Archivos Latinoamericanos de Producción Animal. 2021. 29 (3-4)

Phylogenetic relationships of Argentin ean Creole with other Latin American Creole Cattle as

revealed by a medium density Single Nucleotide Polymorphism microarray

Recceived: 2020-09-25. Accepted: 2021-02-07

Corresponding author: Mario Andrés Poli; e-mail: poli.mario@inta.gob.ar

Mario Andrés Poli

María Agustina Raschia

The demographic history of Creole cattle in Argentina and in Latin America dates back to the time of the Spanish

colonization. This study aimed to investigate the potential use of a medium-density SNP array to describe cattle

from the most representative and oldest herds of the Argentine Creole cattle breed registered at the time of the

constitution of the Argentinean C reo le Cattle Breeders Ass o c iatio n and to explore the phylogenetic relationship with

Creole cattle from other Latin American countries. To achieve this goal, genotypes from 51 animals on 34,008

autosomal SNP were used to generate genetic distance matrices based on the proportion of shared identical-by-state

alleles among individual animals and animals clustered according to their origin, analyzed by the PLINK program.

A neighbor-joining phylogenetic tree based on pairw is e genetic distance was constructed using PHYLIP and was

prepared for visualization using FigTree. A multidimensional scaling analysis was performed to evaluate the lev el

of relationship in terms of genetic distance among the different animal clusters. Genetic distances between animals

varied from 0.186 to 0.357 when considering all pairs of animals, and from 0.186 to 0.338 when considering Creole

pairs. The dendrogram obtained showed three major clusters. Cluster 1 included Latin American Creole cattle from

Colombia, Guadalupe, Paraguay, and Uruguay, and the reference groups of Holstein and Jersey cattle. Cluster 2

contained exclusively Patagonian Creole cattle, while the third cluster included the remaining Argentinean C reo les.

The genetic relationship patterns obtained via multidimensio nal scaling showed a close relationship among four

groups of Creole animals from Argentina. The closeness between clusters can be explained in part on the basis of

early migration of animals that gave rise to founders herds at some Argentinean locations. The outcom es of this

study contribute to a better understanding of the composition of the early founder herds of Creole cattle in

Argentina and the relationship with other Latin America Creole cattle populations .

Keywords: SNP microarray, multidimensional scaling, phylogenetic tree, Argentinean Creole cattle, Latin Am eric a

Creole cattle

Relaciones filogen étic as del Criollo argentino con otros bovinos Criollos de América Latina según lo revelado

por un microarreglo de polimorfismos de nucleótido simple de mediana densidad

La historia demográfica del bovino Criollo en Argentina y en América Latina en general se remonta a la época de

la colonización española. El objetivo de este estudio fue investigar el uso potenc ial de un microarreglo de SNP de

mediana densidad para describir animales de los ro d eo s más representativos y antiguos en el tiempo en que se

constituyó la Asociación Argentina de Criadores de Ganado Bovino Criollo y explorar la relación filogenética con

otros bovinos criollos de Latino am éric a. Con los genotipos de 51 animales en 34 008 SNP autosómicos se generaron

matrices de distancias genéticas basadas en la proporción de alelos idénticos por estado compartidos entre animales

y entre animales agrupados según su origen, con el programa PLINK. S e construyó un árbol filogenético basado en

distancias genéticas entre pares de animales usando PHYLIP, y se preparó para su visualización con FigTree. Se

realizó un análisis de escalamiento multidimensional para evaluar la relación en términos de d is tanc ia genética

entre los diferentes grupos de animales. Las distanc ias genéticas entre animales variaro n de 0.186 a 0.357 al

considerar todos los pares de animales, y de 0.186 a 0.338 al considerar pares de criollos. El dendrograma obtenido

presentó tres agrupamientos. El grupo 1 incluyó ganado criollo de Colombia, Guadalupe, Paraguay y Uruguay, y

los grupos de referencia Holando y Jersey. El grupo 2 c o m prend ió exclusivamente ganado criollo patagónico,

mientras que el tercer grupo incluyó los restantes criollos argentinos. Los patrones de relación genética obtenidos a

través del escalamiento multidimensional demostraron una íntima relación entre criollos de cuatro orígenes de

Argentina. Esto puede explicarse en parte por la migración temprana de animales que o riginaro n los rodeos

Instituto Nacional de Tecnología Agropecuaria, CICVyA-CNIA, Instituto de Genética “Ewald A. Favret”, Nicolás Repetto y de Los Reseros s/n,

Hurlingham (B1686), Buenos Aires, Argentina

Raschia y Poli

Introduction

The current territory of Argentina received the first

cattle through four routes in the 16th century: Bolivia

in 1549, Chile in 1551, Paraguay in 1554, and the South

of Brazil. Later, the introduction o f animals from the

Canary Islands to the Río de la Plata also occurred.

