DOI: https://doi.org/10.52973/rcfcv-e32137
Received: 15/03/2022 Accepted: 30/03/2022 Published: 21/06/2022
1 of 5
Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32137, 1 - 5
ABSTRACT
The purpose of this study was to optimize a real-time PCR-melting
analysis for reliable and economical detection of the 7.5 Kb mutant
insert of the BoERVK bovine transposable element in exon 5 of the
Apolipoprotein B (APOB) gene, which causes cholesterol deciency
— CD — (OMIA 001965-9913). This technique was also used to perform
a preliminary molecular screening to detect this mutation in a DNA
sample of Holstein Friesian cows (HFc) of six commercial dairy farms
from different regions of Uruguay. By amplifying the 170 and 146 bp
PCR products, two genotypes were clearly identied: homozygote (wild
type wt/wt) and heterozygote (carrier of the CD mutation: MUT/wt). The
homozygous wt/wt genotype was detected in the representative sample
of 103 HFc. It is concluded that Real-Time PCR-melting analysis is a
fast, easily interpretable, low cost, and highly accurate technique for
detecting this mutation, which can be implemented in genetic selection
programs to prevent the spread of the disease in HFc.
Key words: Cholesterol deciency; Holstein Friesian; real-time
PCR-melting
RESUMEN
El objetivo de este estudio fue optimizar un análisis mediante PCR en
tiempo real con curvas de disociación para la detección conable y
económica del inserto mutante de 7,5 Kb del elemento transponible
bovino BoERVK en el exón 5 del gen de la Apolipoproteína B (APOB),
determinante de la deciencia de colesterol — CD — (OMIA 001965-
9913). Asimismo, aplicando esta técnica se realizó un cribado molecular
preliminar para determinar la presencia de esta mutación en una
muestra de ADN de vacas Holando (H) pertenecientes a seis tambos
o ncas comerciales de diferentes regiones del Uruguay. A partir
de la amplicación de los productos de PCR de 170 y 146 pb se logró
distinguir claramente dos genotipos: homocigota (tipo silvestre wt/
wt) y heterocigota (portador de la mutación CD: MUT/wt). El genotipo
homocigota wt/wt fue detectado en la muestra representativa de 103
vacas H. Se concluye que el análisis mediante PCR en tiempo real con
curvas de disociación es una técnica rápida, fácilmente interpretable,
de bajo costo y altamente precisa para la detección de esta mutación,
el cual puede ser implementado en programas de selección genética
para evitar la propagación de la enfermedad en bovinos H.
Palabras clave: Deciencia de colesterol; Holando; PCR en tiempo
real con curvas de disociación
Optimization of Real-Time PCR-melting for detection of the Cholesterol-
deciency mutation in Holstein Friesian cattle
Optimización de PCR en tiempo real con curvas de disociación para la detección de la mutación
causante de deciencia de colesterol en bovinos Holando
Andrea Branda-Sica
1
* , Paula Nicolini
2
, Rody Artigas
3
, Maria Teresa Federici
1
y Silva Llambi
3
1
Instituto Nacional de Investigación Agropecuaria (INIA), INIA Las Brujas, Unidad de Biotecnología. Canelones, Uruguay.
2
Universidad de la República, Centro Universitario de Tacuarembó, Instituto Superior de la Carne, Área Biología Molecular. Tacuarembó, Uruguay.
3
Universidad de la República, Facultad de Veterinaria, Unidad Académica de Genética y Mejora Animal. Montevideo, Uruguay.
*Email: abranda@inia.org.uy
Optimization of Real-Time PCR for detection of BoERVK in Holstein cattle / Branda-Sica et al.___________________________________________
2 of 5
INTRODUCTION
Cholesterol deciency — CD — (OMIA 001965-9913) [19] is caused
by a loss-of-function mutation in the Apolipoprotein B (APOB) gene,
which is necessary for liver lipid metabolism, steroid biosynthesis, and
cholesterol absorption in the small intestine [17]. The APOB mutation
influences cattle (Bos taurus) fertility, growth, and health [9]. CD
disease is usually confused with other types of neonatal diarrhea [14].
