Detection of carrier Booroola (FecB) allele in BMPR1B gene of MEGA (Merino × Garut) sheep and its association with growth traits
Journal of Genetic Engineering and Biotechnology volume 21, Article number: 19 (2023)
Bone morphogenetic protein receptor 1B (BMPR1B) gene is one of candidate genes for reproductive and growth traits in sheep. The present study was aimed to detect the Booroola (FecB) allele in BMPR1B gene and its association with growth traits in MEGA (Merino × Garut) sheep. A total of 82DNA samples collected from individual lamb (mixed-sex) blood were genotyped for allelic polymorphism using a PCR–RFLP method.
The PCR analysis in BMPR1B gene resulted the amplicons with size of140 bp. The RFLP analysis with AvaII restriction enzymeresultedtwo allelic types of wildtype (A/Fec+) and mutant or Booroola (G/FecB) with frequency of 0.89 and 0.11, respectively. However, the genetic diversity in BMPR1B/AvaII gene of animal studies was categorized tolow category (PIC = 0.18)and under in a genetic equilibrium (χ2 = 1.25).
Itshowed us that carrying FecB allele in the heterozygous sheep were not associated with growth traits in MEGA sheep.
Bone morphogenetic protein receptor 1B (BMPR1B) gene is one of the candidate genes for prolificacy trait in sheep (Juengel et al. 2013) . The BMPR1B gene with a coding sequence 17 exons has been mapped on sheep chromosome 6 along 451,922 bp (GenBank: NC_056059.1). A transitional mutation (A to G) has been occured in the exon 8 of ovine BMPR1B gene namely Booroola (FecB) mutation (Kumar et al. 2021) . In addition, the FecB mutation was occured at 109th nucleotide (GenBank:GQ863576.1)with an amino acid changes from Glutamine (Q) to Arginine (R).This mutation is located in the kinase highly conserved domain BMPR1B or activin-like kinase 6 (ALK6) and characterized by precocious differentiation of ovarian follicles, leading to the production of large members of ovulatory follicles that are smaller in diameter than wildtype follicles (Souza et al. 2003) .
Interestingly, the FecB mutation also affected to themany productive traits.A previous studies reported that the FecB mutation was significantly associated with ovulation rate (Davis, 2005) , litter size (Mahdevi et al. 2014 ; Maskur et al. 2016) , body weight (Gootwine et al. 2006 ; Guan et al. 2007 ), wool production (Schulze et al. 2003) , lamb survival (Gootwine et al. 2008)  and carcass traits (Fahmy et al. 1992 ; Dimitrov and Nedelchev, 1999 ).
The FecB mutation was firstly detected in Booroola Merino sheep from Australia.Unfortunately, there are a few studies to detect the FecB mutation in Indonesian sheep. Maskur et al. (2016)  reported that the evidence ofFecB mutation was observed in fat-tailed sheep breed and associated with litter size.Merino sheep has been imported in Indonesia to increase meat production through crossbreeding program with local ewes.Garut is one of Indonesian native thin-tailed sheep breeds that potential for crossbreeding program with Merino sheep. Previous studies reported that the average of adult weight in Garut rams was39.53 ± 1.95 kg (Rosmawan et al. 2021)  and carcass weight in Garut ewes was23.63 ± 2.39 kg (Prahasta, 2015) . In addition, Haya et al. (2020)  reported Garut ewes at the breeding station has a type of birth single (34.87%), double/twin (45.16%), triple/triplet (19.26%), quadruple/quartet (0.51%) and pentuple (0.16%).
The cross breeding program between Merino and Garut sheep breeds is potential to produce a crossbred sheep with high meat production and litter size traits. Hence, the selection program in the crossbred sheep (Merino × Garut) is important to improve their productivity. This study was aimed to detect the evidence of Booroola (FecB) mutation in the MEGA (Merino × Garut) sheep using a PCR–RFLP method. In addition, the present study was aimed to observe the effect of FecB mutationonthe growth traits of sheep.The results of this study can be used as the early information to select sheep based on BMPR1B gene polymorphism.
