IQC CONG NGH$ SINH HQC TOAN QUOC 2013
NEW ALLELIC VARIATIONS OF HEART FATTY ACID-BINDING PROTEIN IN INDIGENOUS PIG BREEDS OF VIETNAM
Nguyen Van Cuong*, N g u ^ n Thi Thu, Ta Thi Thoa, Nguyen Thi Thuy, Nguyen Thi Dieu Thuy Institute of Biotechnology. Vietnam Academy of Science and Technology
ABSTRACT
Tbe objectives of this study were to analyse genotype distributons and sequence vanaiions of candidate gene putatively associated with meat quality in indigenous pig breeds. For this puipose, a total of 340 pigs from four mdigenous and two exotic breeds were included in die analysis of tbe polymoiphisms of heart 6tty acid-binding protein (H-FABP) gene by sequencing and PCR-RFLP methods. For H-FABP, 17 SNPs were detected in mdigenous pig breeds by direct sequencing of a ftagment at mtron 2 of H-FABP gene Tbe mutation T1556C created a new restriction site for die enzyme Msp\ which give rise to new allelic variants in three indigenous pig breeds. In indigenous breeds, the frequency ofthe favouriile alleles amdd&t Mspl and MaeUl sites of H-FABP gene were low. Meanwhile, the frequency of die </ allele at Haelll site in exotic breeds was significantly higher ttian those of mdigenous pig breeds. It is suggested that T1556C mutation at H-FABP may be important for genetic improvement of intramuscular fet contenL Key words H-FABP gcne.IMF, PCR-RFLP
Conesponding author Tel: +84-912762015: Email: cuong ibtlSyahQQ.CQm INTRODUCTION
In recent decades, the aim of pig production in Vietnam was directed to increase the lean percentage of carcass and high growth performance by making use of heterosis mainly behween indigenous and imported breeds like the Landraee, Yoritshire, Large White. This effort resulted In Increasing the lean meat proportion and high growth rate in the Vietnamese pig herd. However, this effort also resuKed in a decrease in some economically important traits like adaptation to the indigenous ecologies, meat quality (pork becomes tougher and less favourable) and disease resistance (Hai, 1996) The estimation of the frequency of genete polymorphism existing in different pig breeds allows identification of both males and females carrying beneficial alleles early in life, thereby improving accuracy of selection, reducing the generation interval and accelerating the rate of genetic improvement of the tralL
Intramuscular fat (II^F) content is a major determinant of pork quality which determine the dietetic value and taste of pork. A higher IMF content has a favourable effect on the juiciness, palatability and tenderness of meat (Eikelenboom et al., 1996). The IMF deposition is a complicated process and affecting by genetic, physiological and biochemical factors.
WiUi respect to genetic factors, 4 to 13 QTLs for IMF content have been identified in different pig populations (Gao &
Zhao. 2009). These QTLs for IMF were found on 11 chromosomes (PigQTLdb, httD://www.qenome.iastate.edu/cqi- bin/QTT-db/SS/index). In addition to QTL studies, many candidate gene polymorphisms have been analyzed in tenns of their associations with pig fatness Among them, a confirmed effect on pig fabiess was found for a well-known polymorphism of the heart fatty acid-binding pnjtein (H-FABP) gene (Gerbens ef al., 1999). Heart fatty acid-binding protein plays an important role in the transportation of inb^cellular fatty acids thereby affecting ttie intramuscular fat content (Cameron & Enser, 1991). The H-FABP gene, located on pig chreimosome 6, is regarded as Uie candidate gene for IMF (Gerbens et al., 1999).-Three restriction enzymes (H/nfl, Haelll, Afspl) which cut loci of H-FABP gene were identified as RFLP markers for IMF in pigs. In analysing tiie effects of ttie genotypes of H-FABP gene on IMF content, it has been found that dd>Dd>DD and aa>Aa>AA in terms of the etiect on IMF content (Gerbens e( al., 2001). However, polymoqjhisms in the H-FABP gene in Vietnamese indigenous pig breeds and the association of IMF wiUi different H- FABP genotypes have not been well studied.
