Udzungwa Mountains of Tanzania. Sex determination was performed using the marker genes SRY (Sex-determining Region Y) and DDX3X-DDX3Y (DEAD-Box Helicase 3). Samples were attributed to 34 individuals (19 females and 15 males) belonging to five social groups. Combining the results of sex determination with two amplicon sequencing datasets on bacterial (V1-V3 region of the 16S rRNA gene) and fungal (ITS2) gut communities, we found that overall, baboon females had a significantly higher gut bacterial richness compared to males. Beta diversity estimates indicated that bacterial composition was significantly different between males and females, and this was true for individuals from both well- and less protected forests. Our results highlight the combined role of sex and habitat type in shaping variation in gut microbial communities in wild non-human primates.

A multidisciplinary approach to tackling invasive species: barcoding, morphology, and metataxonomy of the leafhopper Arboridia adanae.

The leafhopper genus Arboridia includes several species that feed on Vitis vinifera and cause leaf chlorosis. We report the first alien Arboridia infestation in Italy in 2021 in an Apulian vineyard. To confirm the taxonomic status of the species responsible for crop damage, and reconstruct its demographic history, we barcoded individuals from Apulia together with Arboridia spp. from Crete (Greece), A. adanae from Central Turkey and other specimens of the presumed sister species, A. dalmatina from Dalmatia (Croatia). Molecular phylogenies and barcoding gap analysis identified clades not associated with sampling locations. This result is incongruent with classical specimen assignment and is further supported by morphological analyses, which did not reveal significant differences among the populations. Therefore, we propose A. dalmatina as a junior synonym of A. adanae, which would become the only grapevine-related Arboridia species in the eastern Mediterranean. To further characterise A. adanae evolution, we performed a molecular clock analysis that suggested a radiation during the Pleistocene glaciations. Finally, to assess whether the Apulian individuals carried microorganisms of agricultural relevance, we sequenced their bacterial microbiota using 16S rRNA amplicon sequencing identifying three phytopathogens not generally associated with Arboridia activities as well as Wolbachia in one Apulian haplogroup. We discuss the agricultural implications of this infestation.

Mock community as an in situ positive control for amplicon sequencing of microbiotas from the same ecosystem.

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Currently available protocols for metataxonomy assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. It has been suggested that the addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases during processing and support direct comparisons of microbiota composition, but the impact of MC on diversity estimates of samples is unknown. Here, large and small aliquots of pulverized bovine fecal samples were extracted with no, low or high doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We demonstrated that sample diversity estimates were distorted only if MC dose was high compared to sample mass (i.e. when MC > 10% of sample reads). We also showed that MC was an informative in situ positive control, permitting an estimation of the sample 16S copy number, and detecting sample outliers. We tested this approach on a range of sample types from a terrestrial ecosystem, including rhizosphere soil, whole invertebrates, and wild vertebrate fecal samples, and discuss possible clinical applications.

A validated protocol for eDNA-based monitoring of within-species genetic diversity in a pond-breeding amphibian.

In light of the dramatic decline in amphibian biodiversity, new cost-efficient tools to rapidly monitor species abundance and population genetic diversity in space and time are urgently needed. It has been amply demonstrated that the use of environmental DNA (eDNA) for single-species detection and characterization of community composition can increase the precision of amphibian monitoring compared to traditional (observational) approaches. However, it has been suggested that the efficiency and accuracy of the eDNA approach could be further improved by more timely sampling; in addition, the quality of genetic diversity data derived from the same DNA has been confirmed in other vertebrate taxa, but not amphibians. Given the availability of previous tissue-based genetic data, here we use the common frog Rana temporaria Linnaeus, 1758 as our target species and an improved eDNA protocol to: (i) investigate differences in species detection between three developmental stages in various freshwater environments; and (ii) study the diversity of mitochondrial DNA (mtDNA) haplotypes detected in eDNA (water) samples, by amplifying a specific fragment of the COI gene (331 base pairs, bp) commonly used as a barcode. Our protocol proved to be a reliable tool for monitoring population genetic diversity of this species, and could be a valuable addition to amphibian conservation and wetland management.

Correction: Transcript Analysis and Regulative Events during Flower Development in Olive (Olea europaea L.).

[This corrects the article DOI: 10.1371/journal.pone.0152943.].

Regulation of Fruit Growth in a Peach Slow Ripening Phenotype.

