CitRWP May Not Be the Major or Only Major Gene That Regulates Apomixis in Citrus and Its Close Related Genus

Abstract Background: Nucellar embryony in citrus and its closely related genera is a form of apomixis that interferes with crossbreeding and impacts genetic evolution and taxonomy in citrus. Numerous hypotheses have been proposed regarding the genetic regulation of apomixis in citrus and its close relatives; however, none have identified the precise regulatory sequence until the recent discovery of the candidate gene, citRWP. Nevertheless, the evidence supporting citRWP as the primary or sole regulatory gene remains inconclusive. Results: First, the M/P (Monoembryonic/Polyembryonic) marker, designed based on the upstream sequence of citRWP, was used to assess whether the marker exhibited consistent segregation across different embryonic genotypes. Results showed that all sweet orange selections, whether monoembryonic or polyembryonic, contained the P-marker. However, the P-marker was not detected in polyembryonic Poncirus, and a novel band pattern appeared in nine polyembryonic progenies of W-Murcott × Flying Dragon trifoliate orange. Subsequently, CitRWP expression levels were analyzed using RT-PCR and Real-time RT-PCR. RT-PCR revealed that CitRWP was expressed in the ovules of all tested mono- or poly-embryonic varieties. Real-time RT-PCR results for ovules were consistent with those from RT-PCR, with the lowest expression observed in monoembryonic pummelo and no significant difference between polyembryonic and monoembryonic sweet orange cultivars. In the hybrid offsprings, the expression of the CitRWP gene showed an extreme two-stage differentiation. In young leaf and ovule of polyembryonic and monoembryonic sweet orange cultivars, the expression of the CitRWP gene were different, with a higher expression level in young leaf and a lower expression level in ovule of polyembryonic cultivars. Finally, CitRWP cDNA sequences from ovules were cloned and sequenced, revealing significant variation in Poncirus, including SNPs in the start or stop codon and fragment insertions/deletions that led to incorrect translation. Similar variations were also observed in monoembryonic/polyembryonic sweet orange cultivars and pummelo varieties. Conclusion and Perspectives: CitRWP was not consistently distributed or expressed across the different embryonic genotypes tested, suggesting it may not be the primary or sole gene regulating apomixis in citrus and its close relatives. To improve our understanding of the complex mechanisms governing nucellar embryony in citrus, a broader range of genotypes and additional candidate genes should be investigated.