Genetic Conservation and Population Management of Non-Human Primates: Parentage Determination Using Seven Microsatellite-Based Multiplexes

Abstract

Conservation of non-human primates receives much attention, with nearly 350 of the more than 520 recorded primate species classified as threatened. To conduct effective population management, monitoring genetic diversity within species is of key importance, as it can offer insights into the levels of inbreeding within groups or populations. To examine kinship within the macaque breeding groups housed at the Biomedical Primate Research Centre, located in Rijswijk, The Netherlands, we have developed seven microsatellite-based multiplexes for parentage analysis. These multiplexes comprise a unique set of 23 short tandem repeats (STR) distributed across 15 chromosomes. Extensive validation has been conducted across 2217 Indian rhesus (Macaca mulatta) and 759 long-tailed macaques (M. fascicularis), demonstrating that these STR markers are highly polymorphic and segregate. Most markers exhibit a polymorphic information content (PIC) value above 0.5, illustrating that they are highly informative and valuable in providing us with a reliable parentage determination. Beyond macaques, we manifested that the multiplexes are also suitable for addressing parentage issues in apes and other Old World monkey species. Furthermore, this assay works on DNA isolated from both invasive and non-invasive derived material (e.g., hair follicles and potentially feces). Thus, we present here seven validated multiplexes suitable for parentage analysis in apes and Old World monkey species. These multiplexes support future colony management objectives for various captive populations and, given the applicability of non-invasive techniques, could also be valuable for monitoring free-ranging primate populations.

Keywords: NHP; Old World monkey; apes; molecular genetics; non‐invasive; parentage.

FIGURE 1

Overview of the five microsatellite multiplexes previously published by the INPRIMAT consortium (left) (Roeder et al. 2009) compared to our current extended and refined microsatellite multiplexes (right). The arrows and color codes depict corresponding short tandem repeat (STR) identification (ID) numbers between both panels. Details regarding the various microsatellite markers included in the multiplexes are provided in (Data S1; Table S1).

FIGURE 2

Overview of the offspring distribution produced by the different males in a breeding group of rhesus macaques. For each animal, their age and rank position within the breeding group are provided, utilizing data collected during the two observational periods, 2006–2007 and 2007–2008 (Massen et al. 2012). Animal r01027 was removed from the breeding group in 2007–2008 due to his age and rank. Highlighted in red is the period between 2007 and 2008, during which animal 95007 maintained his status as alpha male, but offspring were produced by other males in the group.

FIGURE 3

Overview of the STR allele distribution (A) and various calculated parameters (B) of the microsatellite markers included in the seven multiplexes for the rhesus and long‐tailed macaque cohorts. (B) Number of alleles (k), number of typed animals (N), observed (Ho) and expected (He) heterozygosity, polymorphic information content (PIC), average non‐exclusion probability for one candidate parent (NE‐1P), average non‐exclusion probability for one candidate parent given the genotype of a known parent of the opposite sex (NE‐2P), significance of deviation from Hardy–Weinberg equilibrium (HW; NS not significant and *, **, *** significance at the 5%, 1%, 0.1% level, respectively), and estimated null allele frequency (F(Null) are provided. Bold printed numbers highlight microsatellite markers for which a slightly higher Ho is observed as compared to He.

FIGURE 4

Bar charts (A, B) and LOD metrics (C) on parentage analysis in BPRC's rhesus (left panel) and long‐tailed (right panel) macaque cohorts for the years 2019 to 2023. (A) The panels display the percentage of offspring for which the parents were assigned with 0, 1, 2, and 3 mismatches (mm) based on trio loci mismatching. (B) The panels show the number of samples that displayed a mismatch at a corresponding microsatellite marker. (C) The panels present a scatter plot of the trio LOD scores calculated for each offspring, with the median LOD score indicated.

FIGURE 5

Information on birth rate, early death animals, and parent couples involved in recurrent miscarriages at the BPRC facilities from 2019 till 2023 (A) Bar chart showing the total number (#) of birth, # of early death animals, and # parents identified of early death animals for our rhesus and long‐tailed macaques (B) Overview of rhesus macaque parent couples that showed recurrent miscarriages in the period 2020 to 2023.

FIGURE 6

Bar chart illustrating the distribution of the number of STR alleles detected for each microsatellite marker included in the seven multiplexes for the Hamadryas baboon cohort.

FIGURE 7

Bar chart (A) and LOD metrics (B) on the total number of Hamadryas baboon samples (N) for which parentage analysis could be conducted. (A) The number of offspring for which parents are assigned with 0, 1, and 2 mismatches (mm) based on trio loci mismatching are indicated. (B) Scatter plot of the trio LOD scores calculated per offspring plotted together with the median.

FIGURE 8

Graphical illustration of paternity analysis for two newborn chimpanzees, indicating the minimal number of microsatellite markers necessary to assign paternity. The results of the complete microsatellite dataset are provided in Table S5. The STR‐allele length indicated in pink is inherited from the mother, and the length indicated in blue is inherited from the father. When no corresponding STR‐allele length is found between the newborn chimpanzee and the possible father, an “x” is placed in the subsequent row to indicate that the individual in question could not be the father of the child.

FIGURE 9

Overview of the length patterns detected for microsatellite markers in the multiplexes MuA and MuBC, using Moloch gibbon DNA samples isolated from feces. When a microsatellite marker is successfully amplified, the fragment lengths of the alleles are provided. In some instances, both alleles have identical fragment lengths, which may suggest that the gibbon in question is homozygous for the associated microsatellite marker. The last column summarizes the total number (#) of microsatellite markers amplified for a sample.