Sequence characterization and evolutionary analysis of S-RNase gene among five genera Pomoideae

【Objective】In order to avoid self-pollination, self-incompatibility is a common phenomenon in 5 genera in Pyridae fruit trees during evolution. In order to understand the latest isolation and identification of S-RNase gene in pear incompatibility, we analyzed its sequence characteristics and evolutionary rules.【Methods】The keywords Malus S-RNase complete cds, Pyrus S-RNase complete cds, Cydonia S-RNase complete cds, Crataegus S-RNase complete cds and Eriobotrya S-RNase complete cds were searched for the full-length CDS sequence of pear S-RNase gene in the GenBank database of NCBI. And the Blast tool was use to search and supplement. After the initial identification of the searched sequences, the sequence analysis was performed using VectorNTI11.5.3 software, and the duplicate sequences were manually corrected and eliminated. The Find Best DNA/Protein Models program of MEGA11 software was used to find out the optimal model suitable for the sequence of 5 genera of Pyridae S-RNase gene, and the corresponding model and algorithm were used to calculate the differentiation between the sequences. Tajima's Test of Neutrality under Selection was used to calculate genetic polymorphisms, and the sequences of signal peptide, C1-C5, HV region and other relevant regions were analyzed respectively. The nucleotide composition and Compute codon usage bias programs of MEGA11 software were used to calculate the base composition and relative synonymous of the SRNase gene sequence of 5 genera of Pyridae codon usage value. The ClustalW program of MEGA11software was used to compare the CDS full-length sequence of 5 genera in Pyridae S-RNase gene. The phylogenetic tree of gene coding region was constructed by NJ in distance method, and the reliability test was performed 1000 times by Bootsrap. SPSS22 was used to cluster the S-RNase gene RSCU of 5 genera of Pyridae using Euclidean square distance as inter- gene evolutionary distance.【Results】At present, 120 CDS full-length sequences of 5 genera in Pyridae S-RNase gene were included in GenBank database, and 90 CDS sequences were removed. There were 51 genes from pears, 35 genes from apples, 1 gene from quince, 1 gene from loquat and 2 genes from hawthorn. The sequence analysis showed that the total length of CDS sequence of the S-RNase gene in 5 genera in Pyridae was 678- 711 bp. There were 1 gene length of 678 bp, 17 for 681 bp, 26 for 684 bp, 33 for 687 bp, 6 for 690 bp, 7 for 699 bp, and 1for 711 bp. The polymorphism of the S-RNase gene signal peptide, HV, C3-C4 sequence length of pear subfamily showed certain polymorphism, and the HV region had the largest change, followed by the signal peptide region, C3- C4, and the rest of the sequence length did not change. The signal peptide region was mainly replaced, followed by deletion and insertion. The deletion of HV region was much more than the insertion. There was also an insertion of C3-C4. The intron sequence size of the HV region ranged from 109 bp to 3130 bp, which had rich length polymorphism. The A, T, C and G components of the S-RNase gene in 5 genera of Pyridae were significantly different, and the conversion/transmutation ratio was less than 1 in only C2-HV and HV regions, and greater than 1 in other regions. Considering the number of positives, the two indexes of genetic polymorphism Θ, π and Tajima test statistic D, the C2-HV, C4-C5 and C5- regions might be involved in the identification of pollen S gene. The pear subfamily S-RNase did not form distinct genetic differentiation by genus or species. The differentiation distance between apple and pear was smaller than that within the genus, and the distance between apple, pear and hawthorn was the closest. The S-RNase gene of the pear subfamily also did not form a distinct species- specific boundary, and was found in hawthorn, apple, begonia, and flowering begonia P. pyrifolia, P. bretschneideri, P. communis and P. ussuriensis showed that the genetic differentiation distance between species was smaller than that between species. The codon use preference analysis showed that there were 5 high frequency codons and 20 preference codons in 5 genera in Pyridae S-RNase gene, and the stop codon was TAA. The content of bases at the first and second positions of the codon was relatively consistent, that is, A was dominant, and T was dominant at the third position, and the bases at the three positions were A+T greater than C+G. The four bases were not randomly arranged in this gene, and there was a certain bias. The phylogenetic tree constructed by the CDS sequence and codon of S-RNase genes of 5 genera of Pyridae showed that the S-RNases of different genera or species clustered together first, without obvious species or genus clustering.【Conclusion】At present, a total of 90 CDS full-length sequences of the S-RNase genes of 5 genera of Pyridae without duplication were recorded in GenBank. It was found that in addition to the recognized HV region with pollen recognition features, other regions also showed similar features; The phylogenetic tree generated by gene coding sequence and codon preference showed no genus or species-specific clustering, indicating that the S-RNase gene differentiation in 5 genera of Pyridae was earlier than that among different genera. The codon preference reflected the abundance of transfer RNA and the frequency of different nucleotides used in genes to some extent. Most of the S-RNase codons ending with C and G bases had low RSCU value, indicating that the four bases would not be randomly arranged in this gene, and there would be a certain bias, and the codon ending with A/T would be preferred.