药学学报

Species	Family	Genus	GenBank accession number
Ardisia gigantifolia	Myrsinaceae	Ardisia	MN548760.1
Ardisia mamillata	Myrsinaceae	Ardisia	MN136062.1
Ardisia polysticta	Myrsinaceae	Ardisia	KC465962.1
Ardisia fordii	Myrsinaceae	Ardisia	OK514746.1
Ardisia quinquegona	Myrsinaceae	Ardisia	OK514745.1
Ardisia villosa	Myrsinaceae	Ardisia	OK509170.1
Ardisia faberi	Myrsinaceae	Ardisia	OK509169.1
Ardisia japonica	Myrsinaceae	Ardisia	OK148445.1
Ardisia solanacea	Myrsinaceae	Ardisia	MN094783.1
Ardisia argenticaulis	Myrsinaceae	Ardisia	OK509165.1
Ardisia merrillii	Myrsinaceae	Ardisia	OK054493.1
Ardisia crenata	Myrsinaceae	Ardisia	MW929178.1
Ardisia omissa	Myrsinaceae	Ardisia	OK054494.1
Ardisia carnosicaulis	Myrsinaceae	Ardisia	OK054492.1
Ardisia replicate	Myrsinaceae	Ardisia	OK514747.1
Ardisia balansana	Myrsinaceae	Ardisia	OK509167.1
Ardisia sieboldii	Myrsinaceae	Ardisia	OK509163.1
Ardisia obtusa	Myrsinaceae	Ardisia	OK509166.1
Maesa montana	Myrsinaceae	Maesa	KU569490.1
Maesa hupehensis	Myrsinaceae	Maesa	MZ846203.1
Myrsine stolonifera	Myrsinaceae	Myrsine	MN167883.1
Aegiceras corniculatum	Myrsinaceae	Aegiceras	MN167882.1
Embelia scandens	Myrsinaceae	Embelia	MW246148.1
Embelia vestita	Myrsinaceae	Embelia	MN167884.1
Myrsine africana	Myrsinaceae	Myrsine	MN165129.1
Primula handeliana	Primulaceae	Primula	MG181221.1
Primula pulchella	Primulaceae	Primula	KX668179.1
Primula moupinensis	Primulaceae	Primula	KX668175.1

Species	Family	Genus	GenBank accession number
Ardisia gigantifolia	Myrsinaceae	Ardisia	MN548760.1
Ardisia mamillata	Myrsinaceae	Ardisia	MN136062.1
Ardisia polysticta	Myrsinaceae	Ardisia	KC465962.1
Ardisia fordii	Myrsinaceae	Ardisia	OK514746.1
Ardisia quinquegona	Myrsinaceae	Ardisia	OK514745.1
Ardisia villosa	Myrsinaceae	Ardisia	OK509170.1
Ardisia faberi	Myrsinaceae	Ardisia	OK509169.1
Ardisia japonica	Myrsinaceae	Ardisia	OK148445.1
Ardisia solanacea	Myrsinaceae	Ardisia	MN094783.1
Ardisia argenticaulis	Myrsinaceae	Ardisia	OK509165.1
Ardisia merrillii	Myrsinaceae	Ardisia	OK054493.1
Ardisia crenata	Myrsinaceae	Ardisia	MW929178.1
Ardisia omissa	Myrsinaceae	Ardisia	OK054494.1
Ardisia carnosicaulis	Myrsinaceae	Ardisia	OK054492.1
Ardisia replicate	Myrsinaceae	Ardisia	OK514747.1
Ardisia balansana	Myrsinaceae	Ardisia	OK509167.1
Ardisia sieboldii	Myrsinaceae	Ardisia	OK509163.1
Ardisia obtusa	Myrsinaceae	Ardisia	OK509166.1
Maesa montana	Myrsinaceae	Maesa	KU569490.1
Maesa hupehensis	Myrsinaceae	Maesa	MZ846203.1
Myrsine stolonifera	Myrsinaceae	Myrsine	MN167883.1
Aegiceras corniculatum	Myrsinaceae	Aegiceras	MN167882.1
Embelia scandens	Myrsinaceae	Embelia	MW246148.1
Embelia vestita	Myrsinaceae	Embelia	MN167884.1
Myrsine africana	Myrsinaceae	Myrsine	MN165129.1
Primula handeliana	Primulaceae	Primula	MG181221.1
Primula pulchella	Primulaceae	Primula	KX668179.1
Primula moupinensis	Primulaceae	Primula	KX668175.1

Gene name	Primer sequence (5′-3′)	Size/bp
IRa-SSC-F	CCCTTCCATGCCTCATTTCA	1 284
IRa-SSC-R	GGCGGACGCATCTCATCTTA
IRa-LSC-F	TATCGGTATACGCCCATCCA	1 250
IRa-LSC-R	AGGCACGGCCATACATAACA
IRb-SSC-F	TGTGCTCGCATAGACCCTTT	1 418
IRb-SSC-R	GGCAGAATACCGTCACCCAT
IRb-LSC-F	TCTGGGGAGGTCCGTTTGAT	1 306
IRb-LSC-R	TCGGCGGTTCCCTATTCAGT

Gene name	Primer sequence (5′-3′)	Size/bp
IRa-SSC-F	CCCTTCCATGCCTCATTTCA	1 284
IRa-SSC-R	GGCGGACGCATCTCATCTTA
IRa-LSC-F	TATCGGTATACGCCCATCCA	1 250
IRa-LSC-R	AGGCACGGCCATACATAACA
IRb-SSC-F	TGTGCTCGCATAGACCCTTT	1 418
IRb-SSC-R	GGCAGAATACCGTCACCCAT
IRb-LSC-F	TCTGGGGAGGTCCGTTTGAT	1 306
IRb-LSC-R	TCGGCGGTTCCCTATTCAGT

Genomic region	A. crispa var. amplifolia		A. crispa var. dielsii
Total	156 749	37.05		156 748	37.05
LSC	86 303	34.95	86 303	34.95
IR	26 014	42.97	26 014	42.97
SSC	18 418	30.14	18 417	30.14

Genomic region	A. crispa var. amplifolia		A. crispa var. dielsii
Total	156 749	37.05		156 748	37.05
LSC	86 303	34.95	86 303	34.95
IR	26 014	42.97	26 014	42.97
SSC	18 418	30.14	18 417	30.14

Gene group	Gene function	Gene name
Self-replication	Large subunit of ribosone	rpl23(x2), rpl2^*(x2), rpl36, rpl33, rpl32, rpl20, rpl16^, rpl22, rpl14*
	Small subunit of ribosone	rps8, rps12(x2), rps11, rps3, rps2, rps18, rps7(x2), rps4, rps19, rps16^, rps15, rps14*
	RNA polymerase	rpoB, rpoC2, rpoC1^, rpoA*
	rRNA	rrn16S(x2), rrn4.5S(x2), rrn5S(x2), rrn23S(x2)
	tRNA	tRNA-Val(x2), tRNA-Gly(x2), tRNA-Tyr, tRNA-Lys, tRNA-Trp, tRNA-Thr(x2), tRNA-Ser(x3), tRNA-Phe, tRNA-Met(x4), tRNA-His, tRNA-Glu, tRNA-Arg(x3), tRNA-Gln, tRNA-Pro, tRNA-Cys(x2), tRNA-Asp, tRNA-Asn(x2), tRNA-Ala(x2), tRNA-Leu(x4), tRNA-Ile(x2)
Photosynthesis	Photosystem I	psaI, psaB, psaJ, psaC, psaA
	Photosystem II	psbA, psbD, psbC, psbK, psbI, psbM, psbL, psbF, psbZ, psbJ, psbE, psbB, psbT, psbN, psbH
	NADH dehydrogenase	ndhJ, ndhF, ndhD, ndhK, ndhC, ndhG, ndhI, ndhA^*, ndhH, ndhB^(x2), ndhE*
	Cytochrome b/f complex	petB^, petD^, petN, petL, petG, petA
	ATP synthase	atpA, atpE, atpF^, atpI, atpB, atpH*
	Large subunit of rubisco	rbcL
Other genes	Translational initation factor	infA
	Maturase	matK
	Protease	clpP^*
	Envelope membrane protein	cemA
	Subunit of acetyl-carboxylase	accD
	C-type cytochrome synthesis	ccsA
	Open reading frames	ycf15(x2), ycf4, ycf2(x2), ycf3^, ycf1*

