Introduction
The camphor tree (Cinnamomum camphora) is an evergreen broad-leaved tree and distributed in the south of China and Japan (Chen et al., Reference Chen, Zheng, Liu, Zhong, Wu, Xu and Xu2017). This species has been widely cultivated in many countries because of its aromatic oils, insect-repellent effects and ornamental value (Chen et al., Reference Chen, Zheng, Liu, Zhong, Wu, Xu and Xu2017; Kameyama et al., Reference Kameyama, Furumichi, Li and Tseng2017). Despite its vast utilization and cultivation, little is known about its genetic diversity and population genetics. To date, only a few genomic simple sequence repeat (SSR) markers were developed in C. camphora (Kameyama Reference Kameyama2012) and no genic-SSR primers were available yet. Thus, it is necessary to develop more genic-SSR markers, which will facilitate the genetic studies of Cinanamomum.
The SSR markers have become one of the most powerful high-resolution tools for the study of genetic diversity because of abundant polymorphism, good reproducibility, and co-dominant inheritance (Xu et al., Reference Xu, Liu, Wang, Teng, Shi, Li and Huang2017). With the further popularization of second-generation sequencing technology, it has become a high-efficiency method to develop novel SSR markers on the basis of transcriptome data (Xu et al., Reference Xu, Liu, Wang, Teng, Shi, Li and Huang2017). So far, no studies have reported the development of genic-SSR markers in C. camphora. In this study, a set of novel genic-SSR markers were developed on the basis of transcriptome sequencing.
Experimental
Total RNA was extracted from young camphor leaves, with two biological replicates per sample. The paired-end cDNA libraries were generated by using NEBNext Ultra RNA Library Prep Kit (NEB, USA), and were sequenced by using the Illumina HiSeq 2500 sequencing platform. All raw reads have been deposited to NCBI (SRA accession no. SRP127892). Genic-SSR loci were identified using MISA search module (Table S2). The parameters were set to detect perfect mono-, di-, tri-, tetra-, penta- and hexa-nucleotide motifs with a minimum of ten, six, five, five, five and five repeats, respectively. The maximal length of interruptions between two adjacent SSRs was 100 bp.
Of the identified SSRs, 100 SSR loci were selected for designing primer pairs (Table S4). Previous studies revealed that longer repeat length generally had a higher tendency to be polymorphic, thus we would prefer to select longer SSRs for designing primers. DNA was isolated from dried leaves of both 45 camphor trees (Table S1) and 30 individuals from other six species (Table S1), using a modified CTAB extraction method. PCR conditions were as follows: an initial denaturation at 94 °C for 5 min, and then followed by 30 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, followed by a final extension 3 min at 72 °C. A typical 10 µl reaction: 1 × PCR buffer, 0.75 mM MgCl2, 0.1 mM dNTPs, 0.25 U of Taq DNA polymerase, 0.4 µM of each primer and 10–25 ng genomic DNA. The PCR products were resolved on 8% denatured polyacrylamide gels, which were silver-stained for detecting SSR bands. To estimate allele sizes, the length of bands was compared with a 50 bp DNA ladder. The genetic diversity indexes were estimated using Popgene v1.32 and GenAIEx version 6.5.
Discussion
After cDNA library construction, transcriptome sequencing and quality filtering, a total of 237,624,594 clean reads were generated from the cDNA libraries, accounting for approximately 35.64G, and over 95.5 and 89.1% of them had Phred quality scores at the Q20 and Q30 level, respectively. De novo assembly generated 276,189 transcripts with an average length of 688 bp and an N50 length of 1085 bp, and 156,184 unigenes with an average length of 997 bp and an N50 length of 1430 bp.
Finally, 74 primer pairs were successfully amplified under the above condition (Table S4), of which 21 produced polymorphic products (Table 1 and Fig. S1). The Ho and He of the three camphor populations were 0.3449 and 0.4254 at the intraspecific level, respectively (Table S3). The average genetic diversity within populations, H and I was 0.4123 and 0.6837, respectively (Table S3). The 21 loci were further examined for cross-species transferability and polymorphism in other six related species: C. micranthum, C. porrectum, C. subavenium, C. bodinieri, C. japonicum and C. jensenianum (Table 2).
Table 1. Genetic characterization of 21 polymorphic genic-SSR markers for Cinnamomum camphora.
Note: The same melting temperatures for all primer pairs: 58 °C; S (bp): Expected size (bp); A = number of alleles; Ho = observed heterozygosity, He = expected heterozygosity; Fis = inbreeding coefficient; Fst = genetic differentiation coefficient; Nm = gene flow; n = number of individuals sampled.
*Significant deviations from Hardy–Weinberg equilibrium (P < 0.001).
Table 2. Cross-species amplification of 21 polymorphic camphor SSR markers.
Note: n = number of individuals sampled; + successful amplification; – = failed amplification; the number of alleles in parentheses; C. micranthum (Cmi); C. porrectum (Cpo); C. subavenium (Csu); C. bodinieri (Cbo); C. japonicum (Cja); C. jensenianum (Cje).
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S147926211800014X.
Acknowledgements
This study was supported by the Forestry Science and Technology Innovation Special of Jiangxi (201701), and the Postdoctoral Science Foundation of Jiangxi Academy of Sciences.