Animal and DNA extraction
A total of eighty two (82) mixed-sex MEGA (Merino × Garut) sheep kept at the research station (LIPI and BALITVET) of Bogor, West Java-Indonesia were used in this study. About 7–10 mL of blood sample was taken in each animal from jugular veinusing a venoject needle with vacutainer vacum tube containing EDTA and held on ice box until delivery to the laboratory for further experiments. The genomic DNA was extracted using a modified method of Montgomery and Sise (1990) .
The PCR amplification of ovine BMPR1B gene was performed using a primer pair of Forward: 5′-GTC GCT ATG GGG AAG TTT GGA TG-3′ and Reverse: 5′-CAA GAT GTT TTC ATG CCT CAT CAA CAC GGT C-3′ (Wilson et al. 2001) . According to that primer, the target sequence of BMPR1B gene is along 140 bp (Fig. 1). The PCR reaction was performed in a total volume 10 μL containing 1.2 μL of DNA template (2.18 ng/μL), 10 pmol/μL each of primer, 2 × of DreamTaq Green PCR mastermix kit (ThermoScientific, USA) and 3.6 μL of nuclease-free water. The PCR reaction was performed in a Mastercycler Gradient thermocycler (Eppendorf-Germany) with amplification program comprised of pre-denaturation (95 °C at 2 min); followed by 36 cycles of denaturation (95 °C at 30 s), annealing (56.6 °C at 1 min; 30 s), and extension (72 °C at 30 s); and final extension (72 °C at 2 min).
Electrophoresis of PCR product (amplicon) was performed using 1% agarose at 100 V at 30 min. Amplicons were stained with GelRed (Biotium, USA) along 30 min and then visualized using G-Box documentation system (Syngene, UK).
For genotyping, amount of 10 μL of reaction solutions was used for RFLP analysis with containing of 2 μL PCR product, 0.2 μL of AvaII (G*GWCC) restriction enzyme (ThermoScientific, USA), 1 μL of enzyme buffers and 6.8 μL of nuclease-free water and placed in the water bath for 1 h at 37 °C for digestion. Digested samples were then quantified to visualize the amplified fragments by gel electrophoresis (2% agarose). After digestion with AvaII, a wild type animal (AA or Fec+/Fec+genotype) can be signedwith the presence of one DNA fragment with size of 140 bp in an agarose gel. A mutant animal (GG or FecB/FecB genotype) signed with two DNA fragments along 109 bp and 31 bp. Meanwhile, the carrier animal (AG or Fec+/FecB genotype) signed with three DNA fragments of 140 bp, 109 bp, and 31 bp.
A forward sequencing analysis was performed with a carrier sample (30 μL of PCR product) to confirm the Booroola mutation site in the animal study. The sequencing analysis was performed by a commercial laboratoryservices (1st BASE Laboratories Sdn Bhd, Malaysia) with ABI Prism 96-capillary 3730 xl DNA Analyzer (Applied Biosystems, USA).
The data of growth traits was collected from a herd book of year 1999 to 2002. The animal used in this study was born on first parity of dam.Hence, the data correction was performed in body weight to reduce the effect of sire, sex, and type of birth by using an equation as follows (Hardjosubroto, 1994) :
where BWc is the corrected birth weight; WWc is the corrected weaning weight; YWc is the corrected yearling weight; BW is the actual birth weight; WW is the actual weaning weight; W is the actual weight; Tw is the weaning age; T is the period beetwen weaning to weighing times; DGpre is the pre-weaned daily gain; DGpost is the post-weaned daily gain;CFsexis the correction factor for sex; CTB is the constanta for type of birth, i.e., 1.0 (single)and 1.10 (twin). Therefore, the statistical analyses of genotypic and allelic frequencies, observed heterozigosity (Ho), expected heterozigosity (He), number of effective allele (ne), polymorphic informative content (PIC), and chi-square (χ2) value were computed according to Yasuda (1988)  to evaluate the genetic diversity in the BMPR1B gene of animal studies.
