QTL analysis on a lemon population provides novel insights on the genetic regulation of the tolerance to the two-spotted spider mite attack.

BACKGROUND: Among the Citrus species, lemon (Citrus limon Burm f.) is one of the most affected by the two-spotted spider mite (Tetranychus urticae Koch). Moreover, chemical control is hampered by the mite's ability to develop genetic resistance against acaricides. In this context, the identification of the genetic basis of the host resistance could represent a sustainable strategy for spider mite control. In the present study, a marker-trait association analysis was performed on a lemon population employing an association mapping approach. An inter-specific full-sib population composed of 109 accessions was phenotyped through a detached-leaf assays performed in modified Huffaker cells. Those individuals, complemented with two inter-specific segregating populations, were genotyped using a target-sequencing approach called SPET (Single Primer Enrichment Technology), the resulting SNPs were employed for the generation of an integrated genetic map. RESULTS: The percentage of damaged area in the full-sib population showed a quantitative distribution with values ranging from 0.36 to 9.67%. A total of 47,298 SNPs were selected for an association mapping study and a significant marker linked with resistance to spider mite was detected on linkage group 5. In silico gene annotation of the QTL interval enabled the detection of 13 genes involved in immune response to biotic and abiotic stress. Gene expression analysis showed an over expression of the gene encoding for the ethylene-responsive transcription factor ERF098-like, already characterized in Arabidopsis and in rice for its involvement in defense response. CONCLUSION: The identification of a molecular marker linked to the resistance to spider mite attack can pave the way for the development of marker-assisted breeding plan for the development of novel selection coupling favorable agronomical traits (e.g. fruit quality, yield) with a higher resistance toward the mite.

The Role of Italy in the Use of Advanced Plant Genomic Techniques on Fruit Trees: State of the Art and Future Perspectives.

Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the 'Made in Italy' label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The 'new genomic techniques' (NGTs), renamed in Italy as 'technologies for assisted evolution' (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.

Genome-wide footprints in the carob tree (Ceratonia siliqua) unveil a new domestication pattern of a fruit tree in the Mediterranean.

Intense research efforts over the last two decades have renewed our understanding of plant phylogeography and domestication in the Mediterranean basin. Here we aim to investigate the evolutionary history and the origin of domestication of the carob tree (Ceratonia siliqua), which has been cultivated for millennia for food and fodder. We used >1000 microsatellite genotypes to delimit seven carob evolutionary units (CEUs). We investigated genome-wide diversity and evolutionary patterns of the CEUs with 3557 single nucleotide polymorphisms generated by restriction-site associated DNA sequencing (RADseq). To address the complex wild vs. cultivated status of sampled trees, we classified 56 sampled populations across the Mediterranean basin as wild, seminatural or cultivated. Nuclear and cytoplasmic loci were identified from RADseq data and separated for analyses. Phylogenetic analyses of these genomic-wide data allowed us to resolve west-to-east expansions from a single long-term refugium probably located in the foothills of the High Atlas Mountains near the Atlantic coast. Our findings support multiple origins of domestication with a low impact on the genetic diversity at range-wide level. The carob was mostly domesticated from locally selected wild genotypes and scattered long-distance westward dispersals of domesticated varieties by humans, concomitant with major historical migrations by Romans, Greeks and Arabs. Ex situ efforts to preserve carob genetic resources should prioritize accessions from both western and eastern populations, with emphasis on the most differentiated CEUs situated in southwest Morocco, south Spain and eastern Mediterranean. Our study highlights the relevance of wild and seminatural habitats in the conservation of genetic resources for cultivated trees.

Trifolium subterraneum cover cropping for improving the nutritional status of a Mediterranean apricot orchard.

BACKGROUND: The utilization of Trifolium subterraneum L. cover crops may represent an innovative and efficient option in low-input and organic farming, especially in Mediterranean agroecosystems where low and irregular rainfall require frequent soil tillage and use of herbicides to reduce moisture losses and weed competitiveness. Since imbalances of soil macro- and micro-nutrients due to cover cropping establishment could be responsible for numerous problems in specialized orchards, such as disturbances in the normal tree growth and quality of fruits, the objective of this study was to investigate, the cumulative effects of a 3-years established T. subterraneum cover cropping, compared with a spontaneous flora and a conventional management (as a control), on the levels of mineral nutrients in the apricot leaves and fruits. RESULTS: Our findings indicated that T. subterraneum cover cropping tended to stimulate higher leaf macro- and micro-nutrients content than conventional management and flora spontaneous cover cropping. In addition, the presence of T. subterraneum cover cropping, especially with the incorporation of dead mulches into the soil, increased the content of potassium (K), nitrogen (N), calcium (Ca), iron (Fe) and manganese (Mn) in apricot fruits. CONCLUSION: Taking also into account the effects of T. subterraneum cover cropping on both the reduction of soil weed and enhancement of bacteria communities involved in the soil N-cycle, we may suggest its application in Mediterranean orchards as an eco-friendly alternative to synthetic herbicides for weed control and mineral N fertilizers, while enhancing the apricot tree nutritional status and fruit quality. © 2020 Society of Chemical Industry.

