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

<div><p>The pear (genus <i>Pyrus</i>) 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198512#pone.0198512.s006" target="_blank">S4 Table</a>). These results shed light on the genetic relatedness between Italian varieties and wild genotypes. Among the wild species, compared with <i>P</i>. <i>amygdaliformis</i>, few <i>P</i>. <i>pyraster</i> 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.</p></div

Polymorphism information for the nuclear and chloroplast markers.

<p>Polymorphism information for the nuclear and chloroplast markers.</p

STRUCTURE results.

<p>Inferred population structure: Bar plot generated by STRUCTURE according to the K = 2 model based on eight nSSRs.</p

PCA analysis.

<p>Two-dimensional PCA plot depicting the distribution of accessions over the first two PCs. Different colours represent the four groups of pears: international cultivar varieties (ICV, red squares), national cultivar varieties (NCV, orange dots), local varieties (LV, green crosses), and wild related species (RS, blue triangles). RS are further subdivided into <i>P</i>. <i>pyraster</i> (filled triangles) and <i>P</i>. <i>amygdaliformis</i> (empty triangles).</p

SSR markers used in this study.

<p>SSR markers used in this study.</p

Median-joining network analysis reveals relationships among individuals.

<p>Haplotypes have been calculated based on the four cpDNA markers. The pie charts highlight the relative frequency of individuals for each haplotype, exhibiting a clear prevalence (>90%) of one subpopulation (red = wild subpopulation, blue = cultivated subpopulation). Grey represents samples with more evident genetic admixtures.</p

Neighbour-joining analysis.

<p>Nj dendrogram calculated by employing both nSSR and cpSSR. The three major clusters (named A, B and C) are reported.</p

Table_4_The origin and the genetic regulation of the self-compatibility mechanism in clementine (Citrus clementina Hort. ex Tan.).xlsx

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.</p

Comparison of pear germplasm.

<p>Section of fruit and leaves of <i>P</i>. <i>amygdaliformis</i> (a), <i>P</i>. <i>pyraster</i> (b) and widespread cultivated traditional variety (‘Spineddu’; c) on Mount Etna (scale bar = 2 cm).</p

List of genotypes used in this study.

<p>List of genotypes used in this study.</p