Cross-species validation of human specific STR system, SureID®21G and SureID®23comp (Health Gene Technologies) in Chimpanzee (Pan Troglodytes)

Objectives The human specific commercially available STRs system are often not tested in non human primates for their cross applicability. The aim of this study is to test Cross-species validation of two commercially available human specific STR kits i.e. SureID® 21G and SureID® 23comp (Health Gene Technologies) for their positive application in Chimpanzee (Pan troglodytes). Result In SureID® 21G, 19 loci amplified and while 20 loci amplified in SureID® 23comp. All the amplified loci in both STR kits were found polymorphic and the locus Amelogenin showed differential banding patterns between male and female revealing their known gender. The present study validates the applicability of these human specific STR kits in Chimpanzee that can be used in forensics analysis, paternity testing and population genetic studies.


Introduction
Human identification kits based on Short Tandem Repeats (STRs) markers are widely accepted and utilized in several fields of population genetics and forensic case works. Human identification prior to the development of genetic methods was preferably attempted by the serological techniques as they have proven in successfully determining the species of origin through a presumptive test in forensic cases [1]. However, there have been several instances where human antiserum showed crossreactivity with non-human blood. For example, Landsteiner and Miller [2] documented that the anthropoid blood of Gorilla, Orangutan and Chimpanzee showed precipitation with human antiserum. This indicated that serological tests showed less specificity and sensitivity with the phylogenetically close relatives of human. Numerous STR panels are used for human identification in forensics analysis and population genetic studies [3][4][5] but none of the marker systems has been validated for its cross applicability in non-human primate except PowerPlex ® 21 human identification System (Promega, USA) [6]. However, it is important to explore the possibility of cross-species amplification of these marker systems with non-human primates, so as to understand its applicability and link the specific use (possibility of exclusion in cases where samples of human and non-human primates are mixed).
We attempted to check the cross-reactivity of two human-specific STR marker system SureID ® 21G and SureID ® 23comp (Health Gene Technologies) in Chimpanzee following the Thakur et al. [6] which has established applicability of PowerPlex ® 21 human identification System (Promega, USA) in Chimpanzee.

Statistical analysis
Allele calling was manually done using GeneMapper ID version 3.2 (Applied Biosystems, USA). The allelic data was exported into the Microsoft Excel and scoring and re-arrangement of the sizes of alleles were performed for the assessment of genetic diversity indices. Software, GENEALEX version 6.5 [7] was used to estimate the genetic diversity indices such as the observed number of alleles per locus (Na), an effective number of alleles (Ne), Observed heterozygosity (Ho), Expected heterozygosity (He) and Inbreeding Coefficient (F). Allelic data was used in estimating the genealogical relationship or relatedness among the three chimpanzees of unknown ancestry. The computer program, ML-Relate that calculates maximum likelihood estimates of relatedness and relationship was used [8] which does accommodate null alleles which may arise when using heterologous markers.

Applicability of SureID ® 21G Human STR Identification kit
Of the 21 loci in SureID ® 21G Human STR Identification kit, two loci i.e. D12S391 & D21S11 did not amplify in all three chimpanzee samples and other 19 loci were polymorphic. The detail allelic data can be obtained upon request. The locus AMEL correctly assign the sexes of known chimpanzees providing differential banding patterns in males and females (single allele of 112 bp in female and 112, 117 bp alleles in males). This exhibited discrimination power of SureID ® 21G Human STR Identification systems in determining genders of Chimpanzee. In total, 62 alleles were found associated with 19 loci. The number of observed alleles ranged from 6 (D1S1656 & D2S1338) to 1 (D5S818) with 3.26 ± 0.30 mean number of alleles per locus. The effective number of alleles with mean 2.84 ± 0.31 did not exceed the observed number of alleles for all loci. The mean Ho and He were 0.63 ± 0.07 and 0.57 ± 0.05 with mean UHe of 0.68 ± 0.06. A negative mean F value of − 0.13 ± 0.09 were observed representing a high heterozygosity than expected ( Table 1) Table 2).

Applicability of SureID ® 23comp Human STR Identification kit
Of the 23 loci in SureID ® 23comp Human STR Identification kit, three loci i.e. D12S391, D8S1132, and D2S441 did not amplify in all three chimpanzee samples and other 20 loci were polymorphic. The detail allelic data can be obtained upon request. The locus AMEL further validated the known gender of chimpanzees by amplifying alleles of size 108 in female and males showing 113 and 108 bp. This validates the discrimination power of gender identification in chimpanzees using SureID ® 23comp Human STR Identification systems. In total, 75 alleles were found associated with 20 loci. The number of observed alleles ranged from 6 (D1S1656) to 2 (D14S1434, D19S253, D22-GATA198B05) with mean number of alleles 3.75 ± 0.27 per locus. The effective number of alleles with mean 3.31 ± 0.26 did not exceed the observed number of alleles for all loci. The mean Ho and He were 0.77 ± 0.05 and 0.66 ± 0.03 with mean UHe of 0.82 ± 0.03 (Table 3). A negative F value of − 0.17 ± 0.09 was observed that the analyzed chimpanzees plausibly belong to unrelated genetic background. Out of 20 loci, 12 loci were observed relatively more heterozygous with ≥ 4 alleles/locus indicating that the analyzed individuals were genetically unrelated.    Table 3).

Discussion
The study showed the positive application of the SureID ® 21G and SureID ® 23comp STR system with Chimpanzee DNAs which can be further utilized in cases of paternity analysis, gender discrimination, population genetic studies and understanding the evolution and phylogenetic relationships among the human and non-human primates with the common marker systems. Several genetic parameters like higher observed heterozygosity than the expected heterozygosity, a negative fixation index and log-likelihood relationship index suggested that these three individuals were genetically unrelated. Time and efforts spent to explore the applicability of commercially available human STR systems SureID ® 21G and SureID ® 23comp STR system in Chimpanzee has been well justified and raised hope to test the applicability of these marker systems in other non-human primates like Gorilla, Orangutan, Langur and Macaques.

Limitations
The small sample size was a limitation for this study. However present study well explains the Cross-species validation of Human specific STR systems in non human primate (Pan Troglodytes) and would propose further studies with large sample size.