De novo transcriptome assembly and its utility in development and characterization of the first set of genic SSR markers in cashew

Abstract

Cashew is an industrially important nut tree grown in over 30 countries. The demand for cashew is increasing due to its nutritional value and industrial applications. Despite socio-economic importance, genomic sequence and marker resources are very limited in cashew. De novo transcriptome sequencing allows the discovery of genes and molecular markers cost-effectively. In the present study, for the first time, we de novo assembled and characterized the transcriptome of cashew shoots and developed novel genic SSR markers. About 24.78 million high-quality Illumina sequences were used for de novo transcriptome assembly and it resulted in the identification of 71571 transcripts. The size of transcripts ranged from 201 to 23,274 bp with an average size of 687 bp. The N50 value of transcriptome was 1011 bp and the L50 sequence count was 13520. BUSCO analysis showed assembly completeness of 94 %. A total of 39836 transcripts had at least one BLAST hit against the Viridiplantae database. The functional annotation by KAAS analysis identified a total of 203 pathways. In silico SSR mining from assembled transcriptome identified 4271 SSRs. Tri-nucleotide (49.12 %) was the most dominant class of SSRs. The frequency of Class I SSRs (≥20 bp) was 32.1 %. Fifty-four of 80 primers synthesized successfully PCR amplified the cashew genomic DNA and 36 of them were polymorphic. Cross-species amplification rates of SSRs were 100 % in two Anacardium species whereas 61 % in Semecarpus anacardium and 87 % in S. prainii. Nineteen SSR markers were used for genetic diversity analysis in 64 accessions detected a total of 206 alleles and grouped the accessions into three major clusters. PCoA could differentiate the cashew accessions based on geographic regions to a certain extent. The population structure analysis using the Bayesian model-based clustering revealed the existence of three genetic subpopulations (K=3). The annotated transcriptome data and informative genic SSR markers developed in this study provide valuable tools for deciphering agronomically important genes and QTLs, the genetic variations in populations, and molecular breeding in cashew.