Chloroplast Genome Phylogeny and Codon Preference of <i>Anabasis aphylla</i> L.

JIANG Ping; HUANG Xiang; SULAIMAN Shah; HE Meng-yao

doi:10.12403/j.1001-1498.20220578

Volume 36 Issue 4

Aug. 2023

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Chloroplast Genome Phylogeny and Codon Preference of Anabasis aphylla L.

College of Agriculture, Shihezi University, Shihezi　832003, Xinjiang, China

Corresponding author: JIANG Ping, shzujp@163.com ;
Received Date: 2022-11-26
Accepted Date: 2023-02-09

Abstract

Objective To analyze the structural characteristics of Anabasis aphylla chloroplast genome, clarify the taxonomic status of Anabasis in Chenopodiaceae for further exploring its codon preference and determining the optimal codon. Method Total genomic DNA was extracted from fresh assimilation twigs of A. aphylla based on CTAB method. Sequencing was performed using the Illumina Genome Analyzer HiSeq 2000 high-throughput sequencing platform. The chloroplast genome sequence was assembled and annotated by GetOrganelle and Plann. Simple sequence repeat (SSR) in chloroplast genome was analyzed by MISA. Multiple sequence alignment and ML phylogenetic tree construction were analyzed using MAFFT v.7.450 and IQ-TREE v.2.1.1 software. The nucleotide polymorphism values were calculated using DnaSP 6.0 software. The codon preference was studied by CUSP and Codon W 1.4.2. software. Result The full-length chloroplast genome of A. aphylla was 154 084 bp, including a large single copy region (LSC, 85 124 bp), a small single copy region (SSC, 18 934 bp), and a pair of inverted repeat regions (IRa and IRb, 25 013 bp). A total of 132 genes were annotated, including 83 protein-coding genes, 8 rRNA genes, 37 tRNA genes and 4 pseudogenes. The number of SSRs located in the intergenic region was the largest (70.4%), and the number of single-base (A/T) repeat type SSRs was the largest. The optimal model of phylogenetic tree was TVM + F + R3, and Chenopodiaceae was divided into four clustering groups. Among them, A. aphylla showed the closest relationship with Haloxylon and Salsola. TrnS-trnG(exon1)、ndhF-rpl32、rpl32-trnL、rps16(exon1)-trnQ and ycf1 were high nucleotide polymorphism regions. A total of 20 optimal codons (UUU, UAU, UGU, CAU, UCU, UCA, UUA, CUU, CCU, AGA, GAA, ACU, ACA, AAU, GAU, AAA, GUU, GCU, GGU, CAA) were determined, all ending with A/U. The codon usage preference was mainly affected by natural selection, and the influence of mutation and other influencing factors was weak. Conclusion The chloroplast genome structure of A. aphylla is conservative, showing typical quadripartite structure. In the phylogeny of Chenopodiaceae, A. aphylla has the closest relationship with Haloxylon and Salsola. The identified hypervariable regions and SSR loci can be used for molecular identification of intergeneric species in Chenopodiaceae. The codon of the chloroplast genome of A.aphylla prefer endings with A/U, and the 20 optimal codons determined are useful for the optimization of its exogenous codons. The results can provide a reference for molecular marker development, phylogeny and chloroplast gene engineering of A. aphylla.
- Anabasis aphylla L.,
- chloroplast genome,
- phylogenetic analysis,
- codon

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无叶假木贼（Anabasis aphylla L.）隶属于藜科（Chenopodiaceae）假木贼属（Anabasis），半灌木，具有强的抗盐碱能力，在我国主要分布于西北地区^[1-2]。无叶假木贼是荒漠植被的主要建群种和优势种^[3]，常作为防风固沙的植物材料，具有很高的生态价值。同时，其植株提取物包含生物碱、萜类、皂苷类等多种生物活性物质^[4]，有效治疗疥癣、疥疮和湿疹痒痛，还有效防治菜青虫、蚜虫等多种害虫^[5-6]。