The target gene of BMPR1B gene in animal studies was successfully amplified with signed by presence of a DNA fragment size of 140 bp in the 1% of agarose gel (Fig. 2). Therefore, the PCR–RFLP analysis in BMPR1B/AvaII gene reveals of two genotype of AA-wildtype (Fec+/Fec+) and AG-carrier (Fec+/FecB) as shown in Fig. 3. In this study, a 31 bp of DNA fragment was not ilustrated in the 1% agarose gel because of low size. However, absence of 31 bp in this study was not influenced by the genotyping of BMPR1B/AvaII gene. Hence, a carrier sheep in this study signed with two DNA fragements along 140 bp and 109 bp. In addition, the presence of Booroola mutation site (c.109A > G) was confirmed in the carrier MEGA sheep as shown in Fig. 4. In addition, sheep with carrier FecB allele (heterozygous animal) was observed with low frequency (0.22) as presented in Table 1. Moreover, the frequency of FecB allele in animal studies was 0.11 and lower than Fec+ allele (0.89). However, the polymorphism of BMPR1B/AvaII gene was not associated with growth traits in MEGA sheep (Table 2). Interestingly, the MEGA sheep with heterozygote genotype (Fec+/FecB) have the higher of birth weight, yearling weight and post-weaned daily gain values.
Mostly the FecB allele was absence in many sheep breeds as shown in Table 3. However, low frequency of FecB allele has been reported in Indonesian Fat Tail (0.19) , Nilagiri (0.14) , Bayanbulak (0.08) , Kalehkoohi (0.35)  sheep (Table 3). In contrast, high frequency of FecB allele has been reported in Assaf (0.54) , Garole (0.61) , Bonpala (0.87) , Small Tail Han (0.72) , and Hu (0.84)  sheep (Table 3). However, the absence of FecB allele in sheep can be described as genetic drift evidence that caused by selection and migration factors . Furthermore, the polymorphism in the BMPR1B/AvaII gene of animal study belongs to low category, signed with low PIC value (PIC < 0.30). However, the chi-square (χ2) value revealed that the genetic diversity in BMPR1B/AvaII is under the genetic equilibrium (χ2 < 3.84).
Low PIC value in the present study indicated that the BMPR1B/AvaII gene can not be used as molecular selection because of low genetic diversity. According to Table 2, the FecB (Booroola) allele included of a minor allele since the FecB/FecB is typical of rare genotype. There are many factors causing the allelic frequency such as selection, migration, cross breeding and inbreeding Falconer DS (1996) .
The selection may be the main factor that affecting allelic frequency of FecB in MEGA since the farmers prefer to keep sheep with low litter size. According to the farmers expirience, the survival rate of single lambs are better than twin kids or triplet lambs. This statement supported by Sodiq  who reporting the significant effect between litter size with survival rate in sheep.
This preliminary study showed that the carrying FecB allele was not affected by the growth traits in MEGA sheep (Table 2). The similar finding reported by Abella et al. (2005)  that there was no effect of carrying FecB allele in the growth traits in Boorola × Merinos d’Arles sheep. However, present study reported that the average of BW, YW and DGpost in heterozygous sheep (Fec+/FecB) were higer than those in wild type sheep(Fec+/Fec+). Prevous studies reported that Fec+/FecB genotype was as superior genotype for adult weight in Assaf sheep (Gootwine et al. 2006)  and yearling weight in Garole × Malpura sheep (Kumar et al. 2008) .
In the future, study to observe the effect of FecB mutation on reproductive traits of MEGA sheep is important for developing marker assisted selection (MAS). A previous studies reported that carrying FecB allele affected to the litter size of Indonesian Thin Tail (Maskur et al. 2016) , Mehraban (Talebi et al. 2018) , Kalehkoohi (Mahdavi et al. 2014) , Small Tail Han (Chu et al. 2011)  and Colombian Creole (Hernandez et al. 2020) . In addition, a previous studies reported that two novel mutation of c.35 T/A and c.113A/G were detected in BMPR1B gene (GenBank: GQ863576.1) as reported by Farag et al. (2018)  and Talebi et al. (2018) , respectively. In this study, the evidence of mutation c.35 T/A was not detected with the forward sequencing. Meanwhile, the mutation c.113A/G did not occur in the MEGA sheep as shown in Fig. 4. Furthermore, the intronic region of BMP15 gene has the potency as the genetic markers for sheep since it has many insertion/deletion (indel) mutation sites . Despite, a another BMP family genes of BMP2 and BMP7 are potential as the candidate genes for litter size of sheep (Li et al. 2021) .
