RNA-Seq for excavation of genes involved in the biosynthesis of primary active components and identification of new EST-SSR markers in medicinal chrysanthemum

Chrysanthemum morifolium cv. ?Huaihuang? has great medicinal and commercial value. However, limited genomic and transcriptional information restricts the further research of ?Huaihuang?. In this study, using ?Huaihuang? leaf as the sample, we performed RNA-Seq and obtained numerous unigene sequences for functional characterization, and developed new EST-SSR markers. The results show that 176 unigenes were assigned to the ?antioxidant activity? in GO annotations, 998 unigenes were assigned to ?Secondary metabolite biosynthesis, transport and catabolism? in the KOG database, 381 unigenes were assigned to ?Biosynthesis of other secondary metabolites?, and 377 unigenes were assigned to ?Metabolism of terpenoids and polyketides? in the KEGG database. A number of genes involved in the biosynthesis of flavones and terpenes, such as CHS, CHI, FLS, HMGR and MVD, were found in the transcription data of ?Huaihuang?. Also, 36 new polymorphic EST-SSR markers were developed and validated. Using them, the genetic diversity was analyzed and a dendrogram of six clades was constructed among 15 medicinal chrysanthemum varieties. The excavated unigenes and identified new ESTSSR markers will provide useful resources for the study of biosynthesis of active components, genetic diversity analysis, population structure identification and in molecular breeding research of ?Huaihuang? and related species.

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Highly divergent isolates of chrysanthemum virus B and chrysanthemum virus R infecting chrysanthemum in Russia

PeerJ ◽

10.7717/peerj.12607 ◽

2022 ◽

Vol 10 ◽

pp. e12607

Author(s):

Sergei N. Chirkov ◽

Anna Sheveleva ◽

Anastasiya Snezhkina ◽

Anna Kudryavtseva ◽

George Krasnov ◽

...

Keyword(s):

Genetic Diversity ◽

High Throughput Sequencing ◽

Germplasm Collection ◽

Chrysanthemum Morifolium ◽

Chinese Isolate ◽

Replicase Gene ◽

Rna Seq ◽

Botanical Gardens ◽

Complete Genomes ◽

Golden Standard

Background Chrysanthemum is a popular ornamental and medicinal plant that suffers from many viruses and viroids. Among them, chrysanthemum virus B (CVB, genus Carlavirus, family Betaflexiviridae) is widespread in all chrysanthemum-growing regions. Another carlavirus, chrysanthemum virus R (CVR), has been recently discovered in China. Information about chrysanthemum viruses in Russia is very scarce. The objective of this work was to study the prevalence and genetic diversity of CVB and CVR in Russia. Methods We surveyed the chrysanthemum (Chrysanthemum morifolium Ramat.) germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. To detect CVB and CVR, we used RT-PCR with virus-specific primers. To reveal the complete genome sequences of CVB and CVR isolates, metatransciptomic analysis of the cultivars Ribonette, Fiji Yellow, and Golden Standard plants, naturally co-infected with CVB and CVR, was performed using Illumina high-throughput sequencing. The recombination detection tool (RDP4) was employed to search for recombination in assembled genomes. Results A total of 90 plants of 23 local and introduced chrysanthemum cultivars were surveyed. From these, 58 and 43% plants tested positive for CVB and CVR, respectively. RNA-Seq analysis confirmed the presence of CVB and CVR, and revealed tomato aspermy virus in each of the three transcriptomes. Six near complete genomes of CVB and CVR were assembled from the RNA-Seq reads. The CVR isolate X21 from the cultivar Golden Standard was 92% identical to the Chinese isolate BJ. In contrast, genomes of the CVR isolates X6 and X13 (from the cultivars Ribonette and Fiji Yellow, respectively), were only 76% to 77% identical to the X21 and BJ, and shared 95% identity to one another and appear to represent a divergent group of the CVR. Two distantly related CVB isolates, GS1 and GS2, were found in a plant of the cultivar Golden Standard. Their genomes shared from 82% to 87% identity to each other and the CVB genome from the cultivar Fiji Yellow (isolate FY), as well as to CVB isolates from Japan and China. A recombination event of 3,720 nucleotides long was predicted in the replicase gene of the FY genome. It was supported by seven algorithms implemented in RDP4 with statistically significant P-values. The inferred major parent was the Indian isolate Uttar Pradesh (AM765837), and minor parent was unknown. Conclusion We found a wide distribution of CVB and CVR in the chrysanthemum germplasm collection of the Nikita Botanical Gardens, which is the largest in Russia. Six near complete genomes of CVR and CVB isolates from Russia were assembled and characterized for the first time. This is the first report of CVR in Russia and outside of China thus expanding the information on the geographical distribution of the virus. Highly divergent CVB and CVR isolates have been identified that contributes the better understanding the genetic diversity of these viruses.

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