With ttie aim to develop genetic markers for meat quality, and for further research on the characteristics of indigenous pig breeds, a DNA fragment of H-FABP gene were sequenced and genotyped in six pig breeds in Vietnam.
MATERIALS AND METHODS
Samples were collected from four Vietnamese indigenous pig breeds and two introduced Vietnamese Yorkshire and Landraee. Meo pigs (ME) are mainly kept by ethnic Thai and H'mong people in North and NorUi West Vietnam. The Muong Khuong (MK) pig is particularly kept by H'mong farmers in the mountainous areas of North Vietnam. The Muong Khuong pig is planned to be mainly preserved as a sow line for crossbreeding. The Tap Na (TN) has been found in Cao Bang province, kept under low-input condition It is well adapted tia the ecological condition where it is kept. Tap Na is said to be resistant to certain diseases, and to have tasty meat. The number of Tap Na pigs is decreasing and it is at high nsk of extinction. Mong Cai (MC) is the major indigenous breed in North and central Vietnam, originated from ttie Red River delfa and was eommercially used and kept in a sfation. The Mong Cai is adapted to poor quality feed. In the large-frame type, is said to have a higher lean meat and higher growth rate. The Mong Cai was frequentty used to improve indigenous breeds with lower perfonnance. Viettiamese Yoritshire (VV) was inttx>duced to Vietnam in 1936;
meanwhile Vietnamese Landraee (LV) was imported in 1994. Exotte breeds are less adapted to the local environment and husbandry condition than the native breeds and so perform below their genetic potential.
Blood samples were collected from jugular veins of liiflng animals Blood was collected in test tubes confaining an anticoagulant solution. Muscle tissue samples were taken from pig ears. A total of 340 pigs from four indigenous breeds and twfo exotic breeds were included in the analysis. Samples from sows of MC (n = 46), YV (n = 52) and LV (n = 54) were collected at two breeding stations in Hanoi and Lao Cai Province. For ME (n = 47). MK (n = 91) and TN (n =50) approximately equal numbers of males and females were included.
HOI NtiMi M i U A H y u UONG NGH^ SINH HULI TT::.
Genomic DNA from Wood and muscle tissue was isolated according to standaid protocols. For amplification ol iaiyei DNA sequence invohflng a potential polymorphic site, the polymerase chain reaction (PCR) was used. Based on the published nucleotide sequence information of the H-FABP gene the following primers were used to ampli^ ttie faig«
DNA sequences, fonward primer 5"-ATT GCC TTC GGT GTG TTT GAG-3' and reverse pnmer 5'-TCA GGA ATG GGA GTT ATT GG-3' (GenBank accession No:Y16180). PCR ampllfieatiDns were peribnned in a 25 pL reaction solution as follow. H2O deionized: 17.2 pL, Buffer (10X): 2.5 pL, MgCl2 (25 Mm): 2.0 pL, dNTPs (25 mM): 0.2 pL, fonward primer (10 pM): 1.0 pL, reverse primer (10 pM); 1.0 pL, Taq 1 u/pl (Fermenfas, Inwtrogen): 0.1 pL, DNA template (50 ng/pL): 1.0 uL The PCR themial cydes were 94°C (4 min), then followed by 35 cycles of denaturatton 94''C (35 sec); annealing 5 / C (50 sec) for H-FABP, (40 sec) for FUT1 and SS^C (45 see) for BPi; polymerizabon 72°C (45 sec). The final eyde was concluded by an extension intenral of 7 min at 72° G and the PCR products were stored at 4° C.
Fifteen pL of ttie PCR products were digested overnight at 37°C by Haelll (6U), Mspl (6U). The digested products were separated by elechophoresis ttirough a 2.5% agarose gel. The PCR products were purified with the QIAquick PCR Purification kits (QIAGEN). The DNA ft-agment from five individuals per breed was sequenced by using ABI PRISM*
3100 Avant Genetic Analyzer. The sequences were aligned by Clustalx2, bioedit, and Mega 4.1 sofhware Restriction endonudease DNASTARsites were detected by using primer premier 5.0 softvrare (Lalitha, 2000).
Statistical analysis, frequendes of genotypes and alleles were estimated by direct counting in six breeds under shidy.
