Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor β (TGF β) super family that is expressed by oocytes and plays key roles in granulosa cell development and fertility in animal. This study investigated the molecular genetic variation of BMP15 gene of some selected mammalian species with a view to providing relevant genetic information for breeding and selection programmes in the studied species using computational methods. A total of thirty seven (37) BMP15 nucleotide sequences comprising goats (18), sheep (6), cattle (6), swine (4) and chicken (3) were retrieved from the GenBank. Sequence alignment, translation and comparison of the BMP15 gene of the various species were done with ClustalW. High degree of polymorphism of BMP15 gene was observed among the studied species. The significant value (P < 0.01) for relative proportions of non-synonymous substitutions per non-synonymous site (dN) and the number of synonymous substitutions per synonymous site (dS) symbolized that non-synonymous sites evolved faster than the synonymous sites and positive selection effect over shadowed purifying selection. Functional analysis of missense mutations using PROVEAN showed that twelve amino acid substitutions (L10S, W13A, E20L, V28S, P31R, P31G, P40Q, L42W, Q46N, A52V, R58C and G64T) in goats, nine in sheep (H21R, S32R, I33A, A39W, Q46W, E51A, G54S, R61D and E72A), six in cattle (Q30M, T41W, E50R, I62R, H65E, and E72S), seven in swine (I7L, T9I, V33I, L35H, C40P, R46P and Q61R) and five in chickens (A20H, L27H, W43L, A47P and G50Y) appeared beneficial. The phylogenetic trees from nucleotide and amino acid sequences revealed the close relatedness of members of the bovidae family (goat, sheep and cattle). The present information could guide future efforts involving selection of markers of fecundity to improve genetically livestock species in Nigeria.
One of the strides towards increased productivity of protein of animal origin is to have an in-depth knowledge of the genes associated with reproduction especially those related to ovulation and litter size. Identifying genes of major effect is also a prerequisite which offers the opportunity to improve production efficiency, product quality and product diversity in livestock industry, through utilizing them in breeding programs [
A total of thirty seven (37) BMP15 nucleotide sequences comprising goats (18), sheep (6), cattle (6), swine (4) and chicken (3) were retrieved from the GenBank (NCBI) (www.ncbi.nlm.nih.gov). The Genbank accession numbers of the sequences are JF824149.1, JF824148.1, NM001285588.1, JQ350892.1, JQ350891.1, JQ350090.1, HM462258.1, HM462255.1, HM462254.1, FJ429281.1, GU732196.1, EU743938.1, JX860305, JX860304, JN655670.1, JN655669.1, EU095935.1 and EU888137.1 (Goat); KF644561.1, NM001114767.1, EU402923.1, FJ600405.1, JN655672.1 and JN655671.1 (Sheep); DQ463368.1, NM001031752.1, EU712722.1, AY572412.1, DQ489533.1 and AY304484.1 (cattle); NM,001005155.1, AY295074.1, AH012830.1, and AY295073.1 (swine); AY725199.1, NM001006589.2 and AY729025 (chicken).
