Genetic diversity of Calpain 1 gene in Creole, Nellore and Brahman bovine breeds in Bolivia

Archivos Latinoamericanos de Producción Animal. 2022. 30 (2)
www.doi.org/10.53588/alpa.300206
Genetic diversity of Calpain 1 gene in Creole, Nellore and
Brahman bovine breeds in Bolivia
Juan A. C. Pereira
1
Agustin H. Falomir­Lockhart2
Ariel J. Loza
Egle E. Villegas­Castagnasso2
Pedro Rojas
Monica H. Carino2
Rodrigo Hoyos Andres Rogberg­Muñoz2
Guillermo Giovambattista2
1Facultad
2
de Ciencias Veterinarias, Universidad Autónoma Gabriel René Moreno, Santa Cruz de la Sierra, Departamento de Santa Cruz, Bolivia.
IGEVET ® Instituto de Genética Veterianaria "Ing. Fernando N. Dulout (UNLP­ CONICET La Plata), Facultad de Ciencias Veterinarias,
Universidad Nacional de La Plata. Av. 60 y 118 ­ La Plata (1900), Buenos Aires, Argentina.
Abstract. In Bolivia, beef production is mainly based on two genotypes, Bos taurus (Creole cattle) and B. indicus
(zebu), being weight gain the main selection criteria used by farmers. However, meat quality and especially
tenderness must be incorporated in the selection process. Meat tenderness is partly determined by the calpain
(CAPN1)/calpastatin (CAST) protein system. Thus, the objective of the present work was to determine and
compare the genetic variability of the CAPN1 gene in Creole (CreBo), Brahman (BraBo) and Nellore (NelBo) breeds
in Bolivia. DNA was extracted from blood samples from 147 CreBo, 59 BraBo and 93 NelBo, and three
polymorphisms were genotyped using ARMS­PCR (CAPN1­316 and CAPN1­4751) and PCR­RFLP (CAPN1­530).
Furthermore, CAPN1­316 and CAPN1­4751 SNPs were analyzed with Axiom™ Bos 1 Genotyping Array r3 and the
Axiom™ ArBos 1 Genotyping Array. Allele frequencies associated with higher tenderness in CreBo, BraBo and
NelBo were 0.22, 0 and 0.09 (CAPN1­316 C; P < 0.001), 0.76, 0.16 and 0.08 (CAPN1­4751 C; P < 0.001), and 0.77, 0.92
and 0.94 (CAPN1­530 G; P < 0.001). Linkage disequilibrium (LD) analysis revealed the presence of two LD blocks.
Our results evidence that CreBo has a higher frequency of alleles associated with higher meat tenderness than the
cebuine breeds. These markers could be used in breeding programs to improve Bolivian cattle herd meat quality
either by selection within Creole breeds or crosses with cebuine cattle.
Keywords: Bolivian Creole, Brahman, Nellore, tenderness, CAPN1, SNPs.
Diversidad genética del gen Calpaina 1 en las razas bovinas Criollo, Nellore
y Brahman en Bolivia
Resumen. En Bolivia, la producción de carne se basa principalmente en la cría de dos genotipos, Bos taurus (ganado
Criollo) y B. indicus (cebú), siendo la ganancia de peso el principal criterio de selección utilizado por los criadores.
Sin embargo, la calidad y especialmente la terneza de la carne deben ser incorporadas al proceso de selección. La
terneza en parte está determinada por el sistema proteico calpaína (CAPN1)/calpastatina (CAST). Por lo tanto, el
objetivo del presente trabajo fue determinar y comparar la variabilidad genética del gen CAPN1 en las razas Criolla
(CreBo), Brahman (BraBo) y Nelore (NelBo) de Bolivia. El ADN se extrajo de muestras de sangre de 147 CreBo, 59
BraBo y 93 NelBo, y tres polimorfismos se genotipificaron por ARMS­PCR (CAPN1­316 y CAPN1­4751) y PCR­
RFLP (CAPN1­530). Adicionalmente, los SNPs CAPN1­316 y CAPN1­4751 fueron analizados con los microarrays
Axiom™ Bos 1 Genotyping Array r3 y the Axiom™ ArBos 1 Genotyping Array. Las frecuencias de los alelos
asociados con una mayor terneza en CreBo, BraBo y NelBo fueron 0.22, 0 y 0.09 (CAPN1­316 C; P < 0.001), 0.76, 0.16
y 0.08 (CAPN1­4751 C; P < 0.001), y 0.77, 0.92 y 0.94 (CAPN1­530 G; P < 0.001). El análisis del desequilibrio de
ligamiento reveló la presencia de dos bloques. Estos resultados muestran que CreBo presenta una mayor frecuencia
de alelos asociados a mayor terneza en la carne que las razas cebuinas analizadas. Estos marcadores podrían ser
utilizados en los programas de cría para mejorar la calidad de la carne del ganado boliviano, tanto por selección
dentro del ganado Criollo como por cruzamiento con ganado cebú.
