ANALYSIS OF FSHB GENE POLYMORPHISM IN POLISH LANDRACE AND POLISH LARGE WHITE X POLISH LANDRACE SOWS

Daniel Polasik¹, Monika Kumalska¹, Yuri Sawaragi², Grzegorz Żak³, Mirosław Tyra³, Paweł Urbański⁴, Arkadiusz Terman^1
1 Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
² Kyoto University, Kyoto, Japan
³ Department of Animal Genetics and Breeding, National Research Institute of Animal Production, Balice k. Krakowa, Poland
⁴ Department of Molecular Cytogenetics, Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, Jastrzębiec, Poland

ABSTRACT

FSHB gene is considered as a marker for reproductive traits in human as well as in domestic animals. The aim of this study was to estimate associations between polymorphism in FSHB gene and reproductive traits in Polish Landrace breed (n=260) and Polish Large White × Polish Landrace crossbreed (n=381) sows. The following traits were analyzed: total number of piglets born (TNB), number of piglets born alive (NBA) and number of piglets weaned (NW). The polymorphism in FSHB gene was determined by PCR-RFLP method using HaeIII restriction enzymes. Mixed linear model  was applied for association analysis which considered effects of genotype, litter order and year season. Two alleles of FSHB gene were identified – A and B with following frequency: 0.058 and 0.942 (Polish Landrace), 0.150 and 0.850 (Polish Large White × Polish Landrace) respectively. The distribution of genotypes was in Hardy-Weinberg equilibrium in both of analyzed herds. Analysis of relationship between FSHB/HaeIII polymorphism and reproductive traits in sows showed that genotype BB was favorable for all analyzed traits in first and second parity but differences were small and not confirmed statistically. The significant effect (P≤0,01) of litter order on analyzed traits was however observed.

Key words: FSHB gene, pigs, polymorphism, reproductive traits.

INTRODUCTION

Reproductive performance of sows is one of the most economically important attribute taking into consideration in pig production. There are limited number of genes that have proved significant associations with reproductive traits. Among them are genes coding for: estrogen receptor (ESR), prolactin receptor (PRLR), follicle-stimulating hormone β subunit (FSHB), erythropoietin receptor (EPOR) and retinol binding protein 4 (RBP4) [19].

Follicle-stimulating hormone (FSH) is a member of pituitary glycoprotein family which includes  luteinizing hormone, thyroid-stimulating hormone and chorionic gonadotropin. FSH is the major stimulator of follicle development during early antral to preovulatory stages [6]. Recombinant human FSH (r‑hFSH) is frequently used in assisted reproductive technology (ART) mainly to control ovarian hyperstimulation [24]. FSH is made of two subunits – α and β which are encoded by different genes [4]. An α-subunit is shared with luteinizing hormone and thyroid-stimulating hormone, however β-subunit is specific to FSH that confers biological specificity [22]. The porcine FSHB gene has been mapped on chromosome 2 (SScr2: 32.860.737-32.865.005) and consists of three coding regions. Transcript length is 1797bp, however protein – 129aa [3]. FSHB transcription has major control over serum FSH and, therefore, affects reproduction directly [10]. Hirai et al. [5] studied the structure of porcine FSHB gene as well as their flanking regions, spanning 10kbp together. They found that consensus steroid-responsive element was absent in the 5'-flanking region. Authors, however proved the presence of short interspersed repeated sequences (SINES)-type non-viral retroposons and other SINES-like sequences in the analyzed genomic region. Further investigations showed that Chinese pig breeds exhibited a statistically significant higher frequency of the allele with SINE located in the first intron of FSHB gene than the European pig breeds did. Moreover there was proof that SINE insertion negatively regulates the expression of FSHB gene [16]. In the intron 1 of porcine FSHB gene single nucleotide polymorphism (SNP) was also detected (g.5894A>G) [23]. Similar to SINE sequence different genotypes may influence expression of FSHB gene as well.

The aim of this study was to analyze A>G substitution located in FSHB gene and find possible associations between different genotypes and reproductive traits in purebred and crossbred sows.