叶绿体是一种重要的质体，在植物细胞的光合作用等生物过程中起着关键作用^[7]。叶绿体基因组通常比核基因更为保守，对植物系统发育和物种鉴定有重要作用^[8-9]。在物种进化过程中，叶绿体基因组在序列、组成、大小和基因含量方面高度保守^[10]，具有2个反向重复区（inverted repeats, IR）、1个小单拷贝区（small single copy, SSC）和1个大单拷贝区组成的四分体结构（large single copy, LSC）^[11]。IR区域的收缩和扩张是叶绿体基因含量和基因组大小变化的主要影响因素^[12]。叶绿体基因组中存在一些简单重复序列（SSRs）和单核苷酸多态性（SNP）的热点区域，可产生足够的信息用于物种分类和鉴定^[13]。此外，植物叶绿体基因组中的密码子偏好性反映其在进化过程中的分子适应程度和受到的进化压力，同时参与基因的表达^[14]。目前，叶绿体基因组序列作为超级条形码，已经在藜科中多个物种的系统发育研究中得到应用^[15-16]。然而，假木贼属物种的叶绿体基因组尚未被报道，它们的进化特征和遗传多样性尚不清晰。

本研究首次对假木贼属的无叶假木贼的叶绿体基因组进行测序、组装和注释，进一步分析其叶绿体基因组特征和密码子偏好性等；此外，将其与已公布叶绿体基因组的藜科物种构建系统发育树，进一步筛选种间基因组高变区。本研究目的在于：（1）阐明无叶假木贼与其它藜科物种的进化关系及其在系统发育中的地位；（2）筛选有效的候选分子标记序列和最优密码子，以期为无叶假木贼的分子标记开发、系统进化及叶绿体基因工程研究提供参考。

1. 材料与方法

1.1. 试验材料及测序

植物样本来源于新疆准格尔盆地南缘（84°52' E，45°22' N，海拔265 m），经石河子大学楚光明教授鉴定为无叶假木贼（A. aphylla L.）。采集的幼嫩同化枝用液氮处理后置于液氮保温桶，带回实验室放于-80℃冰箱保存。基于改良的CTAB法^[17]提取无叶假木贼总DNA，用超声波将DNA片段化，经过纯化、末端修复、3’端加A、连接测序接头的片段，通过琼脂糖凝胶电泳的方法选择合适长度的片段进行PCR扩增，构建测序文库。文库质检后，基于Illumina Genome Analyzer Hiseq 2000测序平台进行叶绿体基因组测序。

1.2. 叶绿体基因组组装与注释

使用GetOrganelle软件^[18]对无叶假木贼叶绿体基因组序列进行组装；通过Perl语言脚本Plann^[19]对叶绿体基因组进行注释；利用Sequin^[20]检查注释缺失或错误的基因。利用OGDRAW v. 1. 3. 1软件^[21]绘制叶绿体基因组环状结构图。无叶假木贼叶绿体基因组数据已上传GenBank数据库（https://www.ncbi.nlm.nih.gov/genbank/），登录号为OP712667。

1.3. 叶绿体基因组重复序列分析

利用REPuter在线工具对长重复序列进行分析，最大重复序列数为100，最小重复大小为22 bp^[22]。使用MISA在线工具^[23]检测简单重复序列（Simple Sequence Repeats， SSRs），参数设置为：单核苷酸重复次数≥10，二核苷酸重复次数≥5，三核苷酸重复次数≥4，四核苷酸到六核苷酸重复次数≥3。