The spread of cattle before and during that period was

a consequence of the foundation of cities by Spanish

colonizers (Gibert i, 1970).

The geographic conditions, moderate climate,

abundance of pastures , and nearly complete absence

of predators in the central and eastern regions of

Argentina (Pampas) resulted in a great expansion of

livestock. By 1850 the first cattle breed from northern

Europe were imported (Shorthorn, Hereford, A ngus ).

In the following decades, the crossbreeding process

was very extensive in order to obtain animals with a

greater tendency to fatness as required by the export

market. The consequence was a total absorption o f the

Creole cattle of the Pampas region. Creole cattle were

then displaced and confined to regions where other

breeds could not survive, including tropical,

subtropical, arid, and Southwest Patagonia areas

(Martinez et al., 2000).

In 1959, the Instituto Nacional de Tecnología

Agropecuaria (INTA) established the first

experimental Creole cattle herd (consisting of 35 cows

and 2 bulls from the NW region) in Leales in Tucumán

province. During 1959-1970 Creole animals w ere used

as a control “local” breed for the crosses of European

breeds with Zebu cattle. Later (1971-1988), the

objectives were to intensify Creole characterization

and to develop a select nucleus of Creole cattle with

emphasis on the diffusion and insertion of this breed

into the national cattle herds (Holgado and Ortega,

2019). In 1985 and by the joint initiative of private

breeders and INTA, the current Argentinean Creole

Cattle Breeders Assoc iatio n was established.

Creole cattle in Argentina have been studied by

other institutions and their productive and

reproductive behavior has been characterized in

different environments, both in purebred and

crossbred states (Corva et a l., 1995; Holgado and

Rabasa, 2001; Martinez et al., 2003; Rabasa and

Holgado, 2000; Rabasa et al., 2005; Sal Paz et al., 1976).

Early studies involved characterization by blood

groups and other biochemical polymorphisms (Poli,

1986; Poli and Antonini, 1991). Later, studies of genetic

diversity using molecular markers at the DNA level,

including microsatellites and mitochondrial DNA,

were carried out (Giovambattista et al., 1996, 2001;

Lirón et al., 2006; Martinez et al., 2003). Since the

advancement in high-throughput genotyping

techniques such as SNP (single nucleotide

polymorphism) microarrays, several studies using a

relatively large number of SNPs to explore the genetic

diversity, demographic history, and relatedness

between different cattle breeds have been published

(Browett et al., 2018; Mastrangelo et al., 2018;

Sermyagin et al., 2018; Frantz et al., 2020; Meseret et a l.,

2020; Upadhyay et al, 2019). The purpose of this study

was to investigate the potential use o f a medium-

density SNP array to describe cattle from the most

representative and oldest herds of the Argentine

Creole cattle breed registered at the time of the

constitution of the Argentinean Creole Cattle Breeders

Association and to explore the phylogenetic

relationship with Creole cattle from other Latin

American co untries .

Material and Methods

Animals

A total of 42 samples of Creole cattle were used in

this study from the D NA repository of Creole cattle

that INTA has at the Institute of Genetics. Animals

were sampled from 13 herds from 5 countries whose

geographic distribution is shown in Figure 1. Table 1

shows the name of the farm where samples were

collected, the acronyms used in this wo rk, the number

of samples genotyped, the country, and the author and

year of collec tio n. All samples from Argentina were

taken during the phenotypic inspection and

registration process of the first animals in the breeder

association record book. After the Breeders

Association was established and in the following years

a breed standard was drawn up, and training and

dissemination courses on productive and phenotypic

traits of this breed were held. The genotyped samples

were from unrelated animals selected based on the

structure of the herd to which they belonged, their

origins, and the availability of historical, phenotypic,

and pedigree inform atio n. The samples from Paraguay

fundadores en algunas localidades argentinas. Los resultados de este estudio contribuirán a una mejor comprensión

de la formación de los rodeos fundadores de criollos en Argentina y de la relación con otras poblaciones de ganado

criollo de América Latina.

Palabras clave: Microarreglo d e SNP, escalamiento multidimensional, árbol filogenético, bovino Criollo A rgentina,

Bovinos Criollos L at inoam éric a

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Phylogenetic stud y of Latin American Creole cattle

Results

Figure 1. Geographical location of the farms from which samples referenced in Table 1 were taken.

and Uruguay were taken in the framework of the

PROCISUR PTR-Genomics applied to beef production

(2003-2006) project and those from Colombia and

Guadalupe were sent by German Martinez C or real

and Michel Naves, respectively. Furthermore, five

Holstein and five Jers ey bulls samples fro m the central

area of Argentina were included as outgroups.

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Table 1. Farm name, acronym us ed, number of samples genotyped, c o untry and name of the Creole breed and year of sample

collection.

