**4.3. Pollen S structure**

*Pyrus* and *Malus* SRNases as well as the presence of *Prunus* RNase genes which cannot involved with GSI. The first clue for the cloning of *Prunus* S-RNase was obtained when N-terminal sequences of almond (*Prunus dulcis*) SRNase were reported [120]. By the N-terminal amino p sequences, sweet cherry (*Prunus avium*) [120] and almond [125] S-RNases were cloned. Currently, sequences of over 100 *Prunus* S-RNase alleles are actually deposited in GenBank.

The pollen S determinant in the S-RNase-based GSI in Rosaceae, Solanaceae, and Plantagina‐ ceae was discovered decades after the real stylar determinant, S-RNase. The subcentromeric location in the solanaceous and plantaginaceous S locus experienced had long prevented chromosome walking [23, 142]. The first clue for the identification in the pollen S was from the S locus in the Plantaginaceae. Sequencing analysis in the *Antirrhinum hispanicum* S locus exposed the presence of the pollen-expressed F-box gene (AhSLF for a. *hispanicum* S locus F-

Even though it was speculated that the F-box protein gene encoded the real pollen S, only one particular allele has been cloned. The S locus of *Prunus*, which is located right at the end of the linkage group 6 [20], was much more compact than those of Solanaceae and Plantaginaceae. Two separate groups in Japan successfully sequenced the real S locus of *Prunus* beginning from the S-RNase [22, 127]. Ushijima et al. (2003) did DNA sequencing and transcriptional analyses for the genomic areas that flank real almond (*P. dulcis*) S-RNase and identified polymorphic and non-polymorphic S locus F-box genes, called SFB of S haplotype-specific F-box gene and SLF for S locus F-box, respectively. At present, SLF continues to be referred to as SLFL1 [79] after the nomenclature of Entani et al. (2003). The options that come with SFB, including the high degree of allelic polymorphism, pollen-specific appearance, and the close physical distance to the S-RNase many supported that SFB may be the male determinant of GSI in almond. The same research group also found SFB in the cherry (*Prunus*) S locus in their attempt to compare the same S-RNase allele on SC and SI kinds of cherries, *R. cerasus* and *P. avium*, respectively [140]. Another study group in Japan reviewed the S locus place of a couple different S haplotypes in Japanese apricot (*Prunus mume*) and found some F-box genes [22]. Among them, SLF of S locus F-box, that includes a different name but can be orthologous to SFB throughout almond and cherries, shows a high level of allelic string diversity and was supposed to be a prospect of pollen S. The other F-box genes found, SLFL1, SLFL2, SLFL3 of SLF including gene 1, 2 and 3, respectively, showed far lower allelic string diversity. The pollen S candidate of Japanese apricot has been independently cloned in a study by Yamane et al. (2003d) and named differently as PmSFB. Since then, the *Prunus* pollen S was initially referred to by a couple of different words or terms such as ''SFB'' and also ''SLF''. We used ''SFB'' in this particular review to show the various features in *Prunus* SFB than the SLF in Solanacae

Direct evidence that S locus F-box gene adjustments of allele specificity in the pollen was from a transgenic research in *R. inflata* [109]. This kind of experiment employed a well-known

**4.2. Pollen S determinant**

170 Plants for the Future

and Plantaginaceae.

*4.2.1. Identification of F-box gene*

box) located 9 kb downstream of S2-RNase [65].

SFB has just a single intron inside 50 untranslated places, where zero intron was within solanaceous and plantaginaceous SLFs. Although intron dimension varies together with different alleles, the difference inside the intron dimension is too small to be detected the intron length polymorphism for for S genotyping. Hence, fluorescent primers and an automatic sequencer were used to detect the real intron period polymorphism of S genotyping depending on SFB alleles [128]. Nonetheless, because fairly large-scale recombinational research using 1022 meioses confirmed no recombination involving the Prunus S-RNase and SFB [46] and S-RNase genotyping is often much easier, SFB genotyping can be used as a supplementary research for S haplotype determination. SFB research, on the contrary, is usually a powerful tool as well as the sole way to detect SC pollen-part mutant (PPM) S haplotypes, where only the real pollen S continues to be mutated [47-48, 126].

Another essential auxiliary examination of SFB uncovered the event of a few variables (V1 and V2) and two hypervariable (HVa and HVb) territories [47]. These hypervariable regions appeared to be hydrophilic or at least not strongly hydrophobic, which suggests that these regions may be exposed on the surface and function in the allele specificity of the recognition response. The fact that positively selected sites appear to concentrate in the variable and hypervariable regions further supports the possibility that these regions could play an important role in the SC/SI recognition.

Phylogenetic analyses with F-box genes inside *Arabidopsis thaliana* genome indicated that *Prunus* SFB, Petunia SLF, and Antirrhinum SLF might have a monophyletic beginning [134], as continues to be reported for that pistil S-RNase [45, 116]. Vieira et al. (2009) also concluded that there's no effective evidence to declare that the pollen S was independently recruited many times while in evolution. Nonetheless, phylogenetic reconstructions in the pollen S across all three families highlight numerous differences. As opposed to the phylogenetic tree of the S-RNase and S-like RNases of plants, the pollen-determinant F-box genes of a given family or genus are more closely related to its own S locus F-box genes that have no role in SI than to the pollen-determinants of the other families or genera [105, 126, 136, 79].

Another difference is available in the allelic string diversity in SFB of *Prunus* and SLF in the Solanaceae and also Plantaginaceae [92]. Deduced amino p sequences via SLF alleles prove as extremely excessive high pairwise identities of over 90%. When it comes to plantaginaceous alleles, the actual pair wise identities about SLF alleles are 97% and 99% [144], while those of the respective S-RNase alleles are 30% and 60% [139]. On the contrary, the pair wise allelic string identities on Prunus SFB alleles is lower and a lot like those of S-RNase alleles; the degrees of divergence is comparable around 60–90% [45, 92]. Nonetheless, the phylogenetic associations among SFB are generally incongruent together with one of several S-RNases for the same S haplotype, regardless of the expectation of co-evolution in the pollen and pistil determinants [46, 92]. In Solanaceae and Plantaginaceae, it usually is plausible to consider that more allelic string identities can be achieved within SLF compared with SFB of *Prunus* and can show faster evolutionary heritage. Interestingly, the S-RNase of Solanaceae and also Planta‐ ginaceae show higher degrees of allelic string diversity when compared with that of *Prunus*. Therefore, it is clear the *Prunus* S locus and also solanaceous and plantaginaceous S loci evolved in a variety of ways.