The economic impact of CD is very important. A study in Germany
calculated that 3,400 recessive homozygous calves were born per year,
resulting in an annual economic loss of approximately € 1.3 million [13].
Furthermore, in the United States of America (USA), annual losses due
to that disease were calculated at USA$ 1.7 million [3]. In addition to
severe diarrhea, affected calves have hypocholesterolemia and usually
die within the rst weeks (wk) to 6 months (mon) of life [14]. Some
heterozygous calves showed reduced blood cholesterol concentrations,
whereas in recessive homozygous blood cholesterol levels and
triglyceride concentrations were virtually zero [9, 14, 21]. Gross et al.
[8] found that low cholesterol concentrations associated with the APOB
mutation in carriers are not due to primary CD at the cellular level, as
the term “CD” suggests, but a consequence of decreased cholesterol
transport capacity in blood. These authors suggest that, despite the
presence of the APOB mutation, cholesterol does not limit metabolic
adaptation or yield in heterozygous Holstein Friesian cows (HFc) [8]. The
origin of this disease was traced to the American sire Maughlin Storm,
born in 1991 and widely used in the HF population worldwide [14, 23].
This disease is caused by a 1,299 base pairs (bp) insertion of a long
transposable element (LTR_ERV2-1) between nucleotides 24 and 25 of
exon 5 of the APOB gene [17]. This insertion causes a shift in the reading
frame of the APOB gene that leads to the truncation of 97% of the
bovine APOB protein [17]. These ndings were independently conrmed
by other authors [4, 22]. This result was conrmed by Charlier [4],
albeit he estimated the size of the insertion of the bovine endogenous
retroviral element in exon 5 of the APOB gene in 7.5 Kb (BoERV); this
leads to transcriptional termination and loss of protein function. Due
to this, the protein was synthetized to only 3% of its normal size.
Although molecular methods such as Polymerase Chain Reaction
(PCR) and its variants are currently applied to diagnose the CD-causing
mutation [5, 12, 17, 22], there are no published studies in Uruguay on
the application of these techniques for the accurate and effective
detection of these transposable elements. The design of molecular
diagnostic strategies for this mutation would be important for this
Country, in order to achieve immediate results regarding the control of
this disease, since Briano-Rodriguez et al. [2] reported a prevalence
of CD carriers of 2.61% in a population sample of HF calves using the
GeneSeek Genomic Proler — GGP — Bovine 50K genotyping panel.
Hence, the purpose of this study was to optimize and implement
a reliable and economical molecular screening procedure for the
detection of the 7.5 Kb mutant insert (BoERVK) of the APOB gene
through real-time PCR analysis with melting curve analysis (real-
time PCR-melting), as well as to obtain preliminary results on its
presence in a representative sample of HFc from the Dairy Cattle
deoxyribonucleic acid (DNA) Genomic Bank of Uruguay.
MATERIALS AND METHODS
DNA samples and reference population
It worked with a representative sample of 103 second-lactation
HFc of six commercial dairy farms from different Regions of Uruguay.
Genomic deoxyribonucleic acid (gDNA) from these samples was
stored in the Dairy Cattle DNA Genomic Bank of the Biotechnology
Unit (INIA Las Brujas) as reference material for research projects
(INML-UdelaR-INIA agreement). The extraction of these gDNA samples
was initially performed from fresh blood samples at the Nuclear
Techniques Laboratory (Facultad de Veterinaria, UdelaR) in 2008
using a digestion procedure with proteinase K and salting-out [18].
For optimization of the real-time PCR-melting, two gDNA samples
were used as reference controls for comparison with the patterns of
the melting curves to be analyzed. These control samples corresponded
to: (1) gDNA of a bull (ALTALeap 011HO12336) diagnosed as a carrier of
the CD mutation, and (2) gDNA of a bull (ALTABolero 011HO11572) free of
the disease; both from AltaGenetics company (Montevideo, Uruguay).
