无叶假木贼叶绿体基因组特征系统发育及密码子偏好性分析

Chloroplast Genome Phylogeny and Codon Preference of Anabasis aphylla L.

[目的]分析无叶假木贼叶绿体基因组结构特征,阐明假木贼属在藜科中的分类地位,进一步探究其密码子偏好性和确定最优密码子。[方法]基于CTAB法从无叶假木贼幼嫩同化枝中提取总DNA;通过Illumina Genome Analyzer HiSeq 2000高通量测序平台进行测序;使用GetOrganelle和Plann对叶绿体基因组序列进行组装和注释;利用MISA对叶绿体基因组中简单重复序列(SSR)进行分析;使用MAFFT v.7.450和IQTREE v.2.1.1软件进行多序列比对、构建最大似然法(Maximum likelihood,ML)系统进化树;使用DnaSP 6.0软件计算核苷酸多态性值;通过CUSP和Codon W 1.4.2软件进行密码子偏好性研究。[结果]无叶假木贼叶绿体基因组全长为154084 bp,包括1个大单拷贝区(LSC,85124 bp)、1个小单拷贝区(SSC,18934 bp)、1对反向重复序列(IRa和IRb,25013 bp)。共注释到132个基因,包含83个蛋白编码基因,8个rRNA基因,37个tRNA基因和4个假基因。SSR位于基因间区的数量最多(70.4%),单碱基(A/T)重复类型SSR数量最多。系统发育树的最优构树模型为TVM+F+R3,藜科划分为4个聚类组,其中,无叶假木贼与梭梭属和猪毛菜属亲缘关系最近。trnS-trnG(exon1)、ndhF-rpl32、rpl32-trnL、rps16(exon1)-trnQ和ycf1是高核苷酸多态性区域。共确定20个最优密码子(UUU、UAU、UGU、CAU、UCU、UCA、UUA、CUU、CCU、AGA、GAA、ACU、ACA、AAU、GAU、AAA、GUU、GCU、GGU、CAA),均以A/U结尾,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。[结论]无叶假木贼叶绿体基因组结构保守,呈典型的四分体结构。在藜科的系统发育中,无叶假木贼与梭梭属和猪毛菜属亲缘关系最近。鉴定的高变区和SSR位点可用于藜科属间物种的分子鉴定。无叶假木贼叶绿体基因组密码子偏好以A/U结尾,确定的20个最优密码子有助于其外源密码子的优化。研究结果可为无叶假木贼分子标记开发、系统进化及叶绿体基因工程研究提供参考。

[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 154084 bp,including a large single copy region(LSC,85124 bp),a small single copy region(SSC,18934 bp),and a pair of inverted repeat regions(IRa and IRb,25013 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.