Palabras clave: Criollo Boliviano, Brahman, Nelore, terneza, calpaína, SNPs.
1Recibido:
2021­09­03. Aceptado: 2021­12­12
Autores para la correspondencia: [email protected] ; [email protected]
1 IGEVET – Instituto de Genética Veterinaria “Ing. Fernando N. Dulout” (UNLP­CONICET LA PLATA), Facultad de Ciencias
2 Veterinarias, Universidad Nacional de La Plata. Avenida 60 y 118 ­ La Plata (1900), Buenos Aires, Argentina.
2
121
122
Pereira et al.
Abbreviations
Ang, Angus; ARMS­PCR, amplification­refractory mutation system­polymerase chain reaction; BraAr, Argentinean
Brahman; BraBo, Bolivian Brahman; Brf, Braford; Brn, Brangus; CAPN1, calpain; CAST, calpastatin; CreAr,
Argentinean Creole; CreBo, Bolivian Creole; CreCh, Chaqueño Creole; CreSa, Saveedreño Creole; CreVa, Valley
Creole; CreYa, Yacumeño Creole; Hol, Holstein; LD, linkage disequilibrium; NelBo, Bolivian Nellore; PCR­RFLP,
polymerase chain reaction­Restriction Fragment Length Polymorphism.
Introducción
First bovines were introduced to the Americas in the
15th century by the Spanish conquerors, which landed
in the Caribbean Islands and then passed to South
America through Santa Marta (Colombia) (Primo,
1992). In less than 50 years, cattle were introduced to
Venezuela and Peru, which were the main centers of
dispersion. From Peru, bovines were transported to
Bolivia, Paraguay and Chile, finally reaching
Argentina and Uruguay. By the year 1524, the
presence of cattle in all South American countries was
reported. Expansion throughout the continent and
adaptation to different nature conditions allowed a
great phenotypic variability and the creation of the so­
called “Creole” breeds (Primo, 1992).
Since 19th century, the introduction of highly
selected commercial European (Shorthorn, Hereford,
Aberdeen Angus) and Zebu (Nellore, Gir, Guzerat)
breeds led to a drastic reduction of the Creole cattle
populations, which were relegated to marginal
regions. Over time, Creole cattle lost value against the
introduced breeds, and the Creole pure breed mainly
remained where commercial breeds were not
productive. In this context, cattle breeding in Bolivia
was not an exception (de Alba, 2011). Nowadays, beef
cattle industry in Bolivia is mainly located in the
departments of Beni and Santa Cruz, where 80 % of
the eight million Bolivian cattle are bred. Although the
department of Beni has a greater number of heads, the
department of Santa Cruz concentrates the main
breeders (genetics). Most abundant beef breed is
Nellore, followed by Brahman and Creole. These Zebu
breeds were introduced in the mid­20th century and
adapted to their new environment in an almost
natural way, and in few years replaced Creole cattle
from the humid tropical region of Bolivia (Beni and
north of Santa Cruz). However, there is still a
considerable number of Creole cattle in Santa Cruz,
especially in the area called Chaco, characterized by its
dry seasons and silvopastoral systems. Therefore, Bos
indicus and B.taurus breeds coexist in two different
ecosystems, and both should be addressed in the
improvement of Bolivian selection systems.
Creole breeds are taurine bovines characterized by
high longevity and fertility in tropical and subtropical
environments, as well as increased resistance to
endemic diseases (Guglielmone et al., 1991; Hansen,
1994). This contrasts with the majority of European
taurine breeds, which show less adaptation to such
environmental conditions (Bouzat et al., 1998). For this
reason, Creole breeds are considered a valuable
American genetic resource. In addition, from a
productive point of view, Creole cattle have good
carcass performance (Garriz et al., 1993; Holgado et al.,
2021).
The international trend is that consumers are
willing to pay more for higher quality products
(Shackelford et al., 2001), thus leading to an
improvement in selection processes to determine the
animals with the highest genetic merit towards quality
(Dekkers and Hospital, 2002). This selection approach
is most effectively performed for highly heritable traits
that are easily measured before the reproductive age.
In this sense, marker­assisted selection has the
potential to significantly increase the rate of genetic
improvement of traits of interest (MacNeil and Grosz,
2002). Tenderness is one of the most important
qualities in the meat industry, and presents great
differences between taurine and zebuine breeds.