Consumers' choices are mainly based on fruit external characteristics such as the final size, weight, and shape. The majority of edible fruit are by tree fruit species, among which peach is the genomic and genetic reference for Prunus. In this research, we used a peach with a slow ripening (SR) phenotype, identified in the Fantasia (FAN) nectarine, associated with misregulation of genes involved in mesocarp identity and showing a reduction of final fruit size. By investigating the ploidy level, we observed a progressive increase in endoreduplication in mesocarp, which occurred in the late phases of FAN fruit development, but not in SR fruit. During fruit growth, we also detected that genes involved in endoreduplication were differentially modulated in FAN compared to SR. The differential transcriptional outputs were consistent with different chromatin states at loci of endoreduplication genes. The impaired expression of genes controlling cell cycle and endocycle as well as those claimed to play a role in fruit tissue identity result in the small final size of SR fruit.

Population structure and evolution of resistance to acetolactate synthase (ALS)-inhibitors in Amaranthus tuberculatus in Italy.

BACKGROUND: Before 2010, Amaranthus tuberculatus (Moq.) J. D. Sauer was barely known to farmers and stakeholders in Italy. Since then, several populations resistant to acetolactate synthase (ALS)-inhibiting herbicides have been collected. In most populations, a known target site resistance-endowing mutation was found, a Trp to Leu substitution at position 574 of the ALS gene, but it was unclear whether they had evolved resistance independently or not. The aims of the work were (i) to elucidate the population structure of Italian ALS-resistant A. tuberculatus populations, and (ii) to analyze the ALS haplotypes of the various populations to determine whether resistance arose multiple times independently. RESULTS: In order to determine the population structure of eight A. tuberculatus populations, eight previously described microsatellite loci were used. Two ancestors were found: three populations derived from one, and five from the other. In the 4-kb ALS region of the genome, including the 2-kb coding region, 389 single nucleotide polymorphisms were found. In silico haplotype estimation was used to reconstruct the sequence of three distinct haplotypes carrying the Trp574Leu mutation. In addition, no mutation was found in 83% of plants of a single population. CONCLUSIONS: (i) Resistance must have arisen independently at least three times; (ii) at least one population was already resistant to ALS inhibitors when introduced in Italy; (iii) a single haplotype with a Trp574Leu mutation was shared among six populations, probably because of broad seed dispersal; and (iv) one population likely evolved nontarget site ALS inhibitors resistance. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Molecular Determination of Hybridity and Homozygosity Estimates in Breeding Populations of Lettuce (Lactuca sativa L.).

The development of new varieties of horticultural crops benefits from the integration of conventional and molecular marker-assisted breeding schemes in order to combine phenotyping and genotyping information. In this study, a selected panel of 16 microsatellite markers were used in different steps of a breeding programme of lettuce (Lactuca sativa L., 2 n = 18). Molecular markers were first used to genotype 71 putative parental lines and to plan 89 controlled crosses designed to maximise recombination potentials. The resulting 871 progeny plants were then molecularly screened, and their marker allele profiles were compared with the profiles expected based on the parental lines. The average cross-pollination success rate was 68 ± 33%, so 602 F1 hybrids were completely identified. Unexpected genotypes were detected in 5% of cases, consistent with this species' spontaneous out-pollination rate. Finally, in a later step of the breeding programme, 47 different F3 progenies, selected by phenotyping for a number of morphological descriptors, were characterised in terms of their observed homozygosity and within-population genetic uniformity and stability. Ten of these populations had a median homozygosity above 90% and a median genetic similarity above 95% and are, therefore, particularly suitable for pre-commercial trials. In conclusion, this study shows the synergistic effects and advantages of conventional and molecular methods of selection applied in different steps of a breeding programme aimed at developing new varieties of lettuce.

Ovule Gene Expression Analysis in Sexual and Aposporous Apomictic Hypericum perforatum L. (Hypericaceae) Accessions.

