Isolation and Characterization of <i>S-RNase</i>-homologous Genes Expressed in Styles in ‘Hyuganatsu’ (<i>Citrus tamurana</i> hort. ex Tanaka)

Abstract

In this study, S-RNase-homologous genes expressed in styles were isolated from a gametophytic selfincompatible citrus cultivar, 'Hyuganatsu'. Sweet orange and clementine genome databases were searched to identify 13 ribonuclease T2 (T2 RNase) genes. Further blast searches using citrus EST databases were conducted with these 13 sequences as queries to obtain five additional EST sequences. Known T2 RNase genes, including the S-RNases of Rosaceae, Solanaceae, and Plantaginaceae were retrieved from the public database. All data collected from the databases were combined to make a dataset for phylogenetic analysis. From the phylogenetic tree, 10 citrus sequences were found to be monophyletic to the clade of S-RNases. Degenerate primers were designed from these genes to identify similar sequences in cDNA derived from 'Hyuganatsu' styles. RT-PCR and 3' and 5' RACE resulted in the isolation of three S-RNase-like sequences; these sequences were further characterized. Pistil-specific gene expression was confirmed for all sequences by RT-PCR; Citrus tamurana RNase1 (CtRNS1) and RNase3 (CtRNS3) had basic pI values (> 8) and molecular weights of approximately 25 kDa, consistent with S-RNase features of other families. The distribution of CtRNS1, CtRNS2, and CtRNS3 was investigated using 10 citrus cultivars. CtRNS1 and CtRNS2 did not correspond to any S-haplotypes in the 10 cultivars, while CtRNS3 matched the citrus' S 1-haplotype.

Self-incompatibility Related to Seedless Fruit Production in <i>Citrus</i> Plants

“…Thus, they are considered distinct from S-RNases (Honsho et al, 2021). The previously reported homologous genes of S-RNase, CtRNS1 (Honsho et al, 2019b) and CgRNS3 (Liang et al, 2017), were placed in low polymorphic subclusters 1 and 2, respectively, both of which are non-S-RNase subclusters (Fig. 2).…”

“…It is reasonable to hypothesize that citrus SI is based on the S-RNase system because several phenotypic features of citrus SI, such as pollen tube behavior in the SI reaction, are similar to those of known S-RNase-based GSI plants. The S-RNase homologs from citrus plants were cloned after overcoming many challenges (Chai et al, 2011a;Honsho et al, 2019b;Liang et al, 2017;Miao et al, 2011a). Although some genes did not show tissue-specific expression (Chai et al, 2011a;Miao et al, 2011a), indicating that they are inappropriate as S-RNase genes, Liang et al (2017) identified a tissuespecific S-RNase homolog, named CgRNS3, from 'Shatian' pummelo based on information obtained from the genome sequence of sweet oranges (Xu et al, 2013).…”

“…Although some genes did not show tissue-specific expression (Chai et al, 2011a;Miao et al, 2011a), indicating that they are inappropriate as S-RNase genes, Liang et al (2017) identified a tissuespecific S-RNase homolog, named CgRNS3, from 'Shatian' pummelo based on information obtained from the genome sequence of sweet oranges (Xu et al, 2013). Honsho et al (2019b) also cloned three S-RNase homologs from 'Hyuganatsu', CtRNS1-CtRNS3, show-ing specific expression in the female organ.…”

SKP1-like protein, CrSKP1-e, interacts with pollen-specific F-box proteins and assembles into SCF-type E3 complex in ‘Wuzishatangju’ (Citrus reticulata Blanco) pollen

“…In ‘Shatian’ pummelo ( C. grandis ), CgRNS3 possessed several common characteristics of the pistil determinant of SI and was specifically expressed in pistil ( Liang et al, 2017 ), however, it was not anchored into the S-locus ( Liang et al, 2020 ). Pistil-specific CtRNS3 from Citrus tamurana showed S 1 -genotype-dependent in different cultivars, implying that CtRNS3 may serve as the S 1 - gene ( Honsho et al, 2019 ). Liang et al (2020) studied the S-locus in pummelo and concluded that S-RNase and SLF mediate SI reaction in Citrus .…”

Supplemental Information 9: Sequence alignment of CUL1 genes

“…In 'Shatian' pummelo (C. grandis), CgRNS3 possessed several common characteristics of the pistil determinant of SI and was specifically expressed in pistil (Liang et al, 2017), however, it was not anchored into the S-locus (Liang et al, 2020). Pistil-specific CtRNS3 from Citrus tamurana showed S 1 -genotype-dependent in different cultivars, implying that CtRNS3 may serve as the S 1-gene (Honsho et al, 2019). Liang et al (2020) studied the S-locus in pummelo and concluded that S-RNase and SLF mediate SI reaction in Citrus.…”