Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography

Abstract

Y chromosome haplotypes are particularly useful in deciphering human evolutionary history because they accentuate the effects of drift, migration, and range expansion. Significant acceleration of Y biallelic marker discovery and subsequent typing involving heteroduplex detection has been achieved by implementing an innovative and cost-efficient method called denaturing high-performance liquid chromatography (DHPLC). The power of the method resides in its sensitivity and ability to rapidly compare amplified sequences in an automated manner. We have determined the allelic states of 22 Y polymorphisms; 19 of which are unreported, in 718 diverse extant chromosomes; established haplotype frequencies; and deduced a phylogeny. All major geographic regions, including Eurasia, are characterized by mutations reflecting episodes of genetic drift and expansion. Most biallelic markers are localized regionally. However, some show wider dispersal and designate older, core haplotypes. One transversion defines a major haplogroup that distinguishes a previously unknown deep, apparently non-African branch. It provides evidence of an ancient bottleneck event. It is now possible to anticipate the inevitable detailed reconstruction of human Y chromosome genealogy based on several tens to even hundreds of these important polymorphisms.

Figure 1

Representative chromatograms showing resolution of heteroduplex (peaks 1) from homoduplexes (peaks 2) created on reannealing PCR products of a male reference with males having the polymorphisms M4, M7, M9, M18, M19, and M20, respectively. Column—DNASep, 50 × 4.6-mm ID, Transgenomic, Inc.; linear gradient of 0.45% acetonitrile per minute in 0.1 m triethylammonium acetate, at pH 7.0, at a flow-rate of 0.9 ml/min; mobile phase temperatures: 52°C, 56°C, 54°C, 58°C, 58°C, and 57°C, for M4, M7, M9, M18, M19, and M20, respectively. PCR amplicons were injected directly into the preheated mobile phase.

Figure 2

Relative frequencies of 20 human Y chromosome haplotypes in 10 world regions, and total numbers of males studied per region (last column). The three major haplotypes, A1, B1, and C1, are dashed to indicate the uncertainty in the order of branching points. Haplotypes appearing only once are marked with an asterisk. The principles followed in constructing the tree are described in the text. The mutations leading from one haplotype to another are indicated above each horizontal branch of the tree, and are described in Table 1. In the last row are the numbers of populations (totaling 54, listed in Methods) in which each haplotype has been observed.