Development of Simple Sequence Repeats (SSR) by Transcriptome in Chinese Mitten Crab (Eriocheir sinensis H. Milne Edwards)
Development of Simple Sequence Repeats (SSR) by Transcriptome in Chinese Mitten Crab (Eriocheir sinensis H. Milne Edwards)
Fukuan Du1,2, Yan Li2, Zhixin Wen1 , Ruguo Chen1 and Pao Xu2*
1Jiangsu Red Grease Crab Co. Ltd, Xinghua, Jiangsu, 225766, China
2Wuxi Fisheries College, Nanjing Agricultural University, Wuxi, Jiangsu, 214081, China
The Chinese mitten crab (Eriocheir sinensis) is one of the most studied and deconomically important crustaceans in China. However, the production scale does not meet the market demand. This resulted in excessive fishing, which almost caused extinction of the species in the the Yangtze River. Therefore, it is very urgent to carry out genetic breeding to improve yield. In this study, we developed SSR markers by transcriptome, which could be used to genetic research. In this study, we performed transcriptome sequencing of six libraries from hepatopancreas samples in E. sinensis. After removing low-quality reads, short reads and reads belonging to mitochondria, a total of 278,260,960 clean reads corresponding to mRNAs were obtained, these reads covered a total of 34,780,598,273 bases. A total of 48,657 SSR loci were recognized, of which 10,310 unigene sequences contain more than one SSR. These genetic markers are ideal molecular markers for the following investigating genetic diversity, constructing genetic maps, and marker assisted selection.
Received 27 August 2016
Revised 08 October 2016
Accepted 13 October 2016
Available online 08 February 2017
PX and RC designed the study. YL and ZW performed the experimental work. FD performed bioinformatics analysis and wrote the article.
Eriocheir sinensis, Transcriptome, SSR
* Corresponding author: firstname.lastname@example.org
0030-9923/2017/0002-497 $ 8.00/0
Copyright 2016 Zoological Society of Pakistan
The Chinese mitten crab Eriocheir sinensis, native to East Asia, is the most important aquatic economic animals with a high commercial value as a food source throughout the northern and central coastal regions of China (). E. sinensis has received increasing attention over the years and has been cultured by fishermen because of its wonderful flavor and high commercial value. The aquaculture production in China reached 600,000 tons per year (). However, the production scale does not meet the market demand. This resulted in excessive fishing, which almost caused extinction of the species in the Yangtze River. Therefore, it is very urgent to perform genetic research on E. sinensis to protect the wild population and carry out genetic breeding to improve yield.
Currently, transcriptome has been applied in the E. sinensis. Sex-biased genes are considered to account for most of phenotypic differences between males and females. In order to explore the sex-biased gene expression in crab, several studies have been performed the whole-body (), testis (; ) and accessory sex glands () transcriptome analysis in male and female juveniles of E. sinensis using next-generation sequencing technology. detailed the molecular basis of osmoregulation and the stress adaption mechanisms of larvae at key developmental stages with a comparative transcriptomic analysis of E. sinensis megalopae before and after desalination. In the study of , comparative transcriptomic analysis between the fifth zoeae and megalopae of E. sinensis was performed. Patterns of differential gene expression at the two developmental stages were screened in order to reveal the molecular basis of the principal change in the larvae at the two stages. These studies provided the different tissues RNA sequencing for E. sinensis and will facilitate further studies on molecular mechanisms of sex control, stress adaption and development in crab.
DNA markers are useful for the genetic research and the genetic breeding of aquacultured organisms (). Several types of genetic markers, including microsatellites (), SNP (), complete mitochondrial DNA () and AFLP () have been developed to assist in the improvement and enhancement of the economically important traits of aquatic animals. However, the SSR markers development in E. sinensis is limited.
Microsatellites are nuclear genetic markers that are considered an ideal molecular marker system for investigating genetic diversity (), constructing genetic maps (; ), and marker assisted selection (MAS) (; ). So in this study, we will develop SSR markers by transcriptome, which could be used to genetic research.
Materials and Methods
E. sinensis (average weight, 12.4 ± 2.04 g) were adapted to the conditions in a 7.0 × 5.0 × 1.0 m3 aquarium with a water temperature of 28.5 ± 1.0 °C, pH 7.2, and dissolved oxygen concentration of 9.2 ± 0.5 mg O2/L dechlorinated and aerated water. The crabs were fed twice daily, at 7:00 AM and 5:00 PM. At the onset of the experiments, all crabs appeared healthy.