Sample collection and DNA extraction

Fresh b lo od was obtained from the jugular vein of

animals using EDTA as anticoagulant. The procedure

for blood sample collection was approved by the

Institutional Co m mit tee for Care and Use of

Experimental Animals of the National Institute of

Agricultural Technology (CICUAE-INTA) and

followed the guidelines described in the institutional

manual. Genomic DNA was extracted using a

commercial kit (AxyPrep Blood Genomic DNA

Miniprep Kit, Axygen Biosciences , Union City, CA)

according to the protocol supplied by the

manufacturer. Genomic DNA from some bulls was

obtained from semen straws using a phenol-

chloroform extraction method. DNA quantity and

quality were determined by measuring its UV

absorption at 260 and 280 nm and the 260/ 280 and

260/230 abs o rbanc e ratios, using a NanoDropTM 1000

spectrophotometer (Thermo Scientific, Wilmington,

Delaware, USA ).

Genotyping

Creole animals were genotyped with the GeneSeek®

Genome Profiler (GGP) Bovine 150K v2 microarray, while

the reference groups of Holstein and Jersey bulls were

genotyped using the BovineSNP50 v2 BeadChip (Illumina

Inc., San Diego, CA, USA). Genotyping was performed

on all animals by GeneSeek (Neogen Corporation

Company, Linc o ln, NE, USA).

The GGP 150K v2 microarray evaluates 138 892 SNP

distributed over the 29 bovine autosomes, sex

chromosomes, and mitochondrial DNA, spaced on

average 18,868 bp apart throughout autosomes and 38

523 bp apart throughout the genome. The BovineSNP50

v2 BeadChip evaluates 54 609 SNP distributed over the

29 bovine autosomes and sex chromosomes, spaced on

average 48 102 bp apart throughout the genome. SNP

locations were mapped to the bovine assembly ARS-

UCD1.2 (Rosen et al., 2020) for computations.

The quality control of genotype data was performed

using PLINK program (Purcell et al., 2007) and consisted

in the exclusion of SNP with unknown position on the

genome, located on sex chromosomes, with a call rate

lower than 0.90, a minor allele frequency lower than 0.03

or a p-value for the exact test to detect deviations from

the Hardy-Weinberg equilibrium lower than 1.10-4.

Animals with a call rate lower than 0.80 were also

excluded.

Phylogenetic analysis

A matrix of the genetic distances for all pairs of

animals, estimated as 1 – IBS, being I B S the proportion

of alleles identical by state, was o btained with PLINK

San Martin farm had identified animals from different origins as: L P-L o s Planteles, TxC-Tandil x Cerrillada, CR-Carlos Romero, and Ch-

Chasquivil.

Samples from Argentina, Paraguay, Colombia, Uruguay, and Guadalupe were taken by Poli, M., Ferreira, N., Martínez Correal, G., Postiglioni,

A.and Naves, M. , respectively.

Farm´s Name Acronym N Country (Crio llo ´s name) Sample collec tio n

9 de Julio 9J 3 Argentina (Criollo Argentino Patagónico) 1991

Cruz de Guerra CG 5 Argentina (Criollo Argentino) 1992

INTA Leales CERL 4 Argentina (Criollo Argentino) 1992

La Josefina LJ 2 Argentina (Criollo Argentino) 1992

San Martin

LP, TxC,CR , Ch 8 Argentina (Criollo Argentino) 1993

Nueva Valencia NV 1 Argentina (Criollo Argentino) 1993

Las Acac ias LA 4 Argentina (Criollo Argentino) 1995

Bartolomé d e Las

Casas BC 2 Argentina (Criollo Argentino) 1995

San Carlos SC 2 Paraguay (Criollo Pilcomayo) 2007

Santa Gabriela SG 2 Paraguay (Criollo Pampa Chaqueño) 2007

Centro de Investigación

la Libertad de CORPOICA Col 3 Colombia (Sanmartinero) 2000

Reserva Parque Nacional

San Miguel Uy 3 Uruguay (Criollo Uruguayo) 2007

INRA Unit of

Zootechnique Research IG 3 Guadalupe (Criollo ) 2000

Raschia y Poli

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Results and Discus s ion

After genomic data quality control, genotypes from

51 animals on 34 008 autosome SNP shared by both

microarrays used were available for subsequent

analyses. Genotypes on those SNP were used to

calculate the genetic distance for all pairs of animals.

Genetic distances between animals v aried from 0.186

to 0.357 when considering all pairs of animals. T he

genetic distance among all Argentinean creole varied

from 0.186 to 0.338. While the highest genetic distance

(0.357) was found between two bulls, a Holstein and a

Jersey, from the outgroups, among Creole animals the

highest distance (0.338) was ob serv ed between an

animal from Guadalupe and another from 9 de Julio.

The dendrogram in Figure 2 shows that the 51

animals were grouped into three major clusters.

Cluster 1 included Creo le cattle from Colombia,

Guadalupe, Paraguay, Uruguay, and the two

outgroups Holstein and Jersey. Cluster 2 con

tained

only and exclusively anim als from Patagonia (9 de

Julio farm) and cluster 3 all the remaining Argentinean

Creole.

