Genetic characteristics of whole chloroplast genome in <i>Prunus triloba</i> ‘Multiplex’

DUAN Chunyan; WANG Xiaoling

doi:10.11833/j.issn.2095-0756.20230489

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DUAN Chunyan, WANG Xiaoling. Genetic characteristics of whole chloroplast genome in Prunus triloba ‘Multiplex’[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20230489

Citation:

DUAN Chunyan, WANG Xiaoling. Genetic characteristics of whole chloroplast genome in Prunus triloba ‘Multiplex’[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20230489

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Genetic characteristics of whole chloroplast genome in Prunus triloba ‘Multiplex’

doi: 10.11833/j.issn.2095-0756.20230489

College of Agriculture/Peony College, Henan University of Science and Technology, Luoyang 471000, Henan, China

Received Date: 2023-09-30
Accepted Date: 2024-03-06
Rev Recd Date: 2023-12-13

Abstract

Objective To investigate the phylogenetic location of Prunus triloba ‘Multiplex’ and analyze its chloroplast gene composition characteristics, the whole chloroplast genome sequence of P. triloba ‘Multiplex’ was sequenced. Method The leaves of P. triloba ‘Multiplex’ were used as materials to extract chloroplast DNA using 2×CTAB method. The whole chloroplast genome was sequenced by Illumina NovaSeq platform. We then assembled, annotated, and analyzed the genetic characteristics of the chloroplast genomic sequence. Combined with the NCBI database data, the phylogenetic relationship of P. triloba ‘Multiplex’ was constructed based on the whole chloroplast genome sequence. Result The total length of the chloroplast genome of P. triloba ‘Multiplex’ is 157 827 bp, with NCBI registration number MT937181. The structure is a classic tetrad structure, consisting of 1 LSC (large single copy region), 1 SSC (small single copy region), and IRa/IRb (reverse repeat region), with sequence lengths of 86 032, 19 023, 26 386, and 26 386 bp, respectively. The total content of GC and AT is 36.80% and 63.20%, respectively. The complete chloroplast genome sequence of P. triloba ‘Multiplex’ was annotated to 132 genes, including 37 tRNA genes, 87 coding protein genes, and 8 rRNA genes, respectively. The chloroplast genome of P. triloba ‘Multiplex’ encodes a total of 26 672 codons and 236 eligible SSR loci. The A/T base is dominant in the SSR site, and the base preference is very obvious. Conclusion The phylogenetic tree analysis showed that P. triloba ‘Multiplex’ and P. triloba aggregate into a branch structure, and are closely related to P. pedunculata in the same genus. The results of this study will provide new evidence and basic experimental data for species identification, phylogenetic evolution, and breeding and selection of P. triloba ‘Multiplex’. [Ch, 4 fig. 4 tab. 26 ref.]
- Prunus triloba ‘Multiplex’,
- whole chloroplast genome,
- codon preference,
- repeated sequence,
- phylogeny

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重瓣榆叶梅Prunus triloba ‘Multiplex’属蔷薇科Rosaceae李属Prunus灌木或小乔木，早春开花，是中国特有园林植物^[1−2]。重瓣榆叶梅具有较强的抗盐碱能力，广泛栽培于中国东北、华北和西北地区^[3]；重瓣榆叶梅花和花粉富含蛋白质、生物酶、多糖、脂肪、氨基酸和维生素等物质，可以用做保健品、药品、食品和营养性化妆品的原材料^[4−5]。种仁含油量较高，可代郁李仁入药^[6]。重瓣榆叶梅于1855年被引入欧洲、美洲、大洋洲^[7]，广泛用于园林绿化。研究发现：榆叶梅P. triloba与近缘种桃P. persica的核不对称性较接近，榆叶梅为八倍体(2n=64), 核型分类为2B或2A，核不对称系数高达63.08%^[2]。但榆叶梅的物种发生还尚不清楚，关于重瓣榆叶梅和榆叶梅系统发育关系仍是空白。

