Research Article PCR-SSCP Variation of IGF1 and PIT1 Genes and … · 2019. 12. 12. · PCR-SSCP...

Hindawi Publishing CorporationGenetics Research InternationalVolume 2013 Article ID 272346 6 pageshttpdxdoiorg1011552013272346

Research ArticlePCR-SSCP Variation of IGF1 and PIT1 Genes and TheirAssociation with Estimated Breeding Values of Growth Traits inMakooei Sheep

Masoud Negahdary1 Abbas Hajihosseinlo2 and Marziyeh Ajdary3

1 Yazd Cardiovascular Research Center Shahid Sadoughi University of Medical Sciences Yazd Iran2Department of Animal Science Faculty of Agriculture Urmia University Urmia Iran3 Young Researchers and Elites Club Khorasgan Branch Islamic Azad University Isfahan Iran

Correspondence should be addressed to Abbas Hajihosseinlo abbas hajihosseinloyahoocom

Received 1 July 2013 Revised 26 October 2013 Accepted 30 October 2013

Academic Editor Michael Walter

Copyright copy 2013 Masoud Negahdary et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Molecular biology techniques genetic improvement by facilitating identification mapping and analysis of polymorphism of genesby encoding proteins that act on metabolic pathways involved in economically interesting traits This use of genetic markers canaid identification of those animals with the highest breeding values in sheep On the basis of sheep genome mapping informationwas examined on the ovine IGF1 and PIT1 genes as a possible genetic marker for growth traits in sheep The current study wasdesigned to estimate the frequencies of putative IGF-1 and PIT-1 genes SNPs and investigate associations with calculated EBVs ofgrowth traits in Makooei sheep PCR-SSCP analysis of the exon1 of IGF-I gene and include a part of intron2 exon3 and a part ofintron3 and PIT-1 gene revealed the following banding patterns three (AA AG GG) and four AA (p1) AB (p2) CC (p3) CD (p4)banding patterns respectively Results from this study demonstrated higher performance of AA animals in BW and GBW and AGanimal in WW andW6 that may be related to the role of IGF-1 at the pre-puberty and puberty stages Also higher performance ofp3 animals in W9 YW and GSN and p1 animal in GNY may be related to the PIT-1 role in post-puberty

1 Introduction

TheMakooei sheep are one of the Iranian fat-tailed medium-size breeds They are distributed in the mountainous areasof the country especially in West Azerbaijan province Alsothey are found in Turkey and called White Karaman Theyare valuable primarily for meat and also for their wool andmilk The wool produced is coarse and usually used forcarpet weaving [1] The main objective of the application ofmolecular biology techniques to animal genetic improvementprograms currently consists in identifying mapping andanalyzing polymorphisms of the genes involved in the mainmetabolic pathways that are related to animal growth anddistribution of nutrients to the different tissues [2] Recentlyinvestigators and breeders focus onmarker-assisted selection(MAS) and genome analysis MAS may increase annual rate

of genetic gain in livestock by 15 to 30without increasing therisk involved in breeding schemes [3] In the livestock indus-try growth traits that determine economic value of livestockare always of primary concern during breeding [4]Themostsignificant growth traits for birth cohort studies in geneticsare gestation litter size sex and environmental variables [4]In farm animals promising candidate genes for many traitsare in the growth hormone (GH) axis The GH gene pathwaycontains various interdependent genes such as GH insulin-like growth factor1 (IGF1) pituitary specific transcriptionfactor1 (PIT1) growth hormone releasing hormone (GHRH)somatostatin growth hormone releasing hormone receptor(GHRHR) growth hormone receptor (GHR) and others [5]For growth traits GH GHR insulin-like growth factor I(IGF-I) leptin (LEP) caprine-pituitary-specific transcriptionfactor-1 (POU1F1) caprine myostatin (MSTN) and bone

