Identification of sex-specific markers from black tiger shrimp Penaeus monodon using subtraction analysis

Abstract

Generally, female P. monodon attains a relatively larger size than males reflecting the difference on growth rate between male and female P. monodon. However, studies of sex determining mechanisms in crustaceans are not well advanced. Sex chromosomes in P. monodon are not yet cytological identified. Therefore, sex specific markers of P. monodon will be identified by molecular biological techniques. Once sex determining system in the tiger prawn is understood, this basic knowledge will allow the possibility to develop a monosex culture of P. monodon to increase the efficiency of shrimp culture successfully. Genomic DNA subtraction, a powerful technique for isolating the differences of nucleic acid composition from two cell samples was carried out. Nine and four clones were obtained from PERT and RDA male subtractions, respectively while 4 and 4 clones were obtained from PERT and RDA female subtractions, respectively. Of these, 1 clone of PERT subtracted male (PMMSH6) and 2 clones of RDA subtracted female (PMFJ200 and PMFJ800) were sex-specifically verified by PCR. The results showed no difference in PCR product pattern between genders. Further analysis by SSCP revealed the polymorphic but no sex specific properties of these clones. For genome walking analysis, sex-specific characters of some candidates were initially observed. Ultimately, they were proven to be the artifacts caused by individual variation. For Southern blot analysis, 4 PERT-subtracted clones, 2 males (PMMSH3 and PMMSH8) and 2 females (PMFSH26 and PMFSH32), 4 RDA-subtracted clones 2 males (PMMJ200 and PMMN200) and 1 females (PMFJ300) were undetectable from both sexes, assuming that these fragments were parts of single copy genes. Five RDA-subtracted clones, 2 males (PMMJ450 and PMMN450) and 3 females (PMFJ200, PMFJ800 and PMFN300) were hybridized to genomic DNAs from both sexes, indicating no sex specificities of these candidates for sex identification in P. monodon. Male and female genomic DNA libraries of P. monodon were constructed. Titer efficiencies of male and female libraries were 7.3 x 10[superscript 3] and 1.3 x 10[superscript 4] pfu/ml, respectively. The average size of the clones was detected as 20 kb. Furthermore, amplification of estrogen receptor gene (ER) using 4 combinations of degenerated primers was performed, resulting 5 clones from the PCR of ovary cDNA reaction. One clone was used as probe for Southern blot analysis on DraI-digested genomic DNA and plague screening from male and female genomic libraries, the results revealed positive bands and plaques from both male and female samples