Molecular Phylogenetics and Evolution 49 (2008) 663–668

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

Short Communication

A multiplex primer extension assay for the rapid identification of paternallineages in domestic goat (Capra hircus): Laying the foundations for a detailedcaprine Y chromosome phylogeny

Filipe Pereira a,b,*, João Carneiro a,b, Pedro Soares c, Sónia Maciel d, Fouad Nejmeddine a, Johannes A. Lenstra e,Leonor Gusmão a, António Amorim a,b

a Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugalb Faculdade de Ciências da Universidade do Porto, Portugalc Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UKd Agriculture Research Institute of Mozambique, Department of Animal Sciences, Artificial Insemination Centre, Maputo, Mozambiquee Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

a r t i c l e i n f o

Article history:Received 3 April 2008Revised 12 August 2008

1055-7903/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.ympev.2008.08.026

* Corresponding author. Address: Instituto de Patolda Universidade do Porto (IPATIMUP), Rua Dr. RoberPortugal. Fax: +351 22 5570799.

E-mail address: fpereira@ipatimup.pt (F. Pereira).

Accepted 20 August 2008Available online 10 September 2008

Keywords:Capra hircusY chromosome

1. Introduction

Genetic variation on the mammalian Y chromosome is extre-mely useful for understanding global patterns of population diver-sity and sex-specific population parameters such as male-mediateddispersals or introgressions (Hurles and Jobling, 2001; Petit et al.,2002). Its uniparental mode of inheritance and the complete ab-sence of meiotic recombination in almost all its extent imply thateach individual has a single haplotype and that phylogenetic anal-yses are relatively straightforward to interpret. These features mayhelp to unravel the complexity of animal domestication and com-plement the picture emerged from mitochondrial DNA (mtDNA)surveys (Hanotte et al., 2000; Bruford et al., 2003; Pidancieret al., 2006; Meadows et al., 2006). Studies on the Y chromosomeare of particular interest in domesticated species because commonbreeding strategies impose that only a few males contribute genet-ically to the next generation leading to a strong sex-bias in thedomestication process (Lindgren et al., 2004).

The first livestock species to be domesticated in the Fertile Cres-cent of the Near East was the goat (Capra hircus) by taming of the

ll rights reserved.

ogia e Imunologia Molecularto Frias s/n, 4200-465 Porto,

wild bezoar goat (Capra aegagrus) at least 10,000 years ago (Mason,1984; Zeder, 2006). A series of recent genetic studies based onmtDNA sequence variation has revealed a complex pattern of cap-rine domestication (Luikart et al., 2001; Naderi et al., 2007). Sixdivergent mtDNA haplogroups have been found in domestic goatpopulations worldwide, suggesting the incorporation of numerousgenetic lineages into the domestic goat gene pool, most likely fromdifferent populations of the bezoar goat. The development of acoherent phylogeny of Y-chromosomal haplotypes will be particu-larly useful to understand patrilineal contributions to the originand subsequent movements of goat pastoralism worldwide.

Here we present a rapid and robust multiplex PCR/primerextension assay to genotype a set of goat Y chromosome singlenucleotide polymorphisms (SNPs) previously described (Lenstra,2005). Four diagnostic SNPs located in two flanking domains ofthe SRY gene (promoter region and 30 untranslated region) were se-lected to allow the discrimination of the four Y-chromosomal hap-lotypes Y1A, Y1B, Y1C and Y2 identified so far in domestic goatpopulations. The detection of SNP variants is based on the SNaP-shot single-base sequencing (also known as minisequencing),which consists in using dideoxynucleotides (ddNTPs) for single-base extension of an unlabeled primer that anneals one base up-stream to the relevant SNP. Identification of haplotypes is thenpossible by automated capillary electrophoresis (for separation ofextended products of known lengths) and multicolour fluorescence

664 F. Pereira et al. / Molecular Phylogenetics and Evolution 49 (2008) 663–668

detection (the four ddNTPs labelled with different fluorescentdyes) (Quintans et al., 2004; Sanchez et al., 2005).

