Marco Caruso

EST–SSR markers for asparagus genetic diversity evaluation and cultivar identification

Simple sequence repeat (SSR) markers generated from expressed sequence tag (EST) sequences represent useful tools for genotyping and their development is relatively easy because of the public availability of EST databases. We report design and application of EST–SSRs to assess the level of genetic diversity among thirty-five asparagus cultivars and to fingerprint DePaoli, a new variety released by University of California, Riverside. DNA was isolated from bulks of pooled cladophylls coming from five plants of each variety to reduce the number of DNA extractions and PCR reactions. Allele frequencies were estimated from the intensity of the bands in two bulks and two individual plant samples for each variety. Although asparagus varieties derive from a limited germplasm pool, eight EST–SSR loci differentiated all of the analyzed cultivars. Moreover, UPGMA (unweighted pair group method with arithmetic mean) and neighbor-joining trees, as well as principal components analysis separated the cultivars into clusters corresponding to the geographical areas where they originated.

High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR – An Example in Citrus

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.

Microsatellite markers help to assess genetic diversity among Opuntia ficus indica cultivated genotypes and their relation with related species.

Opuntia spp. belong to the Cactaceae family and are native to Central America. The most economically important species is O. ficus indica, cultivated both for fruits and cladodes. The genus includes other important edible species (from diploid to octoploid) that occur worldwide as either wild or cultivated species in many arid or semiarid areas (e.g., the Mediterranean region). Several accessions are cultivated in different growing regions, but little is known about their ancestries and levels of genetic diversity. The aim of this study was to investigate the level of intraspecific genetic diversity among O. ficus indica cultivated varieties and some related species. Specifically, six highly polymorphic simple sequence repeats (SSR) and two expressed sequence tag (EST)-SSR loci were investigated in 62 wild and cultivated genotypes belonging to 16 Opuntia species. The clusters identified by the distance and model-based analyses clearly separated the wild opuntias from the cultivated ones. However, the O. ficus indica accessions did not cluster separately from other arborescent cactus pear species, such as O. amyclaea, O. megacantha, O. streptacantha, O. fusicaulis, and O. albicarpa, indicating that their current taxonomical classifications do not fit with their genetic variability. In general, the genotypes cultivated in Mexico showed high levels of diversity, whereas most of the spineless accessions collected in other countries had a very narrow genetic base. This study increases our knowledge of the variability among some of the most diffused Opuntia cultivated accessions. This study also points to the inconsistencies of previous taxonomical genotype assignments that were based solely on morphological characteristics.

New microsatellite loci for pomegranate, Punica granatum (Lythraceae)

UNLABELLEDnnnnPREMISE OF THE STUDYnA new set of pomegranate microsatellites was selected and characterized to assess the level of genetic diversity among cultivars and wild genotypes. •nnnMETHODS AND RESULTSnNine Simple Sequence Repeat (SSR) markers were obtained using the Microsatellite-AFLP technique and were successfully amplified in 34 genotypes belonging to Italian, Spanish, and Turkish germplasm collections. The number of alleles per locus ranged from 1 to 5, and the total number of alleles was 22. •nnnCONCLUSIONSnBecause only a few codominant markers are available for this species, the newly identified SSRs will facilitate genetic diversity studies, fingerprinting, and mapping. In addition, the 9 loci successfully amplified in P. granatum var. nana. No cross transferability was observed for Cuphea micropetala and Lagerstroemia indica (Lythraceae).

Physiological and Molecular Analysis of the Maturation Process in Fruits of Clementine Mandarin and One of Its Late-Ripening Mutants

Peel color is one of the main features affecting citrus quality. Clementine is a widespread citrus species with several mutants showing a delay in pigmentation and harvesting. This work characterizes the fruit development and ripening of two clementine clones, Comune, a widespread variety, and one of its natural mutations, Tardivo, which differ by a delayed color-break and extended harvest period. Morphological, chemical, and molecular analyses were carried out on fruits of both genotypes during the whole maturation process. Analysis showed that mutation did not affect ripening characteristics such as juice acidity and TSS. However, biochemical and molecular analysis revealed marked differences in the flavedo regarding carotenogenesis and chlorophyllase gene expression. Carotenoid showed quantitative differences at biochemical and molecular levels. Results demonstrated that the mutation in Tardivo influenced the transcriptional activation of PSY, a key step in carotenoid biosynthesis. The differential PSY expression led to a significant quantitative difference in phytoene accumulation between the two genotypes. Also, Tardivo showed delayed accumulation of carotenes, lutein, and beta,beta-xanthophylls. The differential expression of genes involved in ethylene biosynthesis and perception suggested differing responses to ethylene signaling between the two genotypes. Moreover, exogenous application of ethylene revealed a different sensitivity of the two varieties to this hormone. The analysis added new information to better understand the complex process of ripening in citrus.

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.

Comparative transcriptome analysis of stylar canal cells identifies novel candidate genes implicated in the self-incompatibility response of Citrus clementina

BackgroundReproductive biology in citrus is still poorly understood. Although in recent years several efforts have been made to study pollen-pistil interaction and self-incompatibility, little information is available about the molecular mechanisms regulating these processes. Here we report the identification of candidate genes involved in pollen-pistil interaction and self-incompatibility in clementine (Citrus clementina Hort. ex Tan.). These genes have been identified comparing the transcriptomes of laser-microdissected stylar canal cells (SCC) isolated from two genotypes differing for self-incompatibility response (Comune, a self-incompatible cultivar and Monreal, a self- compatible mutation of Comune).ResultsThe transcriptome profiling of SCC indicated that the differential regulation of few specific, mostly uncharacterized transcripts is associated with the breakdown of self-incompatibility in Monreal. Among them, a novel F-box gene showed a drastic up-regulation both in laser microdissected stylar canal cells and in self-pollinated whole styles with stigmas of Comune in concomitance with the arrest of pollen tube growth. Moreover, we identify a non-characterized gene family as closely associated to the self-incompatibility genetic program activated in Comune. Three different aspartic-acid rich (Asp-rich) protein genes, located in tandem in the clementine genome, were over-represented in the transcriptome of Comune. These genes are tightly linked to a DELLA gene, previously found to be up-regulated in the self-incompatible genotype during pollen-pistil interaction.ConclusionThe highly specific transcriptome survey of the stylar canal cells identified novel genes which have not been previously associated with self-pollen rejection in citrus and in other plant species. Bioinformatic and transcriptional analyses suggested that the mutation leading to self-compatibility in Monreal affected the expression of non-homologous genes located in a restricted genome region. Also, we hypothesize that the Asp-rich protein genes may act as Ca2+ entrapping proteins, potentially regulating Ca2+ homeostasis during self-pollen recognition.

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.