不同地理种群腐食酪螨遗传变异和遗传分化分析

doi:10.12140/j.issn.1000-7423.2024.01.010

摘要/Abstract

摘要：

目的分析不同地理种群腐食酪螨遗传多样性及遗传分化。方法 2018年6—7月在安徽省芜湖市（WH）、阜阳市（FY）和河北省石家庄市（SJZ）、张家口市（ZJK）4个地区的面粉厂和米厂采集螨虫标本并经形态学和细胞色素C氧化酶亚基1（cox1）分子鉴定筛选出腐食酪螨，PCR扩增腐食酪螨线粒体细胞色素b（Cytb）和核糖体DNA内转录间隔区（ITS）并测序。使用Chromas 2和DNAStar 1.00软件对基因序列进行校对和拼接，使用DnaSP 5.10.00 软件计算各种群单倍型多样性（Hd）和核苷酸多态性（Pi），用MEGA 10.2 软件包分析种群遗传变异和遗传分化指数（F_st）及基因流（N_m），用Arlequin3.1软件计算Tajima’s D值和Fu’s FS值并进行中性检验和分子方差分析，使用Network 10.2基于Median-joining法构建单倍型网络关系图，采用最大似然法（ML）对单倍型构建系统进化树。结果腐食酪螨呈椭圆形，表皮柔软，乳白色或黄棕色，口器高度变异或退化；本研究获得的cox1与GenBank中腐食酪螨cox1（登录号：LC190838.1）的序列一致性大于98%。腐食酪螨样本Cytb基因长度为372 bp，16个单倍型（H1~H16）中仅H4为共享单倍型（为来自WH和FY种群的9个个体共享），其余为独享单倍型。4个地理种群Hd较高，整体值为0.895（> 0.5），其中以WH种群最高（Hd = 0.867），SJZ种群最低（Hd = 0.464）。基于Cytb序列分析显示腐食酪螨4个地理种群遗传多样性较高（Pi > 0.005）；分子方差分析可见腐食酪螨4个地理种群F_st > 0.15（P < 0.05）；中性检验结果显示，腐食酪螨Tajima’s D值为-0.737 22，Fu’s FS值为2.336 33（均P > 0.05）。单倍型网络图与系统进化树结果一致，4个地理种群个体相互交织分布，仅ZJK种群个别个体聚为另外一支。ITS序列长度为1 259~1 405 bp，32个单倍型（G1~G2）均为独享单倍型。4个地理种群Hd较高，整体值与分别值为1.000（> 0.5）。基于ITS序列分析显示腐食酪螨4个地理种群遗传多样性较高（Pi > 0.005），F_st > 0.25（P < 0.05）；Tajima’s D值和Fu’s FS值分别为2.030 29和3.044 54（均P > 0.05）。单倍型网络图与系统进化树结果一致，WH、FY、SJZ种群单倍型聚为一支，ZJK的单倍型单独聚为一支。结论腐食酪螨4个地理种群遗传多样性较高，地理种群间存在较大遗传分化，FY种群可能有向WH种群扩张的历史，不同腐食酪螨地理种群间发生局部高水平基因交流，未发现明显地理分布格局。

关键词: 腐食酪螨, 细胞色素b, 内转录间隔区, 种群遗传多样性

Abstract:

