JIA-2019-11

2553 LI Liu et al. Journal of Integrative Agriculture 2019, 18(11): 2549–2560 7355 nt), ORF3 (363 nts, 7357 to 7719 nt) andORF4 (228 nts, 7628 to 7855 nt) consisted of a triple gene block (TGB) and encoded proteins (TGBps 1–3) with molecular masses of 25, 12 and 7 kDa, respectively. ORF5 (1 212 nts, 7 927 to 9138 nt), encoded a CP with a molecular mass of 42 kDa. Although the CP sizes among ASPV isolates were highly variable, the CP gene of ASPV-LYC had the same size with that of AGCaV isolates Aurora-1 and CYD. 3.2. Sequence comparison between ASPV-LYC and available ASPV and AGCaV isolates The genome sequence of ASPV-LYC was 77% identical to that of ASPV PA66 (size 9332 nts, accession no. D21829.2), the well-characterized ASPV isolate identified as the type member of the genus (Jelkmann 1994), and shared the highest nucleotide identity of 81.8% with an isolate HB- HN1 from pear and the lowest nucleotide identity of 73.1% with isolate Hannover available in GenBank database (Table 2). Its 5´-UTR was highly conserved and its 3´-UTR was divergent by sharing 82.8–100% and 67.9–83.3% nt identities with the corresponding regions of other isolates, respectively (Table 2). Similarly, its ORF1 also showed the highest nucleotide and amino acid sequence identities of 86.0 and 94.8% with HB-HN1 (Table 2). However, its ORF2–5 always showed the highest nucleotide and amino acid identities with that of AGCaV isolates Aurora-1 or CYD (Table 2). Notably, its ORF5 even showed the lowest nucleotide (66.4%) and amino acid sequence (69.3%) identities with that of HB-HN1. Meanwhile, to understand whether the molecular variants of ASPV similar to isolate LYC presented in any other pear trees grown in China, the complete CP genes of ASPV isolates were amplified using primer set F7/R7 from leaf samples of 17 pear trees grown at Hubei, Fujian, Yunnan and Shandong provinces, China. Sequence comparison of CP genes of these isolates together with isolate LYC, 32 ASPV isolates reported previously from our group (Ma et al . 2016) and all ASPV isolates available in GenBank showed that only one isolate from a pear plant grown in Fujian Province, named as FJ-12 (accession no. MG763896.1), had relatively high similarity with the isolate LYC by sharing 84.3% nt and 87.8% aa sequence identities. The result suggested that ASPV isolates with molecular characteristics like ASPV-LYC were rarely found in other cultivated pear plants. Phylogenetic trees were generated from the nucleotide sequences of full genome and ORFs 1–5 of ASPV-LYC and ASPV isolates referred from GenBank (Appendix A). In the full genome-based (Fig. 1-A) and ORF1-based trees (data not shown), ASPV-LYC showed the closest phylogenetic relationship with a pear isolate HB-HN1 (accession no. KU308398.1). The two isolates clustered into the same clade together with isolates KL1 (accession no. JF946775.1) and KL9 (accession no. JF946772.1) from ‘kurle’ pear ( P . sinkiangensis ), but clearly in two separate sub-clades. In the tree based on the CP gene (ORF5) sequences of ASPV isolates used to generate the full genome-based phylogenetic tree and representative isolates (Fig. 1-B), Table 2 Sequence comparison of complete genome and different genomic regions between Apple stem pitting virus isolate ASPV-LYC and 16 ASPV isolates and two apple green crinkle associated virus (AGCaV) isolates (Aurora-1 and CYD) referred from NCBI database Host Isolate Genome 5´ UTR ORF1 (Rep) ORF2 (TGB1) ORF3 (TGB2) ORF4 (TGB3) ORF5 (CP) 3´ UTR nt % nt % nt% aa% nt% aa% nt% aa% nt% aa% nt% aa% nt % Pear LYC 9 237 – 59 – – – – – – – – – – – 135 – Apple Aurora-1 9 266 79.8 60 100 79.3 92.4 83.8 96.0 84.0 95.0 89.7 87.1 77.4 79.2 131 76.0 Quince CYD 9 266 76.9 60 95 76.1 80.4 86.0 95.1 81.0 85.8 80.3 74.7 74.5 74.2 132 68.2 Apple PB66 9 363 77.0 60 98.3 79.9 92.7 77.1 91.0 79.3 89.2 85.1 89.3 68.3 73.4 135 76.7 IF38 9 293 73.9 60 98.3 76.1 85.1 75.6 89.2 78.5 86.7 85.1 86.7 67.2 72.4 132 83.3 PA66 9 332 76.0 59 98.3 78.5 91.1 77.4 89.7 81.0 90.0 83.6 85.7 67.1 71.4 132 73.8 Palampur 9 267 75.5 29 82.8 78.4 89.9 75.6 89.2 79.3 88.3 85.1 86.7 68.2 72.9 135 67.9 YL 9 262 75.7 33 97.0 77.9 91.6 76.2 90.1 80.2 90.8 86.4 78.7 68.3 73.9 132 72.7 YT 9 270 75.8 59 100 78.0 91.2 76.9 89.7 78.8 88.3 84.5 87.1 68.1 73.1 122 72.7 PM8 9 284 74.3 60 96.6 76.8 86.0 78.4 91.0 79.3 89.2 84.2 82.7 67.8 72.1 131 78.5 Hannover 9 324 73.1 60 94.9 74.3 86.2 79.0 91.9 75.2 85.8 83.6 81.4 66.9 72.1 132 75.6 GA2 9 358 78.9 60 96.6 80.7 92.8 82.7 96.4 80.2 82.7 86.0 82.7 69.0 73.1 134 78.0 GA4 9 276 77.5 59 100 79.4 92.6 79.3 91.5 79.3 84.3 84.0 84.3 71.2 72.6 134 74.2 AKS 9 296 76.4 59 100 78.8 92.1 77.8 91.9 82.6 84.0 83.8 84.0 67.2 69.3 128 75.8 XC 9 286 74.2 60 98.3 75.5 88.6 76.8 89.7 79.6 85.0 82.0 82.7 67.2 71.4 132 78.8 Pear KL9 9 265 77.6 33 100 80.1 92.0 77.8 91.5 77.7 82.5 84.0 81.4 66.9 71.7 130 72.3 KL1 9 265 77.2 33 100 79.7 91.9 77.4 91.0 79.6 86.7 83.6 84.3 67.3 71.9 130 76.2 HB-HN1 9 270 81.8 60 98.3 86.0 94.8 76.2 91.0 78.5 87.5 86.0 85.3 66.4 69.3 129 73.5 YLX 9 291 75.7 27 96.3 77.8 91.0 76.5 90.6 81.0 91.7 85.1 82.7 66.6 68.9 133 76.5