The carrier Booroola (FecB) allele was detected in the MEGA (Merino × Garut) sheep with low frequency and not associated with the growth traits. However, birth weight, yearling weight and post-weaned daily gain in homozygous sheep (Fec+/FecB) were higher than those in wildtype sheep (Fec+/Fec+).
Availability of data and materials
All data are primary data and generated from the research, research materials belong to our laboratory (Laboratory of Animal Molecular Genetics).
- Fec B :
- BMPR 1B:
Bone morphogenetic protein receptor 1B
Crossing Merino × Garut sheep
Polymerase chain reaction
Polymerase chain reaction- restriction fragment length polymorphism
Activin-like kinase 6
Ethyelene diamine tetra acetic acid
- DGpost :
Post-weaned daily gain
Juengel JL, Davis GH, McNatty KP (2013) Using sheep lines with mutation in single genes to better understand ovarian function. Reproduction 146:111–123
Kumar A, Kumar R, Misra SS, Sharma RC (2021) Impact of Booroola fecundity gene introgression on sheep production: Indian perspective. Indian J Anim Sci 91:327–336
Souza CJ, Campbell BK, McNeilly AS, Baird DT (2003) Bone morphogenetic proteins and folliculogenesis: lesson from the Booroola mutation. Reproduction 61:361–370
Davis GH (2005) Major genes affecting ovulation rate in sheep. Genet Sel Evol. 37:s11–s23
Mahdavi M, Nanekarani S, Hosseini SD (2014) Mutation in BMPR-1B gene is associated with litter size in Iranian Kalehkoohi sheep. Anim Rep Sci 147:93–98
Maskur M, Tapaul R, Kasip L (2016) Genetic polymorphism of bone morphogenetic protein receptor 1 B gene and its association with litter size in Indonesian fat-tailed sheep. Afr J Biotechnol 15:1315–1319
Gootwine E, Razov A, Bar A, Reicher S (2006) Carrying the FecB (Booroola) mutation is associated with lower birth weight and slower post-weaning growth rate for lambs, as well as a lighter mature body weight for ewes. Rep Fert Dev 18:433–437
Guan F, Liu SR, Shi GQ, Yang LG (2007) Polymorphism ofFecB gene in nine sheep breeds or strain and its effect onlitter size, lamb growth and development. Anim Rep Sci. 99:44–52
Schulze KS, Waldron DF, Willingham TD, Shelby DR, Engdahl GR, Gootwine E, Yoshefi S, Montgomery GW, Tate ML, andLord EA, (2003) Effects of the FecB gene in half-sib familiesof Rambouillet-cross ewes. Sheep Goat Res J18:83–88
Gootwine E, Reicher S, Rozov A (2008) Prolificacy and lambsurvival at birth in Awassi and Assaf sheep carrying the FecB(Booroola) mutation. Anim Rep Sci 108:402–412
Fahmy MH, Boucher JM, Poste LM, Gregoire R, Butler G, Comeau JE (1992) Feed efficiency, carcass characteristics, and sensory quality of lambs, with or without prolific ancestry, fed diets with different protein supplements. J Anim Sci. 70:1365–1374
Dimitrov D, Nedelchev D (1999) Study on growth intensityand meat quantity of lambs crosses between Booroola and north-east Bulgarian finewool sheep. Zhivotnovdni Nauki 36:11–15
Rosmawan DP, Rahmat D, Ayuningsih B, Dhalika T, Siswanto HI (2021) Body weight growth curve of Garut sheep aged 13–16 months feed ration containing 40% grass and 60% concentrate. JITRO 8:104–108 ((in Indonesian with an abstract in English))
Prahasta BI (2015) Carcass and non-carcass characteristic of Garut ewe by different live weight at slaughter house in Babedahan Garut. Bogor Agricultural University, Bogor, Indonesia, B.Sc ((in Indonesian))