Differences between fi^quendes of genotypes and alleles were estimated by T- test. Hardy-Weinberg equilibrium was tested by comparing expected and observed genotype frequencies using a Chi-square test. If tiie observed and expected counts were not significantty different (p>0.05), the population was in Hardy-Weinberg equilibrium. The expected heterozygosity (He) was calculated according to Nei (1978).
Where Pi is the frequency ofthe i"" of k alleles
Polymorphism information content (PIC) was calculated according to the formula:
n n-l n i=i i=i j=i+i Where'
Pi= The fi^quency of allele i
j = The allele n having codominance with allele i n = The number of individual of one population
A population is said to be highly polymorphic if PIC > 0.5 and lowly polymorphic if PIC < 0.25 (Botstein et at., 1980).
RESULTS
By sequencing an 816 bp fragment at intron 2 of H-FABP gene. 17 SNPs were found in four indigenous pig breeds, of which 14 were substitutions and three are insertions. The frequendes of mutations varied from 0.86% (7/816) for MC lo 1 22% (10/816) for MK. The insertion mutation caused an increased RFLP fragment size of 821 bp in ME, MK, MC and 822 bp in TN The T1556C mutetion created a novel restti'etion site for ttie Mspl. These results coresponded well witti the PCR-RFLP data . In the ME, MK and TN breeds two Mspl recognition sites were found in the RFLP fragments, while in two exotic and MC breeds only one Mspl site was detected.
Figure 1 Mspl polymorphism of the H-FABP gene In pigs. The genetic variations of H-FABP gene were invesljgated by PCR-flFLP.
The digested PCR products with Mspl generated a 816 bp fragment for allele a, two fragments of 727.89 bp for allele A. two fragments
' ^ ^ G O N G N G H E S I N H H Q C T O A N Q U O C 2 0 1 3
<rf660. 1 5 6 b p f o r a i l e t e b : a n d t h r i e e f t a g m e n t s o f 6 6 0 , 89. 67 bp tor aBele B.Lane M: standards for size detenninalion. Lane 1: PCR p n x k i c t D f 8 1 6 b p . L a n e 2 . 3 . 4 : 9 e n o t y p e A A L a n e 5: genotype A a Lane 6. 7: genotype AS. Lane 8: genotype 8 8
T h e R F L P p o t y m o f p h i s m o f H-FABP g e n e w a s detected by P C R - R F I P mettiod u s i n g t h e restriction e n z y m e s M s p l a n d H a e l l l . D i g e s t i o n o f t h e P C R product witti M s p l p r o d u c e d four aBeles a. A. b a n d B. T h e 8 1 6 b p fragment w e r e defined 35 allete a, 7 2 7 a n d 8 9 b p a s allele A . 660 a n d 156 b p a s aHele b a n d 6 6 0 , 8 9 a n d 6 7 b p a s allele B. G e n o t y p i n g 3 4 0 pigs fiwn sbc b r e e d s kJentitied t h r e e alleles a. A a n d S (Figure 1). fiitele a n d genotype f r e q u e n d e s e s t i m a t e d a t M s p l I o d of ttie H-FABP g e n e in sb( b r e e d s a r e g i v & i in Table 1. T h e results s h o w e d that the Atepl polymorphic site of H-FABP gene w a s f o u n d in five of ttie t>reeds, e x c e p t for M C b r e e d . In i n d i g e n o u s pig breeds M E , M K a n d T N (which are m a i n f a i n e d in ttieff original e n v i r o n m e n t s ) t h r e e aHeles A, a a n d 6 w e r e klenttfied witti average f r e q u e n c i e s of 0.95, 0 . 0 0 2 a n d 0.05.
respectiveiy. In exotic b r e e d s Y V , L V a n d indigenous b r e e d M C , w h k * is kept at a breeding s t a t o n , only hvo alleles (A and a) w e r e f o u n d w i t i i a v e r a g e frequendes o f 0.97 a n d 0 . 0 3 . In all populations allele A w a s a major allele. T h e frequency o f f a v o u r a b l e allele a did not differ m u c h b e h v e e n t h e indigenous a n d exotic breeds. T h e PIC at ttie M s p l site was k i w in i n d i g e n o u s (0.09) a n d exotic (0.06) b r e e d s . Except for M C b r e e d , ttie g e n o t y p e frequencies a t M s p l I o d were in HanJy-Weinberg E q u i l i b r i u m .