Figure 2. Dendrogram built based on genetic distances between animals estimated according to the proportion of shared IBS

alleles J, Jersey; H, Holstein; SG (Santa Gabriela - Parag uay); IG, (Isla Guadalupe); SC (San Carlos – Paraguay); Col (Colombia);

Uy (Res erv a Parque Nacional San Miguel, Uruguay); and Argentinean Creole cat tle: 9J (9 de Julio ); LJ (La Josefina); LP (Los

Planteles); TxC (Tandil x Cerrillada); CR (Carlos Romero); NV (Nueva Valencia); B C (Bartolomé de las Casas); Ch (Chasquivil);

CERL (INTA Leales ); CG (Cruz de Guerra); LA (Las Acacias).

Phylogenetic stud y of Latin American Creole cattle

program. This file was adapted to the input format

required by the Neighbor program in PHYLogeny

Inference Package (PHYLIP; Felsenstein, 1989) for

inferring phylogenies. Briefly, the neighbor-joining

method, a distance matrix method producing an

unrooted tree, implemented in Neighbor was used.

The output of this program, a tree file in nested-

parenthesis notation, was used as input for the

Consense program of PHYLIP, which computed the

consensus tree by the majority rule cons ens us tree

method. The consensus tree was prepared for

visualization using FigTree v1.4.3 program (Rambaut,

2009).

Multidimensional scaling

A multidimensional scaling (MDS) analysis on the

mean identical by state (IBS) pairwise genetic

distances between animals clustered according to their

geographical origin was performed with PL I NK

program. Then, a reduced representation of the data in

two dimensio ns was generated. Each point in this plot

represents a group of animals clustered according to

their origin.

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Cluster 1 presented five subgroups of Creo le cattle

from four Latin American countries and both breeds

used as outgroups (Holstein and Jersey), all clearly

differentiated. The phylogenetically closest subgroup

to the Argentinean Creole cattle was that from

Uruguay. Creole animals from these two countries are

phenotypically v ery similar on coat color diversity and

body and horn shape (Personal inspection, October

2019). Genetic distances among the Argentinean and

Uruguayan Creole animals g enot yped varied between

0.294 and 0.32, with a mean of 0.305. Uruguayan

Creole Cattle samples came fro m a single population

of approx imat ely 600 pure individuals restricted to

San Miguel National Park, in the Northeast of

Uruguay. The foundation herd consisted of 35 Creole

cows, b ulls , and calves brought from different

locations around 70 years ago (Armstrong, et al., 2013).

Colombian Creole samples belonged to the

Sanmartinero breed, c harac terized by uniform coat

color, and to the founders’ herds in the La Libertad

Research Center of CORPOICA in Villavicencio. The

samples analyzed from Paraguay did not constitute a

narrow cluster and this is due to the conformat io n of

both groups, Criollo Pilcomayo and Criollo Pampa

Chaqueño. The Criollo Pilcomayo belongs to a single

population and is phenotypically very similar to the

Argentinean Creole cattle due to its body structure,

coat color diversity, and horns. This herd was made

up of animals collected by Mr. E. Prayo nes in the 70s

in the area comprised by the coast of the Pilcomayo

river (border with Argentina) and the E st ero Patiño in

the western region (Mr. Prayones personal

communication to MP during S an Carlos farm visit,

August 2007). On the other hand, the Criollo Pampa

Chaqueño is phenotypically sim ilar to the Hereford

breed regarding coat color and its body distributio n

and conformation. In a comparative study of Pampa

Chaqueño, Criollo Pilcomayo, and Hereford cattle

using microsatellite mo lec ular markers, the genetic

diversity and their profile were clearly different

(Martínez-López, 2019). Ou r SNP analysis results

located Criollo Pampa Chaqueño cattle as a very

distant group from the other Creoles, including those

from Guadalupe, and the closest to the two outgroups

of European breeds.

Cluster 2 contained only animals from Patagonia (9

de Julio farm). These samples belonged to animals that

were obtained by R. Martinez and A. R o drig uez with

the advice of H. Echev erria (9 de Julio farm) from Los

Glaciares National Park in 1990, in the framework of

the Agreement project between the Lomas de Zamora

University and the National Parks Administratio n.

The origin of the Creole cattle of Patagonia was

described in detail by Martinez (2008) in his doctoral

thesis, who concludes that these animals would have

been the descendants of those that between the end of

the 19th century and the beginning of the 20th century

led the first settlers who colonized some of the sectors

from current Los Glaciares National Park.