Objective To analyze the genetic diversity and differentiation of different geographic populations of Tyrophagus putrescentiae. Methods From June to July 2018, mite specimens were collected from flour and rice mills in Wuhu City (WH) and Fuyang City (FY) in Anhui Province, Shijiazhuang City (SJZ) and Zhangjiakou City (ZJK) in Hebei Province, to screen for copra mites identified by morphology and cytochrome C oxidase subunit 1 (cox1) gene sequence. Mitochondrial cytochrome b (Cytb) and ribosomal DNA internal transcriptional spacer (ITS) of the copra mites were amplified by PCR and sequenced. Using Chromas 2 and DNAStar 1.00 software were used to proofread and concatenate gene sequences. DnaSP 5.10.00 software to calculate haplotype diversity (Hd) and nucleotide polymorphism (Pi) of various mite populations, MEGA 10.2 software package to analyze population genetic variation and differentiation index (F_st) and gene flow (N_m), Arlequin 3.1 software to calculate Tajima’s D value, neutral tests to estimate Fu’s FS value, and analysis of molecular variance to assess genetic variation. To construct haplotype network diagram, Network 10.2 program was used based on the median-joining method. Haplotype phylogenetic tree was constructed using maximum likelihood method (ML). Results The T. putrescentiae mite was elliptical in shape, with soft skin, milky white or yellow-brown colour, and highly variable or degraded mouthparts. The consistency between the cox1 sequence obtained in this study and the cox1 sequence in GenBank (login number: LC190838.1) was greater than 98%. The length of the Cytb gene in the sample of T. putrescentiae was 372 bp. Among the 16 haplotypes (H1-H16), only H4 was a shared haplotype (shared by 9 individuals from the WH and FY populations), while the rest were exclusive haplotypes. The Hd of the four geographical populations was relatively high, with an overall value of 0.895 (> 0.5), with the WH population having the highest Hd (0.867) and the SJZ population having the lowest Hd (0.464). Based on Cytb sequence analysis, it was found that the genetic diversity of the four geographical populations of T. putrescentiae was relatively high (Pi > 0.005). Molecular analysis of variance showed that the F_st of the four geographical populations of T. putrescentiae was > 0.15 (P < 0.05). The neutral test results showed that the Tajima’s D value and the Fu’s FS value were -0.737 22 and 2.336 33, respectively (both P > 0.05). The haplotype network diagram was consistent with the results of the phylogenetic tree, where individuals from the four geographic populations were interwoven and distributed, with only a few individuals from the ZJK population clustered into another branch. The length of the ITS gene sequence was 1259-1405 bp, and all 32 haplotypes (G1-G2) were exclusive haplotypes. The Hd of the four geographical populations was relatively high, with an overall value of 1.000 (> 0.5) compared to the respective values. Based on ITS sequence analysis, it was also found that the genetic diversity of the four geographical populations of T. putrescentiae was high (Pi > 0.005), with F_st > 0.25 (P < 0.05). The Tajima’s D value and Fu’s FS value were 2.030 29 and 3.044 54, respectively (both P > 0.05). The haplotype network diagram was consistent with the results of the phylogenetic tree. The haplotypes of WH, FY and SJZ populations were clustered into one branch, while the haplotypes of ZJK were clustered into a single branch. Conclusion 4 geographic populations of T. putrescentiae are highly polymorphic, showing significant genetic differentiation. FY population might expanded to WH population historically, and there have been high level of partial gene exchange between different geographic populations of copra mites, but no obvious geographical distribution pattern was found.

Key words: Tyrophagus putrescentiae, Cytochrome b, Internal transcribed spacer, population genetic diversity

中图分类号:

R384.42

乔婷婷, 陶香林, 叶长江, 李政, 周笑妍, 孙恩涛. 不同地理种群腐食酪螨遗传变异和遗传分化分析[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 69-77.

QIAO Tingting, TAO Xianglin, YE Changjiang, LI Zheng, ZHOU Xiaoyan, SUN Entao. Analysis on genetic variation and differentiation of Tyrophagus putrescentiae in different geographic populations[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(1): 69-77.