M l 2 3 4 5 6 7
500 bp'
F i g u i * 2 N M I I I p o l y m o r p h i s m of the HFABP gene In pigs. The polymorph'tsms of H-FABP gene were investigated by PCR-RFLP using spedfk; piimeis and Ihe restriction enzyme Haelll. Allele D is d i g ^ t e d PCR product at (wo positions to create three fra^wits ot 683,117 and 10 bp Allele d is digested PCR products at three positkjns to create four fragments of 405,278,117 and 16 bp. LaneM standards for size detennination Lane 1: PCR product Lane 2 , 4 : genotype DD. Lane 3, 5, 6: genotype £W. Lane 7. 8: genotype dd Td>le 1 Genotype, allele frequencies and heterozygosity of H4^ABP gene in six |rig breeds at Mspl-RFLP site
Breed*
ME MK TN MC Mean +
IndB' YV LV Mean±
ExoB' Sample size(n) 47 91 50 46
52 54
Genotype frequencies (No.ot pigs)^
AA 0.96 (45) 0.79 (72) 0 88(44) 1.00 (46) 0 91
±0.09 0 94 (49) 0.93 (50) 0.93
± 0 . 0 1 Aa 0.02 (1) 0.00 (0) 0.00 (0) 0.00 (0) 0.01
± 0 . 0 1 0.06 (3) 0.07 (4) 0 0 6
±0.01 AB 0.02(1) 0.20 (18) 0.10(5) 0.00 (0) 0-08
±0.09 0.00 (0) 0.00 (0)
0 0 0 BB 0.00 (0) 0.01 (1) 0.02 (1) 0.00 (0) 0.01
± 0 . 0 0 0.00 (0) 0.00 (0)
0 0 0
Allele frequencies A
0.98 0.89 0 93 1.00 0.95
±0.05 0.97 0.96 0.9?
±0.01 a 0-01 0.00 0.00 0.00 0.00
±0.01 0.03 0-04 0.03
±0.01 B 0.01 0.11 0.07 0.00 0.05
±0.05 0 00 0.00
0.00 He'
0.O4 0.20 0.13 0.00 0 09
±0.09 0.06 0.07 0.06
±0.01 P I C '
0.04 0.18 0.12 0.00 0.09
±0.08 0.05 0-07 0.06
±0.01 X2- n s n s n s
n s n s .
*Pig tjreeds: ME = Meo; MK = Muong Khuong; TN = Tap Na; MC = Mong Cai; YV = Vietnamese Yorttshire and LV = Virtnanese Landraee. * Values in parentheses are the numbers of pigs found in each genotype. " He = Expected heterozygosity; * PIC=
Polymor^rtiism infomialion content; x2* "= chi-square test, n.s = not significant (the population was in HWE); 'Mean values of genotype,
^ e l e frequendes and heterozygosity in indigenous pig breeds. ^Mean v ^ u e s of genotype, allele frequencies and heterozygosity in exotic pig breeds.
