Identification of growth-related SNP markers in the giant tiger shrimp Penaeus monodon

Abstract

Polymorphism of growth-related genes; calponinl (PmCnnl), cyclin C (PmCyC) and cdc25 (PmCdc25) in 3- (BUM03 and SNP3A) and 5-month-old (PM05) juveniles of the giant tiger shrimp (Penaeus monodon) were identified by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. Relationships between SSCP patterns and growth parameters (average body weight, BW; total length, TL; hepatopancreatic weight, HPW and/or hepatosomatic indes, HSI) of the examined shrimp were examined. In the SNP3A sample, shrimp carrying SSCP patterns I and II of PmCnnl₅₃₀ (primers Cnnl-F/R) had a greater average BW and TL than those exhibiting pattern III (N=156, P<0.05). Likewise, juveniles shrimp carrying SSCP pattern II of PmCyC possessed a significantly greater average BW and HPW than those of shrimp carrying patterns I and III (N=145, P<0.05). For PmCdc25, the BW, TL and HPW pf shrimp carrying SSCP pattern I was significantly greater than those of shrimp carrying SSCP pattern II (N=144, P<0.05). Moreover, significant relationships between SSCP patterns of PmCnnl₄₂₅ and average BW and TL were found in the BUM03 sample (P<0.05). Nucleotide sequences of cloned PmCnnl₅₃₀, PmCyC and PmCdc25 gene segments of representative individuals carrying each SSCP genotype were determined. Six intronic SNPs of PmCnnl₅₃₀ were significantly related with growth parameters. Of these, shrimp with each of G/G₂₀₉T/T₂₁₀-/-₂₁₂-/-₂₁₃C/C₂₁₈G/G₂₄₀ (SSCP pattern I) and each of (G/A)₂₀₉(T/A)₂₁₀(-/G)₂₁₂(-/T)₂₁₃(C/T)₂₁₈(G/A)₂₄₀ (SSCP pattern II) had a greater average BW, TL and HPW than those with each of A/A₂₀₉A/A₂₁₀G/G₂₁₂T/T₂₁₃T/T₂₁₈A/T₂₄₀ (SSCP pattern III). For PmCyC, three exonic (A/G₃₁‚G/A₃₇₉‚ and T/C₃₈₂) and two intronic (T/C ₁₃₄ and T/C₁₈₈) SNPs corresponding to SSCP pattern I, II and III were observed, respectively. Each SNP of shrimp with SSCP pattern II: G/G₃₁C/T₁₃₄C/C₁₈₈A/A₃₇₉C/C₃₈₂ had a significantly greater average growth parameters (except HSI) than those with each SNP of shrimp found in SSCP pattern I: A/A₃₁C/C₁₃₄T/T₁₈₈G/G₃₇₉T₃₈₂ and III: A/G₃₁C/T₁₃₄T/C₁₈₈G/A₃₇₉T/C₃₈₂. Only one SNP (A/C₂₄₃) was found in PmCdc25 for which shrimp exhibiting A/C₂₄₃ had a significantly greater average BW, TL and HPW (P<0.05) than those carrying C/C₂₄₃. Simplification of SNP detection of PmCnnl₅₃₀ and PMCdc25 gene segments was successfully developed based on PCR-RFLP. The relative expression level of PmCnnl and PmCdc25 in hepatopancreas of juvenile shrimp (SNP3A) carrying different SSCP pattern were significantly different (P<0.05). The expression level of PmCnnl in shrimp exhibiting SSCP pattern III was significantly greater than those exhibiting pattern I and II (P<0.05) while the expression level of Pmcdc25 in shrimp exhibiting SSCP pattern I was significantly greater than those exhibiting genotypes II (P<0.05). The full-length cDNA of PmCyC was successfully characterized. It was 1443 bp in length containing and ORF of 804 bp corresponding to a polypeptide of 267 amino acids. Moreover, recombinant PmCnnl protein was successfully expressed as the soluble protein in E.coli. The polyclonal antibody against rPmCnnl was successfully produced in rabbit.