2 Genetics Research International

morphogenetic protein (BMP) genes are necessary for boneformation birthweight weaningweight body condition andmuscle growth [6] IGF1 is a mediator of many biologicaleffects it increases absorption of glucose stimulates myoge-nesis inhibits apoptosis participates in the activation of cellcycle genes increases the synthesis of lipids stimulates theproduction of progesterone in granular cells and intervenesin the synthesis of DNA protein RNA and functions in cellproliferation [7]The predicted sequence of amino acid oIGF-I peptide differs from the human bovine and porcine IGF-Isat a single amino acid (at position 66 alanine is substituted forproline) and differs from rat and mouse IGF-Is at positions4 and 5 respectively Ovine IGF-I amino-terminal peptidesare 1 amino acid longer than other mammalian IGFs dueto the presence of an extra amino acid (glutamine) thatis present at the proposed boundary of exon1 and exon2[8] IGF-1 is one of the most potent natural activators ofthe AKT signaling pathway a stimulator of cell growthand multiplication and a potent inhibitor of programmedcell death The transcription factor pituitary-1 (Pit-1 officialnomenclature now POU1F1) plays a major role in pituitarydevelopment and hormone expression It has been shownto be a positive regulator of the growth hormone prolactinand the thyrotropin subunit (TSH120573) in the mammalianpituitary [9] POU1F1 is a member of the POU-domain genefamily The POU-domain is composed of high-affinity DNAbonds the POU-specific domain (POU-SD) and a POU-homeodomain (POU-HD) [10] Mutations of the POU1F1gene led to a deficiency or an absence of GH PRL and TSH-120573 and can result in stunted growth Mammalian growth anddevelopment require steady expression of the POU1F1 gene[10] The ovine POU1F1 gene has a high level of conservationwith its bovine human and rat counterparts showing 982912 and 862 of similarity at coding levels respectively[10] Such findings suggest that variation in IGF-1 and PIT-1 genes in domestic animals may be important contributorsto growth rate However studies on associations of IGF-1 andPIT-1 Polymorphisms with growth traits are mainly carriedout in cattle but examination of associations between SNPsand growth traits in these genes has not been reported insheepTherefore the objective of this study was to determineassociations of genotypes with estimated breeding values ofgrowth traits in Makooei sheep

2 Materials and Methods

21 Collection of Sheep Blood Samples and Extraction ofGenomic DNA Extraction The Makooei breed of sheepwere examined in this study they are fat-tailed sheep withmedium body size white in color with black spots on faceand feet They are farmed in the East and West Azerbaijanprovinces of Iran for meat and wool [11] Blood samples werecollected into a 5mL EDTA vacutainer tube and transferredto the laboratory within 2 hours for DNA extraction TotalDNA extractions were made with a modified salting outmethod [12] from whole fresh blood Quality and quantity ofextracted DNA were measured on 08 agarose gel preparedin 05x TBE buffer (45Mm Tris base 45Mm boric acid

Table 1 Parameters estimated of analyzed traits

Traits 1205902119886

1205902119898

1205902119890

1205902119901ℎ2 1198982

BW 0045 0015 014 022 02 007WW 16 096 490 832 02 0126MW 259 281 648 1295 02 0229MW 235 204 68 1175 02 017YW 329 174 1038 1635 02 011

and 1mM EDTA pH 80) visualized with ethidium bromide(10 120583gmlminus1) and photographed under UV light Estimates ofvariance components and genetic parameters for birth weight(BW) weaning weight (WW) 6-month weight (6MW) 9-month weight (9MW) and yearling weight (YW) fromsingle-trait analyses are presented in Table 1

22 PIT1 Genotyping ThePIT1 gene was genotyped by PCR-SSCP (polymerase chain reaction-single strand conformationpolymorphism) A 295 bp fragmentwas amplified from a partof intron2 exon3 and a part of intron3 The primers usedfor the procedure were those designed by [10] PIT-1-uP 51015840-GAGGGATAATTACAAATGGTCC-3 and PIT-1-down 51015840-TGTTAACAGCTGTGGGACACAC-31015840 PCR contained 25ndash50 ng genomic DNA 10 pmoL of each primer 2 120583L 10xPCR buffer 15mMMgCl2 200Mm dNTP and 1 unit Taq-polymerase in a total volume of 20120583L DNA amplificationswere performed using aMaster cycler (Eppendorf Germany)programmed for a preliminary step of 15min at 95∘C fol-lowed by 34 cycles of 45 s at 94∘C then 1min at 58∘C followedby 45 s at 72∘C and finally an extension of 5min at 72∘C

