Isolation and characterization of a female-specific DNA marker in the giant freshwater prawn Macrobrachium rosenbergii

Abstract

In this study, a female-specific DNA marker in the freshwater prawn Macrobrachium rosenbergii was identified through amplified fragment length polymorphism (AFLP). The AFLPderived sequence-characterized amplified region (SCAR) marker was tested in over 200 individuals, giving reproducible sex identification. Further molecular characterization of the sex-marker's genomic region (B3 kb long) revealed the presence of tandem and inverted repeats. The B3-kb sequence was identified both in male and female prawns, but with subtle differences: a deletion of 3 bp (present in female prawn but absent in male prawn) identified upstream of the SCAR marker sequence and two female-specific single-nucleotide polymorphisms, both indicating that male prawns are homozygous, whereas female prawns are heterozygous in this locus. Fluorescent in situ hybridization showed the B3-kb sequence to be unique: to the best of our knowledge, this is the first report of a unique sex-specific sequence observed in situ in crustaceans. The sex-specific marker identified in M. rosenbergii may have considerable applied merit for crustacean culture in that it will enable the determination of genetic sex at early developmental stages when phenotypic differences are not identifiable.

Identification of Sex-Specific Markers Reveals Male Heterogametic Sex Determination in Pseudobagrus ussuriensis

“…Perhaps, it is the high sequence difference in this region between male and female that makes the AFLP screening to catch the low differentiating sex-specific sequences. AFLP-based screening and PCR-based marker detection has been extensively demonstrated to be an ideal approach for the analysis of whole genome polymorphisms and to allow the detection of small differences between male and female genomes (Ventura et al 2011), because the screening was time-consuming, but the detection was very expedient and inexpensive . In this study, we developed three pairs of sex-specific primers and used them to efficiently identify genetic sex in P. ussuriensis.…”

Male Sexual Development and the Androgenic Gland: Novel Insights through the de novo Assembled Transcriptome of the Eastern Spiny Lobster,<b><i> Sagmariasus verreauxi</i></b>

“…On secretion, it is this hormone that acts on the male gonads and is ultimately responsible for testicular differentiation [Rodríguez et al, 2007]. The recent identification of genetic sex markers [Ventura et al, 2011b] in conjunction with temporal silencing of IAG at the timing when the hormone starts to be expressed [Ventura et al, 2011c] has enabled biotechnology to develop all-male populations in the giant freshwater prawn Macrobrachium rosenbergii Sagi et al, 2013]. Such biotechnology highlights the key role of novel molecular techniques in enhancing the commercial output of aquaculture practices.…”

On the safety of RNAi usage in aquaculture: The case of all-male prawn stocks generated through manipulation of the insulin-like androgenic gland hormone

“…Comparing representative mixed-sex versus neo-female populations 2.6.1. Source of animals M. rosenbergii broodstock populations comprising normal females and neo-females were maintained at Ben-Gurion University of the Negev as previously described (Ventura et al, 2011) and fed daily with Artemia nauplii. Containers were aerated vigorously using air stones on the bottom of the tank.…”

“…The second phase involves crossing the neo-females with normal males to produce all-male progeny. A prerequisite for successful application of these RNAi-based biotechnologies has been the use of a sex-specific marker in M. rosenbergii to enable the identification of genetic sex at early developmental stages when phenotypic differences are not identifiable (Ventura et al, 2011). All the above components have been integrated into a commercialized technology representing the first RNAi-based biotechnology applied in the entire field of aquaculture .…”