多脉铁木叶绿体基因组的序列特征和系统发育

doi:10.11707/j.1001-7488.20200507

摘要/Abstract

摘要：

目的: 多脉铁木是中国特有种，为浙江省珍稀濒危野生植物和极小种群保护对象。在浙江，多脉铁木的野生植株数量极其稀少，低龄个体极少，种群面临衰退。叶绿体基因组在进化过程中相对保守，可为亲缘关系分析和系统发育研究提供有用信息。本研究在组装叶绿体基因组的基础上，对序列特征进行分析，以明确多脉铁木叶绿体基因组的大小、结构和基因组成等信息；通过系统发育分析，明确其与近缘种的关系和分类地位。方法: 利用CTAB法提取多脉铁木的基因组DNA，并构建测序文库，用HiSeq X Ten进行高通量测序，策略为双端测序；借助NovoPlasty组装叶绿体基因组；用PCR方法克隆边界序列并进行测序验证；SSR的检测采用MISA软件；利用PhyML 3.0构建系统发育树。结果: 去除接头和低质量的序列，共得到19 869 412条clean reads。序列组装结果表明，多脉铁木叶绿体基因组的全长为159 583 bp，具一个典型的四分体结构，大单拷贝区、小单拷贝区及反向重复序列的长度分别为88 514、18 953、26 058 bp。多脉铁木叶绿体基因组中共有131个基因，其中蛋白编码基因85个、tRNA基因36个、rRNA基因8个，其中的ycf1和ycf15为假基因，它们的序列长度均短于正常基因。多脉铁木叶绿体基因组上共有58个SSR，其中单碱基重复有53个。序列比对结果表明，多脉铁木与天目铁木的相似性最高，达99.1%，与铁木的相似性最低，为98.7%。构建系统发育树的最佳模型为GTR+G+I，其中的AIC（赤池信息标准）和BIC（贝叶斯信息标准）分别为556 643.629 64和556 954.642 07。系统发育分析结果表明，12种植物在发育树上可分为两大组，桦木族的日本桤木和红桤木聚为一组，榛族的10种植物聚于另一组；多脉铁木与铁木属其他3个种聚于同一分支，支持率均为100%。结论: 在高通量测序的基础上，组装完成多脉铁木的叶绿体基因组，它与天目铁木、毛果铁木和铁木叶绿体基因组的相似性较高，在系统发育树上聚为一组。多脉铁木叶绿体基因组的组装、序列比对和系统发育分析，可为后续开展遗传结构和群体遗传多样性研究奠定基础。

关键词: 多脉铁木, 叶绿体基因组, 组装, 系统发育

Abstract:

Objective: Ostrya multinervis is a species endemic to China, and it is listed as a rare and endangered wild plant, and a species with extremely small population in Zhejiang Province. The wild O. multinervis trees are found to be very rare in Zhejiang Province with very few young-aged plants and a declining population. During the evolution process, the chloroplast genomes are relatively conserved and thus provide useful information for genetic relationship analysis and phylogenetic studies. In the present study, based on the assembly of chloroplast genome, sequence characteristics were determined to confirm its structure and gene organization. By phylogenetic analysis, relationships and taxonomic ranks with its relative were identified. Methods: Genomic DNA of O. multinervis was extracted by using CTAB method, and a library was constructed for high-throughput sequencing with HiSeq X Ten with the paired-end strategy. The chloroplast genome was assembled by using NovoPlasty, and PCR was applied to isolate the border sequences following sequencing verification. SSR loci were detected by MISA and a phylogenetic tree was generated using PhyML 3.0. Results: A total of 19 869 412 clean reads were obtained by getting rid of both adaptors and low quality sequences. The result indicated that the complete O. multinervis chloroplast genome was 159 583 bp in length containing a typical quadripartite structure, and the lengths of large single copy, small single copy, and inverted repeat were 88 514 bp, 18 953 bp, and 26 058 bp, respectively. The O. multinervis chloroplast genome contains 131 genes, with 87 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. Among them, ycf1 and ycf15 were pseudogenes, and their sequences were shorter than normal ones. A total of 58 SSR loci were predicted from O. multinervis plastome, and 53 of which were mononucleotide repeats. Multiple sequence comparison showed the highest similarity between O. multinervis and O. rehderiana (99.1%) and the lowest was between O. multinervis and O. japonica (98.7%). The best fit model for constructing a phylogenetic tree was GTR+G+I, and the Akaike information criterion and Bayesian information criterion were 556 643.629 64 and 556 954.642 07, respectively. The phylogenetic analysis indicated that 12 plants could be divided into two groups, and Alnus japonica and A. rubra from the tribe Betuleae were grouped into one clade, while 10 plants from tribe Coryleae were grouped into another clade. O. multinervis and the other three Ostrya species clustered in the same clade with 100% of bootstrap support. Conclusion: Based on the high-throughput sequencing, the chloroplast genome of O. multinervis was assembled, and its structure and gene organizations were confirmed. Higher similarities in chloroplast genome sequences were found between O. multinervis and other three Ostrya species, and they shared the same clade. The assembly, sequence comparison and phylogenetic analysis provide a basis for genetic structure and population diversity studies in the future.