23 IGF-1 Genotyping As primer pair IGF-1-up (51015840-ATTACAG CTGCCTGCCCCTT-31015840) and IGF-1-down (51015840-CACATCTGCTAATACACCTTACCCG-31015840) targeting a frag-ment of 265 bp were employed in DNA amplifications asdescribed by [13] PCR contained 25ndash50 ng genomic DNA10 pmoL of each primer 2120583L 10x PCR buffer 15mMMgCl2200Mm dNTP and 1 unit Taq-polymerase in a volume of20120583L DNA amplifications were done using a Master cyclerprogrammed for a preliminary step of 2min at 95∘C followedby 31 cycles of 45 s at 94∘C 30 s at 58∘C and 30 s at 72∘C witha final extension of 3min at 72∘C

24 Single Strand Confirmation Polymorphism (SSCP) Twopairs of oligonucleotide primers were designed and a stan-dard PCR protocol was used to amplify two fragments

SSCP analysis several factors were tested to optimize themethodology amount of PCR product (4ndash15120583L) dilutionin denaturing solution (20ndash85) denaturing solution (A95 of formamide 10mMNaOH 005 xylene-cyanol and005bromophenol blue B same asAplus 20mMof EDTA)acrylamide concentration (6ndash14) 6 percentage for PIT1 7percentage for IGF-1 percentage of crosslinking (15 to 5)presence (10) or absence of glycerol voltage (100ndash50V)running time (2ndash12 h) and running temperature (4 6 10 and15∘C) Each PCR reaction was diluted in denaturing solution

Genetics Research International 3

denatured at 95∘C for 5min chilled on ice and resolved ona nondenaturing polyacrylamide gelThe electrophoresis wascarried out in a vertical unit (Payapajoohesh VEU-7350 160times 140 times 075mm) in 1x TBE bufferThe gels were stained withsilver

25 Data Collection Data were collected at the MakooeiSheep Breeding Station at Makoo (36∘ 351015840S and 48∘ 221015840E)in West Azerbaijan province Animals are kept on naturalpasture during spring summer and autumn seasons Rangeconditions are poor during the winter months and thereforeanimals are kept indoors during the winter In general theflock is managed under a semimigratory system

26 Breeding Value Estimation and Association Analysis Theanalysis was conducted using restrictedmaximum likelihood(REML) using a derivative-free (DF) algorithm procedure[14] Data on growth traits were retrieved from the nationalSheep Recording System (SRS) The following fixed effectsmodel was used to calculate BV (breeding value) withDFRIMEL software [14]

119884119894119895119896119897119898119899119900= 120583 + YR

119894+ SX119895+ BT119896

+ AD119897+ AN

119898+119872119899+ 119864119894119895119896119897119898119899119900

(1)

with 120590119886119898= 0 where 120583 is overall mean of population 119884

119894119895119896

is each observation YR119894is fixed effect of year 119894 SX

119895is fixed

effect of sex 119895 BT119896is fixed effect of birth type 119896 AD

119897is fixed

effect of age of dam 119897 AN119898is additive genetic effect of animal

m119872119899is maternal genetic effect 119864

119894119895119896is residual effect of an

observation ijk The estimated parameters according to model were

phenotypic variance (1205902119901) direct additive genetic variance

(1205902119886) maternal genetic variance (1205902

119898) residual variance (1205902

119890)

direct heritability (ℎ2119886) (12059021198861205902119901) and maternal heritability

(ℎ2119898) (12059021198981205902p)

SAS software was used to calculate least squares meansand for multiple comparisons among the different genotypesin ldquoMakooeirdquo sheep Least square analysis using the generallinear model (GLM) procedure was done to identify the fixedeffects on a model that were to be included in the modeland then the model was made using the fixed effects (sex2 classes type of birth 2 classes IGF-1 3 classes PIT-1 4classes) according to the following statistical model

119884119894119895119896119897119898= 120583 + 119866

119894+ 119875119889+ 119878119895+ 119897119904119896+ 119866119894times LS119896+ 119890119894119895119896119897119898 (2)

where 119884119894119895119896119897119898

is growth traits 120583 is the overall mean 119866119894is the

fixed effect of the 119894th genotype for IGF-1 Pd is the fixed effectof the dth genotype for PIT-1 119878

119895is the fixed effect of sex

(119895 = 1 2) 119897119904119896is the fixed effect of litter size (119896 = 1 2) GiLS

is the interaction between genotype for IGF-1 and litter sizeand 119890119894119895119896119897119898

is the random residual error

27 Statistical Analysis All data were stored in SAS foroperating system of Microsoft Co (version 19) Group com-parisons were done using the analysis of variance (ANOVA)

test Significant differences between them were assessed byStudentrsquos t-test All data were expressed as mean plusmn standarderror of mean (SEM) 119875 values less than 005 were consideredsignificant