Sequence alignment, translation and comparison of the BMP15 gene of the various species were done with ClustalW as described by Larkin et al. [
The relative proportion of non-synonymous substitution per non synonymous site (dN) and the number of synonymous substitutions per synonymous site (dS) of the deduced amino sequences of BMP15 gene of the various species were computed by bootstrap method (1000 replicates) using the modified Nei-Gojobori (assumed transition/transversion bias = 2) method [
In silico functional analysis of missense mutations was obtained using PROVEAN with threshold value of −2.5. PROVEAN collects a set of homologous and distantly related sequences from the NCBI NR protein database (released August 2011) using BLASTP (ver.2.2.25) with an E-value threshold of 0.1. The sequences are clustered based on a sequence identity of 80% to remove redundancy using the CD-HIT program (ver.4.5.5) [
Neighbor-Joining (NJ) trees were constructed using maximum composite likelihood method and pairwise deletion gap/missing data treatment as described by Saitou and Nei [
The variation in sequence length in base pair (bp) of BMP15 gene within and among species ranges between 222 bp and 6648 bp (
The predicted amino acid sequences of goat, sheep, cattle, swine and chicken BMP15 showed varying degree of amino acid substitutions in the studied species (
Means of dS, dN and dN/dS with their corresponding Z-scores and P-values are presented in
The results of functional analysis of coding nonsynonymous single nucleotide polymorphism (nsSNP) of BMP15 gene for goats, sheep, cattle, swine and chicken are presented in Tables 3-7, respectively. Fourteen amino acid substitutions of the wild type alleles located in the putative peptide coding region of goats were obtained from the alignment of deduced amino acid sequences of goats. Out of these, twelve amino acid substitutions (L10S, W13A, E20L, V28S, P31R, P31G, P40Q, L42W, Q46N, A52V, R58C, G64T) were returned neutral indicating that the substitutions did not impair protein function, while the remaining two amino acid substitutions (M70F and Y74M) were returned as deleterious, an indication that the substitutions were harmful. In sheep, nine amino acid substitutions (H21R, S32R, I33A, A39W, Q46W, E51A, G54S, R61D and E72A) were returned neutral; an indication that they did not impair protein function while the remaining amino acid substitution (M70V) was predicted to be harmful. Out of the seven amino acid substitutions in cattle, six (Q30M, T41W, E50R, I62R, H65E, and E72S) appeared beneficial while the remaining one (Y74Q) appeared harmful.
For swine, seven amino acid substitutions (I7L, T9I, V33I, L35H, C40P, R46P and Q61R) were obtained and they all appeared beneficial (
The phylogeny based on nucleotide and amino acid sequences of BMP15 (
Specie | Accession Number | Base pair (bp) | Sequence length variation |
---|---|---|---|
Goat | EU095935.1 | 393 | 393 - 6648 |
HM462258.1 | 465 | ||
HM462254.1 | 465 | ||
HM462255.1 | 477 | ||
JX860305.1 | 762 | ||
JX860304.1 | 762 | ||
EU888137.1 | 1182 | ||
JQ350892.1 | 6495 | ||
JQ350891.1 | 6495 | ||
JQ350890.1 | 6495 | ||
GU732196.1 | 6494 | ||
JF824149.1 | 1230 | ||
JF824148.1 | 1230 | ||
NM001285588.1 | 1230 | ||
FJ429281.1 | 1436 | ||
EU743938.1 | 6648 | ||
JN655670.1 | 6648 | ||
JN655669.1 | 6648 | ||
Sheep | KF644561.1 | 222 | |
FJ600405.1 | 222 | 222 - 6641 | |
EU402923.1 | 260 | ||
NM001114767.1 | 1182 | ||
JN655671.1 | 6638 | ||
JN655672.1 | 6641 | ||
Cattle | AY304484.1 | 720 | |
DQ489533.1 | 953 | 720 - 1189 | |
EU712722.1 | 1184 | ||
AY572412.1 | 1185 | ||
NM001031752.1 | 1185 | ||
DQ463368 | 1189 | ||
Swine | AY295074.1 | 988 | |
AY295073.1 | 988 | 988 - 1316 | |
NM001005155.1 | 1194 | ||
AH012830.1 | 1316 | ||
Chicken | AY729025.1 | 1053 | |
AY725199.1 | 1875 | 1051 - 1875 | |
NM001006589.2 | 1875 |
Specie | No. of codons | dS | dN | dN/dS | Z-score | P-value |
---|---|---|---|---|---|---|
Goat | 340 | 2.28 | 2.42 | 1.06 | 10.77 | 0.00 |
Sheep | 353 | 1.46 | 1.41 | 0.97 | −1.232 | 1.00 |
Cattle | 226 | 2.53 | 2.62 | 1.04 | 11.83 | 0.00 |
Swine | 310 | 1.13 | 1.36 | 1.20 | 6.39 | 0.00 |
Chicken | 347 | 0.70 | 0.77 | 1.10 | 7.85 | 0.00 |
dN/dS = ratio of nonsynonymous to synonymous divergence.
