DataSheet_1_The origin and the genetic regulation of the self-compatibility mechanism in clementine (Citrus clementina Hort. ex Tan.).docx

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.

自交不亲和性（Self-incompatibility, SI）是显花植物中普遍存在的一种遗传机制，用于阻止自交受精。在柑橘属物种中，多个柚、柑橘及其近缘种质均表现出自交不亲和特性。在此类物种中，自交不亲和性伴随程度不一的单性结实特性，可产出无籽果实——这一性状深受消费者青睐。
在柑橘属中，近期研究证实存在基于S-核糖核酸酶（S-ribonucleases, S-RNases）的配子体型自交不亲和系统，该系统可抑制自花花粉管在花柱中上段的生长。本研究结合聚合酶链式反应（Polymerase Chain Reaction, PCR）分析与下一代测序技术，对克里曼丁橘（Citrus clementina (Hort. ex Tan.)）、自交不亲和的‘科穆内’克里曼丁橘及其自交亲和自然突变体‘蒙雷阿尔’的S基因型中S7-与S11-核糖核酸酶的存在情况进行了明确。
本研究首次发布了‘蒙雷阿尔’克里曼丁橘的参考基因组，为新发现的S7-核糖核酸酶的遗传序列分析提供了更为可靠的研究基础。通过单核苷酸多态性（Single Nucleotide Polymorphism, SNP）发掘分析结合检测到的变异注释，本研究在‘蒙雷阿尔’克里曼丁橘与克里曼丁橘参考基因组的比对中，共鉴定出7781个单核苷酸多态性位点，这些位点涉及5661个基因。
对两种克里曼丁橘基因型的成熟未授粉雌蕊进行转录组分析后发现，‘蒙雷阿尔’中S7-核糖核酸酶无表达，这表明该酶的缺失与自交不亲和响应的丧失相关。通过RNA测序（RNA-seq）分析结合基因本体（Gene Ontology, GO）注释研究，本研究共鉴定出2680个差异表达基因（differentially expressed genes, DEGs），其中大量基因参与氧化还原酶活性与跨膜转运过程。
整合DNA测序与RNA转录组数据后，本研究鉴定出164个差异表达基因，这些基因均携带至少一个经预测可对其氨基酸序列产生强效影响的单核苷酸多态性位点。其中，针对两个类Agamous MADS-box蛋白、MYB111以及MLO类蛋白12的4个候选基因得到了验证。此外，转录因子MYB111似乎可结合S7-与S11-核糖核酸酶基因上游2.0 kb的序列区域。
本研究结果为自交不亲和性的遗传基础提供了有价值的参考，表明S7-核糖核酸酶序列中存在的单核苷酸多态性位点可能参与调控该酶的差异表达，进而影响自交不亲和机制的运作。