3 Results

31 Genomic Screening PCR-SSCP analysis of the 51015840 flankregion (exon1) of the ovine IGF-I gene and (a part of intron2exon3 and a part of intron3) PIT-1 gene revealed the followingbanding patterns three (AA AG GG) and four (AA (p1)AB (p2) CC (p3) CD (p4)) banding patterns respectivelyChi-square analysis has shown that the genotypic frequencyfor the PIT1 (119875 lt 005) was not in the Hardy-Weinbergequilibrium but the frequency for IGF-1 (119875 gt 005) was inthe Hardy-Weinberg equilibrium

32 Genetic Parameter Estimation Estimates of variance andcovariance components were based on animal models usingthe restricted maximum likelihood (REML) approach ina derivate-free (DF) algorithm [14] The direct heritabilityestimate of the studied traits was fixed while the maternalheritability estimates varied between 007 and 022 Safariet al have reviewed genetic parameter estimates for sheepgrowth traits and the heritability means for growth traitsranged from 015 to 041 Genetic evaluation for body weightsprior to 6months of growth traits needs to adopt amodel thatconsiders genetic effects both direct and maternal geneticones Therefore the current study was designed to estimatethe effect of SSCP patterns on weight estimated matenalbreeding values for bodyweights prior to 6-months of growthtraits Low additive genetic and high residual variances inBW WW 6MW 9MW and YW could be explained by theharsh environmental conditions of the range that coincidedwith these ages The high genetic and phenotypic correlationbetween weaning weight with 6-month weight 9-monthweight and yearling weight indicates that a breeding rammaybe selected at an earlier age for the PIIT-1 and IGF-1 genes(Table 4)

33 Effect of IGF-I SSCP Variants on the Growth TraitsEffects of the tested SNPs on the EBVs for growth traits inMakooei sheep are presented in Table 2 The table demon-strates that we observed a significant effect of this polymor-phism on 6MW yield (119875 lt 005) The genotype AG has thehighest additive estimated breeding value for the trait pre-six month weight (SW) The effect of the IGF-I gene wassignificant (119875 lt 001) for average daily gain from birth toweaning (GBW) birth weight (BW) weaning weight (WW)six-month weight (SW) and average daily gain from sixmonths to nine months (GSN) In the tested Makooei sheeppopulation mean body weight of the genotype AG at 6MW(3112 kg) was about 269 kg and this was 713 higher thanthose of AA (2843 kg) and GG (2399 kg) SSCP patternsrespectively Also mean of wool weights of genotype AG atyearling weight (0517) was about 0031 kg and 0172 higherthan that of AA (0486) and GG (0345) SSCP patternsrespectively Higher performance of AA animals in BW and


Table 2 Least square means and standard errors of the EBVs of Makui sheep according to the different IGF-1 patterns

(a)

IGF-1 Weight estimated maternal breeding values (means plusmn SE kg)BW W

3W6

AA 0006 plusmn 002 0595 plusmn 014 102 plusmn 027ab

AG 0005 plusmn 003 0873 plusmn 017 145 plusmn 034a

GG 0026 plusmn 004 0294 plusmn 024 0158 plusmn 048b

P value 03ns 232ns 293lowast

(b)

IGF-1 Weight estimated breeding values (means plusmn SE kg)BW W

3W6

W9

W12

AA minus0063 plusmn 004 118 plusmn 028 145 plusmn 047ab 0965 plusmn 025 0304 plusmn 030

AG minus0083 plusmn 005 175 plusmn 035 24 plusmn 059a 0945 plusmn 031 0285 plusmn 037

GG minus024 plusmn 007 0588 plusmn 051 0155 plusmn 083b 0527 plusmn 044 0337 plusmn 053

P value 25ns 233ns 295lowast 048ns 00nsnsnot significant lowastsignificant aAG bGG abAA

Table 3 Least square means and standard errors of the EBVs of Makui sheep according to the different PIT-1 patterns

(a)

PIT-1 Weight estimated breeding values (means plusmn SE kg)BW W

3W6

W9

W12

P1 minus0066 plusmn 003 1017 plusmn 023 0963 plusmn 0281 0167 plusmn 020a 0225 plusmn 0595