During the experiments, euthanized crabs were submerged immediately in crushed ice to retard degradation of RNA. Blood was collected into ammonium-heparinized capillary tubes at the arthrodial membrane of the last walking leg. All crabs appeared healthy during dissection and their hepatopancreas were removed and placed in liquid nitrogen. Plasma was separated by centrifugation. Plasma and hepatopancreas samples were stored at −80°C until later analysis.
Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006), and were approved by the animal ethics committee of Chinese Academy of Fishery Sciences.
RNA sequencing and assembly
The samples were used to construct six separate, normalized cDNA libraries. Transcriptome sequencing was carried out on an Illumina HiSeq 2500 platform that generated approximately 278,260,960-bp paired-end (PE) raw reads (OE Biotech Co., Shanghai, China, ). Raw data were deposited in the NCBI Sequence Read Archive under the accession number (PRJNA318957). After removing adaptor sequences, ambiguous ‘N’ nucleotides (with the ratio of ‘N’ greater than 5%) and low quality sequences (with quality score less than 10), the remaining clean reads were assembled using trinity software () as described for de novo transcriptome assembly without a reference genome.
Unigene SSR analysis
SSR detection is done with software Microsatellite (MISA, ) using unigenes as reference. Keep the SSRs which the length of the both ends on the Unigene are more than 150 bp. We use these sequences to design primers using software primer 3-2.3.4. Then, filtering primers by follows: 1) No SSRs in the primer. 2) Align the primers to unigenes sequence with 5’ site allowed 3’ mismatch and 3’ site allowed 1 mismatch. 3) Remove the primers which aligned to more than one unigene. 4) Find SSRs on the product sequences by SSR finder. Keep the product which SSR finder’s result is same to the MISA’s result.
|Clean Nucleotides (nt)||Q30 percentage (%)|
Generation of Coilia nasus transcriptome data
In this study, we performed transcriptome sequencing of six libraries from hepatopancreas samples in E. sinensis via an Illumina HiSeq 2500 platform sequencer: 46.4, 46.4, 46.3, 46.3, 46.5 and 46.3 million reads were obtained from the six libraries. After removing low-quality reads, short reads and reads belonging to mitochondria, a total of 278,260,960 clean reads corresponding to mRNAs were obtained, these reads covered a total of 34,780,598,273 bases ().
|Number of Unigene||99,983|
|Total bases of Unigene (bp)||81,466,380|
|Unigene mean lengths (bp)||814.8|
|Number of Unigene ≥500 bp||48,157|
|Max length (bp)||
De novo assembly of Coilia nasus transcriptome data
Using the Trinity assembly program, we generated a total of 99,983 unigenes (). The length distribution of unigenes larger than 200 bp is shown in . The mean unigene size and N50 were 814.8 bp and 1,074 bp, respectively. About half of the unigenes (48,157; 48.2%) were ≥ 500 bp. The largest unigene was 21,266 bp in length (). The GC content frequency distribution was showed in .
Unigene SSR filter and analysis
SSR detection is done with software MicroSAtellite (MISA) using unigenes as reference. In this study, a total of 48,657 SSR loci were recognized, of which 10,310 unigene sequences contain more than one SSR (). The most common type was di-nucleotide, followed by mono-nucleotide, tri-nucleotide, hexa-nucleotide, penta-nucleotide and tetra-nucleotide (). The number of repeat motifs per locus ranged from 5 to 12, in which SSRs with 10 repeats were the most abundant, followed by loci with six, five, and nine repeats. The number of SSR repeats of > 11 was rare (). The most abundant di-nucleotide repeat motif was AC/GT, followed by AG/CT, AT/AT; CG/GG was the least abundant. Among the tri-nucleotide repeat units, the dominant motif was AGG/CCT, followed by ACC/GCT and AAT/ATT (, placed at the end).
Several studies have been performed focusing on the E. sinensis transcriptome of eyestalk, testis, and the Y-organ. These studies secreted and produced a variety of neuropeptide hormones which regulates important physiological activities of the crustacean animals, such as growth, metabolism and reproduction (). used RNA-Seq to investigate transcriptomic profiles of the hepatopancreas and identified differentially expressed genes at four molting stages of E. sinensis. This study provided novel insights into the functions of the hepatopancreas in energy metabolism and biological processes pertaining to molting in crustaceans. However, these studies did not develop SSR markers in the transcriptome. In our study, similar numbers of unigene sequences were generated after assembly compared to the previous study, which was partly due to the fact the same tissues were collected for sequencing. In parallel, the length distribution of contigs and unigenes in these studies were also coincidental, which indicated that the Illumina-based sequencing technology was successful and the assembly was relatively reliable. Therefore, the large number of unigenes obtained from this work could substantially increase the genomic information of E. sinensis.