Several studies have shown that meat tenderness
decreases as the percentage of inherited Zebu genes
increases (Mazzucco et al., 2010).
Proteolytic system calpain/calpastatin consists of
two proteases (calpains CAPN1 and CAPN2) and an
inhibitor of calpastatin (CAST). It has an important
effect on tenderness since it is responsible for initiating
post mortem degradation of myofibrillar proteins
(Goll et al., 1992; Huff­Lonergan et al., 1996;
Koohmaraie, 1992; Ouali and Talmant, 1990). Given
their biological function, variations in the sequence of
the genes encoding these enzymes affect their activity
and, consequently, tenderness (Ng and Henikoff,
2006). Different studies have analyzed the effect of
single nucleotide polymorphisms (SNPs) on meat
tenderness (Barendse et al., 2008; Chung et al., 2014;
Corva et al., 2007; Curi et al., 2010; Gill et al., 2009;
Juszczuk­Kubiak et al., 2004; López­Rojas et al, 2017;
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Genetic diversity of Calpain 1 gene in bovine breeds in Bolivia
Page et al., 2002; Page et al., 2004; Soria et al., 2010;
White et al., 2005). These studies have reported
significant associations between different SNPs (e.g.,
CAPN1­316, CAPN1­530 and CAPN1­4751, CAST­285)
of the CAPN1, CAPN2 and CAST genes and
tenderness in different taurine, zebuine and composite
breeds. In addition, some of these SNPs have been
associated with other meat quality variables, such as
color measurement (Castro et al., 2016; Mazzucco et al.,
2010) and overall acceptability (Avilés et al., 2015).
Therefore, these genes are currently used as genetic
markers for this and other variables associated with
meat quality, such as weight of the hindquarter and
first calf birth weight (Castro et al., 2016; Gill et al.,
123
2009). Recently, Cui et al. (2016) reported the
association between CAPN1 promoter polymorphism
and semen quality in Chinese cattle.
Although these SNPs have been extensively studied,
so far there are no reports of Bolivian populations. Thus,
the objectives of the present work were to determine and
compare genetic variability of CAPN1­316, CAPN1­4751
and CAPN1­530 polymorphisms of the CAPN1 gene
in Creole, Brahman and Nellore breeds in Department
of Santa Cruz, Bolivia. Futrthermore, the data obtained
were compared with the results obtained in cattle
breeds bred in the region.
Material and Methods
Animal samples
A first set of blood samples from 114 purebred
Bolivian Creole (CreBo), 59 Bolivian Brahman (BraBo)
and 93 Bolivian Nellore (NelBo) animals from herds
located in the Departments of Santa Cruz, Bolivia,
were taken. Bolivian Creole cattle correspond to four
local populations, namely, Yacumeño (CreYa, N = 86),
Saveedreño (CreSa, N = 20), and Chaqueño (CreCh, N
= 8) were obtained to genotyping by ARMS­PCR and
PCR­RFLP methods (Table 1). Furthermore, a second
set of blood samples comprising by unrelated adult
cattle from Argentinean Creole (CreAr, N = 192),
Saveedreño (CreSa, N = 39), Angus (Ang, N = 94) and
Holstein (Hol, N = 88), composite Brangus (Brn, N =
99), Braford (Brf, N = 46), and Argentinean Brahman
(BraAr, N = 45) were included in the study to
genotyping by microarrays. All the animals sampled
correspond to adult bovines in order to avoid
including closely related animals. In cases where
pedigree records were available, this information was
used during sampling.
Table 1. Detailed information on the sampled populations.
Breed
Acronym
N
Yacumeño Creole
CreYa
861
Saveedreño Creole
CreSa
201; 392
Sampling site
Yabaré, departamento de Santa Cruz, Bolivia
Saavedra, Provincia Obispo Santisteban, departamento de Santa
Cruz, Bolivia
Chaqueño Creole
CreCh
81
Muyupampa, municipio Vaca Gusman, Provincia Luis Calvo, de­
partamento de Chuquisaca
N = number of animals; 1Genotyped by PCR­RFLP and ARMS­PCR method; and 2Genotyped using microarrays de SNPs.
2.2 DNA extraction and genotyping
DNA was extracted from blood samples using the
commercial Wizard® Genomic Purification kit
(Promega, Madison, WI, USA) according to the
manufacturer´s
specifications.
CAPN1­316
(rs17872000) (Page et al., 2002) and CAPN1­4751
(rs17872050) SNPs were genotyped with the ARMS­
PCR method (Rincon and Medrano, 2003), whereas the
PCR­RFLP technique (Rincon and Medrano, 2006) was
used for CAPN1­530 (rs17871051) genotyping (Page et
al., 2002). All PCR reactions were performed in a final
volume of 25 µl with the primers described in Rincon
and Medrano (2003) (Table 2). In the case of CAPN1­
316 and CAPN1­4751, the reaction mixture contained
10 and 1 pmol of each internal and external primer,
respectively.
