Sequence polymorphisms within Kalicephalus indicus ribosomal and mitochondrial genes

doi:10.12140/j.issn.1000-7423.2019.01.015

Abstract

Abstract:

Objective To analyze the ribosomal and mitochondrial genes sequences polymorphisms of Kalicephalus indicus, and discuss their application potential as molecular markers. Methods Two wild Elaphe carinata snakes were captured in June 2016, in Zhangli Town, Changsha County, Hunan Province, and seven K. indicus adult worms were isolated from the snakes. Total DNA from the worms was extracted. The sequences of the first and second internal transcribed spacer regions 1 and 2(ITS-1 and ITS-2), 5.8S, mitochondrial cytochrome c oxidase subunit 1 (cox1), small subunit 12S of ribosomal RNA, ATP synthase subunit 6 (atp6), nicotinamide adenine dinucleotide dehydrogenase subunit 4 (nad4) of K. indicus were amplified using PCR and subjected to DNA sequencing. The sequencing results of each gene were aligned using ClustalX 1.83. A phylogenetic tree based on the mitochondrial DNA sequences cox1, 12S, atp6 and nad4 was constructed using Bayesian method, using Onchocerca volvulus as an outgroup. Intraspecific variation in these sequences among the seven K. indicus samples were analyzed. Results The size of the amplified products of ITS-1, 5.8S and ITS-2 was approximately 900 bp. The size of the amplified products of cox1, 12S, atp6 and nad4 was approximately 400, 600, 500 and 400 bp, respectively. The content of A + T of ITS-1, 5.8S and ITS-2 is 52.7%-53.7%, higher than its content of G + C (46.3%-47.4%). The content of A + T of cox1, 12S, atp6 and nad4 is 67.1%-68.9%, 76.9%-77.4%, 73.0%-75.5%, and 70.3%-73.3%, respectively, higher than their content of G + C (31.1%-32.9%, 22.6%-23.1%, 24.5%-27.0% and 26.7%-29.7%). Intraspecific variation in ITS-1, 5.8S, ITS-2, cox1, 12S, atp6 and nad4 were 0-2.5%, 0, 0-5.0%, 0-2.6%, 0-3.8%, 0.7%-8.7% and 0-11.0%, respectively. The phylogenetic tree clustered the seven K. indicus individuals into the same branch, with Ancylostoma ceylanicum being the closest species. Conclusion The ITS-2 sequence with the largest variation is expected to be an ideal molecular marker for studying the intraspecific variation of K. indicus. The degree of its intraspecific variation in mitochondrial gene sequences is as follows: cox1 < 12S < atp6 < nad4, and nad4 is an ideal mitochondrial molecular marker for studying the genetic diversity of K. indicus.

Key words: Kalicephalus indicus, Ribosomal DNA, Mitochondrial DNA, Sequence analysis

CLC Number:

R383.19

Cheng-yan ZHOU, Jun MA, Wei LIU, Qian-ming XU, Xing-quan ZHU. Sequence polymorphisms within Kalicephalus indicus ribosomal and mitochondrial genes[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(1): 81-86.

Figures/Tables 4

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引物Primer	序列(5′→3′) Sequence (5′→3′)
NC5	GTAGGTGAACCTGCGGAAGGATCATT
NC2	TTAGTTTCTTTTCCTCCGCT
JB3	TTTTTTGGGCATCCTGAGGTTTAT
JB4.5	TAAAGAAAGAACATAATGAAAATG
atp6F	AGAATTCAATTACCTTTAAGTTCAGTAA
atp6R	AAATAAATCAAACGAGAAAAAATATATC
12SF	ATAATACTTGTTCCAGAATAATCGGCTA
12SR	AAACCTACTTTACTACAACTTACTCCCC
nad4F	TTTTGGGCTTTGGTTCTCAAATTGAAAA
nad4R	ATGTGTGTGACCGAAGAATATGCCGCCA

种名 Species	分类地位 Taxonomic status	国家 Country	GenBank登录号 GenBank accession number
锡兰钩口线虫Ancylostoma ceylanicum	钩口科Ancylostomatidae	中国China	NC035142
松材线虫Bursaphelenchus xylophilus	滑刃科Aphelenchoididae	法国France	JQ429761
蛔虫属Ascaris sp.	蛔科Ascarididae	中国China	KC839987
似蚓蛔线虫Ascaris lumbricoides	蛔科Ascarididae	中国China	HQ704900
猪蛔虫Ascaris suum	蛔科Ascarididae	中国China	HQ704901
小熊猫贝蛔虫Baylisascaris ailuri	蛔科Ascarididae	中国China	NC015925
马副蛔虫Parascaris univalens	蛔科Ascarididae	澳大利亚Australia	NC024884
简单异尖线虫Anisakis simplex	异尖科Anisakidae	韩国Korea	NC007934
拟地新异尖线虫Pseudoterranova azarasi	异尖科Anisakidae	中国China	NC027163
猫弓首蛔虫Toxocara cati	弓首科Toxocaridae	中国China	NC010773
蠕形住肠线虫Enterobius vermicularis	尖尾科Oxyuridae	韩国Korea	NC011300
旋盘尾丝虫Onchocerca volvulus	盘尾科Onchocercidae	美国USA	NC001861