This approach has been evaluated with samples from Europeanand African goat populations. An auxiliary software was developedto simplify the process of haplotype identification directly from thesample profile. A maximum likelihood estimation of the diver-gence ages for SRY lineages revealed that three of them are datedto hundreds of thousands of years before present, suggesting thatgenetically differentiated populations of the ancestral wild goathave contributed to the domestic goat gene pool.

Similar approaches have been successfully reported for theanalysis of polymorphisms in human mtDNA (Brandstatter et al.,2003; Quintans et al., 2004; Vallone et al., 2004; Salas et al.,2005), Y chromosome (Sanchez et al., 2005; Bouakaze et al.,2007) and autosomes (Dixon et al., 2005). To our knowledge, thisstudy reports the first minisequencing-based assay for SNPs typingin a domestic species.

2. Materials and methods

2.1. DNA samples and extractions

Samples were collected from unrelated domestic male goats(Capra hircus) from five Portuguese breeds (Bravia, n = 5; Serpenti-na, n = 5; Charnequeira, n = 5; Serrana, n = 5 and Algarvia, n = 1) asdescribed in Pereira et al. (2005). Sample from the MozambicanLandim breed (n = 10) and the Black Moroccan goat type (n = 5)were also used. Ten female samples were tested to evaluate theY chromosome-specificity of the reaction. The species-specificityof the assay was confirmed with seven sheep (Ovis aries) and fivecattle (Bos taurus) samples collected in Portugal.

DNA was extracted from dried blood on FTA paper (Whatman,Clifton, NJ, USA), soft tissue and liver using Chelex (Biorad,Hercules, CA, USA) and phenol-chloroform extraction protocols(Sambrook et al., 1989).

2.2. Goat Y-chromosomal single nucleotide polymorphisms

Four Y-chromosomal lineages Y1A, Y1B, Y1C and Y2 were de-scribed in domestic goats from Europe and the Near East (Lenstra,2005). Four diagnostic SNPs located in two regions of the SRY genewere used to identify each lineage: three SNPs (SRY-2711, SRY-2971 and SRY-3098) in the SRY 30 untranslated region (SRY 30UTR)and one (SRY-1876) in the SRY promoter region (Table 1). Thereference sequences from both SRY regions and the SNPs locationsare given in Supplementary Figs. S1 and S2.

2.3. Multiplex PCR primer design and PCR conditions

Specific PCR primers to amplify the two SRY regions were de-signed using the publicly available Primer3 software (Rozen andSkaletsky, 2000). Primers were designed with similar melting tem-peratures (tm of approximately 60 �C) in order to achieve balancedPCR amplifications in the multiplex reaction (Supplementary TableS1, Figs. S1 and S2). Candidate primers were checked for potentialhairpin and primer-dimer interactions using the Oligo Calculator

Table 1SNP states for all SRY haplotypes observed in domestic goats (from Lenstra (2005))

SNP SRY haplotypes

Y2 Y1A Y1B Y1C

SRY-3098 A G G GSRY-2711 A T T TSRY-2971 T T A ASRY-1876 A A A C

version 3.07 software (Kibbe, 2007) and the screening for potentialcross-reactivity among primer pairs was performed using theAutoDimer version 1.0 program (Vallone and Butler, 2004).

Multiplex PCRs were performed by combining 2 ll of extractedDNA, 1 ll of a primer mix (2 lM of each primer) and 5 ll of Mul-tiplex PCR Master Mix (Qiagen GmbH, Germany) carried out in a10 ll final volume. PCRs were performed as follows: initial dena-turation step at 95� C for 15 min, followed by 33 cycles of 30 s at94 �C, 90 s at 56 �C, and 1 min at 72 �C with a final extension stepof 10 min at 72 �C. PCR products were electrophoresed on horizon-tal polyacrylamide nondenaturing gels and silver stained.

PCR primers and unincorporated dNTPs were removed by add-ing 2.5 ll of ExoSAP-IT (E. coli exonuclease I and shrimp alkalinephosphatase; USB Corporation, OH, USA) to each 5 ll PCR. Reac-tions were incubated for 15 min at 37 �C and then for 15 min at80 �C for enzyme deactivation.