MATERIAL AND METHODS

Investigations were carried out on two sow herds composed of purebred Polish Landrace sows (PL), (n=260) and the crossbreed Polish Large White × Polish Landrace (PLW × PL) (n=381). Herds were bred and raised on farms located in Westpomeranian province (Poland). Rearing and feeding conditions were the same for both farms and equalized for all animals.

Genomic DNA was isolated from whole blood using The MasterPure™ DNA Purification Kit for BloodVersion II (Epicentre). FSHB genotypes were determined using the PCR-RFLP method according to Rohrer et al. [25]. PCR was performed with the following pair of primers: F 5’-AGTTCTGAAATGATTTTTCGGG-5’ and R 5’-TTTGCCATTGACTGTCTTAAAGG-3’. Reactions were carried out in total volume of 15 μl containing: 60–90 ng of DNA, 2xPCR Mix (A&A Biotechnology), 10 pmol of each primer and PCR grade water. The PCR cycle consisted of 92°C for 2 min; 30 cycles of 94°C for 1 min, 58°C for 1 min and 72°C for 1 min; and 72°C for 5 min. Obtained amplicons (634bp) were digested with 5U of the HaeIII restriction enzyme (MBI Fermentas) at 37°C overnight. Restriction fragments were separated in 2% agarose gels stained with ethidium bromide. Gels were visualized in UV light and recorded by use Vilber Lourmat system. The lengths of restriction fragments were as follows: 208, 173, 159, 84bp for A allele and 332, 208, 84bp for allele B.

Reproductive traits such as total number of piglets born (TNB), number of piglets born alive (NBA) and number of piglets weaned (NW) were collected from farm records. All data were analyzed with SAS/STAT® Software Ver. 9 [27]. Following ‘mixed’ linear model (procedure REML) was applied for association study:

Y_ijklm  = µ + g_i + y_sj + p_k + d_l + s_m + e_ijklm

where:
Y_ijklm – analyzed trait (TNB, NBA, NW);
µ – population mean;
g_i – effect of i-th genotype (i = 1,2);
y_sj –  effect of j-th year season (j = 1,2,…, 20) ;
p_k – effect of k-th the parity (k = 1, ≥2);
d_l – random polygenic effect of the l-th dam (l = 1,2,…,225);
s_m - random polygenic effect of the m-th sire (k = 1,2,…,39);
e_ijklm - random residual term.

Genotype AA was excluded from association analysis due to very low frequency. Genetic parameters such as genotypes and alleles frequency, gene diversity (expected heterozygosity), Hardy-Weinberg equilibrium (χ²) and PIC (polymorphic information content) for each herd were calculated using PowerMarker ver. 3.25 [15]. The RYR1 genotypes were determined by use Hin6I restriction enzyme according to Brening and Brem [1].

RESULTS AND DISCUSSION

In the analyzed herds of Polish Landrace and Polish Large White × Polish Landrace breeds three genotypes of FSHB gene were identified. AA genotype appeared with lower frequency, however BB with highest. Minor allele frequency was 0.058 for PL and 0.150 for PLW × PL sows. Analysis of genotypes distribution proved that both herds were in Hardy-Weinberg equilibrium. Gene diversity was above two times higher in PLW × PL (0.254) in relation to PL breed. Similar tendency was observed for polymorphic information content. Table 1 presents genetic parameters calculated for analyzed herds. RYR1 gene is very important in breeding of pigs, and their variants could modify the effect of other genes. In the analyzed herds of sows only one genotype – CC was observed.

Table 1. Genetic parameters calculated for investigated herds of sows

Breed	Genotype frequency			Allele frequency		He	PIC	χ2	p
	AA	AB	BB	A	B
PL	0.004	0.108	0.888	0.058	0.942	0.109	0.103	0.024	0.878
PLW × PL	0.021	0.257	0.722	0.150	0.850	0.254	0.222	0.045	0.832

The basic statistical characteristics and results of variance analysis, taking into account fixed effects included in the model are presented in Table 2. The applied model considered 3 fixed effects: genotype, litter order year and season. As shown in Table 2, only the effect of litter was statistically significant (P≤0,01). It resulted in further analysis of this parameter (see Tab. 3). Although the low statistical effect, FSHB genotypes were also analyzed and included in Table 3, because it was the main goal of this study.   

