Identification of FecXO mutation in the BMP15 gene in prolific

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ISSN 1644-0714
ISSN 2300-6145 (online)
www.asp.zut.edu.pl
Acta Sci. Pol. Zootechnica 14(4) 2015, 93–100
IDENTIFICATION OF FECXO MUTATION
IN THE BMP15 GENE IN PROLIFIC OLKUSKA
SHEEP
Mirosław Kucharski1 , Urszula Kaczor1 , Andrzej Kaczor2
1
University of Agriculture in Kraków, Poland
National Research Institute of Animal Production, Balice n. Kraków,
Poland
2
Abstract. The A1009C (N69H) mutation at the bone morphogenetic protein 15 (BMP15) gene locus located on the X chromosome, which is considered a major gene, contributes to an increase in the number of lambs per litter. The aim of the study was
to determine the frequency of A1009C polymorphism at the BMP15 gene locus in
prolific Olkuska ewes from two flocks originating from southern Poland. Flocks with
a similar ewe prolificacy of 2.21 and 2.54 lambs/litter were found to differ in the
frequency of animals with CC and AA homozygous genotypes. In the simulated matings of the ewes over 3 years with a ram carrying the mutation, frequency of the CC
(FecXO /FecXO ) genotype increased by around 14 percentage units without increasing
the number of heterozygous animals. It should be stressed that the rams carrying the
FecXO mutation, due to location of the BMP15 gene on the X chromosome, will give
the valuable gene to every daughter. When choosing breeding rams for their flocks,
breeders of Olkuska sheep should identify their genotype at the BMP15 locus.
Key words: BMP15 gene, FecXO , polymorphism, prolific Olkuska sheep
INTRODUCTION
The main factors determining the profitability of sheep farms are reproductive performance parameters of the ewes. Currently available molecular biology
tools enable effective selection of animals carrying prolificacy traits. In most breeds of sheep, litter size was found to be polygenically determined, although in
Corresponding author: Urszula Kaczor, Department of Animal Biotechnology, University of
Agriculture in Krakow, ul. R˛edzina 1B, 30-248 Kraków, [email protected]
© Copyright by Wydawnictwo Uczelniane Zachodniopomorskiego Uniwersytetu Technologicznego w Szczecinie, Szczecin 2015
94
M. Kucharski et al.
some breeds it results from the mutation of a single Fec gene [Davis et al. 1982].
These mutations (FecXI , FecXH , FecXB , FecXG , FecXL , FecXR , FecXO , FecXGr ,
FecB, FecGH , FecGT , FecGE ) were identified in various breeds at the loci BMP15,
BMPR1B and GDF9 [Demars et al. 2013]. Effects on the magnitude of reproductive parameters were primarily found for polymorphisms within the bone morphogenetic protein 15 (BMP15) gene locus. This protein is responsible for folliculogenesis and ovarian function in mammals. Mutations in the BMP15 sequence
increase ovulation rate or barrenness in ewes [Hanrahan et al. 2004, McNatty et
al. 2004, Shimasaki et al. 2004, McNatty et al. 2005].
Olkuska sheep is the most prolific indigenous breed, in which lambing rate
averages 200%. However, in some flocks prolificacy is in the 300–350% range,
with single individuals achieving the lifetime prolificacy of 400-600% [Sm˛etek
and Korczyński 2011, PZO 2015]. The A1009C mutation (N69H, GeneBank NM_
_001114767) at the BMP15 gene locus located on the X chromosome increases
the number of lambs per litter [Kaczor 2011]. The mutation identified in exon 2,
which results in an asparagine to histidine change at amino acid 69 of the mature protein, increases ewe prolificacy depending on the number of mutant allele
copies. The effect of one mutated allele was estimated at 0.73 and that of two mutated alleles at 1.07 lambs [Kaczor 2011]. The mutation was named FecXO and,
like FecXGr in Grivette sheep, it is characterized, unlike other mutations in the
BMP15 gene, by no barrenness of the ewes with two gene copies [Demars et al.
2013].
Ten years after the entire population of Olkuska sheep had been included in the
Programme for genetic resources conservation, mean prolificacy of the population
entered in herd books showed a downward trend (from 211% to 200%). However,
the published data suggest that in some flocks prolificacy is just 140% [PZO 2006,
2015]. Due to the rapid increase in the Olkuska population, it is necessary to
identify the distribution of genotypes at the BMP15 locus in different flocks.