Changes in physiological and some nutritional, nutraceuticals, chemical-physical, microbiological and sensory quality of minimally processed cactus pears cvs 'Bianca', 'Gialla' and 'Rossa' stored under passive modified atmosphere.

BACKGROUND: The objective of this study was to compare the overall quality changes of minimally processed cactus pears cvs 'Bianca', Gialla' and 'Rossa' stored at 4 °C for 10 days. Periodically in-package CO2 , O2 and C2 H4 were determined and fruit were assessed for overall quality changes (pH, acidity, sugars, phenolics, betacyanins and betaxanthines, antioxidant capacity, colour, firmness, microbiological population and sensory attributes). In a preliminary study three different polymeric films were tested to select the most suitable to design a package with a short lag time to achieve steady-state conditions. RESULTS: Results showed marked differences between measured in-package CO2 and O2 values and those calculated based on respiration of peeled fruit and film permeance to CO2 and O2 provided by manufactures. The sensory evaluation of packed fruit indicated in film BBT-Bolphane, which created a steady-state in-package partial pressure for CO2 of 4.3-4.8 kPa and for O2 of 4.8-5.5 kPa, as the best film. Results of in-package gas composition with the three cultivars were similar to those achieved in cv. 'Gialla' with the preliminary test. All measured qualitative parameters changed slightly over the storage period for all cultivars and followed the same trend, despite significant differences existing among cultivars. CONCLUSION: This study clearly showed a similar physiological behavior of minimally processed 'Bianca', 'Gialla' and 'Rossa' cactus pears. Storage conditions optimal for one cultivar fit well for the others; thus mixing fruit of different cultivars in a package designed for one specific cultivar does not lead to relevant deviation from expected results. © 2017 Society of Chemical Industry.

The origin and the genetic regulation of the self-compatibility mechanism in clementine (Citrus clementina Hort. ex Tan.).

Self-incompatibility (SI) is a genetic mechanism common in flowering plants to prevent self-fertilization. Among citrus species, several pummelo, mandarin, and mandarin-like accessions show SI behavior. In these species, SI is coupled with a variable degree of parthenocarpy ensuring the production of seedless fruits, a trait that is highly appreciated by consumers. In Citrus, recent evidences have shown the presence of a gametophytic SI system based on S-ribonucleases (S-RNases) ability to impair self-pollen tube growth in the upper/middle part of the style. In the present study, we combined PCR analysis and next-generation sequencing technologies, to define the presence of S7- and S11-Rnases in the S-genotype of the Citrus clementina (Hort. ex Tan.), the self-incompatible 'Comune' clementine and its self-compatible natural mutant 'Monreal'. The reference genome of 'Monreal' clementine is presented for the first time, providing more robust results on the genetic sequence of the newly discovered S7-RNase. SNP discovery analysis coupled with the annotation of the variants detected enabled the identification of 7,781 SNPs effecting 5,661 genes in 'Monreal' compared to the reference genome of C. clementina. Transcriptome analysis of unpollinated pistils at the mature stage from both clementine genotypes revealed the lack of expression of S7-RNase in 'Monreal' suggesting its involvement in the loss of the SI response. RNA-seq analysis followed by gene ontology studies enabled the identification of 2,680 differentially expressed genes (DEGs), a significant number of those is involved in oxidoreductase and transmembrane transport activity. Merging of DNA sequencing and RNA data led to the identification of 164 DEGs characterized by the presence of at least one SNP predicted to induce mutations with a high effect on their amino acid sequence. Among them, four candidate genes referring to two Agamous-like MADS-box proteins, to MYB111 and to MLO-like protein 12 were validated. Moreover, the transcription factor MYB111 appeared to contain a binding site for the 2.0-kb upstream sequences of the S7- and S11-RNase genes. These results provide useful information about the genetic bases of SI indicating that SNPs present in their sequence could be responsible for the differential expression and the regulation of S7-RNase and consequently of the SI mechanism.