Although information on the movement of animals

is scarce, the s am e author mentions: “. . . one of the

highlights is the transfer by boat of a group of 80 cattle from

the Estancia "La Cristina" to the Bahía Onelli sector (Tierra

de Nadie) (Echeverría Horacio Sr., personal

communication.). . . . ”. In 1937 the Los Glaciares

National Park was created and their inhabitants were

withdrawn from those territories, however, many of

the cattle could not be removed and they became

"feral" free-range cattle. The prolonged process of

geographic isolat ion and free breeding of these

animals has given rise to what today is the Patagonian

Creole cattle origin. Although they have phenotypic

characteristics almost indistinguis hable from the other

Creoles of Argentina, mostly in coat color, horn, and

body shape, the mean genetic dist ance found in this

study between these animals and the rest of the

Argentinean Creole is 0.303. This result is in

accordance with that found by Martinez (2008) using a

panel of 27 microsatellites on 36 Patagonian cattle and

45 Creole cattle categorized as from NW Argentina.

cluster 3 three major subgroups can be

distinguished. The first one comprised animals

sampled at San Martin farm (L o s Planteles, Tandil x

Cerrillada, and Carlos Romero herds; breeder M.

Pereyra Iraola) and La Josefina farm (Breeder F

Marenco). The animals sampled at San Martin farm

were d es cend ants from thos e brought by Mr. Martín

Pereyra Iraola grandfather from the NW region of

Argentina in 1938, kept until 1949 at “Estancia San

Juan” (current Pereyra Iraola Park), and then

transferred to Tandileoufu ranch near the city of

Tandil (M. Pereyra Iraola personal communication,

1986-7). On the other hand, cattle from La Josefina

have their origin in a herd that in 1920 Mr. Carlos

Romero brought from the “Alto Peru” (Bolivia) to his

farm in the province of Córdoba. The las t animals of

this herd were auctioned in the city of Bahia Blanca

and acquired by Mr. F. Marenco, Mr. M. Eyerabide,

and E..Martinez Reboul (F. Marenco and E. Martinez

Reboul, personal communication, Dec2-4, 1991). The

closeness of the animals in this cluster is due to the

exchange between the herds from Pereyra Iraola and

Carlos Romero, which can be inferred from the names

of t he animals registered in the Breeders Assoc iatio n

phenotypic inspection and the Argentine Rural Society

(SRA) books. Traditionally, pedigree record books

Raschia y Poli

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Figure 3. Multidimensional scaling plot based on the mean IBS pairwise genetic distances among animals from different

geographical origins. Each dot represents a group of animals clustered according to their origin. J, Jersey; H, Holstein; SG (Santa

Gabriela - Paraguay); IG, (Isla Guadalupe); SC (San Carlos – Paraguay); Col (Colom bia); Uy (Reserva Parque Nacional San

Miguel, Uruguay); and Argentin ean Creole cattle: 9J (9 de Julio); LJ (La Josefina); LP (Los Planteles); TxC (Tandil x Cerrillada);

CR (C arlo s Romero); NV (Nueva Valencia); BC (Bartolomé de las Casas); C h (Chasquivil); CERL (INTA Leales); CG (Cruz de

Guerra); LA (Las Acacias).

Phylogenetic stud y of Latin American Creole cattle

present a name for each anim al which is formed in the

first part by the name of the seed stock followed by the

own name and then, depending on available

information, the name of the father, mother, or place

of origin. This can be verified in the name of some

animals, for example a cow born in 1984, with

Particular ID 40 (ID: animal identification, usually is a

tattoo), named “PLANTELERO AVUTARDA” and

sired by "ROMERO", or a bull with Particular ID 197,

named. “PLANTELERO R U M BE A D OR ROMERO

197”. Several examples like these can be seen in the

record boo k.

The intimate genetic relationship among LP, LJ, T xC ,

and CR is very clear. Probably the similarity of the

animals in this cluster was given by several factors,

such as the time that has elapsed since their origins

(approximately 10 generations), the reduced size o f the

herds, mainly in Pereyra Iraola (35-40 females), the use

of few bulls, and the exchange of animals between

herds. Moreover, the genetic distance between two

animals geno typed from Los Planteles was the lowest

(0.186) and thus they had the closest relationship.

The second subgroup in cluster 3 has four samples,

two animals sampled in Bartolomé de las Casas farm,

one in Nueva Valencia farm, and one in S an Martin

farm. The first three belonged to the same o riginal

herd, INTA Estación Experimental El Colorado (S.

Luque, personal communication, April 4, 1993) and

hence the clo s e clustering. The fourth belongs to an

animal originated in the province of Tucuman

(Chasquivil).

The third subgroup comprised animals from CER

Leales, Cruz de Guerra, and Las Acacias farm s. Within

this group, the lowest genetic dist ance (0.208), was

detected between two bulls, one from Las Acacias and

the other from Cruz de Guerra. Both farms, Las

Acacias and Cruz de Guerra, established their first

herds with Creoles from CERL (S. Rabasa, M.

Garciarena, and E. Andreani, personal

communication). CERL was the main reservoir and

point of dissemination of the Creole cattle from the

NW region of Argentina and it has the oldest and most

complete pedigree records since the ‘70s.