Table 2. Results of variance analysis (mean values, standard deviations, coefficient of variation and effect) of examined traits

Trait	Mean ±SD	V	Effect (Femp)
			Genotype	Parity	Season
PL
TNB	8,98 ±2,25	25,05	2,34ns	11,07**	2,97ns
NBA	8,56 ±2,38	27,80	1,93ns	9,78**	1,83ns
NW	8,07 ±2,14	26,51	1,73ns	8,63**	1,67ns
PLW × PL
TNB	8,88 ±2,25	25,33	1,14ns	12,09**	1,97ns
NBA	8,76 ±2,24	25,57	1,19ns	9,25**	1,43ns
NW	8,38 ±2,12	25,29	1,16ns	9,12**	1,73ns
* P≤0,05; **P≤0,01; ns – statistically non-significant differences; TNB – number of piglets born; NBA – number of piglets born alive; NW – number of piglets weaned

Table 3. Association between FSHB genotypes and reproductive traits in analyzed herds of sows

Trait	Parity	Genotype
		AB			BB
		n		SE	n		SE
PL
TNB	I	26	9.05	0.38	227	9.09	0.29
NBA			8.99	0.38		9.03	0.28
NW			8.82	0.39		8.90	0.29
TNB	II	27	9.14	0.35	229	9.22	0.26
NBA			9.03	0.35		9.11	0.25
NW			8.86	0.37		8.91	0.28
PLW × PL
TNB	I	94	8.85	0.34	265	8.94	0.26
NBA			8.80	0.37		8.90	0.26
NW			8.79	0.34		8.87	0.25
TNB	II	96	8.95	0.33	272	9.02	0.22
NBA			8.89	0.55		8.97	0.21
NW			8.83	0.32		8.94	0.21
TNB – number of piglets born; NBA – number of piglets born alive; NW – number of piglets weaned;

For the association study two genotypes were take into consideration – BB and AB. Results showed that sows with BB genotype are characterized by higher values for all reproductive traits, however differences were small and not confirmed by statistical test. It was noticed for I and II parity in both breeds. Relationships between FSHB genotypes and reproductive traits are given in Table 2.

Genetic improvement of litter size has been limited by traditional selection because of the low heritability and sex-limited expression of reproductive traits. The identification of a specific gene or an anonymous genetic marker for litter size traits in pigs could have a major impact on the improvement of reproductive performance by increasing the accuracy of selection  [26]. Due to its function, FSHB gene has been proposed as a candidate gene for reproductive traits in mammals.

In our study we analyzed polymorphism in FSHB gene located in intron 1. We noticed that the frequency of AA genotype was very low (0.004-0.021). Similar frequency was observed by Korwin-Kossakowska et al. [12] in Polish 990 synthetic line (0.01). Humpolicek et al. [7] ivestigated 3 herds of Czech Large White sows. Two of them were also characterized by low freqency (0.02–0.05), however in the third herd markedly higher frequency was noticed – 0.26. Somewhat higher frequency of AA genotype in Czech Large White boars (0.14) was also observed by Milaković et al. [18]. On the other hand Vano et al. [29] did not obtain AA genotype in sows of White Meaty breed and  their two- or three-breed cross, in which Landrance, Duroc and Dutch crossbreed Dalland shared. The PIC value in our study ranged from 0.103–0.222 and according to classification proposed by Xie et al. [30] FSHB gene represented low polymorphism.

