Non-concerted Evolution Of NrDNA ITS In Lycium

In higher plants,nrDNA,a multigene family consisting of several highly repetitive sequences,is often found in tens of thousands of copies which repeated in tandem within the genome.The internal transcribed spacer(ITS),as a part of nrDNA and its generally concerted evolution,which laid a foundation for direct sequencing of PCR products using ITS sequence.In addition,ITS have parental inheritance and moderate fragment length,which can provide suitable variation and information sites.Nowadays,ITS have become one of the most popular molecular markers used in plant phylogenetic and DNA barcode identification studies.However,as research continues,the phenomenon of ITS non-concerted evolution has been revealed in many plants,with significant differences between ITS sequences,and some ITS copies may form pseudogenes due to functional degradation.These pseudogenes accumulate more mutations and sequence diversity than functional copies and may have some effect on plant phylogeny.Therefore,a systematic analysis of the phenomenon of non-concerted evolution caused by ITS polymorphisms and pseudogenes in plant taxa will not only help to further explore the theoretical mechanism of concerted evolution,but also provide some genetic basis for phylogenetic and species evolutionary.Goji berry(also known as wolfberry),comes from the Lycium barbarum L.of the Solanaceae family,which is an important medicinal and edible species in China.Lycium is a worldwide distributed genus with rich germplasm resources,with seven species and three varieties in China.At present,ITS has been successfully applied to the species identification and genetic diversity studies of plants in Lycium.Subsequently,researcher found inaccessible or nested peaks in sequencing PCR products of Lycium and presumed the existence of ITS polymorphisms and pseudogenes in this genus.Although the popularity of wolfberry,Lycium is attracting increasing attention,a gap remains in the systematic study of ITS sequences in the genus.In this study,we carried out a systematically and in-depth analyzed on the nonconcerted evolution caused by ITS polymorphism and pseudogene in Lycium genus,comparing ITS sequences obtained from different primers(specific and universal primers)and different sequencing methods(direct sequencing of PCR products,sequencing of genomic DNA clones and sequencing of cDNA clones),and the main work are as follows:1.ITS polymorphism and non-concerted evolution in LyciumThe sequencing and identification success rates of cultivars and wild samples of Lycium genus were analyzed by PCR product direct sequencing of ITS sequences.The results showed that there was the phenomenon of nested peaks and inaccessible of this genus,which could not be improved by further optimization of conditions,confirming that the identification and sequencing success rates of ITS were low.The success rates of wild samples were significantly lower than those of cultivars(24%vs 71.4%).To obtain more abundant ITS sequence information,we selected six species and two varieties of Lycium for cloning analysis.The results showed that there was difference in ITS sequence lengths and GC contents among different species,intra-species and intra-individuals of this genus,indicating that ITS sequences of this genus are highly polymorphic,as reflected not only in genomic DNA of different species and individuals but also in functional cDNA,indicating non-concerted evolution.This may be one of the reasons for the low success rate in DNA barcoding studies of Lycium.2.Identification of ITS pseudogenes in LyciumBased on sequence diversity,GC content,secondary structure stability,base substitution patterns and the presence or absence of 5.8S conserved motifs,the ITS pseudogenes of Lycium were identified,and it was found that about 58%of the sequences in this study may have lost function and were identified as putative pseudogenes.Surprisingly,pseudogenes were significantly more common in the wild samples than in the cultivars for both primer pairs.Compared with functional ITS copies,putative pseudogenes not only accumulated more sequence diversity and base variation,but also had a significantly lower GC content and secondary structure stability,and tended to evolve neutrally without the restriction of selection pressure during the evolutionary process.The appearance of ITS pseudogenes further suggests the evolutional pattern of non-concerted evolution in Lycium.Several biological processes might retard or disrupt concerted evolution,such as polyploidization,agamospermy,multiple nucleolar organizer regions,and hybridization.Lycium is an allogamous plant,and interspecific hybridization created a similarly high level of intra-individual ITS polymorphism in this genus.Moreover,we first systematic detection of ITS pseudogenes in Lycium,and the criteria that we used to identify pseudogenes were simple and effective and even showed that some of the sequences used in previous phylogenetic studies of Lycium were pseudogenes.At the same time,the identification of ITS pseudogenes in Lycium complements the study of ITS pseodogenes in plant taxa,and lays a foundation for further exploration of the theoretical mechanism of concerted evolution.3.Phylogenetic utility of ITS pseudogenes in LyciumFurther phylogenetic analysis revealed that pseudogenes of Lycium have multiple types and evolved at a faster rate,each forming a separate branch from functional genes in the evolutionary tree.We presumed their origin may be earlier than that of functional genes.Together with the phylogenetic results of 44 published ITS sequences from NCBI,it was found that the presence of ITS pseudogenes did not have a significant effect on the phylogeny of Lycium.Under in the case of small sample,and the formation of phylogenetic clusters in previous studies could be better explained from the perspective of pseudogenes.To further determine the effect of ITS pseudogenes on the phylogeny of this genus,adequate sample analysis of this genus was conducted.It was found that the presence of ITS pseudogenes may obscure the actual phylogenetic relationships of this genus under the condition of sufficient samples,which is detrimental to the phylogenetic study of this genus.In summary,this is the first comprehensive and systematic analysis of ITS polymorphisms and pseudogenes in Lycium.Extensive polymorphism was found in the genomic DNA ITS sequences as well as in the functional cDNA ITS sequences,indicating non-concerted evolution of the ITS sequences.At the same time,the ITS pseudogenes of Lycium were identified,the types of pseudogenes were analyzed,and their possible impact on the phylogeny of Lycium was assessed.This study not only provides a theoretical basis for the phylogeny and genetic origin of Lycium,but also provides a reference for species identification and genetic diversity analysis using ITS molecular markers in this genus.