The amplification products were electrophoresed
in 6 % polyacrylamide gels with TBE 1X. Amplicons
obtained from CAPN1­530 were digested with
restriction enzyme PsyI (Fermentas, Hanover, MD,
USA) overnight at 37 °C and electrophoresed in 6 %
polyacrylamide gels with TBE 1X. Additionally,
CAPN1­316 and CAPN1­4751 SNPs were also
genotyped in a GeneTitan™ platform (Affymetrix, CA,
USA) using the Axiom™ Bos 1 Genotyping Array r3
(Affymetrix), containing 648 855 SNPs, and the
Axiom™ ArBos 1 Genotyping Array (Affymetrix),
which contains 58 936 SNPs. Raw data were processed
using the Axiom™ Analysis Suite software
(Affymetrix) and SNPs were filtered by sample (≥ 97
%) and SNP (≥ 97 %) call rates, and exported in .PED
and .MAP format. None animals were excluded.
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F ­ 5´­CGTTTCTTCTCAGAGAAGAGCGCAGGGA­3´
R ­ 5´­CCTGCGCCATTACTATCGATCGCAAAGT­3´
inner F ­ 5´­GCATCCTCCCCTTGACTGGGGGGAAACCC­3´
inner R ­ 5´­GTCACTTGACACAGCCCTGCGCCGCA­3´
outer F ­ 5´­CCTGGAGTCCTGCCGCAGCATGGTCAAC­3´
outer R ­ 5´­AAGCTGCAGGAGCTGCCCAAAGCCAGGC­3´
Statistical analysis
Allelic and genotypic frequencies were calculated
for each breed using the MS­Tools 3.1 software (Kavan
and Man, 2011). Expected (he) and observed (ho)
heterozygosities, Hardy­Weinberg equilibrium (HWE,
measured through FIS parameter), population structure
(estimated by FST index) and linkage disequilibrium
(LD) were determined with GENEPOP 4.0 software
(Rousset, 2007). Haplotypes for each breed were
reconstructed using Gabriel algorithm and visualized
with Haploview 4.1 (Barrett et al., 2005) software.
Resultados
1
According to Bos_taurus_UMD_3.1.1; 2 Page et al., 2002.
29:44087629
rs17872050
CAPN1­ 530
C/T
rs17871051
CAPN1­ 47512
A/G
29:44085642
Intron 14
inner F ­ 5´­TTTCCTGCAGCTCCTCGGAGTGGAAGGG­3´
inner R ­ 5´­GCTCCCGCATGTAAGGGTCCAGGG­3´
outer F ­ 5´­GCTGTGCCCACCTACCAGCATC­3´
outer R ­ 5´­CAGGTTGCAGATCTCCAGGCGG­3´
exon 9
rs17872000
CAPN1­ 3162
C/G
2007) for further gene and haplotype analyses. Five
samples were genotyped with both assays, obtaining
more than 95 % concordant results.
rs
SNP
Chromosome
Position1
29:44069063
Gene region
Primer ­ Sequence
Pereira et al.
SNP
Table 2. Details of CAPN1­316, CAPN1­4751 and CAPN1­530 genetic markers and the primers used in the PCR reactions.
124
SNP annotation was performed using the
Axiom_GW_Bos_SNP_1.na35.annot
and
Axiom_ArBos_1.1.na35.annot
annotation
files
(Affymetrix) and positions were assigned according to
the bovine genome assembly UMD 3.1. Eleven SNPs
located within CAPN1 genes (from BTA29:44.06 to
44.09 Mb, including CAPN1­316 and CAPN1­4751)
were filtered using PLINK 1.9 software (Purcell et al.,
Results of CAPN1­316, CAPN1­4751 and CAPN1­
530 SNPs genotyping with ARMS­PCR and PCR­RFLP
are presented in Table 3. They show the estimated
allele and genotype frequencies of the analyzed SNPs
in CreBo, NelBo and BraBo populations. In CreBo,
CAPN1­316­G, CAPN1­4751­C and CAPN1­530­G
were the most frequent alleles, while in the zebu
breeds (NelBo and BraBo) the most frequent alleles
were CAPN1­316­G, CAPN1­4751­T and CAPN1­530­
G. In the case of CAPN1­316, the G allele was fixed in
BraBo, resulting in he values between 0.37 for CAPN1­
4751 in CreBo and 0.12 for CAPN1­530 in NelBo
(Table 4). HWE showed that three of the eight
performed tests were in disequilibrium (CAPN1­316
and CAPN1­530 in NelBo, and CAPN1­4751 in BraBo),
in all cases due to a significant increase of homozygote
genotypes (Table 4). The fact that NelBo and BraBo
samples belonged to different farms would suggest
that this ho reduction could be caused by
subpopulation structure (Wahlund effect) and/or
inbreeding. The analysis of population structure
evidenced that inter­population variance accounted
for 9.36 % of the total variance for the CAPN1 gene (p­
value < 0.01). Furthermore, four out of nine
comparisons resulted statistically significant (p­values
< 0.05), with the exception of BraBo­NelBo for
CAPN1­4751 (p­values = 0.192) and CreBo­BraBo for
CAPN1­530
(p­values
=
0.401).
