Isolation and analysis of NBS-LRR analogs in Cerasus clarofolia

LIU Houyu;WU Minfang;QIAO Guang;WEN Xiaopeng;Institute of Agro-bioengineering, Guizhou University/College of Life Sciences, Guizhou University/The Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region [Ministry of Education];College of Forestry, Guizhou University/Research Center for Forest Resources and Environment of Guizhou Province;Agricultural and Rural Affairs Bureau of Qingzhen;

Cerasus clarofolia;;Resistance gene;;Nucleotide binding site-leucine rich repeat(NBSLRR);;Isolation;;Evolution

【Objective】A line of Cerasus clarofolia Schneid originated from Moutain Leigong(Guizhou Province, P. R. of China) demonstrates high resistance to crown gall of cherry. In order to understand the resistance mechanism to this disease, as well as to clone the candidate resistance gene, the NBS-LRR(nucleotide binding site-leucine rich repeat)analogs were cloned, and the evolution pattern was further analyzed.【Methods】Degeneracy primers based on the conserved domain of NBS-LRR were used to isolate NBS-LRR sequences from genomic DNA of C. clarofolia. An approximate 500 bp fragment was obtained by polymerase chain reaction(PCR), and then the aplicons were cloned, sequenced and analyzed. The inserted fragment in the p GEM-T easy vectors sequences was isolated using VecScreen online tools. Nucleotide acid and deduced amino acid sequences were analyzed via BLASTn and BLASTx(https://blast. ncbi. nlm. nih. gov/Blast. cgi). The length of nucleotide sequence and AT/CG proportion were estimated using DNA star software. Multiple sequences alignment was generated by Clustal W and visualized using BoxShade. The phylogenetic tree from nucleotide acid and deduced amino acid sequences was constructed using Mega 6.0 program. The nonsynonymous substitutions(Ka) and synonymous substitutions(Ks) were estimated using Dnasp5.0.【Results】A total of 36 unique clones from NBS-LRR resistance gene analog(RGAs) were isolated, which were named as PpRGA1-PpRGA36.The sequences were submitted to the GenBank with the accessions as ACK102301-ACK102336. Nucleotide sequences ranged in length from 429 bp to 516 bp, and homology of the 36 sequences varied in range of 41.7% to 99.8% and 19.0% to 100.0% among nucleotide sequences and amino acid sequences,respectively, revealing rich diversity of NBS-LRR in this species. Three sequences contained deletion mutation, frameshift mutation or premature stop codons. AT/GC ratio was in range of 1.37(PpRGA18)-1.58(PpRGA10). The phylogenetic tree generated from nucleotide acid sequences might divide NBSLRR into four clades(Ⅰ-Ⅳ), clades Ⅰ-Ⅲ were classified as TIR type, and clade Ⅳ was classified as non-TIR type. As multiple amino acid sequences alignment, the sequences contained conserved domain motifs such as P-Loop, Kinane-2 and GLPL. According to the characteristics of Kinane-2 motif, 24 sequences were grouped into non-TIR, and 12 into TIR type. To learn more about the evolutionary relationships among PpRGAs, a total of 144 NBS-LRRs from GeneBank, whose homology was above 70%with each subclade of PpRGAs, were used to construct the phylogenetic tree with 36 PpRGAs. The result showed that 40 and 104 NBS-LRRs clustered with non-TIR and TIR, respectively. Of the nonTIRs, all the NBS-LRRs were from Rosaceae species such as P. persica, P. mume, C. avium, P. bretscheideri, Fragaria × ananassa, Malus domestica, Rubus galanucus. Of the TIRs, a total of 93 NBSLRRs from Rosaceae, and 11 NBS-LRRs from other families such as Juglandaceae, Leguminosae, Cucurbitaceae and Fagaceae. Among PpRGA specific clades, 17 P. persica, 13 P. mume, 5 P. avium and 1 P. kansuensis NBS-LRRs were grouped with 24 PpRGAs, indicating that C. clarofolia had more close evolutionary distance with P. persica, P. mume than P. avium, and those clades showed Prunus L. species-specific pattern, inferring the genes have undergone gene expansion after speciation of Prunus L.By considering the category of species clustered result among TIR and non-TIR type, we inferred that TIRs were more ancient than non-TIRs. Furthermore, by estimating average of Ka/Ks ratios among each clades, clade Ⅳ(belong to non-TIR) was greater than 1, cladeⅠand Ⅲ were less than 1 and cladeⅡ was approximately equal to 1, suggesting that there was an evolving process under a neofunctionalization, subfunctionalization and nonfunctionalization model driven by positive selection, purification selection and neutral selection, respectively. In addition, the distribution range of Ks values of TIR and non-TIR was calculated, and interestingly, TIR distributed in two regions, which ranged in 0-0.3 and0.8-12, and non-TIR distributed in range of 0-0.3, suggesting that duplication events of TIR occurred earlier than non-TIR. The frequency of Ks values commonly used to measure duplication scale, by calculation of obtained PpRGAs and TIR, was highly greater than that of non-TIR, indicating that TIR had more duplication events than non-TIR. In addition, 81% of Ks value frequency distributed in range of 0-0.3, and 64% of Ks frequency of non-TIR was in range of 0-0.1, suggesting that large-scale duplication occurred both in non-TIR and TIR in recent period. Finally, the relationship between Ks value and Ka/Ks was analyzed, and in each Ks value distribution region, the Ka/Ks value got smaller as Ks value increased.【Conclusion】Three clades of TIR and one clade of non-TIR were found in C. clarofolia NBSLRRs. The phylogenetic tree as well as homology and Ks value analysis showed TIR may originate from more ancient time than non-TIR, TIR of C. clarofolia got revolution relationship with P. persica,and P. mume was more closely related than C. avium. Both TIR and non-TIR had relatively large-scale duplication events that occurred in recent period. Ka/Ks value inferred that different selective pressure and duplication pattern existed between each clade of non-TIR and TIR, suggesting that those might have different evolutionary pattern during long-term history.