叶绿体基因组是植物叶绿体的独立遗传系统，重组率低且保守，一般为四分体结构，由大单拷贝区(LSC)、双向重复区(IR)和小单拷贝区(SSC)组成，在植物物种鉴定、物种分化、DNA条形码开发和系统分类研究中具备可信度^[8-11]。已有研究通过对46个类群的叶绿体atpB-rbcL构建进化树，明确了小麦族猬草属Hystrix和赖草属Leymus的系统发育关系^[12]；基于rbcL + matK + trnH-psbA组合序列和叶绿体全基因组研究白头翁属Pulsatilla物种的系统发育关系，发现变异热点在物种识别和系统发育分析中建树结果相似^[13]；基于叶绿体psbD 基因构建的进化树表明：花叶矢竹Pseudosasa japonica f. akebonosuji与毛竹Phyllostachys edulis的亲缘关系较近^[14]。目前，已获得野生榆叶梅叶绿体基因组序列^[3]，但未见关于重瓣榆叶梅的叶绿体基因组序列报道。本研究通过 Illumina NovaSeq高通量测序平台，首次成功组装重瓣榆叶梅叶绿体全基因组序列结构并分析其遗传多样性，以期为重瓣榆叶梅系统发育、物种鉴定、资源开发研究提供理论依据。

1. 材料与方法

1.1. 材料

2020和2021年3—4月，对河南省洛阳市洛龙区(34°38′9.94″N，112°26′36.61″E)重瓣榆叶梅种质资源进行调查，采集重瓣榆叶梅植株的幼嫩新鲜叶片，用纯净水和纸巾依次清洁叶片表面，装入有变色硅胶颗粒的密封袋，带回实验室保存以备用。

1.2. DNA提取

采用 2×CTAB法提取重瓣榆叶梅总DNA^[15]。叶片于液氮中研磨后用CTAB法提取植物总DNA。以琼脂糖凝胶电泳(琼脂质量分数1%，电压120 V，电泳时间25 min)和微量核酸测定仪(NanoDrop 2000)检测样本DNA的完整性、质量和质量分数。样本DNA质量浓度为56.16 mg·L⁻¹，体积40 μL，总量2.25 μg，D(260/280)为1.89，D( 260/230)为1.02，检测结果为A类。样品质量满足建库测序要求，且总量满足2次或者2次以上建库需要。

1.3. 测序和序列组装、基因注释

已检测为A类的DNA样品用超声波机械打断，经片段纯化、末端修复、3＇端加 A、连接测序接头后，进行PCR 扩增构建文库，在Illumina NovaSeq平台进行测序得到原始序列(raw reads)，对原始序列进行过滤：去除带接头的序列，过滤N含量超过10%的序列，通过BLAST 软件比对核酸序列数据库(NT)等质控得到过滤序列(clean reads)。原始序列上传于数据库GeneBank。

使用软件SPAdes^[16]、aragorn v1.2.38、DOGMA^[17]和CPGview-RSG^[18]进行测序数据组装和预测注释重瓣榆叶梅叶绿体的基因，通过手工纠正，成功获得其全叶绿体基因组序列并完成重瓣榆叶梅叶绿体基因组的基因注释。用在线软件(https://chlorobox.mpimp-golm.mpg.de/OGDraw.html)绘制其叶绿体全基因组物理可视化图谱。

1.4. 密码子

用CodonW 1.4.2对重瓣榆叶梅叶绿体基因组密码子数量和相对同义密码子使用度(relative synonymous codon usage, RSCU)进行分析。

1.5. 重复序列

利用MISA-web^[19]检测重瓣榆叶梅的cpSSR位点，参数设置为单核苷酸-8、双核苷酸-5、三核苷酸-3、四核苷酸-3、五核苷酸-3和其他核苷酸-3。

1.6. 系统发育分析

于美国国家生物技术信息中心(NCBI)数据库中下载如MK764428、HQ336405、MK798146、KF990036、KY101152、MK790138、MG602257、MK434918和MN661138等多个物种的叶绿体基因组序列，构建包括重瓣榆叶梅在内的蔷薇科李属植物的系统发育进化树，观察并分析重瓣榆叶梅和其他物种间的亲缘关系和系统发育地位。系统发育进化树采用全叶绿体基因组数据进行分析：用MAFFT^[20](v7.427，auto模式)进行多序列比对，将比对好的数据用trimAl v1.4.rev15修剪，使用RAxML v8.2.10，选用GTRGAMMA模型，bootstrap=1 000，构建最大似然进化树。

