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.

Generation of Transfer-DNA-Free Base-Edited Citrus Plants.

To recover transgenic citrus plants in the most efficient manner, the use of selection marker genes is essential. In this work, it was shown that the mutated forms of the acetolactate synthase (ALS) gene in combination with the herbicide selection agent imazapyr (IMZ) added to the selection medium may be used to achieve this goal. This approach enables the development of cisgenic regenerants, namely, plants without the incorporation of those bacterial genes currently employed for transgenic selection, and additionally it allows the generation of edited, non-transgenic plants with altered endogenous ALS genes leading to IMZ resistance. In this work, the citrus mutants, in which ALS has been converted into IMZ-resistant forms using a base editor system, were recovered after cocultivation of the explants with Agrobacterium tumefaciens carrying a cytidine deaminase fused to nSpCas9 in the T-DNA and selecting regenerants in the culture medium supplemented with IMZ. Analysis of transgene-free plants indicated that the transient expression of the T-DNA genes was sufficient to induce ALS mutations and thus generate IMZ-resistant shoots at 11.7% frequency. To our knowledge, this is the first report of T-DNA-free edited citrus plants. Although further optimization is required to increase edition efficiency, this methodology will allow generating new citrus varieties with improved organoleptic/agronomic features without the need to use foreign genes.

Cultural Management of Huanglongbing: Current Status and Ongoing Research.

Huanglongbing (HLB), formerly known as greening, is a bacterial disease restricted to some Asian and African regions until two decades ago. Nowadays, associated bacteria and their vectors have spread to almost all citrus-producing regions, and it is currently considered the most devastating citrus disease. HLB management can be approached in terms of prevention, limiting or avoiding pathogen and associated vectors to reach an area, or in terms of control, trying to reduce the impact of the disease by adopting different cultural strategies depending on infestation/infection levels. In both cases, control of psyllid populations is currently the best way to stop HLB spread. Best cultural actions (CHMAs, TPS system) to attain this goal and, thus, able to limit HLB spread, and ongoing research in this regard is summarized in this review.

Engineering of citrus to obtain huanglongbing resistance.

Huanglongbing (HLB) disease is threatening the sustainability of citriculture in affected regions because of its rapid spread and the severity of the symptoms it induces. Herein, we summarise the main research findings that can be exploited to develop HLB-resistant cultivars. A major bottleneck has been the lack of a system for the ex vivo cultivation of HLB-associated bacteria (CLs) in true plant hosts, which precludes the evaluation of target genes/metabolites in reliable plant/pathogen/vector environments. With regard to HLB vectors, several biotechnologies which have been proven in laboratory settings to be effective for insect control are presented. Finally, new genotypes that are resistant to CLs or their insect vectors are described, and the most relevant strategies for fighting HLB are highlighted.

Temperature-Dependent Compatible and Incompatible Pollen-Style Interactions in Citrus clementina Hort. ex Tan. Show Different Transglutaminase Features and Polyamine Pattern.

In clementine, failure of fertilization can result in parthenocarpic fruit development, which has several advantages, such as seedless fruit, longer shelf-life, and greater consumer appeal. Recently, S-RNases have been identified in Citrus grandis, thus revealing that the self-incompatibility (SI) reaction relies on the S-RNase gametophytic mechanism. The fundamental role of environmental factors, mostly temperature, in determining the numbers of pollen tubes reaching the ovary is also well established in Citrus. In the present work, temperature-dependent pollen-pistil interactions in C. clementina were analyzed, focusing on several morphological aspects, as well as on polyamine (PA) content and the activity and distribution of transglutaminase (TGase), both reported to be involved in the SI response in pear and in pummelo. Results clearly indicate that temperature contributed to a different activation of the SI response, which occurs at optimal temperature of 25°C but was by-passed at 15°C. TGase activity was stimulated during the SI response, and it localized differently in the compatible and incompatible interaction: in compatible pollinated styles, TGase localized inside the style canal, while it was detected all around it in incompatible crosses. TGase localization and activity were congruent with the levels of soluble and insoluble conjugated PAs and with morphological evidences, which highlighted cell wall modification occurring as a result of SI.

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.