Gene group	Gene function	Gene name
Self-replication	Large subunit of ribosone	rpl23(x2), rpl2^*(x2), rpl36, rpl33, rpl32, rpl20, rpl16^, rpl22, rpl14*
	Small subunit of ribosone	rps8, rps12(x2), rps11, rps3, rps2, rps18, rps7(x2), rps4, rps19, rps16^, rps15, rps14*
	RNA polymerase	rpoB, rpoC2, rpoC1^, rpoA*
	rRNA	rrn16S(x2), rrn4.5S(x2), rrn5S(x2), rrn23S(x2)
	tRNA	tRNA-Val(x2), tRNA-Gly(x2), tRNA-Tyr, tRNA-Lys, tRNA-Trp, tRNA-Thr(x2), tRNA-Ser(x3), tRNA-Phe, tRNA-Met(x4), tRNA-His, tRNA-Glu, tRNA-Arg(x3), tRNA-Gln, tRNA-Pro, tRNA-Cys(x2), tRNA-Asp, tRNA-Asn(x2), tRNA-Ala(x2), tRNA-Leu(x4), tRNA-Ile(x2)
Photosynthesis	Photosystem I	psaI, psaB, psaJ, psaC, psaA
	Photosystem II	psbA, psbD, psbC, psbK, psbI, psbM, psbL, psbF, psbZ, psbJ, psbE, psbB, psbT, psbN, psbH
	NADH dehydrogenase	ndhJ, ndhF, ndhD, ndhK, ndhC, ndhG, ndhI, ndhA^*, ndhH, ndhB^(x2), ndhE*
	Cytochrome b/f complex	petB^, petD^, petN, petL, petG, petA
	ATP synthase	atpA, atpE, atpF^, atpI, atpB, atpH*
	Large subunit of rubisco	rbcL
Other genes	Translational initation factor	infA
	Maturase	matK
	Protease	clpP^*
	Envelope membrane protein	cemA
	Subunit of acetyl-carboxylase	accD
	C-type cytochrome synthesis	ccsA
	Open reading frames	ycf15(x2), ycf4, ycf2(x2), ycf3^, ycf1*

Species	P	F	R	C	Total
A. crispa var. amplifolia	29	24	1	0	54
A. crispa var. dielsii	29	24	1	0	54

Species	P	F	R	C	Total
A. crispa var. amplifolia	29	24	1	0	54
A. crispa var. dielsii	29	24	1	0	54

SSR type	A. crispa var. amplifolia	A. crispa var. dielsii
Mono	94	94
Di	41	41
Tri	71	71
Tetra	8	8
Penta	2	2
Hexa	0	0
Total	217	217

SSR type	A. crispa var. amplifolia	A. crispa var. dielsii
Mono	94	94
Di	41	41
Tri	71	71
Tetra	8	8
Penta	2	2
Hexa	0	0
Total	217	217

Amino acid	A. crispa var. amplifolia		A. crispa var. dielsii
Phe	UUU	1.20		UUU	1.20
Leu	UUA	1.28	UUA	1.40
UUG	1.29	UUG	1.25
CUU	1.25	CUU	1.23
Ile	AUU	1.24	AUU	1.23
		AUA	1.04
Val	GUU	1.37	GUU	1.35
GUA	1.12	GUA	1.17
Ser	UCU	1.48	UCU	1.50
UCC	1.11	UCC	1.12
UCA	1.11	UCA	1.25
Pro	CCU	1.10	CCU	1.10
CCA	1.28	CCA	1.21
		CCC	1.05
Thr	ACU	1.21	ACU	1.16
ACC	1.06	ACC	1.04
ACA	1.07	ACA	1.12
Ala	GCU	1.35	GCU	1.36
GCA	1.10	GCA	1.11
Tyr	UAU	1.34	UAU	1.33
His	CAU	1.43	CAU	1.44
Gln	CAA	1.39	CAA	1.38
Asn	AAU	1.39	AAU	1.38
Lys	AAA	1.36	AAA	1.37
Asp	GAU	1.40	GAU	1.40
Glu	GAA	1.39	GAA	1.39
Cys	UGU	1.24	UGU	1.23
Arg	CGA	1.07	CGA	1.11
AGA	2.02	AGA	2.04
AGG	1.06	AGG	1.05
Gly	GGA	1.44	GGA	1.36
		GGU	1.02
TER	UAA	1.29	UAA	1.27

Amino acid	A. crispa var. amplifolia		A. crispa var. dielsii
Phe	UUU	1.20		UUU	1.20
Leu	UUA	1.28	UUA	1.40
UUG	1.29	UUG	1.25
CUU	1.25	CUU	1.23
Ile	AUU	1.24	AUU	1.23
		AUA	1.04
Val	GUU	1.37	GUU	1.35
GUA	1.12	GUA	1.17
Ser	UCU	1.48	UCU	1.50
UCC	1.11	UCC	1.12
UCA	1.11	UCA	1.25
Pro	CCU	1.10	CCU	1.10
CCA	1.28	CCA	1.21
		CCC	1.05
Thr	ACU	1.21	ACU	1.16
ACC	1.06	ACC	1.04
ACA	1.07	ACA	1.12
Ala	GCU	1.35	GCU	1.36
GCA	1.10	GCA	1.11
Tyr	UAU	1.34	UAU	1.33
His	CAU	1.43	CAU	1.44
Gln	CAA	1.39	CAA	1.38
Asn	AAU	1.39	AAU	1.38
Lys	AAA	1.36	AAA	1.37
Asp	GAU	1.40	GAU	1.40
Glu	GAA	1.39	GAA	1.39
Cys	UGU	1.24	UGU	1.23
Arg	CGA	1.07	CGA	1.11
AGA	2.02	AGA	2.04
AGG	1.06	AGG	1.05
Gly	GGA	1.44	GGA	1.36
		GGU	1.02
TER	UAA	1.29	UAA	1.27

Intron gene	A. crispa var. amplifolia	A. crispa var. dielsii
rps16	CDS length	267	267
Gene length	1 116	1 116
atpF	CDS length	630	630
Gene length	1 265	1 265
rpoC1	CDS length	2 061	2 061
Gene length	2 812	2 812
ycf3	CDS length	510	510
Gene length	1 952	1 952
clpP	CDS length	588	588
Gene length	2 122	2 122
petB	CDS length	648	648
Gene length	1 408	1 408
petD	CDS length	483	483
Gene length	1 316	1 316
rpl16	CDS length	408	408
Gene length	1 405	1 405
rpl2	CDS length	825/825	825/825
Gene length	1 480/1 480	1 480/1 480
ndhB	CDS length	1 533/1 533	1 533/1 533
Gene length	2 212/2 212	2 212/2 212
ndhA	CDS length	1 092	1 092
Gene length	2 210	2 210

