Sex determination markers and gene expression in androgenic gland of different male morphotypes of the giant freshwater prawn Macrobrachium rosenbergii

Abstract

Sex-specific DNA markers of the giant freshwater prawn (Macrobrachium rosenbergii) were identified by AFLP analysis. A total of 64 primer combinations were primary screened against bulked genomic DNA of the first sample set including small orange claw (SOC1, N = 10) and blue claw (BC1, N = 5) males and female (F1, N = 10). Ninety candidate male- and forty-two candidate female-specific markers from 40 informative primers were secondary tested with both the first and the second (BC2, N = 10; OC1, N = 15; SOC2, N = 5 and F2, N = 20) sample sets. Only 5 and 4 male- and female-specific AFLP markers were consistently found and regarded as the unknown DNA segments after characterization (E > 10[superscript -4]). SCAR markers were developed from candidate sex-specific markers. Among 13 primer pairs tested, 9 pairs of primers generate the expected amplification product in both male and female M. rosenbergii. Further analysis of the product by SSCP analysis revealed polymorphism of all markers except FE[subscript +3]8M[subscript +3]270.2F/R but polymorphic patterns did not show sex-specificity in M. rosenbergii. RT-PCR of an AGH homologue in M. rosenbergii was not successful. Accordingly, primers designed from AFLP of this study and sex-related transcripts from previous studies in the black tiger shrimp (Penaeus monodon) and the tropical abalone (Haliotis asinina) were applied for identification of sex-specific/differential expression markers in M. rosenbergii. A homologue of Sarco / endoplasmic reticulum Ca[superscript 2] + ATPase C (SERCA) were isolated from ME[subscript +3]8M[subscript +3] 1310.1 primers (E = 1 x 10[superscript -103]). An amplified SERCA (279 bp) revealed differential expression patterns between male and female M. rosenbergii. In addition, a female-specific expression marker was observed from a peritrophin homologue. Likewise, DSI was regarded as differential expression marker for the BC morphotype. Additionally, RAP-PCR analysis was also carried out to isolate various types of expression markers in different morphotype of males and between males and females of M. rosenbergii. In a total, 43 and 24 of male- and female-specific and 29 differential expression RAP-PCR markers were found. Moreover, 10, 12 and 13 RAP-PCR fragments exhibiting morphotype-specific expression in BC, OC and SOC males were also isolated. Interestingly, SOC- (340 bp) and female-specific (315 bp) RAP-PCR markers were successfully identified from combination of the first primers and all investigated second primers (30 primer combination) but conversion of these to SCAR markers was not successful. Additionally, 9 primer pairs were designed and tested. Three developed markers (M122/159RAP.2, SOC268/273RAP and BC428/273RAP) illustrated interesting results at the transcriptional level. The expression levels of M122/159RAP.2 (significantly similar to I-connectin mRNA of the crayfish, P. clarkii) clearly showed differential expression patterns in AG of males and oviducts of female M.rosenbergii but no differences were found between different male morphotypes. Conversely, the amplification product of SOC268/273RAP and BC428/273RAP implied possible length polymorphism through alternative splicing. They also provided polymorphic patterns when genomic DNA of different male and female individuals of M. rosenbergii were genotyped.