E. Tribulato

Citrus phylogeny and genetic origin of important species as investigated by molecular markers.

Abstract Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one.

Histological and molecular analysis of pollen-pistil interaction in clementine.

In contrast to model species, the self-incompatibility reaction in citrus has been poorly studied. It is assumed to be gametophytically determined and genetically controlled by the S-locus, which in other species encodes for glycoproteins (S-RNases) showing ribonuclease activity. To investigate pollen–pistil interaction, the pollen tube growth of two clementine varieties, ‘Comune’ (self-incompatible) and ‘Monreal’ (a ‘Comune’ self-compatible mutation) was analysed by histological assays in self- and cross-pollination conditions. Cross-pollination assays demonstrated that the mutation leading to self-compatibility in ‘Monreal’ occurred in the stylar tissues. Similar rates of pollen germination were observed in both genotypes. However, ‘Comune’ pollen tubes showed altered morphology and arrested growth in the upper style while in ‘Monreal’ they grew straight toward the ovary. Moreover, to identify genes putatively involved in pollen–pistil interaction and self-incompatibility, research based on the complementary DNA-amplified fragment length polymorphism technique was carried out to compare the transcript profiles of unpollinated and self-pollinated styles and stigmas of the two cultivars. This analysis identified 96 unigenes such as receptor-like kinases, stress-induced genes, transcripts involved in the phenylpropanoid pathway, transcription factors and genes related to calcium and hormone signalling. Surprisingly, a high percentage of active long terminal repeat (LTR) and non-LTR retrotransposons were identified among the unigenes, indicating their activation in response to pollination and their possible role in the regulation of self-incompatibility genes. The quantitative reverse trascription-polymerase chain reaction analysis of selected gene tags showed transcriptional differences between the two genotypes during pollen germination and pollen tube elongation.

Characterisation and assessment of genetic diversity in cultivated and wild carob (Ceratonia siliqua L.) genotypes using AFLP markers

Summary Carob (Ceratonia siliqua L.) is a trioecious evergreen legume tree native to the Middle East and widespread throughout the Mediterranean as a crop, or as part of the maquis. Amplified Fragment Length Polymorphism (AFLP) markers were used to estimate intra-specific genetic diversity among 41 carob genotypes from different areas of the eastern and western Mediterranean basin. Italian and Spanish cultivated varieties were included in the study for fingerprinting. A total of 96 unambiguous polymorphic loci were scored from seven Eco RI/Mse I primer combinations. Simple matching similarity coefficients between pairs of genotypes ranged from 0.542 to 1.000, indicating a high level of intra-specific diversity. AFLP markers clearly discriminated between all the accessions, except for two Sicilian varieties. An UPGMA (Unweighted Pair Group Method with Arithmetic Mean) dendrogram and a Principal Co-ordinates Analysis revealed a broad relationship between the clusters and the geographic origin of the genotypes, with few exceptions. Most of the hermaphrodite varieties belonged to the same cluster, indicating that these cultivars were probably selected in a specific area, then distributed elsewhere. Moreover, an analysis of molecular variance (AMOVA) revealed a high level of “within geographic area” differences.

Generation of expressed sequence tags from carob (Ceratonia siliqua L.) flowers for gene identification and marker development

Carob (Ceratonia siliqua L.) is a caesalpinoid legume tree showing labile sex expression. With the main aims of identifying flower-expressed genes and of developing specific markers, 1,056 clones from a complementary DNA library of carob flowers were bidirectionally sequenced. A total of 1,377 high-quality expressed sequence tags were clustered into 1,096 unigenes. Basic Local Alignment Search Tool and Gene Ontology functional annotation allowed to identify several agronomically important genes, such as those involved in flower development and sexual reproduction, response to stress, galactomannan synthesis, and hormone pathways. Genes involved in the ethylene biosynthesis and response were quantified in developing flowers of three sex genotypes (male, female, and hermaphrodite) using quantitative reverse transcription polymerase chain reaction. The transcript levels of 1-aminocyclopropane-1-carboxylic acid oxidase, acting downstream in ethylene pathway, and Ethylene Insensitive 3 (EIN3)-like, a transcription factor involved in ethylene signaling, were directly correlated with maleness, indicating a possible role of ethylene in carob sex expression. Furthermore, the first set of carob genic microsatellites was developed, which might be useful for genotyping and genetic diversity analysis.

Recovery of Citrus Somatic Hybrids Tolerant to Phoma Tracheiphila Toxin, Combining Selection and Identification by RAPD Markers

Phoma tracheiphila is a pathogen that causes a serious tracheomycotic disease in commercial lemon cultivars. Somatic hybridization offers the possibility to transfer the trait for tolerance into susceptible cultivars. One of the limitations of this approach is selection and identification of nuclear hybrids at an early stage. Protoplasts isolated from calli of ‘Murcott’ tangor (tolerant) and ‘Messina’ lemon (susceptible) were fused (PEG treatment). Prior to fusion, ‘Murcott’ protoplasts were treated with 0.25 mM iodoacetate. Fusion treated protoplasts were plated in liquid medium for 40 days and then exposed to 0.6 μM of P. tracheiphila toxin. Total DNA was extracted from the selected calli and corresponding embryos and analyzed by RAPDs. With the selected primers, hybrids were successfully identified. In the survey of coculture with 1 μM toxin, the hybrid callus lines showed tolerance to the toxin. Immunoblot analysis of the recovered hybrid calli indicated the presence of chitinase among the extracellular proteins. The combined methods, selection and identification by RAPD markers, resulted in 90% recovery of nuclear hybrids tolerant to the P. tracheiphila toxin.