Mining Of Microsatellite Markers From Transcriptome Sequences In Four Species Of Siniperca

The transcriptome sequences of F1hybrids between Siniperca chuatsi (♀) and Siniperca scherzeri (♂) which were obtained previously in our lab were used for mining SSR markers. A total of388primer pairs were designed. Those primer pairs were then characterized in four species of sinipercine fishes:S. chuatsi, S. scherzeri, Siniperca undulate and Siniperca obscura separately in6batches. The results are as follows:1. Genetic variability of primers1-54was assessed using39S. chuatsi. Twenty-nine loci were polymorphic with moderate to high number of alleles per locus (2-13alleles) and showed observed and expected heterozygosity ranging from0.0500to1.0000and0.1522to0.8846, respectively. Of these SSRs,6had significant homology to known genes by BLASTx (basic local alignment search toolx) search. We also evaluated the cross-amplification of these polymorphic loci in S. scherzeri. Twenty-seven were successfully amplified and23were polymorphic.2. Primers55-108and primers171-180were characterized both in S. scherzeri and S. chuatsi. Thirty-eight polymorphic loci were detected in37individuals from a wild S. scherzeri population with2-10alleles per locus. The values for observed and expected heterozygosities ranged from0.0000to0.8378and from0.4235to0.8771, respectively. Meanwhile,36microsatellite loci were polymorphic in30individuals from a wild S. chuatsi population with2-8alleles per locus. The values for observed and expected heterozygosities ranged from0.0000to0.8667and from0.4624to0.8582, respectively.3. Primers109-170were characterized both in S. scherzeri and S. chuatsi. A total of46novel polymorphic microsatellite loci were developed. Forty-three of these loci were polymorphic in S. chuatsi, and20were polymorphic in S. scherzeri. In S. chuatsi, the number of alleles per locus ranged from2to8, and the observed and expected heterozygosities varied from0.13to1.00and from0.33to0.85, respectively. In S. scherzeri, the number of alleles per locus ranged from3to9, and the observed and expected heterozygosities varied from0.19to1.00and from0.28to0.88, respectively. We also evaluated the cross-amplification of46polymorphic loci in four species of sinipercine fishes:Siniperca kneri, S. undulate, S. obscura, and Coreoperca whiteheadi. The interspecies cross-amplification rate was very high, totaling94%of the184locus/taxon combinations tested.4. Primers181-265were characterized in32S. scherzeri. Twenty-five loci were polymorphic with2-9alleles per locus. The observed and expected heterozygosities ranged from0.0000to0.9688and from0.1726to0.8596, respectively. We also evaluated the cross-amplification of25polymorphic loci in five species of sinipercine fishes. Twenty-two were successfully amplified in S. chuatsi, S. kneri, S. undulate and S. obscura and17in C. whiteheadi.5. The first set of microsatellite markers for S. undulate was developed. The level of genetic variability of primers266-388was assessed for30S. undulate individuals. Twenty-nine loci were polymorphic, with the number of alleles per locus ranging from3to13. The observed and expected heterozygosities ranged from0.0667to1.0000and from0.2130to0.9006, respectively.6. The first set of microsatellite markers for S. obscura was developed. Among50pairs of primers,31loci were polymorphic in28S. obscura individuals with the number of alleles per locus ranging from2to10. Observed and expected heterozygosity ranged from0.0000to0.9643and from0.1149to0.7506, respectivelyThese novel markers will facilitate further studies on genetic diversity and population structure, the construction of high-density linkage map, conservation and molecular marker-assisted breeding in many species of sinipercine fishes. Our results confirm that analyzing the transcriptome is an efficient method to identify SSRs in transcribed regions.