Composition of discarded Sicilian fruits of Opuntia ficus indica L.: Phenolic content, mineral profile and antioxidant activity in peel, seeds and whole fruit.

Sicily (Italy) is the second producer of Opuntia ficus-indica (OFI) fruits after Mexico. To date, huge quantities of fruit are discarded during the selection for the fresh market, generating a large amount of by-product to be valorized. This study aimed to investigate on the composition of OFI discarded fruits from the main Sicilian productive areas, over two harvesting periods. Peel, seeds and whole fruit samples were characterized in terms of minerals and phenolic compounds through ICP-OES and HPLC-DAD-MS. Potassium, calcium and magnesium were the most abundant elements and peel samples showed the highest values. Seventeen phenolic compounds were detected in peel and whole fruit, including flavonoids, phenylpyruvic and hydroxycinnamic acids, while only phenolic acids were found in the seeds. A multivariate chemometric approach highlighted a correlation between the mineral and phenolic content and the different parts of the fruit as well as a significant influence of productive area.

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

BACKGROUND: Reproductive 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'). RESULTS: The 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. CONCLUSION: The 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.

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.

Biotechnological Approaches for Genetic Improvement of Lemon (Citrus limon (L.) Burm. f.) against Mal Secco Disease.

Among Citrus species, lemon is one of the most susceptible to mal secco disease, a tracheomycosis caused by the mitosporic fungus Plenodomus tracheiphilus, which induces chlorosis followed by leaf drop and progressive desiccation of twigs and branches. Severe infection can cause the death of the plant. Since no effective control strategies are available to efficiently control the pathogen spread, host tolerance is the most desirable goal in the struggle against mal secco disease. To date, both traditional breeding programs and biotechnological techniques were not efficient in developing novel varieties coupling tolerance to mal secco with optimal fruit quality. Furthermore, the genetic basis of host resistance has not been fully deciphered yet, hampering the set-up of marker-assisted selection (MAS) schemes. This paper provides an overview of the biotechnological approaches adopted so far for the selection of mal secco tolerant lemon varieties and emphasizes the promising contribution of marker-trait association analysis techniques for both unraveling the genetic determinism of the resistance to mal secco and detecting molecular markers that can be readily used for MAS. Such an approach has already proved its efficiency in several crops and could represent a valuable tool to select novel lemon varieties coupling superior fruit quality traits and resistance to mal secco.

Secondary metabolic profiles and anticancer actions from fruit extracts of immature pomegranates.

Immature fruits from Punica granatum L. thinning are a neglected side product of pomegranate production with cumbersome disposal costs for farmers. To explore value potential of immature fruits from pomegranate 'Wonderful' cultivars, the compositional landscapes and antitumorigenic activities of pomegranate extracts from two different stages of maturation were assessed. Cancer cell proliferation and cytotoxicity was quantified in human lung H1299 and colon HCT116 adenocarcinomas by crystal violet staining, MTS assay and caspase-3 activity. High performance liquid chromatography-diode array detector (HPLC/DAD) and high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC/ESI-MS) analyses indicate that immature fruits are rich sources of gallotannins and ellagitannins, with the highest amounts contained in immature fruit peels. Biological investigations reveal a robust anticancer activity by those immature P. granatum fruit extracts, which reflected induction of tumor cytotoxicity and cell death mechanisms. Together, present observations suggest P. granatum byproducts from the thinning process may provide unexplored values for virtuous circular economy.

Recent Advances of In Vitro Culture for the Application of New Breeding Techniques in Citrus.

Citrus is one of the most important fruit crops in the world. This review will discuss the recent findings related to citrus transformation and regeneration protocols of juvenile and adult explants. Despite the many advances that have been made in the last years (including the use of inducible promoters and site-specific recombination systems), transformation efficiency, and regeneration potential still represent a bottleneck in the application of the new breeding techniques in commercial citrus varieties. The influence of genotype, explant type, and other factors affecting the regeneration and transformation of the most used citrus varieties will be described, as well as some examples of how these processes can be applied to improve fruit quality and resistance to various pathogens and pests, including the potential of using genome editing in citrus. The availability of efficient regeneration and transformation protocols, together with the availability of the source of resistance, is made even more important in light of the fast diffusion of emerging diseases, such as Huanglongbing (HLB), which is seriously challenging citriculture worldwide.

New Plant Breeding Techniques in Citrus for the Improvement of Important Agronomic Traits. A Review.

