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

Sharma, Samriti, Kaur, Rajinder, Solanke, Amol Kumar U., Dubey, Himanshu, Tiwari, Siddharth, Kumar, Krishan
3 Biotech 2019 v.9 no.4 pp. 161
Rubus, apples, cDNA libraries, complementary DNA, computer software, expressed sequence tags, fruits, gene ontology, genetic variation, high-throughput nucleotide sequencing, leaves, microsatellite repeats, oligodeoxyribonucleotides, peaches, pears, quantitative polymerase chain reaction, raspberries, ripening, strawberries, transcription factors, transcriptomics, unigenes, India
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. 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.