Hypericum perforatum L. (2n = 4x = 32) is an attractive model system for the study of aposporous apomixis. The earliest phenotypic features of aposporous apomixis in this species are the mitotic formation of unreduced embryo sacs from a somatic cell of the ovule nucellus and the avoidance of meiosis. In this research we addressed gene expression variation in sexual and apomictic plants, by focusing on the ovule nucellus, which is the cellular domain primarily involved into the differentiation of meiocyte precursors and aposporous embryo sacs, at a pre-meiotic developmental stage. Gene expression analyses performed by RNAseq identified 396 differentially expressed genes and 1834 transcripts displaying phenotype-specific expression. Furthermore, the sequencing and assembly of the genome from a diploid sexual accession allowed the annotation of a 50 kb sequence portion located upstream the HAPPY locus and to address the extent to which single transcripts were assembled in multiple variants and their co-expression levels. About one third of identified DEGs and phenotype-specific transcripts were associated to transcript variants with alternative expression patterns. Additionally, considering DEGs and phenotype-specific transcript, the co-expression level was estimated in about two transcripts per locus. Our gene expression study shows massive differences in the expression of several genes encoding for transposable elements. Transcriptional differences in the ovule nucellus and pistil terminal developmental stages were also found for subset of genes encoding for potentially interacting proteins involved in pre-mRNA splicing. Furthermore, the sexual and aposporous ovule transcriptomes were characterized by differential expression in genes operating in RNA silencing, RNA-mediated DNA methylation (RdDM) and histone and chromatin modifications. These findings are consistent with a role of these processes in regulating cell fate determination in the ovule, as indicated by forward genetic studies in sexual model species. The association between aposporous apomixis, pre-mRNA splicing and DNA methylation mediated by sRNAs, which is supported by expression data and by the enrichment in GO terms related to these processes, is consistent with the massive differential expression of multiple transposon-related sequences observed in ovules collected from both sexual and aposporous apomictic accessions. Overall, our data suggest that phenotypic expression of aposporous apomixis is concomitant with the modulation of key genes involved in the two interconnected processes: RNA splicing and RNA-directed DNA methylation.

Annotation and Expression of IDN2-like and FDM-like Genes in Sexual and Aposporous Hypericum perforatum L. accessions.

The protein IDN2, together with the highly similar interactors FDM1 and FDM2, is required for RNA-directed DNA methylation (RdDM) and siRNA production. Epigenetic regulation of gene expression is required to restrict cell fate determination in A. thaliana ovules. Recently, three transcripts sharing high similarity with the A. thaliana IDN2 and FDM1-2 were found to be differentially expressed in ovules of apomictic Hypericum perforatum L. accessions. To gain further insight into the expression and regulation of these genes in the context of apomixis, we investigated genomic, transcriptional and functional aspects of the gene family in this species. The H. perforatum genome encodes for two IDN2-like and 7 FDM-like genes. Differential and heterochronic expression of FDM4-like genes was found in H. perforatum pistils. The involvement of these genes in reproduction and seed development is consistent with the observed reduction of the seed set and high variability in seed size in A. thaliana IDN2 and FDM-like knockout lines. Differential expression of IDN2-like and FDM-like genes in H. perforatum was predicted to affect the network of potential interactions between these proteins. Furthermore, pistil transcript levels are modulated by cytokinin and auxin but the effect operated by the two hormones depends on the reproductive phenotype.

Karyological Analysis and DNA Barcoding of Pompia Citron: A First Step toward the Identification of Its Relatives.

Pompia is a citrus fruit endemic of Sardinia, Italy, with an essential oil profile showing outstanding anti-inflammatory and anti-microbic properties. Despite its remarkable pharmaceutical potential, little taxonomic and genetic information is available for this species. We applied flow cytometry and classical cytogenetic techniques to assess the DNA content and to reconstruct the karyotype of several Pompia accessions. Molecular data from plastid DNA barcoding and nuclear DNA sequencing were used to study the genetic distance between Pompia and other citrus species. Flow cytometric estimates of DNA content and somatic chromosome counts suggest that Pompia is a regular diploid Citrus species. DNA polymorphisms of nuclear and chloroplast markers allowed us to investigate the genetic relationships between Pompia accessions and other Citrus species. Based on DNA polymorphism data we propose that Pompia is a very recent interspecific hybrid generated by a cross between C. aurantium (as seed bearer) and C. medica (as pollen donor). Our findings pave the way for further and more specific investigations of local Pompia germplasm resources that may help the preservation and valorisation of this valuable citrus fruit tree.

A Portion of the Apomixis Locus of Paspalum Simplex is Microsyntenic with an Unstable Chromosome Segment Highly Conserved Among Poaceae.

The introgression of apomixis in major seed crops, would guarantee self-seeding of superior heterotic seeds over generations. In the grass species Paspalum simplex, apomixis is controlled by a single locus in which recombination is blocked. In the perspective of isolating the genetic determinants of apomixis, we report data on sequencing, in silico mapping and expression analysis of some of the genes contained in two cloned genomic regions of the apomixis locus of P. simplex. In silico mapping allowed us to identify a conserved synteny group homoeologous to the apomixis locus, located on a telomeric position of chromosomes 12, 8, 3 and 4 of rice, Sorghum bicolor, Setaria italica and Brachypodium distachyum, respectively, and on a more centromeric position of maize chromosome 1. Selected genes of the apomixis locus expressed sense and antisense transcripts in reproductively committed cells of sexual and apomictic ovules. Some of the genes considered here expressed apomixis-specific allelic variants which showed partial non-overlapping expression patterns with alleles shared by sexual and apomictic reproductive phenotypes. Our findings open new routes for the isolation of the genetic determinants of apomixis and, in perspective, for its introgression in crop grasses.

