Abstract
Cherry laurel (Prunus laurocerasus L.) is an extreme polyploid (2n = 22x) species of the Rosaceae family where gametophytic self-incompatibility (GSI) prevents inbreeding. This study was carried out to identify the S-ribonuclease alleles (S-RNases) of P. laurocerasus using PCR amplification of the first and second intron region of the S-RNase gene, cloning and sequencing. A total of 23 putative S-RNase alleles (S1–S20, S5m, S13m, and S18m) were sequenced from the second (C2) to the fifth conserved region (C5), and they shared significant homology to other Prunus S-RNases. The length of the sequenced amplicons ranged from 505 to 1,544 bp, and similar sizes prevented the proper discrimination of some alleles based on PCR analysis. We have found three putatively non-functional alleles (S5m, S18m, and S9) coding for truncated proteins. Although firm conclusions cannot be drawn, our data seem to support that heteroallelic pollen cannot induce self-compatibility in this polyploid Prunus species. The identities in the deduced amino acid sequences between the P. laurocerasus and other Prunus S-RNases ranged between 44 and 100%, without a discontinuity gap separating the identity percentages of trans-specific and more distantly related alleles. The phylogenetic position, the identities in nucleotide sequences of the second intron and in deduced amino acid sequences found one or more trans-specific alleles for all but S10, S14, S18, and S20 cherry laurel RNases. The analysis of mutational frequencies in trans-specific allele pairs indicated the region RC4–C5 accepts the most amino acid replacements and hence it may contribute to allele-specificity. Our results form the basis of future studies to confirm the existence and function of the GSI system in this extreme polyploid species and the alleles identified will be also useful for phylogenetic studies of Prunus S-RNases as the number of S-RNase sequences was limited in the Racemose group of Prunus (where P. laurocerasus belongs to).
Highlights
Gametophytic self-incompatibility (GSI) is a genetic mechanism that enables the plant to differentiate among pollen grains to be accepted or rejected for fertilization
Since the second intron of the Prunus S-RNase is characterized by a considerable length polymorphism, the cherry laurel SRNase alleles were first amplified using the consensus primers PaConsII-F and PaConsII-R designed by Sonneveld et al (2003) for sweet cherry
The length of the amplicons ranged from approx. 500 to 3,000 bp and a maximum of seven bands were detected in a sample (Figure 1). It was remarkably lower than expected since owing to the high ploidy level of cherry laurel, a larger number of S-alleles was supposed to be amplified in each genotype
Summary
Gametophytic self-incompatibility (GSI) is a genetic mechanism that enables the plant to differentiate among pollen grains to be accepted or rejected for fertilization. Fertilization may only occur when the pollen S-allele is different from any of the S-alleles carried by the recipient pistil (McCubbin and Kao, 2000). The mechanism involves the interaction between pollen- and pistil-expressed proteins, both of which are encoded by a single polyallelic locus, the so-called S-locus. The protein accumulating in the stylar tissue is a ribonuclease enzyme called S-RNase (McClure et al, 1989), the pollen S-specificity is determined by an F-box containing protein (Tao and Iezzoni, 2010). The S-RNase is responsible for the rejection of the self-pollen by degrading the pollen RNA and hindering the growth of the pollen tube (McClure et al, 1990). Mating selection in the Prunus genus (Rosaceae family) is known to operate by the GSI mechanism, and many economically significant fruitbearing species exhibit self-incompatibility (SI) (Ashkani and Rees, 2016; Sassa, 2016)
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