Development of eight-plex microsatellite PCR for parentage control in deer

Nine loci have been compiled into two multiplex microsatellite polymerase chain reaction (PCR) sets (four and five loci) and used as a tool to determine the most probable hind for each calf. The two sets were suitable to combine them in an eight-plex reaction. The exclusion probabilities of the eight-plex reaction and the nine loci were 99.3 and 99.6 % respectively, which allows the routine application of eight loci in wildlife management – as a first attempt to use molecular genetic information for such a task and it eliminates multiple sample handling in consecutive PCRs. Two loci out of the nine were never been used in deer previously.


Introduction
Molecular genetic information was already gained successfully in different species e.g. for determination of origin of a modern cattle (BEJA-PEREIRA et al. 2006), for analysis of genetic diversities in donkey (JORDANA et al. 2001) or horse (ABERLE and DISTL 2004) and for classification purposes in pig (ZSOLNAI et al. 2006).
In deer, comprehensive studies have been conducted in searching for quantitative trait loci (SLATE et al. 2002), and for determination of inbreeding effect on breeding success (SLATE et al. 2000).Molecular data have helped to confirm genetic integrity of Charpatian red deer (FEULNER et al. 2004) and were used to seek for key genes in antler development (MOLNÁR et al. 2007) which might lead to useful markers for breeders.
One aspect of the methodologies is the application of paternity test which is already routinely implemented for example in cattle and sheep (GLOWATZKI-MULLIS et al. 2005, 2007) in many countries.There is an obvious demand from the wildlife managements including deer breeders, to follow-up the lineage, especially when important traits (antler characteristics, body weight, etc.) are logged in the herdbook.Molecular genetic approach has already been implemented for completing this task previously.BONNET et al. (2002) applied eleven microsatellites in 3 multiplex reactions and HAANES et al. (2005) used 25 loci in 6 reactions for genotyping in deer.
The farms sampled in this study are using single mating system, where few stags are in use.Hinds are monitored visually for pregnancy, but after parturition the fate of a calf can not be followed easily by observation, because the calf sometimes chooses other lactating animal than its own mother.
Here we aimed to test and select markers suitable in both fallow and red deer, to incorporate them in one reaction to reduce time and costs in laboratory and to characterise its efficiency in parentage assignment.

Material and methods
In the summer and fall of 2007, 119 red deers from two farms were selected for parentage analysis.Farm A had 25 hinds including 4 empty ones and 46 calves.Farm B presented 29 hinds including 3 empty ones and 26 calves.Additional 47 fallow-deer samples from two farms were also available.Blood samples were collected into EDTA coated tubes and stored frozen until DNA preparation (Zsolnai et al. 2003).Muscle pieces were stored in Eppendorf tubes under 96 % ethanol on room temperature.Microsatellite loci used for analysis were; -Set1: BM4208, NVHRT 21, RT 1, NVHRT 73; -Set2: CSSM066, DIK082, IDVGA59, NVHRT48, OARCP26.DNA amplification was performed in a Hybaid Thermal Cycler in a total volume of 10 μl.Parameters of Set1 were: 3 mM MgCl2, 200 μM dNTPs, 0.66 U DyNAzyme, and the corresponding primer pairs in concentration of 0.30, 0.15, 1.00 and 1.00 mM, respectively (Table 1).PCR conditions included: a denaturation step of 4 min at 94 °C; 30 cycles of 40 s at 94 °C, 45 s at 54 °C, 45 s at 72 °C and a final extension of 10 min at 72 °C.Parameters of Set2 were: 3.5 mM MgCl2, 200 μM dNTPs, 0.66 U DyNAzyme, and the corresponding primer pairs in concentration of 0.20, 0.70, 1.20, 2.00 and 0.70 mM, respectively (Table 1).PCR conditions included: a denaturation step of 4 min at 94 °C, 2 cycles of 30 s at 94 °C, 30 s at 58 °C, 1 min at 72 °C; 3 cycles of 30 s at 94 °C, 30 s at 57 °C, 1 min at 72 °C; 3 cycles of 30 s at 94 °C, 30 s at 56 °C, 1 min at 72 °C; 11 cycles of 30 s at 94 °C, 30 s at 54 °C, 1 min at 72 °C; 16 cycles of 20 s at 94 °C, 30 s at 52 °C, 1 min at 72 °C; and a final extension of 10 min at 72 °C.
The eight-plex reaction amplifies all loci listed under Set1 and Set2 except OARCP26.Primer concentrations are the same as in Set1 and Set2.MgCl2 concentration is 4.1 mM.PCR cycling conditions is the same as for Set2.Amplified fragments were resolved on ABI310 Automatic Fragment Analyzer.LIZ600 was used as internal standard.Fragment length allocation was accomplished by Genescan and Genotyper programs.
The following softwares were used for data processing and crosschecking the results.: Microsatellite Toolkit (PARK 2001) has been used for data formatting in order to meet the input format of GeneClass (PIRY et al. 2004), WhichRun (BANKS andEICHERT 2000), PAPA (DUCHESNE et al. 2002) and CERVUS (MARSHALL et al. 1998).

