Identifying FecB genotypes in the muscle from sheep breeds indigenous to Xilingol, and establishment of a TaqMan real‐time PCR technique to distinguish FecB alleles

Abstract The muscle from Xilingol indigenous sheep breeds are famous in China, and the FecB genotype in this population remains uncharacterized. In this study, SNPs in the FecB locus were investigated by pyrosequencing, and an optimized PCR‐RFLP technique was generated to identify SNPs. In addition, an efficient technique for high‐throughput identification of SNPs in FecB was optimized using TaqMan real‐time PCR and breed‐conservative primers and SNP‐specific probes. By genotyping the FecB locus in the muscle of Xilingol indigenous sheep breeds using a novel TaqMan real‐time PCR assay, our study has generated the groundwork for the authentication of Xilingol mutton based on the specific gene and the prolificacy‐oriented breeding of Xilingol sheep using marker‐assisted selection strategies in the future.


| INTRODUC TI ON
The Booroola locus (FecB) carries a dominant autosomal mutation that is responsible for the hyperprolific characteristics of Booroola ewes, which was first discovered in the Australian Merino breed (Davis, 2004). The high fecundity of Booroola ewes results from the FecB mutation in the bone morphogenetic protein receptor 1B (BMPR-1B) gene, which encodes a transforming growth factor β gene (TGFβ) (Mulsant et al., 2001;Souza et al., 2001). The FecB mutation leads to higher ovulation rate by affecting follicular fluid and ovarian vein serum (Guo et al., 2018). The point mutation (A turn to G) at base 746 of the coding region of BMPR-1B, changing a glutamine to an arginine, is associated with the hyperprolific profile of Booroola ewes (Souza et al., 2001;Wilson et al., 2001). Thus, the FecB mutation has become one of the candidates for breeding sheep for high prolificacy using marker-assisted selection (Chen et al., 2015;Hua & Yang, 2009).
The Xilingol grassland in China is a natural grazing area, it is known for its muscle and for its high-quality pollution-free and free-range sheep. The average annual output in Xilingol is more than 10 million sheep. The indigenous sheep breeds from Xilingol are Sunit sheep, Ujimuqin sheep, and Chahar sheep. Despite the thriving sheep farming in the Xilingol region, SNPs in the FecB locus remain unknown in the above three breeds. The hyperprolific effect of the FecB mutation can serve as a genetic marker in marker-assisted selection strategies to increase litter size of sheep (Chen et al., 2015;Wang et al., 2018).
The objective of this study was to develop a novel TaqMan realtime PCR protocol that can be used to identify SNPs in the FecB locus in the muscle from Xilingol indigenous sheep breeds using SNP-specific probes targeting the FecB locus. The assay with different TaqMan probes can be further used for the authentication of Xilingol mutton based on the specific gene and genotyping sheep from the Xilingol region for marker-assisted selection.

| TaqMan Real-time PCR
TaqMan real-time PCRs were carried out using primers (LP3 and RP3) and probes (Probe-A and Probe-G) ( Table 1)

| Amplification and sequencing of the FecB locus in the muscle from Mongolian sheep breeds
The FecB locus has two alleles in mutton, where A is the wild-type nucleotide and G is the mutant nucleotide. For the FecB locus, the G variant leads to a change in amino acid from glutamine to arginine (Souza et al., 2001). The presence of the FecB mutation has been investigated in a few prolific breeds, such as Booroola Merino (Mulsant et al., 2001), Indian Bonpala sheep (Roy et al., 2011),

TA B L E 1
The primers and probes that were used for conventional and real-time PCR

Primers Probes
Sequence (

| Optimization of PCR-RFLP to identify FecB alleles
In order to differentiate between the A and G variants of FecB, we PCR-RFLP is regarded as a simple method for identifying SNPs.
The above PCR-RFLP approach using the AvaII restriction site has been used previously to genotype prolific sheep (Chu et al., 2007;El-Seedy et al., 2017;Ganai et al., 2012;Gootwine et al., 2008;Kumar et al., 2008;Mahdavi et al., 2014;Xu et al., 2010). Nevertheless, improvements were needed to better screen for prolific sheep. First, conventional DNA ladders cannot discriminate the 190-and 160bp bands, which can be difficult to distinguish in the agarose gel.
Second, the 190-and 160-bp bands can sometimes appear as one band in gels with low concentration of agarose. Thus, we developed optimized primers (LP2 and RP2) for PCR-RFLP to identify the FecB alleles (A, G, or A/G). RP2 was designed to introduce a point mutation that results in a 220-bp fragment containing the AvaII restriction site (G|GACC) if the fragment contains the G nucleotide. DNA from four individuals each carrying the A, G, and A/G variant in the FecB locus were used to test the effectiveness of the LP2 and RP2 primers ( Figure 2b). PCR products that were restriction digested using AvaII yielded 220-and 170-bp bands for individuals carrying the A or G nucleotides, respectively (Figure 2b). Individuals that were heterozygous yielded both 220-and 170-bp bands (Figure 2b). These results validate the new primer pair, which allowed for easier differentiation of the two genotypes, as it is easier to separate 220-and 170-bp bands, compared to 190 and 160 bp.

| Development and validation of a TaqMan realtime PCR technique to distinguish the FecB alleles
While PCR-RFLP can be carried out using basic laboratory equipment to relatively high levels of success, distinguishing the relative position of the amplified nucleotide sequence in an agarose gel can be prone to error. Error can be introduced through the difference in the quantity of DNA used for restriction digest, the digestion efficiency of the restriction enzyme, the length of digestion, the concentration of the agarose gel, and the volume of sample loaded during electrophoresis. TaqMan real-time PCR provides a more reliable alternative for genotyping SNPs (Gaedigk et al., 2015;Schleinitz et al., 2011;Woodward, 2014). In particular, real-time PCR results can be observed without electrophoresis, and can be used in a highthroughput manner.
For these reasons, we sought to develop an efficient technique for high-throughput identification of the FecB alleles using TaqMan real-time PCR with primers (LP3 and RP3) and SNP-specific probes (Probe-A and Probe-G) ( Table 1) -2021-01 andYB-2022-13).

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Liang Guo
https://orcid.org/0000-0002-1954-1179 F I G U R E 3 TaqMan real-time PCR to identify the A variant (a), G variant (b), and heterozygous A/G variant (c) of the FecB locus. The homozygous variants were confirmed using 10 sheep with known genotypes. FAM or HEX were simultaneously identified in the amplification plots of eight heterozygous sheep in (c)