1.4. 系统发育树构建

从NCBI的GenBank数据库下载19种藜科（Chenopodiaceae）物种的叶绿体基因组序列，登录号分别为：Spinacia oleracea L. （AJ400848）、Dysphania pumilio (R.Br.) Mosyakin & Clemants （MH936550）、Dysphania botrys L. （MH898873）、Dysphania ambrosioides L. （MK182726）、Chenopodium quinoa Willd. （KY419706）、Chenopodium ficifolium Sm. （MK182725）、Chenopodium album L. （KY419707）、Chenopodium acuminatum Willd. （MW057780）、Atriplex centralasiatica Iljin （MK867774）、Atriplex gmelinii C. A. Mey. ex Bong. （MT810472）、Salsola affinis C. A. Mey （ON080842）、Salsola abrotanoides (Bunge) Akhani （MW123092）、Haloxylon ammodendron (C. A. Mey.) Bunge （KF534478）、Haloxylon persicum Bge. （KF534479）、Suaeda glauca L. （MK867773）、Salicornia europaea L. （KJ629116）、Salicornia brachiate Miq. （KJ629115）、Salicornia bigelovii Torr. （KJ629117）、Kalidium foliatum (Pall.) Moq. （MW699755）；以2种苋科物种，Deeringia amaranthoides (Lam.) Merr. （MK397865）和Celosia argentea L. （MK397861）的叶绿体基因组序列作为外群，与测得的无叶假木贼叶绿体基因组序列共同构建系统发育树。基于MAFFT v. 7. 450软件对22个物种的叶绿体基因组序列进行多序列比对^[24]，通过IQ-TREE v. 2. 1. 1软件^[25]构建最大似然法（Maximum likelihood，ML）系统进化树，其中最优构树模型为TVM + F + R3，步长值为1 000。

1.5. 叶绿体基因组突变位点和IR边界分析

通过MAFFT v. 7. 450软件对包括无叶假木贼在内的藜科20个物种的叶绿体基因组序列进行多序列比对，对齐后的序列通过DnaSP 6.0软件^[26]计算核苷酸多态性值（搜索窗口长度为600 bp，步长为200 bp）。将无叶假木贼及其近缘种的genbank格式的叶绿体基因组文件上传至生信云在线分析网站（http://112.86.217.82:9919/#/tool/alltool/detail/296），进行叶绿体基因组IR区边界区域上基因的可视化。

1.6. 密码子使用偏好性分析

在无叶假木贼叶绿体基因组中，筛选长度大于300 bp的基因序列，使用Codon W 1. 4. 2软件和CUSP 在线程序（https://emboss.toulouse.inra.fr/cgi-bin/emboss/cusp）对有效密码子数、同义密码子相对使用度（RSCU）、密码子GC含量和最优密码子进行计算。通过中性绘图、ENC-plot和PR2-plot分析密码子偏好性的影响因素。

3. 讨论

植物叶绿体基因组相对保守，已有研究表明，被子植物叶绿体基因组长度通常在120～180 kb之间，IR区在20～30 kb之间^[27]。本研究中，无叶假木贼叶绿体基因组全长为154 084 bp，IR区长度为25 013 bp，在被子植株叶绿体基因组序列长度范围内。此外，无叶假木贼叶绿体基因组与大多数被子植物的叶绿体基因组有相似的环状四分体结构^[28]。被子植物叶绿体基因组大小与IR区和SC区边界的扩张和收缩密切相关^[29]。本研究中，无叶假木贼及其9种近缘种叶绿体基因组的边界分析显示，IR区长度变化不大（23 701～25 036 bp），表明藜科植物叶绿体基因组结构相对保守；但藜科中一些物种在IR-LSC边界处ycf1和rps19基因的缺失和不同程度的扩张，这导致了藜科物种叶绿体基因组中IR区长度大小的差异^[30]。无叶假木贼叶绿体基因组的平均GC含量为36.25%，这可能与该基因组偏好使用A/U结尾的密码子有关。无叶假木贼叶绿体基因组中共注释到132个基因，这和蒋礼玲等^[31]报道的4种藜属物种的叶绿体基因组编码基因相比，少1个蛋白编码基因，多4个假基因，这可能与假木贼属中物种进化较缓慢有关^[32]；同时，在胡桃科（Juglandaceae）核桃属（Carya）^[33]和蔷薇科（Rosaceae）梨属（Pyrus）^[32]的叶绿体基因组的相关研究中也得到相似的结果，表明藜科中不同物种间叶绿体基因组基因数量存在差异属于正常现象；此外，笔者还推测这与不同研究中的测序平台和注释结果的差异有关。