Variant | PROVEAN Score | Prediction |
---|---|---|
L10S W13A | −0.947 | Neutral |
−0.722 | Neutral | |
E20L | −0.959 | Neutral |
V28S | 0.681 | Neutral |
P31R | −1.217 | Neutral |
P31G | −1.044 | Neutral |
P40Q | −2.283 | Neutral |
L42W | −2.283 | Neutral |
Q46N | −1.158 | Neutral |
A52V | −1.495 | Neutral |
R58C | −2.302 | Neutral |
G64T | −2.116 | Neutral |
M70F | −3.944 | Deleterious |
Y74M | −6.544 | Deleterious |
Default threshold is −2.5, that is; Variants with a PROVEAN score equal to or below −2.5 are considered “deleterious” while Variants with PROVEAN score above −2.5 are considered “neutral”. G = glycine, A = Alanine, L = leucine, M = methionine, F = phenylalanine, W = tryptophan, Q = glutamine, E = glutamic acid, S = serine, P = proline, V = valine, Y = tyrosine, R = arginine, N = asparagine, T = threonine, C = cysteine.
Variant | PROVEAN Score | Prediction |
---|---|---|
H21R | −1.206 | Neutral |
S32R | −1.171 | Neutral |
I33A | −0.057 | Neutral |
A39W | −1.871 | Neutral |
Q46W | −1.941 | Neutral |
E51A | −2.339 | Neutral |
G54S | −1.826 | Neutral |
R61D | −0.479 | Neutral |
M70V | −2.980 | Deleterious |
E72A | −2.313 | Neutral |
Default threshold is −2.5, that is; Variants with a PROVEAN score equal to or below −2.5 are considered “deleterious” while Variants with PROVEAN score above −2.5 are considered “neutral”. G = glycine, A = Alanine, L = leucine, M = methionine, F = phenylalanine, W = tryptophan, K = lysine, Q = glutamine, E = glutamic acid, S = serine, P = proline, V = valine, I = isoleucine, C = cysteine, Y = tyrosine, H = histidine, R = arginine, N = asparagine, D = aspartic acid, T = threonine.
Variant | PROVEAN Score | Prediction |
---|---|---|
Q30M | −1.170 | Neutral |
T41W | −1.491 | Neutral |
E50R | −0.829 | Neutral |
I62R | −0.835 | Neutral |
H65E | −0.220 | Neutral |
E72S | −1.476 | Neutral |
Y74Q | −6.669 | Deleterious |
Default threshold is −2.5, that is; Variants with a PROVEAN score equal to or below −2.5 are considered “deleterious” while Variants with PROVEAN score above −2.5 are considered “neutral”. M = methionine, Q = glutamine, T = threonine W = tryptophan, E = glutamic acid, R = arginine, I = isoleucine, H = histidine, S = serine, Y = tyrosine.
Variant | PROVEAN Score | Prediction |
---|---|---|
I7L | 0.433 | Neutral |
T9I | 0.329 | Neutral |
V33I | 0.101 | Neutral |
L35H | −1.309 | Neutral |
C40P | 3.229 | Neutral |
R46P | −1.053 | Neutral |
Q61R | 0.187 | Neutral |
Default threshold is −2.5, that is; Variants with a PROVEAN score equal to or below −2.5 are considered “deleterious” while Variants with PROVEAN score above −2.5 are considered “neutral”. I = isoleucine, L = leucine, T = threonine, V = valine, H = histidine, C = cysteine, P = proline, Q = glutamine, Y = tyrosine, R = arginine.
Variant | PROVEAN Score | Prediction |
---|---|---|
A20H | −0.392 | Neutral |
L27H | −1.971 | Neutral |
Q31F | −2.946 | Deleterious |
P38Q | −2.944 | Deleterious |
W43L | −2.161 | Neutral |
A47P | 0.089 | Neutral |
G50Y | −2.034 | Neutral |
E67G | −3.811 | Deleterious |
P70F | −8.148 | Deleterious |
R74C | −4.981 | Deleterious |
Default threshold is −2.5, that is; Variants with a PROVEAN score equal to or below −2.5 are considered “deleterious” while Variants with PROVEAN score above −2.5 are considered “neutral”. A = Alanine, H = histidine, L = leucine, Q = glutamine, F = phenylalanine, P = proline, W = tryptophan, G = glycine, Y = tyrosine, E = glutamic acid, R = arginine, C = cysteine.