Results and discussion
Altogether 41 microsatellite loci were tested.
Twenty-two of them were checked in simplex reactions using Taq and DyNAzyme polymerases with different PCR conditions.Eight and eleven loci out of the forty-one used by others for parentage tests of sheep (advised by the International Society for Animal Genetics) and bovine (GLOWATZKI-MULLIS et al. 1995), respectively.These sets have also been investigated on deer samples.Finally nine loci were selected from the 41, which displayed clear electrophoretic patterns on agarose gel or in capillary electrophoretic separation in case of both red and fallowdeer as well.
Based on the observed lengths of the fragments the primer labelling colours were devised to facilitate multiplexing.
In case of red deer the combined exclusion probabilities for the first parents (CEP) of 4-and 5-plex PCR were 92 and 95 %, respectively.If the two reactions were used consecutively, the previous values were changing to 99.6 % (Table 2).Heterozygozity deficits were observable on most loci (Table 3), but deviation from Hardy-Weinberg equilibrium was significant only in one case (FarmB, locus IDVGA59, data not shown).PAPA, Whichrun, GeneClass and CERVUS programs have been compared for the ability to point on possible hinds.Animals selected as parents by the programs were the same in 93 % of the cases.However the genotype assignment was clear, and repeated PCRs gave the same genotypes, different programs displayed different suggestions for a most probable mother in the remaining cases (7 %).In order to resolve these contradictions, genotypes of the animals in question were aligned and checked visually.Since the output interpretation was mostly convenient in case of GeneClass and CERVUS, we suggest using them for parentage control if many samples have to be compared.Additionally CERVUS can correct genotyping errors increasing the success of paternity assignment (KALINOWSKI et al. 2007) and proved to be useful for forensic applications (CASTAGNASSO et al. 2007).
The two PCR sets are suitable for parentage control in red deer and might be used for other species and farms as well.The combined 8-plex PCR is still retaining high, 99.3 % CEP value for the first parent (Table 3), and can be used alone for the same purpose.
Redesign of primer labelling colours might help in creating the 9-plex PCR, which includes the OaerCP26 locus as well.In this study seven loci are identical with those described in the paper of HAANES et al. (2005), however -BM4208, NVHRT48 and OarCP26 were used in simplex, -NVRT73, CSSM066 and NVHRT21 were used in duplex, -RT1 was used in a triplex reaction by them.These seven loci were in our initially tested primer-pool and proved to be suitable for multiplexing.The remaining two loci (DIK082, IDVGA59) were developed by independent authors for cattle (Table 1).
In case of fallow-deer (47 animals from two farms), the investigated populations were quite homogeneous, the inbreeding coefficient (Fis) were 61 and 43 %.In these populations more microsatellite loci should be involved for useful parentage test.
The described microsatellite reactions (especially the 8-plex) support the wildlife management's decisions, help to minimise labour and running costs and minimise the possibility of cross contamination or sample swapping.Since there is a chance that the multiplex reaction works on other deer species (BONNET et al. 2002), it might be interesting for the community working in this field.
The gained genotype data -will be applied to assign lost or strayed individuals to their subpopulation, -hopefully serve as an essential basis of the efforts of trait mapping, -helps to select breeding individuals for maintaining diversity in the red deer populations, -provide basis for the search of populations capable to decrease inbreeding status of the fallow-deer.

Table 1
PCR primers and their concentrations in the Set1 and Set2 reactions PCR Primer und ihre Konzentration in den Reaktionen von Set1 und Set2

Table 2
Characterisation of the multiplex sets used in two farmsBeschreibung der multiplex Sets aus zwei Farmen n number of sampled animals, Ho observed heterozigosity, He expected heterozigosity, PIC polymorphic information content, CnEP combined non-exclusion probability, Fis inbreeding coefficient Table3Observed and expected heterozygosity, number and size range of alleles, PIC, Fis in different loci and farms Erfasste und erwartete Heterozygotie, Anzahl und Größe der Allele, PIC und Fis-Werte bei unterschiedlichen