Genetic differentiation of Bursaphelenchus xylophilus in east China based on single nucleotide polymorphisms (SNP) markers

doi:10.12302/j.issn.1000-2006.202103026

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2022, Vol. 46 ›› Issue (4): 21-28.doi: 10.12302/j.issn.1000-2006.202103026

Special Issue: 松材线虫病致病机理与病害防治

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Genetic differentiation of Bursaphelenchus xylophilus in east China based on single nucleotide polymorphisms (SNP) markers

WANG Qingtong¹^,²(), DING Xiaolei¹(), YE Jianren¹^,^*(), SHI Xiufeng¹

1. Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2. Shanghai Forestry Station, Shanghai 200072, China

Received:2021-03-13 Revised:2021-07-01 Online:2022-07-30 Published:2022-08-01
Contact: YE Jianren E-mail:qtwang@njfu.edu.cn;dxl@njfu.edu.cn;jrye@njfu.com.cn

Abstract

Abstract:

【Objective】 Jiangsu, Zhejiang, Anhui and Shandong, these four provinces in east China, are recognized as the initial occurrence of pine wilt disease in China. And east China is also the most suitable growth area for Bursaphelenchus xylophilus. Studying the genetic differentiation of the B. xylophilus population in those regions will provide important basic genetic information to establish a B. xylophilus epidemic source tracing system in China. 【Method】 Samples of B. xylophilus were collected from pine trees affected by pine wilt disease in east China, and strains were isolated and purified. The genomic deoxyribonucleic acid (DNA) of strains was extracted and sequenced. The number and types of single nucleotide polymorphism (SNP) loci and genotypes in each region were analyzed using bioinformatics software. Cluster analysis was used to compare the genetic differentiation among different strains based on SNP markers, while Treemix was used to detect gene penetration routes. 【Result】 A total of 67 B. xylophilus strains were isolated and collected in 60 county-level administrative regions of Anhui (AH), Fujian (FJ), Jiangsu (JS), Jiangxi (JX), Shandong (SD) and Zhejiang Province (ZJ) in East China. All strains were sequenced through the whole genome; genome calling analysis indicated 12 genotypes, of the 67 strains, where six types (i.e., A→G, C→G, C→T, G→A, G→C and T→C) occurred at a higher frequency than others. A total of 6 531 684 SNP loci were obtained and the number of SNP was different between strains. The number of SNP counts, homozygotes, missing SNPs, and private SNPs showed no significant regional differences among the six provinces, and there was no significant correlation with invasion time. Principal component analysis showed that 67 strains of B. xylophilus may be divided into three groups; each group had a specific correlation with a geographic origin. Most strains belonged to group 1, which included all strains from Zhejiang and Jiangxi, as well as most of strains from the other four provinces. Group 2 included 14 strains from Jiangsu, Anhui, Shandong and Fujian, and group 3 included only seven strains from Anhui, Fujian and Shandong provinces. Two gene migration routes detected by Treemix: ① group 2 of Jiangsu to group 1 of Anhui; and ② group 3 of Shandong to group 2 of Anhui. 【Conclusion】 There were abundant SNP loci in B. xylophilus in east China, where the SNP characteristics of different strains were relatively disparate, and there was no clear regularity between SNP diversity and invasion time. Overall, the B. xylophilus strains in east China may be divided into three groups, and there was a specific correlation between different groups and geographic areas. In the area where the 1-AH and 2-AH groups were located, there may have been re-invasion by strains from groups in other areas.

Key words: Bursaphelenchus xylophilus, single nucleotide polymorphism (SNP), whole-genome resequencing, genetic differentiation, east China region

CLC Number:

S763

WANG Qingtong, DING Xiaolei, YE Jianren, SHI Xiufeng. Genetic differentiation of Bursaphelenchus xylophilus in east China based on single nucleotide polymorphisms (SNP) markers[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(4): 21-28.

Figures/Tables 7

Table 1

Sample information of 67 B. xylophilus strains"

