Identification of three novel S-RNase alleles in Eriobotrya Lindl. and new evidence for the taxonomic status of E. prinoides var. dadunensis

Abstract: 【Objective】Loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus encoding a polymorphic stylar ribonuclease (S-RNase) . The S-genotype is therefore an important consideration in breeding strategies. However, there has been no previous study dealing with assessement of the S-alleles in wild Eriobotrya species. Therefore, the objective of this study is to determine the S-genotypes of three loquat species, including E. japonica, E. prinoides var. dadunensis, and E. prinoides, in order to provide the scientific basis for the germplasm innovation and taxonomic reseach of E. prinoides var. dadunensis.【Methods】Using two pairs of primers designed according to conserved nucleotide sequences of known S-RNase alleles in Malus (apple) . These primers included a forward primer localized in the C1 region (‘FTQQYQ': 5'-TTTACGCAGCAATATCAG-3') , and two reverse primers localized in between the hypervariable region (HV) and the C3 (‘IIWPNV': 5'-ACRTTCGGCCAAATMATT-3') and C5 (‘FI (D/N) CP (H/R) ': 5'-GYGGGGGCARTYTATGAA-3') conserved regions. PCR amplification was carried out of genomic DNAs of three loquat species. Amplified products were separated by electrophoresis on 2.0% agarose gels, stained with ethidium-bromide (0.5%) , photographed using the UVP-gel documentation system (EC3 System; UVPCo., Upland, CA, USA) . A 100-bp DNA ladder (Tiangen Biotech Co. Ltd., Beijing, PR China) was used for estimating the molecular sizes of the amplicons. Reproducible amplified target fragments were purified using a DP210 DNA gel extraction kit (Tiangen Biotech Beijing, China) , were cloned into the pGEM-T Easy Vector (Promega Corporation, Australia) , were transformed into Escherichia coli, identified by colony PCR, and then were bidirectionally sequenced by BGI Life Tech Co., Ltd. (Beijing) . The obtained nucleotide and putative amino acid sequences were searched against NCBI using BLASTn and BLASTx to identify homologous genes and putative intron sequences. The deduced protein sequences between the C1 and the C5 conserved regions were obtained, and the structures were analyzed using DNAMAN and compared with the available Malus sequences on GenBank. The sequences were then aligned using CLUSTALW.【Results】In this investigation, a total of twelve DNA bands, with fragments ranging from 468 to 773 bp in size, were obtained from the sequencing, and were classified into four different S-RNase alleles based on the BLASTn results, two of the twelve DNA fragments were identical to S2. The remaining ten DNA fragments amplified did not match any known reference alleles, but showed 48.64 to 93.60% similarity at the nucleotide level and 51.80 to 99.40% similarity at the amino acid level with S-RNase alleles previously documented in E. japonica species. These sequences possessed the typical structural features of S-RNase alleles in Pyrinae, including an HV region and five conserved regions (C1, C2, C3, RC4, and C5) . Thus, these sequences were deemed to be alleles of S-RNase genes, and were selected for further characterization. The remaining ten DNA fragments were classified into three different S-RNase alleles based on 100% shared sequences identify between the deduced amino acid sequences and the other fragments. The three S-RNase alleles had lengths of 635, and 773 bp, respectively, and the introns of these fragments were detected within the HV region and ranged in size from 147-245 bp, which is consistent with the position of the region in the loquat S-RNase alleles from BLASTn and also confirms that it accord with the GT-AG rule. Additionally, almost all of these S-RNases shared the four cysteine residues characteristic of S-RNase, and two histidine residues essential for RNase in Rosaceae. We thus suggested that these fragments would constitute novel S-RNase alleles and labeled them accordingly S26-RNase, S32-RNase, and S34-RNase, and their accession numbers were MG765271、MG846012 and MG812504 in GeneBank respectively. We detected four S-RNase alleles in the three loquat species, including the three novel alleles. This finding suggests that wild Eriobotrya species might exhibit high levels of S-allele genetic diversity, and the findings would be indicative of the high immigration rate of new S-alleles, implying their potential value in genetic breeding. Furthermore, the wild E. japonica contains S2 and S26, the former being a common S-RNase allele in loquat cultivars, suggesting that it might constitute the ancestor to various loquat cultivars. Interestingly, E. prinoides var. dadunensis contains S26 and S32, two novel alleles that were most probably inherited from E. japonica (wild species) (S2 S26) and E. prinoides (S32 S34) , supporting previous findings that suggested that E. prinoides var. dadunensis might be a hybrid of E. prinoides and E. japonica (wild species) .【Conclusion】Four S-RNase alleles were identified from the isolated S-RNase genes in three loquat species, including one previously reported S-RNases (S2) and three novel S-RNase alleles (S26, S32, and S34) . The total number of alleles observed in the present study indicated higher S-allele diversity than expected. The S-genotypes of the three tested species were identified as follows: E. japonica (S2 S26) , E. prinoides var. dadunensis (S26 S32) , and E. prinoides (S32 S34) . The homology and phylogenetic analysis indicated that the E.prinoides var. dadunensis might be a hybrid from E. japonica and E. prinoides.