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采集地 Location	代码 Code	采集日期 Date of sampling	经纬度 Longitude and latitude	标本数量 No. samples
安徽芜湖Wuhu City, Anhui Province	WH	2018年6月 June, 2018	31.20°N,118.21°E	15
安徽阜阳Fuyang City, Anhui Province	FY	2018年7月 July, 2018	32.90°N,115.82°E	13
河北石家庄Shijiazhuang City, Hebei Province	SJZ	2018年7月 July, 2018	38.05°N,114.52°E	12
河北张家口Zhangjiakou City, Hebei Province	ZJK	2018年6月 June, 2018	40.82°N,114.88°E	8

基因 Gene	种群 Population	基因序列号 GenBank Accession No.	单倍型/样本数 Haplotype/ Sample size	单倍型及其个数 Haplotype and its number	单倍型多样性 Haplotype diversity (Hd)	核苷酸多样性 Nucleotide diversity (Pi)
Cytb	WH	MH715737~MH715746	6月10日	H1(1), H2(1), H3(3), H4(3), H5(1), H6(1)	0.867	0.008 42
	FY	MH715747~MH715755	4月9日	H4(6), H7(1), H8(1), H9(1)	0.583	0.001 79
	SJZ	MH715756~MH715763	3月8日	H10(6), H11(1), H12(1)	0.464	0.001 34
	ZJK	MH715764~MH715773	4月10日	H13(6), H14(1), H15(2), H16(1)	0.644	0.108 90
ITS	WH	MH793968~MH793974	7月7日	G1(1), G2(1), G3(1), G4(1), G5(1), G6(1), G7(1)	1	0.390 66
	FY	MH793975~MH793984	10月10日	G8(1), G9(1), G10(1), G11(1), G12(1), G13(1), G14(1), G15(1), G16(1), G17(1)	1	0.434 40
	SJZ	MH793985~MH793994	10月10日	G18(1), G19(1), G20(1), G21(1), G22(1), G23(1), G24(1), G25(1), G26(1), G27(1)	1	0.454 54
	ZJK	MH793995~MH793999	5月5日	G28(1), G29(1), G30(1), G31(1), G32(1)	1	0.488 14

基因 Gene	遗传差异来源 Source of genetic variation	自由度 Degree of freedom	平方和 Sum of square	变异组分 Variance component	变异百分比/% Percentage of variation/%	F_st值 F_st value
Cytb	种群间 Inter-population	3	68.967	1.832 33	23.14	0.231 42^a
	种群内 Intra-population	33	200.817	6.085 35	78.86
	合计 Total	36	269.784	7.917 69	100
ITS	种群间 Inter-population	3	3 083.412	96.381 49	25.96	0.259 64^a
	种群内 Intra-population	28	7 695.057	274.823 47	74.04
	合计 Total	31	10 778.469	371.204 96	100

基因 Gene	地理种群 Geographical population	地理种群 Geographical population
基因 Gene	地理种群 Geographical population	WH	FY	SJZ	ZJK
Cytb	WH	-	0.747 84	0.661 14	2.550 65
	FY	0.400 69^a	-	0.058 90	3.437 05
	SJZ	0.430 61^a	0.894 61^a	-	1.622 89
	ZJK	0.163 90^a	0.127 00	0.235 53^a	-
ITS	WH	-	1.697 44	1.428 56	0.679 08
	FY	0.227 54^a	-	inf	0.771 05
	SJZ	0.259 26^a	-0.017 52	-	0.831 48
	ZJK	0.424 06^a	0.393 37^a	0.375 52^a	-

基因 Gene	种群 Population	Tajima’s D值 Tajima’s D value	P值 P value	Fu’s FS值 Fu’s FS value	P值 P value
Cytb	WH	-0.066 12	0.493	-0.711 58	0.262
	FY	-1.512 97	0.044	-1.891 65	0.016
	SJZ	-1.310 09	0.094	-0.998 99	0.056
	ZJK	-0.059 71	0.526	12.947 55	1.000
	合计 Total	-0.737 22	0.289	2.336 33	0.334
ITS	WH	0.785 61	0.768	3.138 3	0.625
	FY	2.269 87	0.978	2.463 84	0.675
	SJZ	2.659 63	0.997	2.510 93	0.757
	ZJK	2.406 07	0.998	4.065 11	0.659
	合计 Total	2.030 29	0.935 25	3.044 54	0.679