3. 讨论

被子植物的叶绿体基因组长度约1.60×10⁵ bp，其编码基因数约130个^[21−22]。本研究所获得的重瓣榆叶梅全叶绿体基因组序列MT937181全长为 157 827 bp，其编码基因数为132个，与一般被子植物相符合。重瓣榆叶梅为园林栽培植物，分布广泛；其他种为野生种，分布和自然生境不同，其中蒙古扁桃十分耐寒且仅分布于蒙古高原。将4个不同叶绿体基因组序列进行比较，发现重瓣榆叶梅总序列、LSC和SSC长度最小，蒙古扁桃SSC和IR长度最大。推测可能在人工栽培和植物品种选育中，重瓣榆叶梅由于外在表现性状和生长环境的优越，其叶绿体基因组中基因种类增加，但总序列长度减小；而在野生环境分布狭窄或需要极端耐寒时，蒙古扁桃为适应不良环境，其SSC区和IR长度增大，基因种类和数量发生较大变化，ycf15基因缺失，且增加了1个rps19基因。ycf15属于未知功能基因，在龙葵属Amborella和蔷薇类等原始被子植物中无功能，在毛茛属Ranunculus植物已经完全丢失^[23]。rps4以及rps19为核糖体小亚基类基因，核糖体在细胞中负责完成“中心法则”里“翻译”：由RNA到蛋白质这一过程。核糖体小亚基通过与信使RNA结合，转运RNA运送的氨基酸分子来合成多肽。ycf15、rps4以及rps19等3类基因的变化情况可能与植物对栽培选育、不同环境适应有直接关系。

自然界遗传密码子在mRNA翻译成蛋白质时具有重要决定作用。同义密码子指编码同一种氨基酸的不同密码子，有时具有使用偏好性^{[10, 24]}。重瓣榆叶梅编码基因RSCU＞1密码子的第 3个核苷酸具有明显的A/U偏好。简单重复序列(SSR)广泛分布于真核生物基因组中，重瓣榆叶梅与榆叶梅(MK790138)、长柄扁桃(MG602257)和蒙古扁桃(MG602256)叶绿体基因组的SSR位点均具有富集多聚A或多聚T情况。本研究系统进化分析表明：重瓣榆叶梅与榆叶梅、长柄扁桃亲缘关系最近，进化结果与传统分类学相符^{[2−3，24−26]}。

4. 结论

本研究首次成功获得重瓣榆叶梅全叶绿体基因组序列，其总长度为157 827 bp，具备经典的四分体结构，GeneBank登录号为MT937181，编码基因数为132个。LSC、SSC和IRa/IRb序列长度分别为86 032、19 023和26 386 bp，GC占比在 IR、LSC 和SSC区域存在较大的差异。重瓣榆叶梅叶绿体基因组与其他3种近缘种植物比较中，ycf15、rps4以及rps19等3类基因有较大变化，可能是栽培环境的选择影响。重瓣榆叶梅叶绿体基因组的SSR位点具有富集A/T情况。基于叶绿体基因组数据首次确定重瓣榆叶梅和榆叶梅系统发育关系，系统进化分析结果与传统植物分类学与形态学相符，即重瓣榆叶梅与榆叶梅、长柄扁桃亲缘关系最近。

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区域	全叶绿体基因组		大单拷贝区		小单拷贝区		反向重复区		反向重复区
区域	数量	占比/%	数量	占比/%	数量	占比/%	数量	占比/%	数量	占比/%
A	49 091	31.10	27 334	31.77	6 606	34.73	7 588	28.76	7 563	28.66
C	29 663	18.79	15 381	17.88	3 047	16.02	5 416	20.53	5 819	22.05
G	28 410	18.00	14 423	16.76	2 752	14.47	5 819	22.05	5 416	20.53
T	50 663	32.10	28 894	33.59	6 618	34.79	7 563	28.66	7 588	28.76
GC	58 073	36.80	29 804	34.64	5 799	30.48	11 235	42.58	11 235	42.58
总计	157 827	100.00	86 032	100.00	19 023	100.00	26 386	100.00	26 386	100.00

基因分类	基因分组	基因名称
光合作用相关基因	光合系统Ⅰ基因	psaA, psaB, psaC, psaI, psaJ
	光合系统Ⅱ基因	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	NADH脱氢酶基因	ndhA, ndhB(2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
	细胞色素复合物基因	petA, petB, petD, petG, petL, petN
	ATP合酶基因	atpA, atpB, atpE, atpF, atpH, atpI*
	二磷酸核酮糖羧化酶大亚基基因	rbcL