Many studies have demonstrated that transcriptome sequencing was a powerful tool for identifying SSR markers (; ; ; ). Various SSRs derived from transcriptome sequencing have been extensively used in aquatic animals genetic diversity analyses (; ; ). However, until now no SSRs were identified and used to evaluate genetic diversity for E. sinensis. In this study, a lot of unigene sequences that contained microsatellite loci were detected. The percentage of SSRs contained sequences was higher than Macrobrachium rosenbergii () and Portunus trituberculatus (), but was lower than Macrobrachium nipponense (). The discrepant SSRs frequency might be mainly caused by the parameters of tools for searching microsatellite loci. The mean density of SSR distribution was one microsatellite locus per 1.6 kb. This density was higher than the density in M. nipponense (1/7.8 kb) (). The different distribution frequency of microsatellite loci may be partially related to genome composition, data size, and microsatellite screening criteria. Di-nucleotide repeats were the most abundant repeat type in our study. This finding was in accordance with previous studies using M. rosenbergii, M. nipponense and P. trituberculatus (; ; ). This difference may also be caused by different parameters for detecting microsatellite and different genome composition of each species. The dominant di-nucleotide and tri-nucleotide repeat motif of E. sinensis was AC/GT and AGG/CCT. The lowest frequent motif was CG/CG which was also rare in studies using the other crustacean animals ().
This work was supported by a grant from the China Postdoctoral Science Foundation (2016M590427), the National Key Technology R&D Program (2012BAD25B07); The Jiangsu Postdoctoral Sustentation Fund, China (1501145B).
Availability of supporting data
Conflict of interest statement
We declare that we have no conflict of interest.
Chen, L., Xu, H., Li, H., Wu, J., Ding, H. and Liu, Y., 2011. Isolation and characterization of sixteen polymorphic microsatellite loci in the golden apple snail Pomacea canaliculata. Int. J. mol. Sci., 12: 5993-5998.
Cui, Z., Hui, M., Liu, Y., Song, C., Li, X., Li, Y., Liu, L., Shi, G., Wang, S., Li, F., Zhang, X., Liu, C., Xiang, J. and Chu, K.H., 2015. High-density linkage mapping aided by transcriptomics documents ZW sex determination system in the Chinese mitten crab Eriocheir sinensis. Heredity (Edinb), 115: 206-215.
Dudaniec, R.Y., Storfer, A., Spear, S.F. and Richardson, J.S., 2010. New microsatellite markers for examining genetic variation in peripheral and core populations of the coastal giant salamander (Dicamptodon tenebrosus). PLoS One, 5: e14333.
Fang, D.A., Zhou, Y.F., Duan, J.R., Zhang, M.Y., Xu, D.P., Liu, K., Xu, P. and Wei, Q., 2015. Screening potential SSR markers of the anadromous fish Coilia nasus by de novo transcriptome analysis using Illumina sequencing. Genet. mol. Res., 14: 14181-14188.
Fuji, K., Hasegawa, O., Honda, K., Kumasaka, K., Sakamoto, T. and Okamoto, N., 2007. Marker-assisted breeding of a lymphocystis disease-resistant Japanese flounder (Paralichthys olivaceus). Aquaculture, 272: 291-295.
Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., Di Palma, F., Birren, B.W., Nusbaum, C., Lindblad-Toh, K., Friedman, N. and Regev, A., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol., 29: 644-652.
He, L., Jiang, H., Cao, D., Liu, L., Hu, S. and Wang, Q., 2013. Comparative transcriptome analysis of the accessory sex gland and testis from the Chinese mitten crab (Eriocheir sinensis). PLoS One, 8: e53915.
Huang, S., Wang, J., Yue, W., Chen, J., Gaughan, S., Lu, W., Lu, G. and Wang, C., 2015. Transcriptomic variation of hepatopancreas reveals the energy metabolism and biological processes associated with molting in Chinese mitten crab, Eriocheir sinensis. Sci. Rep., 5: 14015.
Hui, M., Liu, Y., Song, C., Li, Y., Shi, G. and Cui, Z., 2014. Transcriptome changes in Eriocheir sinensis megalopae after desalination provide insights into osmoregulation and stress adaption in larvae. PLoS One, 9: e114187.
Jiang, H., Yin, Y., Zhang, X., Hu, S. and Wang, Q., 2009. Chasing relationships between nutrition and reproduction: A comparative transcriptome analysis of hepatopancreas and testis from Eriocheir sinensis. Comp. Biochem. Physiol., Part D: Genom. Proteom., 4: 227-234.
Jin, S., Fu, H., Zhou, Q., Sun, S., Jiang, S., Xiong, Y., Gong, Y., Qiao, H. and Zhang, W., 2013. Transcriptome analysis of androgenic gland for discovery of novel genes from the oriental river prawn, Macrobrachium nipponense, using Illumina Hiseq 2000. PLoS One, 8: e76840.