These gDNA samples were extracted from semen with the QIAamp DNA
mini kit, according to the manufacturer’s protocol #2.
gDNA was quantied in the NanoDrop equipment (NanoDrop 8000
Spectrophotometer, Thermo Fisher Scientic, USA), obtaining a range
between 1.8 and 2.0 for the OD260/OD280 ratio. The quality of the
gDNA samples was assessed by 1% agarose gel electrophoresis in
TBE 0.5X buffer [7].
Optimization of the genotyping of the BoERVK_APOB insertion
with real-time PCR-melting
Real-time PCR reactions were performed in a RotorGene™ 6000
(Corbett Life Science, Australia) on a nal volume of 25 microliters per
sample containing 50 nanograms of genomic DNA, 1X NZY qPCR Green
Master Mix (NZYTech Genes & Enzymes, Portugal), and 0.5 microMol of
each primer. A combination of three allele-specic primers designed by
Charlier et al. [5] was used. This combination of primers discriminates
the wild type from the mutated sequence and corresponds to a forward
primer (F1: 5’ AAG GAG GCT GCA AAG CCA CCT AG 3’) and two reverse
primers (mutant R1: 5’ CCT TTG TCA CGA GTG GAA TGC CT 3’; and R2:
5’ CCT CTT GAT GTT GAG GAT GTG TT 3’).
Dip tubes without gDNA were used as a negative control to identify
the possible contamination of reagents and the possible formation
of primer dimers in each PCR reaction mix.
The cycling program consisted of a 5 minutes (min) pre-hold at
95°C; and 40 cycles of 45 seconds (s) at 95°C, 40s at 55°C, and 40s at
72°C; with a 5 min stop-hold at 72°C. The annealing temperature was
adjusted to 55°C, with activation of uorescence data in the green
channel (excitation 470; detection 510 nanometers — nm—). The melting
peak was adjusted using 1°C increments with a 5s retention for each
increase from 75 to 95°C. Melting curve analyses were performed with
the Rotor-Gene Q Series Software 2.3.1 (Build 49) of the RotorGene™
6000 thermal cycler.
Electrophoresis was performed on a 3% agarose gel in 0.5X TBE buffer
[7] in order to assess primer function and specicity; upon completion
of the PCR reaction, the PCR products had the expected fragment size.
The expected fragment sizes for each amplicon are 170 bp for the wild-
type allele, and 146 bp and 120 bp for mutant alleles A and B, respectively.
Conrmation of results by sequencing and multiple sequence
alignment
To conrm the sequence identity of the amplicons identied by
real-time PCR-melting, 23 PCR samples were selected and sent
for sequencing (Humanizing Genomics Macrogen, Seoul, Korea).
Sequencing was performed using the primers of Charlier et al. [5].
Homozygous genotype:
Wild type, wt/wt
(CD-free cows)
Heterozygous genotype:
MUT/wt
(control bull carrier of the
CD mutation)
Homozygous genotype:
Wild type, wt/wt
(control bull with no CD)
________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32137, 1 - 5
3 of 5
The obtained sequences were analyzed by multiple alignment with
a reference sequence for the APOB bovine gene (GeneID 494004,
GenBank), using BioEdit free software [11].
RESULTS AND DISCUSSION
The FIG. 1 shows the denaturation curve (-dF/dT vs. temperature)
of the amplicons obtained by real-time PCR for the BoERVK_APOB
insertion in the analyzed genetic materials of each genotype. A peak
was observed at 86.8°C for the homozygous wild type (wt/wt), and at
88°C for the heterozygous control, carrier of the CD mutation (MUT/wt).
The homozygous wt/wt genotype was detected in the representative
sample of 103 HFc.