分布在植物叶绿体基因组上的重复序列和多态性变异位点，目前已广泛应用于多个物种的遗传多样性和系统关系的研究^[34]。本研究在无叶假木贼叶绿体基因组中鉴定到12种类型，共71个SSR位点，其中，单碱基重复（A/T）、二碱基重复（AT/TA）、三碱基重复（TAA/TTA）和一些多碱基重复（AAAT、TAAT、TTAT、TAAAA、TTATT、TTTTA）均为多聚A或多聚T，占所有SSR位点的83.33%，这是假木贼叶绿体基因组中AT含量高的一个重要因素。在真核生物中，大多数SSR分布在非编码序列^[35]，无叶假木贼叶绿体基因组中SSR主要位于基因间区（70.4%），这可能与该物种的叶绿体基因组在遗传进化中较保守有关^[36]。此外，本研究中，LSC和SSC区的核苷酸多态性显著高于IR区，这和蒋礼玲等^[31]在藜属植物叶绿体基因组的研究结果一致。trnS-trnG(exon1)、ndhF-rpl32、rpl32-trnL、rps16(exon1)-trnQ和ycf1是无叶假木贼叶绿体基因组中的高核苷酸多态性区域，这些序列为该科的属间分子鉴定奠定了基础。

植物叶绿体全基因组序列较单个或多个编码序列包含更丰富的信息，基于其构建的系统发育树结果更加准确^[37]。高鸣泽^[38]通过对藜科41个物种的蛋白编码基因构建系统发育树，将盐角草属、碱蓬属、梭梭属聚成一类，表明其亲缘关系较近。本研究得到了相似的结论，同时，本研究结果进一步表明，假木贼属和梭梭属、猪毛菜属有较高的亲缘关系，这和前人基于形态学特征将假木贼属、梭梭属、猪毛菜属归为猪毛菜族（Salsoleae）的研究结果一致^[39]。本研究是首次对藜科中假木贼属物种进行叶绿体基因组测序，明确了假木贼属在藜科中系统发育的位置，但要充分明确藜科物种的系统发育关系，需要更多藜科物种全基因组被测序。

密码子偏好性在植物叶绿体基因组中蛋白质编码基因的过程中发挥重要作用，这与突变、自然选择和随机遗传漂变等分子进化现象密切相关^[40]。本研究中，ENC-plot、ENC分布直方图、PR2-plot和中性绘图分析的综合结果表明，无叶假木贼叶绿体基因组密码子使用偏好性主要受自然选择影响，突变等影响因素对其影响较弱，这可能与荒漠植物在遗传进化过程中的特殊生存环境有关^[41]。前人研究表明，密码子第三个碱基的GC含量在基因组结构进化过程中也起着重要作用^[42]。本研究中，在RSCU值大于1的30个密码子中，除编码亮氨酸的UUG以G结尾外，其它29种密码子均以A/U结尾。当RSCU值<1时，多以G/C结尾。这表明，以A/U结尾的同义密码子更多地参与无叶假木贼叶绿体基因组的蛋白质编码基因的过程，这与前人在双子叶植物中密码子使用偏好性的研究一致^[43]。此外，本研究在无叶假木贼叶绿体基因组中确定了20个最优密码子，均以A/U结尾，这为无叶假木贼中外源基因密码子的优化提供了理论依据。

4. 结论

无叶假木贼叶绿体基因组全长为154 084 bp，包括1个LSC区（85 124 bp）、1个SSC区（18 934 bp）、1对IR区（IRa和IRb，25 013 bp），呈典型的四分体结构。12种类型共71个SSR位点在无叶假木贼叶绿体基因组中被鉴定；trnS-trnG(exon1)、ndhF-rpl32、rpl32-trnL、rps16(exon1)-trnQ和ycf1是无叶假木贼叶及其9种近缘种叶绿体基因组中的高核苷酸多态性区域；这些信息为无叶假木贼今后的分子标记开发提供了科学的依据。系统发育分析中，20个藜科物种被归为4个聚类组，其中，无叶假木贼与梭梭属和猪毛菜属的物种亲缘关系最近；在无叶假木贼叶绿体基因组中确定20个最优密码子，均以A/U结尾；其密码子使用偏好性主要受自然选择影响，突变等影响因素对其影响较弱；研究结果可为无叶假木贼的系统进化及叶绿体基因工程研究提供参考。

