Identification and characteration of hydroperoxide lyase (HPL) gene family in seven species of Rosaceae

Abstract：【Objective】HPL is not only related to the formation of aroma of multiple fruits and vegetables, but also plays a core role in inducing plant defense as part of the metabolic pathway of lipoxygenase, and is related to physiological processes such as plant resistance to disease, insect, injury, stresses , storage and aging. This study used bioinformatics method to identify HPL gene family of Rosaceae and analyze the characteristics of HPL, so as to have a better understanding of the HPL gene family of Rosaceae, and provide clear candidate genes for the improvement of Rosaceae varieties at the molecular level.【Methods】By known Arabidopsis HPL genes and their encoded protein sequence，we retrieved the genome database of 7 Rosaceae species including Pyrus bretschneideri, Malus domestica, Prunus persica, Fragaria vesca, Pyrus communis, Rubus occidentalis and Prunus mume to identify the HPL candidate genes. Then NCBI was used to compare the candidate sequences for conserved domains. Protparam was used to predict the physicochemical properties of HPL proteins. Then all HPL gene structures and motifs were analyzed by online software GSDS 2.0 and MEME. ClustalW was employed to perform multiple sequence comparison analysis on the amino acid sequences of the obtained HPL gene family, and phylogenetic trees was generated with neighbor-joining method by MEGA 6 software. Nine nodes were selected from the protein phylogenetic tree, the value of dN/dS (ω) for each node was calculated by the CODEML program of PAML 4 software, and the“site-specific”model was used to identify the amino acid sites experiencing positive selection. MCScanX was used to detect the collinearity of theHPL gene. Futhermore, HPL gene expression of pear and apple were examined.【Results】In this study we identified 33 HPL homologous genes, 6 from pear, 4 from apple, 3 from peach, 5 from strawberry, 4 from European pear, 6 from black raspberries, 3 from mume and 2 from Arabidopsis thaliana. MEME results showed that 31 HPL genes contained 10 motifs, and only AT4G15440.1 lacked motif1 and motif3, indicating that HPL gene family members were highly conserved during evolution, and member expansion was mainly related to whole genome replication events. Analysis of all HPL gene structures showed that the HPL gene had a simple structure and most HPL genes contained one or three CDS. Phylogenetic trees divided the HPL genes of Rosaceae species and Arabidopsis into three subgroups. Subgroup B and C contained one Arabidopsis HPL gene. Subgroup A and C contained HPL genes from all seven Rosaceae species, and subgroup B contained HPL genes from six Rosaceae species. Among the 9 tested nodes, 7 nodes showed significant positive selection, indicating that positive selection played an important role in the evolution of HPL genes. Among these HPL genes, the distribution of positive selection sites estimated by Bayesian method under the M8 model was very scattered and the rule was not obvious, which indicated that point mutations with positive selection effect played an indispensable role in the evolution of HPL gene family. Nine collinearity relationships between white pear and six other species of Rosaceae were identified by MCScanX, including three pairs of collinearity of peach and mei, two pairs of collinearity of European pear and one pair of collinearity of apple. This was also related to the shorter differentiation time of these species. The expression results of pear and apple showed that the expression level of Pbr041820.1 of pear in subgroup C was 2- 6 times higher than that of Pbr020059.1 of subgroup B in different periods, and 4 HPL in subgroup A was not expressed. In apple HPL expressed more in wild material than that of cultivar. Apple MDP0000132456 was located in the A subfamily, which was hardly expressed. And the MDP0000424398 and MDP0000225501 in the B subfamily were expressed.【Conclusion】In this study, a total of 31 HPL genes were identified from seven species of Rosaceae. Each species no less than 3 genes. Positive selection had significant effect on HPL gene. Mdp0000424398, mdp0000225501 and pbr041820.1 of apple and pear were active expression genes. The active HPL gene of pear belonged to C subgroup, the active HPL gene of Apple belonged to B subgroup.