Low STR variability in the threatened marsh deer, Blastocerus dichotomus, detected through amplicon sequencing in non-invasive samples

Abstract

Blastocerus dichotomus is the largest deer in South America. We have used 25 microsatellite markers detected and genotyped by Next Generation Sequencing to estimate the genetic variability of B. dichotomus in Argentina, where most of its populations are threatened. Primer design was based on the sequence of a shallow partial genome (15,967,456 reads; 16.66% genome coverage, mean depth 1.64) of a single individual. From the thousands of microsatellite loci found, even under high stringency selection, we chose and tested a set of 80 markers on 30 DNA samples extracted from tissue and feces from three Argentinean populations. Heterozygosity levels were low across all loci in all populations (H=0.31 to 0.40). Amplicon sequencing is a fast, easy, and affordable technique that can be very useful for the characterization of microsatellite marker sets for the conservation genetics of non-model organisms. This work is also one of the first ones to use amplicon sequencing in non-invasive samples and represents an important development for the study of threatened species.

A Selection of 14 Tetrameric Microsatellite Markers for Genetic Investigations in Fallow Deer (Dama dama)

“…Polymorph marker names and their number of alleles are in red. References: 1.: Jones et al, 2000 [ 21 ]; 2.: Jones et al, 2001 [ 22 ]; 3.: Meredith et al, 2005 [ 23 ]; 4.: Brinkman et al, 2010 [ 24 ]; 5.: Szabolcsi et al, 2014 [ 25 ]; 6.: Caparroz et al, 2015 [ 26 ]; 7.: Accession numbers JN643715-JN643722, JN563734-JN563735; 8.: Yang et al, 2018 [ 27 ]; 9.: Hill et al, 2021 [ 28 ]; 10.: Morf et al, 2021 [ 29 ]; 11.: Wolfenson et al, 2022 [ 30 ].…”

“…The first step in compiling a microsatellite set for genetic analysis is the search for suitable markers. This can be done by examining the genome sequence of the target species using next-generation sequencing technology [28][29][30] or by testing markers described in previous publications for closely related species [22,23,25,33]. The latter method, traditionally used due to its lower cost, may encounter the problem of primers originally designed for different species not functioning in the target species.…”

“…To estimate genetic diversity in this species, different markers have been tested, such as mitochondrial DNA [ 14 , 17 ], blood and tissue proteins [ 16 , 18 , 19 ], various genes (e.g., PRNP) [ 15 ], as well as di- and tri-nucleotide microsatellites [ 14 , 20 ]. Microsatellite marker-based developments have been predominantly carried out in other deer species to assess genetic diversity [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ], as the number of repeats in microsatellites tends to be highly variable within a population. Microsatellites or short tandem repeats (STRs) are polymorphic fragments of DNA containing a repeated sequence of generally 2–5 nucleotides.…”

“…Tetramer-STR marker sets, which offer numerous advantages over dimeric microsatellites [ 21 , 22 , 23 , 29 , 30 , 39 ], have already been developed for various deer species but are not yet available for fallow deer. The aim of this study was to develop an investigative method to test all available cross-species tetrameric repeat STRs on fallow deer samples and select polymorphic markers to expand the testing options for this globally important game species.…”

Southern marsh deer (Blastocerus dichotomus) populations assessed using Amplicon Sequencing on fecal samples

“…Moreover, it allows the analyses of short target sequences, such as those obtained from gNIS. However, only a few works have sequenced and genotyped microsatellites with Amplicon Sequencing, applied to a low number of non-invasive samples 22 – 24 .…”