**3. The gene mapping and expression analysis of these genes**

Quantitative trait loci (QTLs) are regions of the chromosome that are found to be associated with particular phenotypic traits by statistical analysis. Thus far, the pig QTL database (Pig QTLdb) has collected 6,344 QTLs from 281 publications in the past more than ten years. Any genes in these regions might be the positional candidate genes underlying the respective traits. Mapping the genes of HPT axis would help us to evaluate the potential genetic effects of the variations of these genes.

#### **3.1 The electrical mapping**

548 A Bird's-Eye View of Veterinary Medicine

amino acid identity to human TRHR. An intron disrupts the open reading frame in the sequence encoding the putative third intracellular loop which is between the fifth and sixth transmembrane domain. Besides, alternative spliced transcript variants and multiple transcription start sites have been observed in porcine *TRHR* gene (Jiang, 2011). The

Thyrotropin is a member of the glycoprotein hormone family which consisting thyrotrophin, follicle-stimulating hormone and luteinizing hormone in anterior pituitary, and chorionic gonadotrophin in the placenta. Each of these hormones is a heterodimer composed of a common α subunit and a hormone specific β subunit. The common α subunit is encoded by the *CGA* gene, while the TSH specific β subunit is encoded by the *TSHB* gene. Porcine *CGA* gene is 14 kb in length and consists of four exons and three introns (Kato et al., 1991). The coding region starts from the exon 2, and the premature peptide contains a signal peptide of 24 amino acids. Two intragenic microsatellites were found in intron 1 and intron 2. The microsatellite located in the intron 1 has been named as PGHAS or ALPHA and

The porcine *TSHB* gene has at least two exons and one intron, and is 939 bp in length. Its product consists of a signal peptide of 24 amino acid residues, a mature beta subunit protein of 112 residues and an additional extension of six amino acid residues at the carboxyl terminus (Hirai et al., 1989). The TSH beta subunit noncovalently links to the alpha subunit

TSH β subunit is responsible for the hormone specificity, and its translation rate directly determines the secretion and synthesis rates of the hormone TSH. Additionally, it has been reported that mature Chinese meishan pigs had 3 fold greater expressions of *TSHB* gene and greater plasma TSH concentrations than mature Western white composite pigs (Li et al.,

The receptor of the thyrotorpin,TSHR, belongs to the glycoprotein hormone receptor (GPHR) family which is a subset of the G-protein coupled receptor (GPCR) superfamily. It contains a seven-transmembrane domain and a large ectodomain composed of many

The mRNA sequence of the porcine *TSHR* gene has been cloned in 2003 (Igarashi & Nagata, 2003) and characterized to containing an open reading frame coding 764 amino acids. However, as the genomic sequence of porcine *TSHR* gene is still unavailable so far, the genomic structure of the gene is not clear. Of human, the *TSHR* gene was found to be 190 kb in length, and contains ten exons and nine introns, of which all the first nine exons encodes the large extodomain while the last exon is in charge of the seven transmembrane domains

leucine-rich repeats forming TSH-binding surface (Farid & Szkudlinski, 2004).

by wrapping with its "seat belt" structure around the alpha subunit's long loop αL2.

biological functions of these variants remain investigations.

**2.3 Thyrotropin alpha subunit gene (***CGA***)** 

widely used in genetic studies as genetic marker.

**2.4 Thyrotropin beta subunit gene (***TSHB***)** 

**2.5 Thyrotropin receptor gene (***TSHR***)** 

and the intracellular tail.

1996).

In April 2009, the International Swine Genome Sequencing Consortium has completed and released a 4 sequence depth draft (Sscrofa9) by a minimal tile path BAC by BAC approach. This assembly can be conveniently accessed by the web-based query on the Ensemble (http://www.ensembl.org/Sus\_scrofa/Info/Index). Though a more recent assembly Sscrofa 10, a mixed BAC and WGS-based assembly of the porcine genome, has been released in April 2011, assemble errors in it remain to be resolved. Furthermore, the Pig QTLdb offered GBrowse map view of QTLs (http://www.animalgenome.org/cgi-bin/gbrowse/pig/) based on the Sscrofa 9. Thus, with the chromosome position of the candidate gene obtained by querying the Ensemble, one can easily get the information of the QTLs which were mapped onto the same genomic region (Figure 4).