Haya AK, Anang A, Heriyadi D (2020) Study on the performance of preweaning weight of Garut sheep at UPTD-BPPTDK Margawati Garut. JITP 8:15–21 ((in Indonesian with an abstract in English))
Montgomery GW, Sise JA (1990) Extraction of DNA from sheep white blood cells. New Zealand J Agricult Res 33:437–441
Wilson T, Wu XY, Juengel JL, Ross IK, Lumsden JM, Lord EA, Dodds KG, Walling GA, McEwan JC, O’Connell AR, McNatty KP, Montgomery GW (2001) Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein 1B receptor (ALK-6) that is expressed in both oocytes and granulose cells. Biol Reprod 64:1225–1235
Hardjosubroto W (1994) Aplikasi Pemuliabiakan Ternak di Lapangan. Gramedia Widiasarana, Jakarta, INA ((in Indonesian))
Yasuda N (1988) HLA polymorphism information content (PIC). Jpn J Hum Genet 33:385–387
Sudhakar A, Rajendran R, Rahumathulla PS (2013) Detection of Booroola (FecB) mutation in Indian sheep - Nilagiri. Small Rum Res 113:55–57
Zuo B, Qian H, Wang Z, Wang X, Nisa N, Bayier A, Ying S, Hu X, Gong C, Guo Z, Wang F (2013) A study on BMPR-1B genes of Bayanbulak sheep. Asian-Aust J Anim Sci 26:36–42
Polley S, De S, Brahma B, Mukherjee A, Vinesh PV, Batabyal S, Arora JS, Pan S, Samanta AK, Datta TK, Goswami SL (2010) Polymorphism of BMPR1B, BMP15 and GDF9 fecundity genes in prolific Garole sheep. Trop Anim Health Prod 42:985–993
Roy J, Polley S, De S, Mukherjee A, Batabyal S, Pan S, Brahma B, Datta TK, Goswami SL (2011) Polymorphism of fecundity genes (FecB, FecX, and FecG) in the Indian Bonpala sheep. Anim Biotech 22:151–162
Chu M, Jia L, Zhang Y, Jin M, Chen H, Fang L, Di R, Cao G, Feng T, Tang Q, Ma Y, Li K (2011) Polymorphisms of coding region of BMPR-1B gene and their relationship with litter size in sheep. Mol Biol Rep 38:4071–4076
Star B, Spencer HG (2013) Effects of genetic drift and gene flow on the selective maintenance of genetic variation. Genetics 194:235–244
Borni J, Sonia B, Naouer DM (2011) PCR-RFLP of BMPR-1B gene in North African Barbarine sheep. ROAVS 1:200–203
Georgeseu SE, Gheorghe H, Rebedea M, Costache M (2011) Investigation of FecB mutation in four Romanian sheep breeds. Sci Pap: Anim Sci Biotech 44:219–222
Abouheif MA, Al-Owaimer AN, Shafey TM, AlShaikh MA, Aljumaah RS (2011) Polymorphism of Boorola FecB gene in prolific individuals from Najdi and Naeimi breeds of Saudi Arabia. J Anim Vet Adv 10:1262–1264
Lopez-Ramirez RB, Magana-Sevilla HF, Zamora-Bustillos R, Ramon-Ugalde JP, Gonzalez-Mendoza D (2014) Analysis of the 3’ end regions of the GDF and BMPR1B genes in Blackbelly sheep from Yucatan, Mexico. Cien Inv Agr 41:123–128
Pardeshi VC, Sainani MN, Maddox JF, Ghalsasi PM, Nimbkar C, Gupta VS (2005) Assessing the role of FecB mutation in productvity of Indian sheep. Current Sci 89:887–890
Mohammadi G (2016) Determination of FecX, FecB and FecGHmutations in Iranian Arabic sheep. Basrah J Vet Res 15:435–445
Talebi R, Ahmadi A, Afraz F, Sarry J, Woloszyn F, Fabre S (2018) Detection ofsingle nucleotide polymorphisms at major prolificacy genes in the Mehraban sheep and association with litter size. Ann Anim Sci 18:685–698
Al-Barzinji YMS (2010) Polymorphism in booroola (FecB) gene associated with litter size in Hamdani sheep. Proceedings of 3rd Kurdistan Conference on Biological Sciences; Duhok, Kurdistan, Iran. pp 413–417