Intron gene	A. crispa var. amplifolia	A. crispa var. dielsii
rps16	CDS length	267	267
Gene length	1 116	1 116
atpF	CDS length	630	630
Gene length	1 265	1 265
rpoC1	CDS length	2 061	2 061
Gene length	2 812	2 812
ycf3	CDS length	510	510
Gene length	1 952	1 952
clpP	CDS length	588	588
Gene length	2 122	2 122
petB	CDS length	648	648
Gene length	1 408	1 408
petD	CDS length	483	483
Gene length	1 316	1 316
rpl16	CDS length	408	408
Gene length	1 405	1 405
rpl2	CDS length	825/825	825/825
Gene length	1 480/1 480	1 480/1 480
ndhB	CDS length	1 533/1 533	1 533/1 533
Gene length	2 212/2 212	2 212/2 212
ndhA	CDS length	1 092	1 092
Gene length	2 210	2 210

大叶百两金和细柄百两金叶绿体全基因组解析及系统发育分析

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曾宪法 , 刘畅 , 杨小英 , 余庆 , 扶世伦 , 鄢藤云 , 蒲翔 ^*

药学学报 | 研究论文 2023,58(1): 217-228

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药学学报 |研究论文 2023 , 58 (1) : 217 -228

大叶百两金和细柄百两金叶绿体全基因组解析及系统发育分析

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曾宪法, 刘畅, 杨小英, 余庆, 扶世伦, 鄢藤云, 蒲翔^*

作者信息

贵州中医药大学, 贵州贵阳 550025

通讯作者:

*蒲翔, E-mail: 314745595@qq.com

Chloroplast genome resolution and phylogenetic analysis of Ardisia crispa var. amplifolia and Ardisia crispa var. dielsii

Xian-fa ZENG, Chang LIU, Xiao-ying YANG, Qing YU, Shi-lun FU, Teng-yun YAN, Xiang PU^*

Affiliations

Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China

出版时间: 2023-01-12 doi: 10.16438/j.0513-4870.2022-0874

文章导航

摘要

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百两金Ardisia crispa（Thunb.）A.DC.为传统苗药药材，在治疗咽喉痛、扁桃腺炎、肾炎水肿及跌打风湿等方面具有显著疗效。大叶百两金Ardisia crispa var.amplifolia和细柄百两金Ardisia crispa var.dielsii是百两金的变种，两者在物种进化关系及分类鉴定方面存在争议。本研究利用Illumina平台测序获取两者叶绿体全基因组序列，对其进行组装、注释和特征分析，采用生物信息学方法比较叶绿体基因组间的结构特征与变异程度，同时下载报春花科Primulaceae和紫金牛科Myrsinaceae植物叶绿体全基因组序列信息构建系统发育树解析其系统发育关系。结果表明大叶百两金和细柄百两金完整叶绿体基因组序列全长156 749 bp、156 748 bp，均注释到基因132个，其中蛋白质编码基因87个；密码子偏好性A/U大于G/C；基因组比较分析中两者在rps15和rpoB基因编码区存在差异，为两者分子鉴定提供新的位点；与同属物种相比较叶绿体基因组中ycf1、ycf2、rpoC1、ycf3、petD及rpl16等基因编码区存在差异，可作该属植物的鉴别位点；系统发育结果中，大叶百两金和细柄百两金聚为一束，支持率为100%，表明两者亲缘关系较近。本研究对大叶百两金与细柄百两金叶绿体基因组结构和系统发育关系进行解析，为两者分子鉴别、遗传变异、育种以及系统发育分析等提供重要的理论依据。

关键词

紫金牛属 / 大叶百两金 / 细柄百两金 / 叶绿体基因组 / 系统发育

Abstract

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Ardisia crispa (Thunb.) A. DC. is a traditional Miao medicinal herb with significant therapeutic effects in the treatment of sore throat, tonsillitis, edema of nephritis and bruising and rheumatism, etc. Ardisia crispa var. amplifolia and Ardisia crispa var. dielsii are varieties of A. crispa. A. crispa var. amplifolia and A. crispa var. dielsii are controversial in terms of species evolutionary relationships and taxonomic identification. In this study, we sequenced the whole genome sequences of A. crispa var. amplifolia and A. crispa var. dielsii chloroplasts using Illumina platform, assembled, annotated and characterized them, compared the structural features and degree of variation among chloroplast genomes using bioinformatics methods, and also downloaded constructing phylogenetic trees to analyze the phylogenetic relationships of chloroplasts in Primulaceae and Myrsinaceae using whole genome sequence information. The results showed that the complete chloroplast genome sequences of A. crispa var. amplifolia and A. crispa var. dielsii were 156 749 bp and 156 748 bp in length, with 132 genes annotated, including 87 protein-coding genes; the codon preference of A/U was greater than that of G/C; The differences in the coding regions of rps15 and rpoB genes in the comparative genome analysis can be used as loci for molecular identification of the two species; the differences in the coding regions of ycf1, ycf2, rpoC1, ycf3, petD and rpl16 genes in the chloroplast genome compared with those of the same genus can be used as loci for identification of the genus. In the phylogenetic results, A. crispa var. amplifolia and A. crispa var. dielsii were clustered together with 100% support, indicating that they are closely related. In this research, we analyzed the chloroplast genome structure and phylogenetic relationships of A. crispa var. amplifolia and A. crispa var.dielsii, providing an important theoretical basis for their molecular identification, genetic variation, breeding and phylogenetic analysis.

Key words

Ardisia / A. crispa var. amplifolia / A. crispa var. dielsii / chloroplast genome / phylogeny

引用本文

曾宪法, 刘畅, 杨小英, 余庆, 扶世伦, 鄢藤云, 蒲翔. 大叶百两金和细柄百两金叶绿体全基因组解析及系统发育分析. 药学学报, 2023 , 58 (1) : 217 -228 . DOI: 10.16438/j.0513-4870.2022-0874

Xian-fa ZENG, Chang LIU, Xiao-ying YANG, Qing YU, Shi-lun FU, Teng-yun YAN, Xiang PU. Chloroplast genome resolution and phylogenetic analysis of Ardisia crispa var. amplifolia and Ardisia crispa var. dielsii[J]. Acta Pharmaceutica Sinica, 2023 , 58 (1) : 217 -228 . DOI: 10.16438/j.0513-4870.2022-0874

正文

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紫金牛属Ardisia植物是紫金牛科最大的属, 该属物种较多, 全世界分布广泛, 在我国主要集中分布于长江流域以南各地^[1], 该属植物常用于治疗风湿、跌打、痨咳及各种炎症等; 该属植物形态或功效的相似以及种间变异错综复杂导致本草记载中常出现同名异物或同物异名的情况, 各物种之间亲缘关系有待进一步明晰。百两金为紫金牛科紫金牛属百两金的干燥根或根茎, 具有清咽利喉、散淤消肿和舒筋活血等功效, 可治疗咽喉痛、扁桃体炎、肾炎水肿、跌打损伤、风湿疼痛、白浊、骨结核、劳伤咳血、痈疔和毒蛇咬伤等^[2]。大叶百两金和细柄百两金为百两金变种, 两变种植株外形差别较小, 仅叶、植株大小有所不同(图 1), 仅仅从形态学方面区分大叶百两金和细柄百两金缺乏科学依据。