Fig. 4. Query result of pig QTLdb Gbrowse map view, using TRH as an example.

Porcine *TRH* gene is located between 57,837,217 and 57,838,594 on chromosome 13. Based on the pig QTLdb, there are 20 QTLs including average daily gain (ADG), backfat weight, meat

Molecular Characterization of Hypothalamo-Pituitary-Thyroid Genes in Pig (Sus Scrofa) 551

pituitary, testis and fat tissue. The two subunit genes of hormone TSH showed different tissue distribution patterns. While *CGA* was detectable in almost all the tissues, *TSHB* was mainly existed in the pituitary and undetectable or in extremely low level in the other tissues except in the fat tissue and stomach. Expression of porcine *TSHR* existed in all the fifteen organs/tissues, with highest expression level in the thyroid, then the testis and

Polymorphism in human *TRH* gene has been reported to be associated with blood pressure variations and hypertension (Kokubo et al., 2006). By re-sequencing the whole coding region of porcine *TRH* gene, eight sequence variations were identified (Jiang 2011). Among these polymorphisms, only ss325994920 (Figure 2) is a missense mutation which would bring a

Mutations in the *TRHR* could result in central hypothyroidism which causes growth retardation, pudginess and sluggishness (Collu et a., 1997). Further *TRHR* polymorphism has been identified responsible for human lean body mass variations in a genome-wide association study (Liu et al., 2009). Re-sequencing the coding sequences and flanking regions of porcine *TRHR* gene identified seven polymorphic loci (Figure 5a). Two of them locate in

Mice with *CGA* gene knockout were viable, but exhibited severe growth insufficiency and infertility (Kendall et al., 1995). While no inactivating mutations of *CGA* gene has been detected in humans and mice, cases of nonsense mutations of *TSHB* gene have been continuously reported in humans which cause growth retardation and fat metabolism disorders (McDermott et al., 2002; Baquedano et al., 2010). 14 new polymorphic loci of porcine *CGA* gene (Figure 5b) and 5 polymorphisms of *TSHB* gene (Figure 5c) were identified and confirmed by re-sequencing (Jiang et al., 2011b). Four of the *CGA* polymorphisms locate in the promoter region. The single nucleotide polymorphism ss181129018 of *TSHB* gene locates on the first coding exon and brings amino acid change to

*TSHR* is sensitive to point mutations and most of the reported human mutations existed in the last exon 10 (Davis et al., 2006). Re-sequencing the last exon of porcine *TSHR* gene detected three polymorphisms in the exon (Figure 5d), but all were synonymous mutations.

**4. The association study with economic traits in the crossbred of Jinhua and** 

A crossbred was established by mating the Chinese Jinhua pigs (Central China swine type) with European Pietrain pigs, and obvious segregation of growth, carcass and meat quality characters were observed in this population. Association study of the five HPT key genes' polymorphisms with the economic important traits in the crossbred of Jinhua and Pietrain were carried out by analyzing fourteen polymorphic loci with thirty traits of 463 individuals (Jiang 2011). Both PCR-RFLP and tetra-primer ARMS PCR procedure were utilized for

genotyping, and haplotypes were considered as genetic effects.

the intron 1 which has been proven to encompass important regulatory elements.

spleen.

**3.4 The polymorphisms of these genes** 

the signal peptide of TSH β subunit.

**Pietrain** 

Val to Met amino acid mutation into pFE22 and pSE14 peptides.

quality traits and blood parameters have been reported in this region. Porcine *CGA* gene is mapped between 58,190,063 and 58,192,283 on chromosome 1, and 21 QTLs have been mapped to the corresponding region, most of which are fat related traits. The position of *TSHB* gene on porcine chromosome 4 is from 109,815,329 to 109,816,276. 49 QTLs including ADG, backfat thickness (BFT) and meat quality related traits have been mapped to this position. Unfortunately, *TRHR* and *TSHR* genes had not yet been annotated in Sscrofa 9 in the year 2009.