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基因功能 Gene Function	基因分类 Gene group	基因 Gene
光合作用 Photosynthesis	光合系统I Photosystem I	psaA, psaB, psaC, psaI, psaJ
	光合系统II Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	NADH 脱氢酶 Subunits of NADH dehydrogenase	ndhA, ndhB*(2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK*
	细胞色素复合物 Cytochrome b/f complex	petA, petB, petD*, petG, petL, petN*
	ATP 合成酶 Subunits of ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI*
	二磷酸核酮糖羧化酶大亚基 Large subunit of rubisco	rbcL
自我复制 Self-replication	核糖体大亚基蛋白 Proteins of large ribosomal subunit	#rpl23(2), rpl14, rpl16, rpl2(2), rpl20, rpl22, rpl32, rpl33, rpl36*
	核糖体小亚基蛋白 Proteins of small ribosomal subunit	#rps19, rps11, rps12(2), rps14, rps15, rps16, rps18, rps19, rps2, rps3, rps4, rps7(2), rps8*
	RNA 聚合酶亚基 Subunits of RNA polymerase	rpoA, rpoB, rpoC1, rpoC2*
	核糖体 RNA Ribosomal RNAs	rrn16S(2), rrn23S(2), rrn4.5S(2), rrn5S(2)
	转运RNA Transfer RNAs	trnA-UGC(2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC, trnH-GUG, trnI-GAU(2), trnK-UUU, trnL-CAA(2), trnL-UAA, trnL-UAG, trnM-CAU(3), 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 Other genes	成熟酶 Maturase	matK
	蛋白酶 Protease	clpP**
	膜包被蛋白基因 Envelope membrane protein	cemA
	乙酰辅酶A羧化酶 Acetyl-CoA carboxylase	accD
	c 型细胞色素合成基因 c-type cytochrome synthesis gene	ccsA
	翻译起始因子 Translation initiation factor	infA
未知功能 Unknown function	假想叶绿体读码框 Hypothetical chloroplast reading frames	#ycf1, ycf1, ycf2(2), ycf3*, ycf4*
注：表示含1个内含子的基因；表示含有2个内含子的基因；#表示假基因；(n )表示多拷贝基因拷贝数n。　　Notes: indicates the gene containing one intron; ** indicates the gene containing two introns; # indicates the pseudogene; (n) indicates the copy number of the multi-copy gene.

基因 Gene	位置 Location	外显子I Exon I/bp	内含子I Intron I/bp	外显子II Exon II/bp	内含子II Intron II/bp	外显子III Exon III/bp
trnK-UUU	LSC	37	2 500	35
rps16	LSC	40	892	197
trnG-UCC	LSC	31	708	60
atpF	LSC	145	777	410
rpoC1	LSC	432	789	1 602
ycf3	LSC	126	779	228	790	153
trnL-UAA	LSC	35	525	50
trnI-GAU	LSC	39	576	56
clpP	LSC	71	949	294	605	226
petB	LSC	6	797	642
petD	LSC	8	744	475
rpl16	LSC	9	1 095	360
ndhB	IR	775	673	758
trnV-UAC	IR	32	946	42
trnA-UGC	IR	37	838	36
ndhA	SSC	553	1 101	539