The multidimensional scaling analysis enabled visual

iden

tification of animal clusters and the lev el of

relationship among them in terms of genetic distance

among the anim als from the different origins studied

(Figure 3).

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An intimate genetic relatio ns hip among LP, LJ, TxC,

and CR is suggested from the MDS analysis as well as

from the phylogenetic relatio ns hip reconstruction. This

can be explained, as mentioned above, by the exchange

of animals between the main herds bro ught from the

north by Iraola and Romero. Furthermore, the

breeding practices and the elapsed time probably

made these animals very genetically different from the

other Creole cattle (mean genetic d is tanc e to other

Argentinean Creoles: 0.303), Although it is very likely

that the Patagonian Creoles originated from the large

populations of cattle that existed in the Pampas region,

the great phylogenetic differenc es observed in this

study between them and those sampled in San Martin

(Pampas region) have at least two possible

explanations: 1) although Creole cattle were seen in

Patagonia towards the end of the 18th century

(Martinez, 2008), the sampled animals were

geographically isolated since the late 19th century in

Los Glaciares National Park; 2) the Creole cattle

sampled in San Martin had their origins in the two

herds brought from the NW between 1920 and 1938.

Moreover, in the MDS the Patagonian animals seem to

be more distant from those of San Martin than the rest

of the Argentinean Creole and from those of Uruguay.

Isolation and genetic drift can also contribute to these

differences.

Animals from CG, CERL, and LA clustered into the

same subgroup in the phylogenetic tree, being

intermixed instead of arranged according to their

origin. Moreover, the points corresponding to thes e

three geographical origins were located very close to

each other in the MDS plot. When we investigated the

background of animals from Cruz de Guerra, we

found that all of them came from CER Leales,

confirming the results obtained in our study. T he plot

also clearly separates the Creoles of Co lo mb ia,

Gaudalupe, and Paraguay, from the Creoles from

Argentina and Uruguay.

As mentioned above, the objective of the study was

to investigate the potential use of a medium-density

SNP array to explore the phylogenetic relationship of

Argentinean Creole cattle from founder herds with

other Latin American Creoles. The results obtained

from the analysis of the genetic distance matrix and

showed in the dendrogram and in the MDS plot are in

agreement with the animals’ origins, as well as with

their demographic history and breeding information.

An example of the usefulness of these tools can be

seen with sample ID-Lab 859 (sample identification in

the laboratory) which was sampled at San Martin farm

(near Tandil city - Buenos Aires province) in 1993.

However, in the dendrogram it was positioned in the

same cluster as the animals from Bartolomé de las

Casas (BC) and Nueva Valencia (NV), and the MDS

plot also showed it very distant from the San Martin

farm cluster. T his sample was a young bull brought by

Mr. Pereyra Iraola from Chasquivil (Ch), Tucumán

province, in 1991, about 1300 km away to San Martin

farm (M. Pereyra Iraola, personal communication, July

16, 1993). This bull is registered in the SRA book as:

“Particular ID 001; Birth Date: 00/00/90; sex: M;

Name: “PLANTE L E R O CHASQUIVI L ”; Coat colors

cod 2,51,96; Date of Inspection: 07/16/93”.

Traditionally a breeder association is based on a

phenotypic st and ard and genealogical records. When

there are no kinship records and the animal has

characteristic phenotypic traits, in general the animal

is not accepted. However, some associations accept

such animals as a “base” category and it takes at least

three generations with full ped igree records for their

progeny to be accepted as purebred.

The availability of information on single nucleotide

polymorphisms in bovines has increased dramatically

in the last ten years. These markers have been used to

describe groups of animals and breeds based on

genomic data (Gorbach, et al., 2010; Hulsegge, et al.,

2013; Hulsegge, et al., 2019). However, the assignment

of an animal to a specific breed by means of a genetic

test requires the genotypes o f a reference group of

animals with defined phenotypic characteristics (breed

standard) and known origin, which is called a

"Reference Population". All individuals in a reference

population must represent genetic diversity within the

particular breed. The clusters constituted within

Argentina and the Latin America Creole demonstrate

that the genetic variability is very large and hence

present a big challenge to build C reole reference

populations and to select the minimum number of

SNPs with the highest capability and reliability to

differentiate between Creole breeds.

Conclusion

In conclusion, this study demonstrates the

effectiveness of using genotypes obtained through a

medium-density SNP array to reconstruct the

phylogenetic relationship of animals belonging to

Creole founder herds of Argentina and the genetic

distance among them and Creole cattle from other

Latin American countries. The outcomes from this

study shed light on the genetic differences of the early

founder herds of Creole cattle in Argentina and on

how Creole cattle populations expanded and spread

throughout Latin A m erica, in order to contribute to a

better understanding of the evolution of these cattle.