虫株编号 strains No.	采样地 origin location	寄主 host	采样时间 sampling time	入侵年份 invasive year	虫株编号 strains No.	采样地 origin location	寄主 host	采样时间 sampling time	入侵年份 invasive year
AH01	安徽省芜湖市无为县	马尾松	2014-12	2008	JS14	江苏省南京市浦口区老山林场	马尾松	2015-02	1984
AH02	安徽省宣城市宣州区水东镇	马尾松	2015-03	1999	JS15	江苏省苏州市常熟市虞山林场	马尾松	2015-02	1995
AH03	安徽省滁州市全椒县	未知	2015-03	1996	JS16	江苏省南京市溧水区白马镇	马尾松	2015-02	1987
AH04	安徽省宣城市广德县新杭镇	未知	2015-03	1994	JS17	江苏省南京市高淳区青山林场	马尾松	2015-02	1999
AH05	安徽省合肥市庐江县	马尾松	2015-03	2009	JS18	江苏省常州市溧阳市天目湖镇	马尾松	2015-03	1997
AH06	安徽省马鞍山市当涂青山林场	马尾松	2015-08	1992	JS20	江苏省苏州市常熟市虞山林场	马尾松	2017-10	1995
AH08	安徽省六安市舒城县万佛湖	马尾松	2015-08	2000	JX06	江西省赣州市赣县储潭镇	马尾松	2014-11	2008
AH10	安徽省滁州市来安县复兴林场	马尾松	2015-08	1995	JX07	江西省赣州市南康区	马尾松	2014-12	2012
AH12	安徽省六安市舒城县高峰乡新旗村	马尾松	2015-08	2000	JX08	江西省九江市庐山区威家镇九星村	马尾松	2015-01	2005
AH13	安徽省六安市霍山县磨子潭	马尾松	2015-08	2009	JX09	江西省九江市彭泽县	马尾松	2015-01	2005
AH14	安徽省六安市霍邱县西山林场	马尾松	2016-08	2015	JX10	江西省抚州市广昌县旴江镇大圹村	马尾松	2015-01	2005
FJ01	福建省福州市仓山区	马尾松	2014-12	2007	JX11	江西省九江市都昌县蔡岭镇	马尾松	2015-03	2007
FJ02	福建省福州市晋安区	马尾松	2014-12	2006	SD01	山东省威海市荣成市人和镇	未知	2014-12	2007
FJ03	福建省厦门市沧海区	马尾松	2014-12	2010	SD02	山东省威海市荣成市石岛山	未知	2014-12	2007
FJ04	福建省厦门市同安区	马尾松	2014-12	2001	SD03	山东省威海市荣成市虎山镇	未知	2014-12	2007
FJ05	福建省泉州市丰泽区	马尾松	2014-12	2005	SD04	山东省烟台市长岛县	未知	2014-12	1990
FJ06	福建省漳州市诏安区	马尾松	2014-12	2006	SD05	山东省威海市文登区	黑松	2014-08	2009
FJ07	福建省莆田市涵江区江口镇	马尾松	2015-07	2014	SD06	山东省烟台市芝罘区北岛	马尾松	2015-07	2015
FJ08	福建省三明市沙县凤岗街道龙坑村	马尾松	2015-07	2004	SD07	山东省日照市东港区山海天旅游度假区	黑松	2017-08	2016
FJ10	福建省三明市沙县青州镇洽湖村	马尾松	2015-07	2004	SD08	山东省日照市东港区山海天旅游度假区	黑松	2017-08	2016
FJ11	福建省莆田市仙游县鲤城街道	马尾松	2016-08	2015	SD10	山东省威海市荣成市虎山镇	未知	2014-12	2007
FJ12	福建省三明市梅列区西街道西村	马尾松	2016-08	2003	ZJ01	浙江省宁波市镇海区	马尾松	2014-12	2003
JS02	江苏省镇江市润州区蒋乔街道	马尾松	2014-12	1988	ZJ02	浙江省宁波市北仓区	马尾松	2014-12	1996
JS03	江苏省镇江市丹徒区长山林场	马尾松	2014-12	1987	ZJ03	浙江省宁波市余姚区	马尾松	2014-12	2003
JS04	江苏省镇江市句容市句容林场	马尾松	2014-12	1987	ZJ04	浙江省宁波市奉化区	马尾松	2014-12	2003
JS05	江苏省南京市中山陵	马尾松	2014-12	1982	ZJ05	浙江省宁波市宁海县	马尾松	2014-12	1999
JS06	江苏省无锡市滨湖区	马尾松	2014-12	1993	ZJ06	浙江省温州市鹿城区藤桥镇	马尾松	2014-08	2014
JS07	江苏省无锡市惠山区	马尾松	2014-12	2001	ZJ09	浙江省丽水市缙云县五云街道	马尾松	2015-01	2003
JS08	江苏省无锡市宜兴市	马尾松	2014-12	2003	ZJ10	浙江省丽水市莲都区白云街道	马尾松	2015-01	2013
JS09	江苏省淮安市盱眙县桂五镇	马尾松	2015-01	2000	ZJ12	浙江省绍兴市上虞区百官街道	马尾松	2015-03	2004
JS10	江苏省常州市金坛区	马尾松	2015-01	1997	ZJ13	浙江省台州市天台县	未知	2015-03	2014
JS11	江苏省连云港市海州区花果山街道	赤松	2015-01	2006	ZJ14	浙江省舟山市定海区	未知	2015-03	1992
JS12	江苏省扬州市仪征市白羊山	马尾松	2015-01	1999	ZJ15	浙江省台州市温岭市	未知	2015-03	2000
JS13	江苏省连云港市连云区北固山	马尾松	2015-01	2002