Specialized real-time PCR thermal cyclers, such as the RotorGene™
6000 used in this study was programmed to produce the melting
curve after the amplication cycles have been completed. As the
temperature increases, the double-stranded gDNA denatures
to single-stranded gDNA, and the fluorophore molecules that
had intercalated within the double-stranded gDNA at the end
of amplification begin to separate from it as the temperature
increases, leading to a decrease in uorescence. The melting curve
is obtained by derivation of the denaturation curve (uorescence vs.
temperature), which shows an increase in absorbance, intensity, and
hyperchromicity during heating. The shape of this curve is related
to the guanine-cytosine (GC) base content and the fragment size of
the amplicons. The temperature value at which 50% of the gDNA is
denatured is called melting temperature (Tm) [6]. The Rotor-Gene Q
Series Software 2.3.1 (Build 49) plots the derivative of uorescence
as a function of uorescence (ordinates) vs. temperature (abscissae).
In the plot, it can be seen a maximum peak corresponding to the
Tm for each of the samples. The negative derivative plot of melting
curves (-dF/dT) represents the rate of uorescence change during
the gDNA melting process and allows the identication of the Tm
value by means of the temperature peaks [1].
For the samples analyzed in this work, the observation of a single
temperature peak in the melting curve of each sample conrmed the
specicity of the primers chosen to amplify the 146 bp mutant allele
with a peak at 88°C, and the 170 bp wild type at 86.8°C. In this study,
the primers of the mutant and wild type alleles had a GC content
between 40 and 60%, which was an adequate one (43.48 and 56.52%,
respectively) since no nonspecic amplications or primer dimers
were observed in the negative reaction control. Differences in the
biological origin of the gDNA samples (blood vs. semen, in this case)
caused the use of different DNA extraction protocols. This means that
there may be differences in the chemical solutions used during the
extraction process for each protocol, as well as in the contaminating
residues that could remain in the nal gDNA solution. Since real-
time PCR equipment is highly sensitive to these differences in the
conditions of the gDNA samples, differences could potentially be
observed in the melting curves of amplicons that, despite having the
same genotype, could be detected as different because their gDNA
was extracted following different protocols [24].
However, in this work, it was not detected any effect of the gDNA
extraction procedure on the melting dynamics of the amplicons, since
the melting peak pattern (-dF/dT) of the disease-free control bull
matched that of wild-type homozygous cows (FIG. 1). Furthermore,
at the end of the PCR reaction, i.e., when the reaction reached the
plateau phase, an inection point was observed in the denaturation
curve of the amplified product of the heterozygous control bull
when compared with the curves of the disease-free control bull and
wild type homozygous cows. Obtaining small amplicons increases
the difference in the signals at a given temperature between two
sequences differing in their nucleotide position from the start site
of the BoERVK insertion element. Differences between genotypes
are more clearly visible when amplicons are smaller in size [10]. The
temperature and uorescence resolution of the RotorGene™ 6000
equipment used in this study demonstrated the accuracy of the
melting curves and their ability to differentiate different genotypes.
FIGURE 1. Negative derivative of the uorescence melting curves at temperature (-dF/dT) for wild-type homozygous wt/wt and heterozygous
MUT/wt genotypes. The graph shows the genetic materials of each genotype. Four homozygous wt/wt genotypes (four cows free of the
disease —green curve—) were observed. Two controls were used, one heterozygous MUT/wt genotype (bull carrier of the CD mutation —red
curve—) and one homozygous wt/wt genotype (bull free of the disease —black curve—).
FIGURE 2. Electropherogram of the forward sequence (APOB-UP1-F1)
of a CD disease-free cow showing the absent insertion start site
Optimization of Real-Time PCR for detection of BoERVK in Holstein cattle / Branda-Sica et al.___________________________________________
4 of 5
Therefore, the primer design of Charlier et al. [5] was validated for
the detection of the 7.5 Kb CD mutant insert (BoERVK) in exon 5 of
the APOB gene by real-time PCR-melting on the RotorGene™ 6000.
