Genetic diversity and evolution of Apple stem pitting virus isolates from pear in China

Figures & data

Table 1. ASPV incidence on apple and pear trees surveyed in this study in different areas.

Geographic area	Total samples	ASPV positive	Host	Collection date (month/year)
EnShi, HuBei Province (HB)^a	5	2	Pear	8/2009
WuHan, HB	83	28	Pear	3/2010
ChongYang, HB	38	12	Pear	4/2011
WuHan, HB	23	12	Pear	4/2012
XiangYang, HB	9	9	Pear	6/2012
AnNing, YunNan Province (YN)	4	1	Pear	2/2010
HangZhou, ZheJiang Province (ZJ)	46	2	Pear	5/2010
ZhengZhou, HeNan Province (HN)	25	9	Pear	11/2010
GuiYang, GuiZhou Province (GZ)	9	4	Pear	9/2011
YingTan, JiangXi Province (JX)	10	9	Pear	4/2012
NanChang, JX	10	10	Pear	4/2012
XingCheng, LiaoNing Province (LN)	117	22	Pear	4/2012
JingTai, GanShu Province (GS)	5	1	Pear	4/2012
GanXian, ShanXi Province (SX)	28	6	Pear	5/2012
ChongQing City (CQ)	13	4	Pear	5/2012
Korla, XinJiang Province (XJ)	14	10	Pear	6/2012
YanTai, ShanDong Province (SD)	12	4	Pear	6/2012
Total samples from pear	451	145
XingCheng, LN	3	2	Apple	9/2010
YanTai, SD	3	2	Apple	10/2010
ZhengZhou, HN	1	1	Apple	11/2010
WuHan, HB	3	3	Apple	4/2011
GuiYang, GZ	2	1	Apple	9/2011
YanTai, SD	2	2	Apple	6/2012
Total samples from pear	14	11

^a HB = HuBei Province, YN = YunNan Province, ZJ = ZheJiang Province, HN = HeNan Province, GZ = GuiZhou Province, JX = JiangXi Province, LN = Liaoning Province, GS = GanShu Province, SX = ShanXi Province, CQ = ChonGQing City, XJ = XinJiang Province, SD = ShanDong Province.

Table 2. Genetic distance between and within groups clustered in phylogenetic trees based on ASPV CP and TGB sequences.

Gene	Between and within-group diversity (±SE)
CP		Gp1^a	Gp2	Gp3
	Gp1	0.1822 ± 0.0096
	Gp2	0.2824 ± 0.0164	0.2005 ± 0.0109
	Gp3	0.3736 ± 0.0228	0.3821 ± 0.0234	0.1602 ± 0.0105
		A^b	B	C	D	E	F
	A	0.0586 ± 0.0038
	B	0.1482 ± 0.0129	0.0511 ± 0.0038
	C	0.1718 ± 0.0105	0.1543 ± 0.0092	0.0754 ± 0.0065
	D	0.2186 ± 0.0102	0.2187 ± 0.0109	0.2156 ± 0.0104	0.1475 ± 0.0064
	E	0.2285 ± 0.0117	0.2167 ± 0.0116	0.2307 ± 0.0130	0.1745 ± 0.0082	0.0540 ± 0.0036
	F	0.3071 ± 0.0160	0.2997 ± 0.0165	0.3199 ± 0.0174	0.3023 ± 0.0150	0.3281 ± 0.0178	0.0370 ± 0.0032
TGB		Gp1^c	Gp2	Gp3	Gp4	Gp5
	Gp1	0.1404 ± 0.0086
	Gp2	0.2398 ± 0.0133	0.1653 ± 0.0105
	Gp3	0.2604 ± 0.0150	0.2656 ± 0.0157	0.0906 ± 0.0063
	Gp4	0.2398 ± 0.0137	0.2598 ± 0.0146	0.2721 ± 0.0149	0.1941 ± 0.0112
	Gp5	0.2545 ± 0.0138	0.2653 ± 0.0149	0.2678 ± 0.0158	0.2642 ± 0.0144	0.1690 ± 0.0104

^aGp1–Gp3 for the CP gene in this table indicate the groups formed in the phylogenetic tree shown in Fig. 1a.

^bA–F for the CP gene in this table indicate the subgroups formed in the phylogenetic tree shown in Fig. 1a.

^cGp1–Gp5 for the TGB gene in this table indicate the groups formed in the phylogenetic tree shown in Fig. 1b.

Fig. 1 Phylogenetic trees of complete CP (a) and TGB (b) sequences of ASPV isolates in this study and global isolates available in GenBank. Reference isolates are named according to their isolate name with GenBank accession numbers. Isolates reported herein are indicated by isolate abbreviations and number followed by the sequenced CP or TGB clone number. Sequences isolated from apple are indicated with the symbol ●, whereas sequences from Korla pear are denoted with the symbol ▲ and all other sequences are from pear isolates. The tree was constructed by the neighbour joining method implemented by MEGA6. Bootstrap analysis with 1000 replicates was performed. Only ≥50% bootstrap values are shown, and branch lengths are proportional to the genetic distances. Bar, 0.05 substitutions per site.

