Among Citrus species, the sweet orange (Citrus sinensis (L.) Osbeck) is the most important in terms of production volumes and cultivated areas. Oranges are particularly appreciated for the organoleptic characteristics and the high nutraceutical value of the fruits (thanks especially to their high content of antioxidants). Recent advances in citrus genetic and genomic resources, such as the release of the reference genomes of several sweet orange cultivars, have contributed to (i) understanding the diversification of C. sinensis and its relation with other citrus species, (ii) assessing the molecular mechanisms underlying traits of interest, (iii) identifying and characterizing the candidate genes responsible for important phenotypic traits, and (iv) developing biotechnological methods to incorporate these traits into different citrus genotypes. It has been clarified that all the genetic diversity within the sweet orange species was derived from subsequent mutations starting from a single ancestor and was derived from complex cycles of hybridization and backcrossing between the mandarin (Citrus reticulata Blanco) and the pummelo (Citrus maxima (Burm.) Merr.). This paper provides an overview of the varietal panorama together with a description of the main driving forces in present and future sweet orange breeding. In fact, for the sweet orange, as well as for other citrus species, the release of novel varieties with improved characteristics is being pursued thanks to the employment of conventional and/or innovative (molecular-based) methods. The state of the art methods together with the innovations in genomics and biotechnological tools leading to the so-called new plant breeding technologies were also reviewed and discussed.

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.