Raschia y Poli

ISSN-L 1022-1301. Archivos Latinoamericano s de Producción Animal. 2021. 29 (3-4): 91-100

Armstrong, E., A. Iriarte, A. M. Martínez, M. Feijoo , J.

L. Vega-Pla, J. V. D elgad o and A. Postiglioni. 2013.

Genetic diversity analysis of the Uruguayan Creole

cattle breed using microsatellites and mtDNA

markers. Genet. Mol. Res. 12: 1119-1131.

http://dx.doi.org/10.4238/2013

Browett, S., G. Mc Hugo , I. W. Richardson, D. A.

Magee, S. D. E. Park, A. G. Fahey, J. F. Kearney, C.

N. Correia, I. A. S. Randhawa and D. E. MacHugh.

2018. Genomic characterization of the indigenous

Irish Kerry cattle breed. Front. Genet. 9:51.

https://doi.org/10.3389/fgene.2018.00051

Corva, P. M., E. L. Villarreal, C. A. Mezzadra and L. M.

Merlucci. 1995. Reproductive traits of Angus,

Criollo and reciprocal crossbred females in the

temperate area of Argentina. Anim. Sci. 61: 241-249.

https://doi.org/10.1017/S135772980001376X

Declaration of interest: The authors have no conflicts of interest to declare.

Acknowledgments

To the breeders and INTA for their visio n and

conviction to establish a Creole Breeders Association

and also their hospitality and talks about the history of

their herds and the origin of the animals during

inspection and sampling in the early ’90. To Germán

Martinez Correal and Michel Naves for providing

samples from Sanmartinero and I s la Guadalupe,

respectively and to Kathiravan Periasamy for technical

assistance and guidance and Mateo Poli Tieffemberg

for the edition of Figure 1.

Funding: This study was supported by I ns tituto Nacio nal de Tecnología Agropecuaria (INTA ) grant PE 145; FAO-

IAEA C R P D3.10.28 and PRIN2017 Program of the Italian M inis try of Education, University and Research (MIUR).

Literature Cited

Felsenstein, J. 1989. PHYLIP - Phylogeny Inference

Package (Version 3.2). Cladistics 5: 164-166.

https://evolution.genetics.washington.edu/phylip.

html

Giberti, H. C. E. 1970. Historia económica de la

ganadería argentina. Ediciones Solar/Hachete.

Buenos Aires . 217 páginas.

Giovambattista, G., C. D. Golijow , F. N. Dulout and M.

M. M. Lo j o . 1996. Gene frequencies of DRB3.2 locus

of Argentine Creole cattle. Anim. Genet. 27: 55-56.

https://doi.org/10.1111/j.1365-2052.1996.tb01178.x

Giovambattista, G., M. V. Ripoli, P. Peral-García and J.

L. Bouzat. 2001. Indigenous domes tic breeds as

reservoirs of genetic diversity: The Argentine Creole

Cattle. Anim. Genet. 32: 240-247.

https://doi.org/10.1046/j.1365-2052.2001.00774.x

Gorbach, D. M., M. L. Makgahlela, J. M. Reecy, S. J.

Kemp, I. Baltenweck, R. Ouma, O. Mwai, K.

Marshall, B. Murdoch, S. Moore and M. F.

Rothschild. 2010. Use of SNP genotyping to

determine pedigree and breed composition of dairy

cattle in Kenya. J. Anim. Breed. Genet. 127: 348-351.

https://doi.org/10.1111/j.1439-0388.2010.00864.x

Hulsegge, I., M. Schoon, J. Windig, M. Neuteboom , S. J.

Hiemstra and A. Schurink. 2019. Development of a

genetic tool for determining breed p urity of cattle.

Livestock Science 223: 60-67.

https://doi.org/10.1016/j.livsci.2019.03.002

Holgado, F. D. and A. Rabasa. 2001. Herencia del

carácter “sin cuernos” en el bovino Criollo

Argentino. Zo ot ecnia Trop. 19(2): 185-190.

Holgado, F. D . and M. F. Ortega. 2019. Caracterización

productiva del bovino criollo argentino: período

2006-2016. Buenos Aires: Ediciones INTA. 26p. ISBN

978-987-521-987-8.

Lirón, J. P., P. Peral-García and G. Giovambattista.

2006. Genetic characterization of Argentine and

Bolivian Creole cattle breeds assessed through

microsatellites. J. Hered. 97(4): 331-339.

https://doi.org/10.1093/jhered/esl003

Martínez-López, O. R., S. B. P. Barbosa, A. M. Martínez,

J. V. Delgado and V. Landi. 2019. Perfil genético de

dos poblaciones bovinas criollas de Paraguay. Actas

Iberoamericanas de Conservación Animal -AICA 14:

141-153.

Martínez, R. D., E. N. Fernández, E. R. Género y F. J. L.

Rumiano. 2000. El ganad o bo v ino crio llo en

Argentina. Arch. Zootec. 49: 353-361.