The s a m e 8 1 6 t>p fragment h a d three Waelll sites. Vtfhen ttiis s e g m e n t o f D N A is cut by H a e l l l , four restriction fragments (405. 2 7 8 , 117 a n d 1 6 b p ) are produced. A p o l y m o r p h i s m a t t h e position 1811 b p w o u l d result in t h e d e s t m c t i o n of a Haelll site, m w g i n g t h e 2 7 8 b p a n d 4 0 5 b p into a 6 8 3 bp f r a g m e n t . Allele d w a s defined a s ttie presence o f t h e 4 0 5 , 278, 117 a n d 16 b p f r a g m e n t s . Allele D is a digested P C R product at t w o positions to create ttiree fragments of 6 8 3 . 117 a n d 16 b p (Figure 2 ) . Allele a n d g e n o t y p e frequencies estimated a t H a e l l l I o d are given in Table 2 . In local pig breeds, ttie homozygotes D D were t h e m o s t fiiequent ranging frtim 0.94 i n M E to 1.00 in T N . H o m o z y g o t e dd was not found in any indigenous pig b r e e d . In exotic breeds, ttiree genotypes DD. Dd a n d d d were f o u n d with average f r e q u e n d e s of 0.33, 0.41 a n d 0 . 2 5 , respectively. T h e P I C value at H a e l l l site in exotte b r e e d s w a s higher ( P <0 0 1 ) ttian In indigenous pig breeds. E x c e p t for t h e T N b r e e d , the g w i o t y p e frequencies at H a e l l l I o d were in H a r d y - W e i n b e r g Equilibrium
HOI NGHI KHOA HOC C 6 N G N G H $ SINH HQC Tf,;
Table 2 G e n o ^ p ^ aDefa ftpquBnclei
Breed*
ME MK TN MC Mean±
IndB' YV LV Mean ±
ExoB"
Sample s c e ( n ) 47 91 50 46
52 54
5 and h e t e r o z ^ o s i t y of H-FABP gene in
^Genotype frequencies (No.of pigs)"
OD 0.94 (44) 0 5 6 (87) 1.00(50) 0SB(A5) 0.97
±0.03 0.44(23) 0.22 (12) 0.33
±0.15
CW dd 0.06 (3) 0.00 0.04 (2) 0.00 0.00 (0) 0.00 0.02(1) 0.00 0.03 „ „
±0.03
0.44(23) 0.12(6) 0.39(21) 0.39(21) 0.41 0.25
±0.03 ±0.19
six pig breeds at HaelH-RFLP sHe /Ulele frequendes
D rf 0.97 0.98 1.00 0 9 9 0.98
±0 01 0 6 6 0 42 0.54
±0 17 0.03 0.02 0.00 0.01 0.02
±0.01 0.34 0.58 0.46
±0.17 He'=
0.06 0.04 0-00 0.02 0.03
±0.03 0.45 0-49 0.47
±0.03 P I C "
0.06 0.04 0.00 0.02 0.03
±0.02 0.35 0.37 0.36
±0.01
xr *
n.s n.s
n.s
n s n.s
'Pig breeds: ME = Meo; MK = Muong Khuong; TN = Tap Na; MC = Mong Cai; YV = Vietnamese Yorttshire and LV = Vietnamese Landraee. '" Values in parentheses are the numbers of pigs found in each genotype. '^ He = Expected heterozygosity; '^ PIC= Polymorphism infomiation content; x2° = chi-square test; n.s = not significant (the population was in 1-IWE); 'Mean values of genotype, allele frequencies and heterozygosity in indigenous pig breeds. ^Mean values of genotype, allele frequendes and heterozygosity in exotic pig breeds.
DISCUSSION
Sequencing results for infron 2 of H-FABP gene from four indigenous pig breeds ^ o w e d more than 99 % identity with the reference sequence. Interestingly, a novel resttiction site for ttie Mspl at position 1556 bp was detected for the first time in ttiree Vietnamese indigenous pig breeds, which are maintained in their original environments, whereas in exotic breeds only one Mspl site was detected untti now. This mutation created a second Mspl recognition site in RFLP fragment, resulting in four alleles in the indigenous breeds. The novel restriction site for ttie Mspl may be important for future genetic improvement.
As shown in Table 1 and Table 2 a higher frequency of allele A and allele D at the Mspl and Haell RFLP sites was observed in indigenous pig breeds, with favourable alleles a and d at Mspl and Haell sites present at low frequencies. In ttie exotic breeds, ttie favourable allele d fiwiueney at Haell site was higher (P <0.01) than those in the indigenous pig breeds.
Investigattng ttie Mspl and Haelll pol^orphisms of HFABP gene. Pang etal, (2006) revealed that ttie frequendes of alkte ' A (Afcpl-RFLP) and allele D (Haelll-RFLP) in Landraee breed were 0.912 and 0.328, respectively. Uteanwhite ttie Chinese native pig breeds had only the A and D allele at Mspl- and Haelll-RFLP sites. The values of IMF content in European and Chinese breeds are considerably difterent, alUiough they have the same genotype (Pang ef al., 2006).