FST
values varied between 0 in NelBo­BraBo for CAPN1­
530 and 0.59 in NelBo­CreBo for CAPN1­4751 (Table
5).
After filtering CAPN1­316 and CAPN1­4751
genotypes from the used microarrays data, that
include information from two Creole cattle
populations (CreSa and CreAr), two Taurine
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Genetic diversity of Calpain 1 gene in bovine breeds in Bolivia
European breeds (Ang and Hol), two composite
breeds (Brn and Brf) and one zebuine (BraAr), allele
and genotype frequencies were further estimated
(Table 6). Our results showed that CAPN1­316­G and
CAPN1­4751­C were the most abundant alleles in
analyzed Creole cattle populations, while CAPN1­316­
G and CAPN1­4751­T were the most frequent alleles in
BraAr. In contrast to BraBo, the frequency of CAPN1­
316­C allele in BraAr was 0.04. Compared to Creole
cattle, the British breed Ang, characterized by its
tender meat, presented higher CAPN1­4751­T
125
frequency, while Hol showed the lowest frequency of
this allele. Regarding CAPN1­316 SNPs, Ang had
similar allele frequencies than CreAr cattle, while the
CreSa and Hol exhibited lower frequencies of C
variant. Composite breeds (Brn and Brf) had higher
frequencies of CAPN1­316­G and CAPN1­4751­T than
Creole cattle due to the Zebu influence (Table 6). These
allele frequencies resulted in he values between 0.47
for CAPN1­316 in CreAr and 0.09 for CAPN1­316 and
CAPN1­4751in BraAr (Table 7), while all analyzed
populations were in HWE (Table 7).
Table 3. Gene and genotypic frequencies estimated for CAPN1­316, CAPN1­4751 and CAPN1­530SNPs in the Bolivian populations of Creole
(CreBo), Nellore (NelBo) and Brahman (BraBo) breeds. Number of animals between brackets.
CAPN1­316
Breed
Genotype
Allele Frequency
CC
CG
GG
C
G
CreBo
0.037 (4)
0.374 (40)
0.589 (63)
0.22
0.78
NelBo
0.092 (8)
0.023 (2)
0.885 (98)
0.09
0.91
BraBo
0 (0)
0 (0)
1 (51)
0
1
TT
C
T
CAPN1­4751
Breed
Genotype
Allele Frequency
CC
TC
CreBo
0.558 (63)
0.398 (45)
0.044 (5)
0.76
0.24
NelBo
0.022 (2)
0.167 (15)
0.811 (92)
0.08
0.92
BraBo
0.154 (8)
0.019 (1)
0.827 (43)
0.16
0.84
A
G
CAPN1­530
Breed
Genotype
Allele Frequency
AA
AG
GG
CreBo
0.075 (8)
0.327 (35)
0.598 (64)
0.23
0.77
NelBo
0.057 (5)
0.057 (5)
0.886 (99)
0.06
0.94
BraBo
0.109 (6)
0.164 (9)
0.727 (40)
0.08
0.92
Table 4. Number of detected alleles (na), observed (ho) and expected (he) heterozygosities, and Hardy­Weinberg equilibrium (HWE, measured
through FIS index) for CAPN1­316, CAPN1­4751 and CAPN1­530 SNPs in the Bolivian Creole (CreBo), Bolivian Nellore (NelBo) and Bolivian
Brahman (BraBo). ND: not determined.
CAPN1­316
Population
Creole (CreBo)
Nellore (NelBo)
Brahman (BraBo)
na
2
2
1
ho
0.37
0.02
0
he
0.35
0.17
0
FIS (p value)
­0.070 (0.58)
0.890 (< 0.001)
ND
CAPN1­4751
Population
Creole (CreBo)
Nellore (NelBo)
Brahman (BraBo)
na
2
2
2
ho
0.40
0.12
0.02
he
0.37
0.14
0.28
FIS (p value)
­0.077 (0.61)
0.175 (0.12)
0.931 (< 0.001)
CAPN1­530
Population
Creole (CreBo)
Nellore (NelBo)
Brahman (BraBo)
na
2
2
2
ho
0.32
0.07
0.16
he
0.36
0.12
0.15
FIS (p value)
0.117 (0,28)
0.393 (0.004)
­0.080 (1.00)
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Pereira et al.