2.4. Extension primers design

Extension primers (alternatively termed minisequencing prim-ers) were manually designed with a predicted tm of approximately55 �C in order to hybridize immediately adjacent to the targetpolymorphic position (Supplementary Table S2, Figs. S1 and S2).A poly-C and a non-homologous tail according to Sanchez et al.(2005) were added at the 50 end of two extension primers (forSRY-2971 and SRY-1876, respectively) to allow a clear separationby capillary electrophoresis. The final sizes range from 21 to35 bp, differing from each other by more than 4 bp (SupplementaryTable S2). Potential hairpin and primer–dimer interactions andcross-reactivity among primers were checked as described for mul-tiplex PCR primers. All primers were purchased from Thermo Elec-tron Corp. (Waltham, MA, USA) with Reversed Phase HPLCpurification.

2.5. Single-base sequencing reaction and electrophoresis conditions

The single-base sequencing reactions were carried out in a totalvolume of 5 ll containing 1 ll of SNaPshot Ready Reaction mix(Applied Biosystems, Foster City, CA), 1.5 ll of purified PCR productand 0.8 ll of primer extension mix (0.3 lM SRY-2711, 0.2 lM SRY-2971, 0.2 lM SRY-3098 and 0.1 lM SRY-1876).

The reaction mixture was subjected to 25 single base extensioncycles of denaturation at 96 �C for 10 s, annealing at 50 �C for 5 sand extension at 60 �C during 30 s. Unincorporated ddNTPs wereremoved with 1 U of SAP (USB) by incubation for 1 h at 37 �C fol-lowed by enzyme inactivation by heating at 85 �C for 15 min.

The single-base sequencing products (0.75 ll) were mixed with13.5 ll of HiDiTM formamide (Applied Biosystems) and 0.75 ll ofGeneScan-120 LIZ size standard (Applied Biosystems). The electro-phoresis was performed on a 3130xl Genetic Analyzer using filterset E5 and Performance Optimum Polymer 7 (Applied Biosystems).Positive and negative controls were used in all reactions.

2.6. DNA sequencing

The SRY 30UTR region was amplified and sequenced in sevensheep using goat SRYuF and SRYuR PCR primers (SupplementaryTable S1). PCRs were carried out as previously described for thegoat multiplex PCR. The sequencing reaction was performed bycombining 2.5 ll of amplified DNA, 0.5 ll of primer SRYuF(2.5 lM) and 2 ll of Big Dye Sequencing Kit (Applied Biosystems).The sequencing amplification protocol consisted of one cycle of2 min at 96 �C, followed by 35 cycles of 15 s at 96 �C, 9 s at 50 �Cand 2 min at 60 �C, with a final extension step of 10 min at 60 �C.Sequencing reaction products were purified using Sephadex G-50Fine (GE Healthcare, UK). Electrophoretic separation was carried

F. Pereira et al. / Molecular Phylogenetics and Evolution 49 (2008) 663–668 665

out on a 3130xl Genetic Analyzer. The sequences have beendeposited in GenBank with Accession No. FJ011696–FJ011702.

2.7. Sequence alignment

The two goat SRY regions used in this study (from GenBankAccession No. D82963) were subjected to BLAST searches (BasicLocal Alignment Search Tool; http://www.ncbi.nlm.nih.gov/blast)in order to find related nucleotide sequences from sheep and cattle.The default query parameters were used against the nucleotidecollection (nr) database. All sheep and cattle sequences with thefull query coverage were aligned with the goat reference sequencesusing the Clustal W software implemented by the BioEdit program(http://www.mbio.ncsu.edu/BioEdit/bioedit.html).

2.8. Y-SNP haplotype identification software

The SNPYGoat software was written in PYTHON 2.5 and a graph-ical interface created using the VisualWX Rapid Application Devel-opment (RAD) environment (http://visualwx.altervista.org). Asingle EXE file was created using the Inno Setup software (http://www.jrsoftware.org/isinfo.php) for installation purposes. The soft-ware is available at http://www.portugene.com/software.html.

2.9. Statistical analysis and maximum likelihood age estimates

The probability of two randomly selected individuals from apopulation having identical SRY lineages (matching probability)was calculated according to the formula:

MP ¼Xn

i¼1

pi2

where pi is the frequency of the i-th haplotype and n the total num-ber of different haplotypes.

