| Chrysanthemum(Chrysanthemum × morifolium Ramat)is a perennial herb of Compositae.It is one of the most famous and traditional flowers in China,which has important ornamental and economic value.Chrysanthemum has produced a wide range of variation in the cultivation history of more than 1600 years.The problem of the origin of chrysanthemum variation is one vital proposition in the biological realm.Some present studies have showed that transposon transpositions is able to transpose to cause species variation.In-depth research on origin and evolutionary history of the chrysanthemum transposon has important significance on understanding the evolution of chrysanthemum and cultivating new varieties of materials.Due to the large size,variability and complex structures of the chrysanthemum genome,there is no sequencing data of the ornamental polyploid chrysanthemum genome to utilize,which limits the further study of the chrysanthemum retrotransposon.With the rapid development of high-throughput sequencing technology,single-molecule real-time sequencing technology(this technology can perform longer fragments has become one of the main methods for sequencing full-length transcriptomes)and has been widely applied to sequence the full-length genes transcriptome in recent years.The traditional chrysanthemum variety ‘Jinbeidahong’ was sequenced relied on this technique.Based on sequencing data,using bioinformatics and other methods,this study has analyzed and identified long-repeat retrotransposons(LTR)and terminal-repeat retrotransposons(TRIM)in full-length transcripts,which are used to establish the corresponding molecular marker technology system and cluster analysis of the 103 and 24 different chrysanthemum varieties were clustered and analyzed to establish corresponding molecular marker maps.The main results are summarized as follows:(1)Traditional chrysanthemum varieties ‘Jinbeidahong’ blossom ahead of time by short-day treatment,RNA was extracted from 10 tissues(roots,stems,leaves,petioles,foot buds,lateral buds,buds,petals,stamens and receptacles)to full-length transcriptome sequencing.The average single-molecule ratio of Zero mode waveguide(ZMW)pore was 62.4% and the effective data were 18.3 Gb,937,088 Circular Consensus Sequence(CCS)sequence with an average accuracy of 0.8.After self-correction by subreads,a total of 902,490 CCS were obtained.The sequence of which 372,712 are full-length sequences,accounting for 41.3% of the total number of CCS,and 365,766 are Full-length Non-chimeric(FLNC)sequences,accounting for 40.5% of the total number of CCS.The clusters were de-redundant to obtain 141,817 high quality non-redundant FLNC sequences for subsequent analysis.The above data indicates that the full-length transcriptome sequencing results are relatively reliable and can be used to construct high-quality chrysanthemum reference gene sets,which will be served as valuable resources for future biological research of chrysanthemums.(2)Using the online software LTR_FINDER(http://tlife.fudan.edu.cn/ltr_finder/)results showed that under certain parameters,274 was identified on the long terminal repeat(LTR)reverse transcription transposon.There were 124 in the 5’UTR of the transcript,24 in the 3’UTR,and 126 in the CDS;the highest score was 8,and the lowest score was 5,of which 33 had a similar LTR similarity of 1.Most of the retrotransposons contain PBS and PPT domains but do not have incomplete in structures.The study has found that these retrotransposon inserted genes have many important functions,such as RNA polymerase II transcription initiation factor,different specific dehydrogenases,α/β hydrolase and so on.16 primers were designed based on the LTR sequence of retrotransposons.Then,establishment of polymorphic IRAP and S-SAP molecular marker system of retrotransposons.The molecular-marker system of sequence-specific amplification ploymorphisms(S-SAP)was used to construct genetic maps of 103 species of chrysanthemums.The 103 species of chrysanthemums were divided into 4 groups.(3)Using the online software LTR_FINDER(http://tlife.fudan.edu.cn/ltr_finder/)to find the TRIM reverse transposon,a total of 309 TRIM reverse transposons were obtained,of which 56 had a similar LTR similarity of 1.A partial TRIM retrotransposons was found to have a topoisomerase structure in cloned TRIM retrotransposons.The primers were designed in the LTR region of the TRIM reverse transposon to establish the IRAP and S-SAP molecular marker systems,.and the genetic diversity cluster analysis of 24 chrysanthemum varieties was carried out.In summary,this study found 274 LTR retrotransposons and 309 TRIM retrotransposons were found by sequencing,and IRAP and S-SAP molecular marker analysis were established.which laid a foundation for chrysanthemum genetic breeding and classification construction basis. |
