Development and evaluation of multiplex Y-STR assays for application in molecular genealogy

Surnames are paternally inherited in many societies. This theoretical co-inheritance with the Y chromosome provides the opportunity to determine if two family lineages have a common paternal ancestor by the analysis of STR loci. Use of 12 (PowerPlex^® Y, Promega Corporation, Madison, WI) or 17 Y-STR (AmpFlSTR^® Yfiler™, Applied Biosystems, Foster City, CA) systems provides enough information to exclude a common ancestor for paternally unrelated males. However, identification of closely related paternal lineages requires more Y-STRs. Based on public genealogical databases, we selected 14 single-copy (DYS607, DYS458, DYS456, DYS442, DYS447, DYS448, DYS426, DYS449, DYS455, DYS388, DYS454, GATA H4.1, DYS460 and DYS570) and 4 multi-copy Y-STRs (DYS459, YCAII, DYS464 and DYS724) for a large (>1200) population project aiming at reconstructing the origin and history of surnames (Y chromosomes) in Belgium. Here, we present the results of the development of three Y-STR mutiplex assays and population data on 245 male samples analysed.

Buccal swab samples were collected after informed consent from 245 Belgian and Dutch males representing 229 different surnames. DNA was extracted by using the Maxwell^® 16 System (Promega) and quantified using real-time PCR (Quantifiler™ Human DNA Kit, Applied Biosystems). DNA amplification was done in a volume of 25 μl using Qiagen^® Multiplex PCR Kit (Qiagen, Hilden, Germany), 5 μl of optimized primer-mix (Table 1; forward primers were fluorescently labelled with either FAM, VIC, NED or PET) and 5 μl of DNA (1 ng) for 15 min at 95 °C, 30 cycles of 0.5 min at 94 °C, 1.5 min at 58 °C (MPI) or 62 °C (MPII and MPIII) and 1.5 min at 72 °C (“9600” ramp speed), and a final extension of 10 min at 72 °C on a GeneAmp 9700 PCR System (Applied Biosystems). Primer sequences were obtained from literature [, , ] or selected with Primer3 for DYS724. PCR products were purified with BigDye^® XTerminator (Applied Biosystems) and 1 μl was analyzed on an ABI PRISM 3130 XL Genetic Analyzer (Applied Biosystems) with a 50 cm capillary and using POP-7™ as separation medium []. Fragment size determination and allele designation was done with GeneMapper v3.2 (Applied Biosystems) and GS-500 [LIZ] as the internal size standard. Average allele size and bin sizes were determined by analysing 100 different samples. Allele designation was confirmed with Standard Reference Material 2395 (NIST, Gaithersburg, MD) and by sequencing individual alleles.

Y-STR loci were selected for a large genealogical study involving males with a documented family history originating in Belgium, Northern France (region of French Flanders), Luxembourg or part of the Netherlands (region of North Brabant). One of the aims of the project is to establish high resolution haplotypes for the identification of closely related paternal lineages and to provide the possibility to compare the results with public available genealogical DNA databases. Twenty nine loci including 24 single-copy and 5 multi-copy STRs (Table 1) were selected which resulted for most male individuals in a 37 Y-STR haplotype. Loci not present in PowerPlex^® Y were included in 3 multiplex reactions. In addition, some STRs were included in more than one multiplex in order to have an internal control. The inclusion of DYS464 into two assays facilitated the interpretation of the alleles and peak height ratios []. The selection of the primers were such that the amplification products from two multiplexes (MPI and MPIII: 14 single-copy and 3 multi-copy STRs) could be combined for analysis on the ABI PRISM 3130 XL Genetic Analyzer.

The analysis of 245 DNA samples collected for the project resulted in 244 different high resolution haplotypes with one haplotype observed in two males with the same surname. For 9 surnames with more than one representative, it was possible to exclude a common paternal ancestor with the minimal (172 observed) or the extended haplotype (200 observed). For 2 surnames only a single difference (one repeat) was present in the males tested, respectively, for DYS388 and DYS464. Gene diversity [] was calculated and revealed the lowest figures for DYS454 and DYS455. These loci are therefore not interesting for forensic applications but their low diversity put also some doubts on the use for genealogical Y-STR typing. The highest discrimination was observed for DYS464 and DYS724. These loci are multi-copy STRs and are localized on the same palindromic sequence (Yq11) together with DYS459. DYS464 shows in most individuals 4 copies but some males have 5 or 6 copies. Similarly, DYS459 and DYS724 usually show 2 copies but some individuals have 3 copies. Twelve individuals in this study displayed a haplotype with 38, 39 or 40 alleles which was due to additional copies of DYS464, sometimes in combination with an additional copy at DYS459 or DYS724. This correlation between loci is the result of large segmental duplications by gene conversion [] and the study of these Y-STR haplotypes might give some insights about the evolution of these regions on the human Y chromosome. While DYS464 could be a complex locus (interpretation of the allelic profiles) for application in a forensic context (e.g. mixtures), DYS724 might be a novel highly polymorphic Y-STR for use in criminal cases and paternity investigations.

None.

This study has been funded by the Flemish Society for Genealogical Research (Antwerp) and by a grant from the Flanders Ministry of Culture. The Flemish Society for Genealogical Research was only involved in the collection of the samples and genealogical data from the participants in the study.

The authors thank Lucie Larno, Monique Coomans and Nathalie Thys for excellent technical assistance.