The two goat SRY domains were combined and compared withhomologous segments from O. aries, B. taurus, B. bubalis, B. grunn-iens, B. bison, B. bonasus, B. javanicus, B. frontalis and B. gaurus. Atree was obtained using Network 4 software and the reducedmedian algorithm (Bandelt et al., 1995) with no ambiguity in theobtained output. Maximum likelihood (ML) age estimates (Felsen-stein, 1981) were obtained using PAML 3.13 (Yang, 1997) assumingthree different substitution models (JC69, HKY85, and REV; Jukesand Cantor, 1969; Hasegawa et al., 1985; Yang, 1994) with gam-ma-distributed rates (approximated by a discrete distribution with8 categories) for comparative purposes. An approximation to thefrequency of each SRY lineage from the dataset of Lenstra (2005)was used in the PAML input file (Y2 35.4%, Y1A 31.7%, Y1B 30.9%and Y1C 2%). The tree was run with and without the clock assump-tion. Three calibration points were used independently to calibratethe molecular clock: two estimates for the Caprinae/Bovinaedivergence time and one for the Capra/Ovis. An average of previousmolecular estimates obtained in the Timetree database (Hedgeset al., 2006) was used in this analysis, yielding the values of 35.7million years (My) for the first and 8.5 My for the latter. A secondCaprinae/Bovinae divergence time of 26.2 My was selected basedon the estimates by Hassanin and Douzery (2003).

Likelihood ratio tests were performed to compare the results ofthe three substitution models, JC69, HKY85 and REV. No significantdifferences were found between these models, with all tests pre-senting a P-value of 1. Therefore, the JC69 model was used to esti-mate divergence times, avoiding the use of more complex models(HKY85, REV). All analyses were performed with and without amolecular clock to test if implementing a clock was inappropriatefor the data. As the P-value yields the value of 0.9999, the clockhypothesis could not be rejected.

3. Results and discussion

A number of studies have shown that reduced levels of variabilityin some Y chromosome loci are a typical feature of mammalian gen-omes (Jobling and Tyler-Smith, 2003; Hellborg and Ellegren, 2004).The observed low diversity may be attributed to a greater suscepti-bility to genetic drift due to a small effective population size (in the-ory, Y chromosome population size is four times smaller than that ofthe autosomes) or to selective sweeps resulting from positive or neg-ative selection at any Y chromosome locus. Because of low mutationrate (�2 � 10�8 mutations per nucleotide site per generation), SNPson the human Y chromosome are usually regarded as unique eventsin human evolution (Jobling and Tyler-Smith, 2003). Since the com-binations of allelic states of markers along most part of the Y chro-mosome pass intact to the next generation and recurrentmutations are rare, Y-chromosomal haplotypes can be straightfor-wardly identified by typing diagnostic SNPs.

We developed a minisequencing-based assay that allows theunequivocal identification of all domestic goat patrilineages iden-tified so far. The system was designed to detect four diagnosticSNP sites located in two regions of the Y chromosome-linked SRYgene (promoter region and 30UTR) (Table 1, Supplementary Figs.S1 and S2). Previous studies have revealed a high amino acid iden-tity along the entire length of the SRY polypeptide within the Bovi-dae family in disagreement with the overall poor conservationobserved for other mammalian species (Payen and Cotinot, 1994;Waters et al., 2007). In this study, we could confirm that ruminantspecies show a moderate level of nucleotide conservation acrossdifferent SRY noncoding domains. Successful PCR amplificationswere obtained for all tested sheep individuals (for both SRY re-gions) and four out of six cattle samples (only for the SRY 30UTR re-gion). Optimal amplifications were achieved at an annealingtemperature of 54 �C, while higher temperatures generally yieldedpoor amplification in both sheep and cattle samples. The presencein cattle of a 50-bp insertion within one of the PCR primer bindingsite used to amplify the SRY promoter region explains the lack ofamplification observed (Supplementary Figs. S1 and S3).

