Comparative Analysis Of Complete Chloroplast Genome Of Endemic Pinus Henryi With Two Sibling Species

Pinus henryi Mast.is an endemic pine in China,distributed in the Daba Mountains,Wushan Mountains,Wuling Mountains.It is an important tree species of warm coniferous forest in China.Its distribution aerea overlaps with the southern margin of Pinus tabuliformis Carr.,and the northern margin of Pinus massoniana Lamb.Since it's named,the systematic position of P.henryi has been controversial.Because of the confusion of the classification system for a long time,P.henryi is often regarded as a variety of other species.It has been deforested and lack of effective protection,and has been listed as a rare plant.The number of populations and samples in previous studies were small.In this paper,based on the next-generation sequencing data,205 chloroplast genomes of P.henryi,P.tabulaeformis,and P.massoniana were sequenced to analyze genetic variation and phylogenetic relationships among the species.And we utilize the selected microsatellite data to further explore geographic population relationships.The main results are as follows:(1)The chloroplast genome sequence of P.henryi was determined for the first time.Similar to P.tabulaeformis and P.massoniana,the chloroplast genome sequence of P.henryi showed a typical quadripartite structure.The chloroplast genome size was 119,669 bp,with a large single copy(LSC)of 65,624 bp and a small single copy(SSC)of 53,055 bp,separated by two inverted repeat regions(IRs)of 495 bp.P.henryi consisted of 74protein-coding genes,4 r RNA genes,and 36 t RNA genes.Moreover,the 4 t RNA(trn H-GUG,trn I-CAU,trn S-GCU,trn T-GGU)had two copies.Only the trn I-CAU gene was duplicated in IRs.Like P.massoniana,P.henryi and P.tabulaeformis also lost all 11 ndh genes.In the analysis of simple sequence repeats,no trinucleotide repeat was detected in the three pine species,and single nucleotide accounted for the largest proportion,followed by tetronucleotide.In addition,(T)11 and(AT)5 repeats are the most abundant mononucleotides and dinucleotides,respectively,among the three species.The codon composition and RSCU values of the three pine species were basically same.Leucine(Leu)was the most abundant that percentage was 10.02%(three species)whereas L-Cysteine(Cys)was minimum that was 1.20%(P.tabuliformis and P.henryi)and 1.19%(P.massoniana).The third base of codon preferred to use A or T in the three pine species.IR boundary and sequence variation analysis showed that the genetic composition and distribution of the three pine species were consistent,and the IR region of P.massoniana had a certain degree of shrinkage compared with that of P.tabuliformis and P.henryi.Psa M,psa M-ycf12,rps2-rpo C2,trn E-UUC-clp P,psa B-rps14,trn I-GAU-trn A-UGC showed high variation,which could be further used as potential DNA barcoding research.A total of 11 genes were under positive selection in P.henryi and P.tabulaeformis.Three SSR loci were positive selection and three SSR loci were balanced selection based on selected SSR loci screening result.Two of the selected SSR loci are adjacent to each of the two selected genes,which may interact with the adjacent selected genes through enhancers.(2)Based on the whole chloroplast genome data,we studied the phylogenetic relationships of P.henryi,P.massoniana and P.massoniana.P.henryi and P.tabulaeformis gathered first into a branch,showing that P.henryi was more closely related to P.tabuliformis compared with P.massoniana.We used the SSR data to further explore the geographical population relationship between P.henryi and P.tabulaeformis.The results also showed that P.henryi and P.tabulaeformis were closely related,and almost all the collected P.henryi populations were clustered in one branch.Structure analysis based on SSRs showed that P.tabulaeformis populations in Wuling Mountains and Daba Mountains gradually differentiated with the change of K value,which may be the result of adaptation to the ecological environment.There was obvious genetic differentiation between P.tabulaeformis populations and P.tabulaeformis populations.Chloroplast SSR has a higher mutation rate than chloroplast DNA sequence,suggesting that P.henryi maybe recently diverge from P.tabuliformis.