Stefano La Malfa

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)

UNLABELLED PREMISE OF THE STUDY A new set of pomegranate microsatellites was selected and characterized to assess the level of genetic diversity among cultivars and wild genotypes. • METHODS AND RESULTS Nine 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. • CONCLUSIONS Because 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).

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.

Polyamines and transglutaminase activity are involved in compatible and self-incompatible pollination of Citrus grandis

Pollination of pummelo (Citrus grandis L. Osbeck) pistils has been studied in planta by adding compatible and self-incompatible (SI) pollen to the stigma surface. The pollen germination has been monitored inside the pistil by fluorescent microscopy showing SI altered morphologies with irregular depositions of callose in the tube walls, and heavy callose depositions in enlarged tips. The polyamine (PA) content as free, perchloric acid (PCA)-soluble and -insoluble fractions and transglutaminase (TGase) activity have been analyzed in order to deepen their possible involvement in the progamic phase of plant reproduction. The conjugated PAs in PCA-soluble fraction were definitely higher than the free and the PCA-insoluble forms, in both compatible and SI pollinated pistils. In pistils, pollination caused an early decrease of free PAs and increase of the bound forms. The SI pollination, showed highest values of PCA-soluble and -insoluble PAs with a maximum in concomitance with the pollen tube arrest. As TGase mediates some of the effects of PAs by covalently binding them to proteins, its activity, never checked before in Citrus, was examined with two different assays. In addition, the presence of glutamyl-PAs confirmed the enzyme assay data and excluded the possibility of a misinterpretation. The SI pollination caused an increase in TGase activity, whereas the compatible pollination caused its decrease. Similarly to bound PAs, the glutamyl-PAs and the enzyme activity peaked in the SI pollinated pistils in concomitance with the observed block of the pollen tube growth, suggesting an involvement of TGase in SI response.

EST-SNP genotyping of citrus species using high-resolution melting curve analysis

Citrus taxonomy is very complex mainly due to specific aspects of its reproductive biology. A number of studies have been performed using various molecular markers in order to evaluate the level of genetic variability in Citrus. SNP markers have been used for genetic diversity assessment using a variety of different methods. Recently, the availability of EST database and whole genome sequences has made it possible to develop more markers such as SNPs. In the present study, the high-resolution melting curve analysis (HRM) was used to detect SNPs or INDELs in Citrus genus for the first time. We aimed to develop a panel of SNPs to differentiate Citrus genotypes which can also be applied to Citrus biodiversity studies. The results showed that 21 SNP containing markers produced distinct polymorphic melting curves among the Citrus spp. investigated through HRM analysis. It was proved that HRM is an efficient, cost-effective, and accurate method for discriminating citrus SNPs as well as a method to analyze more polymorphisms in a single PCR amplicon, representing a useful tool for genetic, biodiversity, and breeding studies. SNPs developed based on Citrus sinensis EST database showed a good transferability within the Citrus genus. Moreover, HRM analysis allowed the discrimination of citrus genotypes at specific level and the resulting genetic distance analysis clustered these genotypes into three main branches. The results suggested that the panel of SNP markers could be used in a variety of applications in citrus biodiversity assessment and breeding programs using HRM analysis.

Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

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.

Relationships among cultivated Opuntia ficus-indica genotypes and related species assessed by cytoplasmic markers

The taxonomic classification of the genus Opuntia, which includes several cultivated species, is complicated mainly because of inadequate morphological descriptors, the common intra- and inter-generic hybridization and the relationships between phenotypic variation and ecological conditions. The phylogenetic relationships among 72 cultivated genotypes, either selected for fruit or forage production, and wild accessions belonging to approximately 15 different species of Opuntia were inferred using cytoplasmic markers. Previous studies indicated as the most important cultivated accessions for fruit production have a polyphyletic origin but polyploidy hampered their clear phylogenetic assignment based on nuclear markers. Cytoplasmic markers are considered helpful for their maternal inheritance and for overcoming the multiple gene copy problem in polyploid phylogenetics already reported in the Opuntia genus. In particular, we combined in this work capillary electrophoresis for newly designed cpSSRs and high resolution melting for SNV analyses to identify chloroplast (ndhF-rpl32, psbJ-petA, atpB-rbcL, matK, ycf1) and mitochondrial (rpl5) DNA markers in a selected group of genotypes. The results revealed the presence of polymorphisms in the predicted cpSSRs and SNVs and clearly evidenced that most of the studied genotypes were closely related to Opuntia ficus-indica (L.) Miller. Plastid markers identified 11 chlorotypes and 8 unique genotypes. Interestingly the analysis evidenced a multiple maternal phylogeny for the fleshy fruit varieties classified as O. ficus-indica. These results allow questioning of the reliability of the current classification based on morphological parameters and reveal the narrow genetic base of the most common cultivated opuntias for fruit production, while forage genotypes evidenced greater variability.

Elucidating the contribution of wild related species on autochthonous pear germplasm: A case study from Mount Etna

The pear (genus Pyrus) is one of the most ancient and widely cultivated tree fruit crops in temperate climates. The Mount Etna area claims a large number of pear varieties differentiated due to a long history of cultivation and environmental variability, making this area particularly suitable for genetic studies. Ninety-five pear individuals were genotyped using the simple sequence repeat (SSR) methodology interrogating both the nuclear (nDNA) and chloroplast DNA (cpDNA) to combine an investigation of maternal inheritance of chloroplast SSRs (cpSSRs) with the high informativity of nuclear SSRs (nSSRs). The germplasm was selected ad hoc to include wild genotypes, local varieties, and national and international cultivated varieties. The objectives of this study were as follows: (i) estimate the level of differentiation within local varieties; (ii) elucidate the phylogenetic relationships between the cultivated genotypes and wild accessions; and (iii) estimate the potential genetic flow and the relationship among the germplasms in our analysis. Eight nSSRs detected a total of 136 alleles with an average minor allelic frequency and observed heterozygosity of 0.29 and 0.65, respectively, whereas cpSSRs allowed identification of eight haplotypes (S4 Table). These results shed light on the genetic relatedness between Italian varieties and wild genotypes. Among the wild species, compared with P. amygdaliformis, few P. pyraster genotypes exhibited higher genetic similarity to local pear varieties. Our analysis revealed the presence of genetic stratification with a ‘wild’ subpopulation characterizing the genetic makeup of wild species and the international cultivated varieties exhibiting the predominance of the ‘cultivated’ subpopulation.