The aim of the study was to determine the frequency of FecXO mutation in the
BMP15 gene in two flocks of prolific Olkuska sheep and to show how mating the
ewes with rams carrying the mutation over three successive years will change the
distribution of genotypes at the BMP15 locus.
MATERIAL AND METHODS
The experiment used over 2-year-old Olkuska sheep with known reproductive
performance parameters, belonging to two pedigree flocks from southern Poland
(n1 = n2 = 61). Peripheral blood for analyses (1 ml) was collected once from
external jugular vein into EDTA tubes. Genomic DNA was isolated using a commercial kit (Epicentre, Illumina Company, USA). The polymorphism was deter-
Acta Sci. Pol.
Identification of FecXO mutation in the BMP15 gene. . .
95
mined with TaqMan® Genotyping Master Mix (Thermo Fisher Scientific, USA),
using the allele discrimination method (Applied Biosystems 7300) with probes
and primers designed by Kaczor [2011]. Chi-square test was used to determine if
the population is in Hardy-Weinberg equilibrium. The effect of N69H polymorphism on lifetime prolificacy of the sheep was determined with the Kruskal-Wallis
test (Statistica ver. 10, StatSoft). A simulation of changes in the frequency of genotypes at the BMP15 locus over three years was performed assuming that annual
culling rate of the ewes is 20% and the ewes are only mated to rams carrying the
FecXO mutation. The hypothesis was tested using χ-square test of independence,
separately for each flock and on combined data.
RESULTS
In the studied population of sheep, a missense A1009C mutation (causing
amino acid substitution at position N69H) was confirmed in the BMP15 gene.
A significant difference was found in the expression of homozygous genotypes
in the flocks under study (Table 1). The flocks differed in the frequency of the
wild-type FecX+ /FecX+ genotype (AA) (0.49 vs 0.15) and the homozygotes with
FecXO /FecXO (CC) (0.09 vs 0.38). Frequency of the FecX+ /FecXO genotypes (AC)
on the farms was similar at 0.42 and 0.47, respectively. Frequency of the FecXO
allele in flock II was twice as high as in flock I, amounting to 0.46 in the entire population. At the studied locus, the population was in Hardy-Weinberg equilibrium.
Mean prolificacy of the animals with FecX+ /FecX+ genotype differed between the
flocks (213% vs 156%, P < 0.01), whereas in both flocks, FecXO /FecXO ewes showed a prolificacy of 229 and 334%, respectively (Table 2). The simulated matings
of the ewes over 3 years with a ram carrying the FecXO allele showed that the
frequency of homozygous animals in the population could increase 1.7-fold for
the entire population (2.2-fold for flock I, 1.4-fold for flock II) (Table 3).
DISCUSSION
In the ewes from the flocks of prolific Olkuska sheep, the A1009C polymorphism (unique for this breed) was identified at the BMP15 gene locus (including
the desirable FecXO allele). The proportion of wild-type FecX+ /FecX+ homozygotes (AA) and carriers of the FecX+ /FecXO mutation (AC) in flock I was similar,
with 91% of the ewes having at least one FecX+ copy (allele A). Ewes with individual genotypes were characterized by similar prolificacy, and FecX+ /FecX+ ewes
gave birth to an average of over 2 lambs per litter, which shows that environmental
conditions had a significant effect on the investigated trait. A significant effect of
Zootechnica 14(4) 2015
96
M. Kucharski et al.
environmental factors on litter size of the ewes in flocks where such major genes
as FecB segregate, was also reported by other authors [Guan et al. 2007].
Table 1. The frequency of genotypes at the locus of BMP15 gene
Tabela 1. Frekwencja genotypów w locus genu BMP15
Genotype at locus BMP15 – Genotyp w locus BMP15
Flock – Stado
I (n = 61)
II (n = 61)
Allele – Allel
AA
AC
CC
A
C
0.49A
0.15B
0.42
0.47
0.09A
0.38B
0.70
0.39
0.30
0.61
Total – Razem
0.29
0.50
0.21
0.54
AA – FecX+/FecX+ homozygote, AC – FecX+/FecXO heterozygote, CC – FecXO/FecXO homozygote.