In conducted investigations we found a tendency of BB genotype being favorable for analyzed litter traits. Humpolíček et al. [5] studied FSHB/HaeIII polymorphism in relation to litter traits. The influence of FSHB genotypes was different depending on the herd and set of analyzed litters. Sows with BB genotypes had significantly highest TNB, NBA and NW in first litters in the herd I and III. Luoreng et al. [17], however obtained completely different results during investigation of Beijing Black Pigs. In the first parity pigs with AA genotype had 1.57 and 2.15 TNB more than those with AB and BB genotypes. Moreover sows with AA and AB genotypes had 1.00 and 0.94 NBA more than those with BB genotype, respectively. Further studies of  Humpolíček et al. [8] showed no significant influences of any genotype on the analyzed growth traits as well as on most reproductive traits (age of the first conception, service period, insemination interval, total number of piglets born and number of piglets weaned). Only number of piglets born alive in the second parity was almost significant (P≤0.1). Similarly, no significant effect of FSHB genotypes on litter traits showed Korwin-Kossakowska et al. [12] in Polish synthetic line 990 and Pripwai and Mekchay [23] in Large White × Landrace crossbred sows. Lin et al. [14] also did not confirm influence of FSHB genotypes on sperm quality traits and fertility traits in the purebred Pietrain and the crossbred Pietrain×Hampshire boars. Statistical evaluation conducted by Kernerova et al. [11] showed that the FSHB gene is not a good discriminator for future classification to hyperprolific line (HPL) in Czech Large White sows. BB genotype, however in connection with the of ESR  or PRLR gene variants increased more or less the probability that the adult gilt will be classified to HPL. Genotypes combinations – ESR, FSHB and MYOG (myogenin) were also studied by Humpolíček et al. [9] in relation to growth and reproduction traits in Czech Large White sows. The genotypes combination ESR (BB)/FSHB(AA)affected the mean daily live weight gain but its effect on litter size was not found. Interestingly, Milaković et al. [18] proved that genotype AA was favorable for backfat thickness (P≤0.05), average  daily  gain (P≤0.05),  average  daily  gain  test  (P≤0.01) and  lean  meat (P≤0.05) in Czech Large  White  boars.

On the chromosome 2. in swine few other genes and their variants were studied in relation to litter traits. Stinckens et al. [28] found that polymorphism in the intron 3 of IGF-2 (insulin-like growth factor 2) gene influences on TNB, NBA, NW per litter as well as on litter weaning weight. Another genes identified through whole genome association study were MEF2C (myocyte enhancer factor 2C)and SLC22A18 (solute carrier family 22 member 18). The first one was associated with TNB and NBA in first two parities, however second one with lifetime TNB [20, 21]. Last examples include imprinted gene – DIO3 (thyroxine 5-deiodinase) and regulatory microRNA gene – miR-27a. DIO3 variants affected TNB, NBA and their weights, while miR-27a – TNB in first parity and TBA in all parities [2, 13].

CONCLUSION

Results mentioned above indicate that among analyzed fixed effects only the litter order influenced statistically significant on reproductive traits in sows. Moreover, analysis showed that FSHB/HaeIII polymorphism is not a very informative for these traits. Although many inaccuracies in our results as well as others it may conclude that BB genotype is favorable for reproductive traits, however AA for growth and carcass traits, but this effect is not strong. FSHB/HaeIII polymorphism, however should be not omitted in further investigations. Therefore it seems reasonable to analyze FSHB genotypes in combination with other, newly discovered gene variants, taking into consideration reproductive traits in sows.

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Accepted for print: 24.01.2016

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Daniel Polasik
Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
Doktora Judyma 6, 71-466 Szczecin, Poland
Phone: +48 91 449 67 80
email: daniel.polasik@zut.edu.pl

Monika Kumalska
Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
Doktora Judyma 6, 71-466 Szczecin, Poland

Yuri Sawaragi
Kyoto University, Kyoto, Japan


Grzegorz Żak
Department of Animal Genetics and Breeding, National Research Institute of Animal Production, Balice k. Krakowa, Poland
ul. Krakowska 1, 32-083 Balice, Poland

Mirosław Tyra
Department of Animal Genetics and Breeding, National Research Institute of Animal Production, Balice k. Krakowa, Poland
ul. Krakowska 1, 32-083 Balice, Poland

Paweł Urbański
Department of Molecular Cytogenetics, Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, Jastrzębiec, Poland


Arkadiusz Terman
Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
Doktora Judyma 6, 71-466 Szczecin, Poland
Phone: +48 91 449 67 83
email: Arkadiusz.Terman@zut.edu.pl

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