Martinez, R. D. 2008. Caracterización genética y

morfológica del bovino criollo argentino de origen

patagónico.

http://www.uco.es/conbiand/tesis/Ruen_Martine

z.pdf

Martínez, R. D., G. Giovambattista, M. V. Ripoli, J. C.

DeLuca and F. N. Dulout. 2003. Patagonian

Argentine Creole cattle polymorphism: comparison

with North-West populations of this breed . Res. Vet.

Sci. 74: 287-290. https://doi.org/10.1016/s0034-

5288(02)00190-x

Phylogenetic stud y of Latin American Creole cattle

Hulsegge, B., M. P. L. Calus, J. J. Windig, A. H. Hoving-

-Bolink, M. H. T. Maurice-van Eijndhoven and S. J.

Hiemstra. 2013. Selection of SNP from 50 K and 777

K arrays to predict breed of origin in cattle. J. Anim.

Sci. 91, 5128-5134. https://doi.org/10.2527/jas.2013-

6678

ISSN-L 1022-1301. Archivos Latinoamericano s de Producción Animal. 2021. 29 (3-4): 91-100

Frantz, L.A.F., Bradley, D.G., Larsonand, G., Orlando,

L. 2020. Animal domestication un era of ancient

genomics. Nature reviews. Genetics.

https://doi.org/10.1038/s41576-020-0225-0

100

Rabasa, A. E., F. D. Holgado and M. A . Poli. 2005.

Bovino Criollo de Argentina: diferentes aspectos en

su caracterización. A gro cien cia vol IX N° 2 y 3: 473-

477.

Poli, M. A. 1986. Asociación entre factores sanguíneos,

transferrinas, genes Ps, Bs y aspectos productivos en

bovinos c rio llo s . RIA, XXI, N 2, 67-76.

Poli, M.A. and A. G. Antonini. 1991. Genetic structure

of milk proteins in Argentinian Holstein and

Argentinian Creole cattle. Hereditas 115: 177-182.

Purcell, S., B. Neale, K. Todd-Brown, L. Thomas, M. A.

R. Ferreira, D. Bender, J. Maller, P. Sklar, P. I. W. de

Bakker, M. J. Daly and P. C. Sham. 2007. PLINK: A

Tool Set for Whole-Genome Associ atio n and

Population-Based Linkage Analyses. Am. J. Hum.

Genet. 81(3): 559-575.

https://doi.org/10.1086/519795

Rabasa, A. and F. D. Holgado. 2000. Reproductive

evaluation of complete dialelic crossbreeding creole-

Nelore. Zootec nia Tropical. 18(1): 79-90.

Rambaut, A. 2009. FigTree, a graphical view er of

phylogenetic trees. I nst itute of Evolutionary

Biology, University of Edinburgh, Edinburgh,

United Kingdom .

Rosen, B. D., D. M. Bickhart, R. D. Schnabel, et al., 2020.

De novo assem bly of the cattle reference genome

with single-molecule sequencing. Gigascience.

9(3):giaa021.

https://doi.org/10.1093/gigascience/giaa021

Sal Paz A. R. de, F. Sal Paz, F. Bergmann and S. L.

Rabasa. 1976. Asociació n de la fert ilid ad femenina

con genes m end eliano s mayores en bovinos

Criollos. M end eliana 1 (2): 91-95.

Sermyagin, A. A., A. V. Dotsev, A. A. Gladyr, A. A.

Traspov, T. E. Deniskova, O. V. Kos ty unina, et al.

2018. Whole-genome SNP analysis elucidates the

genetic structure of Russian cattle and its

relationship w ith Eurasian taurine breeds. Genet.

Sel. Evol. 50:37. https://doi.org/10.1186/s12711-

018-0408-8

Raschia y Poli

Mastrangelo, S., E. Ciani, P. Ajmone Marsan, A.

Bagnato, L. Battaglini, R. Bozzi, et al. 2018.

Conservation status and historical relatedness of

Italian cat tle breeds. Genet. Sel. Evol. 50:35.

https://doi.org/10.1186/s12711-018-0406-x

ISSN-L 1022-1301. Archivos Latinoamericano s de Producción Animal. 2021. 29 (3-4): 91-100

Meseret, S., Mekonnenb, Y.A., Brenigc, B., Schtz, E,

Hannotte, O., Gltas, M ., Schm itt, A.O. 2020. Genetic

diversity and population structure of six ethiopian

cattle breeds from different geographical regions

using high density single nucleotide

polymorphisms- Livestock Science 234.103979.

Upadhyay, M., Eriksson, S., Mikko, S., Strandberg, E.,

St. Ihammar, H., Groenen, M.A.M., Crooijmans,

R.P.M.A., Andersson, G. and Johannson, A.M. 2019.

Genomic relat edness and diversity of swedish

native cattle breed s . Genet Sel Evol 51: 56

https://doi.org/10.1186/s12711-019-0496-0