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Euphytica

, 214:26 | Cite as

An efficient method for developing polymorphic microsatellite markers from high-throughput transcriptome sequencing: a case study of hexaploid oil-tea camellia (Camellia oleifera)

  • Xiangyan Cui
  • Xiaomao Huang
  • Jiaming Chen
  • Xiaoqiang Yang
  • Jun Rong
Article
  • 181 Downloads

Abstract

The bottleneck of microsatellite marker development is to determine polymorphisms of microsatellite markers. A large amount of microsatellites can be detected via high-throughput sequencing. However, most previous studies didn’t fully use the high-throughput sequencing data to predict number of alleles at microsatellite loci. Instead, laborious experiments were performed for manually screening microsatellite loci, finding out number of alleles at each microsatellite loci and selecting those with polymorphisms for marker development. In this study, we improved the method for efficient development of polymorphic microsatellite markers from high-throughput transcriptome sequencing, using hexaploid oil-tea camellia as a case study. Leaf transcriptomes were sequenced of eight wild oil-tea camellia samples at different altitudes in Jinggang and Lu Mountains, China. Microsatellites were directly identified in the sequencing reads and primers were designed. Strategies were designed to filtering duplicate and multi-locus markers. For each marker, number of alleles cross samples was predicted and length of the potentially amplifiable sequence was estimated. 153 predicted polymorphic markers were selected and empirically validated in the eight samples. Sixty five markers (42%) were polymorphic (2–12 alleles) and 31 (20%) were highly polymorphic (6–12 alleles). The empirical number of alleles was generally higher than the predicted number of alleles but they were significantly correlated. The predicted allele length was among the empirical allele length range. Compared with most previous studies, the method shows a higher efficiency for developing polymorphic markers and filtering duplicate and multi-locus markers. The polymorphic microsatellite markers developed can be used for analyzing the genetic diversity of oil-tea camellia.

Keywords

Camellia oleifera High throughput sequencing Microsatellite marker development Polymorphism Simple sequence repeat Short tandem repeat 

Notes

Acknowledgements

We thank Dr. Marinus J. M. Smulders for comments on the data analysis. This work was supported by the National Natural Science Foundation of China (NSFC Grant No. 31460072) and the “Gan-Po Talent 555” Project of Jiangxi Province, China.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10681_2018_2114_MOESM1_ESM.zip (11 kb)
Supplementary material 1 (ZIP 11 kb). File S1 R functions. “Simple_SSR” for selecting simple microsatellites. “SSR_A” for detecting different alleles and calculate number of alleles from each primer pair in each sample
10681_2018_2114_MOESM2_ESM.xlsx (30 kb)
Supplementary material 2 (XLSX 29 kb). Table S1 Primers of potentially amplifiable microsatellite loci in oil-tea camellia
10681_2018_2114_MOESM3_ESM.xlsx (15 kb)
Supplementary material 3 (XLSX 15 kb). Table S2 Validated primers of polymorphic microsatellite loci in oil-tea camellia

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Center for Watershed Ecology, Institute of Life Science and School of Life SciencesNanchang UniversityNanchangChina
  2. 2.Key Laboratory of Poyang Lake Environment and Resource Utilization, Ministry of EducationNanchang UniversityNanchangChina

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