Novel Mutations in The Coding Region of The Third Exon of Myostatin (MSTN) Gene in Madura Cattle
Irida Novianti*1), Rafika Febriani Putri1), Doni Herviyanto1), Ahmad Furqon2), Wike Andre Septian1), Kuswati Kuswati1) and Suyadi1)
1) Faculty of Animal Husbandry, Universitas Brawijaya, Jl. Veterans Malang 65145 East Java Indonesia
2) Research Center for Applied Zoology, National Research and Innovation Agency, Bogor 16915 West Java, Indonesia
Submitted: 13 March 2023, Accepted: 4 April 2023
ABSTRACT: Molecular information using gene variants could support livestock selection to improve livestock productivity. This study aimed to identify the variants in part of the MSTN gene (part of the third exon coding region), a gene known for its significant effect on muscle growth. DNA was isolated from 95 Madura cattle blood samples collected in Pamekasan, Madura. PCR was used to amplify to MSTN region in part of the third exon, and one-way sequencing was conducted to obtain the sequence from the amplified product. Six SNPs were identified. Four SNPs were novel mutations, and the other two SNPs were recorded in the Ensembl database (rs466598800 and rs211583837). Among the identified novel mutations, one mutation in c.1301G/T caused a stop codon for the TT genotype cattle. However, no cattle carried the TT genotype. Three other mutations in c.1324, c.1373, and c.1399 were missense mutations that change the amino acids Q311L, A318P, and L335B, respectively. Most of the cattle had heterozygote genotypes for all the identified SNPs. It could also be noted that there were sequence differences in the part of the third exon identified between Madura cattle and Zebu cattle (by Tantia et al., 2006).
Keywords: Single nucleotide polymorphisms; Myostatin; Madura cattle
*Corresponding Author: [email protected]
INTRODUCTION
Madura cattle is one of the Indonesian indigenous cattle which, from its mtDNA found to be crossbred of Zebu cattle and Banteng (Mohammad et al., 2009). Madura cattle became one of the natural resources from Madura Island, Indonesia, which had a variety of functions: cultural function, prestige/social function, supporting the farming sector, and as the source of income or business (Widi et al.,2013). The prominent roles of Madura cattle become the consideration to improve their productivity.
Productivity can be improved by applying good management of the cattle. Moreover, molecular information (marker assisted selection) can increase cattle productivity.
Therefore, investigating gene markers through DNA variants must be conducted first. DNA variants may alter the encoded protein, leading to an increase, a decrease, or a complete loss of protein expression, and finally, may affect the phenotypic traits (Lodish, Berk, and Zipursky, 2000).
Muscularity is one trait that farmer often uses to select their cattle. The Myostatin gene (MSTN) is known to have a significant role in skeletal muscle growth.
Mc Pherron, Lawler, and Lee (1997) first identified the part of MSTN. MSTN is a negative regulator of skeletal muscle.
Therefore, mutation of MSTN could lead to an increase in skeletal muscle mass. Various MSTN mutations were found in some cattle breeds, especially Bos taurus. Belgian blue is the most famous cattle breed that is known to carry a mutation of the MSTN gene and express extreme muscle mass, thus it is called double-muscled cattle. Deletion of 11 base pairs in the coding area of MSTN at the bioactive carboxyl-terminal domain caused the stop codon and led to the inactivation of MSTN (Mc Pherron, Lawler and Lee, 1997;
Grober et al.,1997; Kambadur et al., 1997;
Aiello, Patel and Lasagna, 2018).
Piedmontese is another Bos taurus that carried an MSTN mutation that altered the G base to become A base and caused the substitution of Cysteine by tyrosine at the 313 amino acids (Mc Pherron, Lawler and
Lee, 1997 and Kambadur et al., 1997). An SNP identified in the first exon was found to affect the moderate muscle mass of Limousine (Mc Pherron, Lawler, and Lee, 1997 and Sellick et al., 2007). Meanwhile, fewer studies observed the mutation of MSTN of the Bos indicus. Tantia et al (2006) were the first research team to compare the MSTN sequence between Bos indicus and Bos taurus.
Only a few studies were on the MSTN polymorphisms for Indonesian indigenous cattle. Khasanah, et al. (2016) identified polymorphism at the promoter region in Bali cattle. Moreover, Prihandini, et al. (2021) identified polymorphisms in the first exon and intron in Rambon, Jabres, Galekan, Sragen, Donggala, and Madura. However, no study identified the MSTN gene polymorphism in the Madura cattle's third exon.
Considering the importance of identifying MSTN variants in all MSTN regions, a study on the polymorphisms of MSTN in the third exon must be conducted.
Using these MSTN identified variants, it would be expected that the association study would be able to be conducted and valuable for marker assisted selection.
MATERIAL AND METHODS
Blood samples from 95 Madura Cattle were collected in this study. Those 95 Madura cattle were obtained from 58 bulls and 37 cows of Madura cattle which were located in Pamekasan District, Madura. All cattle were selected based on the special phenotypic characteristics of purebred Madura cattle.
DNA extraction and amplification
The DNA was extracted from the collected blood sample. A tissue/blood DNA mini kit for DNA extraction (GS100) from Geneaid™ was used. Prior to the DNA extraction process, the amplification using PCR was conducted. Before the amplification, the primers were first designed. The primers were designed to obtain PCR product that would cover where the 11 bp deletion of MSTN gene harbors.
