Objective The transcriptome data of Pinus massoniana under drought stress were used to clarify the function distribution of sequences, as well as the characteristics and distribution patterns of SSR loci, and to explore the key SSR loci linked to drought-resistant gene.
Method The P. massoniana needle samples under lingering drought stress for 10, 15, and 25 days and the corresponding samples with sufficient water as the control (CK) were selected to extract the total RNA. Illumina sequencing were performed to generate raw reads. After removal of low-quality data, the transcriptome assembly was conducted using Trinity software. The unigenes of transcriptome were annotated by aligning with several public databases via BLAST program, including GO (Gene Ontology), KOG (eukaryotic orthologous groups), and KEGG (Kyoto Encyclopedia of Genes and Genomes). The SSRs loci were examined using Misa software, and the PCR amplification SSR primers were designed using Primer 3.0 software. GO and KEGG enrichment analysis were implemented using GOSeq (1.10.0) and KOBAS software, respectively, to determine the major process of biological process and metabolic pathways of differentially expressed unigenes contained SSR loci.
Result A total of 101 806 unigenes were annotated from 194 821 unigenes of transcriptome. Among them, 64 973 functional annotations were from GO database, 35 880 from KOG database and 30 882 from KEGG database. Moreover, 6 728 SSR loci were identified and distributed in 6 367 unigenes, and their average frequency of SSRs was 3.45%. Among all the SSR motifs, mononucleotide, trinucleotide and dinucleotide were the major repeated types, with occurrence frequency of 35.82%, 33.03% and 25.22%, respectively; the form of A/T, AT/AT, AG/CT, AGC/CTG, and AAG/CTT were the most frequent motifs, the length from 10 to 20 bp were the most repeat motifs, and the SSR repeat numbers from 5 to 10 were the most repeat numbers of motifs. A total of 13 338 pairs of SSR primers were designed for marker development of P. massoniana. Furthermore, among the 6 367 unigenes containing SSR loci, 422 unigenes were differentially expressed on drought stress versus the control. Enriched analysis of KEGG pathway showed that 11 unigenes containing SSR loci were significantly enriched into three KEGG pathways, including photosynthesis, plant hormone signal transduction and carotenoid biosynthesis, which were linked to the plant response to drought stress.
Conclusion A total of 101 806 unigenes were annotated from a higher quality of transcriptome database in P. massoniana, 6 728 SSR loci were identified and distributed from 6 367 unigenes, 11 SSR loci from 422 differentially expressed genes containing SSR loci were identified linking to the plant response to drought stress. These results can be used for the subsequent study on molecular mechanism for drought resistance and functional gene localization in P. massoniana.
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