126
Table 5. Population structure, measured through pairwise FST
parameter, of CAPN1­316, CAPN1­4751 and CAPN1­530 SNPs in
Bolivian Creole (CreBo), Bolivian Nellore (NelBo) and Brahman
(BraBo). FST and p­values are shown above and below the diagonal,
respectively.
CAPN1­316
CreBo
CreBo
0
NelBo
0.002
BraBo < 0.0001
NelBo
0.058
0
0.0003
Table 6. Allele frequencies of CAPN1­316 and CAPN1­4751
genotyped using Bos 1 microarray (Affymetrix) and the Axiom™
ArBos 1 Genotyping Array (Affymetrix) in Creole Saavedreño
(CreSa), Argentinean Creole (CreAr), Angus (Ang), Holstein (Hol),
Brangus (Brn), Bradford (Brf), and Argentinean Brahman (BraAr).
Population
BraBo
0.170
0.057
0
CAPN1­4751
CreBo
CreBo
0
NelBo < 0.0001
BraBo < 0.0001
NelBo
0.639
0
0.192
BraBo
0.506
0.027
0
CAPN1­530
CreBo
CreBo
0
NelBo < 0.0001
BraBo
0.401
NelBo
0.102
0
0.016
BraBo
0.067
­ 0.006
0
CAPN1­316
CAPN1­4751
C
G
C
CreSa
11.54
88.46
78.95
21.05
CreAr
36.98
63.02
80.79
19.21
Ang
33.87
66.13
62.23
37.77
Hol
27.27
72.73
89.20
10.80
Brn
22.99
77.01
49.85
50.15
Brf
17.39
82.61
34.78
65.22
4.44
95.56
4.44
95.56
BraAr
T
Table 7. a. Number of detected alleles (na), observed (ho) and expected (he) heterozygosities, and Hardy­Weinberg equilibrium (HWE measured
through FIS index) for CAPN1­316 and CAPN1­4751 genotyped using Allele frequencies of CAPN1­316 and CAPN1­4751 genotyped using Bos 1
microarray (Affymetrix) and the Axiom™ ArBos 1 Genotyping Array (Affymetrix) in Creole Saavedreño (CreSa), Argentinean Creole (CreAr),
Angus (Ang), Holstein (Hol), Brangus (Brn), Bradford (Brf), and Argentinean Brahman (BraAr). b. P values of the genetic differentiation for each
studied population pair estimated using exact G test for CAPN1­316 (Above) and CAPN1­4751 (Below).
a.
CAPN1­316
Population
CreSa
CreAr
Ang
Hol
Brn
Brf
BraAr
N
39
192
94
88
99
46
45
ho
0.18
0.46
0.38
0.39
0.35
0.26
0.09
he
0.21
0.47
0.45
0.40
0.35
0.29
0.09
CAPN1­4751
FIS (p value)
0.134 (0.405)
0.019 (0.877)
0.165 (0.162)
0.032 (0.791)
­ 0.035 (1.000)
0.103 (0.601)
0.007 (0.878)
ho
0.32
0.29
0.39
0.19
0.53
0.52
0.09
he
0.34
0.31
0.47
0.19
0.50
0.46
0.09
FIS (p value)
0.063 (0.649)
0.070 (0.351)
0.168 (0.125)
0.003 (1.000)
­ 0.062 (0.273)
­ 0.139 (0.514)
­ 0.035 (1.000)
b.