P2 minus0082 plusmn 006 0708 plusmn 044 0438 plusmn 0321 0651 plusmn 039ab 0338 plusmn 0670

P3 minus0098 plusmn 004 161 plusmn 0 27 140 plusmn 0218 0890 plusmn 024b 0250 plusmn 0654


P value 140ns 192ns 119ns 366lowast 016ns

(b)

PIT-1 Weight estimated maternal breeding values (means plusmn SE kg)BW W

3W6

P1 minus0005 plusmn 001 0510 plusmn 023 0452 plusmn 022


P3 minus0015 plusmn 002 0826 plusmn 0 13 0832 plusmn 026


P value 066ns 130ns 221ns

P value significant level nsnot significant lowastsignificant aAG bGG abAA

GBW also AG animal inWW and1198826(prepuberty ages) may

be related to the IGF-1 role in prepuberty ages We suggestthat decrease weight of animals has been due to change ofgenotype

34 Effect of PIT-1 SSCP Variants on the Growth Traits Theresults showed that the PIT-1 genotypes were associatedwith estimated breeding values of growth traits (Table 3)A significant effect of this polymorphism was observed on9MW yield (119875 lt 001) The p4 genotype had the highestadditive estimated breeding value for nine-month weight(9MW) The p3 genotype had the highest weight for alltraits except 119882

6and GWS The effect of the PIT-1 gene

was significant (119875 lt 001) for nine-month weight (9W)from age six months to nine months (GSN) and from ninemonths to yearling weight (GNY) In the tested Makooeisheep population mean body weights of p3 genotype at9MW (3098 kg) were about 225 kg and 280 kg and 329 kghigher than those of the SSCP patterns p4 (2843 kg) and p2(2818) and p1 (2769) respectively Higher performance of p3animals in119882

9and GSN also p1 animal in GNY (postpuberty

ages) may be related to role of PIT-1 in postpuberty

4 Discussion

Most traits with economic interest in animal productionshowed continuous variation However their underlying


Table 4 Correlation between traits in Iranian ldquoMakooeirdquo sheepbreed resulted from SAS analysis

Trait 1 Trait 2 11990311990112

11990311988612

WW 6MW 077 075WW 9MW 061 056WW YW 054 0326MW 9MW 075 0936MW YW 059 0809MW YW 073 08711990311990112 phenotypic correlation between trait 1 and trait 2 11990311988612 direct additivegenetic correlation between trait 1 and trait 2

genetic nature is very complex It is possible to identify thechromosome regions containing genes affecting these traitsbecause of the current availability of neutral polymorphismsscattered throughout the genome [15] Growth is a compositeof complex developments that are influenced by geneticnutritional and environmental factors Preweaning (BWWWGBW) and Postweaning (6MW 9MW YW GWS GSY)growth traits have an important impact on the profitabilityin the sheep industry Therefore breeding for optimal bodyweight (BW) and larger gains is a major consideration insheep breeding programs

The SSCP analysis of those genes supposed to have anassociation with production traits could be a valuable alter-native approach to establish the allelic variants that are usefulas markers to aid in selectionMany several studies have beendone on the association of IGF-1 PIT-1 genes polymorphismsand other traits in animals Up to now there were very fewreports about IGF-1 PIT-1 genes SNPs and their relationshipwith growth and development traits of animals especially forsheep Genetic variations of the POU1F1 gene on growth traitsof Nanyang Cattle showed that body weight at 12 months washigher in the BB individuals than in the AA individuals (119875 lt005) [16] The statistical analysis showed that polymorphismof the IGF-I gene had a significant association (119875 lt 005)with birth weight (BW) body weight at 6 months (119882

6) and

at 12 months (11988212) in Nanjing Huang goats The goats with

genotype CC had significantly higher BW119882611988212than those

with genotype GC and had significantly higher 11988212

thanthose with genotype GG The body weight at 12 months washigher in the BB individuals than in the AA individuals (119875 lt005) [17]

