Haplotype Diversity Of ITS2 In Related Species Of Corydalis And Its Effects On Phylogeny

Now,internal transcribed spacer 2 of nuclear ribosomal DNA(nrDNA)is the most widely used DNA barcoding markers for plant phylogenetic relationships,especially at the level of species or populations.However,many studies have discovered the multiple copies of ITS2 in fungi,algae and plants using cloning techniques,next-generation sequencing and genome-wide analyses.Studies of ITS2 intragenomic variation has a significant implications for phylogenetic relationships.Here,we performed ITS2 region using Illumina Miseq sequencing in order to characterize intragenomic,intraspecific and interspecific varition of ITS2 regions in 14 related species in Corydalis.Sanger direct sequencing and high-throughput sequencing were used to compare the obtained ITS2 sequences,in order to study the relationship between the successful sequences of Sanger direct sequencing and the polymorphic copies of ITS2 in the ITS2 genome under the condition of sequence polymorphism in ITS2.And the effects of ITS2 sequence diversity on the phylogeny of Corydalis were analyzed.The main results are as follows:1.ITS2 haplotype diversity was common in the genome of Corydalis.We analyzed 24 samples from 14 species and found that these genomes have 19～107 distinct ITS2 haplotypes(relative abundance ≥0.1%)with a mean of 58 haplotypes per species and 6～22 ITS2 main haplotypes.The ITS2 sequence diversity in the genome was mainly composed of major haplotypes,with an average sequence proportion of 63.35%.Distributions of relative abundances for haplotypes in 12 species exhibited divergent forms.The frequency of the numerically dominant haplotype ranged from approximately 5.69%to 63.68%.The genetic distance within the genome ranges from 0.60%to 7.88%,and the genetic distance of different genomes of the same species is different,which leads to the change of the genetic distance within the species.2.There is a deviation between the sequence proportion of mixed samples and the theoretical sequencing proportion,but the change of total DNA quantity does not affect the sequence proportion of each sample.With the decrease of DNA sequencing volume,the number of effective variant types in each sample decreased,and a large number of minor variants could not be sequenced successfully,while maj or haplotypes were retained.The number and relative abundance of major haplotypes remained stable under different DNA quantities.3.Sanger direct sequencing preferred to amplify sequence types with higher relative abundance within the genome.In high-throughput sequencing,there was always a sequece that were 100%consistent with Sanger direct sequencing sequence,which was the first haplotype or the main haplotype of ITS2 sequence in high-throughput sequencing genome.Further study showed that in the ITS2 polymorphic sequences,the sequences with low GC content and highΔG,which affected the amplification efficiency of PCR,were not the main haplotypes or the haplotypes obtained by Sanger sequencing.4.There were shared haplotypes among related species of Corydalis.As many as 16 shared haplotypes were discovered between six related species,including C.ambigua,C.watanabei,C.humilis,C.kiautschouensis,C.linjiangensis and C.fumariifolia.And 3 shared haplotypes were found between C.caudata and C.hunmosa.These ITS2 haplotype sequences form haplogroups in related species.Because of the presence of shared haplotypes,NJ trees show a complex evolutionary relationship between these species.The ITS2 sequence of related species was clustered into one and independent of distant species,indicating that ITS2 sequence diversity of Corydalis affected the relationship between related species,but did not affect the phylogenetic analysis of distant species.In conclusion,the ITS2 haplotype diversity is common in the genome of Corydalis,which is basically composed of major haplotypes and remains stable in high-throughput sequencing.Sanger successfully sequenced the major haplotypes in the ITS2 genome by high-throughput sequencing.ITS2 sequence diversity of Corydalis has influence on phylogenetic studies of related species,and this study demonstrates complex genetic evolutionary relationships among related species.