叶绿体是植物细胞特有的一种半自主性细胞器, 拥有独立的基因组^[3], 存在于所有绿色植物和某些自养生物细胞中, 是它们进行光合作用和能量转换的重要场所, 是植物生命活动代谢中心^[4]; 在裸子植物中多为父系遗传, 而在被子植物中则以母系遗传为主^[5]。叶绿体基因组是叶绿体内带有遗传信息的一套DNA序列, 一般介于120~180 kb, 通常为典型的环状四分体结构, 包括1个大单拷贝区(large single copy region, LSC)、1个小单拷贝区(small single copy region, SSC)和2个反向重复区(inverted repeats region, IRa/IRb)^[6], 因其具有分子质量小、结构稳定、高度保守等特点, 目前已经被广泛应用于植物鉴定、系统进化分析、遗传多样性、种群动态历史及叶绿体基因工程等研究^{[7, 8]}; 随着测序技术的快速发展, 诸多药用植物的叶绿体基因组已被完成测序和数据分析, 取得了较好的研究成效, 如白头翁Pulsatilla chinensis、牛蒡Arctium lappa、罂粟Papaver somniferum^[9-11]等已经完成了叶绿体基因组分析, 确定了各自的系统位置及其与近缘物种的亲缘关系。目前紫金牛属部分物种已经完成叶绿体基因组测序, 如朱砂根Ardisia crenata、紫金牛Ardisia japonica、纽子果Ardisia polysticta等少数物种, 但变种之间及同种不同个体间叶绿体比较基因组学和系统发育研究鲜有报道。

因此, 本研究对大叶百两金和细柄百两金两个紫金牛属物种进行叶绿体基因组测序, 解析两者叶绿体基因组序列与结构特征, 筛选种间高变异序列, 以期为两变种的分子鉴别提供科学依据; 同时下载报春花科报春花属丽花报春Primula pulchella、宝兴报春Primula moupinensis、陕西报春Primula handeliana以及紫金牛科紫金牛属、杜茎山属Maesa、蜡烛果属Aegiceras、酸藤子属Embelia和铁仔属Myrsine已上传至NCBI植物叶绿体全基因组序列信息构建系统进化树, 旨在解析紫金牛科与报春花科以及紫金牛属种间亲缘关系, 为大叶百两金和细柄百两金物种鉴定、药材特异DNA指纹开发、分子植物育种等研究奠定基础。

材料与方法

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材料样品的新鲜叶片采自贵州省贵州中医药大学种质资源圃, 经魏升华教授鉴定为大叶百两金与细柄百两金, 取健康叶片用无菌水冲洗数次, 晾干后置于-80 ℃冰箱保存备用。通过NCBI数据库(https://www.ncbi.nlm.nih.gov/) 检索紫金牛科紫金牛属物种的叶绿体全基因组序列信息。检索到紫金牛科紫金牛属18个物种以及杜茎山属2个、蜡烛果属1个、酸藤子属2个和铁仔属2个物种叶绿体全基因组序列信息, 然后选取与紫金牛科亲缘关系较近的报春花科报春花属常见的3个观赏植物丽花报春、宝兴报春、陕西报春完整叶绿体全基因组序列信息构建系统发育树(表 1)。

基因组DNA提取与测序 利用植物基因组DNA提取试剂盒[天根生化科技(北京) 有限公司, 北京] 提取总DNA, 0.8%琼脂糖电泳检测DNA样品是否有降解及杂质, Nanodrop分光光度计检测样品浓度及纯度, 检测合格的DNA样品根据Illumian DNA文库构建流程, 构建插入片段大小为350 bp的双末端测序文库。对质检合格的高通量测序文库, 采用Illumina Novaseq6000高通量测序平台进行测序。Illumina高通量测序结果经CASAVA软件进行碱基识别(base calling) 后转化为原始测序序列(raw reads), 利用NGS QC ToolKit软件对raw reads进行质控, 过滤去除接头和低质量序列, 得到高质量序列(clean reads)。

叶绿体基因组组装、注释及图谱绘制 利用FastQC对测序所得原始序列进行质量检测, 采用Trimmomatic软件^[12]过滤去除接头序列和低质量区。使用NOVOPlasty^[13]将原始读长组装成完整的叶绿体基因组。采用BWA将高质量读长比对回叶绿体基因组序列, 并在IGV中进行人工检查以确保组装无误; 利用CPGAVAS软件^[14]和GeSeq软件^[15]对完整叶绿体基因组序列进行注释, tRNA基因用tRNAscan-SE软件^[16]进行鉴定及人工校正; 利用Organellar Genome DRAW (http://ogdraw.mpimp-golm.mpg.de/cgi-bin/ogdraw.pl) 在线绘制叶绿体基因组图谱。

边界序列的PCR扩增 将叶绿体基因组4个边界区域序列IRa-SSC、IRa-LSC、IRb-LSC、IRb-SSC采用PCR方法进行鉴别, 用在线软件Primer 3 (https://bioinfo.ut.ee/primer3-0.4.0/) 设计4对PCR扩增所用引物(表 2)。Eppendorf管中, 依次加入8.2 μL dd H₂O、10.0 μL 2×Taq PCR MasterMix II、0.4 μL的上游引物(20 μmol·L^-1)、0.4 μL下游引物(20 μmol·L^-1)、1.0 μL DNA模板(50 ng·μL^-1)。PCR反应在伯乐T100型PCR仪上进行, 程序为: 95 ℃预变性3 min、90 ℃变性3 s; 退火60 ℃ 30 s、延伸72 ℃ 45 s、30个循环。

重复序列和密码子偏向性分析 使用在线软件MISA^[17] (https://webblast.ipk-gatersleben.de/misa/index.php) 分析简单重复序列(SSRs), 参数阈值设置为1、2、3、4、5、6, 核苷酸参数为9、4、3、3、3、3, 且两个SSRs之间的距离不小于100 bp, 并对SSRs的类型、数量等进行比较分析。利用在线软件REPuter^[18] (https://bibiserv.cebitec.uni-biele feld.de/reputer) 对长重复序列(long repeat) 进行分析, 海明距离(Hamming distance)、最大计算重复次数(maximum computed repeats)、最小重复片段大小(minimal repeat size) 参数设置为3、5 000、30。采用软件CodonW^[19] (http://codonw.sourceforge.net) 分析密码子使用情况。

叶绿体基因组比较分析 利用在线软件IRSCOPE^[20] (https://irscope.shinyapps.io/irapp/) 对百两金与其同属6个物种叶绿体基因组4个区域的边界信息进行分析, 并观察收缩与扩张在IRs区域的现象; 采用mVISTA^[21]对全基因组进行比对分析; 使用Geneious10.2.2软件中Mauve多重基因组比对法进行共线性比较。

系统发育分析 本研究选择已发表的紫金牛科和报春花科共30个物种进行系统发育树构建, 利用在线软件MAFFT 7 (https://mafft.cbrc.jp/alignment/server/) 将30个物种叶绿体基因组序列进行比对, 结果采用MEGAX软件^[22]中邻接法(neighbor-joining, NJ) 法构建系统进化树, bootstrap重复值设置为1 000。同时, 采用IQ-TREE 2.0.5 (http://www.iqtree.org/) 软件, 最大似然法(ML法: maximum likelihood method) 构建系统发育树, 构树参数设置为: -m MFP -B 1 000 -alrt 1 000, 优构树模型为: TVM+F+I+G4。

结果与分析

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1 叶绿体基因的边界验证

通过PCR手段对大叶百两金(1~4) 和细柄百两金(5~8) 4个边界进行验证(图 2)。电泳结果表明, 4对引物均能扩增出单一条带, 大小与预期一致。

2 叶绿体基因组结构

大叶百两金与细柄百两金叶绿体基因组大小为156 749 bp、156 748 bp, 呈双链环状四分区域结构, 包括1个大单拷贝区(large single-copy, LSC)、1对反向互补重复区(inverted repeats, IR) 和1个小单拷贝区(small single-copy region, SSC), 其大小分别为86 303 bp、26 014 bp、18 418 bp, G/C与A/T含量均为37.05%、62.95%, 有明显的AT偏向性(图 3)。此外, LSC、IR和SSC区域的GC值存在一定的差异(表 3), IR区的G/C含量最高(42.97%), 其次是SSC区(30.14%) 及LSC区(34.95%)。