A B
, – The values in columns are significantly different (P < 0.01).
AA – homozygota FecX+/FecX+, AC – heterozygota FecX+/FecXO, CC – homozygota FecXO/FecXO.
A B
, – wartości w kolumnach różnią się istotnie (P < 0,01).
0.46
Table 2. The litter size of ewes of different genotypes at the gene locus BMP15
Tabela 2. Średnia wielkość miotu maciorek o różnych genotypach w locus genu BMP15
Genotype at locus BMP15 – Genotyp w locus BMP15
Flock – Stado
AA
I (n = 61)
II (n = 61)
AC
Total
Razem
CC
xx
SD
xx
SD
xx
SD
xx
SD
2.20
1.56
0.26
0.51
2.20
2.26
0.26
0.63
2.29
3.34
0.25
1.57
2.21
2.54
0.26
1.19
Total – Razem
2.14
0.34
2.22
0.41
2.99
1.36
2.30
0.68
Table 3. Simulated distribution of genotypes at the BMP15 gene locus when mating
ewes with rams carrying FecXO over 3 successive generations
Tabela 3. Frekwencja genotypów w locus genu BMP15 w kolejnych pokoleniach, przy
kojarzeniu maciorek z trykami nosicielami FecXO
Generation – Pokolenie
Flock – Stado
I
II
0
F1
F2
F3
AA
AC
CC
AA
AC
CC
AA
AC
CC
AA
AC
CC
0.49
0.15
0.42
0.47
0.09
0.38
0.42
0.11
0.46
0.45
0.12
0.44
0.36
0.08
0.48
0.41
0.16
0.51
0.30
0.08
0.50
0.40
0.20
0.53
Total – Razem
0.29
0.50 0.21 0.24 0.50 0.26 0.20 0.49 0.31 0.17 0.48 0.35
0 – the initial state, F1–F3 – the next generation after mating ewes with rams carrying FecXO,
0 – stan wyjściowy stad, F1–F3 – kolejne pokolenia, po kojarzeniu maciorek z trykami z allelem FecXO,
A different situation occurred in flock II, where 85% of the ewes had at least one copy of the FecXO gene (allele C). In this flock, 38% of the ewes had
Acta Sci. Pol.
97
the FecXO /FecXO genotype and were characterized by the highest prolificacy of
334%. FecX+ /FecX+ homozygotes were very rare (15% of animals) and showed
low prolificacy (156%). However, this difference was not significant. In the entire
population, the proportion of sheep with FecXO mutation was lower by 12 percentage units compared to that reported for Olkuska sheep by Kaczor [2011], who was
the first to describe A1009C mutation (N69H), later confirmed by Demars et al.
[2013]. In the Olkuska sheep population investigated in 2007–2009, the presence
of at least one allele with the FecXO mutation was found to increase the number
of lambs born: FecX+ /FecXO heterozygotes had an average prolificacy of 247%,
and FecXO /FecXO homozygotes – 298%. Frequency of the FecXO allele in the
population was 0.55, whereas frequency of the FecX+ /FecXO and FecXO /FecXO
genotypes was 0.56 and 0.27, respectively [Kaczor 2011].
Research results confirm that FecXO mutation in exon 2 of BMP15 gives a
different phenotypic effect than the other mutations at the locus of this gene.
N69H is responsible for increased litter size in both heterozygotes and homozygotes carrying the mutation, and individuals with CC genotype are characterized by highest prolificacy. This polymorphism was also found not to cause sterility in homozygous individuals [Kaczor 2011, Demars et al. 2013]. Resent research with French Grivette sheep has identified another mutation in the BMP15
gene, namely FecXGr , with T317I mutation. The presence of two copies of this
gene also does not cause ovarian dysfunction and sterility in the ewes, similar to
what was observed for FecXO . The mean prolificacy of homozygotes and heterozygotes with T317I mutation was 328% and 202%, respectively [Demars et al.
2013]. Therefore, to date we know of only 2 mutations (FecXO and FecXGr ) of the
BMP15 gene locus that differ in activity from 6 other mutations that are known:
FecXI , FecXH , FecXB , FecXG , FecXL , FecXR [Kaczor 2011, Demars et al. 2013].
