cDNA-AFLP-based transcript profiling for the identification of genes functionally related to growth in the pond-reared giant tiger shrimp Penaeus monodon

Abstract

Genes exhibiting differential expression patterns between large-sized (average body weight 17.57 ± 1.8 g and 40.97 ± 4.0 g for 3- and 5-month-old juveniles in a total of 342 and 356 samples, respectively) and small-sized (average body weight 10.01 ± 0.76 g and 22.86 ± 2.51 g, respectively) juvenile giant tiger shrimps (Penaeus monodon) were isolated by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP). In total, 368 primer combinations were screened against the first-strand cDNA of the different groups of 3- and 5-month-old P. monodon.

Over 146 differentially expressed or size-specific markers were derived using 59 selective primer combinations. A total of 70 candidate cDNA-AFLP markers—19 size-specific and 51 showing differential expression—were further cloned, sequenced, and blasted against the GenBank database. Twenty-five markers were found to comprise newly identified sequences (E-values >10⁻⁴).

Quantitative real-time PCR and Single-strand conformational polymorphism (SSCP) analyses of the products generated with the SCAR/cDNA-AFLP primers were further undertaken. Interestingly, the relative expression levels of the products of primers E₊₃4M₊₃14-550-PDI, E₊₃5M₊₃10-350-Unk, E₊₂9/M₊₂11-350-CHK, E₊₃4/M₊₃1-380-phosphotysosyl, and E₊₃8/M₊₃15-800-fucosidase were significantly upregulated in the hepatopancreas of large-sized juvenile shrimp compared with that is small-sized animals. In contrast, the relative expression levels of the products generated by E₊₃1M₊₃8-480-InositolOxy and E₊₂9/M₊₂11-330-vitelline membrane were significantly upregulated in small-sized P. monodon.

The consistency of our results was further assessed in 1-month-old aquaculture-raised P. monodon. Single nucleotide polymorphisms were identified using SSCP at different positions in the sequences of fragments of the different groups generated with the primers E₊₃4M₊₃14-550-PDI and E₊₃5M₊₃10-350-Unk. Our results indicated that cDNA-AFLP has the potential for use in the isolation of functionally important transcripts in P. monodon.