The vesicle trafficking regulator PN_SCD1 is demethylated and overexpressed in florets of apomictic Paspalum notatum genotypes.

Apomixis (asexual reproduction through seeds) is considered a deviation of the sexual reproductive pathway leading to the development of clonal progenies genetically identical to the mother plant. Here we used the Methylation-Sensitive Amplification Polymorphism (MSAP) technique to characterize cytosine methylation patterns occurring in florets of sexual and aposporous Paspalum notatum genotypes, in order to identify epigenetically-controlled genes putatively involved in apomixis development. From twelve polymorphic MSAP-derived sequences, one (PN_6.6, later renamed PN_SCD1) was selected due to its relevant annotation and differential representation in apomictic and sexual floral transcriptome libraries. PN_SCD1 encodes the DENN domain/WD repeat-containing protein SCD1, which interacts with RAB GTPases- and/or MAPKs to promote specialized cell division, functions in clathrin-mediated membrane transport and acts as potential substrate receptor of CUL4 E3 ubiquitin ligases. Quantitative RT-PCR and comparative RNAseq analyses of laser microdissected nucellar cells confirmed PN_SCD1 upregulation in florets of apomictic plants and revealed that overexpression takes place just before the onset of apospory initials. Moreover, we found that several SCD1 molecular partners are expressed in P. notatum florets and upregulated in apomictic plants. Our results disclosed a specific vesicle trafficking molecular pathway epigenetically modulated during apomixis.

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.

KEY MESSAGE: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30-60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.

Developing a Molecular Identification Assay of Old Landraces for the Genetic Authentication of Typical Agro-Food Products: The Case Study of the Barley 'Agordino'.

The orzo Agordino is a very old local variety of domesticated barley (Hordeum vulgare ssp. distichum L.) that is native to the Agordo District, Province of Belluno, and is widespread in the Veneto Region, Italy. Seeds of this landrace are widely used for the preparation of very famous dishes of the dolomitic culinary tradition such as barley soup, bakery products and local beer. Understanding the genetic diversity and identity of the Agordino barley landrace is a key step to establish conservation and valorisation strategies of this local variety and also to provide molecular traceability tools useful to ascertain the authenticity of its derivatives. The gene pool of the Agordino barley landrace was reconstructed using 60 phenotypically representative individual plants and its genotypic relationships with commercial varieties were investigated using 21 pure lines widely cultivated in the Veneto Region. For genomic DNA analysis, following an initial screening of 14 mapped microsatellite (SSR) loci, seven discriminant markers were selected on the basis of their genomic position across linkage groups and polymorphic marker alleles per locus. The genetic identity of the local barley landrace was determined by analysing all SSR markers in a single multi-locus PCR assay. Extent of genotypic variation within the Agordino barley landrace and the genotypic differentiation between the landrace individuals and the commercial varieties was determined. Then, as few as four highly informative SSR loci were selected and used to develop a molecular traceability system exploitable to verify the genetic authenticity of food products deriving from the Agordino landrace. This genetic authentication assay was validated using both DNA pools from individual Agordino barley plants and DNA samples from Agordino barley food products. On the whole, our data support the usefulness and robustness of this DNA-based diagnostic tool for the orzo Agordino identification, which could be rapidly and efficiently exploited to guarantee the authenticity of local varieties and the typicality of food products.

Pistil Transcriptome Analysis to Disclose Genes and Gene Products Related to Aposporous Apomixis in Hypericum perforatum L.

