Abstract
Cephalotaxus oliveri is an endemic conifer of China, which has medicinal and ornamental value. However, the limited molecular markers and genetic information are insufficient for further genetic studies of this species. In this study, we characterized and developed the EST-SSRs from transcriptome sequences for the first time. The results showed that a total of 5089 SSRs were identified from 36446 unigenes with a density of one SSR per 11.1 kb. The most common type was trinucleotide repeats, excluding mononucleotide repeats, followed by dinucleotide repeats. AAG/CTT and AT/AT exhibited the highest frequency in the trinucleotide and dinucleotide repeats, respectively. Of the identified SSRs, 671, 1125, and 1958 SSRs were located in CDS, 3′UTR, and 5′UTR, respectively. Functional annotation showed that the SSR-containing unigenes were involved in growth and development with various biological functions. Among successfully designed primer pairs, 238 primer pairs were randomly selected for amplification and validation of EST-SSR markers and 47 primer pairs were identified as polymorphic. Finally, 28 high-polymorphic primers were used for genetic analysis and revealed a moderate level of genetic diversity. Seven natural C. oliveri sampling sites were divided into two genetic groups. Furthermore, the 28 EST-SSRs had 96.43, 71.43, and 78.57% of transferability rate in Cephalotaxus fortune, Ametotaxus argotaenia, and Pseudotaxus chienii, respectively. These markers developed in this study lay the foundation for further genetic and adaptive evolution studies in C. oliveri and related species.
Highlights
Successful conservation strategies require obtaining the genetic information, of which genetic diversity and population structure are essential parts
Of the 36446 unigenes that we identified, 4352 sequences contained 5089 SSRs with 261 sequences in compound formation and 578 sequences containing more than 1 SSR
These results indicated that the markers developed in this study would provide a powerful molecular tool for evolutionary adaptation and genetic relationship analysis in C. fortune, A. argotaenia, and P. chienii
Summary
Successful conservation strategies require obtaining the genetic information, of which genetic diversity and population structure are essential parts. Molecular markers become useful tools to study genetic diversity and population structure of natural germplasm resources in non-model plants with no reference genomes (Parida et al, 2009). SSRs are classed into genomic SSRs (gSSRs) and expressed sequence tag SSRs (EST-SSRs), the latter of which are located in the genic transcribed regions and are identified by NGS technology (Varshney et al, 2005; Wei et al, 2011). EST-SSRs have been found to be more development-inexpensive, more evolutionarily conserved, and have higher transferability to related species than traditional anonymous gSSRs (Scott et al, 2000; Gadissa et al, 2018)
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