云南省西南地区福寿螺COⅠ基因序列单核苷酸多态性分析

doi:10.12140/j.issn.1000-7423.2019.01.014

摘要/Abstract

摘要：

目的为了解云南省西南地区福寿螺的种群结构,对当地福寿螺的细胞色素c氧化酶亚基Ⅰ（cytochrome c oxidase subunitⅠ,COⅠ）基因序列进行单核苷酸多态性分析。方法 2016年11月18日至2017年5月8日,于云南省西双版纳州景洪市和勐腊县、普洱市思茅区和西盟县、临沧市耿马县及德宏州瑞丽市6个调查点采集福寿螺,进行形态学观察。提取福寿螺基因组DNA,PCR扩增COⅠ基因,对将阳性PCR产物进行测序。采用MEGA 6.06软件进行序列特征分析、计算遗传距离。采用邻接法构建系统发育树。结果共采集福寿螺200只,无法通过形态学鉴定进行种类区分。PCR扩增获得129条COⅠ基因序列（小管福寿螺Pomacea canaliculata 123条,孤岛福寿螺P. insularum 6条）,经多序列比对排齐后长度为621 bp,存在76个核苷酸变异位点,76个简约信息位点,无单一变异、插入和缺失位点、无碱基转换和颠换,A、G、T、C的平均含量分别为23.1%、20.5%、39.8%和16.7%;编码的207个氨基酸残基中,保守氨基酸有203个（占98.1%）。COⅠ基因序列分属4个单倍型（HAP）——HAP-1、HAP-2、HAP-3和HAP-4。HAP-1频率最高,占71.3%（92/129）,其次是HAP-3,占17.8%（23/129）,HAP-2和HAP-4频率分别为6.2%（8/129）和4.7%（6/129）。HAP-1、HAP-2和HAP-3与小管福寿螺的遗传距离最短,为0~0.052,碱基差异数为0或31个;与锥形瓶属（Pila conica）的遗传距离最长,为0.214~0.239,碱基差异数为115~126个。HAP-4与小管福寿螺、孤岛福寿螺和锥形瓶属遗传距离分别为0.100、0.060和0.223,碱基差异数为57、35和119个。4个单倍型间的遗传距离为0.021~0.100,碱基差异数为13~57个。其中,HAP-1和HAP-2的遗传距离最近,为0.021;HAP-4与HAP-1、HAP-2和HAP-3的遗传距离最远,为0.100;HAP-3与HAP-1和HAP-2的遗传距离处于中间水平,为0.052。HAP-1、HAP-2和HAP-3与小管福寿螺基因序列（GenBank登录号为AB433769、KT313034、EU523129）的一致性为98%~100%;HAP-4与孤岛福寿螺基因序列（GenBank登录号为EF514942）的一致性为99%。结论云南省西南地区存在小管福寿螺和孤岛福寿螺2种,且小管福寿螺已产生一定的遗传分化。

关键词: 小管福寿螺, 孤岛福寿螺, 细胞色素c氧化酶亚基Ⅰ基因;, 多态性, 云南省

Abstract:

Objective To identify Pomacea spp. based on the polymorphism of mitochondria DNA cytochrome c oxidase subunit I gene (COⅠ) amplified from Pomacea collected from southwest Yunnan Province. Methods The samples of Pomacea snails were collected from Jinghong City and Mengla County of Xishuangbanna, Simao District and Ximeng County of Pu’er, Gengma County of Lincang, Ruili City of Dehong, Yunnan Province, from November 18, 2016 to May 8, 2017, morphological observation was performed. DNA was extracted and COⅠgene was amplified and sequenced. The phylogenetic tree and genetic distances among the snail samples compared with known species of Pomacea were constructed using the neighbour-joining method with MEGA software (version 6.06). Results A total of 200 Pomacea snails were collected. It is difficult to distinguish species by morphological identification. 129 COⅠsequences were amplified, 123 sequences belonged to Pomacea canaliculata, 6 sequences belonged to P. insularum. The final length of amplified sequence was 621 bp, there were 76 nucleotide variation sites and 76 simplified information sites, without single variation, insertion and deletion sites, and without base conversion and transmutation. The average contents of A, G, T and C were 23.1%, 20.5%, 39.8% and 16.7%, respectively. The DNA sequence for 207 amino acids with 203 conserved (98.1%). The results showed that COⅠ gene sequence fell into 4 unique haplotypes based on the single mutations. HAP-1 haplotype was the predominant one (71.3%, 92/129), followed by HAP-3(17.8%, 23/129), HAP-2 (6.2%, 8/129） and HAP-4 (4.7%, 6/129). The sequence data showed that HAP-1, HAP-2, HAP-3 shared closer genetic distance to P. canaliculata ranged from 0.000 to 0.052 (with 0 or 31 nucleotide differences), further genetic distance to Pila conica with distances ranged from 0.214 to 0.239 (with 115-126 nucleotide differences). The genetic distances between HAP-4 and P. canaliculate, P. insularum, Pila conica were 0.100, 0.060 and 0.223, and with nucleotide differences of 57, 35 and 119, respectively. The genetic distances among the four HAPs ranged from 0.021-0.100 with nucleotide differences of 13-57. Among them, HAP-1 is closer to HAP-2 (0.021), HAP-4 is further away from HAP-1, HAP-2 and HAP-3 (0.100). The genetic distance between HAP-3 and HAP-1/HAP-2 was 0.052. HAP-1, HAP-2 and HAP-3 shared 98%-100% sequence identity to Pomacea canaliculate (GenBank accession AB433769, KT313034、EU523129), and HAP-4 shared 99% sequence identity to P. insularum (EF514942). Conclusion The species of Pomacea snails in southwest Yunnan Province are P. canaliculata and P. insularum based on COⅠgene polymorphism with certain genetic diversity amongst the acquired haplotypes of P. canaliculata identified in this study.

