The development of novel EST-SSRs and construction of multiplex PCR panels for parentage assignment in Macrobrachium rosenbergii

Abstract

The giant freshwater prawn (GFP), Macrobrachium rosenbergii, widely distributed in tropical and subtropical areas, possesses enormous commercial potential due to its attractive flavor and high nutritional value. Declining germplasm resources limit the development and negatively affect the sustainability of the M. rosenbergii aquaculture. To enrich the availability of molecular markers and provide an efficient parent assignment tool for M. rosenbergii, we developed novel microsatellites (SSRs) using transcriptome data and validated their application in parentage assignment in this study. A total of 20,587 SSRs were mined in 59,380 transcriptome unigenes, and 19 novel tri-, tetra- or pentanucleotide SSRs were developed. Subsequently, the genetic information of the 19 newly developed and 2 previously published loci was analyzed. The number of alleles per locus ranged from 3 to 12 (average = 6.9), and the polymorphism information content ranged from 0.440 to 0.868 (average = 0.628). The observed and expected heterozygosity varied from 0.25 to 1 (average = 0.680) and 0.481 to 0.894 (average = 0.729), respectively. Finally, the top 10 polymorphic SSRs were selected and grouped into three multiplex PCR panels. Their suitability for M. rosenbergii parentage assignment was validated using Cervus 3.0 and Colony 2.0 in Zhanjiang and Guam populations, consisting of 21 families and 9 families, respectively. The results showed that Cervus 3.0 achieved a 97.15% and 100% accurate assignment rate in the two populations, and Colony 2.0 achieved a 99.50% and 100% accurate assignment rate in the two populations, respectively. Overall, the parentage assignment based on the 10 SSRs in this study provides a powerful tool for pedigree reconstruction in M. rosenbergii.