Comparative Chloroplast Genomics And DNA Barcoding Of Lagerstroemia Species

Lagerstroemia belongs to the Lythraceae family and is mainly distributed in the global temperate and tropical regions.There are about 55 species of this genus in the world and 18 species in China.It is a famous ornamental plant in summer,which has a flowering period of three months,also known as Bairihong(One-hundred-day's-red).Because of its colorful flowers and long flowering period,crape myrtle is widely used in gardens.Due to frequent interspecific hybridization and introgression of genes,the interspecific phylogeny of the genus has long been in doubt.Although the inflorescence of Lagerstroemia makes it easy to distinguish from other taxa,due to the lack of effective morphological classification characteristics,influenced by environmental conditions and different development stages,there are many controversial species in the morphological classification of Lagerstroemia.So it is difficult to classify Lagerstroemia plants at the species level.Chloroplast genome sequence is not only a powerful tool for studying phylogenetic issues,but also the most valuable molecular marker source for assessing genetic diversity.Therefore,we sequenced the chloroplast genomes of 6 Lagerstroemia species,using bioinformatics methods to compare and analyze the genome characteristics and differences of Lagerstroemia,reconstructing the phylogeny and evolutionary relationship of Lagerstroemia.Then,DNA barcode applicability of three international universal barcodes(mat K?psb A-trn H?rbc L)in Lagerstroemia were tested and evaluated using the criteria of sequence variability,intraspecific and interspecific variation,and species identification rate.The hypervariable fragments were selected from the chloroplast genome as Lagerstroemia-specific barcodes,providing a reference for the rapid identification of Lagerstroemia species.The main findings are as follows:1.Comparative analysis of chloroplast genome of Lagerstroemia:Complete chloroplast genomes of six Lagerstroemia species were obtained by high-throughput sequencing.The chloroplast genomes of Lagerstroemia were highly conserved,with a length between 152 049 bp and 152 521 bp,and all of them have a circular quadripartite structure.The gene content and sequence of six complete genome sequences are identical,totally 112 unique genes are encoded,including 78 protein coding genes,4 r RNA genes and 30 t RNA genes.Based on the chloroplast genome sequences of 7 Lagerstroemia species published by NCBI,13 Lagerstroemia species were compared,and we found that(1)The sequence similarity of 13 cp genomes was more than 90% with small variation.(2)Codon usage is similar in 13 cp genomes: methionine and tryptophan are the least common amino acids,while arginine and serine are the most common amino acids.(3)The long and simple repeat sequences of 13 Lagerstroemia species are mostly located in the protein coding regions,and a few in the no-coding regions,such as ycf2;(4)Evolutionary analysis shows that the five genes atp B,psa I,rpl23,rpl33 and rps7 had experienced positive selection in Myrtales.(5)Two species from Geraniaceae are taken as the out group,and a phylogenetic tree is constructed based on 66 common coding genes.The results show that 13 Lagerstroemia species are a monophyletic group and formed into two evolutionary branches: the first branch is composed of Lagerstroemia intermedia,L.speciosa,L.siamica,L.venusta,L.floribunda,L.tomentosa and L.calyculata;the second branch is composed of L.excelsa,L.limii,L.fauriei,L.subcostata,L.guilinensis and L.indica.In Myrtales,the Melastomataceae family is the earliest differentiated,followed by the Myrtaceae family,The Onagraceae family and Lythraceae family differentiated the latest.2.Study on DNA barcode of LagerstroemiaThe applicability of DNA barcodes(mat K,psb A-trn H,rbc L)in Lagerstroemia was evaluated from the aspects of sequence variation,interspecific variation and species identification.The results showed that trn H-psb A had the largest variation in 13 species of Lagerstroemia(12.3% of variation sites,11.6% of information sites),and could be used as specific DNA strip of Lagerstroemia.Matk(1.6% of variation sites,1.53% of information sites)and rbc L(1% of variation sites,1% of information sites)are too small to be used as DNA barcodes of Lagerstroemia.The combined fragments rbc L + trn H-psb A and mat K + rbc L + trn H-psb A have high identification ability and can be used as Lagerstroemia specific barcodes.Through the whole sequence alignment of 13 species of Lagerstroemia,the highly variable fragments were screened from three aspects,namely,the rate of variable outliers,the rate of information sites and the length of sequences.The results showed that the gene regions were not suitable for specific DNA barcodes because of their high sequence similarity.15 highly variable segments with a potential value of 200-800 bp were screened out in the gene interval region(including intron region),with a mutation rate of more than 3.9% and an information site rate of more than 3.3%.An NJ(Neighbor-Joining)tree was constructed to evaluate 15 highly variable fragments,it was found that 5 fragments pet A-psb J,ndh F-rpl32,ndh G-ndh I,trn S-trn G,and trn R-atp A have the strongest identification ability,so they are suggested as candidate DNA barcodes of Lagerstroemia.