Key words: Ostrya multinervis, chloroplast genome, assembly, phylogenetic analysis

中图分类号:

S718.46

蒋明,柯世省,王军峰. 多脉铁木叶绿体基因组的序列特征和系统发育[J]. 林业科学, 2020, 56(5): 60-68.

Ming Jiang,Shisheng Ke,Junfeng Wang. Characterization and Phylogenetic Analysis of Ostrya multinervis Chloroplast Genome[J]. Scientia Silvae Sinicae, 2020, 56(5): 60-68.

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区域Region	A(%)	T(%)	C(%)	G(%)	长度Length/bp	GC(%)
LSC	32.1	33.7	17.6	16.6	88 514	34.2
SSC	34.9	35.2	15.7	14.1	18 953	29.8
IRa/IRb	28.9	28.6	20.5	22.0	26 058	42.5
合计Total	31.4	32.3	18.5	17.8	159 583	36.4

基因类别 Gene category	基因名称 Gene name	数量 Number
转运RNA Transfer RNA	trnA-UGC(×2)^, trnC-GCA,* trnD-GTC, trnE-TTC, trnF-GAA, trnfM-CAT, trnG-GCC, trnH-GTG, trnI-CAT(×2), trnI-GAU(×2)^, trnL-CAA(×2), trnL-TAG,* trnL-UAA^, trnM-CAT,* trnN-GTT(×2), trnP-TGG, trnQ-TTG, trnR-ACG(×2), trnR-TCT, trnS-CGA^, trnS-GCT,* trnS-GGA, trnS-TGA, trnT-GGT, trnT-TGT, trnV-GAC(×2), trnV-UAC^, trnW-CCA,* trnY-GTA	36
核糖体RNA Ribosome RNA	rrn4.5(×2), rrn5(×2), rrn16(×2), rrn23(×2)	8
核糖体蛋白小亚基 Small subunit of ribosome	rps2, rps3, rps4, rps7(×2), rps8, rps11, rps12(×2)^*, rps14,* rps15, rps16^, rps18,* rps19	14
核糖体蛋白大亚基Large subunit of ribosome	rpl2(×2)^, rpl14,* rpl16^, rpl20,* rpl22, rpl23(×2), rpl32, rpl33, rpl36	11
RNA聚合酶亚基RNA polymerase subunits	rpoA, rpoB, rpoC1^, rpoC2*	4
NADH脱氢酶亚基 Subunits of NADH dehydrogenase	ndhA^, ndhB(×2)^, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK	12
光系统I亚基Subunits of photosystem I	psaA, psaB, psaC, psaI, psaJ	5
光系统II亚基Subunits of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ	15
细胞色素亚基Subunits of cytochrome	petA, petB^, petD^, petG, petL, petN	6
ATP合成酶亚基Subunits of ATP synthase	atpA, atpB, atpE, atpF^, atpH,* atpI	6
Rubisco大亚基Large subunit of Rubisco	rbcL	1
成熟酶Maturase	matK	1
蛋白酶Protease	clpP^**	1
被膜蛋白Envelope membrane protein	cemA	1
乙酰CoA亚基Subunit of acetyl-CoA	accD	1
细胞色素C合成酶Synthase of cytochrome C	ccsA	1
翻译起始因子Translational initiation factor	infA	1
未知功能蛋白Unknown function	ycf1, Ψycf1, ycf2(×2), ycf3^*, ycf4,* Ψycf15	7
合计Total		131

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