A BLAST search of the GenBank database did not reveal anysheep sequence homologous to the goat SRY 30UTR region (Supple-mentary Fig. S4). Therefore, seven male sheep from Portugal weresequenced using goat PCR primers for the SRY 30UTR region (Sup-plementary Table S1). Successful amplifications were achieved inall cases with all individual presenting the same sequence (Supple-mentary Fig. S2).

The two SRY domains were also tested for male specificity byPCR using female goat DNA as template. No amplification productswere detected in 10 female goat samples.

In order to assess the robustness of the minisequencing-basedassay, a set of male goat samples from Portuguese, Moroccan andMozambican breeds were genotyped. Different PCR annealing tem-peratures were used to test the specificity and efficiency of thesimultaneous amplification of the two SRY regions. Optimal PCRamplifications were achieved in all tested samples with an anneal-ing temperature of 56 �C. Chelex treated blood samples and phe-nol–chloroform extractions worked equally well.

The products of the multiplex PCR were minisequenced in a sin-gle reaction after enzymatic purification. The concentration of eachextension primer was adjusted to obtain a balanced signal in theelectropherogram of the single-base sequencing reaction. Slightdifferences were observed between the real sizes of extensionprimers and those determined in the automated sequencer(Fig. 1). A similar effect was previously noticed in other assaysand results from variations in the electrophoretic mobility of prim-ers according to their nucleotide composition and attached dye(Quintans et al., 2004; Vallone et al., 2004). However, no overlap

Fig. 1. Schematic electropherograms depicting three goat Y-chromosomal SRY haplotypes Y1A, Y1B and Y2. Each plot illustrates the sizes of extension primers products (x-axis) vs. relative fluorescent units (y-axis). Peaks from left to right in each plot: SRY-3098, SRY-2711, SRY-2971 and SRY-1876. Orange peaks represent the GeneScan-120 LIZinternal size standard.

Table 2Maximum likelihood estimated divergence times for Capra hircus SRY lineages using a JC69 substitution model with gamma-distributed rates with 8 categories(a = 0.4558 ± 0.22985)

Tree bifurcation Divergence time (ky)

Age (Standard Error)a Age (Standard Error)b Age (Standard Error)c

Caprinae/Bovinae 35,700 26,200 22,560 (2970)C. hircus/O. aries 13450 (2830) 9,870 (2080) 8,500C. hircus Y2/C. hircus Y1 1,170 (780) 860 (570) 740 (490)C. hircus Y1A/C. hircus Y1B + Y1C 120 (90) 90 (60) 70 (50)C. hircus Y1B/C. hircus Y1C 40 (40) 30 (30) 30 (30)Bubalus/Bos + Bison 12,950 (2650) 9,500 (1950) 8,180 (1680)B. grunniens/Bison/B. taurus 3,840 (1010) 2,820 (740) 2,430 (640)B. bison/B. bonasus 1,480 (970) 1,080 (710) 930 (610)B. taurus/B. javanicus/(B. frontalis/B. gaurus) 3,060 (870) 2,250 (640) 1,940 (550)B. frontalis/B. gaurus 410 (400) 300 (290) 260 (250)Mutation rate (mutation per site per year) 1.1508 � 10�9 (1.5132 � 10�10) 2.0541 � 10�9 (2.7010 � 10�10) 1.8210 � 10�9 (2.3945 � 10�10)

a Assuming a Caprinae/Bovinae divergence time of 35.7 My.b Assuming a Caprinae/Bovinae divergence time of 26.2 My.c Assuming a C. hircus/O. aries divergence time of 8.5 My.

666 F. Pereira et al. / Molecular Phylogenetics and Evolution 49 (2008) 663–668

between different extended fragments was observed allowing anunequivocal SNP interrogation.

All samples were successfully typed with the minisequencingmultiplex, providing unequivocal results for the four SNPs. As ex-pected, a single peak was observed for each SNP marker in alltested individuals with the exception of an extra blue peak closeto SNP SRY-1876 observed in some profiles (Fig. 1). This unspecificpeak did not affect the haplotype designation because of its local-ization and considerably lower height. Fig. 1 shows a representa-tive electropherogram of typing the four Y-SNPs in individualsfrom Y2, Y1B and Y1A haplotypes.

