Phylogentic Study Pterygiella And Its Related Taxa Based On Chloroplast Genome

Pterygiella,which belongs to Orobanchaceae,is an endemic genus in Southwest China and is a perennial semi-parasitic plant.The genus now includes five species,including Pterygiella nigrescens,Pterygiella duclouxii,Pterygiella cylindrica,Pterygiella luzhijiangensis,Pterygiella trichosepala,and so on.The species of Pterygiella has unique flower pattern and beautiful plants,it has potential garden value and medicinal value.Traditional Phtheirospermum is a related taxa of Pterygiella,Includes Phtheirospermum japonicum,Phtheirospermum parishii,Phtheirospermum glandulosum,Phtheirospermum muliense and Phtheirospermum tenuisectum,which are endemic to East Asia,and all the plants are densely covered with glandular hairs.Based on recent molecular systematics research,Trib.Rhinantheae,where the genus Pterygiella and traditional Phtheirospermum has been relocated from Scrophulariaceae to Orobanchaceae.In recent years,the classification of Phtheirospermum has been controversial.The most widely distributed Phtheirospermum japonica has been proved to have closer genetic relationship with Pedicularis sylvatica and should be placed in Pedicularis.Phtheirospermum tenuisectum complex,are more closely related to the species of Trib.Rhinantheae,so some scholars have merged them into Pterygiella,but they have strong morphological heterogeneity with Pterygiella.In our previous work,combined with the evidence of morphological geography and molecular systematics,a large number of studies have been made on the genus Pterygiella and its related taxa.The results show that Pterygiella,Phtheirospermum japonicum and Phtheirospermum tenuisectum complex should be regarded as independent genera respectively,and Phtheirospermum parishii should be combined into the genus Pseudobartsia.The species classification of Pterygiella was clarified and revised.In previous studies on molecular systematics,phylogenetic trees were mainly constructed based on ITS,mat K,rbc L,rpo C1 and other gene fragments.However,due to the lack of information sites,the phylogenetic positions of phylogenetic trees constructed based on different molecular fragments in the same group are different,and the phylogenetic positions of Pterygiellaand its related taxa have not been scientifically treated.Compared with traditional gene fragments,chloroplast genome has more information sites,which has been proved to be an ideal resource for studying system evolution.On the basis of the previous work of the research group,12 representative materials(11 species)were selected in this study,and the cp DNA of Pterygiella species was determined and assembled for the first time.Bioinformatics and comparative genomics were used to analyze Pterygiella and its related taxa,and their phylogenetic relationship was explored.The main conclusions are as follows:(1)The basic structure analysis showed that the cp DNA of 12 materials was a typical tetrad structure,consisting of two single copy regions(SC)and a pair of reverse repeat regions(IR).The length ranged from 153,321 bp to 154,952 bp,with great differences among genera.The cp DNA of Pterygiella was the longest(154,844 bp to154,884bp),followed by Phtheirospermum tenuisectum(154,475bp),and Phtheirospermum parishii is in the middle(153,965bp).There are small differences in length and GC content among different regions.The genus Pterygiella is the longest(85,319bp~85,400bp)in LSC region,Phtheirospermum tenuisectum complete(85,183bp~85,366bp),and Phtheirospermum japonicum and Phtheirospermum parishii are shorter(84,430 bp,84,653bp),which are finely divided The total GC content of the studied materials is slightly different,except for Phtheirospermum japonicum and Phtheirospermum parishii,which are 36.4% and37.7% respectively,and the rest materials are all 37.9%.All the research materials contain133 genes,including 88 CDS,37 t RNA genes and 8 r RNA genes.(2)The repetitive sequences of the research materials have high fitting degree,and most of them are distributed in the non-coding regions of cp DNA,and a few are distributed in the coding regions of mat K,rpo C1,rpo C2,etc.,which can be used as an important source for developing molecular markers in the next step.There is little difference in quantity among different species.Among them,there are 79~93 pairs of disperser repeats sequence,the most is Pterygiella nigrescens(91~93pairs),followed by pine Phtheirospermum japonicum and Phtheirospermum tenuisectum(87~90 pairs),and the least is Phtheirospermum parishrii(79 pairs);There are 22~31 pairs of tandem repeats sequence,and there is little difference among them.The types of SSRs are the same in all species,and they all contain one,two,three,four and five nucleotide repeats,but the number of SSRs is quite different(35~61),the most is Phtheirospermum parishrii(61),the least is Phtheirospermum japonicum(35),and the rest materials are between 46~53.(3)The codon usage and preference of cp DNA are highly consistent with that of most angiosperms.They all use 61 kinds of codons(excluding terminator),edit 20 kinds of amino acids,and all prefer to choose the codon whose third base is A/U.The 12 material preference codons include 28 codons such as AAA,AAU,ACA,ACU and AGA.