Complex Mutation Of Microsatellite Locus Spl-106 In Acipenseriformes

Complex mutations of the tetranucleotide microsatellite locus Spl-106 were studied in 16 species of Acipenseriformes. Based on the sequencing results, evolutional process and mutation pattern of the locus were summarized preliminarily, and several factors that might affect the reliability of electrophoretogram data from microsatellite markers were discussed. Moreover, phylogenetic relationships of 12 species were reconstructed based on the variations in the flanking sequences of this locus. The main results were summarized as follows. 1. The locus Spl-106 was originally developed from the genome of shovelnose sturgeon (Scaphirhynchus platorhynchus). Cross-species amplifications with a redesigned primer As-100 of this locus were performed in 103 individuals of 15 species (11 Acipenser, two huso and two paddlefishes). Thirteen of the 15 species were amplified successfully, while the amplification was unsuccessful for the rest two species, Psephurus gladius and A. naccarii. The sequencing results of totally 220 PCR products showed that only 11 species presented polymorphic alleles of locus Spl-106, with polymorphic information content (PIC) ranging from 0.320 to 0.969, and with observed heterozygosity ranging from 0.25 to 1.00. The allele sequences of A. sturio and Polyodon spethula were not homologous to that of locus Spl-106. 2. The allele size distribution of locus Spl-106 was discontinuous in most species. Both the longest and smallest alleles in some species may be neglected as non-specific products because they were far away from the main size region. Size homoplasy was extensively observed in seven species, in which PCR products of the same size may consist of 2-7 alleles with different descent. Moreover, more than one locus were amplified by the primer As-100. Except for locus Spl-106, a total of 19 unhomogeneous loci were detected also from the sequenced PCR products of 13 species. The allele sizes of these unhomogeneous loci were distributed among or near the allele size range of locus Spl-106, so that they were probably calculated by error when only electrophoretograms of this microsatellite marker were used for data analysis. 3. Due to the multilocus amplification, six loci were detected by As-100 from A. sturio. Among them, the flanking sequences of two main loci were only 0.3 and 0.26 in similarities to that of locus Spl-106, and repeat motifs of these two loci were (TAAA)n and interrupted poly(A), respectively. Two "sister alleles"with the same size (207bp), one of which belongs to locus Spl-106, were found in an individual of A. fulvescens. There were totally 60 base pair substitutions uniformly distributing on the whole motif segment between the two "sister alleles", and their similarity was 0.7. It seems that the base pair substitution might be an origin of a new microsatellite, or it suggested that A. fulvescens is a heterogenous multi-ploidy species. 4. Both intra-and interspecific variations were detected in the repeat structures of locus Spl-106. Perfect repeats were often interrupted by base substitutions, and insertion or deletion mutations in intraspecific level, and four types of different repeat structures were detected in interspecific level: perfect (TAGA)n repeat in S. platorhynchus, perfect or imperfect (TAGA)n(TAAA)m compound repeats in the 11 sturgeon species, perfect (TAAA)n repeat in one allele of A. sinensis, and perfect (most) or imperfect (TAAA)n(GAAA)m repeats in A. sinensis and A. dabryanus. 5. The number of chromosomes of sturgeons increased along with their evolutional process. It had been assumed that S. platorhynchus, with about 120 chromosomes, arose earlier than those species with about 240 chromosomes. Thus, the allele sequence of locus Spl-106 in S. platorhynchus might be in an ancestral state. Taking account of the phylogenetic relationships constructed previously, it is likely that the locus Spl-106 in Acipenseriformes originates from perfect (TAGA)n, and mutates to perfect or imperfect (TAGA)n(TAAA)m, then to perfect (TAAA)n, and finally to perfect (most) or imperfect(TAAA)n(GAAA)m. 6. The reconstructed phylogenetic relationship based on the flanking sequences of locus Spl-106 revealed closer relationship between A. sinensis and A. dabryanus, and between A. schrenckii and A. transmontanus, respectively, whereas the two huso species were not clustered together. These results are in agreement with other molecular phylogenetic studies in Acipenseriformes and indicate that the flanking sequences of microsatellite could be used as additional molecular evidences for constructing phylogenetic tree of Acipenseriformes.