Unlike sexual reproduction, apomixis encompasses a number of reproductive strategies, which permit maternal genome inheritance without genetic recombination and syngamy. The key biological features of apomixis are the circumvention of meiosis (i.e., apomeiosis), the differentiation of unreduced embryo sacs and egg cells, and their autonomous development in functional embryos through parthenogenesis, and the formation of viable endosperm either via fertilization-independent means or following fertilization with a sperm cell. Despite the importance of apomixis for breeding of crop plants and although much research has been conducted to study this process, the genetic control of apomixis is still not well understood. Hypericum perforatum is becoming an attractive model system for the study of aposporous apomixis. Here we report results from a global gene expression analysis of H. perforatum pistils collected from sexual and aposporous plant accessions for the purpose of identifying genes, biological processes and molecular functions associated with the aposporous apomixis pathway. Across two developmental stages corresponding to the expression of aposporous apomeiosis and parthenogenesis in ovules, a total of 224 and 973 unigenes were found to be significantly up- and down-regulated with a fold change ≥ 2 in at least one comparison, respectively. Differentially expressed genes were enriched for multiple gene ontology (GO) terms, including cell cycle, DNA metabolic process, and single-organism cellular process. For molecular functions, the highest scores were recorded for GO terms associated with DNA binding, DNA (cytosine-5-)-methyltransferase activity and heterocyclic compound binding. As deregulation of single components of the sexual developmental pathway is believed to be a trigger of the apomictic reproductive program, all genes involved in sporogenesis, gametogenesis and response to hormonal stimuli were analyzed in great detail. Overall, our data suggest that phenotypic expression of apospory is concomitant with the modulation of key genes involved in the sexual reproductive pathway. Furthermore, based on gene annotation and co-expression, we underline a putative role of hormones and key actors playing in the RNA-directed DNA methylation pathway in regulating the developmental changes occurring during aposporous apomixis in H. perforatum.

Transcript Analysis and Regulative Events during Flower Development in Olive (Olea europaea L.).

The identification and characterization of transcripts involved in flower organ development, plant reproduction and metabolism represent key steps in plant phenotypic and physiological pathways, and may generate high-quality transcript variants useful for the development of functional markers. This study was aimed at obtaining an extensive characterization of the olive flower transcripts, by providing sound information on the candidate MADS-box genes related to the ABC model of flower development and on the putative genetic and molecular determinants of ovary abortion and pollen-pistil interaction. The overall sequence data, obtained by pyrosequencing of four cDNA libraries from flowers at different developmental stages of three olive varieties with distinct reproductive features (Leccino, Frantoio and Dolce Agogia), included approximately 465,000 ESTs, which gave rise to more than 14,600 contigs and approximately 92,000 singletons. As many as 56,700 unigenes were successfully annotated and provided gene ontology insights into the structural organization and putative molecular function of sequenced transcripts and deduced proteins in the context of their corresponding biological processes. Differentially expressed genes with potential regulatory roles in biosynthetic pathways and metabolic networks during flower development were identified. The gene expression studies allowed us to select the candidate genes that play well-known molecular functions in a number of biosynthetic pathways and specific biological processes that affect olive reproduction. A sound understanding of gene functions and regulatory networks that characterize the olive flower is provided.

A method for genotyping elite breeding stocks of leaf chicory (Cichorium intybus L.) by assaying mapped microsatellite marker loci.

BACKGROUND: Leaf chicory (Cichorium intybus subsp. intybus var. foliosum L.) is a diploid plant species (2n = 18) of the Asteraceae family. The term "chicory" specifies at least two types of cultivated plants: a leafy vegetable, which is highly differentiated with respect to several cultural types, and a root crop, whose current industrial utilization primarily addresses the extraction of inulin or the production of a coffee substitute. The populations grown are generally represented by local varieties (i.e., landraces) with high variation and adaptation to the natural and anthropological environment where they originated, and have been yearly selected and multiplied by farmers. Currently, molecular genetics and biotechnology are widely utilized in marker-assisted breeding programs in this species. In particular, molecular markers are becoming essential tools for developing parental lines with traits of interest and for assessing the specific combining ability of these lines to breed F1 hybrids. RESULTS: The present research deals with the implementation of an efficient method for genotyping elite breeding stocks developed from old landraces of leaf chicory, Radicchio of Chioggia, which are locally dominant in the Veneto region, using 27 microsatellite (SSR) marker loci scattered throughout the linkage groups. Information on the genetic diversity across molecular markers and plant accessions was successfully assessed along with descriptive statistics over all marker loci and inbred lines. Our overall data support an efficient method for assessing a multi-locus genotype of plant individuals and lineages that is useful for the selection of new varieties and the certification of local products derived from Radicchio of Chioggia. CONCLUSIONS: This method proved to be useful for assessing the observed degree of homozygosity of the inbred lines as a measure of their genetic stability; plus it allowed an estimate of the specific combining ability (SCA) between maternal and paternal inbred lines on the basis of their genetic diversity and the predicted degree of heterozygosity of their F1 hybrids. This information could be exploited for planning crosses and predicting plant vigor traits (i.e., heterosis) of experimental F1 hybrids on the basis of the genetic distance and allelic divergence between parental inbred lines. Knowing the parental genotypes would allow us not only to protect newly registered varieties but also to assess the genetic purity and identity of the seed stocks of commercial F1 hybrids, and to certificate the origin of their food derivatives.