3 叶绿体基因组功能及分类

大叶百两金与细柄百两金叶绿体基因组共包含132个基因, 其中编码蛋白基因87个、rRNA基因8个与tRNA基因37个。根据其功能分为3大类: 自我复制有关的基因、光合作用有关的基因和其他基因; 在这些基因中, 蛋白质编码基因10个: ycf2、ycf1、rpl2、rps12、ndhB、rps19、rps7、rpl23、ycf15、ndhF; tRNA编码基因7个: tRNA-Met、tRNA-Val、tRNA-Arg、tRNA-Leu、tRNA-Ile、tRNA-Asn、tRNA-Ala; rRNA编码基因4个: rrn5S、rrn4.5S、rrn16S、rrn23S位于IR区(表 4)。

4 重复序列分析

长重复序列进行分析表明, 大叶百两金和细柄百两金长重复序列皆为54个, 包括正向重复(forward repeats, F)、回文重复(palindrome repeats, P)、反向重复(reverse repeats, R) 和互补重复(complement repeats, C)。其中有24个正向重复F (10个30~39 bp, 7个40~49 bp, 2个50~59 bp, 2个60~69 bp, 3个70~79 bp)、29个回文重复P (12个30~39 bp, 10个40~49 bp, 2个50~59 bp, 1个60~69 bp, 4个70~79 bp)、1个反向重复R (30~39 bp) 和0个互补重复C, 两者叶绿体基因组中大多数重复序列长度都在30~39 bp之间, 均未鉴定到互补重复序列(表 5)。简单重复序列(simple sequence repeat, SSRs) 分析表明, 两者均有217个SSRs位点, 均未发现六核苷酸重复基序(表 6)。

5 叶绿体基因组密码子使用分析

根据RSCU值计算大叶百两金和细柄百两金叶绿体基因组蛋白编码基因的密码子使用模式(表 7)。结果表明两个百两金叶绿体基因组均包含64种密码子, 其中61种密码子编码21种氨基酸, 其余3个为终止密码子; 在所有氨基酸密码子中, 编码亮氨酸(Leu, L) 的密码子数最多。根据RSCU值对其密码子进行统计分析, 大叶百两金RSCU为 > 1的密码子共30个, 其中4个密码子以G/C结尾, 26个密码子以A/U结尾; 细柄百两金RSCU为 > 1的密码子共33个, 其中5个密码子以G/C结尾, 28个密码子以A/U结尾。上述结果表明两物种叶绿体基因组对A/U结尾密码子的偏好性高于G/C结尾密码子。

6 叶绿体基因组内含子分析

内含子长度或编码序列长度等因素均会直接影响到整个基因长度的变化, 大叶百两金和细柄百两金的叶绿体基因组中内含子编码序列(coding sequences, CDS) 长度和基因全长(gene length) 如下(表 8), 两物种的叶绿体基因组中均检测到11个含内含子的蛋白编码基因: petB、petD、rps16、ycf3、ndhA、clpP、rpoC1、rpl2、atpF、ndhB和rpl16。结果表明在这11个含内含子的蛋白编码基因中, 两物种内含子蛋白编码基因在内含子长度和编码序列长度上均一致。

7 叶绿体基因组边界分析

紫金牛属叶绿体基因组虽然在序列长度、基因组成及GC含量等方面相对稳定保守, 但4个边界区的过渡区域却存在有多样性(图 4)。紫金牛8个物种的IRb-LSC边界均位于rps19基因的编码区; 对于IRb-SSC边界, 大叶百两金和细柄百两金边界均位于ndhF基因的编码区, 月月红Ardisia faberi Hemsl.的ndhF基因仅存在于SSC, 灰色紫金牛Ardisia fordii Hemsl.则存在于ycf1基因的编码区, 其余4个物种IRb-SSC边界均在ndhF基因的编码区; 紫金牛8个物种的IRa-SSC边界, 除了灰色紫金牛位于ndhF基因的编码区外, 其他物种均位于ycf1基因编码区; 对于紫金牛属8个物种的IRa-LSC边界, rpl2基因均位于IRa区域, 月月红、酸苔菜Ardisia solanacea Roxb.、纽子果和走马胎Ardisia gigantifolia Stapf位于trnH基因内, 酸苔菜rps19基因存在向LSC区扩张趋势, 纽子果与走马胎rps1基因位于边界处, 大叶百两金与细柄百两金位于tRNA基因编码区。

8 基因组序列变异分析

为了评估紫金牛属叶绿体基因组序列的差异程度, 本研究采用mVISTA软件, 并选用检测基因重排和倒位的全局比对模式(shuffle-LAGAN), 以大叶百两金作为参考基因组, 对细柄百两金、灰色紫金牛、走马胎、罗伞树Ardisia quinquegona Blume、纽子果、酸苔菜、月月红叶绿体全基因组序列同源性进行比较(图 5) 和基因序列比对研究(图 6)。结果表明, 紫金牛属物种叶绿体基因组LSC、IRa、SSC、IRb四个区域排列顺序较为一致、保守性较高, 在非基因编码区存在有较明显的差异, 变异程度较高, 其中SSC区的变异程度最高, 而在基因编码区差异不明显, 在IRa区的变异程度最低, 具有较高的保守性; 紫金牛属叶绿体基因组中ycf1、clpP、rpoC1、ycf3、petD、ycf2、ndhA、rpl16等基因的编码区存在显著差异, 此外, 大叶百两金和细柄百两金两物种高度相似, 仅rps15和rpoB基因的编码区存在差异, 两位点可为大叶百两金和细柄百两金的分子鉴定提供新的位点资源。基因序列比对研究(图 6) 中大叶百两金与细柄百两金仅存在1个碱基的差异, 该变异发生在125 730 bp处, 大叶百两金在该位点有1个碱基C的插入, 两者与同属物种叶绿体基因比对中部分碱基存在差异, 如ycf1基因131 688 bp处大叶百两金与细柄百两金在该位点为碱基A, 同属其他物种该位点为碱基G。

9 叶绿体基因组共线性分析

为了评估大叶百两金与细柄百两金和同属物种在进化上的差异程度, 利用Mauve软件对大叶百两金、细柄百两金和同属的灰色紫金牛、走马胎、罗伞树、纽子果、酸苔菜、月月红的叶绿体基因进行对照分析, 通过多重基因组比对法检测出8个物种的叶绿体基因组之间有3个局部共线块(locally collinear block, LCB) (图 7)。该属8个物种叶绿体全基因组序列的比对显示其物种之间的基因组具有高度的相似性, 叶绿体基因组之间没有重排或倒置, 但在区域(83 000~110 000; 130 000~156 000) 中观察到高频的变异, 其特征是在对齐的叶绿体基因组中基因序列的高度变异。

10 系统发育分析

根据本研究中大叶百两金和细柄百两金的叶绿体基因组, 并选择已发表的紫金牛科紫金牛属、杜茎山属、蜡烛果属、酸藤子属、铁仔属叶绿体基因组和亲缘关系相近的报春花科报春花属丽花报春、宝兴报春、陕西报春构建NJ与ML系统发育树。邻接法结果显示(图 8), 紫金牛科5个属与报春花科报春花属共30个物种之间存在明确的系统进化关系, 可确定为2个主要类群, 紫金牛属、杜茎山属、报春花属、酸藤子属、蜡烛果属和铁仔属为一个类群, 紫金牛属为一个类群; 杜茎山属、报春花属聚为一个支, 支持率100%, 表明2个属之间亲缘关系密切; 大叶百两金和细柄百两金在第二类群中以100%支持率和虎红舌、白花紫金牛两者聚为一束, 亲缘关系相对属间其他物种更近, 该束中大叶百两金和细柄百两金也以100%支持率聚为一小支, 表明两者亲缘关系最近。以报春花属为外类群的最大似然法(ML法) 发育树中(图 9), 紫金牛属为一个大类群, 杜茎山属、蜡烛果属、酸藤子属、铁仔属聚在一起, 各属植物都聚为一支, 杜茎山属和报春花属相隔最近且大叶百两金和细柄百两金依然100%支持率和虎红舌、白花紫金牛两者聚为一束。系统进化树结果表明叶绿体基因组构建的系统发育树可用于紫金牛属物种植物的鉴定。

