| Oriental river prawn, The systematic name Macrobrachium nipponense,is a commercially important freshwater prawn for aquaculture in China. Single nucleotide polymorphism (SNP) marker is considered to be the most promising as the third generation of molecular marker following RFLP and SSR. SNP were studied and applied in aquatic animals such as fish, shrimp, crab,shellfish, etc. However, no work was reported on identification and application of SNP in Macrobrachium nipponense so far.On the basis of screening of optimal PCR amplification primers, DNA sequences of nuclear rDNA internal transcribed spacer (ITS1) were analyzed through direct sequencing of PCR products in order to identify SNP markers of Macrobrachium nipponense in this paper. Totally32wild samples from Tai Lake were analyzed and the results were as follows: the average length of ITS1sequence was1749.8bp, which was the longest among the reported ITS1sequence; the average contents of A, Q T and C was29.9%,28.3%,27.7%and14.0%respectively, and that of G+C was42.3%. By comparison of sequences from different individuals,81SNP loci with an appearance frequency0.0463were found, including36C/T transitions (44.44%),25A/G transitions (30.86%),5A/T transversions (6.17%),8T/G transversions (9.88%),5A/C transversions (6.17%),1C/G transversion (1.23%),1A/T or C transversion (1.23%). Among81SNP Loci of ITS1,80loci were two alleles and1locus was three alleles which was called multiple allelic loci;there were22SNP loci whose frequencies of the most infrequent allele were equal to or higher than0.0625(≧2/32). In addition, we found3SSR loci with polymorphism,1highly variable segment (243-384bp), and a great deal of deletions and insertions in the ITS1sequences of M. nipponense.This paper not only analyzed ITS1sequence of M. nipponense, but also got access to the characteristics of ITS1sequence and acquired acknowledge about the genetic background of M. nipponense. On the basis of the above, a lot of SNP loci were found, which filled in the blank of SNP research of M. nipponense and provided new available molecular markers for genetic breeding of M. nipponense.The SNP markers identified in the above chapter were applied into genetic analysis between three populations of M. nipponense, including Tai Lake population,"Selected population" and their F1hybrid. Totally27individuals were analyzed,9individuals for each population. Results indicated that the average length of hybrids ITS1neared the median of that of their parents;No remarkable difference in A, G, T and C content and G+C content between three populations. By comparison of sequences from27individuals,86SNP loci were found with appearance frequency0.0489, including83loci with2alleles and3loci with3alleles (multiple allelic loci).67of86SNP loci appeared only in one of three populations (Tai Lake population,"Selected population" and F1hybrid), that means only one population appeared polymorphism of bases in the corresponding loci. The remnant19SNP loci appeared in two or three populations, which means more than one population appeared polymorphism of bases in the corresponding loci. The19SNP loci, including7C/T transitions (36.84%),4A/G transitions (21.05%),1A/T transversion (5.26%),3T/G transversions (15.79%),1A/C transversion (5.26%),1C/T or G transversion (5.26%),1A/G or C transversion (5.26%) and1A/G or T transversion (5.26%), can be used in genetic analysis between three populations. In19SNP loci valuable in genetic analysis,13loci were also identified in the work of Chapter2and6loci were newly found. In ITS1sequence, we also found2SSR loci with polymorphism,1highly variable segment and a great deal of deletions and insertions. SNP loci683bp(C/T) and1700bp(A/G) appeared only in female parents (Tai Lake M. nipponense) and hybrids, which indicated the hybrids inherited these two loci from female parents.190bp(T/G),1673bp(C/T) and1765bp(T/G) appeared only in male parents ("selected population") and hybrids, which indicated the hybrids inherited these three loci from male parents. SNP loci A/T1046and A/G/T1768appeared in both female and male parents but were not found in the hybrids, probably because of the effect of genetic drift in small populations.36SNP loci appeared only in the hybrids and no base polymorphism in the corresponding loci of parents, presumably originated from the genetic polymorphism of female and male parents. It was the first time to apply SNP molecular markers into genetic analysis of M. nipponense crossbreeding, and our work laid foundation for identification of different strains of M. mpponense. |
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