By analyzing the electropherogram and multiple alignments of the
sequences obtained with the reference sequence, the location of the
insertion start point was determined at position 468 (26593) of the
APOB-UP1-F1 sequence of the HF bull control carrier of the CD mutation
(MUT/wt or indel_BoERVK_APOB) after the GTTCCTGAAGG fragment on
the left and before CAAGCAAGTTC, as reported by Charlier et al. [5]. The
mutant BoERVK APOB insert was not found in the sequences of HFc
and, therefore, those DNA samples corresponded to cows free of the
CD disease and with a wild-type homozygous genotype (wt/wt, FIG. 2).
Based on these results, no cows carrying the CD mutation were
found in the six dairy farms sampled from different regions of Uruguay.
However, as reported by Briano-Rodriguez et al. [2] a prevalence of
2.61% of CD carriers was found in a population sample of HF calves
from different Regions of Uruguay. These results were similar to those
observed for India (1.67%, [15]) and the USA (2.6%, [3]), but lower than
those reported for other Countries. For example, in Germany, Kipp et
al. [13] calculated the frequency of HF calves born homozygous for
the CD haplotype in approximately 8.7% of 3,400 calves born each
year. However, Kipp et al. [14] reported a maximum carrier frequency
of 12.25% in the German livestock population in 2012, which was
reduced to 7.87% in 2016 due to the genetic identication strategies
applied. In another study in Germany, Schütz et al. [22] found 12.5%
of carriers among HF bulls born between 2012 and 2015. Kamiñski and
Ruoeæ [12] reported a much higher CD carrier frequency (33.33%)
in Polish HFc. In Chinese HFc, a carrier prevalence of 5.07 and 1.11%
was found for bulls and cows, respectively [16]. Pozovnikova et al.
[20] reported CD carrier frequencies in two groups of Russian HFc,
where 23.26% were found in the offspring born between 2016 and
2019 of CD carrier cows, and 8.09% in the offspring born between
2010 and 2017 of CD carrier bulls.
Therefore, before taking mating decisions (to produce disease-
free calves), it is recommended to perform strict monitoring and
control by means of testing for the detection of the CD mutation, thus
preventing the spread of this disease in Uruguayan dairy farms. It is
also recommended the introduction of disease-free bulls into the HF
genetic improvement and semen production programs.
In this study, real-time PCR-melting made possible the clear
identication of two different genotypes, wild type homozygote wt/wt;
and carrier MUT/wt (or InDel_BoERVK_APOB), for the CD mutation using
CD disease-free controls and conrmed carriers for the CD disease
mutation, validating the use of this technique for genotyping of HFc.
CONCLUSIONS
The real-time PCR-melting analysis herein described provides
an alternative approach for genotyping of mutant alleles in cattle.
Real-time PCR testing with the melting application is a fast, easily
interpretable, low cost, and highly accurate technique for the
detection of the BoERVK mutant insert of the APOB gene, allowing
the genotyping of great volumes of HFc for the CD disease.
ACKNOWLEDGMENTS
The results of this publication are part of the research funded by the
National Agency for Research and Innovation of Uruguay (ANII) under
code POS_NAC_2017_1_141239, the National Institute of Agricultural
Research (INIA) and the Sectorial Commission for Scientic Research
of the University of the Republic (CSIC-UdelaR). To the translation
agency DNA Translations for the translation into English.
BIBLIOGRAPHIC REFERENCES
[1] ANSEVIN, A.T.; VIZARD, D.L.: BROWN, B.W.; MCCONATHY, J.
High-resolution thermal denaturation of DNA. I. Theoretical
and practical considerations for the resolution of thermal
subtransitions. Biopolym. 15: 153-174. 1976.
[2] BRIANO-RODRÍGUEZ, C.; ROMERO, A.; LLAMBÍ, S.; BRANDA-
SICA, A.; FEDERICI, M.; GIANNITTI, F.; CAFFARENA, D.; SCHILD,
C.; CASAUX, M.L.; DUTRA, F. Lethal and semi-lethal mutations
in Holstein calves in Uruguay. [Mutações letais e semi-letais em
bezerros da raça Holandesa no Uruguai.] Anim. Prod. Cien. Rural.