自我复制相关基因	核糖体大亚基基因	rpl14, rpl16, rpl2(2), rpl20, rpl22, rpl23(2), rpl32, rpl33, rpl36
	核糖体小亚基基因	rps11, rps12*(2), rps14, rps15, rps16, rps18, rps19, rps2, rps3, rps4, rps7(2), rps8
	RNA聚合酶亚基基因	rpoA, rpoB, rpoC1, rpoC*2
	核糖体RNA基因	rrn16(2), rrn23(2), rrn4.5(2), rrn5(2)
	转运RNA基因	trnA-UGC(2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC*, trnG-UCC, trnH-GUG, trnI-CAU(2), trnI-GAU*(2), trnK-UUU*, trnL-CAA(2), trnL-UAA*, trnL-UAG, trnM-CAU, trnN-GUU(2), trnP-UGG, trnQ-UUG, trnR-ACG(2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(2), trnV-UAC*, trnW-CCA, trnY-GUA, trnfM-CAU*

其他基因	成熟酶基因	matK
	依赖ATP的蛋白酶单元p基因	clpP**
	包裹膜蛋白基因	cemA
	乙酰辅酶A羧化酶亚基基因	accD
	c型细胞色素合成基因	ccsA

未知功能基因	保守开放阅读框	ycf1(2), ycf15(2), ycf2(2), ycf3*, ycf*4
说明：* 代表基因有1个内含子；**代表基因有2个内含子；上#代表假基因；(2)代表基因的拷贝数为2。

符号	氨基酸	密码子	数量/个	RSCU	符号	氨基酸	密码子	数量/个	RSCU
*	Ter	UAA	52	1.793 1	M	Met	GUG	2	0.016 0
*	Ter	UAG	21	0.724 2	M	Met	UUG	1	0.008 0
*	Ter	UGA	14	0.482 7	M	Met	AUG	620	0.495 2
A	Ala	GCA	385	1.096 8	N	Asn	AAC	308	0.470 2
A	Ala	GCC	226	0.644 0	N	Asn	AAU	1002	1.529 8
A	Ala	GCG	155	0.441 6	P	Pro	CCA	313	1.137 2
A	Ala	GCU	638	1.817 6	P	Pro	CCC	208	0.755 6
C	Cys	UGC	78	0.500 0	P	Pro	CCG	151	0.548 4
C	Cys	UGU	234	1.500 0	P	Pro	CCU	429	1.558 4
D	Asp	GAC	213	0.393 4	Q	Gln	CAA	725	1.547 4
D	Asp	GAU	870	1.606 6	Q	Gln	CAG	212	0.452 6
E	Glu	GAA	1 041	1.476 6	R	Arg	AGA	503	1.880 4
E	Glu	GAG	369	0.523 4	R	Arg	AGG	176	0.658 2
F	Phe	UUC	524	0.688 2	R	Arg	CGA	358	1.338 6
F	Phe	UUU	999	1.311 8	R	Arg	CGC	111	0.415 2
G	Gly	GGA	728	1.621 2	R	Arg	CGG	118	0.441 0
G	Gly	GGC	174	0.387 6	R	Arg	CGU	339	1.267 2
G	Gly	GGG	303	0.674 8	S	Ser	AGC	140	0.412 8
G	Gly	GGU	591	1.316 4	S	Ser	AGU	398	1.174 2
H	His	CAC	152	0.471 4	S	Ser	UCA	414	1.221 0
H	His	CAU	493	1.528 6	S	Ser	UCC	327	0.964 8
I	Ile	AUA	732	0.959 1	S	Ser	UCG	187	0.551 4
I	Ile	AUC	450	0.589 5	S	Ser	UCU	568	1.675 8
I	Ile	AUU	1 108	1.451 4	T	Thr	ACA	422	1.241 2
K	Lys	AAA	1 080	1.505 2	T	Thr	ACC	250	0.735 2
K	Lys	AAG	355	0.494 8	T	Thr	ACG	151	0.444 0
L	Leu	CUA	367	0.788 4	T	Thr	ACU	537	1.5796
L	Leu	CUC	186	0.399 6	V	Val	GUA	557	1.537 6
L	Leu	CUG	188	0.403 8	V	Val	GUC	161	0.444 4
L	Leu	CUU	580	1.246 2	V	Val	GUG	206	0.568 8
L	Leu	UUA	909	1.953 6	V	Val	GUU	525	1.449 2
L	Leu	UUG	562	1.207 8	W	Trp	UGG	454	1.000 0
M	Met	AUU	1	0.008 0	Y	Tyr	UAC	205	0.400 0
M	Met	CUG	2	0.016 0	Y	Tyr	UAU	820	1.600 0