讨论

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本研究完成了大叶百两金和细柄百两金叶绿体基因组的测序、组装和注释, 并对其结构、GC含量等进行了分析, 研究表明两者叶绿体基因组呈典型的环状四分体结构, 序列总长度、GC含量、基因数量基本一致。其中, IRs区序列GC含量最高, 各区域序列中GC含量从高到低依次为IRs > LSC > SSC; 进一步分析发现, 其原因可能是IR区含有高GC含量的rRNA基因。此外, 包括本研究新测序的大叶百两金和细柄百两金在内的紫金牛属8个不同个体叶绿体全基因组、LSC、SSC和IRs区长度的种间差异范围分别为1~895 bp、0~1 264 bp、1~325 bp和0~821 bp, 即总长度的差异主要来自于LSC区, 而SSC和IRs区长度相对保守, 这一发现与张明英等^[18]在对北柴胡、紫花阔叶柴胡叶绿体全基因组解析及柴胡属药用植物叶绿体基因组比较与系统发育分析研究中一致。

叶绿体简单序列重复(SSRs) 拷贝数变异是一种重要的分子标记, 已经被用于基因流、种群分化和遗传多样性研究^[23], 此外, 分布于植物叶绿体基因组中的SSRs还同时兼具单亲遗传模式, 被作为分子标记广泛用于群体遗传学、谱系地理学等相关研究^{[24, 25]}。本研究中大叶百两金和细柄百两金叶绿体基因组共检测到217个SSR位点, 均未发现六核苷酸重复基序, SSR均以单核苷酸重复(占总数的43.32%) 为主要类型, 且随着拷贝数目增加, SSRs数量明显减少。单核苷酸和二核苷酸SSRs的重复序列类型分别以A/T和AT/TA为主, 其余核苷酸SSRs的重复单元亦主要由A、T碱基组合构成, 这也进一步验证了叶绿体基因组序列中的SSRs主要由polyA或polyT所构成, 这些SSRs可以为紫金牛属药用植物分子遗传学相关研究提供候选分子标记^{[18, 26]}。

叶绿体基因组IR、LSC和SSC区域扩张和收缩是植物进化中的常见现象, 也是造成叶绿体基因组长度差异与结构变异的主要原因^{[27, 28]}, 通过分析IR-LSC/SSC区域的边界信息, 对研究叶绿体基因组结构的差异、物种进化等具有重要意义^{[29, 30]}, 本文对紫金牛科8个物种植物叶绿体基因组边界分析发现LSC/IR和SSC/IR边界基因排列和类型有差异, 如月月红的ndhF基因仅存在于SSC区, 灰色紫金牛则存在于ycf1基因的编码区; 同时, 酸苔菜、罗伞树、灰色紫金牛的rps19基因向IRb区扩张现象明显, 与已有研究中马兜铃科^[31]、菊科^[32]、禾本科^[33]物种叶绿体基因组边界分析发现叶绿体基因组边界变化不呈现规律性相一致。

叶绿体基因组包括蛋白编码区和非编码区, 编码区具有多种不同的基因, 具有光合、复制等作用^[34], 大叶百两金和细柄百两金叶绿体全基因组序列在非基因编码区存在有较明显的差异, 变异程度较高, 其中SSC区的变异程度最高, 通过基因组比较分析, 得出两物种在rps15和rpoB基因的编码区存在差异, 可作为两者分子鉴定的位点资源; Bretones等^[35]发现rps15基因主要影响mRNA转录和细胞代谢, 叶绿体基因组的特点是具相同或相关功能的基因组成复合操纵子结构, 例如rpoB-rpoC-rpoC2操纵子是由编码RNA聚合酶各个亚基的基因聚合在一起而形成的, 利于叶绿体基因的表达与调控^[36], 大叶百两金和细柄百两金之间存在的差异或是由此产生。基因组序列变异分析亦发现该属8个物种叶绿体基因组中ycf1、clpP、rpoC1、ycf3、petD、ycf2、ndhA、rpl16等基因的编码区存在显著差异, 可作为该属植物的鉴别位点。

紫金牛属植物是传统苗药药材, 属内物种丰富, 种间变异错综复杂, 导致物种鉴定及系统发育存在较多争议。已有研究表明, 叶绿体基因组常作为DNA条形码应用于物种鉴定和系统发育关系研究^[37]。为进一步界定大叶百两金和细柄百两金在紫金牛科紫金牛属中的系统位置, 基于紫金牛科和报春花科30个物种叶绿体基因组全长构建的系统发育树结果表明, 大叶百两金和细柄百两金亲缘关系密切, 此外两者与同属植物中虎红舌、白花紫金牛亲缘关系最为相近, 植物志中形态特征描述也支持四者亲缘关系相近, 如叶片均顶端渐尖、基部楔形、具有边缘腺点、花瓣白色、卵形等特征^[38]。报春花科报春花属3个物种与杜茎山属的亲缘关系比紫金牛科紫金牛属、酸藤子属、蜡烛果属和铁仔属更为密切, 这与Xie等^[39]认为紫金牛属为报春花科、植物化学方面研究中紫金牛科与报春花科在亲缘关系上相近以及目前紫金牛属已合并到报春花科(http://www.iplant.cn/info/Primulaceae) 说法一致, 但亦有说法紫金牛属为紫金牛科, 紫金牛属是否可以划分为报春花科需要进一步结合贝叶斯树及溯祖理论进行分析。

作者贡献: 曾宪法负责实验工作、数据分析、论文撰写与修改; 刘畅参与实验及数据分析; 杨小英、余庆参与稿件修改; 扶世伦参与数据分析; 鄢藤云参与样品采集; 蒲翔负责实验设计、论文指导和稿件修改。

利益冲突: 本文的研究内容无任何利益冲突。

基金

收起

国家重点研发计划项目(2018YFC1708100)
贵州省中医药管理局中医药、民族医药科学技术研究课题(QZYY-2021-098)
贵州中医药大学2021年度科研创新和探索专项(2018YFC170810101)
贵州省“十四五”中医药、民族医药重点学科(QZYYZDXK（JS）-2021-03)

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2023年第58卷第1期

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文章信息

doi: 10.16438/j.0513-4870.2022-0874

接收时间：2022-07-18
首发时间：2025-11-21
出版时间：2023-01-12

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出版历史

收稿日期：2022-07-18
修回日期：2022-08-17

基金

国家重点研发计划项目(2018YFC1708100)

贵州省中医药管理局中医药、民族医药科学技术研究课题(QZYY-2021-098)

贵州中医药大学2021年度科研创新和探索专项(2018YFC170810101)

贵州省“十四五”中医药、民族医药重点学科(QZYYZDXK（JS）-2021-03)

作者信息

贵州中医药大学, 贵州贵阳 550025

通讯作者:

*蒲翔, E-mail: 314745595@qq.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Species	Family	Genus	GenBank accession number
Ardisia gigantifolia	Myrsinaceae	Ardisia	MN548760.1
Ardisia mamillata	Myrsinaceae	Ardisia	MN136062.1
Ardisia polysticta	Myrsinaceae	Ardisia	KC465962.1
Ardisia fordii	Myrsinaceae	Ardisia	OK514746.1
Ardisia quinquegona	Myrsinaceae	Ardisia	OK514745.1
Ardisia villosa	Myrsinaceae	Ardisia	OK509170.1
Ardisia faberi	Myrsinaceae	Ardisia	OK509169.1
Ardisia japonica	Myrsinaceae	Ardisia	OK148445.1
Ardisia solanacea	Myrsinaceae	Ardisia	MN094783.1
Ardisia argenticaulis	Myrsinaceae	Ardisia	OK509165.1
Ardisia merrillii	Myrsinaceae	Ardisia	OK054493.1
Ardisia crenata	Myrsinaceae	Ardisia	MW929178.1
Ardisia omissa	Myrsinaceae	Ardisia	OK054494.1
Ardisia carnosicaulis	Myrsinaceae	Ardisia	OK054492.1
Ardisia replicate	Myrsinaceae	Ardisia	OK514747.1
Ardisia balansana	Myrsinaceae	Ardisia	OK509167.1
Ardisia sieboldii	Myrsinaceae	Ardisia	OK509163.1
Ardisia obtusa	Myrsinaceae	Ardisia	OK509166.1
Maesa montana	Myrsinaceae	Maesa	KU569490.1
Maesa hupehensis	Myrsinaceae	Maesa	MZ846203.1
Myrsine stolonifera	Myrsinaceae	Myrsine	MN167883.1
Aegiceras corniculatum	Myrsinaceae	Aegiceras	MN167882.1
Embelia scandens	Myrsinaceae	Embelia	MW246148.1
Embelia vestita	Myrsinaceae	Embelia	MN167884.1
Myrsine africana	Myrsinaceae	Myrsine	MN165129.1
Primula handeliana	Primulaceae	Primula	MG181221.1
Primula pulchella	Primulaceae	Primula	KX668179.1
Primula moupinensis	Primulaceae	Primula	KX668175.1

Species

Family

Genus

GenBank accession number

Ardisia gigantifolia

Myrsinaceae

Ardisia

MN548760.1

Ardisia mamillata

Myrsinaceae

Ardisia

MN136062.1

Ardisia polysticta

Myrsinaceae

Ardisia

KC465962.1

Ardisia fordii

Myrsinaceae

Ardisia

OK514746.1

Ardisia quinquegona

Myrsinaceae

Ardisia

OK514745.1

Ardisia villosa

Myrsinaceae

Ardisia

OK509170.1

Ardisia faberi

Myrsinaceae

Ardisia

OK509169.1

Ardisia japonica

Myrsinaceae

Ardisia

OK148445.1

Ardisia solanacea

Myrsinaceae

Ardisia

MN094783.1

Ardisia argenticaulis

Myrsinaceae

Ardisia

OK509165.1

Ardisia merrillii

Myrsinaceae

Ardisia

OK054493.1

Ardisia crenata

Myrsinaceae

Ardisia

MW929178.1

Ardisia omissa

Myrsinaceae

Ardisia

OK054494.1

Ardisia carnosicaulis

Myrsinaceae

Ardisia

OK054492.1

Ardisia replicate

Myrsinaceae

Ardisia

OK514747.1

Ardisia balansana

Myrsinaceae

Ardisia

OK509167.1

Ardisia sieboldii

Myrsinaceae

Ardisia

OK509163.1

Ardisia obtusa

Myrsinaceae

Ardisia

OK509166.1

Maesa montana

Myrsinaceae

Maesa

KU569490.1

Maesa hupehensis

Myrsinaceae

Maesa

MZ846203.1

Myrsine stolonifera

Myrsinaceae

Myrsine

MN167883.1

Aegiceras corniculatum

Myrsinaceae

Aegiceras

MN167882.1

Embelia scandens

Myrsinaceae

Embelia

MW246148.1

Embelia vestita

Myrsinaceae

Embelia

MN167884.1

Myrsine africana

Myrsinaceae

Myrsine

MN165129.1

Primula handeliana

Primulaceae

Primula

MG181221.1

Primula pulchella

Primulaceae

Primula

KX668179.1

Primula moupinensis

Primulaceae

Primula

KX668175.1

Gene name	Primer sequence (5′-3′)	Size/bp
IRa-SSC-F	CCCTTCCATGCCTCATTTCA	1 284
IRa-SSC-R	GGCGGACGCATCTCATCTTA
IRa-LSC-F	TATCGGTATACGCCCATCCA	1 250
IRa-LSC-R	AGGCACGGCCATACATAACA
IRb-SSC-F	TGTGCTCGCATAGACCCTTT	1 418
IRb-SSC-R	GGCAGAATACCGTCACCCAT
IRb-LSC-F	TCTGGGGAGGTCCGTTTGAT	1 306
IRb-LSC-R	TCGGCGGTTCCCTATTCAGT

Gene name

Primer sequence (5′-3′)

Size/bp

IRa-SSC-F

CCCTTCCATGCCTCATTTCA

1 284

IRa-SSC-R

GGCGGACGCATCTCATCTTA

IRa-LSC-F

TATCGGTATACGCCCATCCA

1 250

IRa-LSC-R

AGGCACGGCCATACATAACA

IRb-SSC-F

TGTGCTCGCATAGACCCTTT

1 418

IRb-SSC-R

GGCAGAATACCGTCACCCAT

IRb-LSC-F

TCTGGGGAGGTCCGTTTGAT

1 306

IRb-LSC-R

TCGGCGGTTCCCTATTCAGT

Genomic region	A. crispa var. amplifolia		A. crispa var. dielsii
Total	156 749	37.05		156 748	37.05
LSC	86 303	34.95	86 303	34.95
IR	26 014	42.97	26 014	42.97
SSC	18 418	30.14	18 417	30.14

Genomic region

A. crispa var. amplifolia

A. crispa var. dielsii

Length/bp

GC content/%

Length/bp

GC content/%

Total

156 749

37.05

156 748

37.05

LSC

86 303

34.95

86 303

34.95

26 014

42.97

26 014

42.97

SSC

18 418

30.14

18 417

30.14

Gene group	Gene function	Gene name
Self-replication	Large subunit of ribosone	rpl23(x2), rpl2^*(x2), rpl36, rpl33, rpl32, rpl20, rpl16^, rpl22, rpl14*
	Small subunit of ribosone	rps8, rps12(x2), rps11, rps3, rps2, rps18, rps7(x2), rps4, rps19, rps16^, rps15, rps14*
	RNA polymerase	rpoB, rpoC2, rpoC1^, rpoA*
	rRNA	rrn16S(x2), rrn4.5S(x2), rrn5S(x2), rrn23S(x2)
	tRNA	tRNA-Val(x2), tRNA-Gly(x2), tRNA-Tyr, tRNA-Lys, tRNA-Trp, tRNA-Thr(x2), tRNA-Ser(x3), tRNA-Phe, tRNA-Met(x4), tRNA-His, tRNA-Glu, tRNA-Arg(x3), tRNA-Gln, tRNA-Pro, tRNA-Cys(x2), tRNA-Asp, tRNA-Asn(x2), tRNA-Ala(x2), tRNA-Leu(x4), tRNA-Ile(x2)
Photosynthesis	Photosystem I	psaI, psaB, psaJ, psaC, psaA
	Photosystem II	psbA, psbD, psbC, psbK, psbI, psbM, psbL, psbF, psbZ, psbJ, psbE, psbB, psbT, psbN, psbH
	NADH dehydrogenase	ndhJ, ndhF, ndhD, ndhK, ndhC, ndhG, ndhI, ndhA^*, ndhH, ndhB^(x2), ndhE*
	Cytochrome b/f complex	petB^, petD^, petN, petL, petG, petA
	ATP synthase	atpA, atpE, atpF^, atpI, atpB, atpH*
	Large subunit of rubisco	rbcL
Other genes	Translational initation factor	infA
	Maturase	matK
	Protease	clpP^*
	Envelope membrane protein	cemA
	Subunit of acetyl-carboxylase	accD
	C-type cytochrome synthesis	ccsA
	Open reading frames	ycf15(x2), ycf4, ycf2(x2), ycf3^, ycf1*