51(7): e20200734. 2021. https://doi.org/h2gt.
[3] COLE, J.B.; NULL, D.J.; VANRADEN, P.M. Phenotypic and genetic
effects of recessive haplotypes on yield, longevity, and fertility.
J. Dairy Sci. 99(9): 7274-7288. 2016.
[4] CHARLIER, C. The Role of Mobile Genetic Elements in the Bovine
Genome. Plant and Animal Genome Conference XXIV, San Diego,
CA, January 9-13, USA, Abstract W636. 2016. On Line: https://bit.
ly/3HSHy7f. 15/03/2022.
[5] CHARLIER, C.; GEORGES, M.; HARLAND, C.; COPPIETERS, W.
Detecting cholesterol deciency mutation in cattle. European
Patent Application N° EP 3 181697 A1. European Patent Oce.
19pp. 2017. On Line: https://bit.ly/3OqZCHl. 15/03/2022.
[6] CHIEN, A.; EDGAR, D.B.; TRELA, J.M. Deoxyribonucleic acid
polymerase from the extreme thermophile Thermus aquaticus.
J. Bacteriol. 127(3): 1550-1557. 1976.
[7] GREEN, M.R.; SAMBROOK, J. Agarose Gel Electrophoresis.
Chapter 2. In: Green, M.R.; Sambrook, J. (Eds.) Molecular Cloning:
A Laboratory Manual. 4th. Ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York. Pp 94-99. 2012.
[8] GROSS, J.J.; SCHWINN, A.C.; SCHMITZ-HSU, F.; BARENCO, A.;
NEUENSCHWANDER, T.F.; DRÖGEMÜLLER, C.; BRUCKMAIER,
R.M. The APOB loss-of-function mutation of Holstein dairy cattle
does not cause a deciency of cholesterol but decreases the
capacity for cholesterol transport in circulation. J. Dairy Sci.
102: 10564-10572. 2019. https://doi.org/h2gv.
________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32137, 1 - 5
5 of 5
[9] GROSS, J.J.; SCHWINN, A.C.; SCHMITZ-HSU, F.; MENZI, F.;
DRÖGEMÜLLER, C.; ALBRECHT, C.; BRUCKMAIER, R.M. Rapid
Communication: Cholesterol deficiency-associated APOB
mutation impacts lipid metabolism in Holstein calves and breeding
bulls. J. Anim. Sci. 94: 1761-1766. 2016. https://doi.org/f8nndm.
[10] GUNDRY, C.N.; VANDERSTEEN, J.G.; REED, G.H. PRYOR, R.J.;
CHEN, J.; WITTWER, C.T. Amplicon melting analysis with labeled
primers: a closed-tube method for differentiating homozygotes
and heterozygotes. Clin. Chemistry. 49: 396-406. 2003.
[11] HALL, T.A. BioEdit: a user-friendly biological sequence alignment
editor and analysis program for Windows 95/98/NT. Nucl. Acids.
Symp. Ser. 41: 95-98. 1999.
[12] KAMIÑSKI, S.; RUOEÆ, A. Cholesterol Deciency–new genetic
defect transmitted to Polish Holstein-Friesian cattle. Polish J.
Vet. Sci. 19(4): 885–887. 2016.
[13] KIPP, S.; SEGELKE, D.; SCHIERENBECK, S.; REINHARDT, F.;
REENTS, R.; WURMSER, C.; PAUSCH, H.; FRIES, R.; THALLER,
G.; TETENS, J.; POTT, J.; PIECHOTTA, M.; GRÜNBERG, W. A new
Holstein haplotype affecting calf survival. Interbull Annual Meet.
49: 49-53. 2015.
[14] KIPP, S.; SEGELKE, D.; SCHIERENBECK, S.; REINHARDT, F.;
REENTS, R.; WURMSER, C.; PAUSCH, H.; FRIES, R.; THALLER,
G.; TETENS, J.; POTT, J.; HAAS, D.; RADDATZ, B.B.; HEWICKER-
TRAUTWEIN, M.; PROIOS, I.; SCHMICKE, M.; GRÜNBERG, W.