Sulaiman BK, Abbas BK, Nada SM, Ishak MA (2014) Investigation of fecundity FecB gene in Iraqi Awassi ewes. Iraqi J Agric Sci 45:306–312
Al-Barzinji YMS, Taha KM (2017) Molecular characterization on FecB, FecX and FecGH mutations in Iraqi sheep breeds using PCR-RFLP technique. Mal Appl Biol 46:135–144
El-Hanafy AA, El-Saadani MA (2009) Finger printing of FECB gene in five Egyptian sheep breeds. Biotechnol Anim Husb 25:205–212
Ahmed HN, Mahmoud KGM, Kandiel MM, Helmy NA, Ibrahim SS, Nawito MF, Othman OE (2016) Investigation of the Booroola gene in Egyptian ewes with different reproductive status using PCR-RFLP. Egypt J Vet Sci 47:1–12
Othman OE, El-Kader HAA, El-Rahim AHA, El-Moneim OMA, Alam SS (2018) Genetic characterization of three fertility genes in Egyptian sheepand goat breeds. Indian J Anim Sci 88:200–205
Farag IM, Darwish HR, Aboelhassan DM, Aboelenin MM, Shakweer WME (2018) Study of genetic polymorphism in BMPR1B gene and its association with improving twin production in Egyptian sheep and goats. Res J Anim Vet Sci 10:1–5
Hernandez D, Montes D, Jaime OV (2020) Association of the FecB polymorphism with the natural prolificacy of the Colombian Creole sheep. Rev MZV Cordoba 25:e1771
Mohamed SEI, Ahmed RM, Jawasreh KIZ, Salih MAM, Abdelhalim DM, Abdelgadir AW, Obeidat MT, Musa LMA, Ahmed MKA (2020) Genetic polymorphisms of fecundity genes in Watish Sudanese desert sheep. Vet World 13:614–621
Falconer DS (1996) Introduction to Quantitative Genetics. Pearson Education, London, UK
Sodiq A, Yuwono P, Santosa SA (2011) Litter size and lamb survivability of Batur sheep in upland areas of Banjarnegara Regency, Indonesia. Anim Prod 13:166–172
Abella DF, Cognie Y, Thimonier J, Seck M, Blanc MR (2005) Effectsof the FecB gene on birth weight, postnatal growth rate and puberty in Booroola x Mérinos d’Arles ewe lambs. Anim Res, EDP Sci 54:283–288
Kumar S, Mishra AK, Kolte AP, Arora AL, Singh D, Singh VK (2008) Effect of the Booroola (Fec B) genotypes ongrowth performance, ewe’s productivity efficiency and littersize in Garole × Malpura sheep. Anim Rep Sci 105:319–331
Zhang Z, Liu Q, Di R, Hu W, Wang X, He X, Ma L, Chu M (2019) Single nucleotide polymorphisms in BMP2 and BMP7 and the association with litter size in Small Tail Han sheep. Anim Rep Sci 204:183–192
Li H, Xu H, Akhatayeva Z, Liu H, Lin C, Han X, Lu X, Lan X, Zhang Q, Pan C (2021) Novel indel variations of the sheep FecB gene and their effects on litter size. Gene 767:145176
This research was funded by a research project DIPA LIPI 2021 No: 45/A/DH/2021. The DNA samples and growth data records of animal studies were collected from ACIAR (Australian Centre for International Agricultural Research) project AS1/9727.
The financial support to cover all research activities were obtained from a research project DIPA LIPI 2021 No.: 45/A/DH/2021. DNA materials used in this activity were collected from ACIAR (Australian Centre for International Agricultural Research) project AS1/9727.
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Margawati, E.T., Putra, W.P.B., Rizki, M. et al. Detection of carrier Booroola (FecB) allele in BMPR1B gene of MEGA (Merino × Garut) sheep and its association with growth traits. J Genet Eng Biotechnol 21, 19 (2023). https://doi.org/10.1186/s43141-023-00475-z
- BMPR1B gene
- Growth traits
- MEGA sheep