Until now a few polymorphisms of IGF-1 and PIT-1genes have been detected in small ruminants and sheep havebeen studied much less Very little information is currentlyavailable to compare different Iranian sheep breeds To datethis was the (first) study that attempted to detect allelevariation in the ovine IGF-1 PIT-1 genes and its associationwith breeding value of growth traits in Iranian sheep breedsThe previous breeding programs in most research centers ofIran were based on only phenotypic characters The currentstudy confirmed the importance of molecular studies besidethe morphological data in detecting genetic variation amongindividuals in selecting diverse parents to construct a newpopulationThus adding sheep varieties expanding samplesand doing further correlation studies are still needed to

accumulate quantitative molecular genetics data in studyingthe relationship between the IGF-1 PIT-1 genes and growthtraits in sheep

In conclusion these results indicate that this polymor-phism contributed to variation in the traits analyzed andreinforced the possibility of using these polymorphisms inmolecular marker-assisted selection and breeding programs

Acknowledgments

The authors thank the Makooei Sheep Breeding Station stafffor providing us the data used in this study Also they aregrateful to the head of the Institute of Biotechnology UrmiaUniversity for providing laboratory facilities

References

[1] M-A Abbasi and F Ghafouri-Kesbi ldquoGenetic (co)variancecomponents for body weight and body measurements inMakooei SheeprdquoAsian-Australasian Journal of Animal Sciencesvol 24 no 6 pp 739ndash743 2011

[2] C Peter M Bruford T Perez S Dalamitra G Hewitt and GErhardt ldquoGenetic diversity and subdivision of 57 European andMiddle-Eastern sheep breedsrdquo Animal Genetics vol 38 no 1pp 37ndash44 2007

[3] W Ge M E Davis H C Hines K M Irvin and R C MSimmen ldquoAssociation of a genetic marker with blood seruminsulin-like growth factor-I concentration and growth traits inAngus cattlerdquo Journal of Animal Science vol 79 no 7 pp 1757ndash1762 2001

[4] G H Hua S L Chen J N Yu et al ldquoPolymorphism of thegrowth hormone gene and its association with growth traits inBoer goat bucksrdquoMeat Science vol 81 no 2 pp 391ndash395 2009

[5] J D Cogan and J A Phillips III ldquoGrowth disorders caused bygenetic defects in the growth hormone pathwayrdquo Advances inPediatrics vol 45 pp 337ndash361 1998

[6] C Supakorn ldquoThe important candidate genes in goatsmdashareviewrdquo Walailak Journal of Science and Technology vol 6 no1 pp 17ndash36 2009

[7] X F de la Rosa Reyna H M Montoya V V Castrellon A MS Rincon M P Bracamonte and W A Vera ldquoPolymorphismsin the IGF1 gene and their effect on growth traits in mexicanbeef cattlerdquo Genetics and Molecular Research vol 9 no 2 pp875ndash883 2010

[8] E A Wong S M Ohlsen J A Godfredson D M Dean andJ E Wheaton ldquoCloning of ovine insulin-like growth factor-IcDNAs heterogeneity in the mRNA populationrdquo DNA vol 8no 9 pp 649ndash657 1989

[9] K Xue H Chen S Wang et al ldquoEffect of genetic variationsof the POU1F1 gene on growth traits of nanyang cattlerdquo ActaGenetica Sinica vol 33 no 10 pp 901ndash907 2006

[10] E Bastos I Santos I Parmentier et al ldquoOvis aries POU1F1gene cloning characterization and polymorphism analysisrdquoGenetica vol 126 no 3 pp 303ndash314 2006

[11] C J Sanguinetti E D Neto and A J G Simpson ldquoRapidsilver staining and recovery of PCR products separated onpolyacrylamide gelsrdquo BioTechniques vol 17 no 5 pp 914ndash9211994

[12] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988


[13] A Yilmaz M E Davis and H C Hines ldquoA PCR-SSCPpolymorphism detected in the 5 flanking regions of the ovineIGF1 gene Research and reviews beef and sheeprdquo SpecialCircular vol 5 pp 170ndash199 2000

[14] K Meyer ldquoRestricted maximum-likelihood to estimatevariance-components for animal-models with several randomeffects using a derivative-free algorithmrdquo Genetics SelectionEvolution vol 21 pp 317ndash340 1989

[15] V Molaee R Osfoori M P Eskandari Nasab and S QanbarildquoGenetic relationships among six Iranian indigenous sheeppopulations based on microsatellite analysisrdquo Small RuminantResearch vol 84 no 1-3 pp 121ndash124 2009

