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3 Biotech

, 9:161 | Cite as

Transcriptome sequencing of Himalayan Raspberry (Rubus ellipticus) and development of simple sequence repeat markers

  • Samriti SharmaEmail author
  • Rajinder Kaur
  • Amol Kumar U. Solanke
  • Himanshu Dubey
  • Siddharth Tiwari
  • Krishan Kumar
Original Article
  • 64 Downloads

Abstract

Key message

Rubus ellipticus is a wild crop having less number of EST-SSR markers. First attempt was made towards the transcriptomics data analysis and generation of EST-SSR markers which were used in genetic diversity and transferability studies.

Abstract

Rubus ellipticus is a raspberry with yellow fruits, native to tropical and subtropical India and Asia. Leaves of Rubus ellipticus ‘Kumarhatti’ collection were utilized for cDNA library construction. More than 15 million sequencing reads were generated using NextSeq 500 Illumina RNA-seq technology. The DNASTAR software was used for de novo assembly from which 7777 unigenes with an average length of 500 bp was obtained. These unigenes were annotated using public databases, including NCBI non-redundant and gene ontology. De novo assembly of R. ellipticus unigenes found the highest similarity to apple than to other members of Rosaceae. This is the first attempt to use the Illumina platform of RNA sequencing and de novo assembly for R. ellipticus without a reference genome. In this study, unigenes were used for SSR marker development. ESTs containing SSR motifs were extracted using an online Microsatellite Identification Tool (MISA). SSR primers were designed from the SSR containing 704 EST sequences using the Websat software. Total 304 EST-SSRs primers were successfully designed, out of which 68 randomly selected primer pairs were custom synthesized and used for validation. Real-time PCR was also performed to analyze the relationship of transcriptional factors with fruit ripening. Out of 68 primer pairs, 61 were found to be informative in R. ellipticus, whereas 65 primer pairs were informative in the five tested genera of Rosaceae, i.e., pear, peach, apple, rose, and strawberry with 95.3% and 93.5% polymorphism, leading to the conclusion that these marker systems are very efficient to carryout diversity and cross transferability study in Rosaceae genera.

Keywords

Rubus ellipticus Library preparation Molecular markers EST-SSR Polymorphism and transferability study 

Notes

Author contribution statement

Dr. SS designed, performed the experiments, and wrote the manuscript. Dr. RK was involved in planning and supervised the work. Dr. AKUS and Mr. HD contributed to the interpretation of the results, Dr. ST helped to carry out the RT-PCR analysis and interpretation of the RT-PCR results, and Dr. KK provided plant material for research

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyDr. Yashwant Singh Parmar University of Horticulture and ForestrySolanIndia
  2. 2.National Research Center on Plant BiotechnologyNew DelhiIndia
  3. 3.Department of BiotechnologyNational Agri-Food Biotechnology Institute (NABI)MohaliIndia
  4. 4.Department of Fruit ScienceDr. Yashwant Singh Parmar University of Horticulture and ForestrySolanIndia

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