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Development of polymorphic microsatellite markers by using de novo transcriptome assembly of Calanthe masuca and C. sinica (Orchidaceae).

ABSTRACT: BACKGROUND:Calanthe masuca and C. sinica are two genetically closely related species in Orchidaceae. C. masuca is widely distributed in Asia, whereas C. sinica is restricted to Yunnan and Guangxi Provinces in southwest China. Both play important roles in horticulture and are under the pressure of population decline. Understanding their genetic background can greatly help us develop effective conservation strategies for these species. Simple sequence repeats (SSRs) are useful for genetic diversity analysis, presumably providing key information for the study and preservation of the wild populations of the two species we are interested in. RESULTS:In this study, we performed RNA-seq analysis on the leaves of C. masuca and C. sinica, obtaining 40,916 and 71,618 unigenes for each species, respectively. In total, 2,019/3,865 primer pairs were successfully designed from 3,764/7,189 putative SSRs, among which 197 polymorphic SSRs were screened out according to orthologous gene pairs. After mononucleotide exclusion, a subset of 129 SSR primers were analysed, and 13 of them were found to have high polymorphism levels. Further analysis demonstrated that they were feasible and effective against C. masuca and C. sinica as well as transferable to another species in Calanthe. Molecular evolutionary analysis revealed functional pathways commonly enriched in unigenes with similar evolutionary rates in the two species, as well as pathways specific to each species, implicating species-specific adaptation. The divergence time between the two closely related species was tentatively determined to be 3.42?±?1.86 Mya. CONCLUSIONS:We completed and analysed the transcriptomes of C. masuca and C. sinica, assembling large numbers of unigenes and generating effective polymorphic SSR markers. This is the first report of the development of expressed sequence tag (EST)-SSR markers for Calanthe. In addition, our study could enable further genetic diversity analysis and functional and comparative genomic studies on Calanthe.

SUBMITTER: Hu C

PROVIDER: S-EPMC6219035 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Development of polymorphic microsatellite markers by using de novo transcriptome assembly of Calanthe masuca and C. sinica (Orchidaceae).

Hu Chao C Yang Hongxing H Jiang Kai K Wang Ling L Yang Boyun B Hsieh Tungyu T Lan Siren S Huang Weichang W

BMC genomics 20181106 1

<h4>Background</h4>Calanthe masuca and C. sinica are two genetically closely related species in Orchidaceae. C. masuca is widely distributed in Asia, whereas C. sinica is restricted to Yunnan and Guangxi Provinces in southwest China. Both play important roles in horticulture and are under the pressure of population decline. Understanding their genetic background can greatly help us develop effective conservation strategies for these species. Simple sequence repeats (SSRs) are useful for genetic ...[more]

PMID: 30400862

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Project description:Veratrilla baillonii Franch is an important Chinese medicinal herb for treating liver-related diseases, which has been over-collected in the recent decades. However, the effective conservation and related population genetic study has been hindered because of the lack of genome sequences and genetic markers in the natural population. We have conducted RNA-seq on V. baillonii. We performed de novo assembly of these data to characterize the V. baillonii transcriptome, resulting in 133,019 contigs with size >200 bp. These contigs were annotated using the NCBI nonredundant database and Gene Ontology (GO) terms. From these contigs, we developed novel microsatellite simple sequence repeat (SSR) markers, identifying a total of 40,885 SSRs. SSRs with repeat motifs of 1-4 bp (mono-, di-, tri-, and tetranucleotides) accounted for 99.8% of all SSRs, with mononucleotide repeats most common, followed by dinucleotide (16.2%) and trinucleotide repeats (14.7%). We selected 151 SSRs for experimental validation, of which 74 were confirmed by polymerase chain reaction. Fourteen SSRs were determined to be polymorphic by screening 40 individuals from six distant populations. The number of alleles per locus ranged from two to four, and the expected heterozygosity varied from 0.2637 to 0.8571, suggesting that these SSR markers are highly polymorphic and effective for further genetic analysis in the nature population. In addition, we explored the genetic structure of V. baillonii using five SSRs in four geographic populations and found that the identified genotypes were clustered into two phylogenetic clades: the Mekong River clade and Jinsha River clade. This result indicates that these two regions may harbor highly divergent genetic lineages and enriched genetic diversity. The de novo transcriptome sequences and new SSR markers discovered by this study provide an initial step for understanding the population genetics of V. baillonii, and a valuable resource for effective conservation management.

Dataset Information

Development of polymorphic microsatellite markers by using de novo transcriptome assembly of Calanthe masuca and C. sinica (Orchidaceae).

Publications

Development of polymorphic microsatellite markers by using de novo transcriptome assembly of Calanthe masuca and C. sinica (Orchidaceae).

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