氨基酸 Amino acid	密码子 Codon	RSCU			氨基酸 Amino acid	密码子 Codon	RSCU
氨基酸 Amino acid	密码子 Codon	基因组 Genome	高表达基因 High expression gene	低表达基因 Low expression gene	氨基酸 Amino acid	密码子 Codon	基因组 Genome	高表达基因 High expression gene	低表达基因 Low expression gene
苯丙氨酸 Phe/F	UUU*	1.38	1.56	1.06	谷氨酰胺 Ile/I	AUU	1.52	1.46	0.86
苯丙氨酸 Phe/F	UUC	0.62	0.44	0.94		AUC	0.52	0.30	1.50
络氨酸 Tyr/Y	UAU*	1.64	1.74	0.36		AUA	0.97	1.24	0.64
络氨酸 Tyr/Y	UAC	0.36	0.26	1.64	苏氨酸 Thr/T	ACU*	1.63	1.46	0.81
半胱氨酸 Cys/C	UGU*	1.55	1.79	0.63		ACC	0.69	0.53	1.18
半胱氨酸 Cys/C	UGC	0.45	0.21	1.37		ACA*	1.27	1.55	0.91
组氨酸 His/H	CAU*	1.50	1.62	1.06		ACG	0.42	0.47	1.11
组氨酸 His/H	CAC	0.50	0.38	0.94	天冬酰胺 Asn/N	AAU*	1.56	1.61	0.88
丝氨酸 Ser/S	UCU*	1.70	1.87	0.58	天冬酰胺 Asn/N	AAC	0.44	0.39	1.12
	UCC	0.91	0.84	0.89	天冬氨酸 Asp/D	GAU*	1.59	1.56	1.00
	UCA*	1.19	1.36	0.72	天冬氨酸 Asp/D	GAC	0.41	0.44	1.00
	UCG	0.62	0.60	1.27	赖氨酸 Lys/K	AAA*	1.55	1.66	0.86
	AGU	1.24	1.17	1.10	赖氨酸 Lys/K	AAG	0.45	0.34	1.14
	AGC	0.34	0.16	1.44	缬氨酸 Val/V	GUU*	1.55	1.76	0.79
亮氨酸 Leu/L	UUA*	2.07	2.48	0.91		GUC	0.37	0.40	0.59
	UUG	1.17	0.99	0.82		GUA	1.56	1.47	1.10
	CUU*	1.25	1.21	0.56		GUG	0.52	0.37	1.52
	CUC	0.36	0.34	0.99	丙氨酸 Ala/A	GCU*	1.81	2.02	0.66
	CUA	0.83	0.62	1.25		GCC	0.63	0.67	1.04
	CUG	0.32	0.36	1.47		GCA	1.11	1.11	1.21
脯氨酸 Pro/P	CCU*	1.62	1.82	0.94		GCG	0.45	0.19	1.10
	CCC	0.71	0.58	1.17	甘氨酸 Gly/G	GGU*	1.34	1.45	0.64
	CCA	1.18	1.24	0.66		GGC	0.39	0.21	0.86
	CCG	0.49	0.36	1.23		GGA	1.65	1.72	1.22
精氨酸 Arg/R	CGU	1.45	1.16	0.88		GGG	0.62	0.62	1.28
	CGC	0.38	0.10	0.54	谷氨酸 Glu/E	GAA*	1.55	1.76	0.94
	CGA	1.46	1.46	0.85	谷氨酸 Glu/E	GAG	0.45	0.24	1.06
	CGG	0.38	0.15	1.25	色氨酸 Trp/W	UGG	1.00	1.00	1.00
	AGA*	1.70	2.62	0.94	甲硫氨酸 Met/M	AUG	1.00	1.00	1.00
	AGG	0.62	0.50	1.54	终止子 TER	UAA	1.58	2.00	0.88
谷氨酰胺 Gln/Q	GAA*	1.58	1.74	1.08		UAG	0.57	0.00	0.97
谷氨酰胺 Gln/Q	GAG	0.42	0.26	0.92		UGA	0.85	1.00	1.15
注：表示最优密码子，下划线表示叶绿体基因组中密码子RSCU值大于1。　　Notes: indicates the optimal codon, and the underline indicates that the RSCU value of the codon in the chloroplast genome is greater than 1.

Chloroplast Genome Phylogeny and Codon Preference of Anabasis aphylla L.