(4)The similarity,collinearity,IR region contraction and expansion,and selection pressure of cp DNA of 12 materials were analyzed by comparative genomics.The results showed that the cp DNA of Pterygiella and its related taxa were highly similar,and the similarity between Phtheirospermum japonicum complex and Pterygiella was the highest,while that of Phtheirospermum japonicum was the lowest.Although the similarity is very high,there are also small variations,among which the variation in SC region is larger than that in IR region,and the variation mainly occurs in non-coding regions,only a small part occurs in gene coding regions,and the variation in coding regions mainly occurs in psb A,mat K,rop C2,rpo C1,rpo B,ycf1 and ndh F gene coding regions.Cp DNA of Pterygiella and its related groups is highly collinear,and there is no gene rearrangement and inversion,but only some rearrangement phenomena are found in Phtheirospermum parishrii and Phtheirospermum japonicum.The analysis of contraction and expansion of IR/SC boundary region showed that JLB(LSC/IRb)existed in rps19 gene,and Psedupterygiella huizensis,Phtheirospermum japonicum and Phtheirospermum tenuisectum expanded.In JSB region,the length of ycf1 gene is quite different(more than 5,000bp),with more than 4300 bp distributed in SSC,while other species are only 4bp distributed in SSC,and ndh F gene is located in JLB region of Phtheirospermum japonicum,Phtheirospermum parishrii and Phtheirospermum tenuisectum In JSA(SSC/IRa)region,ycf1 gene is distributed in the genus Pterygiellaand Phtheirospermum complex(Pseudopterygiella jinshajiangensis)in the same way,and both of them expanded to IRa region by 5570bp;ycf1 and ndh F genes are just opposite to other species in JSB region and JSA region in Phtheirospermum tenuisectum complex(Pesudopterygiella huizeensis),Phtheirospermum parishrii and Phtheirospermum japonicum.Therefore,the differences in the length of cp DNA in the study materials are mainly caused by the contraction and expansion of IR segments and the insertion and deletion of gene spacer regions.Among them,IR region has little influence on the length of chloroplast genome,so it is speculated that the insertion and deletion in the intergenic region are the main factors affecting the length of cp DNA.(5)The nucleotide polymorphism(pi)analysis of the whole sequence,large single copy region(LSC),small single copy region(SSC)and two reverse repeat regions(IR)of the genus Pterygiella and its related groups by population genomics showed that the polymorphism of SSC region was the highest,followed by LSC region,and the IR region was very conservative,and its pi value was significantly lower than that of the two copy regions and SSC region.In LSC region,there are high polymorphisms in IGS(mat K,rps16),IGS(trn T-UGU,trn L-UAA)and IGS(trn C-GCA,pet N)intergenic regions.ycf1 gene has high nucleotide polymorphism in SSC region,which is also the highest point of nucleotide polymorphism in chloroplast genome of Pterygiella and its related groups,followed by ndh F gene and rpl32 gene.(6)Based on 17 materials(16 species,5 downloaded from NCBI),phylogenetic trees with inconsistent topological structures were obtained from different tree-building materials(cp DNA sequences and CDS sequences),but the topological structures and support rates of phylogenetic trees(ML and BI)constructed by different methods(maximum likelihood method and Bayesian method)based on the same material were highly consistent.On the phylogenetic tree constructed based on CDS sequence,Phtheirospermum japonicum forms sister group relationship with other materials,which reflects that it is far related to the core group of Pterygiella;A sister group is formed by the monoclinic branches of Phtheirospermum parishrii and Pterygiella,and 7 materials(6 species)of Pterygiella and 3 materials of Phtheirospermum tenuisectum complex respectively form a monoclinic group and are sister groups,which reflects that in the related groups,Phtheirospermum tenuisectum complex is the closest relative to Pterygiella,followed by Phtheirospermum parishrii.This result is highly consistent with the research results of our group in 2020,which supports that the three species of Phtheirospermum tenuisectum complex can be separated into one genus(Psedupterygiella).Because there is no cp DNA data of pseudobartsia yunnanensis,it is impossible to determine whether Phtheirospermum parishrii is a new genus or the same genus as Pseudoobarthia yunnanensis.In the genus Pterygiella,the single line formed by Pterygiella duclouxii and Pterygiella luzhijiangensis forms sister groups with the other four species in the genus,which indicates that the genetic relationship between Pterygiella duclouxii and Pterygiella luzhijiangensis is the closest.Pterygiella nigrescens and Pterygiella cylindrica form a sister group,indicating that Pterygiella nigrescens and Pterygiella cylindrica are more closely related,while Pterygiella kumingensis and branches formed by Pterygiella nigrescens,Pterygiella cylindrica and Pterygiella trichosepala form a sister group,indicating that cp DNA systematic data supports Pterygiella kunmingensis as a new species.