Data_Sheet_1_Confirmation of a Gametophytic Self-Incompatibility in Oryza longistaminata.docx

Oryza longistaminata, a wild species of African origin, has been reported to exhibit self-incompatibility (SI). However, the genetic pattern of its SI remained unknown. In this study, we conducted self-pollination and reciprocal cross-pollination experiments to verify that O. longistaminata is a strictly self-incompatible species. The staining of pollen with aniline blue following self-pollination revealed that although pollen could germinate on the stigma, the pollen tube was unable to enter the style to complete pollination, thereby resulting in gametophytic self-incompatibility (GSI). LpSDUF247, a S-locus male determinant in the gametophytic SI system of perennial ryegrass, is predicted to encode a DUF247 protein. On the basic of chromosome alignment with LpSDUF247, we identified OlSS1 and OlSS2 as Self-Incompatibility Stamen candidate genes in O. longistaminata. Chromosome segment analysis revealed that the Self-Incompatibility Pistil candidate gene of O. longistaminata (OlSP) is a polymorphic gene located in a region flanking OlSS1. OlSS1 was expressed mainly in the stamens, whereas OlSS2 was expressed in both the stamens and pistils. OlSP was specifically highly expressed in the pistils, as revealed by RT-PCR and qRT-PCR analyses. Collectively, our observations indicate the occurrence of GSI in O. longistaminata and that this process is potentially controlled by OlSS1, OlSS2, and OlSP. These findings provide further insights into the genetic mechanisms underlying self-compatibility in plants.

非洲长雄蕊野生稻（Oryza longistaminata）是起源于非洲的野生稻种，已有研究表明其具有自交不亲和（self-incompatibility, SI）特性，但其自交不亲和的遗传模式至今尚未明确。本研究通过自交授粉与正反交授粉实验，验证了非洲长雄蕊野生稻为严格自交不亲和物种。对自交授粉后的花粉进行苯胺蓝染色分析发现，尽管花粉可在柱头上萌发，但花粉管无法伸入花柱以完成授粉过程，由此确定其属于配子体自交不亲和（gametophytic self-incompatibility, GSI）类型。LpSDUF247是多年生黑麦草配子体自交不亲和系统中的S位点雄性决定因子，其编码产物被预测为DUF247家族蛋白。基于与LpSDUF247的染色体序列比对，我们在非洲长雄蕊野生稻中鉴定得到两个自交不亲和雄蕊候选基因OlSS1与OlSS2。染色体区段分析显示，非洲长雄蕊野生稻的自交不亲和雌蕊候选基因OlSP为位于OlSS1侧翼区域的多态性基因。OlSS1主要在雄蕊中表达，而OlSS2在雄蕊与雌蕊中均有表达。通过逆转录聚合酶链式反应（RT-PCR）与实时荧光定量逆转录聚合酶链式反应（qRT-PCR）分析发现，OlSP在雌蕊中特异性高表达。综上，本研究结果证实非洲长雄蕊野生稻存在配子体自交不亲和现象，且该过程可能由OlSS1、OlSS2与OlSP共同调控。本研究结果为阐明植物自交亲和性的遗传机制提供了新的理论参考。