Fig. 1 (Continued).

Table 3. List of putative recombination events involving ASPV CP and TGB sequences.

						Recombination detection methods
		Parental isolate		Break Point		RDP	GeneConv	Bootscan	MaxChi	CHIMAERA	SiScan	3SEQ
Gene	Recombinant isolates	Major	Minor	Start	End	Av. P-Value^a
CP	YN-MRS-23	HN-HZT-2	YN-MRS-3	752	1179	7.670 × 10^–15	5.958 × 10^–13	3.498 × 10^–15	7.009 × 10^–10	1.854 × 10^–10	2.871 × 10^–11	6.641 × 10^–21
CP	HB-HN7-18	HN-HZT-2	YN-MRS-3	752	1179	7.670 × 10^–15	5.958 × 10^–13	3.498 × 10^–15	7.009 × 10^–10	1.854 × 10^–10	2.871 × 10^–11	6.641 × 10^–21
TGB	HB-YH15-6	HB-HN6-10	LN-PGL-6	428	1149	2.413 × 10^–16	1.391 × 10^–10	8.021 × 10^–17	5.408 × 10^–14	8.358 × 10^–14	9.612 × 10^–26	7.840 × 10^–29
TGB	HN-BL-2	HN-SJL-1	HB-HN2-23	11	274	2.170 × 10^–34	4.939 × 10^–34	3.315 × 10^–27	1.607 × 10^–10	1.545 × 10^–10	7.422 × 10^–44	1.787 × 10^–14
TGB	PR1-EU095327	XJ-5–5	PA66-D21829	810	1185	2.994 × 10^–13	7.068 × 10^–08	5.037 × 10^–13	3.978 × 10^–15	4.198 × 10^–14	2.343 × 10^–09	5.145 × 10^–35

^aP-values >0.05 were considered significant; ‘Minor’ and ‘Major’ parents refer to the parental isolates contributing the smaller and larger portions of the recombinant’s sequence, respectively.

Table 4. Analysing the selection pressures (dN/dS) of different ASPV genes.

Genes	Number of Sequences	dS	dNS	dNS/dS
CP	95	0.5695	0.1177	0.2067
TGB1	81	0.66430	0.04721	0.07107
TGB2	81	0.66285	0.05170	0.0780
TGB3	81	0.28095	0.07920	0.2819

Table 5. Population genetic parameters and neutrality tests calculated for ASPV CP and TGB based on geographic origins or variant groups.

Genes	Lineage	n	Hd	Pi	Tajima’s D^a	Fu and Li’s D*	Fu and Li’s F*
CP gene host	All	95	1.000	0.1717	−0.2610 ns	−1.1853 ns	−0.8538 ns
	Gp1 (pear)	47	1.000	0.1487	−0.3836 ns	−1.0547 ns	−0.8690 ns
	Gp2 (apple)	43	1.000	0.1396	−0.9266 ns	−0.4101 ns	−0.7193 ns
	Gp3 (Korla pear)	5	0.900	0.1450	−0.8872 ns	−0.7507 ns	−0.8484 ns
CP gene geographic origins	China	76	0.9994	0.16616	−0.2958 ns	−0.6272 ns	−0.5521 ns
	South Korea	8	1.0000	0.15123	−0.9181 ns	−0.6853 ns	−0.8270 ns
	Poland	9	1.0000	0.1915	−0.4401 ns	0.0017 ns	−0.1189 ns
	Germany	2	1.0000	0.00261	-	-	-
TGB genes variant groups	All	81	1.000	0.1834	0.0395 ns	0.3131 ns	0.2295 ns
	Gp1	38	1.000	0.1344	−0.0816 ns	0.3669 ns	0.2607 ns
	Gp2	5	1.000	0.0636	0.0579 ns	0.4967 ns	0.4435 ns
	Gp3	22	1.000	0.0818	−0.5895 ns	−1.3386 ns	−1.2911 ns
	Gp4 (apple)	12	1.000	0.1624	0.2825 ns	0.6697 ns	0.6476 ns
	Gp5 (Korla pear)	4	1.000	0.1504	−0.7348 ns	−0.4073 ns	−0.5034 ns

^aFu & Li’s and Tajima’s D statistic were performed using DnaSP software version 5.10.01. Values for neutrality tests were not significant (ns) in all cases (P > 0.05).

Fig. 2 Multiple alignments of amino acids of 16 representative ASPV CP sequences. 2–3 sequences from each subgroup in the phylogenetic tree in Fig. 1a were selected. LN-AP1-1 (arrow) from an apple isolate was used as a reference sequence. The deleted regions are represented by rectangular boxes and the inserted regions are represented by oval boxes.