The majority of goat samples used in this study belong to the Y2haplotype (n = 28), while Y1B and Y1A haplotype are less frequent(with 5 and 3 individuals, respectively). No Y1C individuals wereidentified in our sample (Table 1). Thus, the probability that twounrelated individuals would have identical Y-chromosomal haplo-type is much higher (PM = 0.63) than in Lenstra (2005) dataset(PM = 0.32).

A software was developed to simplify and expedite the processof haplotype identification in a visually interactive interface (Sup-plementary Fig. S5). The SNPYGoat software can be easily adaptedto incorporate newly discovered Y-specific SNPs allowing a fastand unequivocal haplotype assignment. The software can also han-dle incomplete profiles resulting from detection failure of someSNPs in the single-base sequencing reaction.

Estimated divergence times between goat SRY lineages Y2, Y1Aand Y1B (Table 2 and Fig. 2) greatly predate the nearly 10,000 years

of domestication history (Mason, 1984; Zeder, 2006). The level ofsequence divergence observed between these SRY lineages couldbe interpreted as evidence for either (a) three independent domes-tication events from distinct populations of the wild goat Capraaegagrus, possibly in different regions of the Near East and centralAsia; (b) one (or two) domestication events from an extremelylarge population containing phylogenetically divergent lineagesor (c) post-domestication introgressions from wild male goatsbelonging to different wild populations. Further resolution on theY chromosome phylogeny and additional sampling are necessaryto calculate precise divergence dates of Y-chromosomal lineagesas well as to elucidate the extent of male goat contributions todomesticated stocks.

4. Concluding remarks

The Y chromosome SNP typing assay present here has theadvantage of using multiplex PCR and single-base sequencingtechniques in which multiple SNPs are typed with conventionalmolecular genetic laboratory equipments. It requires low quanti-ties of template DNA since four SNPs are analysed in a singlePCR, avoiding the use of expensive sequencing reactions. This factreduces the costs per sample (particularly when more SNPs havebeen included in the assay) without compromising data quality.Moreover, new informative polymorphisms can be implementedsince at least 35 SNPs can be simultaneously typed in a single

0.041082

0.000046

C. hircus Y1C

C. hircus Y1B

C. hircus Y1A

0.000133

C. hircus Y2

0.001345

O. aries

0.015480

0.014895

B. bubalis

B. frontalis

B. gaurus

0.000470

B. taurus

B. javanicus

0.004418

B. bison

B. bonasus

0.001697

B. grunniens

0.003524

0.004

Fig. 2. Phylogenetic tree obtained by the maximum likelihood method on concatenated SRY 50UTR and promoter region sequences (810 bp) from Capra hircus, Ovis aries, Bostaurus, B. bubalis, B. grunniens, B. bison, B. bonasus, B. javanicus, B. frontalis and B. gaurus. Maximum likelihood genetic distances are shown for each node.

F. Pereira et al. / Molecular Phylogenetics and Evolution 49 (2008) 663–668 667

minisequencing reaction (Sanchez et al., 2003). The flexibility ofthe multiplex system in combination with the versatility of thegenotyping software described here offers a convenient way to in-crease the throughput of Y-SNP typing.

The group of samples used to validate this procedure showedthat it is a useful tool for population genetic studies in caprine spe-cies as it may provide valuable insights into paternal relationshipsin a range of populations. It will also permit to estimate the extentof male-mediated gene flow from the wild goat population to man-aged herds for understanding the historical and cultural develop-ment of breeding practices associated with animal domestication.The development of a detailed phylogenetic tree of Y-chromosomallineages may also permit to trace in detail the routes used to trans-port domestic goats in historical and pre-historical times.

Acknowledgments

We are grateful to Faiq Abdellatif and collaborators from theveterinary service of Taroudannt (Morocco) for technical assistancein the collection of blood samples. We also thank Sara Queirós forlaboratory assistance. This work was partially supported by a re-search Grant to FP (SFRH/BD/19585/2004) from Fundação para aCiência e a Tecnologia. PS was supported by a Marie Curie EarlyStage Training Grant. IPATIMUP is partially supported by ‘‘Progra-ma Operacional Ciência e Inovação 2010” (POCI 2010), VI ProgramaQuadro (2002–2006).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2008.08.026.

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