[16] X Gao M-Y Shi X-R Xu J-Y Li H-Y Ren and S-Z XuldquoSequence variations in the bovine IGF-I and IGFBP3 genes andtheir associationwith growth anddevelopment traits inChinesebeef cattlerdquo Agricultural Sciences in China vol 8 no 6 pp 717ndash722 2009

[17] C Zhang W Zhang H Luo W Yue M Gao and Z Jia ldquoAnew single nucleotide polymorphism in the IGF-I gene andits association with growth traits in the Nanjiang Huang goatrdquoAsian-Australasian Journal of Animal Sciences vol 21 no 8 pp1073ndash1079 2008

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morphogenetic protein (BMP) genes are necessary for boneformation birthweight weaningweight body condition andmuscle growth [6] IGF1 is a mediator of many biologicaleffects it increases absorption of glucose stimulates myoge-nesis inhibits apoptosis participates in the activation of cellcycle genes increases the synthesis of lipids stimulates theproduction of progesterone in granular cells and intervenesin the synthesis of DNA protein RNA and functions in cellproliferation [7]The predicted sequence of amino acid oIGF-I peptide differs from the human bovine and porcine IGF-Isat a single amino acid (at position 66 alanine is substituted forproline) and differs from rat and mouse IGF-Is at positions4 and 5 respectively Ovine IGF-I amino-terminal peptidesare 1 amino acid longer than other mammalian IGFs dueto the presence of an extra amino acid (glutamine) thatis present at the proposed boundary of exon1 and exon2[8] IGF-1 is one of the most potent natural activators ofthe AKT signaling pathway a stimulator of cell growthand multiplication and a potent inhibitor of programmedcell death The transcription factor pituitary-1 (Pit-1 officialnomenclature now POU1F1) plays a major role in pituitarydevelopment and hormone expression It has been shownto be a positive regulator of the growth hormone prolactinand the thyrotropin subunit (TSH120573) in the mammalianpituitary [9] POU1F1 is a member of the POU-domain genefamily The POU-domain is composed of high-affinity DNAbonds the POU-specific domain (POU-SD) and a POU-homeodomain (POU-HD) [10] Mutations of the POU1F1gene led to a deficiency or an absence of GH PRL and TSH-120573 and can result in stunted growth Mammalian growth anddevelopment require steady expression of the POU1F1 gene[10] The ovine POU1F1 gene has a high level of conservationwith its bovine human and rat counterparts showing 982912 and 862 of similarity at coding levels respectively[10] Such findings suggest that variation in IGF-1 and PIT-1 genes in domestic animals may be important contributorsto growth rate However studies on associations of IGF-1 andPIT-1 Polymorphisms with growth traits are mainly carriedout in cattle but examination of associations between SNPsand growth traits in these genes has not been reported insheepTherefore the objective of this study was to determineassociations of genotypes with estimated breeding values ofgrowth traits in Makooei sheep

2 Materials and Methods

21 Collection of Sheep Blood Samples and Extraction ofGenomic DNA Extraction The Makooei breed of sheepwere examined in this study they are fat-tailed sheep withmedium body size white in color with black spots on faceand feet They are farmed in the East and West Azerbaijanprovinces of Iran for meat and wool [11] Blood samples werecollected into a 5mL EDTA vacutainer tube and transferredto the laboratory within 2 hours for DNA extraction TotalDNA extractions were made with a modified salting outmethod [12] from whole fresh blood Quality and quantity ofextracted DNA were measured on 08 agarose gel preparedin 05x TBE buffer (45Mm Tris base 45Mm boric acid

Table 1 Parameters estimated of analyzed traits

Traits 1205902119886

1205902119898

1205902119890

1205902119901ℎ2 1198982

BW 0045 0015 014 022 02 007WW 16 096 490 832 02 0126MW 259 281 648 1295 02 0229MW 235 204 68 1175 02 017YW 329 174 1038 1635 02 011

and 1mM EDTA pH 80) visualized with ethidium bromide(10 120583gmlminus1) and photographed under UV light Estimates ofvariance components and genetic parameters for birth weight(BW) weaning weight (WW) 6-month weight (6MW) 9-month weight (9MW) and yearling weight (YW) fromsingle-trait analyses are presented in Table 1