The FecXI allele in the Inverdale breed with V299D mutation as well as FecXH
with Q291X mutation (STOP codon) results in sterility of homozygous sheep carrying the mutation and increases prolificacy in heterozygous ewes [Galloway et
al. 2000]. Alleles with a similar effect on phenotype were also identified in the
Belclare breed (FecXB mutation with S367I substitution). In the same breed and
in Cambridge sheep, a Q239X mutation (STOP), abbreviated as FecXG , was identified [Hanrahan et al. 2004]. The next mutations identified in the BMP15 gene
are FecXL in Lacaune sheep with C35Y mutation as well as FecXR with a 17 bp
deletion in the Rasa Aragonesa breed [Bodin et al. 2007, Martinez-Royo et al.
2008, Monteagudo et al. 2009]. Attempts to detect the above Fec genes have been
made in a number of prolific breeds around the world, but their absence suggests
the existence of unknown mutations [Polley et al. 2009, Vacca et al. 2010].
A simulation of changes in the frequency of individual genotypes at the BMP15
locus, performed for the studied flocks of sheep, suggests the need to identify the
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M. Kucharski et al.
presence of the mutation in breeding rams. It should be stressed that these rams
will give this valuable gene to every daughter. This leads us to assume that the distribution of genotypes, obtained after 3 or more years of breeding, will contribute
to increasing prolificacy levels in different flocks. Mating ewes to a ram carrying
the mutation is associated with increased frequency of FecXO /FecXO homozygous
genotypes by around 14 percentage units and causes no increase in the number of
heterozygous animals. Therefore, increased incidence of quadruple and larger litters as well as a decrease in the number of twin litters are to be expected [Kaczor
2011].
CONCLUSIONS
In summary, flocks of Olkuska sheep with similar mean prolificacy of the
ewes may differ in frequency of the FecXO allele. The mating of the ewes with
rams carrying the major gene for 3 successive years, coupled with 20% flock
replacement, will first of all increase the number of FecXO /FecXO homozygous
ewes while maintaining the frequency of heterozygotes at a similar level. When
choosing breeding rams, breeders of Olkuska sheep should identify the BMP15
locus genotype, because mating to a ram with a specific genotype may form the
basis of a correct mating scheme over the next years.
ACKNOWLEDGEMENTS
Funded by DS No 3242/2015 and DS. No 06-001.1.
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IDENTYFIKACJA MUTACJI BMP15 W GENIE FECXO U OWIEC RASY
OLKUSKIEJ PLENNEJ
Streszczenie. Mutacja A1009C (N69H) w locus genu białka morfogenetycznego kości 15 (BMP15), uznana za gen główny wpływa na zwi˛ekszenie liczby jagniat
˛ w miocie. Celem doświadczenia było określenie frekwencji wyst˛epowania polimorfizmu
A1009C w locus genu BMP15 u maciorek rasy olkuskiej plennej w dwóch stadach
Polski południowej. Stwierdzono, że stada o zbliżonej średniej plenności matek 2,21
i 2,54 jagni˛ecia/miot, różniły si˛e frekwencja˛ zwierzat
˛ o genotypach homozygotycznych CC i AA. Przeprowadzona symulacja kojarzenia maciorek z trykiem nosicielem mutacji przez 3 lata wskazuje na możliwość wzrostu frekwencji genotypu CC
(FecXO /FecXO ) o ok. 14 j.p. i nie powoduje wzrostu liczby zwierzat
˛ heterozygotycznych. Należy podkreślić, że tryki nosiciele mutacji FecXO , ze wzgl˛edu na lokalizacj˛e
genu BMP15 na chromosomie X, każdej córce przekaża˛ ten cenny gen. Hodowcy
owiec olkuskich wybierajac
˛ tryki rozpłodowe do stad powinni zidentyfikować ich genotyp w locus BMP15.
Słowa kluczowe: gen BMP15, FecXO , polimorfizm, owca olkuska plenna
Accepted for print: 16.12.2015
For citation: Kucharski, M., Kaczor, U., Kaczor, A. (2015). Identification of FecXO mutation in the BMP15 gene in prolific Olkuska sheep. Acta Sci. Pol. Zootechnica, 14(4),
93–100.
Acta Sci. Pol.

Identification of FecXO mutation in the BMP15 gene in prolific

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