Identification of a haplotype associated with cholesterol
deciency and increased juvenile mortality in Holstein cattle. J.
Dairy Sci. 99: 8915-8931. 2016. https://doi.org/h2gz.
[15] KUMAR, A.; GUPTA, I.D.; KUMAR, S.; VINEETH, M.R; KUMAR, D.;
MOHAN, G.; JAYAKUMAR, S.; KUMAR-NIRANJAN, S. First report
of colesterol deciency associated APOB mutation causing calf
mortality in Indian Holstein Fresian population. Indian J. Anim.
Sci. 91(2): 148-150. 2021.
[16] LI, Y.; FANG, L.; LIU, L.; ZHANG, S.; MA, Z.; SUN, D. The cholesterol-
deficiency associated mutation in APOB segregates at low
frequency in Chinese Holstein cattle. Can. J. Anim. Sci. 99(2):
332-335. 2018.
[17] MENZI, F.; BESUCHET-SCHMUTZ, N.; FRAGNIÈRE, M.; HOFSTETTER,
S.; JAGANNATHAN, V.; MOCK, T.; RAEMY, A.; STUDER, E.;
MEHINAGIC, K.; REGENSCHEIT, N.; MEYLAN, M.; SCHMITZ-HSU,
F.; DRÖGEMÜLLER, C. A Transposable element insertion in APOB
causes cholesterol deciency in Holstein cattle. Anim. Genet.
47(2): 253-257. 2016. doi: https://doi.org/f8fjm5.
[18] MILLER, S.A.; DYKES, D.D.; POLESKY, H.F. A simple salting
out procedure for extracting DNA from human nucleated cells.
Nucleic Acids Res. 12:319-325. 1988.
[19] ONLINE MENDELIAN INHERITANCE IN ANIMALS (OMIA). Faculty
of Veterinary Science, University of Sydney, 2011. On line: https://
bit.ly/39GVPHk/. 28/03/2022.
[20] POZOVNIKOVA, M.V.; GLADYR, E.A.; ROMANENKOVA, O.S.;
VASILEVA, O.K.; LEIBOVA, V.B.; TYSHCHENKO, V.I.; DEMENTEVA,
N.V. Screening of haplotype for cholesterol deciency genetic
defect in the Russian Holstein cattle population. Pol. J. Vet. Sci.
23(2): 313-315. 2020. https://doi.org/h2g5.
[21] SALEEM, S.; HEUER, C.; SUN, C.; KENDALL, D.; MORENO,
J.; VISHWANATH, R. Technical Note: The role of circulating
low-density lipoprotein levels as a phenotypic marker for
Holstein cholesterol deciency in dairy cattle. J. Dairy Sci.
99(7): 5545-5550. 2016. https://doi.org/f8s6hv.
[22] SCHÜTZ, E.; WEHRHAHN, C.; WANJEK, M.; BORTFELD, R.;
WEMHEUER, W.E.; BECK, J.; BRENIG, B. The Holstein Friesian
Lethal Haplotype 5 (HH5) Results from a Complete Deletion of
TFB1M and Cholesterol Deciency (CDH) from an ERV-(LTR)
Insertion into the Coding Region of APOB. PLoS One 11(6):
e0154602. 2016. https://doi.org/h2hj.
[23] VANRADEN, P.; NULL, D. Holstein haplotype for cholesterol deciency
(HCD). 2015. On line: https://bit.ly/3xG2fyv. 29/03/2022.
[24] WHITE, H; POTTS, G. Mutation scanning by high resolution melt
analysis. Evaluation of Rotor-Gene 6000 (Corbett Life Science),
HR-1 and 384 well LightScanner (Idaho Technology). 2006.
National Genetics Reference Laboratory. Wessex. On Line:
https://bit.ly/3y4QLWw. 29/03/2022.