The sequence of MSTN gene from the gene bank (JQ7 11180.1) and Ensembl was used to obtain the MSTN sequence in the third exon. Forward and Reverse primers were designed using Molecular Evolutionary Genetic Analysis X (MEGA X) and prime stat application. The primers were 5’- GTCCTTGAGGTAGGAGAGTG-3’
(forward) and 3’-
GACCTCATGAACACCCACAG-5’
(reverse). The expected PCR product was 445 bp.
BIO RAD T100 Thermal Cycler PCR machine was used to amplify the DNA.
1 μl DNA was added into 30 μl reagent amplification that consisted of 15 μl of go taq green master mix, forward and reverse primers respectively 0.3 μl and 14.4 nuclease-free water (NFW) and sample DNA. PCR was conducted under the following condition: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 10 seconds;
primer annealing at 61°C for 20 seconds;
extension at 72°C for 30 seconds and final extension at 72°C for five minutes. Once the amplification process had been completed, the PCR product then needed to be confirmed by electrophoresis using 1.5%
agarose gel added with Nucleic Acid Dye for
40-45 minutes at 100 V. The PCR product was visualized under UV illumination.
Sequencing and polymorphisms identification
One-way sequencing (forward direction) was carried out at 1st Base Sequencing company, Selangor, Malaysia, which used the ABI Prime 3100-Avant Genetic Analyzer machine. The sequencing results were analyzed using BioEdit program and aligned with the Bos taurus and Bos indicus sequence (JQ7 11180.1 and AY794986.1) using MEGA X. Then the identified polymorphisms were counted for their genotype and allele frequency.
RESULTS AND DISCUSSION
Part of the region in exon three was sequenced, and 445 bp sequencing fragments were obtained from 95 Madura Cattle. The obtained sequence was then compared with Bos taurus and Bos Indicus (JQ7 11180.1 and AY794986.1). Six Single Nucleotide polymorphisms (SNPs) were identified (Table 1). Four identified SNPs were considered novel variants since those SNPs had not been recorded in the Ensembl database. In comparison, two other SNPs were synonymous variants recorded in the Ensembl database (rs466598800 and rs211583837).
Table 1. Identified SNPs in Madura cattle
Base Position Bos taurus sequence (JQ7 11180.1)
Bos indicus sequence (AY794986.1)
Madura cattle Sequence
Mutation type
c. 1301 G G G/T Stop codon
c. 1324 A A A/T Missense (Q311L)
c. 1373 G C G/C Missense (A318P)
c. 1399 G C G/C Missense (L335B)
c. 1444 A C A/C Synonymous
(rs466598800)
c.1450 C T C/T Synonymous
(rs211583837)
Table 1 also showed differences between the sequence of Madura cattle and the Bos indicus sequence that had been identified by Tantia et al. (2006). Those differences might be because Madura cattle not originated only from the Zebu breeds but they also had a banteng introgression (Mohammad et al., 2009)
The SNP at c.1301 should be highlighted since it could cause a stop codon when the T base substituted the G base.
However, no cattle carried the TT genotype in the observed Madura cattle. Most of them are heterozygotes (had GT genotype). A stop codon mutation would halt protein synthesis. A missense mutation in c.1324
caused the substitution of Glutamine with Leucine at the 311 amino acids. Those amino acids change was non-conservative change. While for other two missense mutations were conservative changes.
Heterozygote cattle were mainly identified for all the found SNPs (Table. 2). SNP in c.1301 and c.1324 had only two genotypes (homozygote and heterozygote), while the other SNPs had all three genotypes.
Table.2 Genotype and Allele Frequencies of the Identified SNPs
SNP base
location Genotype Number of Genotype Genotype
Frequency Allele Allele Frequency
c. 1301 GG 1 0.01 G 0.51
GT 94 0.99 T 0.49
TT - -
c. 1324 AA 10 0.11 A 0.55
AT 85 0.89 T 0.45
TT - -
c. 1373 GG 2 0.02 G 0.51
GC 92 0.97 C 0.49
CC 1 0.01
c. 1399 GG 31 0.33 G 0.66
GC 63 0.66 C 0.34
CC 1 0.01
c. 1444 AA 26 0.27 A 0.5
AC 43 0.45 C 0.5
CC 26 0.27
c.1450 CC 22 0.23 C 0.44
CT 40 0.42 T 0.56
TT 33 0.35
11 bp deletion of the MSTN gene that caused double muscling in Belgian Blue cattle also recited in the third exon. The primers used in this study also covered those loci, but no deletion mutation was identified in the targeted region. Jakaria et al. (2021) also concluded that no 11 bp deletions were identified in Ongole filial cattle, a crossbred between Ongole (Zebu cattle) and local Indonesian cattle. Moreover, there was no 11 bp deletion in the third exon of Bos indicus MSTN sequenced by Tantia et al.
(2006). It should be noted that no other studies that had observed the novel identified polymorphisms of the MSTN gene as this study.
CONCLUSION
This study identified six polymorphisms (SNPs), and four SNPs were novel mutations. It can also be concluded that differences were found in the part of the third exon sequence between Madura cattle and Zebu cattle. This preliminary result could be used to conduct association studies.
ACKNOWLEDGEMENT
The authors want to acknowledge the Institute of Research and Community Services of Universitas Brawijaya, Malang, Indonesia, for the Beginner Research Grants (HPP) scheme funding (contract number 974.76/UN10. C10/PN/2022) so that the authors could conduct this study.
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