Gene group

Gene function

Gene name

Self-replication

Large subunit of ribosone

rpl23(x2), rpl2^**(x2), rpl36, rpl33, rpl32, rpl20, rpl16^**, rpl22, rpl14

Small subunit of ribosone

rps8, rps12(x2), rps11, rps3, rps2, rps18, rps7(x2), rps4, rps19, rps16^*, rps15, rps14

RNA polymerase

rpoB, rpoC2, rpoC1^*, rpoA

rRNA

rrn16S(x2), rrn4.5S(x2), rrn5S(x2), rrn23S(x2)

tRNA

tRNA-Val(x2), tRNA-Gly(x2), tRNA-Tyr, tRNA-Lys, tRNA-Trp, tRNA-Thr(x2), tRNA-Ser(x3), tRNA-Phe, tRNA-Met(x4), tRNA-His, tRNA-Glu, tRNA-Arg(x3), tRNA-Gln, tRNA-Pro, tRNA-Cys(x2), tRNA-Asp, tRNA-Asn(x2), tRNA-Ala(x2), tRNA-Leu(x4), tRNA-Ile(x2)

Photosynthesis

Photosystem I

psaI, psaB, psaJ, psaC, psaA

Photosystem II

psbA, psbD, psbC, psbK, psbI, psbM, psbL, psbF, psbZ, psbJ, psbE, psbB, psbT, psbN, psbH

NADH dehydrogenase

ndhJ, ndhF, ndhD, ndhK, ndhC, ndhG, ndhI, ndhA^**, ndhH, ndhB^**(x2), ndhE

Cytochrome b/f complex

petB^*, petD^*, petN, petL, petG, petA

ATP synthase

atpA, atpE, atpF^*, atpI, atpB, atpH

Large subunit of rubisco

rbcL

Other genes

Translational initation factor

infA

Maturase

matK

Protease

clpP^*

Envelope membrane protein

cemA

Subunit of acetyl-carboxylase

accD

C-type cytochrome synthesis

ccsA

Open reading frames

ycf15(x2), ycf4, ycf2(x2), ycf3^*, ycf1

Species	P	F	R	C	Total
A. crispa var. amplifolia	29	24	1	0	54
A. crispa var. dielsii	29	24	1	0	54

Species

Total

A. crispa var. amplifolia

A. crispa var. dielsii

SSR type	A. crispa var. amplifolia	A. crispa var. dielsii
Mono	94	94
Di	41	41
Tri	71	71
Tetra	8	8
Penta	2	2
Hexa	0	0
Total	217	217

SSR type

A. crispa var. amplifolia

A. crispa var. dielsii

Mono

Tri

Tetra

Penta

Hexa

Total

217

Amino acid	A. crispa var. amplifolia		A. crispa var. dielsii
Phe	UUU	1.20		UUU	1.20
Leu	UUA	1.28	UUA	1.40
UUG	1.29	UUG	1.25
CUU	1.25	CUU	1.23
Ile	AUU	1.24	AUU	1.23
		AUA	1.04
Val	GUU	1.37	GUU	1.35
GUA	1.12	GUA	1.17
Ser	UCU	1.48	UCU	1.50
UCC	1.11	UCC	1.12
UCA	1.11	UCA	1.25
Pro	CCU	1.10	CCU	1.10
CCA	1.28	CCA	1.21
		CCC	1.05
Thr	ACU	1.21	ACU	1.16
ACC	1.06	ACC	1.04
ACA	1.07	ACA	1.12
Ala	GCU	1.35	GCU	1.36
GCA	1.10	GCA	1.11
Tyr	UAU	1.34	UAU	1.33
His	CAU	1.43	CAU	1.44
Gln	CAA	1.39	CAA	1.38
Asn	AAU	1.39	AAU	1.38
Lys	AAA	1.36	AAA	1.37
Asp	GAU	1.40	GAU	1.40
Glu	GAA	1.39	GAA	1.39
Cys	UGU	1.24	UGU	1.23
Arg	CGA	1.07	CGA	1.11
AGA	2.02	AGA	2.04
AGG	1.06	AGG	1.05
Gly	GGA	1.44	GGA	1.36
		GGU	1.02
TER	UAA	1.29	UAA	1.27

Amino acid

A. crispa var. amplifolia

A. crispa var. dielsii

Codon

RSCU

Codon

RSCU

Phe

UUU

1.20

UUU

1.20

Leu

UUA

1.28

UUA

1.40

UUG

1.29

UUG

1.25

CUU

1.25

CUU

1.23

Ile

AUU

1.24

AUU

1.23

AUA

1.04

Val

GUU

1.37

GUU

1.35

GUA

1.12

GUA

1.17

Ser

UCU

1.48

UCU

1.50

UCC

1.11

UCC

1.12

UCA

1.11

UCA

1.25

Pro

CCU

1.10

CCU

1.10

CCA

1.28

CCA

1.21

CCC

1.05

Thr

ACU

1.21

ACU

1.16

ACC

1.06

ACC

1.04

ACA

1.07

ACA

1.12

Ala

GCU

1.35

GCU

1.36

GCA

1.10

GCA

1.11

Tyr

UAU

1.34

UAU

1.33

His

CAU

1.43

CAU

1.44

Gln

CAA

1.39

CAA

1.38

Asn

AAU

1.39

AAU

1.38

Lys

AAA

1.36

AAA

1.37

Asp

GAU

1.40

GAU

1.40

Glu

GAA

1.39

GAA

1.39

Cys

UGU

1.24

UGU

1.23

Arg

CGA

1.07

CGA

1.11

AGA

2.02

AGA

2.04

AGG

1.06

AGG

1.05

Gly

GGA

1.44

GGA

1.36

GGU

1.02

TER

UAA

1.29

UAA

1.27

Intron gene	A. crispa var. amplifolia	A. crispa var. dielsii
rps16	CDS length	267	267
Gene length	1 116	1 116
atpF	CDS length	630	630
Gene length	1 265	1 265
rpoC1	CDS length	2 061	2 061
Gene length	2 812	2 812
ycf3	CDS length	510	510
Gene length	1 952	1 952
clpP	CDS length	588	588
Gene length	2 122	2 122
petB	CDS length	648	648
Gene length	1 408	1 408
petD	CDS length	483	483
Gene length	1 316	1 316
rpl16	CDS length	408	408
Gene length	1 405	1 405
rpl2	CDS length	825/825	825/825
Gene length	1 480/1 480	1 480/1 480
ndhB	CDS length	1 533/1 533	1 533/1 533
Gene length	2 212/2 212	2 212/2 212
ndhA	CDS length	1 092	1 092
Gene length	2 210	2 210

Intron gene

A. crispa var. amplifolia

A. crispa var. dielsii

rps16

CDS length

267

Gene length

1 116

atpF

CDS length

630

Gene length

1 265

rpoC1

CDS length

2 061

Gene length

2 812

ycf3

CDS length

510

Gene length

1 952

clpP

CDS length

588

Gene length

2 122

petB

CDS length

648

Gene length

1 408

petD

CDS length

483

Gene length

1 316

rpl16

CDS length

408

Gene length

1 405

rpl2

CDS length

825/825

Gene length

1 480/1 480

ndhB

CDS length

1 533/1 533

Gene length

2 212/2 212

ndhA

CDS length

1 092

Gene length

2 210