CreSa
CreAr
Ang
Hol
Brn
Brf
BraAr
CreSa
0
0.753
0.009
0.046
< 0.001
< 0.001
< 0.001
CreAr Ang
Hol
Brn
< 0.001
0.00008
0.006
0.019
0
0.518
0.025 < 0.001
< 0.001
0
0.210
0.004
0.013 < 0.001
0
0.272
< 0.001
0.003
< 0.001
0
< 0.001 < 0.001
< 0.001
0.008
< 0.001 < 0.001 < 0.001 < 0.001
Brf
0.386
0.0002
0.05
0.074
0.234
0
< 0.001
BraAr
0.001
< 0.001
< 0.001
< 0.001
< 0.001
0.007
0
Discussion
The effect of CAPN1 polymorphisms has been
extensively studied in several taurine and zebuine
breeds. The CAPN1­316­C allele has been associated
with higher meat tenderness in breeds such as Nellore,
Brahman, Santa Gertrudis, Canchin, Brangus,
Braunvieh, Angus and Hanwoo, and crossbreeds of
Angus x Nellore, Rubia Gallega x Nellore, Angus x
Hereford and British x Limousin (Barendse et al., 2008;
Chung et al., 2014; Corva et al., 2007; Curi et al., 2010;
Gill et al., 2009; Page et al., 2002; Page et al., 2004; White
ISSN­L 1022­1301. Archivos Latinoamericanos de Producción Animal. 2022. 30 (2): 121­132
Genetic diversity of Calpain 1 gene in bovine breeds in Bolivia
et al., 2005). The results obtained in the present study
show that while the frequency of CAPN1­316­C in
CreBo as well as in CreAr is considerably high (> 20
%), the NelBo and BraBo breeds present a very low
frequency of this variant (< 10 %). These data are in
agreement with previous reports showing that taurine
breeds generally present a higher frequency of this
allele than zebu breeds (Barendse et al., 2008; Corva et
al., 2007; Curi et al., 2010; Gill et al., 2009; López­Rojas
et al., 2017; Page et al., 2002; Soria et al., 2010; White et
al., 2005), while composite breeds often present
intermediate frequencies. In this sense, the high
frequency of the CAPN1­316­C allele in CreBo may be
due to its taurine origin (Liron et al., 2006a,b ; Primo,
1992). The higher gene frequencies of favorable
CAPN1 and CAST alleles in Bolivian Creole cattle
reported in the present work agree with meat quality
analysis performed in Creole breeds. These reports
showed that Creole cattle and their crossbreed had
similar Warner­Blatzer shear force values (3.5 to 5 kg)
to those reported in European taurine breeds,
including Angus (Garriz et al., 1993; Holgado et al.,
2021).
In the case of the CAPN1­4751 SNP, the C allele
has been associated with higher meat tenderness in
breeds such as Nellore, Brahman, Canchin, Brangus,
Braunvieh, Angus, Hanwoo and crossbreeds between
Angus x Nellore and Rubia Gallega x Nellore (Bonilla
et al., 2010; Casas et al., 2006; Chung et al., 2014; Curi et
al., 2009; Gill et al., 2009; Pinto et al., 2010; Soria et al.,
2010; White et al., 2005). As in the first SNP analyzed,
the present results show that CreBo and CreAr have a
significantly higher frequency of CAPN1­4751­C (> 75
%) than NelBo (< 10 %) and BraBo (< 16 %) in Bolivia.
These results also agree with those reported in the
literature showing that taurine breeds present higher
CAPN1­4751­C allele frequency than zebu breeds
(Bonilla et al., 2010; Casas et al., 2006; Curi et al., 2009;
Gill et al., 2009; Pinto et al., 2010; Pinto et al., 2011; Soria
et al., 2010), while composite breeds generally present
intermediate frequencies. Compared with previous
studies, CreBo breeds, as well as CreAr, presented an
exceptionally high CAPN1­4751­C allele frequency.
Interestingly, Creole cattle had similar CAPN1­4751­C
allele frequency than Iberian and African Taurine
breeds (Rodero et al., 2013; Pelayo et al., 2016). The
partial African origin of Creole cattle germoplasm is
already known (Liron et al., 2006a,b). Thus, in the case
of CAPN1­316, this could be a consequence of the
historical origin of Creole cattle that defined the
characteristics of Creole cattle meat (Garriz et al., 1993;
Holgado et al., 2021; Liron et al., 2006a,b; Primo, 1992).
127
Finally, the major allele of CAPN1­530 SNP is
CAPN1­530­G, with frequency values between 0.77 in
CreBo and 0.94 in BraBo. This result also agrees with
previous studies performed in several pure and
composite breeds (Chung et al., 2014; Corva et al.,
2007). Page et al. (2002), proposed that the CAPN1­530­
G allele had a beneficial effect on meat tenderness as
compared with CAPN1­530­A. Interestingly, the
breeds studied so far, including those of the present
study, showed higher frequencies of this allele.
However, subsequent studies showed conflicting
results regarding the effect of this SNP on meat
tenderness.
Genetic differences among breeds may be due to
the presence of different alleles, to different genetic
frequencies or to different haplotype combinations.