Table 1

Table 2

Sequencing quality of 67 B. xylophilus strains"

虫株编号 strains No.	读数 reads	Q30值/% Q30 value	数据量/GB data size	比对率/% mapped	虫株编号 strains No.	读数 reads	Q30值/% Q30 value	数据量/GB data size	比对率/% mapped
AH01	49 497 246	92.37	7.43	98.41	JS14	61 029 972	88.00	9.20	96.62
AH02	60 545 944	90.00	9.10	95.28	JS15	46 839 256	91.94	7.03	96.40
AH03	59 126 706	88.00	8.90	92.89	JS16	55 316 746	92.98	8.30	97.41
AH04	59 665 312	93.12	8.95	85.80	JS17	47 001 548	92.06	7.05	98.50
AH05	59 733 776	88.00	9.00	97.23	JS18	68 072 922	90.00	10.20	98.31
AH06	53 841 996	93.06	8.08	95.13	JS20	41 915 184	93.91	6.20	98.76
AH08	39 032 238	89.65	6.00	95.62	JX06	49 815 662	92.87	7.47	97.41
AH10	43 924 620	89.83	6.74	91.20	JX07	72 405 752	93.80	10.90	97.91
AH12	44 933 466	92.13	6.74	96.19	JX08	92 873 782	91.83	13.90	98.11
AH13	44 261 102	92.45	6.64	98.20	JX09	87 389 668	91.04	13.10	97.84
AH14	48 921 300	92.47	7.11	90.95	JX10	45 192 018	91.92	6.78	94.77
FJ01	62 826 122	89.30	9.40	97.50	JX11	49 044 706	92.99	7.36	95.36
FJ02	57 058 312	89.00	4.40	97.37	SD01	67 473 506	90.00	10.10	97.01
FJ03	58 162 862	90.00	4.50	97.96	SD02	51 848 172	92.50	7.78	98.06
FJ04	56 703 602	88.90	4.60	97.39	SD03	62 217 990	90.00	9.30	87.48
FJ05	62 283 898	90.00	4.80	97.76	SD04	39 915 816	90.00	6.00	95.10
FJ06	59 674 914	89.80	4.70	96.66	SD05	51 344 852	93.02	7.70	93.40
FJ07	80 687 530	91.42	12.10	93.61	SD06	43 155 976	92.06	6.49	94.19
FJ08	43 089 506	92.84	6.46	85.38	SD07	43 766 654	92.60	6.31	84.83
FJ10	44 300 670	92.79	6.65	95.62	SD08	47 394 288	92.90	6.71	82.74
FJ11	41 197 468	92.74	6.10	79.80	SD10	42 047 430	92.82	6.23	97.47
FJ12	41 894 946	87.00	6.11	71.74	ZJ01	55 758 398	92.10	8.36	94.54
JS02	59 325 892	87.00	8.90	94.64	ZJ02	46 175 272	94.34	6.93	96.35
JS03	44 121 294	89.00	6.60	90.62	ZJ03	48 778 170	93.88	7.32	92.37
JS04	49 417 314	92.38	7.41	97.74	ZJ04	67 997 622	88.87	10.20	90.20
JS05	63 702 654	89.00	9.60	85.92	ZJ05	84 773 858	90.14	12.70	96.89
JS06	62 356 498	89.00	9.40	97.20	ZJ06	58 325 204	93.93	8.75	97.34
JS07	47 794 198	90.15	7.17	96.30	ZJ09	77 271 336	91.21	11.60	96.16
JS08	57 569 104	89.00	8.60	94.92	ZJ10	60 964 626	93.45	9.15	97.58
JS09	55 333 634	89.13	8.30	99.01	ZJ12	60 370 312	92.77	9.06	96.09
JS10	41 737 730	91.07	6.26	92.85	ZJ13	60 765 572	93.10	9.12	96.01
JS11	56 671 628	90.48	8.50	91.09	ZJ14	59 620 594	93.30	8.90	96.98
JS12	63 564 370	88.00	9.50	98.32	ZJ15	65 896 918	94.40	9.90	97.95
JS13	60 356 224	88.00	9.10	98.61

Table 2

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

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Genetic differentiation of Bursaphelenchus xylophilus in east China based on single nucleotide polymorphisms (SNP) markers

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