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In the present study, we typed our previously reported two microsatellite markers, DXYS241 and DXYS266 together with a basic set of nine Y-STRs (DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DXYS156Y, DYS413) on Y chromosomes from two Bolivian populations. Unrelated males from communities living at high- (N = 59) and low- (N = 142) altitude, were studied. Combining the alleles into 11 Y-STRs haplotypes revealed that the high-altitude population is significantly less diverse than the low-altitude population. Haplotype diversities of 0.927 ± 0.029 and 0.996 ± 0.002 were found within the high-altitude, and the low-altitude populations, respectively. Within the high-altitude population 40 haplotypes were detected, whereas in the low-altitude population 113 haplotypes were found. Only three haplotypes were shared between both populations.
Haplotyping-based discrimination using the 11 Y-STRs including our new two microsatellite markers DXYS241 and DXYS266 was shown to be powerful than using the conventional 9 Y-STRs, especially for the low-altitude Bolivian population.
This 11 Y-STRs-based haplotyping system shows a very high potential for discrimination and could provide an ideal tool for forensic analysis and population studies. Moreover, this study includes data about two Bolivian populations which were not previously reported, this will help in building a world-wide database for future use in forensic and legal studies.
Blood samples from 59 high-altitude Bolivian males and 142 low-altitude Bolivian males were collected from healthy blood donors. The subjects in the high-altitude group were from typical rural communities of the Andean Altiplano, living at an approximate elevation of 4000–4100 m above sea level, and located 280 km southwest from the city of La Paz. Details of that community were presented elsewhere [1,2]. The subjects from low-altitude were from the Alto Beni, subtropical Amazonian lowland colonies at an elevation of ~300 m located about 200 km southeast of La Paz along Alto Beni River, an upper tributary of the Amazon River. Details of those subjects were presented previously .
Genomic DNA was extracted from the collected blood samples using the QIAamp Blood Kit from Qiagen (Hilden, Germany).
The 11 Y-STR loci (DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DXYS156Y, DYS413, DXYS241 and DXYS266) were amplified by the polymerase chain reaction (PCR) using four multiplex systems (system I: DYS19, DYS389I/II; system II: DYS390, DYS391, DYS392; system III: DYS393, DXYS156Y, YCAIII; system IV: DXYS241 and DXYS266). For primers and PCR conditions see Kayser et al.  and Lee et al. .
For analysis, 2 μl of the PCR products were mixed with 3 μl of formamide buffer and run on denaturing polyacrylamide gels on an ABI 377 sequencer using a TAMRA 500 internal size standard in each lane. Raw genotype data were analyzed using GENESCAN software (ABI). Allele nomenclature follows Kayser et al.  and de Knijff et al. .
Haplotype and allele frequencies were calculated by the counting method. Estimation of haplotype and gene diversities together with intra- and inter-population indices and analysis of molecular variance (AMOVA) were computed using ARLEQUIN version 2.000, http://igb.unige.ch/arlequin, developed by Schneider, Roessli and Excoffier .
See Table 1
We analyzed Bolivian male samples from high- and low-altitudes using our previously reported haplotyping system, which is based on a single multiplex PCR for three polymorphic microsatellite markers, DXYS241, DXYS265 and DXYS266 . Then, we compared the frequencies of the different alleles of these markers in the two Bolivian populations with our previous data for Japanese, Caucasian-American and African-American populations. All populations exhibited alleles 227 bp (CA14) and 162 bp (CA12) of DXYS241 and DXYS265, respectively, as the predominant alleles. However, at DXYS266, the 216 bp (CA18) and 218 bp (CA19) alleles were prevalent within the Bolivian populations, but were not found in other populations. We studied the haplotype diversity in both Bolivian populations using a basic set of nine Y-STRs (DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DXYS156Y, DYS413) only. At this stage of the study we did not combine our data of the three newly developed markers of Lee et al. . When using only the 9 Y-STRs, the results showed a haplotype diversity of 0.91 ± 0.032 and 0.98 ± 0.003 in the low- and high-altitude populations, respectively.
We eliminated the marker DXYS265 from the haplotyping system because of its low diversity in Bolivian population.
Thus, we used haplotypes comprising the above two microsatellite markers (DXYS241 and DXYS266) together with the basic set of the nine Y-STRs (DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DXYS156Y, DYS413), for further studying the genetic diversity within and between the two Bolivian populations. We could detect 150 different haplotypes in both the high- and low-altitude populations. Only three haplotypes were shared between both populations. Within the high-altitude population 40 haplotypes were detected whereas, in the low-altitude population 113 haplotypes were found (Table 1). Haplotype diversities of 0.927 ± 0.029 and 0.996 ± 0.002 were found within the high-altitude, and the low-altitude populations, respectively. This haplotype diversity indicates that the high-altitude population is significantly less diverse than the low-altitude population. Interestingly, haplotype H22, which is present in both populations, exhibited the highest frequency among all haplotypes in the high-altitude population, 27.1% (16/59), while only 2.1% (3/142) of the low-altitude males have this haplotype (H22). However, still the Y-chromosomal haplotypes can differentiate effectively within and between the two Bolivian populations. We conclude that combining our two new microsatellite markers DXYS241 and DXYS266 with the other 9 Y-STRs within a standard haplotyping system improves the power of discrimination, and could provide an ideal tool for forensic analysis and population studies. These results can present a part of the genetic portrait of the peopling of Americas to discover the genetic diversity, genetic distance and genetic structure with estimation of the timing of migration waves of the New World founders and inhabitants . Moreover, this study includes data about two Bolivian populations which were not previously reported, this will help in building a world-wide database for future use in forensic and legal studies.