Isolation and genetic diversity analysis of microsatellite DNA in <I>Macrobrachium nipponense</I>

Abstract

Microsatellite-enhanced genomic library of oriental river prawn Macrobrachium nipponense of Lake Taihu was constructed using repeat-enrichment method with biotin-labeled oligos [(CA)15 and (CT)15]and streptavidin magnetic beads. From a total of randomly selected 120 clones,83 clones with more than ten motif repeats were positive. And the sequence size ranges from 191 to 580 bp with an average of 311 bp. The (CA/ GT)n and (AG/TC)n were the abundant microsatellite motifs with a percentage of 59. 09% and 20. 45% in the isolated microsatellites. Other 17 types of repeat sequences were detected,including repeats consisting of (GC)n,(AAG)n,(ACAA)n and (GGCAGA)n. Characterization of these microsatellites showed that 25. 75% of sequences were classified as perfect type,20. 45% as imperfect type and 53. 80% as compound type. Genetic diversity of 4 wild stocks of M. nipponense in 4 sites of Lake Taihu,i. e. Wujiang (WJ),Binhu (BH),Yixing (YX) and Wuxing (WX),were investigated using 12 microsatellite DNA loci from the isolated primers . The allele numbers,effective allele numbers,observed heterozygosity and expected heterozygosity under Hardy-Weinberg equilibrium were estimated to characterize genetic diversity. The result showed that all the 12 loci in the 4 stocks were highly polymorphic. Tests of departures from Hardy-Weinberg equilibrium indicated the loci except Mni86、Mni103 and Mni114 in each stock with significant heterozygosity deficiency. FST and AMOVA analysis across all stocks and loci indicated that there was low level of divergence among 4 stocks. The UPGMA clustering tree based on DA genetic distance demonstrated that Wujiang and Wuxing stocks have near relationship,and they have a distant relationship with Wuxi and Yixing stocks.