Intramuscular fat content depends on the genotypes and on ttie breeds (Fisher, 1994). Association analysis revealed ttiat ttie H-FABP alleles significantty affected the IMF content. This effed of 0.36% IMF between the homozygous genotypes is high considenng the overall mean of 1.84% IMF in the Meishan crossbred population (Geriaens et al.. 1999). In the Chinese local pig breeds, ttie IMF content onJered by H-FABP genotypes were HH>Hh>hh, DD<Dd<dd, and AA<Aa<aa.
Fat deposition in adipocytes was higher in the HH, dd and aa genotypes ttian in others (Wei-Jun et al., 2006). In Polish native Ztotnicka Spotted pigs, ttie meat with genotype hh were marked by a higher inframuseular fat content (2.64%) compared to HH group (1.99%); PsO.05 (Hanna ef al., 2010). Besides H-FABP gene, there are 29 genes which are associated with IMF content. There are two possibilities to improve IMF content: 1) by enhancing favorable alleles, and 2) by crossbreeding. Pig breeds of Vietnam have been traditionally selected for adaptation to a diet poor in pfotaln content as well as adapted to the local ecologies. The indigenous pig breeds are said to have good meat quality due to' high rating for overall palatebility and tendemess Meat of local pigs are low in moisture, and have low cooking losses and good flavor. Our analysis showed that IMF content in Tap Na breed is signifieantly higher than exotic breeds and ranging from 3.9% to 4.6% (data not shown). Such selection has driven the aeeumulatton of new mutations with favourable phenotypic effects, as well as the development of alleles and haplotypes ttiat differ from exotic breeds. Marker Mspl-RFLP at T1556C position can be used to track ttie genetic materials in hybrids. The values of PIC at the Mspl and Haell RFLP sites in indigenous breeds are very low; this means that local pig breeds lack polymorphism at the two abow sites.
In conclusion, the favourable alleles of H-FABP gene in the exotic breeds are signifieantly higher than ttiose ol indigenous pig breeds. The T1556C mutation at intron 2 of H-FABP gene found in some indigenous pig breeds may be used as Mspl-RFLP maricer to identtfy or truck the source of genette material In IMF improvement by crossbreeding.
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QC CONG NGH$ SINH HOC T O A N Q U O C 2013
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DA HiNH ALEN Mdi C O A GEN HEART FATTY ACID-BINDING PROTEIN TRONG CAC G I O N G L O N NOI VIET NAM
N g u y e n V a n C i p o ^ g , N g u y S n T h j T h u , T ? T h j T h o a , N g u y e n T h j T h u y , N g u y § n T h j D i f u T h u J Vi^n Cdng nghg sinh hgc, Vi^n Hkn ISm Khoa hgc va Cong ngh^ Viet Nam
T 6 M T A T
Mvc dich dat ra ciia n^ien cihi nay li xac d)nh phin b6 kilu gen va da hinh Uinh tv ciia ung gen heart fatty acid-binding protein (H- FABP) lifin quan chk lu<;mg diit d m^t so giong Ipn n$i Vi$l Nam. V6i muc dich tren 340 miu tu hin gi6ag lgn ndi v^ hai gidng Ipn Qgo^i dl dupe sii dyng de phan tich da Mnh gen H-FABP bing phmmg phap PCR-RFLP va giai trinh tu ADN. Ket qua gidi trinh tu doan ADN (816 bp) d intron 2 ciia gen H-FABP da ph^t hi^n 17 dot bien li;n npi. Dac biet dpi biin T1S56C da tao ra d i ^ cSt moi d6i voi enzyme gioi ban Mspl. Oiem cat moi nay da tao ra alen moi d cac giong Ion npi Vi^t Nam. Ket qua phan tich phan bo ki€u gen H'FABP d hai v) tri Mspl vk Hael^i cho thAy tiln su^t cdc aten co xu hudng cho chit lugng th)t tdt a vkdd cac giong tgn odi rat d i ^ . Tan suat alen d d lgn ngoai cao hon l<;rn noi Dpt bien Tl 556C a gen H-FABP c6 kha nang lien quan chat lupng thjt Ipn.