Key words: Pomacea canaliculata, Pomacea insularum, Cytochrome c oxidase subunit I gene, Genetic diversity, Yunnan Province

中图分类号:

R383.19

张苍林, 保雪莹, 彭佳, 字金荣, 冉甄, 卢娜, 杨亚明. 云南省西南地区福寿螺COⅠ基因序列单核苷酸多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(1): 75-81.

Cang-lin ZHANG, Xue-ying BAO, Jia PENG, Jin-rong ZI, Zhen RAN, Na LU, Ya-ming YANG. Species identification of Pomacea snails in southwest Yunan Province based on COⅠgene polymorphism[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(1): 75-81.

图/表 6

图1

表1

云南省西南地区采集的福寿螺基本信息

采集地点 Collection location		采集环境Habitat	采集时间Collection time	样本量Sample size	壳色Shell color	条纹 Sripe	平均螺壳长/mm Average shell length/mm	平均螺壳宽/mm Average shell width/mm	平均体螺层长/mm Average body length/mm	平均体螺层宽/mm Average body width/mm	平均螺口长/mm Average apertural length/mm	平均螺口宽/mm Average apertural width/mm	平均螺口高/mm Average apertural high/mm	平均体质量/g Average weight/g
西双版纳景洪市Jinghong City of Xishuangbanna	曼列村Manlie Village	沟渠Ditch	2017.3.15	10	黑褐色Black brown	有Yes	24.6	21.3	22.0	8.0	17.1	10.6	10.4	2.9
	曼沙村Mansha Village	沟渠Ditch	2017.3.14	10	黑褐色Black brown	有Yes	22.3	19.8	19.7	6.6	14.8	10.5	10.5	2.8
	曼坝河村Manbahe Village	溪流Stream	2017.3.14	10	棕黄色Brownish yellow	有Yes	33.4	28.5	30.8	10.6	23.3	14.8	14.5	8.4
	勐罕三乡Menghansan Village	沟渠Ditch	2017.3.10	10	黑褐色Black brown	有Yes	26.6	22.6	24.2	8.5	17.5	11.9	11.5	3.8
	大鱼塘Dayutang Village	沟渠Ditch	2017.3.10	10	黑褐色Black brown	有Yes	36.0	30.4	32.6	11.4	25.1	16.4	16.4	8.2
	普提岛Putidao Village	香蕉地 Banana Field	2017.3.9	10	黑褐色Black brown	有Yes	28.6	22.8	25.8	9.0	18.7	13.2	12.9	4.4
西双版纳勐腊县Mengla County of Xishuangbanna	关累村Guanlei Village	沟渠Ditch	2017.3.13	10	黑褐色Black brown	有Yes	54.3	48.1	49.9	17.8	36.3	24.9	24.7	31.8
西双版纳勐腊县Mengla County of Xishuangbanna	曼丢村Mandiu Village	沟渠Ditch	2017.3.7	10	棕黄色Brownish yellow	有Yes	31.4	27.2	29.1	9.9	21.5	14.6	14.2	7.6
普洱市思茅区Simao District of Pu’er	三棵桩村Sankeshu Village	水塘Pond	2017.5.8	19	黑褐色Black brown	有Yes	41.6	36.2	37.6	13.4	29.1	19.3	19.3	12.7
普洱市思茅区Simao District of Pu’er	三棵桩村Sankeshu Village	水塘Pond	2017.5.8	11	棕黄色Brownish yellow	有Yes	41.9	36.3	37.7	13.1	29.4	19.4	19.4	13.3
普洱市西盟县Ximeng County of Pu’er	勐梭村Mengjun village	稻田Rice field	2016.10.18	30	棕黄色Brownish yellow	有Yes	30.8	26.7	9.5	14.9	20.8	17.4	26.8	4.6
德宏州瑞丽市Ruili City of Dehong	集贸市场Farmer’s Market	稻田Rice field	2016.11.14	30	棕黑色Brownish black	有Yes	34.2	30.2	10.6	15.1	23.7	15.9	32.2	8.3
临沧市耿马县Gengma County of Lincang	孟定镇^a Mengding Town^a	稻田Rice field	2016.11.3	30	棕黑色Brownish black	有Yes	31.3	27.5	9.6	14.6	22.7	15.4	29.0	6.6

表1

图2

表2

表3

图3

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单倍型 Haplotype	群体单倍型数量 Number of haplotypes							GenBank登录号 GenBank accession number
单倍型 Haplotype	景洪市 Jinghong City	勐腊县 Mengla County	西盟县 Ximeng County	思茅区 Simao District	瑞丽市 Ruili City	耿马县 Gengma County	合计 Total	GenBank登录号 GenBank accession number
HAP-1	35	8	23	23	3	-	92	MH293322~293332, MH293334, MH293338~293344, MH293346~293352, MH293377~293386, MH293389~293390, MH293392~293422, MH293424~293429, MH293431~293438, MH293440~293441, MH293443~293447, MH293449~293450
HAP-2	3	-	-	-	-	5	8	MH293355, MH293359, MH293365, MH293371, MH293374, MH293376, MH293387, MH293391
HAP-3	4	1	-	-	-	18	23	MH293333, MH293336~293337, MH293345, MH293353~293354, MH293356~293358, MH293360~293364, MH293366~293370, MH293372~293373, MH293375, MH293388
HAP-4	2	-	4	-	-	-	6	MH293335, MH293423, MH293430, MH293439, MH293442, MH293450