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De novo transcriptome assembly and development of SSR markers of oaks Quercus austrocochinchinensis and Q. kerrii (Fagaceae)

An, Miao, Deng, Min, Zheng, Si-Si, Song, Yi-Gang
Tree genetics & genomes 2016 v.12 no.6 pp. 103
Castanea mollissima, Quercus robur, algorithms, biosynthesis, genetic markers, genomics, hybridization, leaves, loci, microsatellite repeats, polymerase chain reaction, rare species, roots, sequence analysis, stems, sympatry, transcriptome, unigenes
The commonly found oak species Quercus kerrii and the rare species Quercus austrocochinchinensis (subgenus Cyclobalanopsis) are genetically close and found in sympatry in Indo-China with morphological evidences of hybridization. The two species provide an opportunity to investigate the mechanism of speciation and to study how species integrity is maintained in the subgenus Cyclobalanopsis. However, the genomic resources are lacking in Cyclobalanopsis to produce enough molecular markers. We performed RNA-seq on Q. austrocochinchinensis and Q. kerrii by pooling tissues of new and old leaves, roots, and stems. A total of 14,247,444/12,900,500 (Q. austrocochinchinensis/Q. kerrii) clean reads were obtained from 2 × 300 bp Illumina MiSeq sequencing platform. De novo assembly produced 79,312/81,921 contigs representing 49,845/50,767 unigenes. The Ka/Ks estimation and following enrichment analysis identified 24 genes. Most of them were related to biosynthesis and growth, which may be involved in the process of speciation. 5196/5021 primer pairs were successfully designated from 13,762/13,430 putative loci with microsatellite repeats. We selectively screened 215 polymorphic simple sequence repeat (SSR) loci according to the list of 29,893 pairwise orthologous genes predicted by a reciprocal best hits algorithm. From the 215 loci, we selected 102 well-amplified SSR primers for polymorphic SSR locus analysis. We found 18 highly polymorphic loci and suitable for population genetic analysis, with 10 loci that had a diagnostic power that may be useful in studying hybridization. Finally, in silico PCR was performed using two Fagaceae species Quercus robur and Castanea mollissima. Our study provides a set of useful SSR markers and can enable further functional and comparative genomic research on the Quercus subgenus Cyclobalanopsis.