22 PIT1 Genotyping ThePIT1 gene was genotyped by PCR-SSCP (polymerase chain reaction-single strand conformationpolymorphism) A 295 bp fragmentwas amplified from a partof intron2 exon3 and a part of intron3 The primers usedfor the procedure were those designed by [10] PIT-1-uP 51015840-GAGGGATAATTACAAATGGTCC-3 and PIT-1-down 51015840-TGTTAACAGCTGTGGGACACAC-31015840 PCR contained 25ndash50 ng genomic DNA 10 pmoL of each primer 2 120583L 10xPCR buffer 15mMMgCl2 200Mm dNTP and 1 unit Taq-polymerase in a total volume of 20120583L DNA amplificationswere performed using aMaster cycler (Eppendorf Germany)programmed for a preliminary step of 15min at 95∘C fol-lowed by 34 cycles of 45 s at 94∘C then 1min at 58∘C followedby 45 s at 72∘C and finally an extension of 5min at 72∘C

23 IGF-1 Genotyping As primer pair IGF-1-up (51015840-ATTACAG CTGCCTGCCCCTT-31015840) and IGF-1-down (51015840-CACATCTGCTAATACACCTTACCCG-31015840) targeting a frag-ment of 265 bp were employed in DNA amplifications asdescribed by [13] PCR contained 25ndash50 ng genomic DNA10 pmoL of each primer 2120583L 10x PCR buffer 15mMMgCl2200Mm dNTP and 1 unit Taq-polymerase in a volume of20120583L DNA amplifications were done using a Master cyclerprogrammed for a preliminary step of 2min at 95∘C followedby 31 cycles of 45 s at 94∘C 30 s at 58∘C and 30 s at 72∘C witha final extension of 3min at 72∘C

24 Single Strand Confirmation Polymorphism (SSCP) Twopairs of oligonucleotide primers were designed and a stan-dard PCR protocol was used to amplify two fragments

SSCP analysis several factors were tested to optimize themethodology amount of PCR product (4ndash15120583L) dilutionin denaturing solution (20ndash85) denaturing solution (A95 of formamide 10mMNaOH 005 xylene-cyanol and005bromophenol blue B same asAplus 20mMof EDTA)acrylamide concentration (6ndash14) 6 percentage for PIT1 7percentage for IGF-1 percentage of crosslinking (15 to 5)presence (10) or absence of glycerol voltage (100ndash50V)running time (2ndash12 h) and running temperature (4 6 10 and15∘C) Each PCR reaction was diluted in denaturing solution





119884119894119895119896119897119898119899119900= 120583 + YR

119894+ SX119895+ BT119896

+ AD119897+ AN

119898+119872119899+ 119864119894119895119896119897119898119899119900

(1)


119894119895119896


119895is fixed


119897is fixed








119890)


(ℎ2119898) (12059021198981205902p)


119884119894119895119896119897119898= 120583 + 119866

119894+ 119875119889+ 119878119895+ 119897119904119896+ 119866119894times LS119896+ 119890119894119895119896119897119898 (2)

where 119884119894119895119896119897119898









3 Results






(a)


3W6





(b)


3W6

W9

W12






(a)


3W6

W9

W12






(b)


3W6














4 Discussion




Trait 1 Trait 2 11990311990112

11990311988612




6) and








Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





119884119894119895119896119897119898119899119900= 120583 + YR

119894+ SX119895+ BT119896

+ AD119897+ AN

119898+119872119899+ 119864119894119895119896119897119898119899119900

(1)


119894119895119896


119895is fixed


119897is fixed








119890)


(ℎ2119898) (12059021198981205902p)


119884119894119895119896119897119898= 120583 + 119866

119894+ 119875119889+ 119878119895+ 119897119904119896+ 119866119894times LS119896+ 119890119894119895119896119897119898 (2)

where 119884119894119895119896119897119898









3 Results






(a)


3W6





(b)


3W6

W9

W12






(a)


3W6

W9

W12






(b)


3W6














4 Discussion




Trait 1 Trait 2 11990311990112

11990311988612




6) and








Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)


3W6





(b)


3W6

W9

W12






(a)


3W6

W9

W12






(b)


3W6














4 Discussion




Trait 1 Trait 2 11990311990112

11990311988612




6) and








Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Trait 1 Trait 2 11990311990112

11990311988612




6) and








Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article PCR-SSCP Variation of IGF1 and PIT1 Genes and … · 2019. 12. 12. · PCR-SSCP...

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Transcript of Research Article PCR-SSCP Variation of IGF1 and PIT1 Genes and … · 2019. 12. 12. · PCR-SSCP...