The genetic relationship of BMP15 of the studied species using UPGMA showed that members of the Bovidae family (sheep, goat and cattle) were closer at this locus compared to swine and chicken (
BMP15, also known as GDF9B, is an X-linked gene in nature that is expressed in the oocyte and plays a key role in ovarian folliculogenesis. It is a polymorphic gene whose polymorphism has been proven to be associated with increased ovulation rate, sterility and litter size of farm animals [
The ratio of the number of nonsynonymous substitution per nonsynonymous sites (dN; amino acid altering) to the number of synonymous mutations per synonymous sites (dS; silent mutation) also known as omega (ɷ = dN/dS), is a useful estimate of gene selective pressure [
nucleotide substitutions over the entire sequences across the species excluding sheep. This symbolizes that non-synonymous sites evolved faster than the synonymous sites and positive selection effect over-shadows purifying selection. It is an implication that balancing selection (or positive Darwinian section) favoured new variants
and increased allelic polymorphism [
One of the key issues in biology, is understanding how natural selection drives gene functional diversification across different species and lineages [
[
The main goal in animal breeding is to select individuals that have high breeding values for traits of interest as parents to produce the next generation and to do so as quickly as possible. To date, most programs rely on statistical analysis of large data bases with phenotypes on breeding populations by linear mixed model methodology to estimate breeding values on selection candidates. However, there is a long history of research on the use of genetic markers to identify quantitative trait loci and their use in marker-assisted selection but with limited implementation in practical breeding programs [
The neutral or beneficial amino acid substitutions are those substitutions that help in maintaining the structural integrity of cells and tissues. Also, they affect positively the functional roles of proteins involved in signal transduction of visual, hormonal, and other stimulants. However, the harmful amino acid substitutions could cause amino acid change further altering protein function which may lead to susceptibility to disease. They may modify enzyme activity, destabilize protein structures or disrupt protein interactions. The beneficial nsSNPs obtained in the present study, therefore, offer hope for future genetic improvement of goats, sheep, cattle, swine and chicken at the BMP15 locus. This is due to the fact that nsSNPs have been reported to be linked to economically important traits and disease development [
The dendrograms were constructed to compare the common ancestral nucleotide and amino acid sequences of the species since each tree may give a useful information for proper understanding of the evolutionary relationships. The phylogenetic tree revealed that clustering was species wise with some level of intermingling between species. This is an evidence of trans-species evolution which might be attributed to the coding nature of the sequences. The UPGMA consensus trees based on nucleotide and amino acid sequences revealed that goat, sheep and cattle were closer followed by swine while chicken was farther apart. This is in accordance with classical classification as goat, sheep and cattle are members of the bovidae family, and swine shares the order artiodactyla with the ruminants. The findings of this study also agrees with the submissions of Misra et al. [
The study revealed that the gene BMP15 is a polymorphic gene that has numerous mutations which can be beneficial or harmful. However, the beneficial ones were more than the deleterious ones in the present study. The omega values obtained symbolized that non-synonymous sites evolved faster than the synonymous sites and positive selection effect over-shadowed purifying selection. Also, the phylogeny based on nucleotide and amino acid sequences revealed the close relatedness of members of the bovidae family (goat, sheep and cattle), an indication of their closeness in the evolutionary timescale. The information emanating from this study would be relevant in performing further genotype-phenotype research especially the selection of markers of fecundity to improve genetically livestock species in Nigeria, Sub-Saharan Africa.
Bwaseh S. Bibinu,Abdulmojeed Yakubu,Steven B. Ugbo,Ndu I. Dim, (2016) Computational Molecular Analysis of the Sequences of BMP15 Gene of Ruminants and Non-Ruminants. Open Journal of Genetics,06,39-50. doi: 10.4236/ojgen.2016.62005