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

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Abstract

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.

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Acknowledgments

This work is supported by grants from the National Natural Science Foundation of China (31270267) and the Shanghai Municipal Administration of Forestation and City Appearances (G142430, G162404, G162405). The authors are thankful to Mr. Allen Coombes of Benemerita Universidad Autónoma de Puebla for helping revise the early version of the manuscript and are also thankful to Mr. Federico Sebastiani of Italian National Research Council for kindly providing the unigene data of Quercus pubescens.

Authors’ contributions

MA and MD conceived/designed the experiments, analyzed the data, and wrote the paper. MA and SSZ performed the experiments. MD and YGS were responsible for the field collections and growing the seedlings.

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Authors and Affiliations

  1. Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, 201602, China

    Miao An, Min Deng, Si-Si Zheng & Yi-Gang Song

  2. Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai, 201602, China

    Miao An, Min Deng, Si-Si Zheng & Yi-Gang Song

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  1. Miao An
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  2. Min Deng
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Correspondence to Min Deng.

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The authors declare that they have no competing interests.

Additional information

Communicated by A. Kremer

Data achieving statement

The original RNA-seq data was deposited to the Sequence Read Archive (SRA) of the National Centre of Biotechnology Information (NCBI) under the accession numbers SRP057982 and SRP058226. The Transcriptome Shotgun Assembly projects have been deposited at GenBank under the accession numbers GEUS00000000 and GEUR00000000.

Electronic supplementary material

Fig. S1

Sequence length distribution of the assembled unigenes of Q. austrocochinchinensis and Q. kerrii. The x-axis represents sequence length, and the y-axis represents the number of unigenes. (PDF 172 kb)

Fig. S2

Gene Ontology (GO) annotations of unigenes. Results were summarized into three main categories: biological processes, cellular components and molecular function. (PDF 753 kb)

Table S1

List of Reciprocal Best Hits of Q. austrocochinchinensis and Q. kerrii. (XLSX 681 kb)

Table S2

E-value and similarity distribution of unigenes annotated in the NR database (DOCX 13 kb)

Table S3

Proportion of matched unigenes in the NR database (DOCX 15 kb)

Table S4

List of SSR primer pairs derived from Q. austrocochinchinensis and Q. kerrii transcriptome. (XLSX 58 kb)

Table S5

The statistical parameters and the molecular weights of 102 validated primer pairs using eight individuals of each species. (XLSX 1162 kb)

Table S6

GO annotations of unigenes with Ka/Ks > 0.5, and the results of following enrichment analysis using Fisher’s exact test. (XLSX 22 kb)

Table S7

In silico PCR analysis using the primers of Q. austrocochinchinensis and Q. kerrii against Q. robur and Castanea mollissima. (XLSX 137 kb)

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An, M., Deng, M., Zheng, SS. et al. De novo transcriptome assembly and development of SSR markers of oaks Quercus austrocochinchinensis and Q. kerrii (Fagaceae). Tree Genetics & Genomes 12, 103 (2016). https://doi.org/10.1007/s11295-016-1060-5

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  • DOI: https://doi.org/10.1007/s11295-016-1060-5

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