Jung, H., Lyons, R.E., Dinh, H., Hurwood, D.A., Mcwilliam, S. and Mather, P.B., 2011. Transcriptomics of a giant freshwater prawn (Macrobrachium rosenbergii): de novo assembly, annotation and marker discovery. PLoS One, 6: e27938.
Li, Y., Hui, M., Cui, Z., Liu, Y., Song, C. and Shi, G., 2015b. Comparative transcriptomic analysis provides insights into the molecular basis of the metamorphosis and nutrition metabolism change from zoeae to megalopae in Eriocheir sinensis. Comp. Biochem. Physiol., Part D: Genom. Proteom., 13: 1-9.
Liu, B., Teng, S.S., Shao, Y.Q., Chai, X.L. and Xiao, G.Q., 2011. Isolation and characterization of 39 novel polymorphic EST-SSR loci for the blood clam, Tegillarca granosa. Conserv. Genet. Resour., 4: 375-378.
Liu, Y., Hui, M., Cui, Z., Luo, D., Song, C., Li, Y. and Liu, L., 2015. comparative transcriptome analysis reveals sex-biased gene expression in juvenile Chinese mitten crab Eriocheir sinensis. PLoS One, 10: e0133068.
Long, Y., Wang, Y., Wu, S., Wang, J., Tian, X. and Pei, X., 2015. De novo assembly of transcriptome sequencing in Caragana korshinskii Kom. and characterization of EST-SSR markers. PLoS One, 10: e0115805.
Luo, H., Xiao, S., Ye, H., Zhang, Z., Lv, C., Zheng, S., Wang, Z. and Wang, X., 2016. Identification of immune-related genes and development of SSR/SNP markers from the spleen transcriptome of Schizothorax prenanti. PLoS One, 11: e0152572.
Lv, J., Liu, P., Gao, B., Wang, Y., Wang, Z., Chen, P. and Li, J., 2014. Transcriptome analysis of the Portunus trituberculatus: de novo assembly, growth-related gene identification and marker discovery. PLoS One, 9: e94055.
Ma, H., Chen, S., Yang, J., Chen, S. and Liu, H., 2011a. Genetic linkage maps of barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) based on AFLP and microsatellite markers. Mol. Biol. Rep., 38: 4749-4764.
Ma, H., Jiang, W., Liu, P., Feng, N., Ma, Q., Ma, C., Li, S., Liu, Y., Qiao, Z. and Ma, L., 2014. Identification of transcriptome-derived microsatellite markers and their association with the growth performance of the mud crab (Scylla paramamosain). PLoS One, 9: e89134.
Mu, X., Hou, G., Song, H., Xu, P., Luo, D., Gu, D., Xu, M., Luo, J., Zhang, J. and Hu, Y., 2015. Transcriptome analysis between invasive Pomacea canaliculata and indigenous Cipangopaludina cahayensis reveals genomic divergence and diagnostic microsatellite/SSR markers. BMC Genet., 16: 12.
Qi, D., Guo, S., Tang, J., Zhao, X. and Liu, J., 2007. Mitochondrial DNA phylogeny of two morphologically enigmatic fishes in the subfamily Schizothoracinae (Teleostei: Cyprinidae) in the Qinghai-Tibetan Plateau. J. Fish Biol., 70: 60-74.
Song, W., Li, Y., Zhao, Y., Liu, Y., Niu, Y., Pang, R., Miao, G., Liao, X., Shao, C., Gao, F. and Chen, S., 2012. Construction of a high-density microsatellite genetic linkage map and mapping of sexual and growth-related traits in half-smooth tongue sole (Cynoglossus semilaevis). PLoS One, 7: e52097.
Wang, C., Li, C. and Li, S., 2008. Mitochondrial DNA-inferred population structure and demographic history of the mitten crab (Eriocheir sensu stricto) found along the coast of mainland China. Mol. Ecol., 17: 3515-3527.
Wei, Z., Chen, R.Y., Laghari, M.Y., Xu, D.D., Mao, G.M., Shi, H.L. and Bao, L., 2016. Characterization of Nibea albiflora transcriptome: sequencing, de novo assembly, annotation and comparative genomics. Pakistan J. Zool., 48: 427-434.
Zhou, T., Li, Z.H., Bai, G.Q., Feng, L., Chen, C., Wei, Y., Chang, Y.X. and Zhao, G.F., 2016. Transcriptome sequencing and development of genic SSR markers of an endangered Chinese endemic genus Dipteronia Oliver (Aceraceae). Molecules, 21: 166.