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Phylogenetic assessment of length variation at a microsatellite locus.

ABSTRACT: Sixty-six haplotypes at a locus containing a simple dinucleotide (CA)n microsatellite repeat were isolated by PCR-single-strand conformational polymorphism from populations of the horseshoe crab Limulus polyphemus. These haplotypes were sequenced to assess nucleotide variation directly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the microsatellite motif. In the repeat region itself, CA-number varied in integer values from 5 to 11 across alleles, except that a (CA)8 class was not observed. Differences among alleles were due also to polymorphisms at 22 sites in regions immediately flanking the microsatellite repeats. Nucleotide substitutions in these regions were used to estimate phylogenetic relationships among alleles, and the gene phylogeny was used to trace the evolution of length variation and CA repeat numbers. A low correlation between size variation and genealogical relationships among alleles suggests that absolute fragment size (as normally scored in microsatellite assays) is an unreliable indicator of historical affinities among alleles. This finding on the molecular fine structure of microsatellite variation suggests the need for caution in the use of repeat counts at microsatellite loci as secure indicators of allelic relationships.

SUBMITTER: Orti G

PROVIDER: S-EPMC23472 | biostudies-literature | 1997 Sep

REPOSITORIES: biostudies-literature

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Phylogenetic assessment of length variation at a microsatellite locus.

Ortí G G Pearse D E DE Avise J C JC

Proceedings of the National Academy of Sciences of the United States of America 19970901 20

Sixty-six haplotypes at a locus containing a simple dinucleotide (CA)n microsatellite repeat were isolated by PCR-single-strand conformational polymorphism from populations of the horseshoe crab Limulus polyphemus. These haplotypes were sequenced to assess nucleotide variation directly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the microsatellite motif. In the repeat region itself, CA-number varied in integer values from 5 to 11 across alleles ...[more]

PMID: 9380705

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Project description:BackgroundMicrosatellites are frequently used genetic markers in a wide range of applications, primarily due to their high length polymorphism levels that can easily be genotyped by fragment length analysis. However, the mode of microsatellite evolution is yet not fully understood, and the role of interrupting motifs for the stability of microsatellites remains to be explored in more detail. Here we present a sequence analysis of mutation events and a description of the structure of repeated regions in the hypervariable, pentanucleotide microsatellite locus HrU10 in barn swallows (Hirundo rustica) and tree swallows (Tachycineta bicolor).ResultsIn a large-scale parentage analysis in barn swallows and tree swallows, broods were screened for mutations at the HrU10 locus. In 41 cases in the barn swallows and 15 cases in the tree swallows, mutations corresponding to the loss or gain of one or two repeat units were detected. The parent and mutant offspring alleles were sequenced for 33 of these instances (26 in barn swallows and 7 in tree swallows). Replication slippage was considered the most likely mutational process. We tested the hypothesis that HrU10, a microsatellite with a wide allele size range, has an increased probability of introductions of interruptive motifs (IMs) with increasing length of the repeated region. Indeed, the number and length of the IMs was strongly positively correlated with the total length of the microsatellite. However, there was no significant correlation with the length of the longest stretch of perfectly repeated units, indicating a threshold level for the maximum length of perfectly repeated pentanucleotide motifs in stable HrU10 alleles. The combination of sequence and pedigree data revealed that 15 barn swallow mutations (58%) produced alleles that were size homoplasic to other alleles in the data set.ConclusionOur results give further insights into the mode of microsatellite evolution, and support the assumption of increased slippage rate with increased microsatellite length and a stabilizing effect of interrupting motifs for microsatellite regions consisting of perfect repeats. In addition, the observed extent of size homoplasy may impose a general caution against using hypervariable microsatellites in genetic diversity measures when alleles are identified by fragment length analysis only.

Dataset Information

Phylogenetic assessment of length variation at a microsatellite locus.

Publications

Phylogenetic assessment of length variation at a microsatellite locus.

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