New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for fruit tree species, in order to obtain new varieties with improved organoleptic traits and resistance to biotic and abiotic stress, and to maintain fruit quality achieved over centuries by (clonal) selection. Knowledge on the gene(s) controlling a specific trait is essential for the use of NPBTs, such as genome editing and cisgenesis. In the framework of the international scientific community working on fruit tree species, including citrus, NPBTs have mainly been applied to address pathogen threats. Citrus could take advantage of NPBTs because of its complex species biology (seedlessness, apomixis, high heterozygosity, and long juvenility phase) and aptitude for in vitro manipulation. To our knowledge, genome editing in citrus via transgenesis has successful for induced resistance to Citrus bacterial canker in sweet orange and grapefruit using the resistance gene CsLOB1. In the future, NPBTs will also be used to improve fruit traits, making them healthier. The regeneration of plants following the application of NPBTs is a bottleneck, making it necessary to optimize the efficiency of current protocols. The strengths and weaknesses of using explants from young in vitro plantlets, and from mature plants, will be discussed. Other major issues addressed in this review are related to the requirement for marker-free systems and shortening the long juvenility phase. This review aims to summarize methods and approaches available in the literature that are suitable to citrus, focusing on the principles observed before the use of NPBTs.

Substantial Equivalence of a Transgenic Lemon Fruit Showing Postharvest Fungal Pathogens Resistance.

The development of genetically modified (GM) crops speeds up the obtainment of novel varieties with improved agronomic characteristics. However, the risk evaluation of the use of GMs is mandatory before their release in the market. In this paper, an untargeted and comprehensive nuclear magnetic resonance-based metabolomic study was carried out on the peel and flesh of a transgenic lemon clone (E23) expressing the chit42 gene and exhibiting an increased tolerance to some pathogenic fungi and on its wild type. Results highlighted a substantial equivalence of the metabolomics profile of the transgenic clone compared to the wild type. In addition, an enhanced response of the E23 clone toward fungal pathogens affecting the postharvest management in lemon was evidenced. These results confirm the potential of genetic engineering for the punctual modification of specific agronomic traits without altering the whole pattern of metabolites and open new perspectives for a more sustainable and effective management of specific postharvest diseases 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.

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.

Advances in genotyping microsatellite markers through sequencing and consequences of scoring methods for Ceratonia siliqua (Leguminosae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) or microsatellite markers have been used in a broad range of studies mostly scoring alleles on the basis of amplicon size as a proxy for the number of repeat units of an SSR motif. However, additional sources of variation within the SSR or in the flanking regions have largely remained undetected. METHODS: In this study, we implemented a next-generation sequencing-based genotyping approach in a newly characterized set of 18 nuclear SSR markers for the carob tree, Ceratonia siliqua. Our aim was to evaluate the effect of three different methods of scoring molecular variation present within microsatellite markers on the genetic diversity and structure results. RESULTS: The analysis of the sequences of 77 multilocus genotypes from four populations revealed SSR variation and additional sources of polymorphism in 87% of the loci analyzed (42 single-nucleotide polymorphisms and five insertion/deletion polymorphisms), as well as divergent paralog copies in two loci. Ignoring sequence variation under standard amplicon size genotyping resulted in incorrect identification of 69% of the alleles, with important effects on the genetic diversity and structure estimates. DISCUSSION: Next-generation sequencing allows the detection and scoring of SSRs, single-nucleotide polymorphisms, and insertion/deletion polymorphisms to increase the resolution of population genetic studies.

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.

Rheological properties and sugar composition of locust bean gum from different carob varieties (Ceratonia siliqua L.).

The seeds of the main Italian carob varieties, Latinissima and Tantillo, and those of two selected accessions of Latinissima were evaluated in terms of yield, rheological properties, and sugar composition of the endosperm (LBG). The separation of the seed components in Latinissima and its seedlings yielded meanly 52.2% gum, 17.4% germ, and 30.5% tegument, whereas Tantillo furnished a lower gum yield (38.5%) and a higher yield of tegument (45.8%). The viscosity of 1% LBG aqueous solutions was measured at different shear rates (3-60 rpm), pH values (3.0-6.0), and temperatures (10-60 degrees C). The best results were shown by Latinissima, whereas Tantillo provided always the poorest thickening capacity. The content of free simple sugars and sucrose in the raw flours, the total monosaccharide residues after acidic hydrolysis, the mannose/galactose ratio, and the distribution of polysaccharides by size exclusion chromatography accounted for the observed viscosities. The seeds of Latinissima showed the highest technological potential.

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.