Although CAPN1 polymorphisms have been
extensively reported in several bovine breeds, most
studies have analyzed only one or two of the CAPN1
SNPs included in the present study, and only a few
works
have
reported
LD
among
CAPN1
polymorphisms (Castro et al., 2016; Chung et al., 2014;
White et al., 2005). LD analysis of CAPN1­316¸
CAPN1­530 and CAPN1­530 SNPs within CreBo,
NelBo and BraBo breeds showed that six out of seven
tests exhibited significant LD p­values (Table S2),
excepting CAPN1­316 and CAPN1­530 pairwise
comparison in NelBo. In agreement with the
significant LD previously observed in the three
genotyped SNP, a more detailed analysis of LD within
the CAPN1 gene using the Bos 1 microarray data
showed two LD blocks clearly defined in Angus,
Argentine and Bolivian Creole cattle, and Brahman­
Composite breeds (Figure 1). The first one comprised
four SNPs spanning from 44.067 to 44.072Mb of BTA29
and included CAPN1­316, while the second block
comprised two SNPs and covered the region from
44.085 to 44.087bp and included CAPN1­4751. None of
these blocks included CAPN1­530; however, this
genetic marker was located close to the upstream end
of block 2. As shown in Figure 1, the level of
recombination between two blocks, measured through
multiallelic D' parameter, varied between 0.48 and
0.64. It is noteworthy that the number and population
frequency as well as connections from block 1 to the
next varied among Angus, Argentine and Bolivian
Creole cattle and Brahman­Composite breeds. Similar
results were published by Chung et al. (2014), who
reported an LD block between exon 14 and intron 17.
This is relevant because certain CAPN1 haplotypes
have been associated with meat tenderness (Chung et
al., 2014; Lee et al., 2014; White et al., 2005). For
example, Page et al. (2004), reported that CAPN1­316­
C – CAPN1­530­G haplotype was associated with
higher tenderness among the four possible classes,
ISSN­L 1022­1301. Archivos Latinoamericanos de Producción Animal. 2022. 30 (2): 121­132
128
Pereira et al.
while White et al. (2005), showed that CAPN1­316­C –
CAPN1­530­G – CAPN1­4751­C haplotype presented
the lowest Warner­Bratzler shear force value.
Therefore, it would be necessary to genotype at least
two tag SNPs to determine the haplotype structure of
CAPN1 gene in order to apply this genetic marker in
breeding programs.
Figure 1. Linkage disequilibrium plot (upper panel) and haplotypes (lower panel) within the CAPN1 gene were reconstructed using Gabriel
algorithm implemented in Haploview 4.1 (Barrett et al., 2005) software: a. Angus breed. b. Argentine and Bolivian Creole cattle breeds. c.
Brahman­Composite breeds. R2 values are detailed within squares.
Conclusions
In conclusion, the results obtained in the present
work show that CreBo cattle had a higher frequency of
CAPN1­316­C and CAPN1­4751­C variants associated
with higher meat tenderness than zebu breeds,
probably due to the historical taurine origin of Creole
cattle. In addition, LD analyses using microarray data
revealed two LD block clearly defined. These markers
could be used in breeding programs to enhance meat
quality in the Bolivian herd, either by selection
programs for Creole animals or through crosses with
Zebu cattle.
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Supplementary Material
Table S1. Details of genetic markers located within the CAPN1 gene, which were included in the Bos 1 microarray (Affymetrix).
Probename
AX­24855879
AX­24855886
AX­24855890
AX­24855898
AX­24855900
AX­24855924
AX­24855930
AX­24855932
AX­24855934
­
AX­24855952
AX­28502573
1
rs
Chromosome
29
rs17872000
29
29
29
29
29
29
29
29
rs178710511
29
29
rs17872050
29
Chromosome position
44067234
44069063
44069994
44072107
44072710
44078806
44079956
44080512
44080770
44085642
44085769
44087629
Gene region
exon 6
exon 9
intron 10
intron 10
intron 10
intron 10
intron 10
intron 10
intron 10
exon 14
intron 14
intron 17
Markername
CAPN1­316
LD Block
1
1
1
1
CAPN1­530
CAPN1­4751
2
2
This marker is not included in Bos 1 microarrays.
ISSN­L 1022­1301. Archivos Latinoamericanos de Producción Animal. 2022. 30 (2): 121­132
Pereira et al.
132
Table S2. Linkage disequilibrium (LD, above the diagonal) and p­values (below the diagonal) of CAPN1­316, CAPN1­4751 and CAPN1­530
SNPs in the Bolivian populations of Creole (CreBo), Nellore (NelBo) and Brahman (BraBo) breeds.
CreBo
316
530
4751
316
­
< 0.001
0.005
530
0.45
­
0.0002
4751
0.23
0.002
­
530
0.91
­
0.017
4751
0.58
0.09
­
530
ND
­
< 0.001
4751
ND
0.58
­
NelBo
316
530
4751
316
­
0.132
0.001
BraBo
316
530
4751
316
­
ND
ND
ISSN­L 1022­1301. Archivos Latinoamericanos de Producción Animal. 2022. 30 (2): 121­132