| RNA-seq is more popular in recent years,it can provide valuable resources for the development of molecular markers.Compared with common makers derived from genomic DNA,the RNA-seq-derived maker is a novel type of molecular maker and has remarkable advantages,such as simpler development,more information,highly transferable and so on.These markers were applied in many fields,eg.comparative mapping,genetic diversity evaluation and phylogenetic analysis et al..Howerver,there is still only a few of species whose EST-derived makers have been developed and applied.Cymbidium ensifolium is one of Cymbidium genus,having elegant shape,beautiful appearance and a fragrant aroma.These features provide this species with extremely high ornamental value.But the genomic information in Cymbidium ensifolium is very scarce now.The study analyzed SSR recouses of transcriptome data in oilseed rape,and preliminarily developed some Genic-SSR makers.Moreover,these markers were tested to applied in anlysis of gentic diversity,providing some referecence on rape breeding.The result as below:(1)Here,the transcriptome in C.ensifolium was subject to RNA-seq.High-quality reads were assembled de novo using Trinity with optimized k-mer length of 25.All high-quality reads were assembled into 101,423 isogenes,with total residues of 139,385,689,the isogenes averaged 1,374 bp.In total,17,793 SSRs were identified within transcriptomic database.The density of SSR was 12.82 SSRs/10 kb,respectively.Among these SSRs,tri-SSR was the most types,followed by bi-SSR,except mono-SSR.Bi-SSR and tri-SSR accounted for 20.46%and 21.98%in all SSRs.The number of isogenes containing SSR was 13,768.These isogenes were annotated against Clusters of Orthologous Groups(COG),Gene Ontology database(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database,respectively.There were 1,748 SSR isogenes assigned into 23 COG classifications,respectively.There were 4,994 SSR isogenes classified into 80 and 78 GO terms,respectively.There were 2,107 SSR isogenes involved in 300 and 308 KEGG pathways.(2)A total of 80 genic-SSRs were chose to design primers,based on their motif,size and location.Of 80 genic-SSR primers,61 succeeded in PCR amplification and 39 showed polymorphism among 12 C.ensifolium accessions.The polymorphism information content(PIC)averaged 0.348,ranging from 0.141 to 0.746.Additionally,Unigenes containing the 61 genic-SSRs were searched against Non-redundant(Nr),Gene Ontology database(GO),Eukaryotic orthologous groups(KOGs)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database,respectively.The search resulted in that 53 hit Nr sequences,38 had GO terms,18 were assigned with KOG,15 were annotated with KEGG.The 61 genic-SSRs with PCR product were also endowed with valuable biological information,which could be used in C.ensifolium genetic diversity,linkages construction and gene mapping.(3)A total of 62 genic-SSRs develop in our lab were used for genetic analysis.The results exhibited that 62 were amplified in C.ensifolium successfully,and 52 showed polymorphism when cross-tested among 4 Cymbidium species comprising 35 accessions.The results suggested these genic-SSRs had a remarkable advantage in high transferability across these related species.The genetic distance averaged 0.389,ranging from 0.016 to 0.600.The polymorphic index content(PIC)of 52 polymorphic markers averaged 0.415,ranging from 0.054 to 0.824.The phylogenetic analysis exhibited that the accessions derived from the same species were grouped together,with only a few exceptions.The results indicated that these genic-SSRs were relatively highly polymorphic and powerful in species discrimination.These genic-SSR markers increase the number of current SSR markers in Chinese cymbidiums.They are very useful in the evaluation of genetic diversity among Chinese cymbidiums.The high transferability and relatively high polymorphism can also help genic-SSRs to discriminate the species efficiently. |
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